Additional exemplary Megalin binding moi eties or ligands are disclosed in U.S. Patent 7,560,431, U.S. Patent 8,877,714, U.S. Patent 8,795,627; International Patent Application WO 2006/138343, U.S. Patent 9,388,418, U.S. Patent 10,065,993, International Patent Application WO 2017/100700, and International Patent Application WO 2018/232122, the entire contents of each of which are hereby incorporated by reference.

In some embodiments, a targeted kidney cell surface factor for delivery of a targeting moiety-associated modulatory nucleic acid agent of the instant disclosure is Megalin, or a fragment, or a variant thereof.

In some embodiments, a targeting moiety is or comprises a megalin-binding moiety. In particular embodiments, a targeting moiety binds an extracellular domain (e.g., to a site on the extracellular domain, e.g., a site that is exposed when megalin is on a cell surface) of megalin. In other particular embodiments, a conjugate comprises a targeting moiety that binds an extracellular domain (e.g., to a site on an extracellular domain, e.g., a site that is exposed when megalin is on a cell surface) of megalin and, upon binding to megalin, causes the internalization of megalin. Cubilin-Binding Moieties

Cubilin is a receptor of about 460kDa. Cubilin is also known as IFCR, Gp280, Intrinsic Factor- Vitamin B12 Receptor, MGA1, or IGS1. As an extracellular protein, Cubilin can interact with other membrane proteins, e.g., Megalin. One of the functions of Cubilin is as a receptor for intrinsic factor-vitamin B12 complexes.

Human Cubilin protein sequence:

MMNMSLPFLWSLLTLLIFAEVNGEAGELELQRQKRSINLQQPRMATERGNLVFLTGSAQ NIEFRTGSLGKIKLNDEDLSECLHQIQKNKEDIIELKGSAIGLPQNISSQIYQLNSKLVDLE RKFQGLQQTVDKKVCSSNPCQNGGTCLNLHDSFFCICPPQWKGPLCSADVNECEIYSGT PLSCQNGGTCVNTMGSYSCHCPPETYGPQCASKYDDCEGGSVARCVHGICEDLMREQA GEPKYSCVCDAGWMFSPNSPACTLDRDECSFQPGPCSTLVQCFNTQGSFYCGACPTGW QGNGYICEDINECEINNGGCSVAPPVECVNTPGSSHCQACPPGYQGDGRVCTLTDICSVS NGGCHPDASCSSTLGSLPLCTCLPGYTGNGYGPNGCVQLSNICLSHPCLNGQCIDTVSGY FCKCDSGWTGVNCTENINECLSNPCLNGGTCVDGVDSFSCECTRLWTGALCQVPQQVC GESLSGINGSFSYRSPDVGYVHDVNCFWVIKTEMGKVLRITFTFFRLESMDNCPHEFLQV YDGDSSSAFQLGRFCGSSLPHELLSSDNALYFHLYSEHLRNGRGFTVRWETQQPECGGIL TGPYGSIKSPGYPGNYPPGRDCVWIVVTSPDLLVTFTFGTLSLEHHDDCNKDYLEIRDGP LYQDPLLGKFCTTFSVPPLQTTGPFARIHFHSDSQISDQGFHITYLTSPSDLRCGGNYTDP EGELFLPELSGPFTHTRQCVYMMKQPQGEQIQINFTHVELQCQSDSSQNYIEVRDGETLL GKVCGNGTISHIKSITNSVWIRFKIDASVEKASFRAVYQVACGDELTGEGVIRSPFFPNVY PGERTCRWTIHQPQSQVILLNFTVFEIGS S AHCETDYVEIGS S SILGSPENKKYCGTDIPSFI

TSVYNFLYVTFVKSSSTENHGFMAKFSAEDLACGEILTESTGTIQSPGHPNVYPHGINCT WHILVQPNHLIHLMFETFHLEFHYNCTNDYLEVYDTDSETSLGRYCGKSIPPSLTSSGNS LMLVF VTD SDL AYEGFLINYEAIS AATACLQD YTDDLGTFT SPNFPNNYPNNWECI YRIT VRTGQLIAVHFTNFSLEEAIGNYYTDFLEIRDGGYEKSPLLGIFYGSNLPPTIISHSNKLWL KFKSDQIDTRSGFSAYWDGSSTGCGGNLTTSSGTFISPNYPMPYYHSSECYWWLKSSHG SAFELEFKDFHLEHHPNCTLDYLAVYDGPSSNSHLLTQLCGDEKPPLIRSSGDSMFIKLRT DEGQQGRGFKAEYRQTCENVVIVNQTYGILESIGYPNPYSENQHCNWTIRATTGNTVNY TFLAFDLEHHINCSTDYLELYDGPRQMGRYCGVDLPPPGSTTSSKLQVLLLTDGVGRRE KGFQMQWFVYGCGGELSGATGSFSSPGFPNRYPPNKECIWYIRTDPGSSIQLTIHDFDVE YHSRCNFDVLEIYGGPDFHSPRIAQLCTQRSPENPMQVSSTGNELAIRFKTDLSINGRGFN ASWQAVTGGCGGIFQAPSGEIHSPNYPSPYRSNTDCSWVIRVDRNHRVLLNFTDFDLEP QDSCIMAYDGLSSTMSRLARTCGREQLANPIVSSGNSLFLRFQSGPSRQNRGFRAQFRQA CGGHILTS SFDTVS SPRFPANYPNNQNC SWIIQ AQPPLNHITLSFTHFELERSTTC ARDF VE ILDGGHEDAPLRGRYCGTDMPHPITSFSSALTLRFVSDSSISAGGFHTTVTASVSACGGTF YMAEGIFNSPGYPDIYPPNVECVWNIVSSPGNRLQLSFISFQLEDSQDCSRDFVEIREGNA TGHLVGRYCGNSFPLNYSSIVGHTLWVRFISDGSGSGTGFQATFMKIFGNDNIVGTHGK VASPFWPENYPHNSNYQWTVNVNASHVVHGRILEMDIEEIQNCYYDKLRIYDGPSIHAR LIGAYCGTQTESFSSTGNSLTFHFYSDSSISGKGFLLEWFAVDAPDGVLPTIAPGACGGFL RTGDAPVFLFSPGWPDSYSNRVDCTWLIQAPDSTVELNILSLDIESHRTCAYDSLVIRDG DNNLAQQLAVLCGREIPGPIRSTGEYMFIRFTSDSSVTRAGFNASFHKSCGGYLHADRGII T SPK YPET YP SNLNC S WHVL VQ S GLTI A VHFEQPF QIPNGD S S CNQGD YL VLRNGPDIC S PPLGPPGGNGHFCGSHASSTLFTSDNQMFVQFISDHSNEGQGFKIKYEAKSLACGGNVYI HD AD S AGYVT SPNHPHN YPPH A DC I W IL AAPPETRIQLQFEDRFDIE VTPNC TSNYLELR DGVDSDAPILSKFCGTSLPSSQWSSGEVMYLRFRSDNSPTHVGFKAKYSIAQCGGRVPG QSGVVESIGHPTLPYRDNLFCEWHLQGLSGHYLTISFEDFNLQNSSGCEKDFVEIWDNHT SGNILGRYCGNTIPDSIDTSSNTAVVRFVTDGSVTASGFRLRFESSMEECGGDLQGSIGTF TSPNYPNPNPHGRICEWRITAPEGRRITLMFNNLRLATHPSCNNEHVIVFNGIRSNSPQLE KLCSSVNVSNEIKSSGNTMKVIFFTDGSRPYGGFTASYTSSEDAVCGGSLPNTPEGNFTSP GYDGVRNYSRNLNCEWTLSNPNQGNSSISIHFEDFYLESHQDCQFDVLEFRVGDADGPL MWRLCGPSKPTLPLVIPYSQVWIHFVTNERVEHIGFHAKYSFTDCGGIQIGDSGVITSPNY PNAYD SLTHC S SLLE APQGHTITLTF SDFDIEPHTTC AWD S VT VRNGGSPE SPIIGQ YCGN SNPRTIQSGSNQLVVTFNSDHSLQGGGFYATWNTQTLGCGGIFHSDNGTIRSPHWPQNFP ENSRCSWTAITHKSKHLEISFDNNFLIPSGDGQCQNSFVKVWAGTEEVDKALLATGCGN VAPGPVITPSNTFTAVFQSQEAPAQGFSASFVSRCGSNFTGPSGYIISPNYPKQYDNNMN CTYVIEANPLSVVLLTFVSFHLEARSAVTGSCVNDGVHIIRGYSVMSTPFATVCGDEMPA PLTIAGPVLLNFYSNEQITDFGFKFSYRIISCGGVFNFSSGIITSPAYSYADYPNDMHCLYTI TVSDDKVIELKFSDFDVVPSTSCSHDYLAIYDGANTSDPLLGKFCGSKRPPNVKSSNNSM LLVFKTDSFQTAKGWKMSFRQTLGPQQGCGGYLTGSNNTFASPDSDSNGMYDKNLNC

VWIIIAPVNKVIHLTFNTFALEAASTRQRCLYDYVKLYDGDSENANLAGTFCGSTVPAPF ISSGNFLTVQFISDLTLEREGFNATYTIMDMPCGGTYNATWTPQNISSPNSSDPDVPFSICT WVIDSPPHQQVKITVWALQLTSQDCTQNYLQLQDSPQGHGNSRFQFCGRNASAVPVFY SSMSTAMVIFKSGVVNRNSRMSFTYQIADCNRDYHKAFGNLRSPGWPDNYDNDKDCT VTLTAPQNHTISLFFHSLGIENSVECRNDFLEVRNGSNSNSPLLGKYCGTLLPNPVFSQNN ELYLRFKSDSVTSDRGYEHWTSSPSGCGGTLYGDRGSFTSPGYPGTYPNNTYCEWVLVA PAGRLVTINFYFISIDDPGDCVQNYLTLYDGPNASSPSSGPYCGGDTSIAPFVASSNQVFI KFHADYARRPSAFRLTWDS (SEQ ID NO: 6)

The extracellular domain of Cubilin includes repeats of CUB domains (complement Clr/Cls, Uegf [epidermal growth factor-related sea urchin protein], and bone morphogenic protein 1) and EGF-type repeats. A typical structure of Cubilin is disclosed in Figure 1 of Marzolo and Farfan (2011), Biol Res 44: 89-105, the entire contents of which are hereby incorporated by reference. The extracellular domain of Cubilin may also include one or more post-translational modifications, such as glycosylation.

Cubilin has been reported to be found on surfaces of one or more of the following tissues and/or cells: immune cells (e.g., bone marrow cells, lymph node cells, thymic cells, peripheral blood mononuclear cells [e.g., myeloid and/or lymphoid cells], erythrocytes, eosinophils, neutrophils, and/or platelets); nervous system (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons, and/or supporting cells; endothelial cells; muscle (e.g., heart muscle, smooth muscle, and/or skeletal muscle); small intestine; colon; adipocytes; kidney; liver; lung; splenic; stomach; esophagus; bladder; pancreas; thyroid; salivary gland; adrenal gland; pituitary gland; breast; skin; ovary; uterus; placenta; prostate; and testis. In kidney tissue, Cubilin has been reported to be found on the surface of proximal tubular epithelial cells and podocytes. Several ligands of Cubilin have been identified, some of which are disclosed in Nielsen et al. 2016.

Table 2: Exemplary Cubilin Ligands

Additional exemplary Cubilin binding moieties or ligands are disclosed in U.S. Patent 10,065,993, International Patent Application WO 2017/100700, International Patent Application WO 2018/232122, and International Patent Application WO 2015/027205, the entire contents of each of which are hereby incorporated by reference.

In some embodiments, a kidney cell surface factor is Cubilin, or a fragment, or a variant thereof.

In some embodiments, a targeting moiety is or comprises a Cubilin-binding moiety.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is chosen from: a peptide, an aminoglycoside, an endogenous ligand (e.g., a ligand disclosed in Table 1 or Table 2, or an analog or variant thereof), a xenobiotic, an antibody or a fragment thereof, or any combination thereof.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises a peptide. In some embodiments, a peptide is chosen from: a fragment of receptor associated protein (RAP), e.g., a RAP fragment comprising residues 219-323); a peptide derived from a radiopharmaceutical conjugate such as ocreotide, ocreotate, exendin, minigastrin, and/or neurotensin; or any combination thereof.

RAP (receptor-associated protein) is a cellular protein comprising about 300 amino acids and is encoded by the LRPAP1 gene. An exemplary RAP sequence is provided by NP_002328.1, and encoded by NM_002337.4. RAP has been shown to bind to Megalin to suppress the interaction of the Megalin receptor with one or more ligands (Willnow et al., EMBO J. 15, 2632-2639, 1996). Studies have shown that a minimal functional domain of RAP comprising about 104 amino acids retains RAP’s receptor binding and inhibition.

RAP protein sequence as provided by NM_002337.4:

MAPRRVRSFLRGLPALLLLLLFLGPWPAASHGGKYSREKNQPKPSPKRESGEEFRMEKL NQLWEKAQRLHLPPVRLAELHADLKIQERDELAWKKLKLDGLDEDGEKEARLIRNLNV ILAKYGLDGKKDARQVTSNSLSGTQEDGLDDPRLEKLWHKAKTSGKFSGEELDKLWRE FLHHKEKVHEYNVLLETLSRTEEIHENVISPSDLSDIKGSVLHSRHTELKEKLRSINQGLD RLRRVSHQGYSTEAEFEEPRVIDLWDLAQSANLTDKELEAFREELKHFEAKIEKHNHYQ KQLEIAHEKLRHAESVGDGERVSRSREKHALLEGRTKELGYTVKKHLQDLSGRISRARH NEL (SEQ ID NO: 7)

In some embodiments, a RAP fragment comprises a fragment of SEQ ID NO: 7, or a sequence with at least 90% identity thereto. In some embodiments, a RAP fragment comprises an LDL receptor binding domain of RAP. In some embodiments, a RAP fragment comprises a fragment of about -104 amino acids. In some embodiments, a RAP fragment is or comprises residues 219-323 of RAP.

Exemplary RAP fragments are disclosed in U.S. Patent Application US 2008/0153753A1, the entire contents of which are hereby incorporated by reference.

In some embodiments, a peptide disclosed herein further comprise one or more fragments, domains, and/or residues.

In some embodiments, a peptide disclosed herein comprises one or more modified amino acids.

In some embodiments, a peptide has one or more, or all of the following characteristics: (i) low molecular weight, e.g., 0.5kDa to lOkDa; (ii) limited charge at pH 7, e.g., -10 to +10; (iii) binding to a cell surface receptor, e g., Megalin, Cubilin, or both. Exemplary characteristics of peptides that may be useful in a targeting moiety are disclosed in Vegt, et al. Eur J Nucl Med Mol Imaging. 2011, and Wischnjow et al, Bioconjugate Chem. 2016, 27, 1050-1057, the entire contents of each of which are incorporated by reference herein.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises an aminoglycoside. In some embodiments, an aminoglycoside is chosen from one or more, or all of: streptomycin, neomycin, kanamycin, paromomycin, gentamicin, G-418 (geneticin), ELX-02, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekacin, isepamicin, framycetin, paromomycin, apramycin, fradiomycin, arbekacin, plazomicin, or a derivative or a variant thereof.

