WO2025024334A1 - Hematopoietic cell targeting conjugates and related methods - Google Patents

Abstract

Provided herein are,inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFR1)); operably connected to (b) at least one oligonucleotide that modulates the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a target cell; and methods of manufacturing and pharmaceutical compositions comprising the same. Further provided herein are methods of utilizing the conjugates, including,e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., α-thalassemia, β-thalassemia, δ-thalassemia, or γ-thalassemia).

Description

HEMATOPOIETIC CELL TARGETING CONJUGATES AND RELATED METHODS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No.: 63/514,956, filed July 21, 2023, the entire contents of which is incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 16, 2024, is named 62992_6WO01_SL.xml and is 2,241 ,941 bytes in size.

1. FIELD

[0003] This disclosure relates to, inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); and an oligonucleotide that modulates the expression and/or activity of a target gene expressed by a target cell. The disclosure further relates to pharmaceutical compositions comprising the same; and methods of utilizing the same, including, e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., a-thalassemia, 0- thalassemia, 5-thalassemia, or y-thalassemia)).

2. BACKGROUND

[0004] The bone marrow is a soft gelatinous tissue that fills the cavities of the bones. In adults bone marrow is either red or yellow, depending upon the preponderance of hematopoietic (red) or fatty (yellow) tissue. Genetic perturbations, including, e.g., gene mutations, overexpression of a gene, deficiency of a gene (and in turn the encoded product (e.g., protein)) in subsets of cells within the bone marrow are associated with various genetic disorders, including, e.g., genetic blood disorders (e.g., hemoglobinopathies and inherited bone marrow failure syndromes).

[0005] In humans the red bone marrow forms all of the blood cells, including red blood cells, with the exception of the lymphocytes, which are produced in the marrow and reach their mature form in the lymphoid organs. Normal red blood cells contain the protein hemoglobin, which functions to transport oxygen (O2) from the lungs to peripheral tissues and carbon dioxide (CO2) from the tissues to the lungs. Hemoglobin is a heterotetramer composed of a-like and (3-like globin subunits, each bound to a heme prosthetic group. Hemoglobin is synthesized from separate a-likc and P-like globin gene clusters, with different types of hemoglobin produced through different subunit combinations. Fetal hemoglobin is the primary hemoglobin produced by the fetus. In healthy humans, a shift from y-globin to p-globin gene expression around birth underlies a switch from fetal hemoglobin to adult hemoglobin production, such that by 6 months of age the major hemoglobin adult hemoglobin. The hemoglobin switch, is not total or irreversible; as adults retain the ability to produce residual levels of fetal hemoglobin (<1% of total hemoglobin). The switch from fetal to adult hemoglobin relies on repression or silencing of the upstream y-globin gene, through a network of repressor proteins. Hemoglobinopathies are a class of genetic diseases associated with the abnormal production and/or structure of hemoglobin and are the most common inherited blood diseases.

3. SUMMARY

[0006] Provided herein are, inter alia, conjugates comprising a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent) that comprises a protein (e.g., an antibody) that specifically binds to TFR (e.g., hTFR (e.g., hTFRl)); and an oligonucleotide that modulates the expression and/or activity of a target gene expressed by a target cell; and methods of manufacturing and pharmaceutical compositions comprising the same. Further provided herein are methods of utilizing the conjugates including, e.g., methods of treating hemoglobinopathies (e.g., sickle cell disease (SCD) or a thalassemia (e.g., a-thalassemia, P-thalassemia, 5-thalassemia, or y-thalassemia (e.g., P-thalassemia)).

[0007] Accordingly, in one aspect provided herein are conjugates comprising: (a) a hematopoietic cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to (b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by the hematopoietic cell.

[0008] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

[0009] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR 1)) expressed on the surface of a hematopoietic cell, the conjugate docs not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0010] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0011] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the hematopoietic cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0012] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1)) is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody. Tn some embodiments, the antibody does not (or docs not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl). In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv) -Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)z (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., a human IgG (hlgG)) antibody. In some embodiments, the antibody is a hlgGl, hIgG2, hIgG3, or hIgG4 antibody (e.g., a hlgGl or hIgG4 antibody).

[0013] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region. In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is a hlg. In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl or hIgG4.

[0014] In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and/or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))). In some embodiments, the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and/or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))).

[0015] In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and/or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat. [0016] In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is h!gG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and/or an alanine at amino acid position L235, numbering according to EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0017] In some embodiments, the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond. In some embodiments, the antibody the amino acid sequence of the first Fc region and/or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

[0018] In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

[0019] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof). In some embodiments, the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0020] In some embodiments, the oligonucleotide enhances the expression and/or activity of the target gene, nucleic acid e.g., mRNA), and/or protein. In some embodiments, the oligonucleotide inhibits the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the oligonucleotide modulates (e.g., enhances or inhibits) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)). In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing. [0021] In some embodiments, the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein). In some embodiments, the oligonucleotide is single stranded or double stranded. In some embodiments, the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region. In some embodiments, the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule. In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region). In some embodiments, the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide pairs in length.

[0022] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified. In some embodiments, substantially all (or all) of the nucleotides in the oligonucleotide are modified. In some embodiments, at least one of the modified nucleotides comprises a modified sugar (e.g., ribose moiety). In some embodiments, at least one of the modified nucleotides comprises a modified nucleobase. In some embodiments, the oligonucleotide comprises at least one modified intemucleoside linkage (e.g., at least one phosphoro thioate intemucleoside linkage). In some embodiments, the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'- F), 2'-O-methyl (2'-0-Me), 2'-O-methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

[0023] In some embodiments, the protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with one or more hemoglobinopathy. In some embodiments, inhibition of or a reduction in expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and/or an increase in the ratio of fetal hemoglobin to adult hemoglobin. In some embodiments, expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and/or an increase in ratio of adult hemoglobin to fetal hemoglobin. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is a transcription factor. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0024] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S 17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1) (e.g., human HAX1), Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) (e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPL Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Like (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

[0025] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), or KLF Transcription Factor 1 (KLF1) (e.g., hKLFl).

[0026] In some embodiments, (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker. In some embodiments, the linker is cleavable or non-cleavable.

[0027] In some embodiments, the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides. In some embodiments, each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

[0028] In one aspect, provided herein are conjugates comprising: (a) an erythroid precursor cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to (b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by the erythroid precursor cell.

[0029] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

[0030] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0031] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0032] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[0033] In some embodiments, the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1)) is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody. In some embodiments, the antibody does not (or does not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl). In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv)i-Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)2 (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the antibody is an IgG (e.g., a human IgG (hlgG)) antibody. In some embodiments, the antibody is a hlgGl, hIgG2, hIgG3, or hIgG4 antibody (e.g., a hlgGl or hIgG4 antibody).

[0034] In some embodiments, the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region. In some embodiments, the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc). In some embodiments, the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig is a hlg. In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl or hIgG4.

[0035] In some embodiments, the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and/or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fey I Ila))). In some embodiments, the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and/or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))).

[0036] In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and/or an alanine at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat. In some embodiments, the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat. [0037] In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and/or an alanine at amino acid position L235, numbering according to EU index of Kabat. In some embodiments, the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

[0038] In some embodiments, the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond. In some embodiments, the antibody the amino acid sequence of the first Fc region and/or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

[0039] In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat. In some embodiments, the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

[0040] In some embodiments, the protein that specifically binds TFR (e.g., hTFR e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof/). In some embodiments, the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

[0041] In some embodiments, the oligonucleotide enhances the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the oligonucleotide inhibits the expression and/or activity of the target gene, nucleic acid e.g., mRNA), and/or protein. In some embodiments, the oligonucleotide modulates (e.g., enhances or inhibits) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)). In some embodiments, the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule). In some embodiments, the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing. [0042] In some embodiments, the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein). In some embodiments, the oligonucleotide is single stranded or double stranded. In some embodiments, the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region. In some embodiments, the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule. In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region). In some embodiments, the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide pairs in length.

[0043] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified. In some embodiments, substantially all (or all) of the nucleotides in the oligonucleotide are modified. In some embodiments, at least one of the modified nucleotides comprises a modified sugar- (e.g., ribose moiety). In some embodiments, at least one of the modified nucleotides comprises a modified nucleobase. In some embodiments, the oligonucleotide comprises at least one modified intemucleoside linkage (e.g., at least one phosphoro thioate intemucleoside linkage). In some embodiments, the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'- F), 2'-O-methyl (2'-0-Me), 2'-O-methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-0-DMAE0E), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

[0044] In some embodiments, the protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with one or more hemoglobinopathy. In some embodiments, inhibition of or a reduction in expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and/or an increase in the ratio of fetal hemoglobin to adult hemoglobin. In some embodiments, expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and/or an increase in ratio of adult hemoglobin to fetal hemoglobin. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is a transcription factor. [0045] In some embodiments, the target gene and/or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

[0046] In some embodiments, the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11 A (e.g., hBCLl 1 A)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1) (e.g., human HAX1), Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) (e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPL Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Like (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

[0047] In some embodiments, the target gene is BCL11A (e.g., hBCLUA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

[0048] In some embodiments, (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide. In some embodiments, (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker. In some embodiments, the linker is cleavable or non-cleavable.

[0049] In some embodiments, the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides. In some embodiments, each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

[0050] In one aspect, provided herein are cells comprising a conjugate described herein. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

[0051] In one aspect, provided herein are pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable excipient. [0052] In one aspect, provided herein are kits comprising a conjugate described herein or a pharmaceutical composition described herein.

[0053] In one aspect, provided herein are methods of delivering a conjugate or pharmaceutical composition to a cell, the method comprising introducing into a cell a conjugate described herein, or a pharmaceutical composition described herein, to thereby deliver the conjugate or pharmaceutical composition into the cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0054] In one aspect, provided herein are methods of delivering a conjugate, cell, or pharmaceutical composition to a subject, the method comprising administering to the subject a conjugate described herein, a cell described herein, or a pharmaceutical composition described herein, to thereby deliver the conjugate, cell, or pharmaceutical composition to the subject.

[0055] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0056] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0057] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. [0058] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

[0059] In one aspect, provided herein are methods of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject).

[0060] In one aspect, provided herein are methods of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a subject (e.g., a human subject). In some embodiments, the target is BCL11A (e.g., hBCLHA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

[0061] In one aspect, provided herein are methods of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

[0062] In one aspect, provided herein are methods of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

[0063] In one aspect, provided herein are methods of inducing expression of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby induce expression of fetal hemoglobin the subject.

[0064] In one aspect, provided herein are methods of increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby increase the level of fetal hemoglobin the subject.

[0065] In one aspect, provided herein are methods of increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby increase the ratio of fetal hemoglobin to adult hemoglobin in the subject.

[0066] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited blood disorder in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited blood disorder in the subject. In some embodiments, the inherited blood disorder is a hemoglobinopathy or an inherited bone marrow failure syndrome.

[0067] In one aspect, provided herein are methods of treating, ameliorating, or preventing a hemoglobinopathy in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the hemoglobinopathy in the subject. In some embodiments, the subject is a human.

[0068] In some embodiments, the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

[0069] In some embodiments, the hemoglobinopathy is sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or '/-thalassemia). [0070] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, P- thalassemia, 8-thalassemia, or ^-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

[0071] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia).

[0072] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited bone marrow failure syndrome in a subject, the method comprising administering to the subject a conjugate described herein, or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject.

[0073] In some embodiments, the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, or thrombocytopenia absent radii (TAR).

[0074] In one aspect, provided herein are conjugates described herein, cells described herein, or pharmaceutical compositions described herein for use in the treatment of a disease in a subject in need thereof.

[0075] In one aspect, provided herein are conjugates described herein, cells described herein, or pharmaceutical compositions described herein for use as a medicament.

[0076] In one aspect, provided herein are uses of a conjugate described herein, a cell described herein, or a pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIGS. 1A-1D are line graphs showing the binding of recombinantly expressed anti- TFR1 monoclonal antibodies (mAbs) (set forth in Example 1) (or isotype control) to soluble TFR1 (using Octet BLI). FIG. 1A is a line graph showing the binding (showing a lack of binding) of isotype control antibody to soluble TFR1. FIG. IB is a line graph showing the binding of anti- TFR1 mAb2 to soluble TFR1. FIG. 1C is a line graph showing the binding of anti-TFRl mAb3 to soluble TFR1. FIG. ID is a line graph showing the binding of anti-TFRl Fab to soluble TFR1. [0078] FIGS. 2A-2D are line graphs showing the binding of anti-TFRl mAbs (set forth in Example 1) to the surface of erythroid-progenitor cells. FIG. 2A is a line graph showing the percent of positive cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 2B is a line graph showing the MFI of cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 2C is a line graph showing the percent of positive cells treated with anti-TFRl Fab (at the indicated concentration). FIG. 2D is a line graph showing the MFI of cells treated with anti-TFRl Fab (at the indicated concentration).

[0079] FIGS. 3A-3D are graphs showing the internalization of anti-TFRl mAbs (set forth in Example 1) (or isotype control) into erythroid-progenitor cells. FIG. 3A is a graph showing the percent positive cells treated with anti-TFRl mAb2 or anti-TFRl mAb3 (at the indicated concentration). FIG. 3B is a graph showing the MFI of cells treated with anti-TFRl mAb2 or anti- TFRl mAb3 (at the indicated concentration). FIG. 3C is a graph showing the percent positive cells treated with anti-TFRl Fab (at the indicated concentration). FIG. 3D is a graph showing the MFI of cells treated with anti-TFRl Fab (at the indicated concentration).

[0080] FIGS. 4A-B are line graphs showing the extent of mRNA knock-down of the indicated target gene by the indicated siRNA in erythroid-progenitor cells. FIG. 4A is a line graph showing HPRT1-2 siRNA mediated HPRT1 mRNA knockdown (at the indicated concentration of siRNA). FIG. 4B is a line graph showing BCL11A-11 siRNA or BCL11A-5 siRNA mediated BCL11A mRNA knockdown (at the indicated concentration of siRNA).

[0081] FIGS. 5A-5B are bar graphs showing the extent of the decrease in protein levels of the indicated target by the indicated siRNA in erythroid-progenitor cells. FIG. 5A is a bar graph showing the BCL11A-11 siRNA or BCA11A-5 siRNA mediated decrease in the level of BCL11A protein (at the indicated concentration of siRNA). FIG. SB is a bar graph showing the HPRT1-2 siRNA mediated decrease in the level of HPRT1 protein (at the indicated concentration of siRNA). [0082] FIGS. 6A-6K are line graphs showing the binding of each of the anti-TFRl antibodysiRNA conjugates (AOCs#l-l l) binding to recombinant TFR1 protein (using Octet BLI). FIG. 6A is a line graph showing the binding of AOC#1 to recombinant TFR1. FIG. 6B is a line graph showing the binding of AOC#2 to recombinant TFR1. FIG. 6C is a line graph showing the binding of A0C#3 to recombinant TFR1. FIG. 6D is a line graph showing the binding of A0C#4 to recombinant TFR1. FIG. 6E is a line graph showing the binding of A0C#5 to recombinant TFR1. FIG. 6F is a line graph showing the binding of A0C#6 to recombinant TFR1. FIG. 6G is a line graph showing the binding of A0C#7 to recombinant TFR1. FIG. 6H is a line graph showing the binding of A0C#8 to recombinant TFR1. FIG. 61 is a line graph showing the binding of A0C#9 to recombinant TFR1. FIG. 6 J is a line graph showing the binding of AOC#10 to recombinant TFR1. FIG. 6K is a line graph showing the binding of AOC#11 to recombinant TFR1.

[0083] FIGS. 7A-7B are line graphs showing the % viability of erythroid precursor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIG. 7A is a line graph showing the % viability of erythroid precursor cells 48 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIG. 7B is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC at the indicated concentration.

[0084] FIGS. 7C-7D are line graphs showing the knockdown of the HPRT1 transcript in erythroid-progenitor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIGS. 7C is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 48 after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration. FIGS. 7D is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (AOC#1 or AOC#2) at the indicated concentration.

[0085] FIGS. 8A-8B are line graphs showing the % viability of erythroid precursor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8A is a line graph showing the % viability of erythroid precursor cells 48 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8B is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration.

[0086] FIGS. 8C-8D are line graphs showing the knockdown of the HPRT 1 mRNA transcript in erythroid-progenitor cells 48 and 72 hours after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8C is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 48 after the first dose with the indicated AOC (AOC#3 or AOC#4) at the indicated concentration. FIG. 8D is a line graph showing the knockdown of the HPRT1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (A0C#3 or A0C#4) at the indicated concentration.

[0087] FIG. 9 is a line graph showing knockdown of the HPRT1 protein in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#2 or AOC#4).

[0088] FIG. 10A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#5) at the indicated concentration. FIG. 10B is a line graph showing the knockdown of the HPRT 1 mRNA transcript in erythroid-progenitor cells 72 after the first dose with the indicated AOC (AOC#5) at the indicated concentration.

[0089] FIG. 11A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#10 or AOC#11) at the indicated concentration. FIG. 11B is a line graph showing the knockdown of the BCL11 A mRNA transcript in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#10 or AOC#11) at the indicated concentration.

[0090] FIG. 12A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#9) at the indicated concentration. FIG. 12B is a line graph showing the knockdown of the BCL11 A transcript in erythroid-progenitor cells 72 hours after the first dose with the indicated AOC (AOC#9) at the indicated concentration.

[0091] FIG. 13A is a line graph showing the % viability of erythroid precursor cells 72 hours after the first dose with the indicated AOC (AOC#7 or AOC#8) at the indicated concentration. FIG. 13B is a line graph showing the knockdown of the BCL11A mRNA transcript in erythroid- progenitor cells 72 hours after the first dose with the indicated AOC (AOC#7 or AOC#8) at the indicated concentration.

5. DETAILED DESCRIPTION

[0092] The inventors have, inter alia, discovered that molecular payloads (e.g., oligonucleotides) capable of modulating (e.g., inhibiting) expression and/or activity of genes capable of modulating (e.g., repressing) e.g., fetal hemoglobin production, can be specifically targeted to hematopoietic cells (e.g., erythroid precursor cells) through conjugation to a targeting agent (e.g., an anti-TFR antibody). As such, the conjugates described herein are useful, inter alia, for the treatment of hemoglobinopathies (including, e.g., sickle cell disease and thalassemias). As such, the current disclosure provides conjugates; and their use in, inter alia, pharmaceutical compositions, and methods of treating diseases (e.g., hemoglobinopathies).

TABLE OF CONTENTS

5.1 Definitions

5.2 Conjugates

5.3 Hematopoietic Cell Targeting Agents

5.3.1 TFR Targeting Agents

5.3.1.1 TF Proteins

5.3.1. l(i) Exemplary hTF Variant Proteins

5.3.1. l(ii) Heterologous Moieties

5.3.1.2 TFR Binding Peptides and Antibody-Like Scaffolds

5.3.1.2(i) Exemplary TFR Binding Peptides and Antibody-Like Scaffolds

5.3.1.2(ii) Heterologous Moieties

5.3.1.3 Anti-TFR (e.g., Anti-TFRl) Antibodies

5.3.1.3(i) Exemplary Anti-TFR (e.g., Anti-TFRl) Antibodies

5.3.1.4 Ig Effector Function

5.3.1.5 Promotion of Heterodimerization

5.3.1.6 Ig Constant Region Variations for Site Specific Conjugation

5.3.L7 Exemplary Variant Fc Regions

5.4 Methods of Making Proteins (e.g., Targeting Agents)

5.5 Molecular Payloads

5.5.1 Oligonucleotides

5.5.1.1 Overall Length

5.5.1.2 Targeting Region

5.5.1.3 Antisense Oligonucleotides

5.5.1.3(i) Overall Length

5.5.1.3(H) Targeting Region

5.5.1.4 RNAi Agents

5.5.1.4(i) Antisense Strand

5.5.1.4(i)(a) Overall Length

5.5.1.4(i)(b) Targeting Region 5.12.2 Methods of Modulating Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and/or Protein

5.12.3 Methods of Reducing or Inhibiting Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and/or Protein

5.12.4 Methods of Modulating Splicing of a Target mRNA

5.12.5 Methods of Inducing Expression of Fetal Hemoglobin

5.12.6 Methods of Treating an Inherited Blood Disorder

5.12.7 Methods of Treating Hemoglobinopathies

5.12.8 Methods of Treating an Inherited Bone Marrow Failure Syndrome

5.13 Kits

5.1 Definitions

[0093] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0095] In this disclosure, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0096] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and “consisting essentially of’ are also provided herein.

[0097] The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0098] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0099] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the ait, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular values or compositions are provided in the disclosure, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition.

[00100] Where proteins are described herein, it is understood that nucleic acid molecules (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) encoding the protein are also provided herein. [00101] Where proteins, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, nucleic acid molecules, vectors, carriers, etc. are also provided herein.

[00102] Where proteins, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, nucleic acid molecules, etc. are also provided herein.

[00103] Where polypeptides or sets of polypeptides are described herein, it is understood that proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa.

[00104] Where proteins are described herein, it is understood that polypeptides comprising the same amino acid sequence either linear or folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein.

[00105] As used herein, the term “administering” refers to the physical introduction of an agent (e.g., a conjugate described herein), e.g., a therapeutic agent (or a precursor of an agent (e.g., a precursor of a therapeutic agent) that is metabolized or altered within the body of the subject to produce the agent (e.g., therapeutic agent) in vivo) to a subject, using any of the various methods and delivery systems known to those skilled in the ait. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. Administering includes self-administration.

[00106] The terms “agent” and “moiety” arc used interchangeably herein and arc used generically to describe any macro or micro molecule (and any combination thereof). Exemplary agents include, but are not limited proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG), conjugates (e.g., described herein), and any combination of the foregoing. Agents may contain more than one individual agent (wherein the individual agents are the same or different). For example, an agent may comprise an antibody (e.g., a targeting agent described herein) and an oligonucleotide (e.g., an oligonucleotide described herein). Agents as defined herein include e.g., conjugates described herein.

[00107] As used herein, the term “affinity” refers to the strength of the binding of one protein (e.g., an Antibody) to another protein (e.g., an Antigen). The affinity of a protein is measured by the dissociation constant Kd, defined as [Antibody] x [Antigen] / [Antibody-Antigen] where [Antibody-Antigen] is the molar concentration of the Antibody-Antigen complex, [Antibody] is the molar concentration of the unbound Antibody and [Ligand] is the molar concentration of the unbound Antigen. The affinity constant Ka is defined by 1/Kd. Standard methods of measuring affinity are known to the person of ordinary skill in the art. Exemplary methods of measuring affinity include, surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the ait (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes).

[00108] As used herein, the term “antibody” or “antibodies” is used in the broadest sense and encompasses various immunoglobulin (Ig) (e.g., human Ig (hlg), murine Ig (mlg)) structures, including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity (i.e., antigen binding fragments or variants). The term antibody thus includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and/or VL regions; and antibody-like scaffolds (e.g., fibronectins). Examples of antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), affybodies, diabodies, tribodies, hctcroconjugatc antibodies, antibody-drug conjugates, single domain antibodies (e.g.,VHH, (VHH)2), single chain antibodies, single-chain Fvs (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab’)2 fragments, disulfide-linked Fvs (sdFv), Fc fusions (e.g., Fab-Fc, scFv- Fc, VHH-Fc, (scFv)₂-Fc, (VHH)2-FC), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. Antibodies can be of Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class (e.g., IgGi, IgG2, IgG , IgG4, IgAi or IgA2), or any subclass (e.g., IgG2a or IgG2b) of Ig). In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgGi or IgG4) or subclass thereof. In certain embodiments, antibodies described herein are mlgG antibodies, or a class (e.g., mlgGl or mIgG2a) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgGi or IgG4 monoclonal antibody. In some embodiments, the term antibodies refers to a monoclonal or polyclonal antibody population. Antibodies described herein can be produced by any standard methods known in the art, e.g., recombinant production in host cells, see, e.g., § 5.4; or synthetic production.

[00109] As used herein, the term “antibody-like scaffold” refers to non-Ig based antigen binding domain. Various antibody-like scaffolds are known in the art. For example, 10th type III domain of fibronectin (e.g., AdNectins®) and designed ankyrin repeat proteins (e.g., DARPins®) have been used as alternative scaffolds for antigen -binding domains, see, e.g., Gebauer and Skcrra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008), the full contents of each of which is incorporated by reference herein for all purposes. Exemplary antibody-like scaffolds include, but are not limited to, lipocalins (see, e.g., US7250297) (e.g., Anticalin®), protein A-derived molecules such as z- domains of protein a (see, e.g., US5831012) (e.g., Affibody®), A domains of membrane receptors stabilized by disulfide bonds and Ca2+ (see, e.g., US7803907) (e.g., Avimer/Maxibody®), a serum transferrin (see, e.g., US2004023334) (e.g., Transbody®); a designed ankyrin repeat protein (see, e.g., US7417130) (e.g., DARPin®), a fibronectin (see, e.g., US6818418) (e.g., AdNectin®), a C- type lectin domain (see, e.g., US2004132094) (e.g., Tetranectin®); a human gamma-crystallin or ubiquitin (see, e.g., US7838629) (e.g., Affilin®); a kunitz type domain of human protease inhibitors (see, e.g., US2004209243), C-Type Lectins (see, e.g., US2004132094) (e.g., Tetranectins®), cysteine knots or knottins (see, e.g., US7186524) (e.g., Microbodies®), nucleic acid aptamers (see, e.g., US5475096), thioredoxin A scaffold (see, e.g., US6004746) (peptide aptamers), and 10th type III domain of fibronectin (see, e.g., US6818418) (e.g., AdNectins®), and cystine-dense peptides (see, e.g., W02023023031). Additional exemplary antibody-like scaffolds are known in the art and for example described in Storz U. Intellectual property protection: strategies for antibody inventions. MAbs. 2011 ;3(3):310-317. doi:10.4161/mabs.3.3.15530. The entire contents of each of the foregoing references is incorporated herein by reference for all purposes. Antibody like scaffolds include e.g., naturally occurring antigen binders, variant (e.g., functional variants) of naturally occurring antigen binders, fragments (e.g., functional fragments) of naturally occurring antigen binders, and synthetic antigen binders (i.e., not naturally occurring antigen binders).

[00110] As used herein, the term “antibody dependent cell mediated cytotoxicity” or “ADCC” refers to an immune mechanism leading to the lysis of antibody (or an Fc region containing protein) (e.g., an Ig Fc containing fusion protein described herein)-coated target cells by immune effector cells (e.g., NK cells). As used herein, the term “reduced ADCC” and the like refers to either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody (or an Ig Fc region containing protein) (e.g., an Fc region containing fusion protein described herein) in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC defined above. The reduction in ADCC is relative to the ADCC mediated by the same antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered (e.g., does not comprise one or more amino acid variation, e.g., amino acid substitution, that mediates a decrease in ADCC). For example the reduction in ADCC mediated by an antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) comprising in its Fc region an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody (or an Fc region containing protein) (e.g., an Fc containing fusion protein described herein) without said amino acid substitution in the Fc region. [00111] As used herein, the term “antisense oligonucleotide” or “ASO” refer to the standard single stranded oligonucleotides known in the art that arc e.g., capable of modulating expression of a target gene (or protein) by hybridizing to a target nucleic acid (e.g., an mRNA encoded by the target gene and encoding the target protein), in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides include DNA, RNA, and hybrid DNA/RNA oligonucleotides.

[00112] As used herein, the term “antisense strand” with reference to an oligonucleotide described herein (e.g., an RNAi agent (e.g., siRNA agent), an antisense oligonucleotide) refers to an oligonucleotide that comprises a region of complementarity comprising a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to a target nucleic acid sequence (e.g., a target mRNA (e.g., a portion of a target mRNA). In the case of single stranded oligonucleotides (e.g., antisense oligonucleotides) the antisense strand will be the only strand. In the case of double stranded oligonucleotides (e.g., siRNAs) the sense strand will typically be paired with a sense strand (as described herein).

[00113] As used herein, the term “ BCL11 Transcription Factor A” or “BCL11A” refers to the C2H2 type zinc-finger transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human BCL11A (hBCLUA) protein is set forth in SEQ ID NO: 291 (NCBI Ref.: NP_075044.2).

[00114] As used herein, the term “bicyclic sugar” refers to a modified sugar (e.g., ribose, deoxyribose) moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In some embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In some embodiments, the furanosyl sugar moiety is a ribosyl moiety.

[00115] As used herein, the term “bicyclic nucleoside” (“BNA”) is a nucleoside comprising a bicyclic sugar.

[00116] As used herein, the term “blunt end” refers to a double stranded oligonucleotide that does not contain any unpaired nucleotides at the end (e.g., 3' terminus, 5' terminus) of the double stranded oligonucleotide (i.e., no nucleotide overhang(s)). The double stranded oligonucleotide can have, for example, a blunt end at the 3' end, 5' end, or both the 3' and 5' end of the molecule.

[00117] As used herein, the term “CDR” or “complementarity determining region” refers to the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chcm. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), the entire contents of each of which is incorporated herein by reference for all purposes. Unless otherwise specified, the term “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). A person of ordinary skill in the art would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the ail.

[00118] The terms “CHI” and “CHI region” are used interchangeably herein and refer to the first constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CHI region is set forth in SEQ ID NO: 171; and the amino acid sequence of an exemplary reference h!gG4 CHI region is set forth in SEQ ID NO: 184.

[00119] The terms “CH2” and “CH2 region” are used interchangeably herein and refer to the second constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH2 region is set forth in SEQ ID NO: 173; and the amino acid sequence of an exemplary reference hIgG4 CH2 region is set forth in SEQ ID NO: 186.

[00120] The terms “CH3” and “CH3 region” are used interchangeably herein and refer to the third constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl CH3 region is set forth in SEQ ID NO: 174; and the amino acid sequence of an exemplary reference hIgG4 CH3 region is set forth in SEQ ID NO: 187.

[00121] As used herein, the term “complementary” in reference to a first nucleotide sequence (e.g., a sense strand or a target mRNA) in relation to a second nucleotide sequence (e.g., an antisense strand or an antisense oligonucleotide), refers to the ability of a nucleic acid molecule comprising the first nucleotide sequence to hybridize to a nucleic acid molecule comprising the second nucleotide sequence and form a double stranded region (through base pair hydrogen bonds) under suitable in vivo or vitro conditions (e.g., under certain standard conditions, under mammalian (e.g., human) physiological conditions). A person of ordinary skill in the ail would be able to select the set of conditions most appropriate for a hybridization test. Complementary sequences include, e.g., Watson-Crick base pairs. For example, complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); and cytosine (C) and guanine (G). Complementary nucleobase pairs include natural and modified nucleotides, and nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. As such, determinations of complementarity (as described herein) arc independent of nucleotide chemical modifications (e.g., as described herein). For example, (C) and 5-methyl cytosine (mC) are both complementary to (G).

[00122] As used herein, the term “conjugation” refers to the operable connection (e.g., chemical conjugation) of at least a first agent (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)) with a second agent (e.g., a protein (e.g., a targeting agent (e.g., a targeting agent described herein (e.g., a hematopoietic cell targeting agent (erythroid precursor cell targeting agent described herein (e.g., an anti-TFR (e.g., anti-hTFR (e.g., anti-hTFRi)) antibody described herein))))). The first agent can be directly connected to the second agent or indirectly connected (e.g., through a linker (e.g., as described herein)). Methods of operably connected two agents (e.g., chemical conjugation methods) are well known in the art, as are commercially available conjugation reagents and kits, with detailed instructions for their use readily available from the commercial suppliers. Operable connection (e.g., chemical conjugation) includes both covalent and non- covalent conjugation. In some embodiments, the operable connection (e.g., chemical conjugation) comprises the covalent linkage of the first agent (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)) with the second agent (e.g., a protein (e.g., a targeting agent (e.g., a targeting agent described herein (e.g., a hematopoietic cell targeting agent (erythroid precursor cell targeting agent described herein (e.g., an anti-TFR (e.g., anti-hTFR (e.g., anti-hTFRi)) antibody described herein))))).

[00123] The terms “constant region” and “constant domain” are used interchangeably herein and refer to a carboxyl terminal portion of a light and/or heavy chain of a full-length antibody which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with an Ig Fc receptor (e.g., Fc gamma receptor). The constant region of an Ig molecule generally has a more conserved amino acid sequence relative to an Ig variable domain.

[00124] As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology. The term disease includes infection (e.g., a viral, bacterial, fungal, protozoal infection). [00125] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple dcoxyribonuclcotidcs that arc polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[00126] As used herein, the term “double stranded oligonucleotide” refers to a complex of two nucleic acid molecules comprising a double stranded region comprising two anti-parallel and at least partially (e.g., substantially, fully) complementary nucleic acid sequences that form the double stranded region. For example, in some embodiments, the double stranded oligonucleotide comprises a sense strand and an antisense strand.

[00127] The term “effector function” when used in reference to an Ig Fc region or a protein comprising an Ig Fc region (e.g., a full-length antibody) refers to those biological activities attributable to the Ig Fc region of a typical full-length antibody, which therefore vary with the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla)), and Clq binding.

[00128] As used herein, the term “erythroid precursor cell” refers to any precursor of a mature erythrocyte (i.e., a mature enucleated red blood cell). As such, erythroid precursor cells include, but are not limited to, megakaryocyte erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, late erythroblast cells, and reticulocytes. In some embodiments, erythroid precursor cells include megakaryocyte erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, and late erythroblast cells. In some embodiments, erythroid precursor cells include proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, and late erythroblast cells.

[00129] As used herein, the term “erythroid precursor cell targeting agent” refers to an agent that specifically binds to an antigen expressed on an erythroid precursor cell (or a subset thereof). For example, the antigen expressed in or on the erythroid precursor cell may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, an erythroid precursor cell targeting agent specifically binds to an antigen on the erythroid precursor cell that facilitates internalization of the erythroid precursor cell targeting agent (and any associated molecular payload) into the erythroid precursor cell. In some embodiments, an ery throid precursor cell targeting agent specifically binds to an internalizing, cell surface receptor on the erythroid precursor cell and is capable of being internalized into the erythroid precursor cell through receptor mediated internalization. In some embodiments, the erythroid precursor cell targeting agent is a protein (e.g., antibody), a peptide, a nucleic acid (e.g., an aptamer), or small molecule. In some embodiments, the erythroid precursor cell targeting agent is linked to a molecular payload.

[00130] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, the entire contents of each of which is incorporated herein by reference for all purposes.

[00131] As used herein, the term “Fab” refers to an antigen binding domain that comprises a Fab heavy chain that comprises from N- to C-terminus a VH region and a CHI region; and a light chain comprising from N- to C-terminus a VL region and a CL region; and wherein the Fab heavy chain and the light chain associate to form an antigen binding domain.

[00132] The term “Fab-Fc” as used herein refers to an antibody that comprises a Fab operably linked to an Fc region.

[00133] As used herein, the term “Fc region” refers to the C-terminal region of a Ig (e.g., a human Ig) heavy chain that comprises from N- to C-terminus at least a CH2 region operably connected to a CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region (e.g., comprises one or more amino acid modification), see, e.g., §§ 5.3.2.1, 5.3.2.2, 5.3.2.3, 5.3.2.4. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (K. O. Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” 2019, Frontiers in Immunology, V. 10, Art. 1296, pp. 1-20, the entire contents of which is incorporated herein by reference for all purposes).

[00134] As used herein, the terms “first” and “second” with respect to Fc regions etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation in the protein unless explicitly so stated. For example, an antibody described herein (e.g., in the case of a full-length antibody) may contain two Fc regions that associate e.g., via one or more covalent (e.g., disulfide) bond. [00135] As used herein, the term “framework region” or “FR region” refers to the amino acid residues that arc part of the variable region of an antibody, but arc not part of the CDRs (e.g., using the Kabat definition of CDRs).

[00136] As used herein, the term “full-length antibody” refers to an antibody having a structure substantially similar’ to a native antibody structure (i) a first Ig light chain comprising from N- to C-terminus a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising from N- to C-terminus a heavy chain variable region (VH) region, a CHI region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising from N- to C-terminus a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region; (iv) a second Ig light chain comprising from N- to C-terminus a VL region and a VH region; wherein said first light chain and said first heavy chain associate to form a first antigen binding domain; wherein said second light chain and said second heavy chain associate to form a second antigen binding domain; and wherein said first heavy chain and said second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise a substantially identical amino acid sequence; and the two light chains comprise a substantially identical amino acid sequence. In some embodiments, the two heavy chains comprise a substantially identical amino acid sequence except for one or more amino acid modifications that promote heterodimerization of the correct heavy chains (e.g., as described herein); and the two light chains comprise a substantially identical amino acid sequence. Antibody chains may be substantially identical but not entirely identical if they differ due to post-translational modifications, such as C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.

[00137] As used herein, the term “fully complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, 100% (all), of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of the first and/or second nucleic acid molecule.

[00138] The term “functional variant” as used herein in reference to a protein refers to a protein that comprises at least one but no more than 20%, not more than 15%, not more than 12%, no more than 10%, no more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, wherein the protein retains at least one particular function of the reference protein. Not all functions of the reference protein (e.g., wild type) need be retained by the functional variant of the protein. In some instances, one or more functions arc selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein.

[00139] The term “functional fragment” as used herein in reference to a protein refers to a fragment of a reference protein that retains at least one particular function. Not all functions of the reference polypeptide or protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein.

[00140] As used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a one polypeptide derived from a first polypeptide to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are different. The term fuse encompasses both a direct connection of the at least two polypeptides through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).

[00141] As used herein, the term “fusion protein” and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide derived from a first polypeptide operably connected to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are wherein the first and second polypeptides are not naturally found operably connected together. The at least two polypeptides of the fusion protein can be directly operably connected through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Therefore, for example, the term fusion protein encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A - Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A - peptide linker - Polypeptide B). In some embodiments, the first polypeptide and the second polypeptide are different.

[00142] As used herein, the term “half-life extension moiety” refers to a moiety (e.g., small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that when conjugated or otherwise operably connected (e.g., fused) to a polypeptide or protein (the subject polypeptide or protein), increases the half-life of the subject polypeptide or protein in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated utilizing in vitro models known in the art. [00143] As used herein, the term “half-life extension polypeptide” refers to a polypeptide that when operably connected to another polypeptide (the subject polypeptide), increases the half-life of the subject polypeptide in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the polypeptide or protein can be evaluated utilizing in vitro models known in the art.

[00144] As used herein, the term “heavy chain” refers to the portion of an immunoglobulin (e.g., a human Ig) that typically comprises from N- to C-terminus a heavy chain variable region (VH), a CHI region, a hinge region, a CH2 region, and a CH3 region. The constant regions of the heavy chain (i.e., the CHI region, the hinge region, the CH2 region, and the CH3 region) can be any distinct isotype, for example, human alpha (a), delta (5), epsilon (E), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to the hlgA, hlgD, IgE, hlgG, and hlgM classes of human antibodies, respectively, including subclasses of hlgG, e.g., hlgGi, h!gG2, hlgGa, and h!gG4. As used herein, the term “heavy chain” when used in reference to a human antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (E), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to human IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of human IgG, e.g., IgGi, IgG₂, IgG?. and IgG4.

[00145] As used herein, the term “hematopoietic cell” refers to any blood cell. As such, the term hematopoietic cell, includes, but is not limited to, hematopoietic pluripotent stem cells (HSPCs), common myeloid progenitor cells, megakaryocyte erythroid progenitor cells, erythroid progenitor cells, proerythroblast cells, early erythroblast cells, intermediate erythroblast cells, late erythroblast cells, reticulocytes, megakaryocytes, platelets, granulocyte monocyte progenitor cells, monoblasts, promonocytes, monocytes, macrophages, myeloblasts, promyelocytes, myelocytes, eosinophils, basophils, neutrophils, common lymphoid progenitor cells, pro-NK lymphoblasts, NK cells, pro-B lymphoblasts, B lymphocytes, pro-T lymphoblasts, T lymphoblasts, and plasma cells. [00146] As used herein, the term “hematopoietic cell targeting agent” refers to an agent that specifically binds to an antigen expressed on a hematopoietic cell (or a subset thereof). For example, the antigen expressed in or on the hematopoietic cell may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, a hematopoietic cell targeting agent specifically binds to an antigen on the hematopoietic cell that facilitates internalization of the hematopoietic cell targeting agent (and any associated molecular payload) into the hematopoietic cell. In some embodiments, a hematopoietic cell targeting agent specifically binds to an internalizing, cell surface receptor on the hematopoietic cell and is capable of being internalized into the hematopoietic cell through receptor mediated internalization. In some embodiments, the hematopoietic cell targeting agent is a protein (e.g., antibody), a peptide, a nucleic acid (e.g., an aptamer), or small molecule. In some embodiments, the hematopoietic cell targeting agent is linked to a molecular payload.

[00147] As used herein, the term “heterologous,” when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a nucleic acid molecule comprising a “heterologous moiety” means a nucleic acid molecule that is joined to a moiety (e.g., carbohydrate, small molecule, polypeptide, polynucleotide, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the nucleic acid molecule in nature.

[00148] The terms “hinge” or “hinge region” are used interchangeably herein and refer to the hinge region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hlgGl hinge region is set forth in SEQ ID NO: 172; and the amino acid sequence of an exemplary reference hIgG4 hinge region is set forth in SEQ ID NO: 185.

[00149] As used herein, the term “isolated” with reference to an agent (e.g., a protein, nucleic acid molecule, etc.) refers to an agent (e.g., a protein, nucleic acid molecule, etc.) that is substantially free of other cellular components with which it is associated in the natural state.

[00150] As used herein, the term “KLF Transcription Factor 1” or “KLF1” refers to the zinc finger transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human KLF1 (hKLFl) protein is set forth in SEQ ID NO: 297 (NCBI Ref.: NP_006554.1).

[00151] As used herein, the term “modified nucleotide,” “nucleotide modification,” or use of the term “modification” and the like in reference to a nucleotide or nucleic acid sequence refers to a nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and/or a modified internucleoside linkage, or any combination thereof. Exemplary modifications are provided herein, see, e.g., § 5.5.1.5. In certain embodiments of the instant disclosure, inclusion of a deoxynucleotide - which is acknowledged as a naturally occurring form of nucleotide - if present within an RNA oligonucleotide is considered to constitute a modified nucleotide. [00152] As used herein, the term “molecular payload” refers to an agent that functions to modulate a biological outcome. In some embodiments, a molecular payload is operably connected to a targeting agent (e. ., a targeting agent described herein (e.g., an anti-TFR antibody)). In some embodiments, the molecular payload is a small molecule, a protein, a peptide, or an oligonucleotide. In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide described herein, see, e.g., § 5.5.1). In some embodiments, the molecular payload functions to modulate (e.g., inhibit) the transcription of a DNA molecule, to modulate (e.g., inhibit) the translation of an RNA (e.g., mRNA) molecule, to modulate (e.g., inhibit) the expression of a protein, or to modulate (e.g., inhibit) the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a target nucleic acid molecule (e.g., an RNA molecule encoded by a target gene (e.g., an mRNA molecule encoded by a target gene)).

[00153] As used herein, the term, “non-complementary nucleotide mismatch” refers to a nucleotide within a region of complementarity (as described herein) that is not complementary to the corresponding nucleotide in the target nucleic acid molecule.

[00154] The terms “nucleic acid molecule,” “polynucleotide,” and “oligonucleotide” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single- stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non-natural, or altered internucleoside linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U). [00155] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that extends from the double stranded region of a double stranded nucleic acid molecule. For example, when a 3'-end of one strand of a double stranded nucleic acid molecule extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang.

[00156] As used herein, the term “operably connected” refers to the linkage of two agents in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein). Or for example, a transcription regulatory polynucleotide e.g., a promoter, enhancer, or other expression control element is operably linked to a polynucleotide that encodes a protein if it affects the transcription of the polynucleotide that encodes the protein. The term “operably connected” also refers for example to the conjugation of a first agent (e.g., a protein (e.g., an antibody)) to a second agent (e.g., an oligonucleotide) wherein the first and second agent are both capable of mediating their function.

[00157] As used herein, “partially complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, at least 70%, but not all, of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of a first or second nucleic acid molecule.

[00158] The determination of “percent identity” between two sequences (e.g., protein (amino acid sequences) or oligonucleotide (nucleic acid sequences)) can be accomplished using a mathematical algorithm. Determinations of identity (as described herein) are independent of nucleotide chemical modifications (e.g., as described herein). For example, (mC) is identical to (C) for the purposes of determining identity. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[00159] As used herein, the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent (e.g., a conjugate described herein) and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance intended for use in contact with the tissues of human beings and/or non-human animals, and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit/risk ratio.

[00160] As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).

[00161] As used herein, the terms “protein”, “polypeptide”, and peptide refers to a polymer of at least 2 (e.g., at least 5) amino acids linked by a peptide bond. The term “polypeptide” does not denote a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” and “protein” are used interchangeably herein. In some embodiments, the protein is folded into its three-dimensional structure. Where polypeptides (e.g., in a linear (i.e., primary) structure arc contemplated herein, it should be understood that proteins folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa. Proteins include e.g., naturally occurring proteins, variant (e.g., functional variants) of naturally occurring proteins, fragments (e.g., functional fragments) of naturally occurring proteins, and synthetic proteins (i.e., not naturally occurring proteins).

[00162] As used herein, the term “region of complementarity” refers to a portion of a first nucleic acid molecule comprising a nucleotide sequence that is at least partially complementary to the nucleotide sequence of at least a portion of a second nucleic acid molecule.

[00163] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar’ is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered intemucleoside linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.

[00164] As used herein, the term “RNAi agent” refers to an agent that contains one or more RNA molecules which can mediate the targeted cleavage of an RNA molecule (e.g., an mRNA molecule) via an RNA-induced silencing complex (RISC) pathway. The RNAi agent is thereby capable of e.g., modulating, e.g., inhibiting, the expression of a target gene or protein in a cell, e.g., a cell within a subject, such as a mammalian subject. RNAi agents include, for example, siRNAs, miRNAs, and shRNAs.

[00165] The term “scFv” or “single chain variable fragment” refers to an antibody that comprises a VH region operably connected via a peptide linker to a VL region, wherein the VH and VL regions associate to specifically bind an antigen (e.g., form an antigen binding domain). In some embodiments, the scFv comprises from N- to C-terminus an VH region, a peptide linker, and an VL region. In some embodiments, the scFv comprises from N- to C-terminus an VL region, a peptide linker, and an VH region.

[00166] The term “(scFv ” as used herein refers to an antibody that comprises a first and a second scFv operably connected (e.g., via a peptide linker). The first and second scFv can specifically bind the same or different antigens. In some embodiments, the first and second scFv are operably connected by a peptide linker.

[00167] The term “scFv-Fc” as used herein refers to an antibody that comprises a scFv operably linked (e.g., via a peptide linker) to an Fc domain or subunit of an Fc domain. In some embodiments, a scFv is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first scFv is operably connected to a first Fc domain and a second scFv is operably connected to a second Fc domain of a first and second Fc domain pair.

[00168] The term “(scFv)2-Fc” as used herein refers to a (scFv)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (scFv)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (scFv)2 is operably connected to a first Fc domain and a second (scFv)2 is operably connected to a second Fc domain of a first and second Fc domain pair.

[00169] As used herein, the term “sense strand” refers to an RNA molecule (e.g., part of an RNAi agent (e.g., described herein), part of a dsRNA agent (e.g., described herein)) that comprises a region that is at least partially (e.g., substantially, fully) complementary to a region of the antisense strand (as defined herein). The sense strand is often referred to as such with reference to the orientation of the sequence of the sense strand being the same with respect to a target RNA (e.g., mRNA sequence).

[00170] As used herein, the term “single domain antibody” or “sdAb” refers to an antibody having a single monomeric variable antibody domain. A sdAb is able to specifically bind to a specific antigen. A VHH (as defined herein) is an example of a sdAb.

[00171] As used herein, the term “specifically binds” refers to preferential interaction, i.e., significantly higher binding affinity, between a first protein (e.g., an antibody) and a second protein (e.g., an antigen) relative to other amino acid sequences. Herein, when a first protein is said to “specifically bind” to a second protein, it is understood that the first protein specifically binds to an epitope of the second protein. The term “epitope” refers to the portion of the second protein that the first protein specifically recognizes. The term specifically binds includes molecules that are cross reactive with the same epitope of a different species. For example, an antibody that specifically binds human TFR may be cross reactive with TFR of another species (e.g., cynomolgus, murine, etc.), and still be considered herein to specifically bind human TFR. A protein can specifically bind more than one different protein. Specific binding can be measured, e.g., through measuring binding affinity (e.g., using standard methods known in the art and described herein (e.g., surface plasmon resonance (SPR) (e.g., BIAcore®-based assay), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff ct al., Cancer Res. 55:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, the full contents of each of which are incorporated by reference herein for all purposes).

[00172] As used herein, the term “subject” includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[00173] As used herein, “substantially complementary” means that in a hybridized pair of a first nucleic acid molecule and a second nucleic acid molecule, at least 85%, but not all, of the bases in a contiguous sequence of the first nucleic acid molecule will hybridize with the same number of bases in a contiguous sequence of the second nucleic acid molecule. The contiguous sequence may comprise all or a part of a first or second nucleic acid molecule.

[00174] As used herein, the term “target nucleic acid sequence” refers to a contiguous portion of the nucleotide sequence of a nucleic acid sequence (e.g., an mRNA molecule formed during the transcription of a target gene). In some embodiments, the target nucleic acid sequence is an mRNA molecule formed during the transcription of a target gene. In some embodiments, the target nucleic acid molecule comprises an mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence (e.g., mRNA) will be at least long enough to serve as a substrate for an oligonucleotide described herein (e.g., an antisense oligonucleotide, an RNAi agent, etc.).

[00175] As used herein, the term “therapeutic agent” refers to an agent (e.g., a conjugate described herein) capable of achieving a desired therapeutic result in a subject or ex vivo (e.g., capable of treating a disease as defined herein) when administered at a therapeutically effective amount. [00176] As used herein, the term “therapeutically effective amount” of a therapeutic agent refers to any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, improves a disease condition, e.g., protects a subject against the onset of a disease (or infection); improves a symptom of disease or infection, e.g., decreases severity of disease or infection symptoms, decreases frequency or duration of disease or infection symptoms, increases disease or infection symptom- free periods; prevents or reduces impairment or disability due to the disease or infection; or promotes disease (or infection) regression. The ability of a therapeutic agent to improve a disease condition can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. [00177] As used herein, the term “translatable RNA” refers to any RNA that encodes at least one peptide or protein and can be translated to produce the encoded peptide or protein in vitro, in vitro, in situ or ex vivo. This includes, e.g., messenger RNA (mRNA).

[00178] As used herein, the term “transferrin” or “TF” refers to the plasma glycoprotein transferrin that functions, inter alia, in iron metabolism and the transport of iron through the blood to various tissues, such as the liver, spleen, and bone marrow. The amino acid sequence of a reference human TF (hTF) protein is set forth in SEQ ID NO: 3 (UniProt Accession P02787).

[00179] As used herein, the term “transfenin receptor” or “TFR” refers to the transmembrane homodimeric glycoprotein that functions, inter alia, in the cellular uptake of iron from the plasma glycoprotein transferrin. The term TFR includes, where applicable, multiple isoforms and homologs. For example, human TFR (hTFR) includes homologs hTFRl and hTFR2. The amino acid sequence of a reference hTFRl protein is set forth in SEQ ID NO: 1 (UniProt Accession P02786|). TFR1 is also commonly known in the art as CD71. The terms TFR1 and CD71 are used interchangeably herein. The amino acid sequence of a reference hTFR2 protein is set forth in SEQ ID NO: 2 (UniProt Accession Q9UP52).

[00180] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease and/or symptom(s) associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disease does not require that the disease, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and/or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of e.g., a conjugate described herein (or a carrier, pharmaceutical composition, etc. comprising the same).

[00181] As used herein, the term “variation” or “variant” or use the like in reference to a nucleotide or nucleic acid sequence refers to a nucleic acid molecule that comprises at least one substitution, addition, deletion, or inversion of one or more nucleotide compared to a reference nucleic acid molecule. Likewise, as used herein, the term “variation” or “variant” or use the like with reference to a peptide or protein refers to a peptide or protein that comprises at least one substitution, addition, deletion, or inversion of an amino acid residue compared to a reference peptide or protein.

[00182] A “variation that promotes heterodimerization of a first Fc region and a second Fc region” (or similar phrasing) is a manipulation of the peptide backbone or the post-translational modifications of an Fc region that reduces or prevents the association of a polypeptide comprising the Fc region with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc regions desired to associate i.e., a first Fc region and a second Fc region), wherein the modifications are complementary to each other so as to promote association of the two Fc regions. For example, a modification promoting association may alter the structure or charge of one or both of the Fc regions so as to make their association sterically or electrostatically favorable, respectively. Thus, heterodimerization occurs between a polypeptide comprising the first Fc region and a polypeptide comprising the second Fc region, which might be non-identical in the sense that further components fused to each of the Fc regions {e.g., antigen binding domains) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc region, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically one or more amino acid substitution, in each of the first Fc region and the second Fc region. See, e.g., § 5.3.2.2.

[00183] As used herein, the term “variable region” refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and arc used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[00184] The terms “VL” and “VL region” are used interchangeably to refer to an immunoglobulin light chain variable region. A VL region can be incorporated into an antibody, e.g., a scFv, a Fab, a full-length antibody. For example, a scFv comprises a VL region operably connected via a peptide linker to a VH region.

[00185] The terms “VH” and “VH region” are used interchangeably to refer to an immunoglobulin heavy chain variable region. A VH region can be incorporated into an antibody, e.g., a scFv, a Fab, a full-length antibody. For example, a scFv comprises a VH region operably connected via a peptide linker to a VL region.

[00186] The term “VHH” as used herein refers to a type of single domain antibody (sdAb) that has a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) which are naturally devoid of light chains or synthetically produced.

[00187] The term “(VHHh” as used herein refers to an antibody that comprises a first and a second VHH operably connected (e.g., via a peptide linker). The first and the second VHH can specifically bind the same or different antigens. In some embodiments, the first and second VHH are operably connected by a peptide linker.

[00188] The term “VHH-Fc” as used herein refers to an antibody that comprises a VHH operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a VHH is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first VHH is operably connected to a first Fc domain and a second VHH is operably connected to a second Fc domain of a first Fc and a second Fc pair.

[00189] The term “(VHH)2-Fc” as used herein refers to (VHH)2 operably linked (e.g., via a peptide linker) to an Fc domain or a subunit of an Fc domain. In some embodiments, a (VHH)2 is operably connected to only a first Fc domain of a first and a second Fc domain pair. In some embodiments, a first (VHH)2 is operably connected to a first Fc domain and a second (VHH)2 is operably connected to a second Fc domain of a first Fc and a second Fc pair.

[00190] As used herein, the term “ZBTB7A” or “Zinc Finger And BTB Domain Containing 7 A” refers to the transcription factor that functions, inter alia, in the repression of fetal hemoglobin and the switch from fetal hemoglobin to adult hemoglobin. The amino acid sequence of a reference human ZBTB7A (hZBTB7A) protein is set forth in SEQ ID NO: 294 (NCBI Ref.: NP_056982.1).

5.2 Conjugates

[00191] Provided herein are, inter alia, conjugates (e.g., antibody-oligonucleotide conjugates), useful in, inter alia, modulating (e.g., inhibiting, reducing, enhancing) expression and/or activity of a target gene or protein (e.g., within a cell (e.g., an erythroid precursor cell), e.g., within a cell in a subject, e.g., a mammalian subject, e.g., a human subject) (e.g., through binding to a target nucleic acid molecule (e.g., an mRNA molecule)).

[00192] The conjugates described herein comprise a targeting agent (e.g., a hematopoietic cell targeting agent (e.g., described herein)) and a molecular payload (e.g., an oligonucleotide described herein (e.g., an oligonucleotide that alters (e.g., inhibits or reduces) expression or activity of a target gene or protein (e.g., a target gene or protein expressed by a hematopoietic cell (e.g., an erythroid precursor cell)).

[00193] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

[00194] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFRI)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[00195] In some embodiments, upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

[00196] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[00197] In some embodiments, the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[00198] In some embodiments, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRI)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

[00199] In some embodiments, the conjugates described herein exhibit one or more of the following properties: (a) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate does not induce death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the target cell remains viable; (d) upon internalization into a target cell (e.g., an erythroid precursor cell), the conjugate does not induce death of the target cell; and/or (e) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the conjugate does not induce degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

5.3 Hematopoietic Cell Targeting Agents

[00200] As described above, the conjugates described herein comprise a targeting agent (e.g., for targ eting a molecular pay load (e.g., an oligonucleotide described herein) to a specific cell or cells (e.g., within a subject)). In some embodiments, the targeting agent is a hematopoietic cell (e.g., an erythroid precursor cell) targeting agent. In some embodiments, the targeting agent is capable of targeting a molecular payload (e.g., an oligonucleotide described herein) to a hematopoietic cell (e.g., an erythroid precursor cell). In some embodiments, the hematopoietic cell (e.g., an erythroid precursor cell) is present in the bone marrow (e.g., of a subject). In some embodiments, the targeting agent is capable of targeting a molecular payload to one or more cell within the bone marrow. In some embodiments the targeting agent specifically targets erythroid precursor cells (e.g., within the bone marrow (e.g., within a subject)). In some embodiments the targeting agent specifically targets erythroid precursor cells (e.g., within the bone marrow (e.g., within a subject)) through specific binding to an antigen expressed on the surface of the erythroid precursor cells (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

[00201] It should be appreciated that various types of targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) may be used in accordance with the disclosure. For example, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) may comprise (or consist of) a small molecule, an oligonucleotide (e.g., DNA, RNA, RNA/DNA hybrid) (e.g., an aptamer), a protein (e.g., an antibody, a peptide), a lipid (e.g., a microvesicle), or a carbohydrate (e.g., a polysaccharide). In some embodiments, the targeting agent is a protein. In some embodiments, the targeting agent is a peptide. In some embodiments, the targeting agent is an antibody. In some embodiments, the targeting agent is an antibody-like scaffold (e.g., as described herein). Exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) are described in further detail herein, however, it should be appreciated that the exemplary targeting agents (e.g., hematopoietic cell (e.g., erythroid precursor cell) targeting agents) provided herein are not meant to be limiting.

[00202] In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on surface of hematopoietic cells (or one or more subset thereof) (e.g., erythroid precursor cells) (e.g., within the bone marrow) (e.g., TFR (e.g., hTFR (e.g., hTFRl))). In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on the surface of erythroid precursor cells (e.g., TFR (e.g., hTFR (e.g., hTFRl))). In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent specifically binds to an antigen expressed on the surface of erythroid precursor cells within the bone marrow (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

[00203] By interacting with one or more molecule (e.g., protein) expressed on the surface of a target hematopoietic cell (e.g., erythroid precursor cell) both tissue (e.g., bone marrow) localization and selective or preferred uptake into hematopoietic cells (e.g., erythroid precursor cells) can be achieved. In some embodiments, molecules (e.g., proteins) that are substrates for hematopoietic cell (e.g., erythroid precursor cell) uptake transporters are useful for delivering a molecular- payload (e.g., an oligonucleotide described herein) into hematopoietic cells (e.g., erythroid precursor cells). Binding to molecules (e.g., proteins) expressed on the surface of hematopoietic cells (e.g., erythroid precursor cells) followed by endocytosis can allow large molecules such as antibodies to enter the hematopoietic cells (e.g., erythroid precursor cells). For example, as described in detail below, molecular payloads (e.g., an oligonucleotide described herein) conjugated to transferrin (or a functional fragment or functional variant thereof) or anti- TFR (e.g., hTFR (e.g., hTFRl)) antibodies can be taken up by hematopoietic cells (e.g., erythroid precursor cells) via binding to TFR (e.g., hTFR (e.g., hTFRl)), which may then be endocytosed, e.g., via endocytosis, e.g., clathrin-mediated endocytosis.

[00204] The use of hematopoietic cell (e.g., erythroid precursor cell) targeting agents may be useful for concentrating a molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cells (e.g., within the bone marrow (e.g., within a subject))) while reducing toxicity associated with effects in other cells or tissues. In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent concentrates a bound molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cell (e.g., within the bone marrow (e.g., within a subject))) as compared to another tissue or cell type within a subject. In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent concentrates a bound molecular payload (e.g., an oligonucleotide described herein) in hematopoietic cells (e.g., erythroid precursor cells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than an amount in non-hematopoietic cells (e.g., non-erythroid precursor cells). In some embodiments, a toxicity of the molecular payload (e.g., an oligonucleotide described herein) in a subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered to the subject when bound to the hematopoietic cell (e.g., erythroid precursor cell) targeting agent.

[00205] In some embodiments, the hematopoietic cell (e.g., erythroid precursor cell) targeting agent exhibits one or more of the following properties: (a) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent is internalized into the target cell; (b) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce death of the target cell; (c) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the target cell remains viable; (d) upon internalization into a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce death of the target cell; and/or (e) upon binding to a target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))) expressed on the surface of a target cell (e.g., an erythroid precursor cell), the hematopoietic cell (e.g., erythroid precursor cell) targeting agent does not induce degradation of the target molecule (e.g., TFR (e.g., hTFR (e.g., hTFRl))).

5.3.1 TFR Targeting Agents

[00206] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to the transferrin receptor (TFR) (e.g., hTFR (e.g., hTFRl)). In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds to hTFR. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds hTFRl.

[00207] TFR1 is a transmembrane homodimeric glycoprotein that functions, inter alia, in the cellular uptake of iron from the plasma glycoprotein transferrin (TF). Iron uptake from transferrin involves the binding of TF to TFR (e.g., TFR1), internalization of TF within an endocytic vesicle by receptor-mediated endocytosis and the release of iron induced by a decrease in endosomal pH. TFR1 is expressed by e.g., placental syncytiotrophoblasts, myocytes, basal keratinocytes, hepatocytes, endocrine pancreas, spermatocytes, and erythroid precursor cells. TFR expression, while known to be highly expressed by erythroid precursor cells, is not expressed by mature erythrocytes. TFR2 is a known homolog of TFR1, but TFR1 is considered the major protein responsible for iron uptake owing to its higher affinity and expression pattern. See, e.g., Derek K. Mar.sec, et al., CD71 (Transferrin Receptor): An Effective Marker for Erythroid Precursors in Bone Marrow Biopsy Specimens, American Journal of Clinical Pathology, Volume 134, Issue 3, September 2010, Pages 429-435, https://doi.org/10.1309/AJCPCRK3MOAOJ6AT; C. Sieff et al., Changes in Cell Surface Antigen Expression During Hemopoietic Differentiation, Blood, Vol 60 (3), 1982, Pages 703-713, https://doi.org/10.1182/blood.V60.3.703.703; the entire contents of each of which are incorporated herein by reference for all purposes.

[00208] The amino acid sequence of a reference hTFRl protein is set forth in SEQ ID NO: 1. The amino acid sequence of a reference hTFR2 protein is set forth in SEQ ID NO: 2. The amino acid sequence of a reference hTF protein is set forth in SEQ ID NO: 3. See Table 1, herein. Table 1. The Amino Acid Sequence of a Reference hTFRl., hTFR2, and hTF Protein.

[00209] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 and TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 but does not specifically bind TFR2. In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds TFR1 and binds to TFR2 with significantly lower affinity.

[00210] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell) targeting agent) specifically binds hTFRl and one or more of murine TFR1, rat TFR1, a non-human primate TFR1 (e.g., cynomolgus TFR1).

[00211] In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent enhances the distribution and/or uptake (e.g., into a cell, e.g., into a cell in a subject, e.g., a cell that expresses TFR (e.g., hTFR (e.g., hTFRl)) (e.g., an erythroid precursor cell (e.g., in the bone marrow))) of a molecular payload (e.g., an oligonucleotide described herein) (e.g., RNAi agent (e.g., siRNA), ASO, etc.) (e.g., as compared to an oligonucleotide that lacks the targeting moiety). In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent alters (e.g., extends) the lifetime (e.g., in vivo) of the molecular payload (e.g., an oligonucleotide described herein) (e.g., RNAi agent (e.g., siRNA), ASO, etc.) (e.g., as compared to an oligonucleotide that lacks the targeting moiety). In some embodiments, the TFR (e.g., hTFR (e.g., hTFRl)) targeting agent provides an enhanced affinity for a selected target, e.g., a selected cell type, compartment (e.g., cell type, tissue, organ or region of the body) (e.g., as compared to an oligonucleotide that lacks the targeting moiety) (e.g., erythroid precursor cells (e.g., in the bone marrow)).

[00212] In some embodiments, the TFR targeting agent does not (or does not significantly) interfere with TF binding to TFR. In some embodiments, the TFR targeting agent does not (or does not significantly) compete for binding to TFR with TF.

[00213] In some embodiments, the TFR targeting agent is a protein. In some embodiments, the targeting agent is a transferrin protein. In some embodiments, the targeting agent is an antibody. In some embodiments, the targeting agent comprises an antibody-like scaffold (e.g., a cysteine dense peptide, see, e.g., W02023023031, the entire contents of which is incorporated by reference herein for all purposes).

5.3.1.1 TF Proteins

[00214] In some embodiments, the TFR targeting agent comprises TF (e.g., hTF) (or a functional fragment or variant thereof). In some embodiments, the TFR targeting agent comprises the TFR1 binding domain of TF (e.g., hTF) (or a functional fragment or variant thereof). In some embodiments, the TF (e.g., hTF) (or a functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3.

[00215] Variant hTF proteins are known in the art, see, e.g., W02009019314A1, W02008152140A2, EP2216341A1, WO2009149393A2, US8158579B2, the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the variant hTF protein exhibits increased stability and/or long plasma half-life (e.g., relative to a reference hTF protein) that does not comprise the one or more variation). In some embodiments, the variant hTF protein comprises a substitution of a non-cysteine amino acid residue with a cysteine (e.g., at position VI, P2, D3, K4 T5, H14, Q20, S21, D24, K27, S28, V29, P31, S32, D33, A43, E89, D104, G106, G1 I4, LI22, G123, P145, SI55, D163, T165, DI66, P168, PI75, GI76, G178, C179, S180, T181, L182, Q184, F187, S189, D197, G198, E212, A215, N216, A218, D221, D229, G257, N268, D277, K278, K280, E281, S287, P288, H289, K291, S298, P307, L326, T33O, P335, T336, N413, S415, D416, D420, K434, S435, A436, S437, D438, D442, N443, G446, N469, N472, G487, K489, D491, S501, G502, L503, N510, T518, P539, Q540, G543, G544, K545, P547, D548, P549, K552, N553, N555, D558, D565, T567, P570, N576, A595, S610, N611, V612, T613, D614, S616, G617, T626, D634, D643, S666, T667 or S669, numbering according to SEQ ID NO: 3) (e.g., as described in W02009019314A1). In some embodiments, the variant hTF protein comprises the addition of a cysteine residue (e.g., as described in W02009019314A1).

(i) Exemplary hTF Variant Proteins

[00216] The amino acid sequence of exemplary hTF variants is provided in Table 2.

Table 2. The Amino Acid Sequence of Exemplary hTF Variants.

[00217] In some embodiments, the TFR targeting agent comprises a TF valiant (e.g., a hTF variant) (or a functional fragment or variant thereof). In some embodiments, TF variant (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a TF variant set forth in Table 2, wherein the amino acid sequence of the TF variant comprises at least one amino acid variation compared to the amino acid sequence of a reference TF protein (e.g., SEQ ID NO: 3). In some embodiments, TF variant (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 4-12, wherein the amino acid sequence of the TF variant comprises at least one amino acid variation compared to the amino acid sequence of a reference TF protein (e.g., SEQ ID NO: 3).

(ii) Heterologous Moieties

[00218] In some embodiments, the TF (e.g., hTF) is operably connected to a heterologous moiety (e.g., an Fc region (e.g., an Fc region described herein (see, e.g., § 5.3.2))). In some embodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moictics include, but arc not limited to, an immunoglobulin (e.g., human Ig (hlg)), a fragment of an Ig (e.g., hlg), an Ig (e.g., hlg) constant region, a fragment of an Ig (e.g., hlg) constant region, an Ig (e.g., hlg) Fc region, human serum albumin (HSA), an HSA binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hlg, a fragment of a hlg, one or more hlg heavy chain constant region, a fragment of a hlg constant region, a hlg Fc region, human serum albumin (HSA), and an HSA binding protein or peptide. The immunomodulatory protein or polypeptide described herein fused or conjugated to a half-life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art.

[00219] In some embodiments, the heterologous moiety is a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises one or more Ig heavy chain constant region (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgGl, IgG2, IgG3, or IgG4.

[00220] In some embodiments, the heterologous polypeptide comprises or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. [00221] In some embodiments, the heterologous polypeptide comprises or consists of an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[00222] In some embodiments, the heterologous polypeptide comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[00223] In some embodiments, the heterologous polypeptide comprises or consists of a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 hinge region, h!gG4 CH2 region, and hIgG4 CH3 region.