In some embodiments, an aminoglycoside disclosed herein has minimal bactericidal activity and/or toxicity, e.g., nephrotoxicity.

In some embodiments, an aminoglycoside comprises a variant having reduced toxicity, e.g., reduced nephrotoxicity as compared to an aminoglycoside without a variant. In some embodiments, an aminoglycoside comprises a variant having reduced bactericidal activity as compared to an aminoglycoside without a variant. In some embodiments, an aminoglycoside comprises a variant which retains activity, e.g., readthrough activity of premature termination codons, as compared to an aminoglycoside without a variant. In some embodiments, a variant of an aminoglycoside has reduced overall cationic charge as compared to an aminoglycoside without a variant. Exemplary aminoglycosides and variants thereof are disclosed in: Popadynec M. et al., (2021) ACS Med. Chem. Lett. 12( 9), 1486-1492; and in Brasell EJ et al., (2019), PLoS ONE 14(12): e0223954; the entire contents of each of which is hereby incorporated by reference.

In some embodiments, an aminoglycoside comprises an analog of an aminoglycoside having reduced antimicrobial activity (e.g., an aminoglycoside produced by resistance mutations in bacteria), and/or reduced endosomal or lysosomal stability, or both.

In some embodiments, an aminoglycoside has one or more, or all of the following characteristics: (i) high potency for binding to a cell surface factor, e.g., Megalin, Cubilin, or both; (ii) low nephrotoxicity; (iii) low ototoxicity; (iv) reduced endosomal or lysosomal stability; (v) reduced antimicrobial activity; or (vi) a combination of any one or all of (i) to (v).

In some embodiments, an aminoglycoside disclosed herein binds to one or more extracellular domains of a cell surface factor (e.g., Megalin, Cubilin, or both). In some embodiments, an aminoglycoside disclosed herein binds a cell surface receptor at or near one or more complement type repeats. Exemplary binding of an aminoglycoside to human Megalin is disclosed in Dagil R et al., (2013) Journal of Biological Chemistry; 288(6); 4424-4435; the entire contents of which are hereby incorporated by reference.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises an endogenous ligand, e.g., a ligand disclosed in Table 1. In some embodiments, an endogenous ligand is chosen from: vitamin carrier proteins, apolioproteins, peptide hormones, or any combination thereof.

In some embodiments, when a targeting moiety (e.g., a megalin binding moiety) is or comprises a vitamin carrier protein, a vitamin carrier protein comprises a vitamin carried by a vitamin carrier protein. For example, for a vitamin carrier protein that carries Vitamin D, in some embodiments such a vitamin carrier protein comprises both a vitamin carrier protein and Vitamin D when used in a kidney-specific binding moiety described herein.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises a ligand or substrate that binds to or is carried by another protein, e.g., a receptor or a carrier protein. Exemplary endogenous ligands are disclosed in Nielsen R. et al. (2016), Kidney Int. 89(l):58-67 the entire contents of which are hereby incorporated by reference.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises a xenobiotic. In some embodiments, a xenobiotic is chosen from: Polymixins, aprotinin, trichosanthin, or any combination thereof.

In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is or comprises an antibody or a fragment thereof. In some embodiments, a targeting moiety (e.g., a megalin binding moiety) is an antibody that binds to a relevant cell surface factor, e.g., Megalin. In some embodiments, an anti-Megalin antibody is a monoclonal antibody or a fragment thereof. In some embodiments, an anti-Megalin antibody is a polyclonal antibody or a fragment thereof. In some embodiments, an anti-Megalin antibody is a bispecific or multispecific antibody, or a fragment thereof. In some embodiments, a bispecific or multispecific antibody binds to Megalin and one or more additional antigens, e.g., a polypeptide present in podocytes.

Exemplary antibodies that bind Megalin include anti-Megalin autoantibodies found in patients having antibrush border antibodies and renal failure (ABBA disease), see e.g., Larsen C. et al., (2018) J Am Soc Nephrol. 29(2): 644-653. Anti-Megalin antibodies are also disclosed in: Perez-Gomez MV et al., (2020) Clin Kidney J., 13(3):281 -286; Dinesh KP et al., (2019) Am J Kidney Dis., 74(1): 132-137, Larsen CP et al., (2018) J Am Soc Nephrol., (2):644-653, and Gamayo A et al., (2019) Clin Kidney J., 13(3):468-472, the entire contents of each of which is hereby incorporated by reference.

Those skilled in the art would also appreciate various commercially available anti-Megalin antibodies (or fragments thereof) that can be utilized as a targeting moiety in a conjugate agent disclosed herein.

In some embodiments, an antibody used in a conjugate agent disclosed herein include one or more modifications of an Fc domain, e.g., an Fc variant. In some embodiments, an Fc variant comprises an effector null mutation. In some embodiments, an Fc variant has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP and/or CDC); (2) reduced binding to one or more Fc receptors; and/or (3) reduced binding to Clq complement. In some embodiments, the reduction in any one, or all of properties (l)-(3 ) is compared to an otherwise similar antibody with a wildtype Fc region. In some embodiments, an Activin A antibody agent comprising a variant Fc region has reduced affinity to a human Fc receptor, e.g., FcyR I, FcyR II and/or FcyR III. Exemplary Fc region variants are disclosed in Saunders K.O., (2019) Frontiers in Immunology; vol 10, Article 296, the entire contents of which is hereby incorporated by reference. For example, a Fc region variant is or comprises a modification provided in Table 3 of Saunders KO (2019).

In some embodiments, an antibody used in a conjugate agent disclosed herein is a neutral binder, e.g., an antibody having no antagonism or blockage of binding sites for other substrates.

In some embodiments, an antibody which binds to Megalin and is used in a conjugate agent disclosed herein can be trafficked intracellularly with Megalin. An exemplary anti-Megalin antibody with such properties is the 20B monoclonal antibody disclosed in Shah M. et al. (2013), Journal of Cell Biology 202(1): 113-127, the entire contents of which are herein incorporated by reference.

In some embodiments, an antibody used in a conjugate agent disclosed herein comprises an Fc variant and is a neutral binder.

Linkers

In some embodiments, a conjugate agent has the structure of the following formula:

wherein a is 1-8; and each of the binding moiety, linker, and modulatory nucleic acid is as defined above and described herein.

The synthesis and application of conjugate agents (e.g., “bioconjugates”) as tools for life science research, as diagnostic reagents, and as therapeutic agents has exploded in recent years and development of conjugate agents remains an area of intensive activity. In some embodiments, a bioconjugate or conjugate agent comprises a modulatory nucleic acid that is chemically conjugated or linked covalently to a binding (e.g., targeting) moiety.

In some embodiments, a conjugate agent is prepared by conjugating or covalently linking a modulatory nucleic acid agent to a binding moiety. In some embodiments, the modulatory nucleic acid may be linked to a binding moiety by, for example, reaction of the modulatory nucleic acid in-solution with a binding moiety. The conjugate agent may also be prepared in a single synthesis, for example using known synthesis methods for preparation of GalNAc-conjugated nucleic acids by solid-phase means. (For example, U.S. Pat. No. 9,422,562, W02009073809, U.S. Pat. No. 8,106,022, U.S. Pat. No. 8,828,956, U.S. Pat. No. 9,133,461, and U.S. Pat. No., 10,131,907, each of which is incorporated by reference in its entirety).

Regardless of how produced and depending on the desired properties of the conjugate agent, it may or may not be advantageous to include a spacer or linker between the modulatory nucleic acid and the binding moiety. If it is advantageous to include a linker, then linkers can be of many different types and chemical compositions.

Generally, linkers are designated as “cleavable” or “non-cleavable”. Cleavable linkers are typically employed when it is desired that the payload and binding moiety to which it is conjugated be released so that either or both can better carry out their function (For example, U.S. Pat. No. 10,808,039 and U.S. Pat. No. 9,463,252, each of which is incorporated by reference in its entirety.) Non-cleavable linkers are typically employed to maintain the desired activity, performance and stability of the conjugate agent, for example enzymes linked to probes or (m)Abs to facilitate ELISA assays, to increase affinity, or bi-specificity, etc. Amongst the cleavable linkers are those that are cleaved chemically, for example by hydrolysis, change in pH, reduction or oxidation, and those that are cleaved enzymatically, for example by action of a protease, an esterase, a glucosidase, a glucuronidase, galactosidase, a phosphatase, phosphodiesterase, nuclease, lipase or any enzyme that is capable of cleaving a linker to liberate the biomolecule from the other compound.

In some embodiments, a cleavable linker is or comprises a disulfide linkage, an ester, a phosphodiester, a saccharide, or a lipid.

In some embodiments, a non-cleavable linker is chemically, enzymatically, or otherwise biochemically and physiologically stable. As such, a non-cleavable linker does not contain linkages that are chemically, biochemically, enzymatically cleavable or are otherwise physiologically unstable.

In some embodiments, whether cleavable or non-cleavable, a linker can be installed by a chemical linking reaction between the modulatory nucleic acid and the binding moiety to which it is being conjugated. The modulatory nucleic acid and binding moiety may or may not be first modified to increase or facilitate reactivity towards one another. Such modification can also increase or improve the specificity of the conjugation reaction and degree of conjugation when that is desired. The linkers may be installed in a single reaction or by stepwise reactions until the desired linker and modulatory nucleic acid have been prepared.

Non-limiting examples of chemical linking reactions to form conjugate agents include reaction of various thiols to form disulfides, reaction between thiols and alkyl halides or mal eimides to form thioethers, reaction of alkynes with azides to form triazoles (“Click Reaction”), reaction between aldehydes and hydrazides or amines, or aminoxy compounds to form hydrazones, imines and oxy imines, reaction between carboxylic acids and amines, thiols or alcohols (i.e., nucleophiles) to form amides, thioesters and esters. The carboxylic acids may be activated in situ in the presence of the amines, thiols or alcohols so as to be made reactive or may be pre-activated prior to addition of the nucleophile, for example by converting to activated esters of N- hydroxysuccinimide (NHS) or sulfonated-NHS. Many reviews of chemical linking reactions exist for example Spicer et al. (2018) Chem. Rev. 2018, 118, 16, 7702-7743.

The reaction of thiols with maleimides is very widely used, see for example (Revasco et al (2018) Chem. Eur. J. 10.1002/chem.201803174) as is the Click Reaction see for example (Fantoni et al., (2021) Chem. Rev., 121, 12, 7122-7154), as is hydrazide formation (See HyNic Peptide Conjugation Protocol, Dirksen et al (2006) J. Am. Chem. Soc., 128, 49, 15602-15603, Kozlov et al. (2004)Biopolymers73(5):621-30. doi: 10.1002/bip.20009). Numerous companies sell chemical compounds and kits with protocols that enable conjugate agents comprising various linkers to be prepared in a straightforward fashion.

As defined generally above and described herein, the linker is a bivalent group that connects or links the binding moiety to the modulatory nucleic acid moiety.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-40 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CH(R¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1 -4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein:

R¹ is an amino acid side chain; and

R is selected from hydrogen or an optionally substituted Ci-6 aliphatic, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3 - to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-35 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-30 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-25 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -QR¹)!-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-20 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-15 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched C1-10 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a bivalent straight or branched Ci-5 aliphatic chain, wherein one or more methylene units of the aliphatic chain are replaced by a group selected from -CHCR¹)-, -C(R¹)₂-, -O-, -S-, -N(R)-, -C(=O)-, -C(=S)-, -C(=NR), -N(R)C(=O)-, -C(=O)N(R)-, -N(R)C(=S)-, -C(=S)N(R)-, -OC(=O)-, -C(=O)O-, -SC(=O)-, -C(=O)S-, -N(R)C(=O)N(R)-, -N(R)C(=O)O-, -OC(=O)N(R)-, -N(R)C(=O)S-, -SC(=O)N(R)-, -OC(=O)O-, -N(R)C(=NR)-, -N(R)C(=NR)N(R)-, a 3- to 6-membered saturated or partially unsaturated carbocyclic ring, phenyl, a 3- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5- to 6-membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, the linker is or comprises a structure selected from

In some embodiments, the cleavable linker is a cathepsin-cleavable linker. In some such embodiments, the linker is or comprises a valine-citrulline (Val-Cit) motif:

wherein R is hydrogen or Ci-6 aliphatic.

In some embodiments, the valine-citrulline linker is or comprises

wherein R is hydrogen or Ci-6 aliphatic.

In some embodiments, the linker comprises a disulfide linkage. In some embodiments, the linker comprises a poly (ethyleneglycol) moiety (e.g., -(CH2CH2O)b-), wherein b is 1-50.

In some embodiments, the linker is or comprises a group selected from

wherein each of k, m, n, p, q, r, s, t, u, v, w, x, y, and z is 1-20; and

R is hydrogen or Ci-io aliphatic.

In some embodiments, k is 3.

In some embodiments, m is 3.

In some embodiments, n is 2. In some embodiments, n is 12.

In some embodiments, p is 3.

In some embodiments, each of m and p is 3.

In some embodiments, q is 1.

In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 6.

In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 6.

In some embodiments, each of r and s is 3. In some embodiments, each of r and s is 4. In some embodiments, each of r and s is 6.

In some embodiments, t is 3. In some embodiments, t is 5.

In some embodiments, u is 3. In some embodiments, u is 5.

In some embodiments, each of t and u is 3. In some embodiments, each of t and u is 5. In some embodiments, v is 3.

In some embodiments, w is 4.

In some embodiments, x is 8.

In some embodiments, y is 2.

In some embodiments, z is 1.

Other Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule Such a lipid or lipid-based molecule optionally binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

.A lipid-based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid-based ligand binds HSA. Optionally, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue.

However, it is certain that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In other embodiments, the lipid-based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of, or in addition to, the lipid-based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells.

I l l Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B 12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, optionally a helical cell-permeation agent. Optionally, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use ofD-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to ASO or iRNA agents can affect pharmacokinetic distribution of the ASO or iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin Pl), a disulfide bond-containing peptide (e g., a -defensin, P-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31 :2717-2724, 2003). Carbohydrate Conjugates

In some embodiments of the compositions and methods of the present disclosure, an ASO or iRNA agent further comprises a carbohydrate. The carbohydrate conjugated ASO or iRNA agent is advantageous for the in vivo deliver}? of nucleic acids, as well as compositions suitable for in vivo therapeutic use. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and tri saccharides include sugars having two or three monosaccharide units (e.g.. C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the present disclosure is a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in US 8, 106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the ASO or iRNA agent to particular cells. In some embodiments, the GalNAc conjugate targets the ASO or iRNA agent to liver cell s, e.g , by serving as a ligand for the asial oglycoprotein receptor of liver cells (e.g., hepatocytes).

Other Nucleic Acid Modifications

In certain aspects, the kidney cell -targeting moieties and other modifications referenced herein can be used in combination with one or more of a variety of other nucleic acid modifications known in the art. Specific nucleic acid modifications explicitly contemplated for use with the SLC6A19-targeting nucleic acids of the instant disclosure (optionally in combination with other modifications as disclosed herein and/or as known in the art) include, without limitation, "extended nucleic acid" ("exNA") modifications, and a range of phosphoryl guanidine-containing backbone linkages ("PN chemistry"), among others.