[00224] In some embodiments, the heterologous polypeptide comprises or consists of a hlg Fc region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a h!gG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

5.3.1.2 TFR Binding Peptides and Antibody-Like Scaffolds

[00225] In some embodiments, the TFR targeting agent comprises a peptide that specifically binds TFR (e.g., hTFR). TFR (e.g., hTFR (e.g., hTFRl)) binding peptides that can be employed in the conjugates described herein are known in the art. For example, see, e.g., US6743893 and US8399653, the entire contents of each of which is incorporated herein by reference for all purposes.

[00226] In some embodiments, the TFR targeting agent comprises an antibody-like scaffold. Anti-TFR antibody like scaffolds that can be employed in the conjugates described herein are known in the art. See, e.g., W02023023031 (describing anti-TFR cysteine dense peptides) and WO2021076546 (describing TFR binding fibronectin type III domains), the entire contents of each of which is incorporated herein by reference for all purposes.

(i) Exemplary TFR Binding Peptides and Antibody-Like Scaffolds

[00227] The amino acid sequence of exemplary TFR specific antibody like scaffolds is provided in Table 3.

Table 3. The Amino Acid Sequence of Exemplary TFR Specific Peptides and Antibody Like Scaffolds.

[00228] In some embodiments, the TFR targeting agent comprises a TFR specific peptide or an antibody like scaffold (or a functional fragment or variant thereof). In some embodiments, the TFR targeting agent comprises a TFR specific peptide (or a functional fragment or valiant thereof). In some embodiments, the TFR specific peptide (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a TFR specific peptide (or the functional fragment or variant thereof) set forth in Table 3. In some embodiments, the TFR specific peptide (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 13-41. In some embodiments, the TFR targeting agent comprises an antibody like scaffold (or a functional fragment or variant thereof). In some embodiments, the antibody like scaffold (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of antibody like scaffold (or the functional fragment or variant thereof) set forth in Table 3. In some embodiments, the antibody like scaffold (or the functional fragment or variant thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 42-61.

(ii) Heterologous Moieties

[00229] In some embodiments, the TFR binding peptide or antibody-like scaffold is operably connected to a heterologous moiety (e.g., an Fc region (e.g., an Fc region described herein (see, e.g., § 5.3.2))). In some embodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human Ig (hlg)), a fragment of an Ig (e.g., hlg), an Ig (e.g., hlg) constant region, a fragment of an Ig (e.g., hlg) constant region, an Ig (e.g., hlg) Fc region human transferrin, human serum albumin (HS A), an HSA binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig, a fragment of an Ig, one or more Ig heavy chain constant region, a fragment of an Ig constant region, an Ig Fc region, a hlg, a fragment of a hlg, one or more hlg heavy chain constant region, a fragment of a hlg constant region, a hlg Fc region, human serum albumin (HSA), and an HSA binding protein or peptide. The immunomodulatory protein or polypeptide described herein fused or conjugated to a half-life extending moiety or a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art.

[00230] In some embodiments, the heterologous moiety is a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises one or more Ig heavy chain constant region (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the Ig is an IgG. In some embodiments, the IgG is IgGl, IgG2, IgG3, or IgG4.

[00231] In some embodiments, the heterologous polypeptide comprises or consists of an IgG CH2 region and an IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[00232] In some embodiments, the heterologous polypeptide comprises or consists of an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[00233] In some embodiments, the heterologous polypeptide comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[00234] In some embodiments, the heterologous polypeptide comprises or consists of a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the heterologous polypeptide comprises or consists of a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[00235] In some embodiments, the heterologous polypeptide comprises or consists of a hlg Fc region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. 5.3.L3 Anti-TFR (e.g., Anti-TFRl) Antibodies

[00236] In some embodiments, the TFR targeting agent is an anti-TFR antibody (e.g., an anti- hTFR antibody) (e.g., an anti-hTFRl antibody)). In some embodiments, the antibody comprises or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), (sdAb)2 (e.g., a (VHH)2, or a (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody comprises or consists of a full-length antibody, Fab, Fab', F(ab')2, Fab-Fc, scFv, scFv-Fc, (scFv)2-Fc, sdAb-Fc (e.g., a VHH-Fc), or (sdAb)2-Fc (e.g., (VHH)2-Fc). In some embodiments, the antibody comprises or consists of a full-length antibody. In some embodiments, the antibody comprises or consists of a Fab. In some embodiments, the antibody comprises or consists of a F(ab')2. In some embodiments, the antibody comprises or consists of a Fab-Fc. In some embodiments, the antibody comprises or consists of a scFv-Fc. In some embodiments, the antibody comprises or consists of a (SCFV)₂-FC. In some embodiments, the antibody comprises or consists of a sdAb-Fc (e.g., a VHH- Fc). In some embodiments, the antibody comprises or consists of a o(sdAb)2-Fc (e.g., (VHH)2-Fc) [00237] In some embodiments, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is an IgGl or IgG4 antibody. In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a hlgGl, h!gG2, h!gG3, or hIgG4 antibody. In some embodiments, the antibody is a hlgGl or hIgG4 antibody. In some embodiments, the antibody is a hlgGl antibody. In some embodiments, the antibody is a hIgG4 antibody.

(i) Exemplary Anti-TFR (e.g., Anti-TFRl) Antibodies

[00238] In some embodiments, the targeting agent (e.g., the hematopoietic cell (e.g., erythroid precursor cell)-targeting agent) comprises an anti-TFR antibody. Anti-hTFRl antibodies that can be employed in the conjugates described herein are known in the art.

[00239] Exemplary anti-TFRl antibodies known in the ail that can be employed in the conjugates described herein include, but are not limited to, e.g., OKT9 (see, e.g., US4364934); MH, M23, M27, B84 (see, e.g., WO2015098989 and US9994641); 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, and 13D4 (see, e.g., WO2016081643 and US9708406); 8D3 (see, e.g., US2010/077498 and Lee et al. “Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052); 0X26 (see, e.g., Haobam, B. et al. 2014. Rabl7- mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion. Cellular microbiology. 16: 1806-21); DF1513 (see, e.g., Ortiz-Zapatcr E et al. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006)); the following commercially available clones e.g., Novus Biologicals) 1A1B2,661G1, MEM-189, JF0956, 29806, 1A1B2, TFRC/1818, 1E6, 66Igl0, TFRC/1059, Ql/71, 23D10, 13E4, TFRC/1149, ER-MP21, YTA74.4, BU54, 2B6, RI7 217; BA120g (see, e.g., US20110311544A1 and US7572895); B3/25 and T58/30 (see, e.g., Trowbridge, I.S. et al. “Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumour cells.” Nature, 1981, volume 294, pages 171-173); the following commercially available clones (e.g., BioXcell) R17 217.1.3, 5E9C11, OKT9 (BE0023 clone), BK19.9, B3/25, T56/14 and T58/1 (see, e.g., Gatter, K.C. et al. “Transferrin receptors in human tissues: their distribution and possible clinical relevance.” J Clin Pathol. 1983 May; 36(5):539-45); 5E9C11; R17 217.1.3 (available from BioXcell), BE0175 (available from BioXcell); the entire contents of each of which is incorporated herein by reference for all purposes.

[00240] Exemplary anti-TFR (e.g., hTFR (e.g., hTFRl)) antibodies that can be employed in the conjugates described herein are described in e.g., WO2023283531; WO2021154477A1;

WO2020132584A1; WO2021154476A1; WO2021150382A1; W02023023031A2;

WO2021146256A1; WO2021142275A1; US20220017635A1; W02016207240A1;

US11267896B2; US20220143206A1, US11028179B2; US11286305B2; W02023087017A1; WO2023086864A1; WO2023044398A1; WO2023039611A2; W02023034409A1;

WO2023283623A1; WO2023283624A2; WO2023283619A2; WO2023283620A1;

WO2023283615A1; WO2023283613A1; WO2023283614A2; US11672872B2; US11648318B2; WO2022271549A1; WO2022201122A1; WO2022174114A1; WO2022026152A2;

W02022020107A1; W02022020106A1; W02022020105A1; W02022020108A;

W02022020109A1; WO2021205358A1; US20230174646A1; US20210299266A1;

WO2021195469A1; US11446387B2; US20220409735A1; US20210301290A1;

US20210369762A1; US11525137B2; US11555190B2; US11111308B2; US10550188B2; US10508151B2; US20160208008A1; US20150291697A1; US20130171061A1; US9562230B2; US7976841B2; US4364934; WO2015098989; US9994641; WO2016081643; US9708406; US2010077498; US20110311544; US7572895; WO2019075417; US20060286030A1; US20190240346A1 ; US20130216476A1 ; WO2023283531 ; US20130177579A1 ; US9598496B2;

US20130045206A1; US20060039908A1; US6015555A; US6008326A; US5648469A; EP79696B1; W02023034409A1; US4364934; US8409573; US9708406; US9611323; WO2015098989; Schneider C. et al. “Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody 0KT9.” J Biol Chern. 1982, 257:14, 8516-8522.; Lee et al. “Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052; Lee et al. “Targeting Rat Anti-8D3 Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther., 292: 1048-1052; Haobam, B. et al. 2014. Rabl7-mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion. Cellular microbiology. 16: 1806-21; Ortiz-Zapater E et al. Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006); Trowbridge, I.S. et al. “Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumour cells.” Nature, 1981, volume 294, pages 171-173; Gatter, K.C. et al. “Transferrin receptors in human tissues: their distribution and possible clinical relevance.” J Clin Pathol. 1983 May; 36(5) :539-4; the entire contents of each of which are incorporated herein by reference for all purposes.

[00241] The amino acid sequence of exemplary anti-hTFR antibodies that can be utilized in the conjugates described herein is provided in Table 4. The CDRs of the anti-hTFR antibodies in Table 4, are denoted according to Kabat. A person of ordinary skill in the art would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the art.

Table 4. The Amino Acid Sequence of Exemplary Anti-hTFR Antibodies.

[00242] The amino acid sequence of additional exemplary anti-hTFRl antibodies that can be utilized in the conjugates described herein is provided in Table 26. The CDRs of the anti-hTFR antibodies in Table 26, are denoted according to Kabat. A person of ordinary skill in the ail would be able to determine the CDRs as defined by another scheme, e.g., Chothia, IMGT, using ordinary methods known in the art.

Table 26. Amino Acid Sequence of Exemplary Anti-TFRl Antibodies.

[00243] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR (e.g., hTFR, e.g., hTFRl) antibody named and/or incorporated by reference herein).

[00244] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[00245] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00246] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00247] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00248] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00249] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[00250] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[00251] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[00252] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti- TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations {e.g., substitution, deletion, addition, etc.).

[00253] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00254] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[00255] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of a VH of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of a VH of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of a VH of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR1 of a VL of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00256] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR1 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR2 of an anti- TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VH CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence CDR1 of a VL of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR2 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein), or the amino acid sequence of a VL CDR3 of an anti-TFR antibody named and/or incorporated by reference herein) comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00257] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH of an anti-TFR antibody named and/or incorporated by reference herein). In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL of an anti-TFR antibody named and/or incorporated by reference herein). In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH of an anti-TFR antibody named and/or incorporated by reference herein); and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL of an anti-TFR antibody named and/or incorporated by reference herein).

[00258] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR e.g., hTFR, e.g., hTFRl) antibody provided in Table 4.

[00259] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[00260] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00261] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00262] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 4, or the amino acid sequence of a VH CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00263] In some embodiments, the amino acid sequence of VH CDRI comprises or consists of the amino acid sequence of a VH CDRI set forth in Table 4, or the amino acid sequence of a VH CDRI set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Tabic 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00264] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[00265] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00266] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00267] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence of a VL CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e. ., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00268] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence of a VL CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00269] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[00270] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 4, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 4 comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 4, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00271] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 4, or the amino acid sequence of a VH CDR1 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 4, or the amino acid sequence of a VH CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 4, or the amino acid sequence of a VH CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 4, or the amino acid sequence CDR1 of a VL set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 4, or the amino acid sequence of a VL CDR2 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 4, or the amino acid sequence of a VL CDR3 set forth in Table 4 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00272] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 4. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 4. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 4; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 4.

[00273] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) antibody comprises an anti-TFR (e.g., hTFR, e.g., hTFRl) antibody provided in Table 26.

[00274] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: a VH CDR1, a VH CDR2, and a VH CDR3.

[00275] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00276] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDRI of a VH set forth in Table 26, or the amino acid sequence of a VH CDRI of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00277] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00278] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00279] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VL that comprises: VL CDRI, VL CDR2, and VL CDR3.

[00280] In some embodiments, the amino acid sequence of VL CDRI comprises or consists of the amino acid sequence of a VL CDRI of a VL set forth in Table 26, or the amino acid sequence of a VL CDRI of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26 comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00281] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00282] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence of a VL CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of a VL CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.). In some embodiments, the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00283] In some embodiments, the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence of a VL CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of a VL CDR2 set forth in Table 26 comprising or consisting of 1 , 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00284] In some embodiments, the anti-TFR (e.g., hTFR, e.g., hTFRl) comprises a VH that comprises: VH CDR1, VH CDR2, and VH CDR3; and a VL that comprises: VL CDR1, VL CDR2, and VL CDR3.

[00285] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR1 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR2 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 of a VH set forth in Table 26, or the amino acid sequence of a VH CDR3 of a VH set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR2 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 of a VL set forth in Table 26, or the amino acid sequence of a VL CDR3 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00286] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of a VH CDR1 set forth in Table 26, or the amino acid sequence of a VH CDR1 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of a VH CDR2 set forth in Table 26, or the amino acid sequence of a VH CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e. ., substitution, deletion, addition, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of a VH CDR3 set forth in Table 26, or the amino acid sequence of a VH CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of a VL CDR1 set forth in Table 26, or the amino acid sequence CDR1 of a VL set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); the amino acid sequence of VL CDR2 comprises or consists of the amino acid sequence of a VL CDR2 set forth in Table 26, or the amino acid sequence of a VL CDR2 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.); and the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence of a VL CDR3 set forth in Table 26, or the amino acid sequence of a VL CDR3 set forth in Table 26 comprising or consisting of 1, 2, or 3 amino acid variations (e.g., substitution, deletion, addition, etc.).

[00287] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 26. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 26. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH set forth in Table 26; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL set forth in Table 26.

5.3.2 Ig Constant Regions

[00288] In some embodiments, the antibody (or heterologous polypeptide (e.g., operably connected to a protein (e.g., hTF) that specifically binds TFR (see, e.g., § 5.3. 1.3)) comprises an IgG CH2 region and an IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises of an IgG hinge region, IgG CH2 region, and IgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgGl CH2 region and an IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgGl hinge region, IgGl CH2 region, and IgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgG4 CH2 region and an IgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises an IgG4 hinge region, IgG4 CH2 region, and IgG4 CH3 region.

[00289] In some embodiments, the antibody (or heterologous polypeptide) comprises an Ig Fc region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[00290] In some embodiments, the antibody (or heterologous polypeptide) comprises a first Ig Fc region and a second Ig Fc region. In some embodiments, the first and/or second Ig Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of at least a portion of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of an IgG hinge region, an IgG CH2 region, and an IgG CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of at least a portion of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of an IgGl hinge region, an IgGl CH2 region, and an IgGl CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of at least a portion of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region. In some embodiments, the first and/or second Ig Fc region comprises or consists of an IgG4 hinge region, an IgG4 CH2 region, and an IgG4 CH3 region.

[00291] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more hlg heavy chain constant regions (e.g., a CH2 region, a CH3 region, a hinge region, an Fc region). In some embodiments, the hlg is a human IgG (hlgG). In some embodiments, the hlgG is hlgGl, IgG2, IgG3, or IgG4. In some embodiments, the hlgG is IgGl or IgG4. In some embodiments, the hlgG is hlgGl. In some embodiments, the hlgG is hIgG4.

[00292] In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgG CH2 region and a hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgG hinge region, hlgG CH2 region, and hlgG CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgGl CH2 region and a hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hlgGl hinge region, hlgGl CH2 region, and hlgGl CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hIgG4 CH2 region and a hIgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a partial hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region. In some embodiments, the antibody (or heterologous polypeptide) comprises a hIgG4 hinge region, hIgG4 CH2 region, and hIgG4 CH3 region.

[00293] In some embodiments, the antibody (or heterologous polypeptide) comprises a hlg Fc region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[00294] In some embodiments, the antibody (or heterologous polypeptide) comprises a first hlg Fc region and a second hlg Fc region. In some embodiments, the first and/or second hlg Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of at least a portion of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of a hlgG hinge region, a hlgG CH2 region, and a hlgG CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of at least a portion of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of a hlgGl hinge region, a hlgGl CH2 region, and a hlgGl CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a h!gG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the first and/or second hlg Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[00295] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more Ig (e.g., hlg) light chain constant region (e.g., a hlg light chain kappa constant region (KCL) or a hlg light chain lambda constant region ( ZCL).

[00296] The amino acid sequence of exemplary reference hlgGl and hIgG4 heavy chain and light chain constant regions, which can be incorporated in one or more of the embodiments described herein (e.g., anti-TFR (e.g., hTFR (e.g., hTFRl)) antibodies and heterologous polypeptides), is provided in Table 5.

Table 5. The Amino Acid Sequence of Exemplary hlg Heavy Chain and Light Chain

Constant Regions and Components Thereof.

[00297] In some embodiments, the antibody (or heterologous polypeptide) comprises one or more hlg constant region, wherein the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 5. In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5. [00298] In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant region comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[00299] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence set forth in Table 5, comprising or consisting of about no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[00300] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 171-198. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198.

[00301] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., amino acid substitutions, deletions, or additions).

[00302] In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the one or more hlg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 171-198, comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

5.3.2.1 Ig Effector Function

[00303] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises an Fc region (see, e.g., § 5.3.2). In some embodiments, the Fc region of an antibody (or heterologous polypeptide) described herein exhibits a decrease in one or more Fc effector function relative to a reference (e.g., wild type) Fc region. Exemplary Fc effector functions include, but are not limited to, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))).

[00304] Standard in vitro and/or in vivo assays known in the art can be conducted to evaluate Fc effector function, including, any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fey receptor) binding affinity, and Clq binding affinity.

[00305] For example, ADCC activity can be assessed utilizing standard (radioactive and nonradioactive) methods known in the art (see, e.g., W02006/082515, W02012/130831), the entire contents of each of which is incorporated by reference herein for all purposes). For example, ADCC activity can be assessed using a chromium-5 (⁵¹Cr) assay. Briefly,⁵¹Cr is pre-loaded into target cells, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicative of lysis of the target cells by the NK cells). Similar non-radioactive assays can also be utilized that employ a similar method, but the target cells are pre-loaded with fluorescent dyes, such as calcein-AM, CFSE, BCECF, or lanthanide fhirophore (Europium). See, e.g., Parekh, Bhavin S et al. “Development and validation of an antibody-dependent cell-mediated cytotoxicityreporter gene assay.” mAbs vol. 4,3 (2012): 310-8. Doi: 10.4161/mabs.19873, the entire contents of which is incorporated by reference herein for all purposes. Exemplary commercially available non-radioactive assays include, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Additional non-limiting examples of in vitro assays that can be used to assess ADCC activity of a fusion protein described herein include those described in US5500362; US5821337; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat’l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggcmann, M., ct al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which is incorporated by reference herein. Alternatively, or additionally, ADCC activity of a fusion protein described herein may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, et al., Proc. Nat’l Acad. Sci. USA 95 (1998) 652-656, the entire contents of which is incorporated by reference herein for all purposes.

[00306] C Iq binding assays can be utilized to assess the ability of a an antibody (or heterologous polypeptide) described herein to bind Clq (or bind with less affinity than a reference fusion protein) and hence lack (or have decreased) CDC activity. The binding of an antibody (or heterologous polypeptide) described herein to Clq can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Clq interactions, including e.g., equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4^th Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein. For example, see, e.g., Clq and C3c binding ELIS As described in W02006/029879 and W02005/100402, the entire contents of each of which is incorporated by reference herein for all purposes. Additional CDC activity assays include those described in e.g., Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and Cragg, M. S., and Glennie, M. J., Blood 103 (2004) 2738-2743), the entire contents of each of which is incorporated by reference herein for all purposes.

[00307] ADCP activity can be measured by in vitro or in vivo methods known in the ail and also commercially available assays (see, e.g., van de Donk NW, Moreau P, Plesner T, et al. “Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma,” Blood, 127(6):681-695 (2016), the entire contents of each of which is incorporated by reference herein for all purposes). For example, a primary cell based ADCP assay can be used in which fresh human peripheral blood mononuclear cells (PBMCs) are isolated, monocytes isolated and differentiated in culture to macrophages using standard procedures. The macrophages are fluorescently labeled added to cultures containing fluorescently labeled target cells. Phagocytosis events can be analyzed using FACS screening and/or microscopy. A modified reporter version of the above described assay can also be used that employs an engineered cell line that stably expresses FcyRIIa (CD32a) as the effector cell line (e.g., an engineered T cell line, e.g., THP-1), removing the requirement for primary cells. Exemplary ADCP assays are described in e.g., Ackerman, M. E. el al. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J. Immunol. Methods 366, 8-19 (2011); and Mcandrew, E. G. et al. Determining the phagocytic activity of clinical antibody samples. J. Vis. Exp. 3588 (2011). Doi: 10.3791/3588; the entire contents of each of which is incorporated by reference herein.

[00308] Binding of an antibody (or heterologous polypeptide) described herein to an Fc receptor can be determined by a variety of in vitro assays (e.g., biochemical or immunological based assays) known in the art for determining Fc-Fc receptor interactions, i.e., specific binding of an Fc region to an Fc receptor. Common assays include equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in e.g., Paul, W. E., ed., Fundamental Immunology, 4” Ed., Lippincott-Raven, Philadelphia (1999), the entire contents of which is incorporated by reference herein for all purposes.

[00309] In some embodiments, the Fc region of an antibody (or heterologous polypeptide) described herein is varied (e.g., comprises one or more amino acid variation (e.g., one or more amino acid substitution, deletion, addition, etc.)) (referred to herein as a “varied Fc region”), relative to the amino acid sequence of a reference Fc region (e.g., a wild type Fc region, e.g., Table 5 herein (e.g., SEQ ID NOS: 178, 180, 191, or 195). In some embodiments, the one or more amino acid variation (e.g., the one or more amino acid substitution, deletion, addition, etc.)) decreases or abolishes one or more Fc effector function, relative to a reference Fc that does not comprise the variation (e.g., the one or more variation (e.g., the one or more amino acid substitution, deletion, addition, etc.)).

[00310] In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits no detectable or decreased ADCC compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation e.g., the one or more amino acid variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits no detectable or decreased CDC compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more amino acid variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a modified Fc region exhibits no detectable or decreased ADCP compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))) compared to a reference antibody (or heterologous polypeptide)that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to FcyRI, Fcylla, and/or Fey I Ha compared to an antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc exhibits decreased or no detectable specific binding affinity to FcyRI compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc exhibits decreased or no detectable specific binding affinity to Fcylla compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to Fcyllla compared to an antibody (or heterologous polypeptide) that docs not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). In some embodiments, the antibody (or heterologous polypeptide) comprising a varied Fc region exhibits decreased or no detectable specific binding affinity to Clq compared to a reference antibody (or heterologous polypeptide) that does not comprise the Fc region variation (e.g., the one or more variation (e.g., one or more amino acid substitution, deletion, or addition)). [00311] In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable ADCC. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable CDC. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable ADCP. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))). In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to FcyRI, Fcylla, and/or. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc exhibits no detectable specific binding affinity to FcyRI. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc exhibits no detectable specific binding affinity to Fcylla. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to Fcyllla. In some embodiments, the antibody (or heterologous polypeptide) comprising an Fc region exhibits no detectable specific binding affinity to Clq.

[00312] Amino acid substitutions that decrease or abolish one or more Fc effector function are known in the ait. See for example, Saunders Kevin, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology, vIO (June 7, 2019) DOI=10.3389/fimmu.2019.01296, the full contents of which is incorporated by reference herein for all purposes, see more particularly for example, e.g., Table 4 of Saunders.

[00313] In some embodiments, the varied Fc comprises a hlgGl Fc region comprising one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hlgGl Fc region comprises an amino acid substitution at amino acid positions L234, L235, and/or P329, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A and/or L235A, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329G, EU numbering according to Kabat. In some embodiments, the hlgGl Fc region comprises the following amino acid substitutions L234A, L235A, and P329A, EU numbering according to Kabat.

[00314] In some embodiments, the varied Fc region comprises a hlg4 Fc region comprising one or more amino acid variations (e.g., one or more amino acid substitutions). In some embodiments, the hIgG4 Fc region comprises an amino acid substitution at amino acid positions S228, F234, and/or L235, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and/or L235A, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc region comprises the following amino acid substitutions S228P, F234A, and/or L235E, EU numbering according to Kabat. In some embodiments, the hIgG4 Fc comprises the following amino acid substitutions S228P and/or L235E, EU numbering according to Kabat.

[00315] The amino acid sequence of exemplary varied Fc regions that are known in the art to exhibit a decrease in one more effector function is provided in Table 6.

Table 6. The Amino Acid Sequence of Exemplary Varied Fc Regions.

[00316] In some embodiments, the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[00317] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.). [00318] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of a polypeptide set forth in Table 6, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[00319] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[00320] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[00321] In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of the variant hlg Fc fusion protein or polypeptide comprises a hlg Fc region that comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 199-224, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[00322] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234 and/or an alanine at position E235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234 and an alanine at position E235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises of an alanine, glycine, or serine at position P329, EU numbering according to Kabat.

[00323] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234; an alanine at position E235; and an alanine, glycine, or serine at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises of an alanine at position E234; an alanine at position E235; and an alanine at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position E234; an alanine at position E235; and a glycine amino acid at position P329 EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234; an alanine at position L235; and a serine amino acid at position P329 EU numbering according to Kabat.

[00324] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and/or an alanine, glycine, or serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[00325] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and/or an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199- 224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[00326] In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and/or an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199- 224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234 and an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine, glycine, or serine amino acid at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and an alanine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a glycine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hlgGl Fc region comprises an alanine at position L234, an alanine at position L235, and a serine at position P329, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[00327] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and an alanine at position L235, EU numbering according to Kabat. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a proline at position S228, EU numbering according to Kabat.

[00328] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234; an alanine at position L235; and a proline at position S228, EU numbering according to Kabat.

[00329] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and/or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[00330] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and/or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

[00331] In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and/or a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234 and/or an alanine at position L235, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224. In some embodiments, the amino acid sequence of the hIgG4 Fc region comprises a phenylalanine at position L234, an alanine at position L235, and a proline at position S228, EU numbering according to Kabat; and comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 199-224.

5.3.2.2 Promotion of Heterodimerization

[00332] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises a first and second Fc region (see, e.g., § 5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise one or more amino acid modifications relative to each other to promote heterodimerization. IgG derived heterodimeric formats can be generated by methods known in the art, e.g., by forced heavy chain heterodimerization. Forced heavy chain heterodimerization can be obtained using known methods in the art, e.g., knob-in-hole or strand exchange engineered domains (SEED), see, e.g., Ji-Hee et al., “Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins” Frontiers in Immunology, v7(article 394) (2016) DOI=10.3389/fimmu.2016.00394 (hereinafter “Ji-Hee 2016”), the entire contents of which is incorporated by reference herein for all purposes.

[00333] In some embodiments, an interface of the first and the second Ig Fc regions is varied, e.g., introduction of an amino acid substitution, to increase heterodimerization, e.g., relative to a non-modified interface, e.g., a naturally occurring interface. For example, dimerization of the first and second Ig Fc regions can be enhanced by providing an Ig Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a non-modified interface.

[00334] Knob-in-Hole amino acid pairing modifications are known in the art, and described in e.g., US5731116; US7476724; Ji-Hee 2016; and Ridgway, J. “'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization” etal. Prot. Engineering 9(7): 617-621 (1996), the full contents of each of which is incorporated by reference herein. Generally, Knob-in- Hole comprises 1) introducing one or more amino acid substitutions in the CH3 domain of one or both of the first and second subject Ig Fc regions to promote heterodimerization; and 2) combining the modified Ig Fc regions under conditions that promote heterodimerization. “Knobs” are typically created by substituting a small amino acid in a parental Ig Fc region with a larger amino acid (e.g., T366Y or T366W); “holes” are created by substituting a larger residue in a parental Ig Fc region with a smaller amino acid (e.g., Y407T, T366S, 11368A, or Y407V). Exemplary Knob- in-Hole mutations include S354C, T366W in the “knob” Ig Fc region and Y349C, T366S, E368A, Y407V in the “hole” Ig Fc region. Other exemplary Knob-in-Hole mutations, which can be incorporated into any one or more of the embodiments, are provided in Table 7, with additional exemplary optional stabilizing Ig Fc cysteine mutations.

Table 7. Exemplary Knob-in-hole and Stabilizing Cysteine Modifications.

[00335] The amino acid sequence of exemplary Fc regions that are known in the ail to promote heterodimerization is provided in Table 8.

Table 8. The Amino Acid Sequence of Exemplary Pairs of Varied Heterodimeric Fc

Regions.

[00336] As described herein, in some embodiments, the antibody (or heterologous polypeptide) comprises a first Fc region and a second 1g Fc region.

[00337] In some embodiments, the amino acid sequence of the first Fc region comprises a T366W amino acid substitution, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, and Y407V, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5). In some embodiments, the amino acid sequence of the first hlg further comprises a S354C amino acid substitution, EU numbering according to Kabat; and the amino acid sequence of the second Fc region comprises a Y349C amino acid substitution, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[00338] In some embodiments, the amino acid sequence of the first Fc region comprises each of the following amino acid substitutions: T366W and a S354C, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, Y407V, and Y349C, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[00339] In some embodiments, the amino acid sequence of the second Fc region comprises a T366W amino acid substitution, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, and Y407V, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5). In some embodiments, the amino acid sequence of the second hlg further comprises a S354C amino acid substitution, EU numbering according to Kabat; and the amino acid sequence of the second Fc region comprises a Y349C amino acid substitution, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[00340] In some embodiments, the amino acid sequence of the second Fc region comprises each of the following amino acid substitutions: T366W and a S354C, EU numbering according to Kabat; and the second the amino acid sequence of the Fc region comprises each of the following amino acid substitutions: T366S, L368A, Y407V, and Y349C, EU numbering according to Kabat; each relative to the amino acid sequence of an exemplary reference Ig Fc region (e.g., a reference Ig Fc region set forth in Table 5).

[00341] In some embodiments, the amino acid sequence of the first Ig Fc region comprises a W amino acid at position T366, EU numbering according to Kabat; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, EU numbering according to Kabat.

[00342] In some embodiments, the amino acid sequence of the first Ig Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat.

[00343] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 225-232; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 241-248.

[00344] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 233-240; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 249-256.

[00345] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 225; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 241.

[00346] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 226; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 242.

[00347] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 227; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 243.

[00348] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 228; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 244.

[00349] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 229; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 245.

[00350] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 230; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 246.

[00351] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 231; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 247.

[00352] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 232; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a

V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 248.

[00353] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 233; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 249.

[00354] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 234; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 250.

[00355] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 235; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 251.

[00356] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 236; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 252.

[00357] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 237; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 253.

[00358] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 238; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 254.

[00359] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 239; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 255.

[00360] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366 and a C amino acid at position S354, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 240; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 256.

5.3.2.3 Ig Constant Region Variations for Site Specific Conjugation

[00361] In some embodiments, the molecular payload is conjugated (e.g., directly, or indirectly through a linker) to the Ig constant region. In some embodiments, the molecular payload (or a linker) is conjugated directly to an amino acid (e.g., a naturally occurring amino acid or an engineered (z.e., variant) amino acid) within the Ig constant region.

[00362] In some embodiments, the molecular payload (or the linker) is conjugated directly to an engineered lysine, cysteine, or tyrosine amino acid residue within the Ig constant region. In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more naturally occurring amino acid residue with a lysine, cysteine, or tyrosine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more non-cysteine amino acid residue with a cysteine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more non-lysine amino acid residue with a lysine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the substitution of one or more nontyrosine amino acid residue with a tyrosine amino acid residue (e.g., to mediate conjugation).

[00363] In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more lysine, cysteine, or tyrosine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more lysine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more cysteine amino acid residue (e.g., to mediate conjugation). In some embodiments, the amino acid sequence of the Ig constant region comprises the addition of one or more tyrosine amino acid residue (e.g., to mediate conjugation).

5.3.2.4 Exemplary Variant Fc Regions

[00364] As described herein, in some embodiments, the antibody (or heterologous moiety) comprises a first and second Ig Fc region (see, e.g., § 5.3.2). In some embodiments, the first Ig Fc region and the second Ig Fc region each comprise multiple amino acid variations described herein, e.g., one or more amino acid variation that decreases or abolishes one or more Ig Fc effector function (e.g., ADCC, ADCP, CDC, and binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRl, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRl, Fcylla, and/or Fcyllla))) (see, e.g., § 5.3.2.1); and one or more amino acid modification that promote heterodimerization of the first and second Fc regions (see, e.g., § 5.3.2.2).

[00365] In some embodiments, the first and second Fc region each comprise one or more amino acid variation that decreases or abolishes one or more Fc effector function (e.g., ADCC, ADCP, CDC, binding affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRl, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRl, Fcylla, and/or Fcyllla))) (see, e.g., § 5.3.2.1); and one or more amino acid variation that promote heterodimerization of the first and second Fc regions (see, e.g., § 5.3.2.2).

[00366] The amino acid sequence of exemplary variant Fc regions is provided in Table 9.

Table 9. The Amino Acid Sequence of Exemplary Variant Fc Regions.

[00367] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid residue at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 257-264; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, and a valine amino acid at position Y407, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 265-272.

[00368] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 273-280; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, a valine amino acid at position Y407, and a cysteine amino acid at position Y349, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOS: 281-288. [00369] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 257; and the amino acid sequence of the second Ig Fc region comprises a serine amino acid at position T366, an alanine amino acid at position L368, a valine amino acid at position Y407, a cysteine amino acid residue at position Y349, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 265.

[00370] In some embodiments, the amino acid sequence of the first Fc region comprises a tryptophan amino acid at position T366, a cysteine amino acid residue at position S354, a leucine amino acid residue at position L234, a leucine amino acid residue at position L235, and an alanine amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 258; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 266.

[00371] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 259; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:

267.

[00372] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 260; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, an A amino acid at position L368, and a V amino acid at position Y407, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:

268.

[00373] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 261; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:

269.

[00374] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 262; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:

270.

[00375] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 263; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO:

271.

[00376] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 264; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, and a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an A amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 272.

[00377] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 273; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, a V amino acid at position Y407, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 281.

[00378] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 274; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 282.

[00379] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 275; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, and a C amino acid at position Y349, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 283.

[00380] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 276; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 284.

[00381] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 277; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 285.

[00382] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 278; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 286.

[00383] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 279; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position L368, a V amino acid at position Y407, and a C amino acid at position Y349, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 287.

[00384] In some embodiments, the amino acid sequence of the first Fc region comprises a W amino acid at position T366, a C amino acid residue at position S354, an L amino acid residue at position L234, an L amino acid residue at position L235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 280; and the amino acid sequence of the second Ig Fc region comprises a S amino acid at position T366, an A amino acid at position E368, a V amino acid at position Y407, and a C amino acid at position Y349, a C amino acid residue at position Y349, an L amino acid residue at position L234, an L amino acid residue at position E235, and an G amino acid residue at position P329, EU numbering according to Kabat, and is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NO: 288.

5.4 Methods of Making Proteins (e.g., Targeting Agents)

[00385] Any of the proteins (e.g., antibodies), peptides, fusion proteins, etc., including the targeting moieties described herein, may be produced using standard methods known in the art. For example, each may be produced by recombinant technology in host cells (e.g., insect cells, mammalian cells, bacteria) that have been transfected or transduced with a nucleic acid expression vector (e.g., plasmid, viral vector (e.g., a baculoviral expression vector)) encoding the protein (e.g., antibody). Such general methods are common knowledge in the art. The expression vector typically contains an expression cassette that includes nucleic acid sequences capable of bringing about expression of the nucleic acid molecule encoding the protein interest, such as promoter(s), enhancer(s), polyadenylation signals, and the like. The person of ordinary skill in the art is aware that various promoter and enhancer elements can be used to obtain expression of a nucleic acid molecule in a host cell. For example, promoters can be constitutive or regulated, and can be obtained from various sources, e.g., viruses, prokaryotic or eukaryotic sources, or artificially designed. Post transfection or transduction, host cells containing the expression vector encoding the protein of interest are cultured under conditions conducive to expression of the nucleic acid molecule encoding the protein. Culture media is available from various vendors, and a suitable medium can be routinely chosen for a host cell to express a protein or polypeptide of interest. Host cells can be adherent or suspension cultures, and a person of ordinary skill in the art can optimize culture methods for specific host cells selected. For example, suspension cells can be cultured in, for example, bioreactors in e.g., a batch process or a fed-batch process. The produced protein or polypeptide may be isolated from the cell cultures, by, for example, column chromatography in either flow-flow through or bind-and-elute modes. Examples include, but are not limited to, ion exchange resins and affinity resins, such as lentil lectin Sepharose, and mixed mode cation exchange-hydrophobic interaction columns (CEX-HIC). The protein or polypeptide may be concentrated, buffer exchanged by ultrafiltration, and the retentate from the ultrafiltration may be filtered through an appropriate filter, e.g., a 0.22pm filter. See, e.g., Hacker, David (Ed.), Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology), Humana Press (2018); and McPherson et al., “Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant,” Chapter 4, in Sunil Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases, Methods in Molecular’ Biology, Springer, New York, 2016. See also U.S. Pat. 5,762,939, the entire contents of each of which is incorporated by reference herein for all purposes.

[00386] The proteins (e.g., antibodies), peptides, fusion proteins, etc. described herein may also be produced synthetically.

5.5 Molecular Payloads

[00387] As described above, the conjugates described herein comprise at least one molecular payload e.g., oligonucleotide (e.g., an oligonucleotide described herein)). In some embodiments, the molecular’ payload (e.g., oligonucleotide (e.g., an oligonucleotide described herein)) modulates (e.g., inhibits, enhances (e.g., inhibits)) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., BCAL11A (e.g., hBCLUA)) (e.g., within a cell, e.g., within a cell in a subject, e.g., a mammalian subject, e.g., a human subject) (e.g., through the degradation of the target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., BCAL1 1 A (e.g., hBCLl 1 A)) mRNA). In some embodiments, the molecular payload (e.g., oligonucleotide (e.g., an oligonucleotide described herein)) inhibits expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., BCAL11A (e.g., hBCLUA)) (e.g., within a cell, e.g., within a cell in a subject, e.g., a mammalian subject, e.g., a human subject) (e.g., through the degradation of the target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., BCAL11 AA (e.g., hBCLl 1 A)) mRNA).

[00388] In some embodiments, the molecular payload (e.g., oligonucleotide (e.g., an oligonucleotide described herein)) mediates the modulation (e.g., inhibition, enhancement (e.g., inhibition)) of the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein through binding to the target mRNA (e.g., a BCL11A mRNA) (e.g., a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments wherein the molecular payload (e.g., oligonucleotide (e.g., an oligonucleotide described herein)) mediates the modulation (e.g., inhibition, enhancement (e.g., inhibition)) of the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein through binding to the target mRNA (e.g., a BCL11A mRNA) (e.g., a portion of a target mRNA (e.g., a BCL11A mRNA)) the downstream mechanism by which the modulation (e.g., inhibition) is achieved may differ (e.g., depending on the form of the molecular payload (e.g., oligonucleotide)). For example, in some embodiments, the molecular payload (e.g., oligonucleotide) is designed such that the molecular payload (e.g., oligonucleotide) causes any one or more of the degradation of a target RNA (e.g., mRNA), disabling of a target RNA (e.g., mRNA), modification of a target RNA (e.g., mRNA), a correction of aberrant splicing of a target RNA (e.g., mRNA), alteration in the splicing of a target RNA (e.g., mRNA), alteration (e.g., decrease) in the stability of a target RNA (e.g., mRNA), a block in translation of a target RNA (e.g., mRNA), and/or the promotion of exon skipping in a target RNA (e.g., mRNA) sequence.

[00389] In some embodiments, the molecular payload is an oligonucleotide (e.g., described herein), a protein, a peptide, a small molecule, a carbohydrate, lipid, or synthetic polymer. In some embodiments, the molecular payload is an oligonucleotide (e.g., described herein). In some embodiments, the molecular payload is a protein. In some embodiments, the molecular payload is a peptide. In some embodiments, the molecular payload is a small molecule.

5.5.1 Oligonucleotides

[00390] In some embodiments, the molecular' payload is an oligonucleotide. In some embodiments, the oligonucleotide comprises or consists of RNA, DNA, or both RNA and DNA. In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is double stranded. In some embodiments, the oligonucleotide is a single stranded DNA (ssDNA) oligonucleotide. In some embodiments, the oligonucleotide is a single stranded RNA (ssRNA) oligonucleotide. In some embodiments, the oligonucleotide is a double stranded DNA (dsDNA) oligonucleotide. In some embodiments, the oligonucleotide is a double stranded RNA (dsRNA) oligonucleotide.

[00391] While the term oligonucleotide is singular, it includes, for example, double stranded oligonucleotides that are connected only through the double stranded region (i.e., are not part of a single nucleic acid molecule) (e.g., such as siRNA molecules that comprise a sense strand and an antisense strand that hybridize to form a double stranded region and are not part of the same single larger nucleic acid molecule).

[00392] Any suitable format of oligonucleotide may be utilized in the conjugates described herein. Various oligonucleotides are known in the ail, including, but not limited to, e.g., RNAi agents, including e.g., small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and microRNAs (miRNAs); antisense oligonucleotides (ASOs), U1 adaptors (see, e.g., US20130137749, US9078823, US2011021736, and US8343941, the entire contents of each of which is incorporated by reference herein for all purposes), gapmers, mixmers, ribozyme, or an aptamer e.g., an aptamer that mediates target degradation), a guide oligonucleotide (e.g., a guide oligonucleotide (e.g., RNA) designed to direct activity of an enzyme (e.g., a CRISPR enzyme). In some embodiments, the oligonucleotide comprises an activating RNA (RNAa) or a small activating RNA (saRNA) (see, e.g., Tan CP, Sinigaglia L, Gomez V, Nicholls J, Habib NA. RNA Activation-A Novel Approach to Therapeutically Upregulate Gene Transcription. Molecules. 2021 ;26(21):6530. Published 2021 Oct 28. doi:10.3390/molecules26216530; Ghanbarian H, Aghamiri S, Eftckhary M, Wagner N, Wagner KD. Small Activating RNAs: Towards the Development of New Therapeutic Agents and Clinical Treatments. Cells. 2021;10(3):591. Published 2021 Mar- 8. Doi: 10.3390/cells 10030591; Albert Kwok, Nina Raulf, and Nagy Habib, Developing small activating RNA as a therapeutic: current challenges and promises, Therapeutic Delivery 2019 10:3, 151-164; WO2022140264A1; WO2023099884A1; W02023024230A1).

[00393] In some embodiments, the oligonucleotide comprises or consists of an siRNA, a shRNA, a miRNA, or an ASO. In some embodiments, the oligonucleotide comprises or consists of a siRNA. In some embodiments, the oligonucleotide comprises or consists of an ASO. In some embodiments, the oligonucleotide comprises or consists of a shRNA. In some embodiments, the oligonucleotide comprises or consists of a miRNA.

[00394] Various salts, mixed salts and free acid forms of the oligonucleotides are also provided herein. In some embodiments, the oligonucleotide is in a free acid form. In some embodiments, the oligonucleotide is in a salt form. In some embodiments, the oligonucleotide is in a sodium salt form. In some embodiments, wherein the oligonucleotide is in the sodium salt form, sodium ions are present in the composition comprising the oligonucleotide as counterions for substantially all of the phosphodiester or phosphoro thioate groups present in the oligonucleotide. In some embodiments, wherein the oligonucleotide is in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester or phosphorothioate groups present in the oligonucleotide.

5.5.1.1 Overall Length

[00395] The overall length of the oligonucleotide may vary depending on the specific format of the oligonucleotide (e.g., siRNA, shRNA, ASO, etc.).

[00396] In some embodiments, the oligonucleotide comprises or consists of from about 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15, 15-100, 15-90, 15-80, 15-70, 15- 60, 15-50, 15-40, 15-30, 15-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30- 40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-

90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 nucleotides.

[00397] In some embodiments, the oligonucleotide comprises or consists of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[00398] In some embodiments, the oligonucleotide comprises or consists of from about 15-50 nucleotides (e.g., 15-49, 15-48, 15-47, 15- 46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15- 36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15- 26, 15- 25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26,

18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,

19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides). In some embodiments, the oligonucleotide comprises or consists of from about 15-50, 15-49, 15-48, 15-47, 15- 46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15- 36, 15-35, 15-34, 15-33, 15-32, 15- 31, 15-30, 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29,

19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,

20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21- 22 nucleotides.

[00399] In some embodiments, the oligonucleotide comprises or consists of from about 15-30 nucleotides (e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-30,

19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,

20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides). In some embodiments, the oligonucleotide comprises or consists of from about 18-25 nucleotides (e.g., 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19- 21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides). In some embodiments, the oligonucleotide comprises or consists of from about 19-25 nucleotide (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 20-21, 20-22, 20-23, 20-24, 20-25, 21-22, 21-23, 21-24, 21-25, 22-23, 22-24, 22-25, 23-24, 23-25, 24-25 nucleotides). In some embodiments, the oligonucleotide comprises or consists of from about 15- 30, 16-30, 17-30, 18-30, 19-30 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 36-30, 27-30, 28-30-, 29-30, 19-20, 19-21, 19-22, 19-23, 19-24, or 19-25 nucleotides.

[00400] In some embodiments, the oligonucleotide comprises or consists of about 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 15 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 16 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 17 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 18 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 19 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 20 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 21 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 22 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 23 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 24 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 25 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 26 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 27 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 28 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 29 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 30 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 31 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 32 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 33 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 34 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 35 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 36 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 37 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 38 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 39 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 40 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 41 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 42 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 43 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 44 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 45 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 46 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 47 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 48 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 49 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 50 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 51 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 52 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 53 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 54 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 55 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 56 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 57 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 58 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 59 nucleotides. In some embodiments, the oligonucleotide comprises or consists of about 60 nucleotides.

5.5.1.2 Targeting Region

[00401] In some embodiments, the oligonucleotide comprises a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of a target nucleic acid molecule (e.g., a target mRNA e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)).

[00402] In some embodiments, the targeting region comprises the full overall length of the oligonucleotide. In some embodiments, the targeting region is shorter than the full overall length of the oligonucleotide. In embodiments wherein the oligonucleotide is double stranded, only one strand may comprise a targeting region (e.g., an siRNA comprising a sense and antisense strand, wherein the antisense strand comprises the targeting region).

[00403] In some embodiments, the nucleotide sequence of the region of complementarity is at least substantially complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is fully complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)).

[00404] In some embodiments, the nucleotide sequence of the region of complementarity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)).

[00405] In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of one or more non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL1 1A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 5 (e.g., 4, 3, 2, 1, or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 3 (e.g., 2, 1, or 0) non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 2 (e.g., 1 or 0) non-complementar y nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 1 (e.g., 0) non-complementary nucleotide mismatch relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises 0 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3'-end of the region of complementarity. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3'-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3'- end of the region of complementarity. Methods known in the art and described herein can be utilized to evaluate the effect of any non-complementary mismatches between an antisense strand and a target nucleic acid molecule on functional properties (e.g., inhibition of expression of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA))).

[00406] In some embodiments, the region of complementarity comprises or consists of from about 15-30 nucleotides, e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-

20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,

20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,

21-24, 21-23, or 21-22 nucleotides. In some embodiments, the region of complementarity comprises from about 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-

21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides. In some embodiments, the region of complementarity comprises from about 19-21 (e.g., 19-20) nucleotides. In some embodiments, the region of complementarity comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 20 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 21 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 22 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

[00407] In some embodiments, the target nucleic acid molecule is part (e.g., a contiguous portion) of a larger nucleic acid molecule. For example, in some embodiments, the target nucleic acid molecule is a portion (e.g., a contiguous portion) of a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is a contiguous nucleotide sequence of a target mRNA (e.g., a BCL11A mRNA) of sufficient length to allow .

[00408] In some embodiments, the target nucleic acid molecule is a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA (e.g., a BCL11A mRNA) formed in the expression of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a mammalian, primate, human, non-human primate, mouse, and/or rat gene) (e.g., a BCL11A gene). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a BCL11A (e.g., hBCLUA) mRNA. In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA formed in the expression of a BCL11A (e.g., hBCLUA) gene. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of the nucleotide sequence set forth in SEQ ID NO: 289 (or a variant or fragment thereof). In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoded by a target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoding a BCL11A (e.g., hBCLUA) protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant or fragment thereof).

[00409] In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-30 nucleotides, e.g., 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-30, 23-29, 23-28, 23-27, 23-26, 23-27, 23-26, 23-25, or 23-24 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 20 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 21 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths arc also contemplated to be pail of the disclosure.

5.5.1.3 Antisense Oligonucleotides

[00410] In some embodiments, the oligonucleotide comprises or consists of an ASO. ASOs are generally known in the art and described above. ASOs are generally single stranded RNA or DNA. In some embodiments, the ASO is a DNA oligonucleotide. In some embodiments, the ASO is a ssDNA oligonucleotide. In some embodiments, the ASO is an RNA oligonucleotide. In some embodiments, the ASO is a ssRNA oligonucleotide.

(i) Overall Length

[00411] In some embodiments, the ASO comprises or consists of from about 10-50, 10-40, 10- 30, 10-20, 10-15, 15-50, 15-40, 15-30, 15-20, 20-50, 20-40, 20-30, 25-50, 25-40, or 25-30 nucleotides. In some embodiments, the ASO comprises or consists of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the ASO comprises or consists of from about 15-30 nucleotides (e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23,

18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,

20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,

21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides). In some embodiments, the ASO comprises or consists of from about 18-25 nucleotides (e.g., 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24,

19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25,

22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides). In some embodiments, the ASO comprises or consists of from about 19-25 nucleotide (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 20-21,

20-22, 20-23, 20-24, 20-25, 21-22, 21-23, 21-24, 21-25, 22-23, 22-24, 22-25, 23-24, 23-25, 24-25 nucleotides). In some embodiments, the ASO comprises or consists of from about 15-30, 16-30, 17-30, 18-30, 19-30 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 36-30, 27-30, 28-30-, 29-30, 19-

20, 19-21, 19-22, 19-23, 19-24, or 19-25 nucleotides.

[00412] In some embodiments, the ASO comprises or consists of about 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the ASO comprises or consists of about 15 nucleotides. In some embodiments, the ASO comprises or consists of about 16 nucleotides. In some embodiments, the ASO comprises or consists of about 17 nucleotides. In some embodiments, the ASO comprises or consists of about 18 nucleotides. In some embodiments, the ASO comprises or consists of about 19 nucleotides. In some embodiments, the ASO comprises or consists of about 20 nucleotides. In some embodiments, the ASO comprises or consists of about 21 nucleotides. In some embodiments, the ASO comprises or consists of about 22 nucleotides. In some embodiments, the ASO comprises or consists of about 23 nucleotides. In some embodiments, the ASO comprises or consists of about 24 nucleotides. In some embodiments, the ASO comprises or consists of about 25 nucleotides. In some embodiments, the ASO comprises or consists of about 26 nucleotides. In some embodiments, the ASO comprises or consists of about 27 nucleotides. In some embodiments, the ASO comprises or consists of about 28 nucleotides. In some embodiments, the ASO comprises or consists of about 29 nucleotides. In some embodiments, the ASO comprises or consists of about 30 nucleotides.

(ii) Targeting Region

[00413] As described above, ASOs (e.g., described herein) comprise a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of a target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)).

[00414] Typically, the targeting region comprises the full overall length of the ASO (e.g., as described herein). However, in some embodiments, the targeting region may be shorter than the full overall length of the ASO.

[00415] In some embodiments, the nucleotide sequence of the region of complementarity is at least substantially complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is fully complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)).

[00416] In some embodiments, the nucleotide sequence of the region of complementarity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)).

[00417] In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of one or more non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 5 (e.g., 4, 3, 2, 1, or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 3 (e.g., 2, 1, or 0) non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 2 (e.g., 1 or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 1 (e.g., 0) non-complementary nucleotide mismatch relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises 0 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3'-cnd of the region of complementarity. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3'- end of the region of complementarity. Methods known in the ail and described herein can be utilized to evaluate the effect of any non-complementary mismatches between an antisense strand and a target nucleic acid molecule on functional properties (e.g., inhibition of expression of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA))).

[00418] In some embodiments, the region of complementarity comprises or consists of from about 15-30 nucleotides, e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-

20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,

20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,

21-24, 21-23, or 21-22 nucleotides. In some embodiments, the region of complementarity comprises from about 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-

21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides. In some embodiments, the region of complementarity comprises from about 19-21 (e.g., 19-20) nucleotides. In some embodiments, the region of complementarity comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 20 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 21 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 22 nucleotides. Tn some embodiments, the region of complementarity comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

[00419] In some embodiments, the target nucleic acid molecule is part (e.g., a contiguous portion) of a larger nucleic acid molecule. For example, in some embodiments, the target nucleic acid molecule is a portion (e.g., a contiguous portion) of a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is a contiguous nucleotide sequence of a target mRNA (e.g., a BCL11A mRNA) of sufficient length to allow .

[00420] In some embodiments, the target nucleic acid molecule is a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a target mRNA (e.g., a BCL11A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA (e.g., a BCL11A mRNA) formed in the expression of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a mammalian, primate, human, non-human primate, mouse, and/or rat gene) (e.g., a BCL11A gene). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a BCL11A (e.g., hBCLUA) mRNA. In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA formed in the expression of a BCL11A (e.g., hBCLUA) gene. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of the nucleotide sequence set forth in SEQ ID NO: 289, 292, or 295 (or a variant or fragment thereof). In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoded by a target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoding a BCL11 A (e.g., hBCLl 1 A) protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 291, 294, or 297 (or a variant or fragment thereof).

[00421] In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-30 nucleotides, e.g., 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-30, 23-29, 23-28, 23-27, 23-26, 23-27, 23-26, 23-25, or 23-24 nucleotides. Tn some embodiments, the target nucleic acid molecule comprises or consists of from about 19-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 20 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 21 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

5.5.1.4 RNAi Agents

[00422] In some embodiments, the oligonucleotide comprises or consists of an RNAi agent. As described above, RNAi agents include, e.g., siRNA molecules, shRNA molecules, and miRNA molecules. In some embodiments, the RNAi agent is a siRNA. In some embodiments, the RNAi agent is a miRNA. In some embodiments, the RNAi agent is a shRNA.

[00423] In some embodiments, the RNAi agent comprises one or more RNA molecule. In some embodiments, the RNAi agent comprises an antisense strand. In some embodiments, the RNAi agent comprises a sense strand. In some embodiments, the agent comprises one or more single stranded RNA (ssRNA) molecules. In some embodiments, the RNAi agent comprises a dsRNA agent.

[00424] In some embodiments, the RNAi agent comprises a dsRNA agent comprising a sense strand and an antisense strand. In some embodiments, the RNAi agent comprises a dsRNA agent comprising a sense strand and an antisense strand that form a double stranded region. In some embodiments, the RNAi agent comprises a dsRNA agent comprising a sense strand and an antisense strand that hybridize to form a double stranded region. In some embodiments, the sense strand and the antisense strand are part of a single nucleic acid molecule (e.g., a single nucleic acid molecule comprising a hairpin loop). In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules. (i) Antisense Strand

(a) Overall Length

[00425] In some embodiments, the antisense strand comprises or consists of from about 15-30 nucleotides (e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,

19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21 , 19-20, 20-30, 20-29, 20-28, 20-27,

20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides). In some embodiments, the antisense strand comprises or consists of from about 18-25 nucleotides (e.g., 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19- 21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides). In some embodiments, the antisense strand comprises or consists of from about 19-25 nucleotide (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 20-21, 20-22, 20-23, 20-24, 20-25, 21-22, 21-23, 21-24, 21-25, 22-23, 22-24, 22-25, 23-24, 23-25, 24-25 nucleotides). In some embodiments, the antisense strand comprises or consists of from about 15- 30, 16-30, 17-30, 18-30, 19-30 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 36-30, 27-30, 28-30-, 29-30, 19-20, 19-21, 19-22, 19-23, 19-24, or 19-25 nucleotides.

[00426] In some embodiments, the antisense strand comprises or consists of not more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the antisense strand comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the antisense strand comprises or consists of about 21 nucleotides. In some embodiments, the antisense strand comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

(b) Targeting Region

[00427] As described above, antisense strands (e.g., described herein) comprise a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of a target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least substantially complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is fully complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)).

[00428] In some embodiments, the nucleotide sequence of the region of complementarity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is at least 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity is 100% complementary to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11 A mRNA), a portion of a target mRNA (e.g., a BCL11 A mRNA)).

[00429] In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of one or more non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)). In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 5 (e.g., 4, 3, 2, 1, or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 3 (e.g., 2, 1, or 0) non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 2 (e.g., 1 or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 1 (e.g., 0) non-complementary nucleotide mismatch relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the nucleotide sequence of the region of complementarity comprises 0 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non- complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3'-end of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 non-complementary nucleotide mismatches relative to the nucleotide sequence of the target nucleic acid molecule, wherein the one or more non-complementary nucleotide mismatches are within the last 3 (e.g., 2 or 1) nucleotides from either the 5'- and/or 3'- end of the region of complementarity. Methods known in the art and described herein can be utilized to evaluate the effect of any non-complementary mismatches between an antisense strand and a target nucleic acid molecule on functional properties (e.g., inhibition of expression of the target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA))).

[00430] In some embodiments, the region of complementarity comprises or consists of from about 15-30 nucleotides, e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15- 20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,

20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,

21-24, 21-23, or 21-22 nucleotides. In some embodiments, the region of complementarity comprises from about 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-25, 19-24, 19-23, 19-22, 19- 21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides. In some embodiments, the region of complementarity comprises from about 19-21 (e.g., 19-20) nucleotides. In some embodiments, the region of complementarity comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 20 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 21 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 22 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths arc also contemplated to be part of the disclosure.

[00431] In some embodiments, the target nucleic acid molecule is part (e.g., a contiguous portion) of a larger nucleic acid molecule. For example, in some embodiments, the target nucleic acid molecule is a portion (e.g., a contiguous portion) of a target mRNA (e.g., a BCL11 A mRNA). In some embodiments, the target nucleic acid molecule is a contiguous nucleotide sequence of a target mRNA (e.g., a BCL11 A mRNA) of sufficient length to allow it to be a substrate for cleavage directed by an RNAi agent (e.g., an RNAi agent described herein, e.g., a dsRNA agent (e.g., described herein)) (i.e., cleavage through a RISC pathway).

[00432] In some embodiments, the target nucleic acid molecule is a target mRNA (e.g., a BCL11A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a target mRNA (e.g., a BCL11A mRNA). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA (e.g., a BCL11 A mRNA) formed in the expression of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a mammalian, primate, human, non-human primate, mouse, and/or rat gene) (e.g., a BCL11A gene). In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of a BCL11A (e.g., hBCLUA) mRNA. In some embodiments, the target nucleic acid molecule is at least a portion (e.g., a portion) of an mRNA formed in the expression of a BCL11A (e.g., hBCLUA) gene. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of the nucleotide sequence set forth in SEQ ID NO: 1 (or a variant or fragment thereof). In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoded by a target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA encoding a BCL11A (e.g., hBCLUA) protein. In some embodiments, the target nucleic acid molecule comprises at least a portion (e.g., a portion) of an mRNA sequence encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant or fragment thereof).

[00433] In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-30 nucleotides, e.g., 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-30, 23-29, 23-28, 23-27, 23-26, 23-27, 23-26, 23-25, or 23-24 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 19-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-25 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of from about 21-23 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 19 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 20 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 21 nucleotides. In some embodiments, the target nucleic acid molecule comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

(ii) Sense Strand

(a) Antisense Strand Complementarity

[00434] As described above, sense strands (e.g., described herein) comprise a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of at least a portion of an antisense strand. As such, pairs of sense and antisense strands can hybridize to form a double stranded region (e.g., under conditions in which the pairs will be used).

[00435] In some embodiments, the nucleotide sequence of the region of complementarity is at least substantially complementary to the nucleotide sequence of at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity is fully complementary to the nucleotide sequence of at least a portion of an antisense strand.

[00436] In some embodiments, the nucleotide sequence of the region of complementarity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity is at least 95%, 96%, 97%, 98%, 99%, or 100% complementary to the nucleotide sequence of at least a portion of an antisense strand.

[00437] In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of one or more non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 5 (e.g., 4, 3, 2, 1, or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 3 (e.g., 2, 1, or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 2 (e.g., 1 or 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity comprises or consists of no more than 1 (e.g., 0) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the nucleotide sequence of the region of complementarity comprises 0 non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand. In some embodiments, the region of complementarity comprises one or more (e.g., 2, 3, or more) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand, wherein the one or more non-complementary nucleotide mismatch is within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from cither the 5'- and/or 3'-cnd of the region of complementarity. In some embodiments, the region of complementarity comprises at least one but not more than 3 (e.g., 1, 2, or 3) non-complementary nucleotide mismatches relative to the nucleotide sequence of the at least a portion of an antisense strand, wherein the one or more non- complementary nucleotide mismatch is within the last 5 (e.g., 4, 3, 2, or 1) nucleotides from either the 5'- and/or 3 '-end of the region of complementarity.

[00438] In some embodiments, the region of complementarity comprises from about 15-30 nucleotides, e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,

19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,

20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. In some embodiments, the region of complementarity comprises from about

18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20- 24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides. In some embodiments, the region of complementarity comprises from about 19-21 (e.g., 19-20) nucleotides. In some embodiments, the region of complementarity comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19, 20, or 21 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 19 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 20 nucleotides. In some embodiments, the region of complementarity comprises or consists of about 21 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be pail of the disclosure.

(b) Overall Length

[00439] In some embodiments, the sense strand comprises or consists of from about 15-30 nucleotides (e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,

19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,

20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides). In some embodiments, the sense strand comprises or consists of from about 18-25 nucleotides (e.g., 18-24, 18-23, 18-22, 18-21 , 18-20, 19-25, 19-24, 19-23, 19-22, 19-21 , 19- 20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 nucleotides). In some embodiments, the sense strand comprises or consists of from about 19-25 nucleotide (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 20-21, 20-22, 20-23, 20- 24, 20-25, 21-22, 21-23, 21-24, 21-25, 22-23, 22-24, 22-25, 23-24, 23-25, 24-25 nucleotides). In some embodiments, the sense strand comprises or consists of from about 15-30, 16-30, 17-30, 18- 30, 19-30 20-30, 21-30, 22-30, 23-30, 24-30, 25-30, 36-30, 27-30, 28-30-, 29-30, 19-20, 19-21,

19-22, 19-23, 19-24, or 19-25 nucleotides.

[00440] In some embodiments, the sense strand comprises or consists of not more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the sense strand comprises or consists of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the sense strand comprises or consists of about 19, 20, 21, 22, 23 nucleotides. In some embodiments, the sense strand comprises or consists of about 19, 20, 21 nucleotides. In some embodiments, the sense strand comprises or consists of about 20 nucleotides. In some embodiments, the sense strand comprises or consists of about 21 nucleotides. In some embodiments, the sense strand comprises or consists of about 21 nucleotides. In some embodiments, the sense strand comprises or consists of about 23 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

(iii) dsRNA Agents

[00441] In some embodiments, the oligonucleotide (e.g., RNAi agent) comprises a dsRNA agent comprising an antisense strand (e.g., described herein) and a sense strand (e.g., described herein) that hybridize to form a double stranded region (e.g., under conditions in which the dsRNA will be used (e.g., under physiological (e.g., mammalian, e.g., human) conditions within a cell)).

[00442] As described above, antisense strands (e.g., described herein) comprise a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of a target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA), a portion of a target mRNA (e.g., a BCL11A mRNA)); and the sense strands comprise a region of complementarity that comprises a nucleotide sequence that is at least partially (e.g., substantially, fully) complementary to the nucleotide sequence of at least a portion of an antisense strand. (a) Single & Multiple Nucleic Acid Molecules

[00443] As described herein, and known in the art, the sense strand and the antisense strand can be part of a single larger nucleic acid molecule (connected as a single stranded nucleic acid molecule) or separate nucleic acid molecules (only connected through the double stranded region). In some embodiments, the sense strand and the antisense strand are separate nucleic acid molecules. In some embodiments, sense strand and the antisense strand are pail of a single larger nucleic acid molecule.

[00444] In embodiments wherein the sense and antisense strands are part of a single nucleic acid molecule, the nucleic acid molecule may comprise a hairpin loop between the antisense strand and the sense strand to allow for formation of the double stranded region. In some embodiments, the hairpin loop comprises at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, 25 or more) unpaired nucleotides (non-complementary nucleotide mismatches). In some embodiments, the hairpin loop comprises at least one but less than 25, 23, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 unpaired nucleotides (non-complementary nucleotide mismatches). In some embodiments, the hairpin loop comprises about 25, 23, 20, 9, 8, 7, 6, 5, 4, 3, or 1 unpaired nucleotide (non-complementary nucleotide mismatch).

[00445] Without wishing to be bound by theory, in embodiments wherein the sense strand and the antisense strand are part of a single nucleic acid molecule, after introduction into a suitable cell (e.g., a mammalian cell, e.g., a human cell), the nucleic acid molecule may be cleaved into a dsRNA molecule wherein the two strands of the dsRNA molecule are no longer part of the same nucleic acid molecule e.g., by a Type III endonuclease (e.g., Dicer) (see, e.g., Sharp et al. (2001) Genes Dev. 15:485, the entire contents of which are incorporated by herein by reference for all purposes).

(b) Length of Double Stranded Region

[00446] In some embodiments, the double stranded region is about 15-30 base pairs in length (e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17,

18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,

19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25,

20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length). In some embodiments, the double stranded region is about 18-25 base pairs in length (e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21 , 21-25, 21-24, 21 -23, 21-22, 22- 25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length (e.g., 19-21 base pairs in length)). In some embodiments, the double stranded region is about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15- 20, 15-19, 15-18, 15-17, 15-16, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26,

21-25, 21-24, 21-23, or 21-22 base pairs in length. In some embodiments, the double stranded region is about 19-21 (e.g., 19-20) base pairs in length.

[00447] In some embodiments, the double stranded region is not more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, the double stranded region is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, the double stranded region is about 19, 20, or 21 base pairs in length. In some embodiments, the double stranded region is about 19 base pairs in length. In some embodiments, the double stranded region is about 20 base pairs in length. In some embodiments, the double stranded region is about 21 base pairs in length. In some embodiments, the double stranded region is about 23 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

(c) Nucleotide Overhangs & Blunt Ends

[00448] In some embodiments, the dsRNA agent comprises one or more (e.g., 1 or 2) nucleotide overhang. As is clear from the disclosure, but for the sake of clarity, the nucleotides of a nucleotide overhang can include one or more a modified (e.g., chemically modified) nucleotide (e.g., described herein, e.g., described in § 5.5.1.5).

[00449] In some embodiments, the nucleotide overhang comprises from about 1-5 nucleotides, e.g., 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, 4-5 nucleotides. In some embodiments, the nucleotide overhang comprises or consists of about 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the nucleotide overhang comprises or consists of about 1 nucleotide. In some embodiments, the nucleotide overhang comprises or consists of about 2 nucleotides.

[00450] The nucleotide overhang(s) can be on the sense strand, the antisense strand, or both the sense strand and the antisense strand. In some embodiments, the sense strand comprises a nucleotide overhang. In some embodiments, the antisense strand comprises a nucleotide overhang. In some embodiments, the sense strand and the antisense strand both comprise a nucleotide overhang. [00451] Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'- end, or both the 5'-cnd, 3'- end of an antisense or sense strand. In some embodiments, the sense strand comprises a nucleotide overhang at the 5'-end. In some embodiments, the sense strand comprises a nucleotide overhang at the 3'-end. In some embodiments, the sense strand comprises a nucleotide overhang at the 5'-end and the 3 '-end. In some embodiments, the antisense strand comprises a nucleotide overhang at the 5'-end. In some embodiments, the antisense strand comprises a nucleotide overhang at the 3'-end. In some embodiments, the antisense strand comprises a nucleotide overhang at the 5'-end and the 3 '-end. In some embodiments, the antisense strand comprises a nucleotide overhang at the 3'-end; and the sense strand comprises a nucleotide overhang at the 3'-end. In some embodiments, the antisense strand comprises a nucleotide overhang at the 5'-end; and the sense strand comprises a nucleotide overhang at the 5'-end.