"Extended nucleic acid" ("exNA") modifications typically refer to a form of modification that inserts a methyl group between the 3'-phosphate group of a first nucleoside and the 5'-carbon of a ribose of the next nucleoside (progressing from 5' to 3') in an oligonucleotide chain. Such canonical "exNA" modifications therefore have the following generic structure:

. In certain embodiments, "exNA" modifications that are optionally abasic and/or that possess longer extensions than methyl, and/or that are optionally substituted at one or more of the base, 2'-substituent and/or extended carbon chain, are also expressly contemplated. In some embodiments of the instant disclosure, the term "exNA" can therefore refer to any moiety having the structure:

, where: R¹ is a nucleobase or is an abasic structure (e.g., H, CH3, or other modified structures, which are optionally substituted); R² is a 2' substituent, including, e.g., without limitation, 2'-O-alkyl (e.g., 2'-O-methyl, etc.), 2'-F, etc.; and C1-10 refers to a 1-10 carbon chain that is optionally saturated or unsaturated and that harbors an optionally substituted alkyl (e g., optionally substituted methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl), alkenyl (e.g., optionally substituted methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, or decylene), or alkynyl (e.g., ethynyl, optionally substituted propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, or decynyl) chain.

In related embodiments, branching of the C1-10 region shown above is also contemplated for "exNA" modifications. For example, in some embodiments, an "exNA" modification of the

instant disclosure can also be represented by the formula , where R¹ is a nucleobase or is an abasic structure (e.g., H, CH3, or other modified structures, which are optionally substituted); R² is a 2' substituent, including, e.g., without limitation, 2'-O-alkyl (e.g., 2'-O-methyl, etc.), 2'-F, etc.; and the side substitution on the 5’- of the ribose can be 2-11 atoms, with various amounts of substituents at X and Y (from X=Y=H to long n- or branched alkyl structures).

In certain embodiments, "exNA" and/or other internucleoside modifications can be positioned at or near the 3'-end of an oligonucleotide therapeutic (e.g., at or near the 3'-end of the guide strand of a siRNA, at or near the 3'-end of an antisense agent, etc.), where, without wishing to be bound by theory, the exNA modification(s) can confer an exNA-modified nucleic acid payload with resistance against 3 '-exonuclease-mediated digestion (Yamada, K. et al., Nature Biotechnology 2024). In some embodiments, exNA-phosphorothioate internucleoside linkages can be positioned at the ultimate and optionally also at the penultimate intemucleoside linkage(s) of the 3'-terminus of a nucleic acid payload (e g., exNA-PS modification(s) positioned at the 3'- terminal region of the guide strand of a siRNA and/or ex-NA-PS modification(s) positioned at the 3'-terminal region of an antisense oligonucleotide). As disclosed in WO 2021/195533, exNA modifications can be used in combination with a range of other commonly used oligonucleotide modifications, including, without limitation, the following:

In such embodiments, it is also expressly contemplated that such exNA modifications can be used in concert with effectively any art-recognized base at the above-noted "Base" or R¹ position, including, without limitation, the following representative natural and artificially modified bases known in the art:

Phosphoryl guanidine-containing backbone ("PN backbone") linkages are also expressly contemplated for use in combination with the kidney cell-targeting moieties of the instant disclosure. Such PN backbone modifications were initially identified in WO 2023/201095 as

members of the following genus: . Exemplified versions of PN backbone modifications include, without limitation, the following:

O or S. In particular embodiments, the PN backbone modifications are selected from among:

"nOOl" modification of WO 2023/201095). Exemplary PN backbone linkages are also disclosed, e.g., in U.S. Patent No. 11,208,430.

In addition to expressly contemplating herein use of the above-noted phosphoryl guanidine structures for modification of internucleoside linkages of the nucleic acid agents of the instant disclosure, a range of other guanidine-based moieties is also expressly contemplated herein for attachment to linkage phosphorous groups. Such other guanidine-based moieties include, without limitation, the following:

1z3l

R^LS is independently -Cl, -Br, -F, N(Me)2, or NHCOCO3.

Other backbone modifications known in the art are also contemplated for use with the targeting moieties and payloads of the instant disclosure. In particular, inclusion of one or more mesyl phosphoramidate modification(s) and/or busyl phosphorami date (and/or other structurally related modification(s)) is expressly contemplated for the nucleic acid agents of the instant disclosure. Mesyl phosphoramidate and busyl phosphoramidate modifications have the following structure, as compared to phosphorothioate modifications (see Sergeeva et al. (2024) Front Chem., 12 - 2024 doi.org/10.3389/fchem.2024.1342178):

. In certain embodiments, oligomeric compounds

(including, e ,g., antisense oligonucleotides, siRNAs, etc., or portions thereof) comprise or consist of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of the following formula:

, where X is selected from O or S, and R is selected from and, a substituted and, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6 alkoxy, C1-C20 alkyl, C1-C6 alkenyl. C l- C6 alkynyl, substituted C1-C20 alkyl, substituted C1-C6 alkenyl substituted C1-C6 alkynyl, and a conjugate group. In certain embodiments, X is O and R is methyl, and the internucleoside linking group of the above formula is an internucleoside linking group of the formula immediately below.

In certain embodiments, oligomeric compounds (including, e.g., antisense oligonucleotides. siRNAs, etc , or portions thereof) comprise or consist of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of the fol I owing formul a:

(a mesyl phosphorami date internucleoside linkage, see, e.g., WO

2023/278589).

In certain embodiments, oligomeric compounds (including, e.g., antisense oligonucleotides, siRNAs, etc., or portions thereof) comprise or consist of oligonucleotides consisting of linked nucleosides and having at least one internucleoside linking group of the fol 1 owi n g form ul a:

In certain embodiments, oligomeric compounds (including, e.g., antisense oligonucleotides, siRNAs, etc., or portions thereof) comprise or consist of oligonucleotides consisting of linked nucleosides and having at least one internucleoside linking group of the following formula:

. Modifications to the phosphate backbone may be positioned within an oligonucleotide agent of the instant disclosure in any position(s) known in the art and/or disclosed herein.

Modified oligonucleotides of the instant disclosure comprise at least one modification relative to an unmodified oligonucleotide (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety, a stereo-non-standard nucleoside, and/or a modified nucleobase) and/or at least one modified internucleoside linkage). In certain embodiments, the modified internucleoside linkage is a modified internucleoside linking group having any of the above formulas. In certain embodiments, compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents, siRNAs, etc., or portions thereof) having at least one modified internucleoside linking group having any of the above formulas.

Assaying Modulation of Expression

Modulation of SLC6A19 and/or B°AT1 expression can be assayed in a variety of ways known in the art. SLC6A19 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume

1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.

Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif, and used according to manufacturer's instructions. The method of analysis of modulation of RNA levels is not a limitation of the instant invention.

Levels of a B°AT1 protein encoded by SLC6A19 mRNA or DNA can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a B°AT1 protein encoded by SLC6A19 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume

2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Active Target Segments

The locations on the target nucleic acid (i.e., SLC6A19 mRNA) defined by having one or more active modulatory RNA compounds targeted thereto are referred to as “active target segments.” There may be substantial variation in activity (e.g., as defined by percent inhibition) of the modulatory RNA compounds within an active target segment. Active modulatory RNA compounds are those that are determined to modulate the expression of their target RNA. In some embodiments, active modulatory RNA compounds are inhibitory, optionally inhibiting expression of their target RNA at least about 50%, optionally at least about 70% and optionally at least about 80%, or more. In some embodiments, the level of inhibition required to define an active inhibitory RNA compound is defined based on the results from a screen used to define the active target segments. Those skilled in the art understand that the percent inhibition by an inhibitory RNA compound on a target mRNA will vary between assays due to factors relating to assay conditions.

Hybridization

As used herein, “hybridization” means the pairing of complementary strands of antisense compounds to their target sequence and/or the pairing of complementary strands of doublestranded nucleic acid molecules with one another. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is complementary to the natural base 5-methyl cytosine and the artificial base known as a G-clamp. Hybridization can occur under varying circumstances.

A modulatory RNA compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the modulatory RNA compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a modulatory RNA compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which modulatory RNA compounds hybridize to a target sequence are determined by the nature and composition of the modulatory RNA compounds and the assays in which they are being investigated.

Complementarity

“Complementarity,” as used herein, refers to the capacity for precise pairing between two nucleobases on either two oligomeric compound strands or an antisense compound with its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The antisense compound and the further DNA or RNA are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the antisense compound and a target nucleic acid, or between respective sense and antisense strands (or subsequences thereof) of a double-stranded nucleic acid molecule.

Identity

Double-stranded RNA compounds, antisense compounds, or portions thereof, may have a defined percent identity to a target sequence and/or complement thereof. As used herein, a sequence is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in the disclosed sequences would be considered identical as they both pair with adenine. This identity may be over the entire length of the oligomeric compound, or in a portion of a given strand, e.g., in a portion of an antisense compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the 27-mer across residues 1-20 to the comparator 20-mer (or to a defined 20 nucleotide sequence within a target nucleic acid molecule (e.g., SLC6A19 mRNA)). It is understood by those skilled in the art that a modulatory RNA compound (e.g., an siRNA compound or an antisense compound) need not have an identical sequence to those described herein to function similarly to the modulatory RNA compound described herein. Shortened or extended versions - on one or both strands where an siRNA compound - of modulatory RNA compounds taught herein, or nonidentical versions of the modulatory RNA compounds taught herein fall within the scope of this disclosure. Non-identical versions are those wherein each base does not have the same pairing activity as the modulatory RNA compounds disclosed herein. Bases do not have the same pairing activity by being shorter or having at least one abasic site. Alternatively, a non-identical version can include at least one base replaced with a different base with different pairing activity (e.g., G can be replaced by C, A, or T). Percent identity is calculated according to the number of bases that have identical base pairing corresponding to the reference sequence (be it a target sequence, a complementary strand of a dsRNA, an antisense oligonucleotide and/or antisense strand sequence, etc.) to which it is being compared. The non-identical bases may be adjacent to each other, dispersed throughout the oligonucleotide, or both.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer across the entire length of the 20-mer. Alternatively, a 20-mer containing four nucleobases not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase antisense compound comprising the full sequence of the complement of a 20 nucleobase active target segment would have a portion of 100% identity with the complement of the 20 nucleobase active target segment, while further comprising an additional 10 nucleobase portion. The complement of an active target segment may constitute a single portion. In certain embodiments, the oligonucleotides are at least about 80%, optionally at least about 85%, even more preferably at least about 90%, most preferably at least 95% identical to at least a portion of the complement of the active target segments presented herein.

It is well known by those skilled in the art that it is possible to increase or decrease the length of a modulatory RNA compound and/or introduce mismatch bases (with either target sequence, complementary strand(s) of a double- stranded RNA, or both) without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992, incorporated herein by reference), a series of ASOs 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA. ASOs 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the ASOs were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the ASOs that contained no mismatches. Modulatory RNA compounds having a contiguous nucleobase composition that is shorter or longer (independently on either or both strands for double-stranded nucleic acid compounds) or that comprises mismatches are contemplated in the instant disclosure so long as the modulatory RNA activity towards target sequence (here, SLC6A19) is maintained.

Therapeutics

Modulatory nucleic acid agents of the instant disclosure can be used to modulate the expression of SLC6A19 and/or B°AT1 in an animal, such as a human. In one non-limiting embodiment, the methods comprise the step of administering to said animal in need of therapy for a disease or condition associated with SLC6A19 and/or B°AT1 an effective amount of a modulatory RNA compound that inhibits expression of SLC6A19 and/or B°AT1. A disease or condition associated with SLC6A19 and/or B°AT1 includes, but is not limited to, a glomerular disorder, a renal tubular disorder, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or combinations thereof. Phenylketonuria (PKU) and urea cycle disorders are specifically described in additional detail elsewhere herein.

In one embodiment, the modulatory RNA compounds effectively inhibit the levels or function of SLC6A19 mRNA. Because reduction in SLC6A19 mRNA levels can lead to alteration in B°AT1 protein products of expression as well, such resultant alterations can also be measured. Modulatory RNA compounds that effectively inhibit the level or function of SLC6A19 RNA or B°AT1 protein products of expression are considered active inhibitory RNA compounds. In one embodiment, the modulatory RNA compounds of the instant disclosure inhibit the expression of SLC6A19 causing a reduction of RNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of SLC6A19 and/or B°AT1 can be measured in a bodily fluid, tissue or organ of the animal. Methods of obtaining samples for analysis, such as body fluids (e.g., blood, plasma, urine, saliva, etc.), tissues (e.g., biopsy), or organs, and methods of preparation of the samples to allow for analysis are well known to those skilled in the art. Methods for analysis of RNA and protein levels are discussed above and are well known to those skilled in the art. The effects of treatment can be assessed by measuring biomarkers associated with the SLC6A19 and/or B°AT1 expression in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds, by routine clinical methods known in the art.

The modulatory nucleic acid agents of the instant disclosure can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Acceptable carriers and diluents are well known to those skilled in the art. Selection of a diluent or carrier is based on a number of factors, including, but not limited to, the solubility of the compound and the desired route of administration. Such considerations are well understood by those skilled in the art. In one aspect, the compounds inhibit the expression of SLC6A19 and/or B°AT1. The modulatory nucleic acid agents can also be used in the manufacture of a medicament for the treatment of diseases and conditions related to SLC6A19 and/or B° ATI expression.

Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the modulatory RNA compounds or compositions are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds disclosed herein resulting in modulation of SLC6A19 and/or B°AT1 expression in the cells of bodily fluids, organs or tissues.

Thus, provided herein is the use of an isolated single- or double-stranded modulatory nucleic acid agent targeted to SLC6A19 and/or B°AT1 in the manufacture of a medicament for the treatment of a disease or disorder by means of the method described above. In certain embodiments, the modulatory nucleic acid agent is a single-stranded antisense compound. In other embodiments, the modulatory nucleic acid agent is a double-stranded inhibitory RNA compound.

In some embodiments, a modulatory nucleic acid agent of the disclosure, e.g., an oligonucleotide, is characterized in that when a modulatory nucleic acid agent of the disclosure, a composition comprising a modulatory nucleic acid agent of the disclosure, or a conjugate agent comprising a modulatory nucleic acid agent of the disclosure is delivered to a cell, tissue, or organism expressing a target, reduced expression and/or activity of a target is observed as compared to a cell, tissue or organism which has not been delivered a modulatory nucleic acid agent, a composition comprising a modulatory nucleic acid agent, or a conjugate agent comprising a modulatory nucleic acid agent of the disclosure.

In some embodiments, a modulatory nucleic acid agent of the disclosure, e.g., an oligonucleotide, is characterized in that when a modulatory nucleic acid agent of the disclosure, a composition comprising a modulatory nucleic acid agent of the disclosure, or a conjugate agent comprising a modulatory nucleic acid agent of the disclosure is delivered to a cell, tissue, or organism expressing a target, reduced expression and/or activity of a target is observed as compared to a cell, tissue or organism which does not express a target (e.g., which has no detectable expression of a target).

In some embodiments, a modulatory nucleic acid agent of the disclosure, e.g., an oligonucleotide, is characterized in that when a modulatory nucleic acid agent of the disclosure, a composition comprising a modulatory nucleic acid agent of the disclosure, or a conjugate agent comprising a modulatory nucleic acid agent of the disclosure is delivered to a cell, tissue, or organism expressing a target, altered expression and/or activity of a target is observed relative to that observed with an appropriate reference agent known to have a specified impact on the target. In some embodiments, expression and/or activity of a target is altered in a manner and/or to an extent reasonably comparable to, or otherwise determined relative to, that observed with an appropriate reference agent known to have a specified impact on the target. In some embodiments, a reference agent may be a positive control reference agent. In some embodiments, a reference may be a negative control reference agent.

In some embodiments, a modulatory nucleic acid agent of the disclosure, e.g., an oligonucleotide, dsRNA, etc., is characterized in that when delivered to a cell, tissue, or organism expressing a target, expression and/or activity of a target is modulated, e.g., reduced, as compared to a cell, tissue, or organism, which has not been delivered a modulatory nucleic acid agent of the disclosure.

Without wishing to be bound by theory, it is believed that in some embodiments, a targeting moiety, e.g., a peptide as disclosed herein, can be conjugated to a modulatory nucleic acid agent of the disclosure, e.g., an oligonucleotide, dsRNA, etc.

Characterization of Conjugate Agents

In some embodiments, conjugate agent(s) as provided and/or utilized in accordance with the present disclosure are characterized in that, for example, when they are provided to a relevant system (e.g., comprising one or more cell(s), tissue(s), organ(s), or organism(s)) they impact expression and/or activity of one or more targets or form(s) thereof.

In some embodiments, a relevant agent is characterized by its impact on RNA (e.g., mRNA) and/or protein (e.g., encoded by an mRNA) targeted by its nucleic acid payload. In some such embodiments, such impact is assessed in vivo (i.e., in an organism). Alternatively or additionally, in some such embodiments, impact is assessed in vitro (e.g., in cell lines).

In some embodiments, conjugate agent(s) as described and/or utilized in accordance with the present disclosure are characterized relative to an unconjugated nucleic acid agent (as payload). In some embodiments, when assessed under comparable conditions, significantly greater impact is observed when an appropriate in vivo or in vitro system is contacted with a conjugate agent described herein than is observed when the system is contacted with an unconjugated nucleic acid agent under otherwise comparable conditions.

Pharmaceutical Compositions

The present disclosure, among other things, provides pharmaceutical compositions that comprise or otherwise deliver a modulatory nucleic acid agent; typically, such pharmaceutical compositions comprise an active agent (e g., an ASO or iRNA agent or a composition comprising the same) and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

The pharmaceutical compositions containing the iRNA are useful for preventing or treating an SLC6A19-associated disorder, e.g., metabolic disorders, including nephropathy and various other kidney diseases or conditions. Such pharmaceutical compositions are formulated based on the mode of delivery'. One example is compositions that are formulated for systemic administration via parenteral delivery', e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the present disclosure may be administered in dosages sufficient to inhibit expression of a SLC6A19 gene.

In some embodiments, the pharmaceutical compositions of the present disclosure are sterile. In another embodiment, the pharmaceutical compositions of the present disclosure are pyrogen free.

The pharmaceutical compositions of the present disclosure may be administered in dosages sufficient to inhibit expression of a SLC6A19 gene. In general, a suitable dose of an ASO or iRNA agent of the present disclosure will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an ASO or iRNA agent of the present disclosure will be in the range of about 0.1 mg/kg to about 5 0 mg/kg, optionally about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimen may include administration of a therapeutic amount of ASO or iRNA agent on a regular basis, such as every month, once every' 3-6 months, or once a year. In certain embodiments, the ASO or iRNA agent is administered about once per month to about once per six months.

After an initial treatment regimen, the treatments can be administered on a less frequent basis. Duration of treatment can be determined based on the severity of disease.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that doses are administered at not more than 1, 2, 3, or 4 month intervals. In some embodiments of the present disclosure, a single dose of the pharmaceutical compositions of the present disclosure is administered about once per month. In other embodiments of the present disclosure, a single dose of the pharmaceutical compositions of the present disclosure is administered quarterly (i.e., about every three months). In other embodiments of the present disclosure, a single dose of the pharmaceutical compositions of the present disclosure is administered twice per year (i.e., about once every' six months).

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to mutations present in the subject, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a prophy lacti cal ly or therapeutically effective amount, as appropriate, of a composition can include a single treatment or a series of treatments.

The iRNA can be delivered in a manner to target a particular tissue (e.g., kidney cells).

Pharmaceutical compositions of the instant disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, selfemulsifying solids, and self-emulsifying semisolids. Formulations include those that target the kidneys.

The pharmaceutical formulations of the instant disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers. In some embodiments, pharmaceutical compositions described herein may comprise buffers including neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextrans, or mannitol; proteins, polypeptides, or amino acids (e.g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, a pharmaceutical composition is substantially free of contaminants, e.g., there are no detectable levels of a contaminant (e.g., an endotoxin).

In some embodiments, pharmaceutical compositions described herein may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. In some embodiments, quantity and/or frequency of administration may be determined by such factors as condition of a patient, and/or type and/or severity of a patient’s disease, disorder, or condition, although appropriate dosages may be determined by clinical trials.

In some embodiments, a pharmaceutical composition provided by the present disclosure may be in a form such as, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Typically, pharmaceutical compositions that comprise or deliver antibody agents are injectable or infusible solutions; in some such embodiments, such compositions can be formulated for administration intravenously, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, transarterially, sublingually, intranasally, topically or intraperitoneally. In some embodiments, provided pharmaceutical compositions are formulated for intravenous administration. In some embodiments, provided pharmaceutical compositions are formulated for subcutaneous administration.

Pharmaceutical compositions described herein can be formulated for administration by using infusion techniques that are commonly known in the field (See, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988, which is hereby incorporated by reference in its entirety).

In some embodiments, pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously, or following) an additional therapy for a symptom, disease or disorder, e.g., a SOC therapy for a symptom, disease or disorder. In some embodiments, pharmaceutical compositions described herein may be administered before or following surgery. In some embodiments, a dosage of any aforementioned therapy to be administered to a subject will vary with a disease, disorder, or condition being treated and based on a specific subject. Scaling of dosages for human administration can be performed according to art-accepted practices.

Use of Modulatory Nucleic Acid Agents, Including Conjugate Agents

Cell type for delivery

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is delivered to a cell, e.g., of a tissue, optionally in which a cell surface factor is present.

In some embodiments, a cell is or comprises a cell (e.g., of a tissue) chosen from: immune cells (e.g., bone marrow cells, lymph node cells, thymic cells, peripheral blood mononuclear cells [e.g., myeloid and/or lymphoid cells], erythrocytes, eosinophils, neutrophils, and/or platelets); nervous system cells (e.g., brain tissue, cortex, cerebellum, retinal cells, spinal cord cells, nerve cells, neurons, and/or supporting cells; endothelial cells; muscle (e.g., heart muscle, smooth muscle, and/or skeletal muscle); small intestine cells; colon cells; adipocytes; kidney cells; liver cells; lung cells; splenic cells; stomach cells; esophagus cells; bladder cells; pancreas cells; thyroid cells; salivary gland cells; adrenal gland cells; pituitary gland cells; breast cells; skin cells; ovary cells; uterus cells; placenta cells; prostate cells; or testis cells, or any combination thereof.

In some embodiments, a cell is or comprises a cell (e.g., of a tissue) chosen from: renal cells, thyroid cells, parathyroid cells, cells of the inner ear, or nervous system cells.

In some embodiments, a cell is or comprises a kidney cell, e.g., as described herein. In some embodiments, a cell is or comprises a proximal tubular epithelial cell, a podocyte, or both.

In some embodiments, a cell to which a conjugate disclosed herein is delivered expresses both a cell surface factor (e.g., Megalin and/or Cubilin) and a target of a payload moiety.

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is administered to a subject having a disease or disorder, e.g., as disclosed herein. In some embodiments, a disease or disorder comprises a cell in which a surface cell factor (e.g., Megalin and/or Cubilin) and/or a SLC6A19 target RNA is present. Indications

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is used to treat and/or prevent a symptom of, a disease or disorder disclosed herein.

In some embodiments, a disease or disorder to which a modulatory nucleic acid agent disclosed herein is provided, has elevated or aberrant expression of a cell surface factor such as Megalin and/or Cubilin.

In some embodiments, Megalin expression is reported to be enriched in the following tissues and/or cells in particular: renal tissue, thyroid tissue, parathyroid tissue, cells of the inner ear, and nervous system tissue. In some embodiments, Megalin is expressed (e.g., at relatively high level(s)) on surfaces of kidney cells such as proximal tubular epithelial cells and podocytes.

In some embodiments, a disease or disorder is chosen from: a glomerular disorder, a renal tubular disorder, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a glomerular disorder. In some embodiments, a glomerular disorder is chosen from: Lupus nephritis, Goodpasture syndrome, IgA nephropathy, Alport syndrome, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, ApoLl nephropathy, post-infection glomerulonephritis, membranoproliferative glomerulonephritis, mesangioproliferative glomerulonephritis, nephrotic syndrome, nephritic syndrome, Anti-LRP2 nephropathy, C3 glomerulopathy, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a renal tubular disorder. In some embodiments, a renal tubular disorder is chosen from: Fanconi syndrome, cystinuria, Lowe syndrome, Dent syndrome, Light Chain Proximal Tubulopathy, Gitelman syndrome, renal tubular acidosis, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, hereditary aminoaciduria, hereditary salt wasting disorders, hereditary phosphate wasting disorders, porphyria associated renal disease, nephropathic cystinosis, autosomal dominant tubulointerstitial kidney disease, or any combination thereof. In some embodiments, a disease or disorder is or comprises other renal disorders. In some embodiments, other renal disoders are chosen from: ADPKD, ARPKD, Nephronophthisis, Chronic Kidney Disease, nephrolithiasis, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, or any combination thereof.

In some embodiments, a disease or disorder is or comprises an inborn error of metabolism. In some embodiments, an inborn error of metabolism is chosen from: phenylketonuria, urea cycle disorder, maple syrup urine disease, galactosemia, hereditary tyrosinemia, glutamic academia, isovaleric acidemia, very long/long/medium/short chain acyl-CoA dehydrogenase deficiency, methylmalonic academia, primary hyperoxaluria, propionic academia, porphyria, Wilson disease, Pyruvate dehydrogenase deficiency, homocystinuria, hereditary fructose intolerance, nonketotic hyperglycinemia, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a systemic metabolic disorder. In some embodiments, a systemic metabolic disorder is chosen from: diabetes, obesity, hypertension, gout, polyneuropathy, hypoglycemia, vitamin B deficiencies, liver cirrhosis, coronary heart disease, stroke, lipodystrophy, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a disorder of the thyroid. In some embodiments, a disorder of the thyroid is chosen from: Hashimoto disease, Graves' disease, hypothyroidism, hyperthyroidism, goiter, thyroid nodules, thyroiditis, thyroid cancer, thyrotropinoma, thyroid hormone resistance, MCT8 deficiency, Riedel’s thyroiditis, Pendred syndrome, sarcoidosis, McCune- Albright syndrome, familial dysalbuminemic hyperthyroxinemia, thyroxin binding globulin (TBG) deficiency, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a disorder of the parathyroid. In some embodiments, a disorder of the parathyroid is chosen from: hyperparathyroidism/hypercalcemia, hypoparathyroidism/hypocalcemia, nephrolithiasis (kidney stone), pancreatitis, granulomatous disease, Addison’s disease, pernicious anemia (many of these belong to hyperparathyroidism and hypoparathyroidism).

In some embodiments, a disease or disorder is or comprises a disorder of the inner ear. In some embodiments, a disorder of the inner ear is chosen from: inherited sensorineural hearing loss, vestibular neuritis, Meniere’s syndrome, benign paroxysmal positional vertigo, tinnitus, age related hearing loss, bilateral vestibular loss, perilymphatic fistula (PLF), superior semicircular canal dehiscence syndrome (SCD), drug-induced ototoxicity, herpes zoster oticus, purulent labyrinthitis, vestibular schwannoma.

In some embodiments, a disease or disorder is or comprises a neurological disorder, e.g., a neurodegenerative disease. In some embodiments, a neurological disorder is chosen from: Alzheimer's disease, Parkinson's disease, Huntington's disease, A.L.S., multiple sclerosis, neuro- AIDS, brain cancer, stroke, brain injury, spinal cord injury, autism, lysosomal storage disorders, fragile X syndrome, inherited mental retardation, inherited ataxias, blindness, paralysis, stroke, traumatic brain injury and spinal cord injury, and lysosomal storage diseases such as MPS I, MPS II, MPS III A, MPS III B, Metachromatic Leukodystrophy, Gaucher, Krabbe, Pompe, CLN2, Niemann-Pick and Tay-Sachs disease, or any combination thereof.

In some embodiments, a disease or disorder is or comprises a viral infection. In some embodiments, a viral infection comprises a polyoma virus (e g., BK virusj-mediated nephropathy.

In certain embodiments, the instant disclosure provides a therapeutic agent for treatment of phenylketonuria (PKU) or a urea cycle disorder.

Phenylketonuria (PKU)

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH), the enzyme responsible for metabolizing phenylalanine. PKU is an autosomal recessive metabolic disorder in which phenylalanine is not properly metabolized and results in abnormally high levels of plasma phenylalanine. People who have PKU have abnormally high blood levels of phenylalanine, which if untreated can lead to irreversible neurological damage resulting in a spectrum of complications such as intellectual disabilities, seizures, neurodevelopmental and behavioral disorders. PKU is difficult to treat because blood levels of phenylalanine are directly related to diet. Patients must adhere to a life-long and strict diet that impacts all aspects of patients’ lives. Current standard of care are enzyme co-factor and enzyme substitution therapy; but these therapies are not effective in all patients, and also carry potential risk for adverse events.

The enzyme responsible for metabolizing phenylalanine, and thus maintaining phenylalanine homeostasis is phenylalanine hydroxylase (PAH). Loss-of-function (LOF) mutations at PAH gene at chromosome 12q23.2 are known to cause most forms of PKU. These LOF mutations resulting in PKU can be diagnosed as classical PKU (the most severe form), and “mild PKU” or “hyperphe” a less severe form. In addition to PAH, mutations in other enyzmes that affect phenylalanine metabolism, such as dihydropteridine reductase (DHPR), the enzyme responsible for synthesis of co-factors required for PAH activity, may also result in elevated levels of phenylalanine. In addition to diet, blood amino acid levels, including levels of phenylalanine, are regulated by SLC6A19. SLC6A19 is located in the proximal tubule of the kidney and is responsible for reabsorption of amino acids back into the blood. Certain aspects of the instant disclosure therefore provide targeting of SLC6A19 in the kidney of a subject (e.g., in the proximal tubule of the kidney).

Urea Cycle Disorders

Urea cycle disorders are inborn errors of metabolism resulting from defects in one of the enzymes or transporter molecules involved in the hepatic removal of ammonia from the bloodstream. Removal of ammonia from the bloodstream normally occurs via its conversion to urea, which is then excreted by the kidneys. Consequently, urea cycle disorders lead to an accumulation of ammonia. Ammonia is extremely toxic, particularly to the central nervous system. Newborns with severe mutations in any one of the first four enzymes of the urea cycle can become catastrophically ill within 36 to 48 hours of birth despite appearing normal at birth. It is therefore possible for a newborn to be discharged from the hospital before signs of urea cycle disorders develop. The newborn may subsequently develop signs that go unrecognized at home. Hyperammonemia is key to the diagnosis of urea cycle disorders and treatment should not be delayed while a definitive diagnosis is sought.