[00452] In some embodiments, the dsRNA agent comprises one or more blunt end. In some embodiments, the dsRNA agent comprises a blunt end at the end of the agent comprising the 3 'end of the sense strand and the 5' end of the antisense strand. In some embodiments, the dsRNA agent comprises a blunt end at the end of the agent comprising the 5 'end of the sense strand and the 3' end of the antisense strand. In some embodiments, both ends of the dsRNA agent are blunt ends.

(d) Exemplary Structural Combinations of Sense & Antisense Strands

[00453] In some embodiments, the antisense strand and the sense strand contain the same number of nucleotides. In some embodiments, the antisense strand and the sense strand contain different numbers of nucleotides. In some embodiments, the nucleotide sequence of the sense strand is from about 1-5, 1-3, or 1-2 nucleotides shorter than the nucleotide sequence of the antisense strand. In some embodiments, the nucleotide sequence of the sense strand is about 1, 2, 3, 4, or 5 nucleotides shorter than the nucleotide sequence of the antisense strand. In some embodiments, the nucleotide sequence of the sense strand is about 2 nucleotides shorter than the nucleotide sequence of the antisense strand. In some embodiments, the nucleotide sequence of the antisense strand is from about 1-5, 1-3, or 1-2 nucleotides shorter than the nucleotide sequence of the sense strand. In some embodiments, the nucleotide sequence of the antisense strand is about 1, 2, 3, 4, or 5 nucleotides shorter than the nucleotide sequence of the sense strand. In some embodiments, the nucleotide sequence of the antisense strand is about 2 nucleotides shorter than the nucleotide sequence of the sense strand.

[00454] In some embodiments, the sense strand comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises or consists of 23 nucleotides. In some embodiments, the sense strand comprises or consists of 21 nucleotides; and the antisense strand comprises or consists of 23 nucleotides. In some embodiments, the double stranded region comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises a 2-nucleotide overhang at the 3'end. In some embodiments, the 5' end of the antisense strand and 3' end of the sense strand form a blunt end. In some embodiments, the sense strand comprises or consists of 21 nucleotides; the antisense strand comprises or consists of 23 nucleotides; the double stranded region comprises or consists of 21 nucleotides; the antisense strand comprises a 2-nucleotide overhang at the 3'end; and the 5' end of the antisense strand and 3' end of the sense strand form a blunt end.

[00455] In some embodiments, the sense strand comprises or consists of 19 nucleotides. In some embodiments, the antisense strand comprises or consists of 21 nucleotides. In some embodiments, the sense strand comprises or consists of 19 nucleotides; and the antisense strand comprises or consists of 21 nucleotides. In some embodiments, the double stranded region comprises or consists of 19 nucleotides. In some embodiments, the antisense strand comprises a 2-nucleotide overhang at the 3'end. In some embodiments, the 5' end of the antisense strand and 3' end of the sense strand form a blunt end. In some embodiments, the sense strand comprises or consists of 19 nucleotides; the antisense strand comprises or consists of 21 nucleotides; the double stranded region comprises or consists of 19 nucleotides; the antisense strand comprises a 2-nucleotide overhang at the 3'end; and the 5' end of the antisense strand and 3' end of the sense strand form a blunt end.

[00456] In some embodiments, the sense strand comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises or consists of 21 nucleotides. In some embodiments, the sense strand comprises or consists of 21 nucleotides; and the antisense strand comprises or consists of 21 nucleotides. In some embodiments, the double stranded region comprises or consists of 19 nucleotides. In some embodiments, the antisense strand comprises a 2-nucleotide overhang at the 3'end. In some embodiments, the sense strand comprises a 2-nucleotide overhang at the 3'end. In some embodiments, the sense strand comprises or consists of 21 nucleotides; the antisense strand comprises or consists of 21 nucleotides; the double stranded region comprises or consists of 19 nucleotides; the antisense strand comprises a 2-nucleotide overhang at the 3'end; and the sense strand comprises a 2-nucleotide overhang at the 3'end.

[00457] In some embodiments, the sense strand comprises or consists of 20 nucleotides. In some embodiments, the antisense strand comprises or consists of 19 nucleotides. In some embodiments, the sense strand comprises or consists of 20 nucleotides; and the antisense strand comprises or consists of 19 nucleotides. In some embodiments, the double stranded region comprises or consists of 20 nucleotides. In some embodiments, the sense strand comprises a 1 -nucleotide overhang at the 5' end. In some embodiments, the 5' end of the antisense strand and 3' end of the sense strand form a blunt end. In some embodiments, the sense strand comprises or consists of 20 nucleotides; the antisense strand comprises or consists of 19 nucleotides; the double stranded region comprises or consists of 20 nucleotides; the sense strand comprises a 1 -nucleotide overhang at the 5 'end; and the 5' end of the antisense strand and 3' end of the sense strand form a blunt end.

[00458] In some embodiments, the sense strand comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises or consists of 19 nucleotides. In some embodiments, the sense strand comprises or consists of 21 nucleotides; and the antisense strand comprises or consists of 19 nucleotides. In some embodiments, the double stranded region comprises or consists of 19 nucleotides. In some embodiments, the sense strand comprises a 1-nucleotide overhang at the 3'end. In some embodiments, the sense strand comprises a 1-nucleotide overhang at the 5'end. In some embodiments, the sense strand comprises or consists of 21 nucleotides; the antisense strand comprises or consists of 19 nucleotides; the double stranded region comprises or consists of 19 nucleotides; the sense strand comprises a 1-nucleotide overhang at the 3'end; and the sense strand comprises a 1-nucleotide overhang at the 5' end.

[00459] In some embodiments, the sense strand comprises or consists of 24 nucleotides. In some embodiments, the antisense strand comprises or consists of 23 nucleotides. In some embodiments, the sense strand comprises or consists of 24 nucleotides; and the antisense strand comprises or consists of 23 nucleotides. In some embodiments, the double stranded region comprises or consists of 21 nucleotides. In some embodiments, the antisense strand comprises a 2-nucleotide overhang at the 3'end. In some embodiments, the sense strand comprises a 3-nucleotide overhang at the 3'end. In some embodiments, the sense strand comprises or consists of 24 nucleotides; the antisense strand comprises or consists of 23 nucleotides; the double stranded region comprises or consists of 21 nucleotides; the antisense strand comprises a 2-nucleotide overhang at the 3'end; and the sense strand comprises a 3-nucleotide overhang at the 3' end.

[00460] In some embodiments, the sense strand comprises or consists of 19 nucleotides. In some embodiments, the antisense strand comprises or consists of 19 nucleotides. In some embodiments, the sense strand comprises or consists of 19 nucleotides; and the antisense strand comprises or consists of 19 nucleotides. Tn some embodiments, the double stranded region comprises or consists of 19 nucleotides. In some embodiments, the 5' end of the antisense strand (and 3' end of the sense strand) form a blunt end. In some embodiments, the 3' end of the antisense strand (and 5' end of the sense strand) form a blunt end. In some embodiments, the sense strand comprises or consists of 19 nucleotides; the antisense strand comprises or consists of 19 nucleotides; the double stranded region comprises or consists of 19 nucleotides; the 5' end of the antisense strand (and 3' end of the sense strand) form a blunt end; and the 3' end of the antisense strand (and 5' end of the sense strand) form a blunt end

[00461] In some embodiments, the antisense strand and the sense strand are part of the same larger nucleic acid molecule, wherein the nucleic acid molecule comprises or consists of 44 nucleotides, the antisense portion comprises or consists of 21 nucleotides, the sense strand portion of the nucleic acid molecule comprises 19 nucleotides, the double stranded region comprises or consists of 19 nucleotides, the antisense strand comprises a 2-nucleotide overhang at the 3 'end, and the intervening nucleotide sequence between the antisense strand and the sense strand comprises or consists of 4 unpaired nucleotides that create a hairpin loop.

5.5.1.5 Modified Oligonucleotides

[00462] In some embodiments, the oligonucleotide (or any component thereof (e.g., any nucleic acid molecule thereof)) comprises one or more modified nucleotide(s) (as defined herein). The nature, percentage, and arrangement of the modified nucleotides within any given oligonucleotide may vary depending on the format of the oligonucleotide (e.g., siRNA, shRNA, ASO, etc.) The modified oligonucleotide may have one or more different (e.g., improved) properties relative to a corresponding unmodified oligonucleotide. For example, the modified oligonucleotide may exhibit decreased immuno stimulatory activity (e.g., when administered to a subject), increased stability (e.g., in vivo, in a cell, when administered to a subject), and/or increased inhibition of expression of a target nucleic acid molecule (e.g., a target mRNA (e.g., a BCL11A mRNA)), or any combination thereof.

(i) Nature of Nucleotide Modifications

[00463] Nucleotide modifications can include modification to any one of more of the nucleoside and/or the internucleoside linkage. Nucleoside modifications include modification to the sugar (e.g., ribose) moiety and/or the nucleobase. In some embodiments, the modified oligonucleotide (or component thereof) (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotides comprising a modified sugar moiety. In some embodiments, the modified oligonucleotide (or component thereof) (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotides comprising a modified nucleobase. In some embodiments, the modified oligonucleotide (or component thereof (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotides comprising a modified internucleoside linkage. In some embodiments, the modified oligonucleotide (or component thereof) (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotides comprising one, two, or three of a modified sugar moiety, a modified nucleobase, and/or a modified intemucleoside linkage.

[00464] Exemplary nucleotide modifications are described below and also known in the art, see, e.g., WO2021257782, W02013075035, WO2022246251, and WO2022271573, the entire contents of each of which is incorporated by reference herein for all purposes.

(a) Modified Nucleosides

[00465] In some embodiments, the modified oligonucleotide (or any component thereof) (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotide comprising a modified nucleoside. As discussed above, nucleoside modifications can include modification of the sugar (e.g., ribose) moiety and/or modification of the nucleobase.

• Sugar Modifications

[00466] In some embodiments, the modified oligonucleotide (or any component thereof) (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more nucleotides comprising a modified sugar (e.g., ribose) moiety.

[00467] The modified sugar (e.g., ribose) moiety can comprise, for example, a substituent at any one or more position of the sugar' (e.g., ribose), including e.g., positions 2', 4', and/or 5'. In some embodiments, the modified sugar (e.g., ribose) comprises a substituent at 2' position of the sugar (e.g., ribose). In some embodiments, the modified sugar (e.g., ribose) comprises a substituent at 5' position of the sugar (e.g., ribose). In some embodiments, the modified sugar (e.g., ribose) comprises a substituent at 5' position of the sugar (e.g., ribose).

[00468] In some embodiments, the agent (or any component thereof comprises any one or more of the following substituents (e.g., at any position of the sugar (e.g., ribose) (e.g., at position 2')): a group for improving the stability of the agent, a group for improving the pharmacokinetic properties of the agent, or a group for improving the pharmacodynamic properties of the agent, an RNA cleaving group, a reporter group, an intercalator, or other substituents having similar properties.

[00469] Exemplary substituents include, for example, but are not limited to, substitution (e.g., at any position of the sugar (e.g., ribose) (e.g., at position 2')) with any one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to Cio alkyl or C₂ to C10 alkenyl and alkynyl. Additional exemplary substitutions (e.g., at any position of the sugar (e.g., ribose) (e.g., at position 2')) include, for example, but are not limited to, substitution with any one of the following: 0[(CH₂)_n0]m, CH₃, O(CH₂)_nOCH₃, O(CH₂)„NH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)nON[(CH₂)nCH₃)]₂, where n and m are from 1 to about 10.

[00470] In some embodiments, the modified sugar (e.g., ribose) comprises any one of the following modifications: 2'-O-methyl (2'-0Me), 2'O-methoxyethyl (2'-0-M0E), 2'deoxy-2'- fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, 2'-O-methyl-4-pyridine (2-O-methyl-4- pyridine (2'-O-CH2Py(4)).

[00471] In some embodiments, the agent (or any component thereof) comprises any of the following substituents at the 2'-position of the sugar (e.g., ribose): Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, or a substituted silyl. In some embodiments, the agent (or any component thereof) comprises a 2 '-methoxy ethoxy (2'-0 — CH₂CH₂OCH₃, also known as 2'-O-(2-methoxyethyl) or 2'- MOE) (see, e.g., Martin et al., Helv. Chim. Acta, 1995, 78:486-504, the entire contents of which is incorporated by reference herein for all purposes) (i.e., an alkoxy-alkoxy group). In some embodiments, the agent (or any component thereof) comprises a 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE; a 2 '-dimethylaminoethoxy ethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0 — CH₂ — O — CH₂ — N(CH₃)₂; a 5'-Me-2'-F nucleotide, a 5'-Me-2'-OMe nucleotide, a 5'-Me-2'-deoxynucleotide, (both R and S isomers in these three families); a 2'-alkoxyalkyl; and 2'-NMA (N- methylacetamide). [00472] Exemplary US patents that describe the preparation of such modified sugar structures include, but arc not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920; the entire contents of each of the foregoing are hereby incorporated herein by reference for all purposes. o Non-Bicyclic Sugar Modifications

[00473] In some embodiments, the modified sugar (e.g., ribose) moiety comprises a non- bicyclic modified sugar' (e.g., ribose) moiety. In some embodiments, the modified sugar' (e.g., ribose) moiety comprises a furanosyl ring comprising one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. In some embodiments one or more non-bridging substituent of a non-bicyclic modified sugar moiety is branched. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2', 4', and/or 5' positions.

[00474] In some embodiments, non-bicyclic modified sugar moiety comprises a substituent group at the 2'-position of the sugar (e.g., ribose). Examples of 2'- substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2'-O-methyl (2'-0Me), 2'0- methoxyethyl (2'-0-M0E), 2'deoxy-2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, 2'- O-methyl-4-pyridine (2-O-methyl-4-pyridine (2'-O-CH2Py(4)), and 2'-O-N-alkyl acetamide (e.g., 2'-O-N-methyl acetamide (“NMA”), 2'-O-N-dimethyl acetamide, 2'-O-N-ethyl acetamide, and 2'- O-N-propyl acetamide). For example, see, e.g., U.S. 6,147,200, Prakash et al., 2003, Org. Lett., 5, 403-6, the entire contents of which is incorporated by reference herein for all purposes.

[00475] In some embodiments, the 2'-substituent group is a halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, O-C1-C10 alkyl, O-C1-C10 substituted alkyl, S-alkyl, N(R_m)-alkyl, O-alkenyl, S-alkenyl, N(R_m)-alkenyl, O-alkynyl, S- alkynyl, N(R_m)-alkynyl, O-alkylenyl-O- alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3,0(CH2)2ON(Rm)(Rn) or OCH2C(=O)- N(Rm)(Rn), where each R_m and R_n is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, or a 2'- substituent group described in any one of the following: Cook et al., U.S. 6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087, the entire contents of which are incorporated herein by reference for all purposes. In some embodiments, these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.

[00476] In some embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, NH2, N3, OCF3, OCH₃, O(CH2)₃NH₂, CH₂CH=CH₂, OCH₂CH=CH₂, OCH2CH2OCH3, O(CH2)₂SCH3, O(CH₂)2ON(R_m)(Rn), O(CH₂)₂O(CH2)2N(CH3)2, and N-substituted acetamide (OCH₂C(=O)- N(R_m)(Rn)), where each R_m and R_n is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl. In some embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2 '-substituent group selected from: F, OCF, OCH3, OCH2CH2OCH3, O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)2, O(CH₂)₂O(CH₂)₂N(CH₃)₂, and OCH₂C(=O)-N(H)CH₃ (“NMA”). In some embodiments, a 2'- substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, OCH3, OCH2CH2OCH3, and OCH₂C(=O)-N(H)CH3.

[00477] In some embodiments, non-bicyclic modified sugar’ moiety comprises a substituent group at the 3 '-position of the sugar (e.g., ribose). Examples of substituent groups suitable for the 3 '-position of modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl).

[00478] In some embodiments, non-bicyclic modified sugar moiety comprises a substituent group at the 4'-position of the sugar (e.g., ribose). Examples of 4'-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., W02015106128.

[00479] In some embodiments, non-bicyclic modified sugar moiety comprises a substituent group at the 5 '-position of the sugar (e.g., ribose). Examples of substituent groups suitable for the 5'-position of modified sugar moieties include, but are not limited to, vinyl (e.g., 5'-vinyl), alkoxy (e.g., methoxy (e.g., 5'-methoxy)), and alkyl (e.g., methyl (R or S) (e.g., 5'-methyl (R or S)), ethyl). [00480] In some embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2'-F-5 '-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836, the entire contents of each of which is incorporated herein by reference for all purposes. [00481] In some embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties arc further defined by isomeric configuration. For example, a 2'- deoxyfuranosyl sugar moiety may be in seven isomeric configurations other than the naturally occurring 0-D- deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2019/157531, the entire contents of which are incorporated by reference herein for all purposes.

[00482] In some embodiments, the sugar (e.g., ribose) modification comprises an unlocked nucleotide (UNA). UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked sugar (e.g., ribose) residue. For example, in some embodiments, the bonds between Cl'-C4' have been removed (i.e., the covalent carbon-oxygen- carbon bond between the Cl' and C4' carbons). In some embodiments, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar (e.g., ribose) have been removed. See, e.g., Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al.. Mol. Biosyst., 2009, 10, 1039, the entire contents of which are incorporated herein by reference. UNAs and methods of making are known in the art. See, e.g., US8314227; and US20130096289; US20130011922; and US20110313020, the entire contents of each of which are hereby incorporated herein by reference. o Bicyclic Sugar Modifications

[00483] In some embodiments, the modified sugar (e.g., ribose) moiety comprises a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar (e.g., ribose) moiety. In some embodiments, the bicyclic sugar (e.g., ribose) moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include but are not limited to: 4’-CH2-2’, 4’-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (“LNA”), 4'-CH2-S-2', 4'-(CH2)2-O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt”), 4'-CH₂- O-CH₂-2', 4'-CH₂-N(R)-2', 4'-CH(CH₂OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al., U.S. 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'- CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. 8,278,425), 4'-CH2-O-N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou, et al., J. Org. Chem.,2QQ9, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,426), 4'-C(R_aR_b)-N(R)-O-2', 4'-C(R_aRb)-O-N(R)-2', 4'-CH₂-O-N(R)-2', and 4'-CH₂-N(R)- 0-2', wherein each R, R_a, and Rb is, independently, H, a protecting group, or C1-C12 alkyl (see, c.g. Imanishi et al., U.S. 7,427,672). The entire contents of all of the foregoing references is incorporated by reference herein for all purposes.

[00484] In some embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(R_a)(Rb)]n-, -[C(R_a)(Rb)]n-O-, -C(R_a)=C(Rb)-, -C(R_a)=N-, -C(=NR_a)-, -C(=O)-, -C(=S)-, -O-, -Si(R_a)₂-, -S(=O)X-, and -N(R_a)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R_a and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C₂-Ci₂ alkenyl, substituted C₂-Ci₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C₁₂ alkynyl, Cs-C₂o aryl, substituted Cs-C₂o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, C00J1, acyl (C(=O)- H), substituted acyl, CN, sulfonyl (S(=0)2-Jl), or sulfoxyl (S(=O)-J1); and each JI and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C₂-Ci₂ alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C₂-Ci₂ alkynyl, Cs-C₂o aryl, substituted C5-C20 aryl, acyl (C(=O)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[00485] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731- 7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastavaet al., J. Am. Chem. Soc., 2007, 129, 8362-8379;Wengel et a., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al. U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727. The entire contents of all of the foregoing references is incorporated by reference herein for all purposes.

[00486] In some embodiments, the modified sugar’ (e.g., ribose) comprises a constrained ethyl nucleotide comprising a 4'-CH(CH3) — 0-2' bridge. In some embodiments, the constrained ethyl nucleotide is in the S conformation (S-cEt). In some embodiments, the modified sugar (e.g., ribose) comprises a conformationally restricted nucleotide (CRN). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and — C5' carbons of ribose. Representative publications that teach the preparation of certain of the above include, but are not limited to, US2013/0190383; and WO2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

[00487] In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the 0-D configuration. Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and -D-ribofuranose (see, e.g., WO 99/14226, the entire contents of which are incorporated herein by reference for all purposes).

[00488] Additional representative U.S. Patents and U.S. Patent Publications that teach the preparation of bicyclic nucleosides (e.g., locked nucleic acid) include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

• Nucleobase Modifications

[00489] In some embodiments, the modified oligonucleotide (or any component thereof) (e.g., antisense strand, sense strand, dsRNA agent, etc.) comprises one or more nucleotides comprising a modified nucleobase.

[00490] As used herein, “unmodified” nucleobases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases.

[00491] Modified nucleobases include, but are not limited to, 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 5- mcthylcytosinc, 2-aminopropyladcninc, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, deoxythymidine (dT), 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2- propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C=C-CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines,

5-halo, particularly 5-bromo, 5-trifhioromethyl, 5-halouracil, and 5-halocytosine, 7- methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N- benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-Nbenzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine- 2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one (G- clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. 3,687,808; The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442-443; the entire contents of each of which is incorporated herein by reference for all purposes.

[00492] In some embodiments, the modified nucleobase comprises a pseudouridine, 2'thiouridine (s2U), N6'-methyladenosine, 5 'methylcytidine (m⁵C), 5 'fluoro-2 'deoxy uridine, N- ethylpiperidine 7-EAA triazole modified adenine, N-ethylpiperidine 6 'triazole modified adenine,

6-phenylpyrrolo-cytosine (PhpC), 2',4'-difluorotoluyl ribonucleoside (rF), or 5 'nitroindole. In some embodiments, the modified nucleobase comprises a 5-substituted pyrimidine; 6- azapyrimidine; or N-2, N-6 and 0-6 substituted purines (including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine). 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[00493] Representative U.S. Patents an published applications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;

5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;

6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;

7,427,672; 7,495,088; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257;

5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; U.S. 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,811,534; 5,750,692; 5,948,903; 5,587,470;

5,457,191; 5,763,588; 5,830,653; 5,808,027; 6,166,199; and 6,005,096, the entire contents of each of which is hereby incorporated herein by reference for all purposes.

(b) Internucleoside Linkage Modifications

[00494] In some embodiments, the modified oligonucleotide (or any component thereof) (e. ., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.) comprises one or more modified intemucleoside linkage. Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of an agent (e.g., described herein).

[00495] The naturally occurring internucleoside linkage of RNA and DNA is a 3’ to 5' phosphodiester linkage. In some embodiments, the modified internucleoside linkage contains a normal 3'-5' linkage. In some embodiments, the modified intemucleoside linkage contains a 2'-5' linkage. In some embodiments, the modified intemucleoside linkage has an inverted polarity wherein the adjacent pairs of nucleoside units are linked e.g., 3'-5' to 5'-3' or 2'-5 ' to 5'-2'.

[00496] The two main classes of modified intemucleoside linking can be defined by the presence or absence of a phosphorous atom.

• Modified Phosphorous Containing Internucleoside Linkages

[00497] In some embodiments, the modified intemucleoside linkage comprises a phosphorous atom. Representative modified phosphorus-containing intemucleoside linkages include but are not limited to phosphorothioates (PS (Rp isomer or Sp isomer)) (e.g., 5'phosphorothioate), phosphotriesters, phosphoramidates (e.g., 3'-amino phosphoramidate and aminoalkylphosphoramidates), chiral phosphorothioates, phosphorodithioates (PS2), aminoalkylphosphotricstcrs, methyl and other alkyl phosphonates (e.g., mcthylphosphonatc (MP), 3'-alkylene phosphonates), methpxypropyl-phosphonates (MOP), 5'-(E)-vinylphosphonates, 5'methyl phosphonates, (S)-5'C-methyl with phosphates, phosphinates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, boranophosphates, and peptide nucleic acids (PNAs).

[00498] Methods of preparing polynucleotides containing one or more modified phosphorus- containing internucleoside linkage are known in the ail. See, e.g., U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;

5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;

5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;

6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;

6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference for all purposes.

• Modified Non-Phosphorous Containing Internucleoside Linkages

[00499] In some embodiments, the modified intemucleoside linkage does not contain a phosphorous atom. Modified internucleoside linkages that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component pails.

[00500] Representative non-phosphorous containing intemucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (- O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). [00501] Methods of preparing polynucleotides comprising modified intemucleoside linkages do not contain a phosphorous atom are known in the art. See, e.g., U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

(c) Additional Exemplary Nucleotide Modifications

[00502] In some embodiments, the modified oligonucleotide comprises one or more RNA mimetic in which both the sugar and the intemucleoside linkage of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate nucleic acid target (e.g., a target mRNA). In some embodiments, the RNA mimetic is a peptide nucleic acid (PNA). In PNAs, the ribose moiety of the RNA nucleotide is replaced with an amide containing moiety (e.g., an aminoethylglycine). The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the agents described herein are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500, the entire contents of which is incorporated by reference herein for all purposes.

[00503] Potentially stabilizing modifications to the terminal ends of the agents (e.g., described herein) can also be incorporated to agents described herein. For example, N-(acetylaminocaproyl)- 4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4- hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxy thymidine (ether), N-(aminocaproyl)-4- hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT(idT) and others. Such modifications are known in the art. See, e.g., WO2011/005861, the entire contents of which is incorporated herein by reference.

[00504] In some embodiments, the oligonucleotide may be a morpholino-based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991, the entire contents of each of which is incorporated herein by reference for all purposes. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Then, 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the entire contents of each of which is incorporated herein by reference for all purposes).

(ii) Extent of Modified Nucleotides

[00505] In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein (e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.)) are modified. In some embodiments, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., antisense strand, sense strand, dsRNA agent, RNAi agent (e.g., siRNA, shRNA, miRNA), ASO, etc.)) are modified. In some embodiments, at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified. In some embodiments, substantially all of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified. In some embodiments, all of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified.

[00506] In some embodiments, not more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the nucleotides of the of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. In some embodiments, not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. In some embodiments, not more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. [00507] In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent) are unmodified. In some embodiments, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. In some embodiments, substantially all of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified. In some embodiments, all of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are unmodified.

[00508] In some embodiments, not more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the nucleotides of the of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified. In some embodiments, not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified. In some embodiments, not more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the agent (or any component (e.g., nucleic acid molecule) thereof) (e.g., described herein, e.g., an antisense strand, a sense strand, a dsRNA agent, RNAi agent, etc.) are modified.

[00509] In some embodiments, the RNAi agent (e.g., antisense strand, sense strand, dsRNA agent (e.g., described herein)) comprises one or more non-naturally internucleoside linkage. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the intemucleoside linkages of the RNAi agent (e.g., antisense strand, sense strand, dsRNA agent (e.g., described herein)) are non-naturally occurring. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the or intcmuclcosidc linkages of the RNAi agent (e.g., antisense strand, sense strand, dsRNA agent (e.g., described herein)) are chemically modified.

5.5.1.6 Exemplary Oligonucleotide Targets

[00510] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with a genetic disorder. In some embodiment, the gene, nucleic acid (e.g., mRNA), and/or protein is associated with an inherited blood disorder. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with a bone marrow genetic disorder. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an hemoglobinopathy. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an inherited bone marrow syndrome.

[00511] In some embodiments, inhibition or a reduction in expression or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an increase in the level of fetal hemoglobin. In some embodiments, inhibition or a reduction in expression or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein is associated with the induction of expression of fetal hemoglobin. In some embodiments, inhibition or a reduction in expression or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an increase in the ratio of fetal hemoglobin to adult hemoglobin. In some embodiments, a target gene, nucleic acid (e.g., mRNA), and/or protein is associated with repression of fetal hemoglobin. In some embodiments, a target gene, nucleic acid (e.g., mRNA), and/or protein functions in the switch from fetal hemoglobin to adult hemoglobin. In some embodiments, a target gene, nucleic acid (e.g., mRNA), and/or protein functions in the repression of fetal hemoglobin.

[00512] Exemplary targets include, but are not limited to, B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCLUA)), ZBTB7A (e.g., human ZBTB7A (hZBTB7A)), KLF1 (e.g., human KLF1 (hFLFl)). BCL11A, ZBTB7A, and KLF1 are transcription factors that function, inter alia, in the silencing of fetal hemoglobin and the fetal-to- adult hemoglobin switch. See, e.g., Shen Y, Li R, Teichert K, Montbleau KE, Verboon JM, Voit RA, et al. (2021) Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing. PLoS Genet 17(10): el009835. https://doi.org/10.1371/joumal.pgen.1009835; Stuart H. Orkin and Daniel E. Bauer, Emerging Genetic Therapy for Sickle Cell Disease, Annual Review of Medicine 2019 70:1, 257-271; Miroslawa Siatecka, James J. Bieker; The multifunctional role of EKLF/KLF1 during erythropoiesis. Blood 2011; 118 (8): 2044-2054. doi: https://doi.org/10.1182/blood-2011-03- 331371 , the entire contents of each of which are incorporated by reference herein for all purposes . [00513] In some embodiments, the target is BCL11A (e.g., hBCLl 1A). In some embodiments, the target is ZBTB7A (e.g., hZBTB7A), KLF1 (e.g., hKLFl), or KLF1 (e.g., hKLFl).

[00514] The mRNA sequence of a reference hBCLl 1 A gene is set forth in SEQ ID NO: 289. The reverse complement sequence of the hBCLl 1A mRNA is set forth in SEQ ID NO: 290. The amino acid sequence of the hBCLl 1 A protein encoded by the hBCLl 1A reference gene is set forth in SEQ ID NO: 291. The mRNA sequence of a reference hZBTB7A gene is set forth in SEQ ID NO: 292. The reverse complement sequence of the hZBTB7A mRNA is set forth in SEQ ID NO: 293. The amino acid sequence of the hZBTB7A protein encoded by the hZBTB7A reference gene is set forth in SEQ ID NO: 294. The mRNA sequence of a reference hFLFl gene is set forth in SEQ ID NO: 295. The reverse complement sequence of the hFLFl mRNA is set forth in SEQ ID NO: 296. The amino acid sequence of the hFLFl protein encoded by the hFLFl reference gene is set forth in SEQ ID NO: 297. See Table 10, herein.

Table 10. The Nucleic Acid and Amino Acid Sequences of Reference hBCL 11A, hZBTB7A, and hKLFl.

(i) Exemplary BCL11A Targeting Oligonucleotides

[00515] In some embodiments, the oligonucleotide targets BCL11A (e.g., hBCLUA). Examples of oligonucleotides useful in targeting BCL11A (e.g., hBCLUA) are known in the art and described in for example, e.g., WO2020193434A1; W02019213013A1; WO2015164750A2; W02014188001A1; W02012079046A2; US10570392B2; W02010030963; He, D„ Wu, H„ Ding, L. et al. Combination of BCL11A siRNA with vincristine increases the apoptosis of SUDHL6 cells. EurJ Med Res 19, 34 (2014). https://doi.org/10.1186/2047-783X-19-34, the entire contents of each of which is incorporated by reference herein for all purposes.

[00516] In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a hBCLl 1 A mRNA sequence (e.g., as set forth in Table 10, SEQ ID NO: 289). In some embodiments, the oligonucleotide comprises a region of complementarity to a hBCLUA mRNA sequence set forth in Table 10. In some embodiments, the oligonucleotide comprises a region of complementarity to a hBCLUA mRNA sequence set forth in SEQ ID NO: 289. In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a hBCLl 1 A mRNA sequence set forth in SEQ ID NO: 289.

[00517] BCL11A targeting siRNA agents useful in the conjugates described herein are known in the art, see, e.g., W02012079046. The nucleotide sequence of exemplary unmodified hBCLl 1 A targeting siRNAs comprising a sense and antisense strand (e.g., suitable for targeting hBCLUA, suitable for inhibiting hBCLl 1 A expression)), is provided in Table 11 and SEQ ID NOS: 298-301. More specifically, Table 11 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and siRNA pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 11, the disclosure encompasses siRNA agents comprising any sense strand and any antisense set forth in Table 11 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). It is to be understood that while the nucleotide sequence of the sense strands and antisense strands in Table 11 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the sense and antisense sense strands set forth in Table 11 comprising one or more modified nucleotide (e.g., as described herein).

Table 11. Unmodified Sense and Antisense Strand Sequences of hBCLUA siRNA Agents.

[00518] The nucleotide sequence of exemplary modified hBCLUA targeting siRNAs comprising a sense and antisense strand (e.g., suitable for targeting hBCLUA, suitable for inhibiting hBCLUA expression)), is provided in Table 12 and SEQ ID NOS: 302-339. More specifically, Table 12 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and siRNA pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 12, the disclosure encompasses siRNA agents comprising any sense strand and any antisense set forth in Table 12 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). The following nucleotide abbreviations are utilized in Table 12: a (2'-O-methyladenosine-3'- phosphate); as (2'-O-methyladenosine-3'-phosphorothioate); c (2'-O-methylcytidine-3'- phosphate); cs (2'-O-methylcytidine-3'-phosphorothioate); g (2'-O-methylguanosine-3'- phosphate); gs (2'-O-methylguanosine-3'- phosphorothioate); t (2'-O-methyl-5-methy luridine-3' -phosphate); ts (2'-O-methyl-5 -methyluridine-3'-phosphorothioate); u (2'-O-methyluridine-3'- phosphate); us (2'-O-methyluridine-3'-phosphorothioate); s (phosphorothioate linkage); A (Adenosine -3'-phosphate); Af (2'-fluoroadenosine-3 ’-phosphate); Afs (2 ’-fluoroadenosine-3 ’- phosphorothioate); As (adenosine-3'-phosphorothioate); C (cytidine-3'-phosphate); Cf (2'- fluorocytidinc-3'-phosphatc); Cfs (2'-fluorocytidinc-3'-phosphorothioatc); Cs (cytidinc-3¹- phosphorothioate); G (guanosine-3'-phosphate); Gf (2'-fluoroguanosine-3'-phosphate); Gfs (2'- fluoroguanosine-3'-phosphorothioate); Gs (guanosine-3'-phosphorothioate); U (Uridine-3¹- phosphate); Uf (2'-fhiorouridine-3'-phosphate); Ufs (2'-fluorouridine-3'-phosphorothioate); and U s (uridine-3'-phosphorothioate).

Table 12. Modified Sense and Antisense Strand Sequences of hBCLUA siRNA Agents.