The etiology of UCDs is complex since multiple proteins and two subcellular compartments are involved, i.e., the mitochondrial matrix and cytoplasm. The first two enzymatic steps in the urea cycle take place in the mitochondrial matrix. In step 1, carbamoylphosphate synthetase I (CPS1) ligates ammonia with bicarbonate forming carbamoyl phosphate which enters the urea cycle. Stressors such as sepsis can easily initiate defects in CPS1, which result in the most severe form of UCDs and hyperammonemia. It should be noted that N-acetylglutamate is an allosteric activator of CPS1. N-acetylglutamate is formed from acetyl-CoA and glutamate in a reaction catalyzed by N-acetylglutamate synthase (NAG), a mitochondrial enzyme. Defects in NAG, although very rare, mimic CPS1 UCDs and result in hyperammonemia.

In step 2, the liver enzyme, ornithine transcarb amylase (OTC) catalyzes the reaction of carbamoyl phosphate with ornithine to form citrulline. The OTC gene (SLC25A15) is located on the X chromosome, and OTC defects are inherited in an X-linked fashion. Defects in OTC are the most common cause of UCDs. OTC deficiency in males results in a severe form of UCD. In females, the severity is variable ranging from asymptomatic to severe depending upon the random nature of X chromosome inactivation. The ornithine needed for OTC must be transported into the mitochondrial matrix, and this task is accomplished by the mitochondrial ornithine translocase (0RNT1) protein. Defective 0RNT1, although very rare, can result in hyperammonemia which often manifests itself later in life.

Step 3 and all remaining enzymatic steps take place in the cytoplasm. In step 3, argininosuccinate synthase 1 (ASS1) catalyzes the formation of argininosuccinate from citrulline and aspartic acid. Defects in ASS1 result in both citrullinemia and hyperammonemia. The second mitochondrial transporter, citrin, contributes to the cytoplasmic pool of aspartic acid by transporting aspartic out of the mitochondria and into the cytoplasm where it is needed for the urea cycle. Citrin also transports glutamate from the cytoplasm into the mitochondria. Gene SLC25A13 codes for citrin and defects in this gene can result in both citrullinemia and hyperammonemia.

Step 4 of the urea cycle involves the cleavage of argininosuccinate to form fumaric acid and arginine, and this reaction is catalyzed by argininosuccinate lyase (ASL). Defects in ASL can cause a severe UCD in neonates as well as an adult-onset form. In contrast to the adult-onset form in which hyperammonemia is episodic, the neonatal form results in persistent hyperammonemia if left untreated. Elevated plasma levels of both citrulline and argininosuccinate are characteristic of ASL deficiency.

In the last step of the urea cycle, the arginine from step 4 is cleaved into urea and ornithine by the arginase enzyme which is coded for by the ARG1 gene. Defects in arginase can result in both argininemia and hyperammonemia. The signs and symptoms of arginase deficiency usually appear by 3 years of age and without a rapid onset of hyperammonemia.

Overall, the incidence of UCDs has recently been determined to be 1 in 35,000 births. Most State Screening Laboratories currently detect only 2 of all the conditions that cause UCDs. About two-thirds of all USDs are due to mutations in OTC, one-fifth due to ASS1, and one-tenth due to ASL.

While OTC deficiency (OTCD) is inherited in an X-linked manner, all other UCDs follow an autosomal recessive pattern. Moreover, there is evidence that even asymptomatic female carriers of OTCD can develop cognitive defects as a result of episodic hyperammonemia. Newborns with UCDs typically appear normal at birth and shortly after that can present with nonspecific signs and symptoms similar to many IEMS or an infection, i.e., lethargy, poor appetite, vomiting, and irritability. IEMs, in general, should be considered in the differential diagnosis of any infant with such signs. Except for arginase deficiency, most infants with severe UCDs rapidly develop cerebral edema, and subsequent neurological problems, resulting from acute hyperammonemia. Unless treated, hyperammonemia from severe UCDs can progress to coma and death. In cases of mild UCDs, hyperammonemia can result from a variety of stressors such as illness and surgery.

Accordingly, several enzyme deficiencies have been noted as contributing to urea cycle disorders. These include:

(a) N-acetyl glutamate synthetase deficiency, which causes neurologic deterioration due to elevated blood ammonia.

(b) Carbamyl phosphate synthetase (CPS) deficiency, which is often a lethal disease with death occurring in the first weeks of life.

(c) Ornithine transcarbamylase deficiency (OTID), which is inherited in a sexdinked dominant manner and is generally fatal in the newborn male.

(d) Argininosuccinic acid synthetase, which typically results in severe neurological impairment leading to mental retardation or death.

(e) Argininosuccinate lyase deficiencies, which result in clinical manifestations of retardation, spasticity, and episodes of convulsions. Plasma ammonia level are greatly elevated.

(f) Arginase deficiency, which results in severe neurologic deterioration over time. Plasma arginine concentrations are greatly elevated.

All of these disorders respond to some degree to restriction of protein intake. Acute episodes are usually precipitated by an increased protein intake, an infection or any incident that leads to a negative nitrogen balance. These acute episodes are best handled by the omission of protein and intravenous fluid therapy. Prolonged treatment of children by limiting protein intake to the minimal requirement together with adequate energy intake and supplements of essential amino acids has resulted in control of the plasma ammonia levels and alleviation of the clinical symptoms. No single panacea is available and nutritional support is specific to the individual disorder. A need therefore exists for targeted molecular treatments that are effective for managem ent/ cure ofUCDs.

Delivery of a Modulatory Nucleic Acid Agent

The delivery' of a modulatory' nucleic acid agent of the present disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject susceptible to or diagnosed with an SLC6A19-associated disorder, e.g., metabolic disorders, including nephropathy and various other kidney diseases or conditions) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an ASO or iRNA agent of the present disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an ASO or iRNA agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery'’ may be performed indirectly by administering one or more vectors that encode and direct the expression of the ASO or iRNA agent.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an ASO or iRNA agent of the present disclosure (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol.2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery', factors to consider in order to deliver an ASO or iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also shown success with local delivery' to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther.12:59-66; Makimura, H., et al (2002) BMC Neurosci.3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528, Thakker, ER., et al (2004) Proc. Natl. Acad. Sei. U.S.A. 101 : 17270-17275; Akaneya,Y., et al (2005) J. Neurophysiol.93: 594-602). Modification of the RNA or the pharmaceutical carrier can also permit targeting of the ASO or iRNzX agent to the target tissue and avoid undesirable off-target effects. ASO or iRNA agent molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an .ASO or iRNA agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemical ly into mice and resulted in knockdown of apoB rn RNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432: 173-178). In an alternative embodiment, the ASO or iRNA agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic deliver}- systems facilitate binding of an ASO or iRNA agent molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an ASO or iRNA agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an ASO or iRNA agent, or induced to form a vesicle or micelle (see e.g , Kim SH, et al (2008) Journal of Controlled Release 129(2): 107-1 16) that encases an ASO or iRNA agent. The formation of vesicles or micelles further prevents degradation of the ASO or iRNA agent when administered systemically. Methods for making and administering cationic- ASO or iRNA agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res.9: 1291 -1300; Arnold, AS et al (2007) J. Hypertens.25: 197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery- systems useful for systemic delivery of ASOs or iRNA agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN, et al (2003), supra), "solid nucleic acid lipid particles" (Zimmermann, TS, et al (2006) Nature 441 : 111-114), cardiolipin (Chien, PY, et al (2005) Cancer Gene Ther.12:321-328; Pal, A, et al (2005) Int J. Oncol.26: 1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA, et al (2007) Biochem. Soc. Trans.35:61-67; Yoo, H , et al (1999) Pharm Res 16: 1799- 1804). In some embodiments, an ASO or iRNA agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of ASOs and iRNA agents and cyclodextrins can be found in U.S. Patent No.7, 427, 605, which is herein incorporated by reference in its entirety.

Vector-Encoded Modulatory Nucleic Acids

In embodiments, modulatory nucleic acid agents targeting the SLC6A19 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillem, A, et al., International PCT Publication No WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U S Patent No.6,054, 299) Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci USA (1995) 92: 1292).

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lenti viral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors, (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, if desired.

Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. Constructs for the recombinant expression of an ASO or iRNA agent will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the ASO or iRNA agent in target cells. Other aspects to consider for vectors and constructs are known in the art.

Dosing regimens

Those skilled in the art will be able to determine, according to known methods, the appropriate amount, dose or dosage of a conjugate agent, to administer to a patient, taking into account factors such as age, weight, general health, the route of administration, the nature of the symptom, disease or disorder requiring treatment, and the presence of other medications. For example, various dosing regimens for antibodies are disclosed in Hendrikx J et al. (2017) Oncologist 22(10): 1212-1221, PMID: 28754722, the entire contents of which is hereby incorporated by reference.

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is administered at a fixed dose, i.e. independent of body weight. In some embodiments, a fixed dose reduces interpatient variability, e.g., efficacy and/or PK/PD parameters. In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is administered based on body weight, e.g., in a mg/kg dosing.

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein - is administered at an initial dose. In some embodiments, an initial dose may be followed by one or more subsequent doses. In some embodiments, one or more subsequent dose may be administered daily, weekly, or monthly, or at other intervals in between. In some embodiments, a dosing regimen disclosed herein may be repeated for one or more times.

Methods For Inhibiting SLC6A 19 Expression

The instant disclosure also provides methods of inhibiting expression of a SLC6A19 gene in a cell. The methods include contacting a cell with an ASO or RNAi agent, e.g., a double-stranded RNA agent, in an amount effective to inhibit, expression of SLC6A19 in the cell, thereby inhibiting expression of SLC6A19 in the cell.

Contacting of a cell with an ASO or iRNA agent, e.g., a double stranded RNA agent, may be done in vitro or in vivo. Contacting a cell in vivo with the ASO or iRNA agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the ASO or iRNA agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as di scussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In certain embodiments, the targeting ligand is a binder of a cell surface factor (e.g., binder of megalin or cubilin), or any other ligand that directs the ASO or i RNA agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,”' “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a SLC6A19” is intended to refer to inhibition of expression of any SLC6A19 gene (such as, e.g., a mouse SLC6A19 gene, a rat SLC6A19 gene, a monkey SLC6.A19 gene, or a human SLC6A19 gene) as well as variants or mutants of a SLC6A19 gene. Thus, the SLC6A19 gene may be a wild-type SLC6A19 gene, a mutant SLC6A19 gene, or a transgenic SLC6A19 gene in the context of a genetically manipulated cell, group of cells, or organism. “Inhibiting expression of a SLC6A19 gene” includes any level of inhibition of a SLC6A19 gene, e.g., at least partial suppression of the expression of a SLC6A19 gene. The expression of the SLC6A19 gene may be assessed based on the level, or the change in the level, of any variable associated with SLC6A19 gene expression, e.g., SLC6A19 mRNA level or B°AT1 protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject. It is understood that SLC6A19/B°AT1 is expressed in the kidney, as well as in various other tissues.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with SLC6A19 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g , a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control)

In some embodiments of the methods of the present disclosure, expression of a SLC6A19 gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below' the level of detection of the assay. In certain embodiments, expression of a SLC6A19 gene is inhibited by at least 70%. It is further understood that inhibition of SLC6A19 expression in certain tissues, e.g., in liver, without a significant inhibition of expression in other tissues, e.g , brain, may be desirable.

In certain embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e g., an AAV-infected mouse expressing the human target gene (i.e., SLC6A19), e.g., when administered as a single dose, e.g., at 3 mg/kg at the nadir of RNA expression. Knockdown of expression of an endogenous gene in a model animal system can also be determined, e.g., after administration of a single dose at, e.g , 3 mg/kg at the nadir of RNA expression. Such systems are useful when the nucleic acid sequence of the human gene and the model animal gene are sufficiently close such that the human iRNA provides effective knockdown of the model animal gene. RNA expression in kidney is determined using art-recognized approaches.

Inhibition of the expression of a SLC6A19 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a SLC6A19 gene is transcribed and which has or have been treated (e g., by contacting the cell or cells with an ASO or iRNA agent of the present disclosure, or by administering an ASO or iRNA agent of the present disclosure to a subject in which the cells are or were present) such that the expression of a SLC6A19 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an ASO or iRNA agent or not treated with an ASO or iRNA agent targeted to SLC6A19).

In certain embodiments, inhibition of the expression of a SLC6A19 gene may be assessed in terms of a reduction of a parameter that is functionally linked to SLC6A19 gene expression, e.g., B°AT1 protein level in kidney cells or in a non-kidney tissue from a subject. SLC6A19 gene silencing may be determined in any cell expressing SLC6A19, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of a B°AT1 protein may be manifested by a reduction in the level of the B°AT1 protein that is expressed by a cell or group of cells or in a subject sample (e.g , the level of protein in a kidney cell or other sample derived from a subject). For the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells, or the change in the level of protein in a subject sample, e.g., kidney cells or other cells derived therefrom .

A control cell, a group of cells, or subject sample that may be used to assess the inhibition of the expression of a SLC6A19 gene includes a cell, group of cells, or subject sample that has not yet been contacted with an ASO or RNAi agent of the present disclosure. For example, the control cell, group of cells, or subject sample may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an ASO or RNAi agent or an appropriately matched population control.

The level of SLC6A19 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of SLC6A19 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the SLC6A19 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phen ol/guani dine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy^IM RNA preparation kits (Qiagen®) or PAXgene^1M (PreAnalytix^1M, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis.

In some embodiments, the level of expression of SLC6A19 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific SLC6A19. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to. Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to SLC6A19 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of SLC6A19 mRNA.

An alternative method for determining the level of expression of SLC6A19 in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No.4, 683, 202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), self sustained sequence replication (Guatelli et al (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), rolling circle replication (Lizardi et al., U.S Patent No.5, 854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low⁷ numbers. In particular aspects of the present disclosure, the level of expression of SLC6A19 is determined by quantitative Anorogenic RT-PCR (i.e., the TaqMan^{1 M} System). In certain embodiments, expression level is determined by the method provided in the Examples using, e.g., a lOnM siRNA concentration, in the species matched cell line.

The expression levels of SLC6AJ 9 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U S. Patent Nos.5, 770, 722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of SLC6A19 expression level may also comprise using nucleic acid probes in solution.

In certain embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein. In certain embodiments, expression level is determined by the method provided in the Examples using a lOnM siRNA concentration in the species matched cell line.

The level of B°AT1 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western bloting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the present disclosure are assessed by a decrease in SLC6A19 mRNA or B°AT1 protein level (e.g., in a kidney biopsy).

In some embodiments of the methods of the present disclosure, the ASO or iRNA agent is administered to a subject such that the ASO or iRNA agent is delivered to a specific site within the subject. The inhibition of expression of SLC6A19 may be assessed using measurements of the level or change in the level of SLC6A19 mRNA or B°AT1 protein in a sample derived from fluid or tissue from the specific site within the subject (e.g., kidney or blood).

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

Prophylactic and Treatment Methods of the Disclosure

The instant disclosure also provides methods of using an ASO or iRNA agent of the present disclosure or a composition containing an ASO or iRNA agent of the present disclosure to inhibit expression of SLC6A19, thereby preventing or treating an SLC6A19-associated disorder, e.g., metabolic disorders, including a glomerular disorder, a renal tubular disorder, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof. Targeting of SLC6A19 in kidney via ASO was identified in a mouse model of phenylketonuria (PKU) to reduce circulating levels of phenylalanine (Phe) in such mice, indicating a role for inhibition of SLC6A19 in kidney as a therapy for PKU and related aminoacidopathies (Belanger et al. JCI Insight 3(14): el21762). In the methods of the present disclosure, the cell may be contacted with the ASO or siRNA in vitro or in vivo, i.e., the cell may be within a subject

.A cell suitable for treatment using the methods of the present disclosure may be any cell that expresses a SLC6A19 gene, e.g., a kidney cell, a urinary bladder cell, a gastrointestinal tract cell (e.g., a duodenum or small intestine cell), or a gall bladder cell, but optionally a kidney cell. A cell suitable for use in the methods of the present disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell, including human cell in a chimeric non-human animal, or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), or a non-primate cell. In certain embodiments, the cell is a human cell, e.g., a human kidney cell. In the methods of the present disclosure, SLC6A19 expression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the level of detection of the assay.

The in vivo methods of the present disclosure may include administering to a subject a composition containing an ASO or iRNA agent, where the ASO or iRNA agent includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the SLC6A19 gene of the mammal to which the ASO or iRNA agent is to be administered. The composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol ), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intramuscular injection.

In one aspect, the instant disclosure also provides methods for inhibiting the expression of a SLC6A19 gene in a mammal. The methods include administering to the mammal a composition comprising an ASO or dsRNA that targets a SLC6A19 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the niRNA transcript of the SLC6A19 gene, thereby inhibiting expression of the SLC6A19 gene in the cell.

Reduction in gene expression can be assessed by any methods known in the art and by methods, e.g., qRT-PCR, described herein, e.g., in the Examples. Reduction in protein production can be assessed by any methods known it the art, e.g , ELISA. In certain embodiments, a kidney biopsy sample serves as the tissue material for monitoring the reduction in the SLC6A19 gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the B°AT1 protein expression.

The instant disclosure further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with an SLC6A19-associated disorder, such as, a glomerular disorder, a renal tubular disorder, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof.

The instant disclosure further provides methods of prophylaxis in a subject in need thereof. The treatment methods of the present disclosure include administering an ASO or iRNA agent of the present disclosure to a subject, e.g , a subject that would benefit from a reduction of SLC6A19 expression, in a prophylactically effective amount of an ASO or iRNA agent targeting a SLC6A19 gene or a pharmaceutical composition comprising an ASO or iRNA agent targeting a SLC6A19 gene.

In one embodiment, an SLC6A19-associated disease is selected from the group consisting of kidney diseases or conditions, including glomerular disorders, renal tubular disorders, other renal disorders, inborn errors of metabolism, systemic metabolic disorders, disorders of the thyroid, disorders of the parathyroid, disorders of the inner ear, neurological disorders, or viral infections. In one embodiment, a SLC6A 19-associated disease is a glomerular disorder, including Lupus nephritis, Goodpasture syndrome, IgA nephropathy, Alport syndrome, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, ApoLl nephropathy, post-infection glomerulonephritis, membranoproliferative glomerulonephritis, mesangioproliferative glomerulonephritis, nephrotic syndrome, nephritic syndrome, Anti-LRP2 nephropathy, C3 glomerulopathy, or any combination thereof.

In one embodiment, a SLC6A19-associated disease is a renal tubular disorder, including Fanconi syndrome, cystinuria, Lowe syndrome, Dent syndrome, Light Chain Proximal Tubulopathy, Gitelman syndrome, renal tubular acidosis, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, hereditary aminoaciduria, hereditary salt wasting disorders, hereditary phosphate wasting disorders, porphyria associated renal disease, nephropathic cystinosis, autosomal dominant tubulointerstitial kidney disease, or any combination thereof.

In another embodiment, a SLC6A 19-associated disease is an other renal disorder, including Autosomal dominant polycystic kidney disease (ADPKD), Autosomal recessive polycystic kidney disease (ARPKD), Nephronophthisis, Chronic Kidney Disease, nephrolithiasis, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, or any combination thereof.

In a further embodiment, a SLC6A19-associated disease is an inborn error of metabolism, including phenylketonuria, urea cycle disorder, maple syrup urine disease, galactosemia, hereditary tyrosinemia, glutamic academia, isovaleric acidemia, very long/long/medium/short chain acyl-CoA dehydrogenase deficiency, methylmalonic academia, primary hyperoxaluria, propionic academia, porphyria, Wilson disease, Pyruvate dehydrogenase deficiency, homocystinuria, hereditary fructose intolerance, nonketotic hyperglycinemia, or any combination thereof.

Any disorder that may be a cause of kidney disease or disorder, including a glomerular disorder, a renal tubular disorder, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof, as well as various other kidney diseases or conditions is encompassed by the term ‘^:SLC6A 19-associated disease”. Non-limiting examples of SLC6A 19-associated diseases include Lupus nephritis, Goodpasture syndrome, IgA nephropathy, Alport syndrome, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, minimal change disease, ApoLl nephropathy, post-infection glomerulonephritis, membranoproliferative glomerulonephritis, mesangioproliferative glomerulonephritis, nephrotic syndrome, nephritic syndrome, Anti-LRP2 nephropathy, C3 glomerulopathy, Fanconi syndrome, cystinuria, Lowe syndrome, Dent syndrome, Light Chain Proximal Tubulopathy, Gitelman syndrome, renal tubular acidosis, nephrogenic diabetes insipidus, Bartter syndrome, Liddle syndrome, hereditary aminoaciduria, hereditary salt wasting disorders, hereditary phosphate wasting disorders, porphyria associated renal disease, nephropathic cystinosis, autosomal dominant tubulointerstitial kidney disease, Autosomal dominant polycystic kidney disease (ADPKD), Autosomal recessive polycystic kidney disease (ARPKD), Nephronophthisis, Chronic Kidney Disease, nephrolithiasis, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, Autosomal dominant polycystic kidney disease (ADPKD), Autosomal recessive polycystic kidney disease (ARPKD), Nephronophthisis, Chronic Kidney Disease, nephrolithiasis, acute kidney injury, Alagille syndrome, cardiorenal syndrome, renal cell carcinoma, renal osteodystrophy, or any combination thereof.

An ASO or iRNA agent of the present disclosure may be administered as a "free ASO" or a ‘‘free iRNA.’¹ A free ASO or free iRNA is administered in the absence of a pharmaceutical composition. The naked ASO or iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the ASO or iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an ASO or iRNA agent of the present disclosure may be administered as a pharmaceutical composition, such as an ASO or dsRNA liposomal formulation.

Subjects that would benefit from an inhibition of SLC6A19 gene expression are subjects susceptible to or diagnosed with a SLC6A19-associated disorder, such as metabolic disorders, including nephropathy and various other kidney diseases or conditions, etc.

In an embodiment, the method includes administering a composition featured herein such that expression of the target SLC6A19 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 1-6, 1- 3, or 3-6 months per dose. In certain embodiments, the composition is administered once every 3- 6 months. Optionally, the ASOs or iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target SLC6A19 gene. Compositions and methods for inhibiting the expression of genes using ASOs or iRNAs can be prepared and performed as described herein.

Administration of the ASO or iRNA agent according to the methods of the present disclosure may result in prevention or treatment of an SLC6A19-associated disorder, e.g., metabolic disorders, including nephropathy and various other kidney diseases or conditions.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The ASO or iRNA agent is optionally administered intravenously or subcutaneously, i.e., by intravenous or subcutaneous injection. One or more injections may be used to deliver the desired dose of ASO or iRNA agent to a subject. The injections may be repeated over a period of time.

The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeatdose regimen may include administration of a therapeutic amount of ASO or iRNA agent on a regular basis, such as once per month to once a year. In certain embodiments, the ASO or iRNA agent is administered about once per month to about once every' three months, or about once every' three months to about once every six months.

The present disclosure provides methods and uses of an ASO or iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of SLC6A19 gene expression, e.g., a subject having a SLC6A19-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

Accordingly, in some aspects of the present disclosure, the methods which include either a single ASO or iRNA agent of the present disclosure, further include administering to the subject one or more additional therapeutic agents.

The ASO or iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

Examples of additional therapeutic agents include those known to treat metabolic disorders, including nephropathy and various other kidney diseases or conditions and other diseases that are caused by, associ ated with or are a consequence of metabolic disorders, including nephropathy and various other kidney diseases or conditions.

The ASO or iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art. or described herein.

Combination Therapies

In some embodiments, a modulatory nucleic acid agent of the instant disclosure - e.g., an ASO, a dsRNA and/or a conjugate agent that includes one or more modulatory nucleic acid agents disclosed herein, or a composition comprising the same - is administered in combination with an additional agent, e.g., additional therapy. In some embodiments, an additional therapy comprises a therapy for a disease or disorder, e.g., a standard of care (SOC) therapy, for a symptom, disease or disorder. In some embodiments, a modulatory nucleic acid agent is administered before, concurrently with or after administration of an additional therapy, e.g., a SOC therapy.

Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of an ASO or iRNA agent compound, e.g., an ASO, a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an ASO or dsRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof)

Such kits include one or more dsRNA agent(s) and instructions for use, e.g., instructions for administering a prophy lacti cal ly or therapeutically effective amount of an ASO or dsRNA agent(s). The ASO or dsRNA agent may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the ASO or dsRNA agent, (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of SLC6A19 (e.g , means for measuring the inhibition of SLC6A19 inRNA, SLC6A19 protein, and/or SLC6A19 activity) Such means for measuring the inhibition of SLC6A19 may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the present disclosure may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.

In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container, e.g., a vial or a pre-filled syringe. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an ASO or siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition The kit can also include a delivery⁷ device.

INCORPORATION BY REFERENCE

All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

This present disclosure is further illustrated by the following examples which should not be construed as limiting the scope or content of the disclosure in any way. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal Sequence Listing and Figures, are hereby incorporated herein by reference. EXAMPLES

Example 1: Materials and Methods

Screening of Unconjugated Modulatory Nucleic Acid Agents (e.g., ASOs, siRNAs, PMOs, exonskipping oligonucleotides)

HEP3B217 cells (ATCC, cat # HB-8064) were grown to near confluence at 37°C in an atmosphere of 5% CO?, in Eagle’s Minimum Essential Medium (Gibco® ) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Cells were counted and reseeded in a 96-well plate. Transfection was carried out by adding 0.2 pL of Lipofectamine¹*¹ RNAiMax (Invitrogeifo, Carlsbad, CA. cat # 13778-150) plus 1 nM and 10 nM of modulatory nucleic acid agent (e g., siRNA, as shown in FIG. 1, or ASC) or other agent(s)) in Opti-MEM. The mixture was incubated at room temperature for 20 minutes. Eighty pl of complete growth media without antibiotic containing ~2 xlO⁴ HEP3B217 cells was then added to 20 uL of modulatory nucleic acid agent mixture. Cells were placed in the incubator at 37°C and 5% CO?, and allowed to incubate for 48 hours. Primary screening was performed at 1 nM and 10 nM (FIG. 1), and doseresponse studies w⁷ere carried out at 9-point half-log dilution between 0.03 nM and 100 nM. A known human SLC6A19-targeting siRNA (Horizon: L-007352-01-0050) was used as a positive control of transfection

In alternative embodiments, for screening of PMOs, 6 pM Endo-Porter (GeneTools), a reagent for delivering Morpholino oligonucleotides into the cytosol of cultured cells, is used as a transfection agent, while other methods described above remain the same.

RT-PCR Readout

To prepare samples for RT-qPCR, the cells were washed with PBS and lysed using the Cells-to-CT™ 1-Step TaqMan™ Kit (Thermo Fi sher Scientific™). 50 pL of lysis buffer / DNase I reagent mixture was added into each well, mixed thoroughly and incubated for 5-10 minutes at room temperature. 5 uL of Stop Solution was added to each well and incubated for 5 minutes. The cell lysates were used for RT-qPCR analysis, to evaluate mRNA expression. RT-qPCR was performed on a Quant Studio 7 Pro Real-Time PCR System using a Cells-to-Cells-to-CT™ 1- Step TaqMan™ kit and TaqMan™ probes (Thermo Fisher Scientific™). Cell lysates from treatment and control wells were probed with Human SLC6A19-FAM (Thermo: HS01384157 M1, 4448489) and Human HMBS-VIC (Thermo: MM01143545 MI, 4448489). HMBS was designated as the house keeping gene for normalization The fold change of target gene relative to Non-targeting control wells (Horizon: D-001810-01-05) was calculated as 2^A(- AACt). Relative expression was compiled and analyzed in GraphPad Prism.

Protein Readout

To prepare samples for western blotting, cells are washed with ice-cold PBS and 200 pL chilled complete RIP A lysis buffer (Thermo Scientific®, Cat. #89901) plus Hall Protease/Phosphatase Inhibitor Cocktail (Thermo Scientific®, Cat. #78440) is added to lyse the cells. Samples are mechanically sheared by passing the sample 5-6 times through a 21 -gauge needle. The lysates are incubated for 20 min in a cold room with gentle shaking and centrifuged at 14,000 g for 10 min at 4 °C. The supernatants are transferred to a pre-chilled microcentrifuge tube. Total protein is quantified using Pierce^1M BCA Protein Assay Kit (Thermo Scientific®, Cat #23221) according to manufacturer’s instructions. Samples are prepared by adding NuPAGE^1M LDS sample buffer (Thermo Scientific®, Cat #NP0007) and NuPAGE™ sample Reducing Agent (Thermo Scientific®, Cat. &NP0004) to the cell lysates. The mixture is heated at 95 °C for 10 min to denature proteins, spun down briefly and samples are used for western blotting or stored at -80 °C. Twenty pL of samples are loaded on NuPAGE™ 4 to 12%, Bis-Tris 1.0-1.5 mm, Mini Protein Gels along with 3 pL of Duo Pre-stained Protein Ladder (LI-COR Cat. #988-15653) and run at constant 200V for 45 min The gels are transferred to membrane using Invitrogen™ iBlot™ 2 Dry Blotting System. The membrane is blocked with Intercept®' Blocking Buffer (LI-COR™ Cat. #927-60001 ) for 1 hour at RT or 4 °C for overnight with gentle shaking, followed by incubation with anti-SLC6A19 primary antibody and appropriate secondary’ antibody. The antibody solutions are washed off' with TBST, and membranes are read on a LI-COR™ Odyssey® imaging system. Screening of Conjugated Modulatory Nucleic Acid Agents (e.g., ASOs, siRNAs, PMOs, exonskipping oligonucleotides)

For screening of conjugated modulatory nucleic acid agents, similar methods are followed as described above. However, the conjugated molecules are not subjected to transfection, but are instead administered to allow' free uptake. For free uptake cell culture experiments, cells are seeded in serum-free media and treated with conjugated modulatory nucleic acid agent without any transfection reagent for up to 4 hours. The media is then aspirated, cells are washed with PBS, then incubated in complete media for a further 24 -72 hours. The cells are then harvested for either RNA or protein detection according to the methods mentioned above. Example 2: Design and Screening of SLC6A19-Targeting Antisense Oligonucleotides and siRNAs

Human SLC6A19-targeting ASOs (including exon-skipping ASOs) and siRNAs were designed by art-recognized processes and are shown in Tables 3-15. Initial assessment of human SLC6A19-targeting siRNAs was performed using the methods of the above Example, and multiple human SLC6A19-targeting siRNAs that exhibited significant knockdown of SLC6A19 expression were thereby identified (FIG. 1). Dose-responsive behavior was also observed for many such siRNAs, when comparing between 10 nM and 1 nM administrations of respective siRNAs in HEP3B cell culture.