[00519] The nucleotide sequence of additional exemplary unmodified and modified hBCLl 1 A targeting siRNAs comprising a sense and antisense strand (e.g., suitable for targeting hBCLl 1 A, suitable for inhibiting hBCLl 1A expression)), is provided in Table 24 and SEQ ID NOS: 835-838 and 845-848. More specifically, Table 24 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and siRNA pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 24, the disclosure encompasses siRNA agents comprising any sense strand and any antisense set forth in Table 24 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). In the nucleotide sequences of the modified siRNAs set forth in Table 24 (SEQ ID NOS: 835-838), the following applies: Lower case (x) = 2’-0Me; UPPER CASE (X) = native RNA (2'-OH); dX = DNA; * = PS bond.

Table 24. Exemplary Unmodified and Modified Sense and Antisense Strand Sequences of hBCLUA siRNA Agents.

[00520] In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in Table 24. In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of SEQ ID NOS: 835-838 or 845-848.

[00521] In some embodiments, the oligonucleotide comprises a double stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of any one of the antisense strands set forth in any one of Tables 11-12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21 ) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in any one of Tables 11-12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in any one of Tables 11-12 or 24.

[00522] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of dsRNA agents 1-228 set forth in any one of Tables 1 1-12 or 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in any one of Tables 11- 12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in any one of Tables 11-12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in any one of Tables 11-12 or 24.

[00523] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of the dsRNA agents set forth in any one of Tables 11-12 or 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in any one of Tables 11- 12 or 24.

[00524] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of any one of the antisense strands set forth in any one of Tables 11-12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in any one of Tables 11-12 or 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in any one of Tables 11-12 or 24.

[00525] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21 , 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of the dsRNA agents set forth in any one of Tables 11-12 or 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in any one of Tables 11- 12 or 24.

[00526] In some embodiments, the oligonucleotide comprises a double stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of any one of the antisense strands set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in Table 24.

[00527] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of dsRNA agents 1-228 set forth in Table 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in Table 24.

[00528] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of the dsRNA agents set forth in Table 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in Table 24.

[00529] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of any one of the antisense strands set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from any sense strand set forth in Table 24. In some embodiments, the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the corresponding sense strand set forth in Table 24.

[00530] In some embodiments, the oligonucleotide comprises a dsRNA agent comprising a sense strand and an antisense strand forming a double stranded region, and wherein the antisense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the antisense strands of any one of the dsRNA agents set forth in Table 24; and wherein the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) contiguous nucleotides of and differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence of the sense strand of the corresponding dsRNA agent set forth in Table 24.

[00531] hBCLUA targeting shRNAs are known in the art. See, e.g., WO2010/030963, the entire contents of which is incorporated herein by reference for all purposes. The nucleotide sequence of exemplary unmodified hBCLl 1 A targeting shRNA agents (e.g., suitable for targeting hBCLUA, suitable for inhibiting hBCLl 1 A expression)), is provided in Table 13 and SEQ ID NOS: 340-341. It is to be understood that while the nucleotide sequence of the shRNAs in Table 13 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the shRNAs set forth in Table 13 comprising one or more modified nucleotide (e.g., as described herein).

Table 13. Exemplary Unmodified Sequences of hBCLUA Targeting shRNAs.

[00532] hBCLUA targeting ASOs arc known in the art. See, e.g., W02014188001, the entire contents of which is incorporated herein by reference for all purposes. The nucleotide sequence of exemplary unmodified hBCLUA targeting ASOs (e.g., suitable for targeting hBCLUA, suitable for inhibiting hBCLl 1A expression)), is provided in Table 14 and SEQ ID NOS: 342-368. It is to be understood that while the nucleotide sequence of the ASOs in Table 14 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the ASOs set forth in Table 14 comprising one or more modified nucleotide (e.g., as described herein).

Table 14. Exemplary Unmodified Sequences of hBCLUA Targeting ASOs.

[00533] The nucleotide sequence of exemplary unmodified hBCLl 1 A targeting miRNAs (e.g., suitable for targeting hBCLl 1A, suitable for inhibiting hBCLl 1A expression)), is provided in Table 15 and SEQ ID NOS: 369-284. It is to be understood that while the nucleotide sequence of the miRNAs in Table 15 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the miRNAs set forth in Table 15 comprising one or more modified nucleotide e.g., as described herein).

Table 15. Exemplary Unmodified Sequences of BCL11A Targeting miRNAs.

[00534] In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of Tables 10-14 or 24. In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of SEQ ID NOS: 298-384 or 835-838 or 845-848.

(ii) Exemplary ZBTB7A Targeting Oligonucleotides

[00535] In some embodiments, the oligonucleotide targets ZBTB7A. Examples of oligonucleotides useful in targeting ZBTB7A are described in, e.g., US20160145624 and W02023081003, the entire contents of each of which is incorporated by reference herein for all purposes.

[00536] In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a human ZBTB7A mRNA sequence (e.g., as set forth in Table 10, SEQ ID NO: 292). In some embodiments, the oligonucleotide comprises a region of complementarity to a human ZBTB7A mRNA sequence set forth in Table 10. In some embodiments, the oligonucleotide comprises a region of complementarity to a human ZBTB7A mRNA sequence set forth in SEQ ID NO: 292. In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a human ZBTB7A mRNA sequence set forth in SEQ ID NO: 292.

[00537] The nucleotide sequence of exemplary unmodified ZBTB7A targeting oligonucleotides (e.g., suitable for targeting hFASN, suitable for inhibiting ZBTB7A expression)), including dsRNA agents (e.g., siRNAs), shRNAs, and ASOs is provided in Tables 15-16 and SEQ ID NOS: 385-586.

[00538] ZBTB7A targeting siRNA agents are known in the ail, see, e.g., US20160145624, the entire contents of which is incorporated herein by reference for all purposes. The nucleotide sequence of exemplary unmodified ZBTB7A targeting dsRNA agents (e.g., siRNAs) comprising a sense and antisense strand (e.g., suitable for targeting hBCLl 1 A, suitable for inhibiting ZBTB7A expression)), is provided in Table 16 and SEQ ID NOS: 385-584. More specifically, Table 16 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and dsRNA agent pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 16, the disclosure encompasses dsRNA agents comprising any sense strand and any antisense set forth in Table 16 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). It is to be understood that while the nucleotide sequence of the sense strands and antisense strands in Table 16 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the sense and antisense sense strands set forth in Table 16 comprising one or more modified nucleotide (e.g., as described herein).

Table 16. Unmodified Sense and Antisense Strand Sequences of ZBTB7A siRNA Agents.

[00539] hZBTB7A targeting shRNA agents are known in the art, see, e.g., W02023081003, the entire contents of which is incorporated herein by reference for all purposes. The nucleotide sequence of exemplary unmodified hZBTB7A targeting shRNA agents (e.g., suitable for targeting hZBTB7A, suitable for inhibiting hZBTB7A expression)), is provided in Table 17 and SEQ ID NOS: 585-586. It is to be understood that while the nucleotide sequence of the shRNAs in Table 17 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the shRNAs set forth in Table 17 comprising one or more modified nucleotide (e.g., as described herein).

Table 17. Exemplary Unmodified Sequences of hZBTB7A Targeting shRNAs.

[00540] In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of Tables 15-16. In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of SEQ ID NOS: 385-586.

(iii) Exemplary KLEI Targeting Oligonucleotides

[00541] In some embodiments, the oligonucleotide targets KLF1. Examples of oligonucleotides useful in targeting KLF1 are described in, e.g., WO2012079046, the entire contents of which is incorporated by reference herein for all purposes.

[00542] In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a human KLF1 mRNA sequence (e.g., as set forth in Table 10, SEQ ID NO: 295). In some embodiments, the oligonucleotide comprises a region of complementarity to a human KLF1 mRNA sequence set forth in Table 10. In some embodiments, the oligonucleotide comprises a region of complementarity to a human KLF1 mRNA sequence set forth in SEQ ID NO: 295. In some embodiments, the oligonucleotide comprises a region of complementarity (e.g., as described herein) to a human KLF1 mRNA sequence set forth in SEQ ID NO: 295.

[00543] The nucleotide sequence of exemplary unmodified KLF1 targeting oligonucleotides (e.g., suitable for targeting KLF1, suitable for inhibiting h KLF1 expression)), including dsRNA agents (e.g., siRNAs), shRNAs, and ASOs is provided in Tables 17-18 and SEQ ID NOS: 587- 805.

[00544] KLF1 targeting siRNA agents are known in the art, see, e.g., W02012079046. The nucleotide sequence of exemplary unmodified KLF1 targeting dsRNA agents (e.g., siRNAs) comprising a sense and antisense strand (e.g., suitable for targeting h KLF1, suitable for inhibiting h KLF1 expression)), is provided in Table 18 and SEQ ID NOS: 287-681. More specifically, Table 18 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and dsRNA agent pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 18, the disclosure encompasses dsRNA agents comprising any sense strand and any antisense set forth in Table 18 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). It is to be understood that while the nucleotide sequence of the sense strands and antisense strands in Table 18 are set forth as unmodified (not containing any modified nucleotides), the disclosure encompasses the sense and antisense sense strands set forth in Table 18 comprising one or more modified nucleotide (e.g., as described herein).

Table 18. Unmodified Sense and Antisense Strand Sequences of hKLFl siRNA Agents.

[00545] KLF1 targeting modified siRNA agents are known in the art, see, e.g., WO2012079046. The nucleotide sequence of exemplary modified hKLFl targeting modified siRNAs comprising a sense and antisense strand (e.g., suitable for targeting hKLFl, suitable for inhibiting hKLFl expression)), is provided in Table 19 and SEQ ID NOS: 682-813. More specifically, Table 19 sets forth the nucleotide sequence of exemplary sense strands, antisense strands, and siRNA pairs of sense and antisense strands. It is to be understood that while the sense and antisense strands are set forth in pairs in Table 19, the disclosure encompasses siRNA agents comprising any sense strand and any antisense set forth in Table 19 (e.g., that are at least partially complementary (e.g., as could be determined by a person of ordinary skill in the art)). The following nucleotide abbreviations are utilized in Table 19: a (2'-O-methyladenosine-3'- phosphate); as (2'-O-methyladenosine-3'-phosphorothioate); c (2'-O-methylcytidine-3'- phosphate); cs (2'-O-methylcytidine-3'-phosphorothioate); g (2'-O-methylguanosine-3'- phosphate); gs (2'-O-methylguanosine-3'- phosphoro thioate); t (2'-O-methyl-5-methyluridine-3'- phosphate); ts (2'-O-methyl-5-methyluridine-3'-phosphorothioate); u (2'-O-methyluridine-3'- phosphate); us (2'-O-methyluridine-3'-phosphorothioate); s (phosphorothioate linkage); A (Adenosine -3'-phosphate); Af (2'-fluoroadenosine-3 ’-phosphate); Afs (2 ’-fluoroadenosine-3 ’- phosphorothioate); As (adenosine-3'-phosphorothioate); C (cytidine-3'-phosphate); Cf (2'- fluorocytidine-3'-phosphate); Cfs (2'-fluorocytidine-3 '-phosphorothioate); Cs (cytidine-3¹- phosphorothioate); G (guanosine-3'-phosphate); Gf (2'-fluoroguanosine-3'-phosphate); Gfs (2'- fluoroguanosine-3'-phosphorothioate); Gs (guanosine-3'-phosphorothioate); U (Uridine-3'- phosphate); Uf (2'-fluorouridine-3'-phosphate); Ufs (2'-fluorouridine-3'-phosphorothioate); Us (uridine-3'-phosphorothioate), dT (2' -deoxy thymidine) .

Table 19. Modified Sense and Antisense Strand Sequences of hKLFl siRNA Agents.

[00546] In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of Tables 17-18. In some embodiments, the oligonucleotide comprises or consists of a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of an oligonucleotide set forth in any one of SEQ ID NOS: 587-813.

(iv) Additional Exemplary Oligonucleotide Targets

[00547] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an inherited bone marrow failure syndrome. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with any one or more of amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, or thrombocytopenia absent radii (TAR).

[00548] Exemplary gcncs/protcins associated with exemplary inherited bone marrow failure syndromes are set forth in Table 20. The genes (or encoded protein) listed in Table 20 are exemplary only and it is known in the art that other genes (and encoded proteins) may be associated with the specified inherited bone marrow failure syndrome. See, e.g., Park M. Overview of inherited bone marrow failure syndromes. Blood Res. 2022 Apr 30;57(Sl):49-54. doi: 10.5045/br.2022.2022012. PMID: 35483926; PMCID: PMC9057667; Shimamura A, Alter BP. Pathophysiology and management of inherited bone marrow failure syndromes. Blood Rev. 2010 May;24(3): 101-22. doi: 10.1016/j.blre.2010.03.002. Epub 2010 Apr 24. Erratum in: Blood Rev. 2010 Jul-Sep;24(4-5):201. PMID: 20417588; PMCID: PMC3733544; Dokal I, Vulliamy T. Inherited bone marrow failure syndromes. Haematologica. 2010 Aug;95(8): 1236-40. doi: 10.3324/haematol.2010.025619. PMID: 20675743; PMCID: PMC2913069, the entire contents of each of which is incorporated by reference herein for all purposes. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is one set forth in Table 20.

Table 20. Exemplary Genes/Proteins Associated with Exemplary Inherited Bone Marrow Failure Syndromes.

[00549] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCLUA)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1 ) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pscudouridinc Synthase 1 (DKC1) e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear' Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1) (e.g., human HAX1), Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) (e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPL Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Like (SAMD9L) (e.g., human SAMD9L), or MDS 1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

[00550] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00551] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELA2 (e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

5.6 Methods of Making Oligonucleotide Molecular Payloads [00552] An oligonucleotide (e.g., described herein) (e.g., RNAi agent, ASO, etc.) can be synthesized by standard methods known in the ail (e.g., chemical synthesis (e.g., solid phase synthesis)). See, e.g., “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, and See, e.g., Dong Y, Siegwart DJ, Anderson DG. Strategies, design, and chemistry in siRNA delivery systems. Adv Drug Deliv Rev. 2019 Apr; 144: 133- 147. doi: 10.1016/j.addr.2019.05.004. Epub 2019 May 15. PMID: 31102606; PMCID: PMC6745264, the entire contents of each of which is incorporated by reference herein for all purposes. As such, further provided herein are methods of making an agent described herein) (e.g., RNAi agent, dsRNA agent, antisense strand, sense strand).

[00553] For example, single stranded oligonucleotide (e.g., described herein) (e.g., antisense strands, sense strands, ASOs, etc.) can be prepared using solution-phase or solid-phase organic synthesis or both. Double stranded oligonucleotides (e.g., described herein (e.g., siRNA agents)) can be prepared using a two-step procedure, wherein the individual strands of the double stranded oligonucleotide are prepared separately and subsequently annealed. The individual strands of the double stranded oligonucleotide can be prepared using solution-phase or solid-phase organic synthesis or both. Regardless of the method of synthesis, the oligonucleotides (e.g., described herein) can be prepared in a solution (e.g., an aqueous or organic solution) that is appropriate for formulation. For example, an oligonucleotide can be precipitated and redissolved in pure doubledistilled water, and lyophilized. The lyophilized oligonucleotide can be resuspended in a solution appropriate for the intended formulation process.

5.7 Linkers

[00554] In some embodiments, the molecular' payload (e.g., oligonucleotide (e.g., described herein) (RNAi agent (e.g., siRNA), dsRNA agent, antisense strand, sense strand, ASO, etc.) is directly attached to the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) (e.g., directly attached through a single chemical bond). In some embodiments, the molecular payload (e.g., oligonucleotide (e.g., described herein) is indirectly attached to the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))). In some embodiments, the molecular payload (e.g., oligonucleotide (e.g., described herein) is indirectly attached to the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) via a linker.

[00555] Suitable linkers for use in the conjugates described herein and methods of synthesis are known in the art and can be evaluated by a person of ordinary skill in the ail using standard methods. See, e.g., (see, e.g., Kline, T. et al. “Methods to Make Homogenous Antibody Drug Conjugates.” Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al. “Current ADC Linker Chemistry” Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J.R. and Owen, S.C. “Antibody Drug Conjugates: Design and Selection of Linker, Pay load and Conjugation Chemistry” AAPS J. 2015, 17:2, 339-351.), the entire contents of each of which is incorporated herein by reference for all purposes. A suitable linker will not interfere with the function of the components of the conjugate. Exemplary linkers and components thereof for use in the conjugates described herein are also described below.

[00556] Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkeny lary lalky 1, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl. alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkeny lheterocy cly lalky 1 alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl. alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[00557] In some embodiments, the linker comprises ethylene glycol, nucleosides, or amino acid units. In some embodiments, the linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thiocthcr, and hydroxylamino. In some embodiments, the linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In some embodiments, the linker comprises groups selected from alkyl and amide groups. In some embodiments, the linker comprises groups selected from alkyl and ether groups. In some embodiments, the linker comprises at least one phosphorus moiety. In some embodiments, the linker comprises at least one phosphate group. In some embodiments, the linker comprises at least one neutral linking group. Exemplary linkers include but are not limited to pyrrolidine, 8-amino- 3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA). Additional exemplary linkers include but are not limited to substituted or unsubstituted Ci-Cw alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[00558] In some embodiments, the linker is bifunctional. In general, a bifunctional linker comprises at least two functional groups. One of the functional groups is selected to react with a particular site on an agent (e.g., described herein) and the other is selected to react with a TFR targeting domain (e.g., described herein (e.g., an anti-TFR antibody)). Examples of functional groups used in a bifunctional linkers include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[00559] In some embodiments, the linker is a monovalent linker, a bivalent linker, a trivalent linker, or a tetravalent linker.

5.7.1 Cleavable Linkers

[00560] In some embodiments, the linker is cleavable. Cleavable linkers contain at least one (or a plurality of) cleavable bonds that are susceptible to one or more cleavage agent. Exemplary classes of cleavable linkers include, but are not limited to, redox cleavable linkers, phosphate based cleavable linkers, acid cleavable linkers, ester-based cleavable linkers, and peptide-based cleavable linkers. In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. [00561] Clcavablc linkers may be advantageous when a stable conjugate is desired under a first set of conditions but under a second set of conditions it is advantageous to release the molecular payload (e.g., oligonucleotide (e.g., described herein)) from the targeting moiety (e.g., described herein). For example, in some embodiments, it may be desirable to have a sufficiently stable conjugate outside of a cell (e.g., within a subject (e.g., within the blood or serum of a subject)), and upon entry into a cell (e.g., a target cell (e.g., a target cell within a subject)) have the linker cleaved to release the molecular’ payload (e.g., oligonucleotide (e.g., described herein)) from the targeting moiety. In some embodiments, the linker is not cleaved (or is cleaved at a lower rate) under a first condition relative to under a second condition. In some embodiments, the first condition is within the blood (e.g., of a subject) (or in an in vitro system sufficient to mimic the conditions of the blood within a subject) and the second condition is with a cell (e.g., a cell within a subject) (or in an in vitro system sufficient to mimic the conditions of a cell within a subject). [00562] The suitability of a cleavable linker can be assessed by standard methods known in the art. In general, the suitability of a cleavable linker can be evaluated by testing the ability of a cleavage agent (or condition) to cleave the candidate linker (e.g., the cleavage bond(s)). In some embodiments, it may be desirable to further test the ability of the linker to resist cleavage under a certain condition (e.g., within the blood or serum of subject, when in contact with a non-target cell, tissue, organ).

[00563] In some embodiments, the linker is a redox cleavable linker that is cleaved upon reduction or oxidation. An example of a reductively cleavable linker is a disulphide (-S-S-) containing linker. Redox cleavable linkers can be evaluated using methods analogous to those described above.

[00564] In some embodiments, the linker is a phosphate-based cleavable linker. A phosphate- based cleavable linker is cleaved by agents that degrade or hydrolyze the phosphate group. For example, in cells, enzymes such as phosphatases are capable of cleaving phosphate groups. Examples of phosphate -based linkers include those comprising any of the following -O- P(O)(ORk)-O-, -OP(S)(ORk)-O-„ -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S- P(O)(ORk)-S-, -OP(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S- P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S-, wherein Rk at each occurrence can be, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include are -OP(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S- P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)- O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, -S-P(O)(H)-S-, or -O-P(S)(H)-S-. Phosphate based cleavable linker can be evaluated using methods analogous to those described above.

[00565] In some embodiments, the linker is an acid cleavable linker. An acid cleavable linker is cleaved under acidic conditions. For example, in some embodiments the acid cleavable linker can be cleaved in an acidic environment with a pH of about 6.5 or less e.g., about 6.0, 5.5, 5.0, or less). In some embodiments the acid cleavable linker can be cleaved by enzymes that can act as a general acid. In a cell (e.g., within a subject), specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linkers. Examples of acid cleavable linkers include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Acid cleavable linkers can be evaluated using methods analogous to those described above.

[00566] In some embodiments, the linker is an ester-based cleavable linker. An ester-based cleavable linker is cleaved by enzymes such as esterases and amidases in cells. Examples of ester- based cleavable include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. The cleavable bonds of ester cleavable linkers have the general formula -C(O)O- or - OC(O)-. Ester-based cleavable linkers can be evaluated using methods analogous to those described above.

[00567] In some embodiments, the linker is a peptide-based cleavable linker. A peptide-based cleavable linker is cleaved by enzymes such as peptidases and proteases (e.g., present in cells (e.g., cells within a subject)). Peptide-based cleavable linkers comprise peptide bonds formed between amino acids to yield polypeptides e.g., dipeptides, tripeptides, etc.). As known in the art, peptide bonds. The peptide bonds (i.e., the amide bond) of the peptide linker is generally the site of cleavage. Peptide-based cleavable linkers can be evaluated using methods analogous to those described above.

5.7.2 Non-Cleavable Linkers

[00568] In some embodiments, the linker is non-cleavable. Generally, a non-cleavable linker cannot be readily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitutions may include halogens, hydroxyl groups, oxygen species, and other common substitutions. In some embodiments, a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising a LPXT sequence, a thioether, a biotin, a biphenyl, repeating units of polyethylene glycol or equivalent compounds, acid esters, acid amides, sulfamides, and/or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation can be utilized to covalently link an antibody (e.g., an anti-TFR antibody) comprising a LPXT sequence to a molecular payload comprising a (G)n sequence (see, e.g., Proft T. Sortase- mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10., the entire contents of which is incorporated by reference herein for all purposes).

[00569] In some embodiments, a linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S,; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S, an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), e.g. polyethylene oxide or polypropylene oxide. In some embodiments, a linker may be a non- cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker.

5.7.3 Linker Conjugation

[00570] Methods of attaching a linker to a targeting agent e.g., described herein) and/or a molecular payload (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)) are known in the art. In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide described herein) via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, a linker is covalently linked to an oligonucleotide (e.g., described herein) through a phosphate or phosphorothioate group, e.g., a terminal phosphate of an oligonucleotide backbone. In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))), through a lysine or cysteine residue present on the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))).

[00571] In some embodiments, a linker, or a portion thereof is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide described herein) by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide or the alkyne may be located on the hematopoietic cell (e.g., erythroid precursor cell)-targeting agent described herein (e.g., an anti-TFR (e.g., hTFR (e.g., hTFRl)) antibody), molecular payload (e.g., an oligonucleotide (e.g., described herein)), or the linker. In some embodiments, an alkyne may be a cyclic alkyne, e.g., a cyclooctyne. In some embodiments, an alkyne may be bicyclononyne (also known as bicyclo [6.1.0] nonyne or BCN) or substituted bicyclononyne. In some embodiments, a cyclooctyne is as described in WO2011136645, the entire contents of which is incorporated by reference herein for all purposes. In some embodiments, an azide may be a sugar or carbohydrate molecule that comprises an azide. In some embodiments, an azide may be 6-azido-6- deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, a sugar or carbohydrate molecule that comprises an azide is as described in W02016170186, the entire contents of which is incorporated by reference herein for all purposes. In some embodiments, a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide or the alkyne may be located on the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))), molecular payload (e.g., an oligonucleotide (e.g., described herein)), or the linker is as described in WO20I4065661 or W02016170186, the entire contents of each of which is incorporated by reference herein for all purposes.

[00572] In some embodiments, a linker comprises a spacer, e.g., a polyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, a spacer is as described in Verkade, J.M.M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody- Drug Conjugates”, Antibodies, 2018, 7, 12, the entire contents of which is incorporated by reference herein for all purposes.

[00573] In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)) by the Diels-Alder reaction between a dienophile and a diene/hetero-diene, wherein the dienophile or the diene/hetero-diene may be located on the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))), a molecular payload (e.g., an oligonucleotide (e.g., described herein)), or the linker. In some embodiments a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)) by other pericyclic reactions such as an ene reaction. In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)) by an amide, thioamide, or sulfonamide bond reaction. In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)) by a condensation reaction to form an oxime, hydrazone, or semicarbazide group existing between the linker and the targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)).

[00574] In some embodiments, a linker is covalently linked to a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or (e.g., and) a molecular payload (e.g., an oligonucleotide (e.g., described herein)) by a conjugate addition reaction between a nucleophile, e.g., an amine or a hydroxyl group, and an electrophile, e.g., a carboxylic acid, carbonate, or an aldehyde. In some embodiments, a nucleophile may exist on a linker and an electrophile may exist on a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or a molecular payload (e.g., an oligonucleotide (e.g., described herein)) prior to a reaction between a linker and a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) or a molecular payload (e.g., an oligonucleotide (e.g., described herein)). In some embodiments, an electrophile may exist on a linker and a nucleophile may exist on a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or a molecular payload (e.g., an oligonucleotide (e.g., described herein)) load prior to a reaction between a linker and a targeting agent (e.g., a hematopoietic cell (e.g., erythroid precursor cell) targeting agent (e.g., an anti-TFR antibody (e.g., described herein)))) and/or a molecular payload (e.g., an oligonucleotide (e.g., described herein)). In some embodiments, an electrophile may be an azide, pentafluorophenyl, a silicon centers, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or an activated sulfur center. In some embodiments, a nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilido group, and/or a thiol group.

5.8 Conjugation

[00575] Various methods of conjugating a targeting agent (e.g., a targeting agent described herein) to a molecular payload (e.g., a molecular payload described herein) are well known in the art. See, e.g., Datta-Mannan A, Choi H, Stokell D, et al. The Properties of Cysteine-Conjugated Antibody-Drug Conjugates Are Impacted by the IgG Subclass. AAPS J. 2018;20(6):103. Published 2018 Sep 25. doi: 10.1208/s 12248-018-0263-0 (hereinafter “Datta-Mannan 2018”); Dugal-Tessier J, Thirumalairajan S, Jain N. Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates. J Clin Med. 2021 ; 10(4):838. Published 2021 Feb 18. doi:10.3390/jcml0040838 (hereinafter “Dugal-Tessier 2021”), the entire contents of each of which are incorporated herein by reference for all purposes. The method of conjugation will necessarily depend on the nature of the targeting agent (e.g., a protein (e.g., an anti-TFR antibody described herein)) and the nature of the molecular payload (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)).

[00576] For example, methods of conjugating a protein-based targeting agent (e.g., an antibody (e.g., an antibody described herein)) to an oligonucleotide-based molecular payload (e.g., an oligonucleotide described herein) arc known in the art. See, e.g., Dugal-Tcssicr 2021 and Datta- Mannan 2018. For example, an oligonucleotide can be conjugated to a protein (e.g., an antibody) utilizing a direct conjugation method, wherein a linkable group is added to the oligonucleotide and conjugated directly to an amino acid residue (e.g., a lysine, cysteine, tyrosine) or an engineered amino acid (e.g., as described herein) of the protein (e.g., an antibody). As described above, one way to have greater control over the ratio of protein (e.g., an antibody) to oligonucleotide is to utilize engineered amino acids (e.g., lysine, cysteine, tyrosine) in the amino acid sequence of the protein (e.g., an antibody). In some embodiments, the conjugation method utilizes the introduction of an unnatural amino acid (e.g., an acetylphenylalanine (pAcPhe)) into the targeting agent (e.g., antibody). See, e.g., Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012), the entire contents of which is incorporated herein by reference for all purposes. A variety of chemistries can be utilized in direct conjugation, including, e.g., DBCO-azide click chemistry, transglutaminase-click chemistry, conjugation through the phosphate backbone (e.g., utilizing a maleimide linker, a direct disulfide linker, a maleimide disulfide linker), disuccinimidyl linker conjugation, and beta- Lactam conjugation. See, e.g., Dugal-Tessier 2021.

[00577] Another exemplary method of protein (e.g., antibody) oligonucleotide conjugation includes the use of ionic interactions, wherein initial fusion or modification of the protein (e.g., antibody) with a multi-cationic moiety such as protamine or polyarginine allows for conjugation of the oligonucleotide wherein the negative charge of the oligonucleotide backbone and positive charge of the protamine binds the oligonucleotide and protein (e.g., antibody) strongly via ionic interactions. See, e.g., Dugal-Tessier 2021 (and references cited therein). A similar conjugation approach utilizes the strong interactions between a biotin-labeled oligonucleotide and an avidin- labeled protein (e.g., antibody). See, e.g., Dugal-Tessier 2021 (and references cited therein). Hybridization conjugation methods can be utilized for double stranded oligonucleotides (e.g., an siRNA), wherein a single strand oligonucleotide is first conjugated to a protein and a complementary strand is hybridized to form a double-stranded oligonucleotide.

[00578] Another method of protein (e.g., antibody) oligonucleotide conjugation overcomes issues of stereoselectivity and regioselectivity by utilizing the specificity of enzymatic reactions. See, e.g., Hess G.T., Guimaraes C.P., et al. Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self- Assemble End-to-End Multiphage Structures. ACS Synth. Biol. 2013;2:490- 496. doi: 10.1021/sb400019s (hereinafter “Hess 2013”); Duckworth B.P., Chen Y., ct al. A Universal Method for the Preparation of Covalent Protein-DNA Conjugates for Use in Creating Protein Nanostructures. Angew. Chem. Int. Ed. 2007;46:8819-8822. doi: 10.1002/anie.200701942 (hereinafter “Duckworth 2007”), the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide is labeled with a small molecular tag that is recognized by the enzyme to initiate the conjugation mechanism. One of the most favored enzymes for this type of conjugation is Sortase A, which recognizes a LPXTG tag on the C-terminus of a protein target (e.g., an antibody (e.g., an antibody described herein)) or a molecular payload (e.g., an oligonucleotide (e.g., an oligonucleotide described herein)). See, e.g., Hess 2013 (and references cited therein). Once recognized, the enzyme catalyzes a transpeptidation reaction with an N-terminal glycine tag on the protein target (e.g., an antibody) Additionally, some bioconjugation techniques rely on post-translational modifications, such as protein farnesyl transferase which can transfer an oligonucleotide labelled with a farnesyl pyrophosphate to the cysteine residue of a CVIA sequence within a large protein structure (e.g., an antibody). See e.g., Duckworth 2007 (and references cited therein).

[00579] Another way to access conjugation of a protein target (e.g., antibody) and a molecular payload (e.g., an oligonucleotide) is through self-labeling with a small molecule tag, such as a HaloTag which comprises a chloroalkane linker attached to a molecular payload (e.g., an oligonucleotide). See e.g., Sacca B., Meyer D.-C.R., et al. Orthogonal Protein Decoration of DNA Origami. Angew. Chem. Int. Ed. 2010;49:9378-9383. doi: 10.1002/anie.201005931 (hereinafter “Sacca 2010”), the entire contents of which are incorporated herein by reference for all purposes. Furthermore, a SnapTag utilizes a modified human-derived O⁶-benzylguanine nucleobase to form covalent linkages between a molecular payload (e.g., an oligonucleotide) and a protein target (e.g., an antibody). See e.g., Gholami Z., Hanley Q. Controlled Assembly of SNAP-PNA-Fluorophore Systems on DNA Templates to Produce Fluorescence Resonance Energy Transfer. Bioconjug. Chem. 2014;25:1820-1828. doi: 10.1021/bc500319p (hereinafter “Gholami 2014”), the entire contents of which are incorporated herein by reference for all purposes.

5.9 Activity of Molecular Payloads (e.g., Oligonucleotides) & Conjugates Thereof

[00580] In some embodiments, the conjugate (or the molecular payload thereof (e.g., an oligonucleotide (e.g., described herein)) modulates (e.g., inhibits, reduces, enhances) the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)). In some embodiments, the conjugate (or the molecular payload thereof (e.g., an oligonucleotide (e.g., described herein)) modulates (e.g., inhibits, reduces, enhances) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)) in a cell. In some embodiments, the conjugate (or the molecular’ payload thereof (e.g., an oligonucleotide (e.g., described herein)) modulates (e.g., inhibits, reduces, enhances) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)) in a cell in a subject (e.g., a mammalian subject, e.g., a primate, human, non-human primate, mouse, rat, etc.). In some embodiments, the conjugate (or the molecular’ payload thereof (e.g., an oligonucleotide (e.g., described herein)) modulates (e.g., inhibits, reduces, enhances) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)) in a cell in a subject (e.g., a mammalian subject, e.g., a primate, human, non-human primate, mouse, rat, etc.) by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[00581] In some embodiments, the conjugate (or the molecular- payload thereof (e.g., an oligonucleotide (e.g., described herein)) inhibits the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)). In some embodiments, the conjugate (or the molecular’ payload thereof (e.g., an oligonucleotide (e.g., described herein)) inhibits expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)) in a cell. In some embodiments, the conjugate (or the molecular- payload thereof (e.g., an oligonucleotide (e.g., described herein)) inhibits expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11 A, ZBTB7A, KLF1)) in a cell in a subject (e.g., a mammalian subject, e.g., a primate, human, non-human primate, mouse, rat, etc.). In some embodiments, the conjugate (or the molecular payload thereof (e.g., an oligonucleotide (e.g., described herein)) inhibits expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., e.g., a target gene, nucleic acid (e.g., mRNA), and/or protein described herein (e.g., BCL11A, ZBTB7A, KLF1)) in a cell in a subject (e.g., a mammalian subject, e.g., a primate, human, nonhuman primate, mouse, rat, etc.) by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the conjugate (or the molecular payload thereof (e.g., an oligonucleotide (e.g., described herein)) mediates degradation of a target mRNA (e.g., a BCL11A, ZBTB7A, or KLF1 mRNA).

[00582] Any one or more of the above activities can be evaluated in vitro, ex vivo, or in vivo. Any one or more of the above activities can be evaluated by standard methods known in the art. For example, by PCR (e.g., qPCR), branched DNA assays, or by a protein-based methods (such as immunofluorescence analysis (using, e.g., western blotting or flow cytometric techniques). In some embodiments, modulation (e.g., inhibition) of gene (e.g., BCL11A, ZBTB7A, or KLF1) expression is determined by qPCR.

5.10 Cells

[00583] In one aspect, provided herein are cells (e.g., host cells) comprising a conjugate described herein (or any component thereof). In some embodiments, the host cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is within a subject (e.g., a subject described herein).