ASOs tested in screening experiments are shown in Table 5, while siRNAs tested in such screening assays are shown in Tables 10 and 11, and exon-skipping antisense oligonucleotides are presented in Tables 12 and 13. Highly effective inhibitory ASOs (including exon-skipping .ASOs) and additional siRNAs targeting SLC6A19 for knockdown are thereby identified.

Table 3. SLC6A19~Targeting Antisense Oligonucleotides

|

777 ~|

Table 4. Selected SLC6A19-Targeting Antisense Oligonuekotides

Table 5. Further Selected SLC6A 19- Targeting Antisense Oligonucleotides

Table 6. Screening Set Selection of SLC6A19-Targeting Antisense OHgonncleotides

Table 7. Selected SLC6A19-Targeting siRNAs

Table 8. Further Selected SLC6A19-Targeting siRNAs

Table 9. Additionally Selected SLC6A19-Targeting siRNAs

Tabk 10. Sub-sekrtion of SLC6A19~Targeting siRNAs

Table 11. Alternative Selection of SLC6A19"Targeting siRNAs

Table 12. SLC6A19 Exon-Skipping Antisense Oligonucleotides

m = 2'-methoxyethyl nucleotide

Table 13. Selected SLC6A19 Exon-Skipping Antisense Oligonucleotides

m = 2'-methoxyethyl nucleotide

Example 3: Screening of dsRNA Duplexes using Hep3B cells

Hep3B cells (ATCC Cat#: HB-8064) were cultured rn vitro Cells were seeded in 96-well plates using MEM (Gibco Cat#: 10370-021) supplemented with GlutaMAX and 10% FBS. Following plating, cells were treated with siRNA/transfection reagent (RNAiMAX) complexes (Table 15), at final concentrations of 1 nM or 10 nM, or with a control treatment. Cells were harvested 72 hours post-treatment for RNA isolation and cDNA synthesis, utilizing the TaqMan™ Fast Advanced Cells-to-CT™ Kit (ThermoFisher Cat#: A35378). Quantitative PCR (QPCR) analysis w-as performed using a Taqman probe at a concentration of 60X to measure SLC6A19 (ThermoFisher Cat#: Hs01384157_ml) expression. The expression levels of SLC6A19 wese normalized to ACTB (ThermoFisher Cat#: Hs01060665 m l) levels for analysis (Table 16).

Table 14. SLC6A19-Targeting siRNAs

Table 15. SLC6A19-Targeting siRNAs

modifications; and f and upper case letters denote 2' fluoro modifications

Table 16. SLC6A19-Targeting siRNAs Screening Results

Example 4: Screening of dsRNA Duplexes using Li-7 ceils

Li-7 cells (Riken, RCB1941) were cultured at 37°C in 5% CO2 using RPMI 1640 medium (Gibco, Cat #: C22400500CP) supplemented with 10% FBS (ExCell-Bio-FSP500) and 1% Penicillin-Streptomycin (Gibco, Cat #: 15140122). After plating, transfection was performed by adding RNAiMax (Invitrogen, Carlsbad, CA, Cat # 13778-150) along with 1 nM and 10 nM siRNA duplexes (Table 17). Cells were harvested 24 hours post-treatment for RNA analysis. Total RNA was extracted using the EZ-Press 96 RNA Purification Kit (EZBioscience, Cat #: EZ4001- L), and cDNA was generated with HiScript III RT SuperMix (Vazyme, Cat #: R323-01). Quantitative PCR (QPCR) analysis was conducted using a custom Taqman probe for SLC6A19 (Sangon Biotech, Shanghai, China), normalized to ACTB (Sangon Biotech, Shanghai, China) for evaluation (Table 19).

Table 17. SLC6A19-Targeting siRNAs

Table 18. SLC6A19-Targeting siRNAs

Asterisks (*) denote phosphorothioate linkages; lower case letters denote 2'-oMe modifications; and f and upper case letters denote 2' fluoro modifications

Table 19. SLC6A19-Targetmg siRNAs Screening Results

Example 5; In vivo screening of dsRNA Duplexes in Mice

Duplexes of interest, identified from the above in vitro studies, are evaluated in vivo.

At day 0, groups of three mice expressing human SLC6A19 mRNA are administered a single 3 mg/kg dose of the agents of interest (including conjugate agents as described herein) or PBS control via intravenous injection. At day 7 or day 14 post-dose, animals are sacrificed, kidney samples are collected and snap-frozen in liquid nitrogen. Kidney mRNA is extracted and analyzed by the RT-QPCR method.

Human SLC6A19 mRNA levels are compared to a housekeeping genes, like GAPDH, PPIB. The values are then normalized to the average of PBS vehicle control group. The data are expressed as percent of baseline value, and are presented as mean plus standard deviation. Results are expected to demonstrate that the exemplary duplex agents tested effectively reduce the level of the human SLC6A19 messenger RNA in vivo in targeted kidney cells.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

We claim:

1. A modulatory' nucleic acid agent comprising a first strand of 12 to 60 linked nucleosides in length targeted to a human solute carrier family 6 member 19 (SLC6A19) RNA, wherein the human SLC6A19 RNA is the human SLC6A19 RNA of SEQ ID NO: 1.

2. The modulatory nucleic acid agent of claim 1, wherein the modulatory nucleic acid agent comprises an antisense oligonucleotide (ASO).

3. The ASO of claim 2, having at least about 80% complementarity to a target region of the human SLC6A19 RNA.

4. The antisense compound of claim 2, wherein the ASO hybridizes with 12 or more nucleotides of nucleotides 3 to 25, nucleotides 8 to 30, nucleotides 13 to 35, nucleotides 38 to 60, nucleotides 43 to 65, nucleotides 48 to 70, nucleotides 93 to 115, nucleotides 98 to 120, nucleotides

103 to 125, nucleotides 108 to 130, nucleotides 111 to 133, nucleotides 113 to 135, nucleotides 118 to 140, nucleotides 214 to 236, nucleotides 219 to 241, nucleotides 244 to 266, nucleotides 249 to 271, nucleotides 252 to 274, nucleotides 252 to 274, nucleotides 253 to 275, nucleotides 254 to 276, nucleotides 257 to 286, nucleotides 259 to 281, nucleotides 264 to 286, nucleotides 289 to 311, nucleotides 294 to 316, nucleotides 339 to 361, nucleotides 397 to 419, nucleotides 460 to 480, nucleotides 461 to 481, nucleotides 481 to 503, nucleotides 486 to 508, nucleotides 491 to 513, nucleotides 557 to 579, nucleotides 561 to 583, nucleotides 562 to 584, nucleotides 564 to 586, nucleotides 566 to 586, nucleotides 567 to 589, nucleotides 616 to 638, nucleotides 621 to 643, nucleotides 626 to 648, nucleotides 670 to 692, nucleotides 671 to 691, nucleotides 672 to 694, nucleotides 674 to 694, nucleotides 675 to 697, nucleotides 680 to 702, nucleotides 685 to 707, nucleotides 690 to 712, nucleotides 695 to 717, nucleotides 700 to 722, nucleotides 705 to 727, nucleotides 710 to 732, nucleotides 715 to 737, nucleotides 720 to 742, nucleotides 725 to 747, nucleotides 730 to 752, nucleotides 735 to 757, nucleotides 740 to 762, nucleotides 745 to 767, nucleotides 750 to 772, nucleotides 751 to 776, nucleotides 755 to 777, nucleotides 760 to 782, nucleotides 765 to 787, nucleotides 770 to 792, nucleotides 775 to 797, nucleotides 798 to 823, nucleotides 800 to 822, nucleotides 825 to 847, nucleotides 890 to 912, nucleotides 895 to 917, nucleotides 900 to 922, nucleotides 905 to 927, nucleotides 910 to 932, nucleotides 911 to 937, nucleotides 915 to 937, nucleotides 972 to 994, nucleotides 973 to 995, nucleotides 977 to 1000, nucleotides 977 to 999, nucleotides 978 to 1000, nucleotides 983 to 1005, nucleotides 1035 to 1057, nucleotides 1058 to 1080, nucleotides 1059 to 1081, nucleotides 1060 to 1082, nucleotides 1063 to 1082, nucleotides 1065 to 1087, nucleotides 1068 to 1087, nucleotides 1070 to 1094, nucleotides 1070 to 1092, nucleotides 1074 to 1096, nucleotides 1075 to 1097, nucleotides 1077 to 1096, nucleotides 1080 to 1102, nucleotides 1085 to 1107, nucleotides 1086 to 1106, nucleotides 1090 to 1112, nucleotides 1095 to 1117, nucleotides 1100 to 1122, nucleotides 1105 to 1127, nucleotides 1110 to 1132, nucleotides 1115 to 1137, nucleotides 1115 to 1137, nucleotides 1117 to 1139, nucleotides 1177 to 1199, nucleotides 1182 to 1204, nucleotides 1187 to 1209, nucleotides 1191 to 1214, nucleotides 1192 to 1214, nucleotides 1195 to 1217, nucleotides 1234 to 1256, nucleotides 1239 to 1261, nucleotides 1242 to 1264, nucleotides 1244 to 1266, nucleotides 1247 to 1268, nucleotides 1249 to 1271, nucleotides 1251 to 1270, nucleotides 1254 to 1276, nucleotides 1259 to 1281, nucleotides 1263 to 1282, nucleotides 1264 to 1286, nucleotides 1266 to 1285, nucleotides 1269 to 1291, nucleotides 1326 to 1348, nucleotides 1331 to 1353, nucleotides 1334 to 1361, nucleotides 1334 to 1356, nucleotides 1334 to 1356, nucleotides 1335 to 1357, nucleotides 1336 to 1358, nucleotides 1337 to 1359, nucleotides 1337 to 1359, nucleotides 1338 to 1360, nucleotides 1340 to 1362, nucleotides 1341 to 1363, nucleotides 1346 to 1368, nucleotides 1351 to 1373, nucleotides 1353 to 1372, nucleotides 1377 to 1399, nucleotides 1382 to 1404, nucleotides 1383 to 1403, nucleotides 1387 to 1409, nucleotides 1392 to 1414, nucleotides 1397 to 1419, nucleotides 1402 to 1424, nucleotides 1407 to 1429, nucleotides 1412 to 1434, nucleotides 1417 to 1439, nucleotides 1456 to 1478, nucleotides 1458 to 1477, nucleotides 1481 to 1503, nucleotides 1486 to 1508, nucleotides 1491 to 1513, nucleotides 1496 to 1518, nucleotides 1521 to 1543, nucleotides 1568 to 1590, nucleotides 1569 to 1591, nucleotides 1574 to 1596, nucleotides 1578 to 1621, nucleotides 1579 to 1601, nucleotides 1583 to 1605, nucleotides 1583 to 1605, nucleotides 1584 to 1606, nucleotides 1584 to 1606, nucleotides 1585 to 1607, nucleotides 1585 to 1607, nucleotides 1587 to 1609, nucleotides 1588 to 1610, nucleotides 1589 to 1611, nucleotides 1594 to 1616, nucleotides 1595 to 1617, nucleotides 1599 to 1621, nucleotides 1601 to 1620, nucleotides 1602 to 1624, nucleotides 1642 to 1664, nucleotides 1749 to 1775, nucleotides 1750 to 1772, nucleotides 1755 to 1777, nucleotides 1757 to 1779, nucleotides 1758 to 1780, nucleotides 1760 to 1782, nucleotides 1761 to 1792, nucleotides 1764 to 1783, nucleotides 1765 to 1787, nucleotides 1770 to 1792, nucleotides 1809 to 1831, nucleotides 1810 to 1829, nucleotides 1812 to 1832, nucleotides 1814 to 1836, nucleotides 1852 to 1881, nucleotides 1853 to 1875, nucleotides 1854 to 1876, nucleotides 1858 to 1880, nucleotides 2168 to 2188, nucleotides 2333 to 2353, nucleotides 2454 to 2474, nucleotides 2507 to 2527, nucleotides 2593 to 2613, nucleotides 3244 to 3264, nucleotides 3288 to 3308, nucleotides 3304 to 3324, nucleotides 3332 to 3352, nucleotides 3333 to 3353, nucleotides 3501 to 3523, nucleotides 3595 to 3615, nucleotides 3595 to 3617, nucleotides 3643 to 3671, nucleotides 3646 to 3668, nucleotides 3646 to 3668, nucleotides 3647 to 3669, nucleotides 3648 to 3668, nucleotides 3648 to 3670, nucleotides 3648 to 3670, nucleotides 3649 to 3671, nucleotides 3651 to 3670, nucleotides 3651 to 3673, nucleotides 3651 to 3673, nucleotides 3652 to 3671, nucleotides 3712 to 3733, nucleotides 3713 to 3735, nucleotides 3763 to 3786, nucleotides 3763 to 3785, nucleotides 3764 to 3786, nucleotides 3791 to 3811, nucleotides 3796 to 3816, nucleotides 3801 to 3829, nucleotides 3801 to 3820, nucleotides 3802 to 3821, nucleotides 3804 to 3826, nucleotides 3809 to 3831, nucleotides 3810 to 3832, nucleotides 3812 to 3833, nucleotides 3812 to 3834, nucleotides 3813 to 3835, nucleotides 3814 to 3833, nucleotides 3814 to 3836, nucleotides 3814 to 3836, nucleotides 3839 to 3859, nucleotides 3840 to 3862, nucleotides 3902 to 3924, nucleotides 3965 to 3987, nucleotides 3995 to 4015, nucleotides 3998 to 4020, nucleotides 4100 to 4124, nucleotides 4102 to 4124, nucleotides 4141 to 4163, nucleotides 4141 to 4163, nucleotides 4142 to 4164, nucleotides 4143 to 4165, nucleotides 4144 to 4166, nucleotides 4146 to 4168, nucleotides 4150 to 4172, nucleotides 4151 to 4173, nucleotides 4192 to 4214, nucleotides 4197 to 4219, nucleotides 4201 to 4223, nucleotides 4265 to 4287, nucleotides 4270 to 4292, nucleotides 4318 to 4338, nucleotides 4319 to 4341, nucleotides 4329 to 4349, nucleotides 4348 to 4386, nucleotides 4349 to 4371, nucleotides 4350 to 4369, nucleotides 4350 to 4372, nucleotides 4350 to 4372, nucleotides 4351 to 4370, nucleotides 4352 to 4371, nucleotides 4352 to 4374, nucleotides 4353 to 4372, nucleotides 4353 to 4375, nucleotides 4354 to 4376, nucleotides 4359 to 4381, nucleotides 4361 to 4383, nucleotides 4362 to 4384, nucleotides 4362 to4384, nucleotides 4364 to 4386, nucleotides 4365 to 4387, nucleotides 4395 to 4415, nucleotides 4396 to 4416, nucleotides 4409 to 4431, nucleotides 4410 to 4432, nucleotides 4412 to 4434, nucleotides 4637 to 4657, nucleotides 4638 to 4658, nucleotides 4640 to 4660, nucleotides 5103 to 5123, nucleotides 5124 to 5144, nucleotides 5126 to 5146, or nucleotides 5127 to 5147 of SEQ ID NO: 1.