5.11 Pharmaceutical Compositions

[00584] In one aspect, provided herein are pharmaceutical compositions comprising a conjugate described herein (or any component thereof) and/or a host cell described herein; and a pharmaceutically acceptable excipient (see, e.g., Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, the entire contents of which is incorporated by reference herein for all purposes).

[00585] In one aspect, also provided herein are methods of making pharmaceutical compositions described herein comprising providing a conjugate described herein and/or a host cell described herein; and formulating it into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipient.

[00586] Also provided herein are pharmaceutical compositions comprising a conjugate described herein and/or cell described herein, wherein the pharmaceutical composition lacks a predetermined threshold amount or a detectable amount of a process impurity or contaminant, e.g., lacks a predetermined threshold amount or a detectable amount of a process-related impurity such as host cell proteins, host cell DNA, or a cell culture component (e.g., inducers, antibiotics, or media components); a product-related impurity (e.g., precursors, fragments, aggregates, degradation products); or a contaminant, e.g., endotoxin, bacteria, viral contaminant.

[00587] Acceptable excipients (e.g., carriers and stabilizers) are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid or methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol;or m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00588] A pharmaceutical composition may be formulated for any route of administration to a subject. Non-limiting embodiments include parenteral administration, such as intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal.

[00589] In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular’, intradermal, or subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular- injection. In one embodiment, the pharmaceutical composition is formulated for administration by intradermal injection. In one embodiment, the pharmaceutical composition is formulated for administration by subcutaneous injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions. The injectables can contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as for example, sodium acetate, sorbitan monolauratc, triethanolamine oleate or cyclodextrins. In some embodiments, the pharmaceutical composition is formulated in a single dose. In some embodiments, the pharmaceutical compositions if formulated as a multi-dose. [00590] Pharmaceutically acceptable excipients used in the parenteral preparations described herein include for example, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents or other pharmaceutically acceptable substances. Examples of aqueous vehicles, which can be incorporated in one or more of the formulations described herein, include sodium chloride injection, Ringer’s injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer’s injection. Nonaqueous parenteral vehicles, which can be incorporated in one or more of the formulations described herein, include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to the parenteral preparations described herein and packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride or benzethonium chloride. Isotonic agents, which can be incorporated in one or more of the formulations described herein, include sodium chloride or dextrose. Buffers, which can be incorporated in one or more of the formulations described herein, include phosphate or citrate. Antioxidants, which can be incorporated in one or more of the formulations described herein, include sodium bisulfate. Local anesthetics, which can be incorporated in one or more of the formulations described herein, include procaine hydrochloride. Suspending and dispersing agents, which can be incorporated in one or more of the formulations described herein, include sodium carboxymethylcelluose, hydroxypropyl methylcellulose or polyvinylpyrrolidone. Emulsifying agents, which can be incorporated in one or more of the formulations described herein, include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions, which can be incorporated in one or more of the formulations described herein, is EDTA. Pharmaceutical carriers, which can be incorporated in one or more of the formulations described herein, also include ethyl alcohol, polyethylene glycol or propylene glycol for water miscible vehicles; orsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[00591] The precise dose to be employed in a pharmaceutical composition will also depend on the route of administration, and the seriousness of the condition caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), other medications administered, or whether therapy is prophylactic or therapeutic. Therapeutic dosages are preferably titrated to optimize safety and efficacy.

5.12 Methods of Use

[00592] Provided herein are various methods of utilizing a conjugate described herein; a host cell described herein; and/or a pharmaceutical composition described herein; or any combination thereof.

[00593] In some aspects, the methods described herein comprise administering one or more of the foregoing to a subject. Exemplary subjects include mammals, e.g., humans, non-human mammals, e.g., non-human primates. In some embodiments, the subject is a human.

[00594] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, P- thalassemia, 5- thalassemia, or y-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof. In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y-thalassemia).

[00595] The dosage of any of the foregoing, to be administered to a subject in accordance with any of the methods described herein can be determined in accordance with standard techniques known to those of ordinary skill in the art, including the route of administration, the age and weight of the subject.

5.12.1 Methods of Delivery

[00596] Provided herein are, inter alia, various methods of delivering a conjugate described herein; a host cell described herein; and/or a pharmaceutical composition described herein; or any combination thereof to e.g., a cell, subject, a cell within a subject. [00597] In one aspect, provided herein are methods of delivering a conjugate described herein or a pharmaceutical composition described herein; or any combination thereof to a cell, the method comprising introducing into a cell a conjugate described herein, or a pharmaceutical composition described herein into the cell, to thereby deliver the conjugate or pharmaceutical composition into the cell. In some embodiments, the conjugate or pharmaceutical composition is introduced in an amount and for a time sufficient to deliver the conjugate or pharmaceutical composition into the cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject).

[00598] In one aspect, provided herein are methods of delivering a conjugate described herein; a host cell described herein; or a pharmaceutical composition described herein; or any combination thereof to a subject, the method comprising administering to a subject a conjugate described herein; a host cell described herein; or a pharmaceutical composition described herein, to thereby deliver the conjugate, host cell, or pharmaceutical composition to the subject. In some embodiments, the conjugate, host cell, or pharmaceutical composition is administered in an amount and for a time sufficient to deliver the conjugate, host cell, or pharmaceutical composition to the subject.

[00599] In one aspect, provided herein are methods of delivering a conjugate described herein; or a pharmaceutical composition described herein; or any combination thereof to a cell within a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby deliver the conjugate or pharmaceutical composition to a cell within the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to deliver the conjugate or pharmaceutical composition to the cell within the subject. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject).

5.12.2 Methods of Modulating Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and/or Protein

[00600] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the target gene is one listed in Table 20.

[00601] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the target gene is one listed in Table 20.

[00602] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene is one listed in Table 20.

[00603] In one aspect, provided herein are methods of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene is one listed in Table 20.

[00604] In one aspect, provided herein arc methods of inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the target gene is one listed in Table 20.

[00605] In one aspect, provided herein are methods of inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the target gene is one listed in Table 20.

[00606] In one aspect, provided herein are methods of inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene is one listed in Table 20.

[00607] In one aspect, provided herein are methods of inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene is one listed in Table 20.

[00608] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00609] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1 ). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELA2 (e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL (e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

[00610] Standard assays to measure the modulation (e.g., inhibiting or enhancing) of a target gene, nucleic acid (e.g., mRNA), and/or protein are known in the art. For example, immunoassays such as a western blot and an enzyme-linked immunosorbent assay (ELISA) can be utilized to evaluate protein levels. mRNA levels can be measured by, e.g., a northern blot or by RT-qPCR, a nuclease protection assay, or in situ hybridization.

5.12.3 Methods of Reducing or Inhibiting Expression of a Target Gene, Nucleic Acid (e.g., mRNA), and/or Protein

[00611] In one aspect, provided herein are methods of reducing or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the level of expression of the target gene, nucleic acid (e.g., mRNA), and/or protein in the cell is reduced by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., as compared to suitable control) (e.g., as measured by an assay known in the art or described herein, e.g., RT-qPCR). In some embodiments, the target gene is one listed in Table 20.

[00612] In one aspect, provided herein are methods of reducing or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising delivering into the cell a conjugate described herein, or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the level of expression of the target gene, nucleic acid (e.g., mRNA), and/or protein in the cell is reduced by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., as compared to suitable control) (e.g., as measured by an assay known in the art or described herein, e.g., RT-qPCR). In some embodiments, the target gene is one listed in Table 20.

[00613] In one aspect, provided herein are methods of reducing or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the level of expression of the target gene, nucleic acid (e.g., mRNA), and/or protein in the cell is reduced by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., as compared to suitable control) (e.g., as measured by an assay known in the ail or described herein, e.g., RT-qPCR). In some embodiments, the target gene is one listed in Table 20. [00614] In one aspect, provided herein are methods of reducing or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to reduce or inhibit the expression and/or activity of the target gene, nucleic acid e.g., mRNA), and/or protein. In some embodiments, the level of expression of the target gene, nucleic acid (e.g., mRNA), and/or protein in the cell is reduced by at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., as compared to suitable control) (e.g., as measured by an assay known in the ail or described herein, e.g., RT-qPCR). In some embodiments, the target gene is one listed in Table 20. [00615] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00616] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELA2 (e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL (e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

[00617] Standard assays to measure the reducing or inhibiting expression of a target gene, nucleic acid (e.g., mRNA), and/or protein are known in the art. For example, immunoassays such as a western blot and an enzyme-linked immunosorbent assay (ELISA) can be utilized to evaluate protein levels. mRNA levels can be measured by, e.g., a northern blot or by RT-qPCR, a nuclease protection assay, or in situ hybridization.

5.12.4 Methods of Modulating Splicing of a Target mRNA

[00618] In one aspect, provided herein are methods of modulating splicing of a target mRNA expressed by a hematopoietic cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate splicing of the target mRNA. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate splicing of the target mRNA. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the target gene is one listed in Table 20.

[00619] In one aspect, provided herein are methods of modulating splicing of a target mRNA expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate splicing of the target mRNA. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to modulate splicing of the target mRNA in the cell in the subject. In some embodiments, the target gene is one listed in Table 20.

[00620] In one aspect, provided herein are methods of correcting aberrant splicing of a target mRNA expressed by a hematopoietic cell in a cell, the method comprising delivering into the cell a conjugate described herein or a pharmaceutical composition described herein, to thereby correct aberrant splicing of the target mRNA. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to correct aberrant splicing of the target mRNA. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the target gene is one listed in Table 20.

[00621] In one aspect, provided herein are methods of correcting aberrant splicing of a target mRNA expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby modulate splicing of the target mRNA in the cell in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to correct aberrant splicing of the target mRNA in the cell in the subject. In some embodiments, the target gene is one listed in Table 20.

[00622] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL1 IA, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00623] In certain embodiments, the target mRNA is BCL11A (e.g., human BCL11A). In certain embodiments, the target mRNA is ZBTB7A e.g., human ZBTB7A). In certain embodiments, the target mRNA is KLF1 (e.g., human KLF1). In certain embodiments, the target mRNA is FANCA (e.g., human FANCA). In certain embodiments, the target mRNA is DKC1 (e.g., human DKC1). In certain embodiments, the target mRNA is RTEL1 (e.g., human RTEL1). In certain embodiments, the target mRNA is TERT (e.g., human TERT). In certain embodiments, the target mRNA is TERC (e.g., human TERC). In certain embodiments, the target mRNA is TINF2 (e.g., human TINF2). In certain embodiments, the target mRNA is RPS19 (e.g., human RPS19). In certain embodiments, the target mRNA is RPL11 (e.g., human RPL11). In certain embodiments, the target mRNA is RPS26 (e.g., human RPS26). In certain embodiments, the target mRNA is RPS 10 (e.g., human RPS 10). In certain embodiments, the target mRNA is RPL35A (e.g., human RPL35A). In certain embodiments, the target mRNA is RPS24 (e.g., human RPS24). In certain embodiments, the target mRNA is RPS 17 (e.g., human RPS 17). In certain embodiments, the target mRNA is SBDS (e.g., human SBDS). In certain embodiments, the target mRNA is SRP54 (e.g., human SRP54). In certain embodiments, the target mRNA is ELF1 (e.g., human ELF1). In certain embodiments, the target mRNA is ELA2 (e.g., human ELA2). In certain embodiments, the target mRNA is HAX1 (e.g., human HAX1). In certain embodiments, the target mRNA is G6PC3 (e.g., human G6PC3). In certain embodiments, the target mRNA is GFI1 (e.g., human GFI1). In certain embodiments, the target mRNA is WAS (e.g., human WAS). In certain embodiments, the target mRNA is CSF3R (e.g., human CSF3R). In certain embodiments, the target mRNA is MPL (e.g., human MPL). In certain embodiments, the target mRNA is GATA2 (e.g., human GATA2). In certain embodiments, the target mRNA is SAMD9 (e.g., human SAMD9). In certain embodiments, the target mRNA is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target mRNA is MECOM (e.g., human MECOM).

[00624] Standard assays to assess the splicing of an mRNA are known in the art. For example, RT-PCR (e.g., nested RT-PCR) and RNA sequencing. See, e.g., Singh NN, Seo J, Rahn SJ, Singh RN (2012) A Multi-Exon-Skipping Detection Assay Reveals Surprising Diversity of Splice Isoforms of Spinal Muscular' Atrophy Genes. PLOS ONE 7(11): e49595. https://doi.org/10.1371/joumal.pone.0049595, the entire contents of which are incorporated herein by reference for all purposes.

5.12.5 Methods of Inducing Expression of Fetal Hemoglobin

[00625] In one aspect, provided herein are methods of inducing expression of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby induce expression of fetal hemoglobin the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to induce expression of fetal hemoglobin the subject.

[00626] In one aspect, provided herein are methods of increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby increase the level of fetal hemoglobin the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to increase the level of fetal hemoglobin the subject.

[00627] In one aspect, provided herein are methods of increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby increase the ratio of fetal hemoglobin to adult hemoglobin in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to increase the ratio of fetal hemoglobin to adult hemoglobin in the subject.

[00628] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting a target gene, nucleic acid (e.g., mRNA), and/or protein.

[00629] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is one listed in Table 20.

[00630] In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00631] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELA2 e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL (e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

[00632] Standard assays to measure the level of fetal hemoglobin are known in the art. For example, immunoassays such as a western blot and an enzyme-linked immunosorbent assay (ELISA) can be utilized to evaluate protein levels. mRNA levels can be measured by, e.g., a northern blot or by RT-qPCR, a nuclease protection assay, or in situ hybridization. Fetal hemoglobin can be measured in, e.g., sample from a subject (e.g., a blood or tissue sample).

5.12.6 Methods of Treating an Inherited Blood Disorder

[00633] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited blood disorder in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited blood disorder in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to treat, ameliorate, or prevent the inherited blood disorder in the subject.

[00634] In some embodiments, the inherited blood disorder is a hemoglobinopathy (e.g., a hemoglobinopathy described herein (see, e.g., § 5.12.7)). In some embodiments, the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, P -thalassemia, 8-thalasscmia, or y-thalasscmia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, or methemoglobinemia.

[00635] In some embodiments, the inherited blood disorder is an inherited bone marrow failure syndrome (e.g., an inherited bone marrow failure syndrome described herein (see, e.g., § 5.12.8)). In some embodiments, inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, thrombocytopenia absent radii (TAR), a SAMD9/SAMD9L disorder, or a MECOM-associated syndrome.

[00636] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting BCL11 A, ZBTB7A, or KLF1 and the disease is an inherited blood disorder is a hemoglobinopathy (e.g., a hemoglobinopathy described herein (see, e.g., § 5.12.7)). In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting BCL11A, ZBTB7A, or KLF1 and the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, - thalassemia, 6-thalassemia, or y-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, or methemoglobinemia.

[00637] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM; and the inherited blood disorder is an inherited bone marrow failure syndrome (e.g., an inherited bone marrow failure syndrome described herein (see, e.g., § 5.12.8)).

[00638] In some embodiments, the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega) and the payload targets MPL. In some embodiments, the inherited bone marrow failure syndrome is diamond blackfan anemia (DBA) and the payload targets RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, or RPS17. In some embodiments, the inherited bone marrow failure syndrome is dyskeratosis congenita (DC) and the payload targets DKC1, RTEL1, TERT, TERC, or TINF2. In some embodiments, the inherited bone marrow failure syndrome is Fanconi anemia (FA) and the payload targets FANCA. In some embodiments, the inherited bone marrow failure syndrome is severe congenital neutropenia (SCN) and the payload targets ELA2, HAX1, G6PC3, GFI1, WAS, or CSF3R. In some embodiments, the inherited bone marrow failure syndrome is Schwachman diamond syndrome (SDS) and the payload targets SBDS, SRP54, or ELFE In some embodiments, the inherited bone marrow failure syndrome is GATA2 deficiency and the payload targets GATA2. In some embodiments, the inherited bone marrow failure syndrome is a SAMD9/SAMD9L disorders and the payload targets SAMD9 or SAMD9L. In some embodiments, the inherited bone marrow failure syndrome is a MECOM- associated syndrome and the payload targets MECOM.

[00639] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting a target gene, nucleic acid e.g., mRNA), and/or protein. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00640] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11 A (e.g., human BCL11 A). Tn certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1). In certain embodiments, the target gene, nucleic acid e.g., mRNA), and/or protein is ELA2 (e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL (e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

5.12.7 Methods of Treating Hemoglobinopathies

[00641] In one aspect, provided herein are methods of treating, ameliorating, or preventing a hemoglobinopathy in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the hemoglobinopathy in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to treat, ameliorate, or prevent the hemoglobinopathy in the subject.

[00642] In some embodiments, the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof. In some embodiments, the hemoglobinopathy is sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia). In some embodiments, the hemoglobinopathy is sickle cell disease. In some embodiments, the hemoglobinopathy is P-thalassemia.

[00643] In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P- thalassemia, 8-thalassemia, or y-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof. In some embodiments, the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y-thalassemia).

[00644] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting BCL11 A, ZBTB7A, or KLF1 and the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a- thalassemia, P- thalassemia, 8-thalassemia, or ^-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, or methemoglobinemia.

[00645] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting a target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, or KLF1. In certain embodiments, the target gene, nucleic acid e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1).

5.12.8 Methods of Treating an Inherited Bone Marrow Failure Syndrome

[00646] In one aspect, provided herein are methods of treating, ameliorating, or preventing an inherited bone marrow failure syndrome in a subject, the method comprising administering to the subject a conjugate described herein or a pharmaceutical composition described herein, to thereby treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject. In some embodiments, the conjugate or pharmaceutical composition is administered in an amount and for a time sufficient to treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject.

[00647] In some embodiments, the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, thrombocytopenia absent radii (TAR), a SAMD9/SAMD9L disorder, or a MECOM-associated syndrome.

[00648] In some embodiments, the subject is suspected of having or has been diagnosed with amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Duhowitz syndrome, Kostmann syndrome, refractory cytopcnia, or thrombocytopenia absent radii (TAR).

[00649] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM; and the inherited blood disorder is an inherited bone marrow failure syndrome (e.g., an inherited bone marrow failure syndrome described herein (see, e.g., § 5.12.8)).

[00650] In some embodiments, the conjugate (or pharmaceutical composition comprising the same) comprises a payload targeting a target gene, nucleic acid (e.g., mRNA), and/or protein. In some embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A, ZBTB7A, KLF1, FANCA, DKC1, RTEL1, TERT, TERC, TINF2, RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, RPS17, SBDS, SRP54, ELF1, ELA2, HAX1, G6PC3, GFI1, WAS, CSF3R, MPL, GATA2, SAMD9, SAMD9L, or MECOM.

[00651] In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is BCL11A (e.g., human BCL11A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ZBTB7A (e.g., human ZBTB7A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is KLF1 (e.g., human KLF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is FANCA (e.g., human FANCA). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is DKC1 (e.g., human DKC1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RTEL1 (e.g., human RTEL1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERT (e.g., human TERT). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TERC (e.g., human TERC). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is TINF2 (e.g., human TINF2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS19 (e.g., human RPS19). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL11 (e.g., human RPL11). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS26 (e.g., human RPS26). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS10 (e.g., human RPS10). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPL35A (e.g., human RPL35A). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS24 (e.g., human RPS24). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is RPS17 (e.g., human RPS17). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SBDS (e.g., human SBDS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SRP54 (e.g., human SRP54). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELF1 (e.g., human ELF1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is ELA2 (e.g., human ELA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is HAX1 (e.g., human HAX1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is G6PC3 (e.g., human G6PC3). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GFI1 (e.g., human GFI1). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is WAS (e.g., human WAS). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is CSF3R (e.g., human CSF3R). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MPL (e.g., human MPL). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is GATA2 (e.g., human GATA2). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9 (e.g., human SAMD9). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is SAMD9L (e.g., human SAMD9L). In certain embodiments, the target gene, nucleic acid (e.g., mRNA), and/or protein is MECOM (e.g., human MECOM).

[00652] In some embodiments, the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega) and the payload targets MPL. In some embodiments, the inherited bone marrow failure syndrome is diamond blackfan anemia (DBA) and the payload targets RPS19, RPL11, RPS26, RPS10, RPL35A, RPS24, or RPS17. In some embodiments, the inherited bone marrow failure syndrome is dyskeratosis congenita (DC) and the payload targets DKC1, RTEL1, TERT, TERC, or TINF2. In some embodiments, the inherited bone marrow failure syndrome is Fanconi anemia (FA) and the payload targets FANCA. In some embodiments, the inherited bone marrow failure syndrome is severe congenital neutropenia (SCN) and the payload targets ELA2, HAX1, G6PC3, GFI1, WAS, or CSF3R. In some embodiments, the inherited bone marrow failure syndrome is Schwachman diamond syndrome (SDS) and the payload targets SBDS, SRP54, or ELFE In some embodiments, the inherited bone marrow failure syndrome is GATA2 deficiency and the payload targets GATA2. In some embodiments, the inherited bone marrow failure syndrome is a SAMD9/SAMD9L disorders and the payload targets SAMD9 or SAMD9L. In some embodiments, the inherited bone marrow failure syndrome is a MECOM- associated syndrome and the payload targets MECOM.

5.13 Kits

[00653] In a one aspect, provided herein are kits comprising a conjugate described; a host cell described herein; and/or a pharmaceutical composition described herein; or any combination thereof. In addition, the kit may comprise a liquid vehicle for solubilizing or diluting, and/or technical instructions. The technical instructions of the kit may contain information about administration and dosage and subject groups.

[00654] In some embodiments, the conjugate (e.g., described herein), host cell (e.g., described herein), and/or pharmaceutical composition (e.g., described herein) is provided in a separate part of the kit. In some embodiments, the conjugate (e.g., described herein), host cell (e.g., described herein), and/or pharmaceutical composition (e.g., described herein) is optionally lyophilized, spray-dried, or spray-freeze dried. The kit may further contain as a part a vehicle (e.g., buffer solution) for solubilizing the dried or lyophilized conjugate (e.g., described herein), host cell (e.g., described herein), and/or pharmaceutical composition (e.g., described herein).

[00655] In some embodiments, the kit comprises a single dose container. In some embodiments, the kit comprises a multi-dose container. In some embodiments, the kit comprises an administration device (e.g., an injector for intradermal injection or a syringe for intramuscular injection).

[00656] Any of the kits described herein may be used in any of the methods described herein (see, e.g., § 5.12).

6. EXAMPLES

TABLE OF CONTENTS

6.1 Example 1. Anti-TFRl Antibody Binding to Recombinant TFR1.

6.2 Example 2. Anti-TFRl Antibody Binding to Erythroid-Progenitor Cells.

6.3 Example 3. Internalization of anti-TFRl Antibodies by Erythroid-Progenitor Cells. 6.4 Example 4. siRNA Mediated Knockdown of Target mRNA in Erythroid-Progenitor Cells.

6.5 Example 5. siRNA Mediated Knockdown of Target Protein Expression in Erythroid-Progenitor Cells.

6.6 Example 6. Generation of Anti-TFRl Antibody-siRNA Conjugates (AOCs).

6.7. Example 7. Anti-TFRl Antibody-siRNA Conjugate (AOC) Binding to Recombinant TFR1 Protein.

6.8 Example 8. Target Gene Transcript Knock-Down in Erythroid-Progenitor Cells After Treatment with Antibody-siRNA Conjugates (AOCs).

6.9 Example 9. Target Protein Knock-Down in Erythroid-Progenitor Cells After Treatment with Antibody-siRNA Conjugates (AOCs).

6.1 Example 1. Anti-TFRl Antibody Binding to Recombinant TFR1.

[00657] The ability of multiple unrelated anti-TFRl antibodies of different format and sequence were assessed for their ability to bind recombinant TFR1.

[00658] Briefly, two full-length monoclonal anti-TFRl antibodies (mAb2 and mAb3) and an anti-TFRl Fab (Fab) were recombinantly produced according to standard methods known in the art. The amino acid sequence of the anti-TFRl antibodies is set forth in Table 21 below.

Table 21. Amino Acid Sequence of Exemplary Anti-TFRl Antibodies.

[00659] The amino acid sequence of an isotype control antibody (and several components thereof) utilized in Examples 1-3 is provided below in Table 22.

Table 22. Amino Acid Sequence of Exemplary Isotype Control Antibody.

[00660] The ability of each of the anti-TFRl antibodies (mAb2, mAb3, and Fab) to bind recombinant TFR1 was assessed using OCTET/BLI. Briefly, a Sartorius Octet R8 system was utilized. The anti-TFRl antibody, ranging from a concentration between 50-100nM was bound to either anti-Human Fc capture (AHC) biosensor (to capture mAbs) or anti-human CHI region (FAB2G) biosensors (to capture Fab molecules). The antigen utilized in the binding assays was the human CD71/TFRl-6xHis (“6xHis” disclosed as SEQ ID NO: 853) (ACROBiosystems, cat#CDl-H5243). The antigen concentration tested in all assays was lOOnM. The loading of the antibodies onto the respective biosensor was performed for 180-300s, the association step was performed by dipping the loaded biosensors into wells containing lOOnM of the antigen and the dissociation step was carried out by dipping the biosensors into wells containing assay buffer (PBS + 0.1% BSA) for a time ranging from 300-600s. All baseline steps were performed using assay buffer (PBS + 0.1% BS A).

[00661] As shown in FIGS. 1A-1E, each of the anti-TFRl antibodies, mAb2 (FIG. IB), mAb3 (FIG. 1C), and the Fab (FIG. ID), bound to the TFR1 protein whereas the isotype control antibody (FIG. 1A) failed to bind to the TFR1 protein.

6.2 Example 2. Anti-TFRl Antibody Binding to Erythroid-Progenitor Cells.

[00662] The anti-TFRl antibodies set forth in Example 1 (mAb2, mAb3, and Fab) were assessed for their ability to bind erythroid-progenitor cells.

[00663] Briefly, 1 million erythroid-progenitor cells were collected per test and washed with lx PBS. Cells were blocked with Fc block from Thermo (Catalog #14-9161-73). Cells were incubated on ice with anti-TFRl antibodies. Cells were washed with 0.5% BSA in lx PBS. Secondary detection antibodies were incubated to detect the primary antibodies on ice. Cells were washed with 0.5% BSA in lx PBS. Cells were incubated with Live/Dead stain from Thermo (Catalog #14-9161-73). Cells were washed with 0.5% BSA in lx PBS. Cells were passed through a Falcon round bottom tube with a cell strainer cap prior to analysis on an Attune FACS machine. 100,000 events were collected per test. Cells were gated based on forward/side scatter, forward scatter height vs width, live cells and then cells positive for TFR1 expression. The percent positive cells and the mean fluorescent intensity (MFI) were measured and reported using Flow Jo.

[00664] As shown in FIGS. 2A-2D, the anti-TFRl antibodies bound to TFR1 expressed on the surface of erythroid-progenitor cells. FIGS. 2A-2B show the percentage positive cells (FIG. 2A) and MFI (FIG. 2B) of mAb2 and mAb3 binding to TFR1 on erythroid-progenitor cells. FIGS. 2C-2D shows the percentage positive cells (FIG. 2C) and MFI (FIG. 2D) of Fab binding to TFR1 on erythroid-progenitor cells.

6.3 Example 3. Internalization of anti-TFRl Antibodies by Erythroid-Progenitor Cells.

[00665] The internalization of anti-TFRl antibodies set forth in Example 1 (mAb2, mAb3, and Fab) by erythroid-progenitor cells was assessed.

[00666] Briefly, the anti-TFRl antibodies were with labeled with amine-reactive pHrodo™ dyes (Molecular Probes, Life Technologies; Catalog Nos. P36600) according to manufacturer’s protocol. The DOL for the various binding antibodies ranged between 1-3. To measure TFR1 mediated internalization of the antibodies, two million erythroid-progenitor cells were plated per well of a 6-well plate. Various amounts of the labeled antibody was added to each well. Cells and antibodies were incubated at 37 °C for either 1 hr or 4hrs. 1 million cells were collected after Ihr of incubation. The remaining cells were collected after 4hrs. Cells were washed with lx PBS and then with 0.5% BSA in lx PBS. Cells were incubated on ice with Live/Dead stain from Thermo (Catalog #14-9161-73). Cells were washed with ice cold lx PBS and passed through a Falcon round tube with a cell strainer cap prior to analysis on an Attune FACS machine. 100,000 events were collected per test. Cells were gated based on forward/side scatter, forward scatter height vs width, live cells and then cells positive for TFR1 expression. The percent positive cells and the mean fluorescent intensity (MFI) were measured and reported using FlowJo.

[00667] FIGS. 3A-3D show that the anti-TFRl antibodies rapidly internalize into erythroid- progenitor cells. FIGS. 3A-3B show pHrodo dye labeled mAb2 & mAb3 are internalized into erythroid-progenitor cells as measured by percentage positive cells (FIG. 3A) and total MFI (FIG. 3B) after 1 hour of incubation with the cells. FIGS. 3C-3D show pHrodo dye labeled Fab internalized into erythroid-progenitor cells as measured by percentage positive cells (FIG. 3C) and total MFI (FIG. 3D) after 1 hour of incubation with the cells.

6.4 Example 4. siRNA Mediated Knockdown of Target mRNA in Erythroid-Progenitor Cells.

[00668] The ability of a series of siRNAs to knockdown mRNA expression of HPRT1 and BCL11A in erythroid-progenitor cells was assessed.

[00669] The RNA sequence of siRNAs targeting HPRT1 or BCL11A is set forth in Table 23 below, as well as a control scrambled siRNA.

Table 23. RNA Sequence of Exemplary Modified siRNAs.

[00670] In the nucleotide sequences set forth in Table 23 (SEQ ID NOS: 833-838 and 843-844), the following applies: Lower case (x) = 2’-0Me; UPPER CASE (X) = native RNA (2'-OH); dX = DNA; * - PS bond.

[00671] Briefly, all siRNAs were resuspended in sterile distilled water. 24-well plates were prepared with 500uL of complete growth media and incubated at 37°C prior to electroporation. 500,000 cells were collected per electroporation, washed with pre-warmed lx PBS and resuspended in electroporation buffer to a density of 5xlO^A7. siRNA was added to cells not to exceed 1/10^th the total electroporation volume. The Neon NxT electroporator and kit (Catalog #N1025) was used to electroporate cells. The electroporation program used was identified from a previous screen (TB_12: 1200V 20ms 2 pulses). After electroporation, the cells and siRNA was added to one well of a 24-well plate and incubated for various time points (48 or 72hrs post electroporation). Cell numbers and viability were counted with C-Chip disposable hemacytometer and trypan blue staining. Cells were collected via centrifugation, washed with lx PBS, centrifuged again and the PBS was removed. Cell Pellets were stored at -80 degrees. RNA was isolated using the Qiagen RNeasy Plus Mini Kit (Catalog #74134). Cells were lysed using the Qiagen QIAshredder columns (Catalog #79654). RNA isolation was performed using manufacturer’s protocol. RNA was normalized to lug and cDNA was synthesized using the Superscript VILO kit from Thermo (Catalog #11754-050). 20ng of cDNA was used to evaluate transcripts via qPCR. The detection assays were labeled with either FAM or SUN for detection using the Quantstudio 6 or 7. Each sample was run in either duplicate or triplicate. Ct values were determined by the Quantstudio program. Ct values for each sample was averaged for both the target and endogenous genes. The delta-delta Ct method (2"^AACt method) was used to calculate the relative fold gene expression of samples relative to non -electroporated erythroid-progenitor cells. Percent knockdown relative to non-clcctroporatcd erythroid-progenitor cells is shown in the graphs.

[00672] FIGS. 4A-B show the extent of mRNA knock-down of the indicated target genes (FIG. 4A - HPRT1 knockdown; FIG. 4B - BCL11A knockdown) upon electroporation of the target gene specific siRNA(s) into erythroid-progenitor cells.

6.5 Example 5. siRNA Mediated Knockdown of Target Protein Expression in Erythroid- Progenitor Cells.

[00673] The ability of a series of siRNAs (a subset set forth in Example 4) to knockdown protein expression of BCL11A in erythroid-progenitor cells was assessed.

[00674] Briefly, all siRNA were resuspended in sterile dH20. 24-well plates were prepared with 500uL of complete growth media and incubated at 37 degrees prior to electroporation. 500,000 cells were collected per electroporation, washed with pre-warmed lx PBS and resuspended in electroporation buffer to a density of 5xl0^A7. siRNA was added to cells not to exceed 1 / 10^th the total electroporation volume. The Neon NxT electroporator and kit (Catalog #N1025) was used to electroporate cells. The electroporation program used was identified from a previous screen (TB_12: 1200V 20ms 2 pulses). After electroporation, the cells and siRNA was added to one well of a 24-well plate and incubated for various time points (48 or 72hrs post electroporation). Cell numbers and viability were counted with C-Chip disposable hemacytometer and trypan blue staining. Cells were collected via centrifugation, washed with lx PBS, centrifuged again and the PBS was removed. RIPA buffer from Thermo (Catalog #89900) including Halt Protease and Phosphatase Inhibitor cocktail (lOOx, Cat#78440) was added to each pellet at a density of 5xl0^A6 cells/mL to create lysates. Total protein concentrations were measured using the Pierce BCA protein assay kit (Catalog # 23227). 5ug of total protein was loaded into each well of 4-12% gradient Bis-Tris gel. Proteins were transferred to a nitrocellulose membrane, blocked and probed for either BCLlla or HPRT1 protein. Beta- Actin was used as a loading control. Secondary detection antibodies had either IRDye 680 or IRDye 800 and were used to detect the primary antibodies. The membrane was washed with lx PBS + Tween 20 in between antibody incubations. The LiCor Odyssey machine was used to develop the nitrocellulose membrane the software was used to calculate band intensity and normalize signal to the Beta- Actin loading control. I reported the amount of protein relative to non-electroporated erythroid-progenitor cells. [00675] FIGS. 5A-5B show the extent of the decrease in protein levels of BCL11 A (FIG. 5A) and HPRTl (FIG. 5B) in erythroid-progenitor cells electroporated with indicated concentration of respective siRNA.

6.6 Example 6. Generation of Anti-TFRl Antibody-siRNA Conjugates (AOCs).

[00676] A series of anti-TFRl antibody-siRNA conjugates (AOCs) were generated.

[00677] Briefly, all siRNAs used to generate the conjugates were synthesized as double stranded RNA with either an amine or azide group attached to either the 3' or 5' end of the sense strand.

[00678] To generate the MCC-siRNA conjugate, to a solution of siRNA (45.0 mg) in RNAse free water (0.707 mL) was added Succinimidyl-4-(N-Maleimidomethyl) Cyclohexane- 1 - Carboxylate (SMCC) in DMSO (30.0 eq., 0.921 mL, 32.24 mg), additional DMSO (1.328 mL) and borate buffer (0.900 mL) to a final concentration of 100 mM and pH 8.0. The reaction was allowed to proceed at 22°C. Reaction progress was monitored by Reverse-Phase HPLC analysis (RP-HPLC). Complete conversion to functionalised siRNA was observed shortly after addition of the reagents. Functionalised MCC-siRNA was purified by preparative Size Exclusion Chromatography (SEC) using aHiLoad® 16/600 Superdex® 200 pg column with isocratic elution in Dulbecco’s PBS. Fractions containing functionalised MCC-siRNA were pooled and concentrated by UF/DF using a 3 kDa MWCO, polyethersulfone (PES) membrane centrifugal concentrator. Purified MCC-siRNA was characterised by RP-HPLC and analytical SEC with UV detection for purity, and by UV-Vis at 260 nm for quantification. The sample was stored at -80°C. Analysis by RP-HPLC: 90% purity; analytical SEC: 100% purity.

[00679] To generate the Val-Cit-PAB siRNA conjugate, to a solution of siRNA (72.0 mg) in RNAse free water (1.131 mL) was added MC-Val-Cit-PAB-PNP in DMSO (30.0 eq., 3.252 mL, 113.83 mg), additional DMSO (0.047 mL) and borate buffer (1.2 mL) to a final concentration of 100 mM and pH 8.0. The reaction was allowed to proceed at 22°C. Reaction progress was monitored by RP-HPLC. 75% conversion to functionalised siRNA was observed after 5 minutes. Functionalised MC-Val-Cit-PAB-siRNA was purified by preparative SEC using a HiLoad® 16/600 Superdex® 200 pg column with isocratic elution in Dulbecco’s PBS. Fractions containing functionalised MC-Val-Cit-PAB-siRNA were pooled and concentrated by UF/DF using a 3 kDa MWCO, PES membrane, centrifugal concentrator. Purified MC-Val-Cit-PAB-siRNA was characterised by RP-HPLC and analytical SEC with UV detection for purity, and by UV-Vis at 260 nm for quantification. The sample was stored at -80°C. Analysis by RP-HPLC: 68% purity; analytical SEC: 100% purity.

[00680] The linker modified siRNAs were next used to generate the antibody-siRNA conjugates (AOC). To generate the mAb (mAb2)-MCC- siRNA or mAb (mAb2 or isotype control)-MC-Val- Cit-PAB-siRNA conjugates with average DAR 2, the antibody at 9.5 mg/mL in Dulbecco’s PBS, pH 7.1-7.4 was reduced with TCEP (10.0 eq.) at 40 °C for 1 h. After 1 h, the reduced antibody solution was diluted to 6.0 mg/mL with Dulbecco’s PBS, pH 7.5 and functionalised linker-siRNA conjugate (MCC-siRNA or MC-Val-Cit-PAB-siRNA conjugate) solution at 19.5 mg/mL in Dulbecco’s PBS (4.0 eq.) was added to the reduced antibody solution. The conjugation reaction was allowed to proceed at 22°C for Ih. After Ih, the reaction mixture was purified by preparative SEC using a HiLoad® 16/600 Superdex® 200 pg column with isocratic elution in Dulbecco’s PBS, pH 7.4. Fractions containing the antibody oligonucleotide conjugate were pooled and concentrated using 30 kDa MWCO, PES membrane, centrifugal concentrators, and sterile filtered through a 0.22 pm pore size, PVDF membrane filter. The final sample was analysed by analytical hydrophobic interaction chromatography (HIC), analytical SEC, analytical strong anion exchange chromatography (SAX) and SDS-PAGE. To generate the Fab-MCC-siRNA or Fab-MC-VaLCit- PAB-siRNA conjugates with an average DAR 2, the Fab antibody fragment at 9.5 mg/mL in Dulbecco’s PBS, pH 7.1-7.4 was reduced with 10 mM DTT for 1 h at 40 °C. Full reduction was confirmed by LC-MS and then by SDS-PAGE analysis. The reduced Fab was purified by ultrafiltration and diafiltration (UF/DF) in Dulbecco’s PBS to remove excess reducing agent and diluted to 6.0 mg/mL. Functionalised linker-siRNA conjugate (MCC-siRNA or MC-Val-Cit-PAB- siRNA) solution at 7.8 mg/mL in Dulbecco’s PBS (4.0 eq.) was added to the reduced antibody solution. The conjugation reaction was allowed to proceed at 22 °C for Ih. After Ih, the reaction mixture was purified by preparative SEC using a HiLoad® 16/600 Superdex® 200 pg column with isocratic elution in Dulbecco’s PBS, pH 7.4. Fractions containing the Fab oligonucleotide conjugate were pooled and concentrated using 10 kDa MWCO, PES membrane, centrifugal concentrators, and sterile filtered through a 0.22 pm pore size, PVDF membrane filter. The final sample was analysed by analytical HIC, SEC, SAX and by SDS-PAGE.

[00681] To generate antibody-siRNA conjugates using the TFP-PEG4-DBCO [tetrafLuorophenyl-PEG4-dibenzocyclooctyne] linker, the purified protein (Fab or mAb3) in PBS pH 7.4 was reacted with a 10 to 20-fold molar excess of DMSO-reconstituted TFP-PEG4-DBCO bifunctional linker (ThermoFisher Scientific, catalog no. C20043) for 1 hour at room temperature. Unreacted linker was separated from the protein by gel filtration chromatography (HiTrap G25 column) using PBS pH 7.4 as mobile phase. The level of linker modification was determined by absorbance. The linker-modified protein was then incubated with a 3-10 fold excess of azide- modified siRNA and allowed to incubate at RT for 1-2 hours followed by incubation at 4C for 24- 48 h. The reaction mixture was purified by preparative size exclusion chromatography (Superdex 200 16/60 Hi Load column) with PBS pH 7.4 as mobile phase. For Fab conjugates, DARI and DAR2 species were effectively separated by this step. Fractions were analyzed by SDS-PAGE and pooled based on level of siRNA incorporation. The pooled SEC fractions were concentrated using Amicon Ultra 4k ultrafiltration devices with either a lOkDa or 30kDa MWCO membrane. Measurement of protein concentration was performed using BCA assay, with a standard curve generated by the known concentrations of the parent (unconjugated) protein. Final product analysis included reducing and non-reducing SDS-PAGE, analytical SEC and endotoxin assay. DAR (Drug- antibody ratio) was approximated based on SEC peak area.

[00682] Table 25 below describes the series of anti-TFRl antibody-siRNA conjugates generated according to the methods described above.

Table 25. Anti-TFRl Antibody-siRNA Conjugates.

6.7 Example 7. Anti-TFRl Antibody-siRNA Conjugate (AOC) Binding to Recombinant TFR1 Protein.

[00683] The binding of anti-TFRl antibody-siRNA conjugates generated in Example 6 to recombinant TFR1 protein was assessed using OCTET/BLI.

[00684] Briefly, a Sartorius Octet R8 system was used for binding studies. The anti-TFRl antibody-siRNA conjugates (AOCs), ranging from a concentration between 50-100nM was bound to either Anti-Human Fc capture (AHC) biosensor (to capture mAbs) or anti-human CHI region (FAB2G) biosensors (to capture Fab molecules). The antigen utilized in the binding assays was the human CD71/TFRl-6xHis (ACROBiosystems, cat#CDl-H5243). The antigen concentration tested in all assays was lOOnM. The loading of the antibodies onto the respective biosensor was performed for 180-300s, the association step was performed by dipping the loaded biosensors into wells containing lOOnM of the antigen and the dissociation step was carried out by dipping the biosensors into wells containing assay buffer (PBS + 0.1% BSA) for a time ranging from 300- 600s. All baseline steps were performed using assay buffer (PBS + 0.1% BSA).

[00685] As shown in FIGS. 6A-6E and 6G-6K, each of the anti-TFRl antibody- siRNA conjugates bound to the TFR1 protein (AOC#1 - FIG. 6A; AOC#2 - FIG. 6B; AOC#3 - FIG. 6C; AOC#4 - FIG. 6D; AOC#5 - FIG. 6E; AOC#7 - FIG. 6G; AOC#8 - FIG. 6H; AOC#9 - FIG. 61; AOC#10 - FIG. 6J; and AOC#11 - FIG. 6K). FIG. 6F presents the binding (nonbinding) of the control AOC (AOC#6) to the TFR1 protein.

6.8 Example 8. Target Gene Transcript Knock-Down in Erythroid-Progenitor Cells After Treatment with Antibody-siRNA Conjugates (AOCs).

[00686] The ability of antibody-siRNA conjugates to knock-down target gene transcripts in erythroid-progenitor cells was assessed.

[00687] Briefly, 250,000 erythroid-progenitor cells were plated in 24-well plates to a density of 5xl0^A5 cells per mL. AOCs were diluted in lx PBS to lOx final concentrations. Half of the final concentration was given on the day of cell plating and the other half was given the next day keeping AOC dosing volumes to 1/10* of the cell culture media volume for each concentration tested. 48hrs and 72hrs post the initial dose, cell numbers and viability were determined with C-Chip disposable hemacytometers and trypan blue staining. Cells were collected via centrifugation, washed with lx PBS, centrifuged again and the PBS was removed. Cell Pellets were stored at -80 degrees. RNA was isolated using the Qiagen RNeasy Plus Mini Kit (Catalog #74134). Cells were lysed using the Qiagen QIAshrcddcr columns (Catalog #79654) using manufacturer’s protocol. RNA concentrations were evaluated using the Nanodrop. RNA was normalized to lug and cDNA was synthesized using the Superscript VILO kit from Thermo (Catalog #11754-050). 20ng of cDNA was used to evaluate transcripts via qPCR. The detection assays were labeled with either FAM or SUN for detection using the Quantstudio 6 or 7. Each sample was run in either duplicate or triplicate. Ct values were determined by the Quantstudio program. Ct values for each sample was averaged for both the target and endogenous gene (b2M). The delta-delta Ct method (2'^AACt method) was used to calculate the relative fold gene expression of samples relative to nonelectroporated erythroid-progenitor cells. The percent knockdown relative to non-AOC treated erythroid-progenitor cells is reported.

[00688] The results are summarized in FIGS. 7A-8D and 10A-14B and below.

[00689] 48 (FIG. 7A) and 72 (FIG. 7B) hours post the first dose with AOC#1 or AOC#2, the erythroid-progenitor cells cultures maintained a high percentage of viability. Both AOC#1 and AOC#2 showed significant knockdown of the target gene transcript - HPRT1 (FIG. 7C (48 hours post first dose) and FIG. 7D (72 hours post first dose)).

[00690] 48 (FIG. 8A) and 72 (FIG. 8B) hours post the first dose with AOC#3 or AOC#4, the erythroid-progenitor cells maintained a high percentage of viability. Both AOC#3 and AOC#4 showed significant knockdown of the target gene transcript - HPRT-1 (FIG. 8C (48 hours post first dose) and FIG. 8D (72 hours post first dose)).

[00691] 72 hours post the first dose with AOC#2, the erythroid-progenitor cells maintained a high percentage of viability (FIG. 10A). AOC#2 showed significant knockdown of the target gene transcript, HPRT1, 72 hours post the first dose (FIG. 10B).

[00692] 72 hours post the first dose with AOC#10 or AOC#11, the erythroid-progenitor cells maintained a high percentage of viability (FIG. 11A). Both AOC#10 and AOC#11 showed significant knockdown of the target gene transcript, BCL11 A, hours post first dose (FIG. 11B).

[00693] 72 hours post the first dose with AOC#9, the erythroid-progenitor cells maintained a high percentage of viability (FIG. 12A). AOC#9 showed significant knockdown of the target gene transcript, BCL11A, 72 hours post first dose (FIG. 12B).

[00694] 72 hours post the first dose with either AOC#7 or AOC#8, the erythroid-progenitor cells maintained a high percentage of viability (FIG. 13A). AOC#7 and AOC#8 showed significant knockdown of the target gene transcript, BCL11 A, 72 hours post first dose (FIG. 13B).

6.9 Example 9. Target Protein Knock-Down in Erythroid-Progenitor Cells After Treatment with Antibody-siRNA Conjugates (AOCs).

[00695] The ability of antibody- siRNA conjugates (AOCs) to knock-down target protein in erythroid-progenitor cells was assessed.

[00696] Briefly, 1 million erythroid-progenitor cells were plated into 6-well plates at a density of 6.7xl0^A5 cells/mL. AOCs were diluted in lx PBS to lOx final concentrations. Half of the total final concentration of the AOC was given on the day of cell plating and the other half was given the next day keeping AOC dosing volumes to 1710^th of the cell volume for every concentration. 72hrs post the initial dose, cell numbers and viability were determined with C-Chip disposable hemacytometers and trypan blue staining. Cells were collected via centrifugation, washed with lx PBS, centrifuged again and the PBS was removed. Cells were lysed with RIPA buffer + proteinase/phosphatase inhibitors. Total protein in the cell lysate was concentrated using Amicon Ultra-4 concentrators from Millipore with a 10k molecular’ weight cutoff. Total protein content was determined using a Pierce BCA protein assay kit using Albumin as a standard curve.

[00697] 5ug of total protein was loaded per well of a 4-12% Gradient Bis-Tris gel. The gel was run in lx MES running buffer until the protein bands were separated. The proteins from the gel were transferred to a nitrocellulose membrane using the iBlot 2 transfer system. The blot was blocked with Blocker FL Fluorescent blocking buffer. The blot was then stained with primary antibodies against human HPRT1 (Thermo; Cat# PA5-22281) and Beta- Actin (Thermo; Catalog# AM4302). The membrane was washed with lx PBS + Tween 20. The primary antibodies were detected with fluorescently labeled secondary antibodies from LiCor. The membrane was washed with lx PBS + Tween 20 prior to imaging on the LiCor Odyssey. Band intensity was determined using the LiCor Odyssey software. The signal from HPRT1 was normalized to Beta Actin in the same lane. The normalized signal was used to calculate percent knockdown relative to untreated erythroid-progenitor cells.

[00698] Both AOC#2 and AOC#4 also showed significant knockdown of the target protein, HPRT1 72, hours post the first dose (FIG. 9).

* * *

[00699] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00700] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[00701] Other embodiments are within the following claims.

Claims

CLAIMS What is claimed is:

1. A conjugate comprising:

(a) a hematopoietic cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transferrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to

(b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by the hematopoietic cell.

2. The conjugate of claim 1, wherein upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell.

3. The conjugate of claim 1 or 2, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (c) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

4. The conjugate of any one of claims 1-3, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the conjugate does not induce death of the hematopoietic cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

5. The conjugate of any one of claims 1-4, wherein the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR 1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the hematopoietic cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the hematopoietic cell remains viable; (d) upon internalization into a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the hematopoietic cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of a hematopoietic cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

6. A conjugate comprising:

(a) an erythroid precursor cell targeting agent that comprises a protein (e.g., an antibody) that specifically binds to the transl'crrin receptor (TFR) (e.g., human TFR (hTFR) (e.g., hTFRl)); operably connected to

(b) at least one oligonucleotide that modulates (e.g., inhibits) the expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by the erythroid precursor cell.

7. The conjugate of claim 6, wherein upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell.

8. The conjugate of claim 6 or 7, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (c) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (d) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

9. The conjugate of any one of claims 6-9, wherein the conjugate exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the conjugate does not induce death of the erythroid precursor cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

10. The conjugate of any one of claims 6-10, wherein the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR e.g., hTFRl)) exhibits one or more of the following properties: (a) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate is internalized into the erythroid precursor cell; (b) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the target cell; (c) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the erythroid precursor cell remains viable; (d) upon internalization into an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce death of the erythroid precursor cell; and/or (e) upon binding to TFR (e.g., hTFR (e.g., TFR1)) expressed on the surface of an erythroid precursor cell, the protein (e.g., an antibody) that specifically binds to the TFR (e.g., hTFR (e.g., hTFRl)) or conjugate does not induce degradation of TFR (e.g., hTFR (e.g., TFR1)).

11. The conjugate of any one of the preceding claims, wherein the protein that specifically binds TFR (e.g., hTFR (e.g., TFR1)) is an anti-TFR (e.g., hTFR (e.g., TFR1)) antibody.

12. The conjugate of claim 11, wherein the antibody comprises or consists of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fab-Fc, a scFv, a scFv-Fc, a (scFv)2-Fc, an Fv, a single domain antibody (sdAb) (e.g., a VHH), a sdAb-Fc (e.g., a VHH-Fc), a (sdAb)2 (e.g., a (VHH)2), or a (sdAb)2-Fc (e.g., a (VHH)2-Fc).

13. The conjugate of any one of claims 11 -12, wherein the antibody is an IgG (e.g., a human IgG (hlgG)) antibody.

14. The conjugate of any one of claims 11-13, wherein the antibody is a hlgGl, h!gG2, h!gG3, or h!gG4 antibody (e.g., a hlgGl or h!gG4 antibody).

15. The conjugate of any one of claims 11-14, wherein the antibody comprises an immunoglobulin (Ig) (e.g., a human Ig (hlg)) Fc region.

16. The conjugate of any one of claims 11-15, wherein the antibody comprises or consists of a full-length antibody, a Fab-Fc, a scFv-Fc, a (scFv)2-Fc, a sdAb-Fc (e.g., a VHH-Fc), or a (sdAb)₂-Fc (e.g., a (VHH)₂-Fc).

17. The conjugate of any one of claims 15-16, wherein the Ig (e.g., hlg) Fc region comprises at least a portion of a hinge region, a CH2 region, and a CH3 region.

18. The conjugate of any one of claims 15-17, wherein the Ig (e.g., hlg) Fc region comprises a hinge region, a CH2 region, and a CH3 region.

19. The conjugate of any one of claims 15-18, wherein the Ig is a hlg.

20. The conjugate of claim 19, wherein the hlg is a human IgG (hlgG).

21. The conjugate of claim 20, wherein the hlgG is hlgGl or hIgG4.

22. The conjugate of any one of claims 15-21, wherein the Ig (e.g., hlg) Fc region comprises one or more amino acid substitutions relative to a reference Ig (e.g., hlg) Fc region that reduces or abolishes one or more of the following effector functions relative to the reference hlg Fc region: antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and/or affinity to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))).

23. The conjugate of any one of claims 15-22, wherein the Ig (e.g., hlg) Fc region does not substantially mediate ADCC, does not substantially mediate CDC, and/or does not bind to one or more human Fc receptor (e.g., an Fey receptor (e.g., FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb (e.g., FcyRI, Fcylla, and/or Fcyllla))).

24. The conjugate of any one of claims 15-23, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position L234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

25. The conjugate of any one of claims 15-24, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234 and/or an alanine at amino acid position L235, numbering according to the EU index of Kabat.

26. The conjugate of any one of claims 15-25, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, an alanine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat.

27. The conjugate of any one of claims 15-26, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position L234, a serine at amino acid position L235, and/or a glycine, an alanine, or a serine at position P329 numbering according to the EU index of Kabat.

28. The conjugate of any one of claims 15-27, wherein the Ig is hlgGl and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

29. The conjugate of any one of claims 15-23, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an amino acid substitution at amino acid position S228, an amino acid substitution at amino acid position F234, and/or an amino acid substitution at amino acid position L235, numbering according to the EU index of Kabat.

30. The conjugate of any one of claims 1-23 or 29, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises a proline at amino acid position S228, an alanine at amino acid position F234, and/or an alanine at amino acid position L235, numbering according to EU index of Kabat.

31. The conjugate of any one of claims 15-23 or 29-30, wherein the Ig is hIgG4 and the amino acid sequence of the Fc region comprises an alanine at amino acid position N297, numbering according to the EU index of Kabat.

32. The conjugate of any one of claims 11-31, wherein the antibody comprises a first Fc region and a second Fc region that associate via at least one covalent (e.g., disulfide) bond.

33. The conjugate of any one of claims 11-32, wherein the antibody the amino acid sequence of the first Fc region and/or the amino acid sequence of the second Fc region comprise one or more amino acid substitution that promotes the association (e.g., heterodimerization) of the first and second Fc regions.

34. The conjugate of any one of claims 11 -33, wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid positions T366, L368, and Y407, numbering according to the EU index of Kabat.

35. The conjugate of any one of claims 11-34, wherein the amino acid sequence of the first Fc comprises a serine at amino acid position T366, an alanine at amino acid position L368, and a valine at amino acid position Y407, numbering according to the EU index of Kabat.

36. The conjugate of any one of claims 11-35, wherein the amino acid sequence of the first Fc region comprises an amino acid substitution at amino acid position Y349, numbering according to the EU index of Kabat.

37. The conjugate of any one of claims 11-36, wherein the amino acid sequence of the first Fc region comprises a cysteine at amino acid position Y349, numbering according to the EU index of Kabat.

38. The conjugate of any one of claims 11-37, wherein the amino acid sequence of the second Fc region comprises an amino acid substitution at amino acid position T366, numbering according to the EU index of Kabat.

39. The conjugate of any one of claims 11-38, wherein the amino acid sequence of the second Fc region comprises a tryptophan at amino acid position T366, numbering according to the EU index of Kabat.

40. The conjugate of any one of claims 11-39, wherein the amino acid sequence of the second Fc region of the antibody comprises an amino acid substitution at amino acid position S354, numbering according to the EU index of Kabat.

41. The conjugate of any one of claims 11-40, wherein the amino acid sequence of the second Fc region of the antibody comprises a cysteine at amino acid position S354, numbering according to the EU index of Kabat.

42. The conjugate of any one of claims 11-41, wherein the antibody does not (or does not substantially) block binding of TF (e.g., hTF) to the TFR (e.g., hTFRl).

43. The conjugate of any one of the preceding claims, wherein the protein that specifically binds TFR (e.g., hTFR (e.g., hTFRl)) is a TFR ligand (or a functional fragment or functional variant thereof).

44. The conjugate of claim 43, wherein the TFR ligand comprises transferrin (TF) (e.g., human transferrin (hTF)) (or a functional fragment or functional variant thereof).

45. The conjugate of any one of the preceding claims, wherein the oligonucleotide enhances the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein.

46. The conjugate of any one of the preceding claims, wherein the oligonucleotide inhibits the expression and/or activity of the target gene, nucleic acid e.g., mRNA), and/or protein.

47. The conjugate of any one of the preceding claims, wherein the oligonucleotide modulates (e.g., enhances or inhibits) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein through binding to a target nucleic acid molecule encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein (e.g., target mRNA molecule (e.g., a portion of a target mRNA molecule)).

48. The conjugate of any one of the preceding claims, wherein the target nucleic acid molecule is a target mRNA molecule (e.g., a portion of a target mRNA molecule).

49. The conjugate of any one of the preceding claims, wherein the oligonucleotide mediates one or more of the following degradation of the target nucleic acid molecule (e.g., mRNA), disabling of the target nucleic acid molecule (e.g., mRNA), modification of the target nucleic acid molecule (e.g., mRNA), alteration in the splicing of the target nucleic acid molecule (e.g., mRNA), alteration (e.g., a decrease) in the stability of the target nucleic acid molecule (e.g., mRNA), or a block in the translation of the target nucleic acid molecule (e.g., mRNA), or any combination of the foregoing.

50. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises or consists of an antisense oligonucleotide (ASO), small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

51. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises or consists of an antisense strand comprising a region of complementarity to a target sequence (e.g., an mRNA sequence encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein).

52. The conjugate of any one of the preceding claims, wherein the oligonucleotide is single stranded or double stranded.

53. The conjugate of any one of the preceding claims, wherein the oligonucleotide is a DNA, RNA, or RNA and RNA hybrid molecule.

54. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises a sense strand and an antisense strand forming a double stranded region.

55. The conjugate of any one of the preceding claims, wherein the sense strand and the antisense strand arc part of a single nucleic acid molecule (e.g., wherein a hairpin loop is between the sense strand and the antisense strand of the single nucleic acid molecule.

56. The conjugate of any one of the preceding claims, wherein the sense strand and the antisense strand are separate nucleic acid molecules (i.e., connected only through the double stranded region).

57. The conjugate of any one of the preceding claims, wherein the double stranded region is from about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-20, 19-21, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21- 23, or 21-22 nucleotide pairs in length.

58. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide.

59. The conjugate of any one of the preceding claims, wherein at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the oligonucleotide are modified.

60. The conjugate of any one of the preceding claims, wherein substantially all (or all) of the nucleotides in the oligonucleotide are modified.

61. The conjugate of any one of the preceding claims, wherein at least one of the modified nucleotides comprises a modified sugar (e.g., ribose moiety).

62. The conjugate of any one of the preceding claims, wherein at least one of the modified nucleotides comprises a modified nucleobase.

63. The conjugate of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleoside linkage (e.g., at least one phosphorothioate intemucleoside linkage).

64. The conjugate of any one of the preceding claims, wherein the at least one modified nucleotide is a 2’ modified nucleotide (e.g., a 2'-fluoro (2'-F), 2'-O-methyl (2'-0-Me), 2'-O- methoxyethyl (2'-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), ethylene -bridged nucleic acid (ENA), and (S)- constrained ethyl-bridged nucleic acid (cEt) (e.g., a 2' modified nucleotide is 2'- O-methyl or 2'-fluoro (2'-F))).

65. The conjugate of any one of the preceding claims, wherein the protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with one or more hemoglobinopathy .

66. The conjugate of any one of the preceding claims, wherein inhibition of or a reduction in expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with an increase in the level of fetal hemoglobin, the induction of expression of fetal hemoglobin, and/or an increase in the ratio of fetal hemoglobin to adult hemoglobin.

67. The conjugate of any one of the preceding claims, wherein expression and/or activity of a protein encoded by the target gene, nucleic acid (e.g., mRNA), and/or protein is associated with a repression of fetal hemoglobin, a decrease in the level of fetal hemoglobin, an increase in the level of adult hemoglobin, and/or an increase in ratio of adult hemoglobin to fetal hemoglobin.

68. The conjugate of any one of the preceding claims, wherein the target gene, nucleic acid (e.g., mRNA), and/or protein is a transcription factor.

69. The conjugate of any one of the preceding claims, wherein the target gene, nucleic acid (e.g., mRNA), and/or protein is highly expressed in erythroid precursor cells (relative to other non-erythroid precursor cell types).

70. The conjugate of any one of the preceding claims, wherein the target gene is B cell lymphoma leukemia 11A (BCL11A) (e.g., human BCL11A (e.g., hBCLUA)), Zinc Finger and BTB Domain Containing 7A (ZBTB7A) (e.g., hZBTB7A), KLF Transcription Factor 1 (KLF1) (e.g., hKLFl), FA Complementation Group A (FANCA) (e.g., human FANCA), Dyskerin Pseudouridine Synthase 1 (DKC1) (e.g., human DKC1), Regulator of Telomere Elongation Helicase 1 (RTEL1) (e.g., human RTEL1), Telomerase Reverse Transcriptase (TERT) (e.g., human TERT), Telomerase RNA Component (TERC) (e.g., human TERC), TERFI Interacting Nuclear Factor 2 (TINF2) (e.g., human TINF2), Ribosomal Protein S19 (RPS19) (e.g., human RPS19), Ribosomal Protein Li l (RPL11) (e.g., human RPL11), Ribosomal Protein S26 (RPS26) (e.g., human RPS26), Ribosomal Protein S10 (RPS10) (e.g., human RPS10), Ribosomal Protein L35A (RPL35A) (e.g., human RPL35A), Ribosomal Protein S24 (RPS24) (e.g., human RPS24), Ribosomal Protein S17 (RPS17) (e.g., human RPS17), SBDS Ribosome Maturation Factor (SBDS) (e.g., human SBDS), Signal Recognition Particle 54 (SRP54) (e.g., human SRP54), E74 Like ETS Transcription Factor 1 (ELF1) (e.g., human ELF1), Elastase Neutrophil Expressed (ELA2) (e.g., human ELA2), HCLS1 Associated Protein X-l (HAX1 ) (e.g., human HAX1), Glucosc-6-Phosphatasc Catalytic Subunit 3 (G6PC3) (e.g., human G6PC3), Growth Factor Independent 1 Transcriptional Repressor (GFI1) e.g., human GFI1), WASP Actin Nucleation Promoting Factor (WAS) (e.g., human WAS), Colony Stimulating Factor 3 Receptor (CSF3R) (e.g., human CSF3R), MPE Proto-Oncogene Thrombopoietin Receptor (MPL) (e.g., human MPL), GATA Binding Protein 2 (GATA2) (e.g., human GATA2), Sterile Alpha Motif Domain Containing 9 (SAMD9) (e.g., human SAMD9), Sterile Alpha Motif Domain Containing 9 Eike (SAMD9L) (e.g., human SAMD9L), or MDS1 And EVI1 Complex Locus (MECOM) (e.g., human MECOM).

71. The conjugate of any one of the preceding claims, wherein the target gene is BCL11A (e.g., hBCLUA), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

72. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is non-covalently conjugated to (b) the at least one oligonucleotide.

73. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is covalently conjugated to (b) the at least one oligonucleotide.

74. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is directly conjugated to (b) the at least one oligonucleotide.

75. The conjugate of any one of the preceding claims, wherein (a) the protein that specifically binds TFR is indirectly conjugated to (b) the at least one oligonucleotide through (c) a linker.

76. The conjugate of any one of the preceding claims, wherein the linker is cleavable or non- cleavable.

77. The conjugate of any one of the preceding claims, wherein the wherein (b) comprises at least 2, 3, 4, 5, 6, or more oligonucleotides.

78. The conjugate of any one of the preceding claims, wherein each of the at least 2, 3, 4, 5, 6, or more oligonucleotides are individually conjugated to (a) the protein (e.g., antibody) that specifically binds TFR (e.g., as described herein).

79. A cell comprising the conjugate of any one of claims 1-78.

80. The cell of claim 79, wherein the cell is in vitro, ex vivo, or in vivo.

81. A pharmaceutical composition comprising the conjugate of any one of claims 1-78 and a pharmaceutically acceptable excipient.

82. A kit comprising the conjugate of any one of claims 1 -78 or the pharmaceutical composition of any one of claims 81.

83. A method of delivering a conjugate or pharmaceutical composition to a cell, the method comprising introducing into a cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby deliver the conjugate or pharmaceutical composition into the cell.

84. The method of claim 83, wherein the cell is in vitro, ex vivo, or in vivo.

85. The method of claim 83 or 84, wherein the cell is a subject (e.g., a human subject).

86. A method of delivering a conjugate, cell, or pharmaceutical composition to a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81, to thereby deliver the conjugate, cell, or pharmaceutical composition to the subject.

87. A method of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein.

88. A method of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein.

89. The method of any one of claims 87-88, wherein the cell is in vitro, ex vivo, or in vivo.

90. The method of any one of claims 87-89, wherein the cell is a subject (e.g., a human subject).

91. A method of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

92. A method of modulating (e.g., inhibiting or enhancing) expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby modulate (e.g., inhibit or enhance) the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

93. A method of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein.

94. A method of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell, the method comprising introducing into the cell the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein.

95. The method of any one of claims 93-94, wherein the cell is in vitro, ex vivo, or in vivo.

96. The method of any one of claims 93-95, wherein the cell is a subject (e.g., a human subject).

97. The method of any one of claims 93-96, wherein the target is BCL11 A (e.g., hBCLl 1 A), ZBTB7A (e.g., hZBTB7A), or KLF1 (e.g., hKLFl).

98. A method of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by a hematopoietic cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

99. A method of reducing and/or inhibiting expression and/or activity of a target gene, nucleic acid (e.g., mRNA), and/or protein expressed by an erythroid precursor cell in a cell in a subject, the method comprising administering to the subject the conjugate of any one of claims 1- 78 or the pharmaceutical composition of claim 81, to thereby reduce or inhibit the expression and/or activity of the target gene, nucleic acid (e.g., mRNA), and/or protein in the subject.

100. A method of inducing expression of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby induce expression of fetal hemoglobin the subject.

101. A method of increasing the level of fetal hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby increase the level of fetal hemoglobin the subject.

102. A method of increasing the ratio of fetal hemoglobin to adult hemoglobin in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby increase the ratio of fetal hemoglobin to adult hemoglobin in the subject.

103. A method of treating, ameliorating, or preventing an inherited blood disorder in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the inherited blood disorder in the subject.

104. The method of claim 103, wherein the inherited blood disorder is a hemoglobinopathy or an inherited bone marrow failure syndrome.

105. A method of treating, ameliorating, or preventing a hemoglobinopathy in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the hemoglobinopathy in the subject.

106. The method of claim 105, wherein the hemoglobinopathy is sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y- thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

107. The method of claim 105 or 106, wherein the hemoglobinopathy is sickle cell disease or a thalassemia (e.g., a-thalassemia, P-thalassemia, 8-thalassemia, or y-thalassemia).

108. The method of any one of claims 105-107, wherein the subject is a human.

109. The method of any one of claims 105-108, wherein the subject is suspected of having or has been diagnosed with sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia (e.g., a-thalasscmia, P-thalasscmia, 8-thalasscmia, or '/-thalassemia), a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, methemoglobinemia, or any combination thereof.

110. The method of any one of claims 105-109, wherein the subject is suspected of having or has been diagnosed with sickle cell disease or a thalassemia (e.g., a- thalassemia, P-thalassemia, 6-thalassemia, or '/-thalassemia).

111. A method of treating, ameliorating, or preventing an inherited bone marrow failure syndrome in a subject, the method comprising administering to the subject the conjugate of any one of claims 1-78 or the pharmaceutical composition of claim 81, to thereby treat, ameliorate, or prevent the inherited bone marrow failure syndrome in the subject.

112. The method of claim 111, wherein the inherited bone marrow failure syndrome is amegakaryocytic thrombocytopenia (Amega), diamond blackfan anemia (DBA), dyskeratosis congenita (DC), Fanconi anemia (FA), Pearson syndrome, severe congenital neutropenia (SCN), Schwachman diamond syndrome (SDS), GATA2 deficiency, cyclic neutropenia, Dubowitz syndrome, Kostmann syndrome, refractory cytopenia, thrombocytopenia absent radii (TAR) a SAMD9/SAMD9E disorder, or a MECOM-associated syndrome.

113. The conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for use in the treatment of a disease in a subject in need thereof.

114. The conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for use as a medicament.

115. Use of the conjugate of any one of claims 1-78, the cell of any one of claims 79-80, or the pharmaceutical composition of claim 81 for the manufacture of a medicament for the treatment of a disease in a subject in need thereof.