5. The ASO of any one of the preceding claims, wherein the compound includes a second strand of 15 to 60 nucleobases in length and complementary to said first strand

6. The ASO of any one of claims 2-4-, wherein the compound is an antisense oligonucleotide.

7. The ASO of claim 6 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.

8. The ASO of claim 7, wherein the modified intemucleoside linkage comprises a phosphorothi oate linkage.

9. The ASO of claim 8, wherein the at least one modified internucleoside linkage, sugar moiety, or nucleobase comprises a modification selected from the group consisting of a deoxynucleoside, a 3 ’-terminal deoxy-thymine (d'T) nucleoside, a 2'-O-methyl (2'-0Me) modified nucleoside, a 2'-fluoro (2'-F) modified nucleoside, a 2'-deoxy-modified nucleoside, a locked nucleotide (LNA), an unlocked nucleoside, a conformationally restricted nucleoside, a constrained ethyl nucleoside, an abasic nucleoside, a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), a 2’- amino-modified nucleoside, a2’-O-allyl-modified nucleoside, 2’-C-alkyl-modified nucleoside, 2’- hydroxly-modified nucleoside, a 2’-methoxyethyl (2 -MOE) modified nucleoside, a 2’-O- alkylmodified nucleoside, a morpholino nucleoside, a phosphoramidate, a non-natural base comprising nucleoside, a tetrahydropyran modified nucleoside, a 1,5-anhydrohexitol modified nucleoside, a cyclohexenyl modified nucleoside, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5’-methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleoside comprising adenosine-glycol nucleic acid (GNA), a nucleoside comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymetbyl-tetrahydrofurane-5- phosphate, a nucleotide comprising 2’deoxythymidine-3 ’phosphate, a nucleotide comprising 2 deoxyguanosine-3 ’-phosphate, and a terminal nucleoside linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

10. The ASO of claim 7 wherein the modified nucleobase comprises 5-methylcytosine.

11. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of human solute carrier family 6 member 19 (SLC6A19), wherein said dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to a SLC6A19 RNA, and wherein the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences of Tables 7-11, 14 or 17.

12. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of human solute carrier family 6 member 19 (SLC6A19) in a cell, wherein said dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from SEQ ID NO: 1.

13. The dsRNA agent of claim I I or 12, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 3 to 25, nucleotides 8 to 30, nucleotides 13 to 35, nucleotides 38 to 60, nucleotides 43 to 65, nucleotides 48 to 70, nucleotides 93 to 115, nucleotides 98 to 120, nucleotides 103 to 125, nucleotides 108 to 130, nucleotides 111 to 133, nucleotides 113 to 135, nucleotides

118 to 140, nucleotides 214 to 236, nucleotides 219 to 241, nucleotides 244 to 266, nucleotides

249 to 271, nucleotides 252 to 274, nucleotides 252 to 274, nucleotides 253 to 275, nucleotides

254 to 276, nucleotides 257 to 286, nucleotides 259 to 281, nucleotides 264 to 286, nucleotides

289 to 311, nucleotides 294 to 316, nucleotides 339 to 361, nucleotides 397 to 419, nucleotides

460 to 480, nucleotides 461 to 481, nucleotides 481 to 503, nucleotides 486 to 508, nucleotides

491 to 513, nucleotides 557 to 579, nucleotides 561 to 583, nucleotides 562 to 584, nucleotides

564 to 586, nucleotides 566 to 586, nucleotides 567 to 589, nucleotides 616 to 638, nucleotides

621 to 643, nucleotides 626 to 648, nucleotides 670 to 692, nucleotides 671 to 691, nucleotides

672 to 694, nucleotides 674 to 694, nucleotides 675 to 697, nucleotides 680 to 702, nucleotides

685 to 707, nucleotides 690 to 712, nucleotides 695 to 717, nucleotides 700 to 722, nucleotides

705 to 727, nucleotides 710 to 732, nucleotides 715 to 737, nucleotides 720 to 742, nucleotides

725 to 747, nucleotides 730 to 752, nucleotides 735 to 757, nucleotides 740 to 762, nucleotides

745 to 767, nucleotides 750 to 772, nucleotides 751 to 776, nucleotides 755 to 777, nucleotides

760 to 782, nucleotides 765 to 787, nucleotides 770 to 792, nucleotides 775 to 797, nucleotides

798 to 823, nucleotides 800 to 822, nucleotides 825 to 847, nucleotides 890 to 912, nucleotides

895 to 917, nucleotides 900 to 922, nucleotides 905 to 927, nucleotides 910 to 932, nucleotides

911 to 937, nucleotides 915 to 937, nucleotides 972 to 994, nucleotides 973 to 995, nucleotides

977 to 1000, nucleotides 977 to 999, nucleotides 978 to 1000, nucleotides 983 to 1005, nucleotides 1035 to 1057, nucleotides 1058 to 1080, nucleotides 1059 to 1081, nucleotides 1060 to 1082, nucleotides 1063 to 1082, nucleotides 1065 to 1087, nucleotides 1068 to 1087, nucleotides 1070 to 1094, nucleotides 1070 to 1092, nucleotides 1074 to 1096, nucleotides 1075 to 1097, nucleotides 1077 to 1096, nucleotides 1080 to 1102, nucleotides 1085 to 1107, nucleotides 1086 to 1106, nucleotides 1090 to 1112, nucleotides 1095 to 1117, nucleotides 1100 to 1122, nucleotides 1105 to 1127, nucleotides 1110 to 1132, nucleotides 1115 to 1137, nucleotides 1115 to 1137, nucleotides 1117 to 1139, nucleotides 1177 to 1199, nucleotides 1182 to 1204, nucleotides 1187 to 1209, nucleotides 1191 to 1214, nucleotides 1192 to 1214, nucleotides 1195 to 1217, nucleotides 1234 to 1256, nucleotides 1239 to 1261, nucleotides 1242 to 1264, nucleotides 1244 to 1266, nucleotides 1247 to 1268, nucleotides 1249 to 1271, nucleotides 1251 to 1270, nucleotides 1254 to 1276, nucleotides 1259 to 1281, nucleotides 1263 to 1282, nucleotides 1264 to 1286, nucleotides 1266 to 1285, nucleotides 1269 to 1291, nucleotides 1326 to 1348, nucleotides 1331 to 1353, nucleotides 1334 to 1361, nucleotides 1334 to 1356, nucleotides 1334 to 1356, nucleotides 1335 to 1357, nucleotides 1336 to 1358, nucleotides 1337 to 1359, nucleotides 1337 to 1359, nucleotides 1338 to 1360, nucleotides 1340 to 1362, nucleotides 1341 to 1363, nucleotides 1346 to 1368, nucleotides 1351 to 1373, nucleotides 1353 to 1372, nucleotides 1377 to 1399, nucleotides 1382 to 1404, nucleotides 1383 to 1403, nucleotides 1387 to 1409, nucleotides 1392 to 1414, nucleotides 1397 to 1419, nucleotides 1402 to 1424, nucleotides 1407 to 1429, nucleotides 1412 to 1434, nucleotides 1417 to 1439, nucleotides 1456 to 1478, nucleotides 1458 to 1477, nucleotides 1481 to 1503, nucleotides 1486 to 1508, nucleotides 1491 to 1513, nucleotides 1496 to 1518, nucleotides 1521 to 1543, nucleotides 1568 to 1590, nucleotides 1569 to 1591, nucleotides 1574 to 1596, nucleotides 1578 to 1621, nucleotides 1579 to 1601, nucleotides 1583 to 1605, nucleotides 1583 to 1605, nucleotides 1584 to 1606, nucleotides 1584 to 1606, nucleotides 1585 to 1607, nucleotides 1585 to 1607, nucleotides 1587 to 1609, nucleotides 1588 to 1610, nucleotides 1589 to 1611, nucleotides 1594 to 1616, nucleotides 1595 to 1617, nucleotides 1599 to 1621, nucleotides 1601 to 1620, nucleotides 1602 to 1624, nucleotides 1642 to 1664, nucleotides 1749 to 1775, nucleotides 1750 to 1772, nucleotides 1755 to 1777, nucleotides 1757 to 1779, nucleotides 1758 to 1780, nucleotides 1760 to 1782, nucleotides 1761 to 1792, nucleotides 1764 to 1783, nucleotides 1765 to 1787, nucleotides 1770 to 1792, nucleotides 1809 to 1831, nucleotides 1810 to 1829, nucleotides 1812 to 1832, nucleotides 1814 to 1836, nucleotides 1852 to 1881, nucleotides 1853 to 1875, nucleotides 1854 to 1876, nucleotides 1858 to 1880, nucleotides 2168 to 2188, nucleotides 2333 to 2353, nucleotides 2454 to 2474, nucleotides 2507 to 2527, nucleotides 2593 to 2613, nucleotides 3244 to 3264, nucleotides 3288 to 3308, nucleotides 3304 to 3324, nucleotides 3332 to 3352, nucleotides 3333 to 3353, nucleotides 3501 to 3523, nucleotides 3595 to 3615, nucleotides 3595 to 3617, nucleotides 3643 to 3671, nucleotides 3646 to 3668, nucleotides 3646 to 3668, nucleotides 3647 to 3669, nucleotides 3648 to 3668, nucleotides 3648 to 3670, nucleotides 3648 to 3670, nucleotides 3649 to 3671, nucleotides 3651 to 3670, nucleotides 3651 to 3673, nucleotides 3651 to 3673, nucleotides 3652 to 3671, nucleotides 3712 to 3733, nucleotides 3713 to 3735, nucleotides 3763 to 3786, nucleotides 3763 to 3785, nucleotides 3764 to 3786, nucleotides 3791 to 3811, nucleotides 3796 to 3816, nucleotides 3801 to 3829, nucleotides 3801 to 3820, nucleotides 3802 to 3821, nucleotides 3804 to 3826, nucleotides 3809 to 3831, nucleotides 3810 to 3832, nucleotides 3812 to 3833, nucleotides 3812 to 3834, nucleotides 3813 to 3835, nucleotides 3814 to 3833, nucleotides 3814 to 3836, nucleotides 3814 to 3836, nucleotides 3839 to 3859, nucleotides 3840 to 3862, nucleotides 3902 to 3924, nucleotides 3965 to 3987, nucleotides 3995 to 4015, nucleotides 3998 to 4020, nucleotides 4100 to 4124, nucleotides 4102 to 4124, nucleotides 4141 to 4163, nucleotides 4141 to 4163, nucleotides 4142 to 4164, nucleotides 4143 to 4165, nucleotides 4144 to 4166, nucleotides 4146 to 4168, nucleotides 4150 to 4172, nucleotides 4151 to 4173, nucleotides 4192 to 4214, nucleotides 4197 to 4219, nucleotides 4201 to 4223, nucleotides 4265 to 4287, nucleotides 4270 to 4292, nucleotides 4318 to 4338, nucleotides 4319 to 4341, nucleotides 4329 to 4349, nucleotides 4348 to 4386, nucleotides 4349 to 4371, nucleotides 4350 to 4369, nucleotides 4350 to 4372, nucleotides 4350 to 4372, nucleotides 4351 to 4370, nucleotides 4352 to 4371, nucleotides 4352 to 4374, nucleotides 4353 to 4372, nucleotides 4353 to 4375, nucleotides 4354 to 4376, nucleotides 4359 to 4381, nucleotides 4361 to 4383, nucleotides 4362 to 4384, nucleotides 4362 to 4384, nucleotides 4364 to 4386, nucleotides 4365 to 4387, nucleotides 4395 to 4415, nucleotides 4396 to 4416, nucleotides 4409 to 4431, nucleotides 4410 to 4432, nucleotides 4412 to 4434, nucleotides 4637 to 4657, nucleotides 4638 to 4658, nucleotides 4640 to 4660, nucleotides 5103 to 5123, nucleotides 5124 to 5144, nucleotides 5126 to 5146, or nucleotides 5127 to 5147 of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

14. The dsRNA agent of any one of claims 11-13, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by not more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of SEQ ID NOs: 2318 and 2430; SEQ ID NOs: 2319 and 2431 ; SEQ ID NOs: 2320 and 2432; SEQ ID NOs: 2321 and 2433; SEQ ID NOs: 2322 and 2434; SEQ ID NOs: 2323 and 2435; SEQ ID NOs: 2324 and 2436; SEQ ID NOs: 2325 and 2437; SEQ ID NOs: 2326 and 2438; SEQ ID NOs: 2327 and 2439; SEQ ID NOs: 2328 and 2440; SEQ ID NOs: 2329 and 2441; SEQ ID NOs: 2330 and 2442; SEQ ID NOs: 2331 and 2443; SEQ ID NOs: 2332 and 2444; SEQ ID NOs: 2333 and 2445; SEQ ID NOs: 2334 and 2446; SEQ ID NOs: 2335 and 2447; SEQ ID NOs: 2336 and 2448; SEQ ID NOs: 2337 and 2449; SEQ ID NOs: 2338 and 2450; SEQ ID NOs: 2339 and 2451; SEQ ID NOs: 2340 and 2452; SEQ ID NOs: 2341 and 2453; SEQ ID NOs: 2342 and 2454; SEQ ID NOs: 2343 and 2455; SEQ ID NOs: 2344 and 2456; SEQ ID NOs: 2345 and 2457; SEQ ID NOs: 2346 and 2458; SEQ ID NOs: 2347 and 2459; SEQ ID NOs: 2348 and 2460; SEQ ID NOs: 2349 and 2461; SEQ ID NOs: 2350 and 2462; SEQ ID NOs: 2351 and 2463; SEQ ID NOs: 2352 and 2464; SEQ ID NOs: 2353 and 2465; SEQ ID NOs: 2354 and 2466; SEQ ID NOs: 2355 and 2467; SEQ ID NOs: 2356 and 2468; SEQ ID NOs: 2357 and 2469; SEQ ID NOs: 2358 and 2470; SEQ ID NOs: 2359 and 2471; SEQ ID NOs: 2360 and 2472; SEQ ID NOs: 2361 and 2473; SEQ ID NOs: 2362 and 2474; SEQ ID NOs: 2363 and 2475; SEQ ID NOs: 2364 and 2476; SEQ ID NOs: 2365 and 2477; SEQ ID NOs: 2366 and 2478; SEQ ID NOs: 2367 and 2479; SEQ ID NOs: 2368 and 2480; SEQ ID NOs: 2369 and 2481; SEQ ID NOs: 2370 and 2482; SEQ ID NOs: 2371 and 2483; SEQ ID NOs: 2372 and 2484; SEQ ID NOs: 2373 and 2485; SEQ ID NOs: 2374 and 2486; SEQ ID NOs: 2375 and 2487; SEQ ID NOs: 2376 and 2488; SEQ ID NOs: 2377 and 2489; SEQ ID NOs: 2378 and 2490; SEQ ID NOs: 2379 and 2491; SEQ ID NOs: 2380 and 2492; SEQ ID NOs: 2381 and 2493; SEQ ID NOs: 2382 and 2494; SEQ ID NOs: 2383 and 2495; SEQ ID NOs: 2384 and 2496; SEQ ID NOs: 2385 and 2497; SEQ ID NOs: 2386 and 2498; SEQ ID NOs: 2387 and 2499; SEQ ID NOs: 2388 and 2500; SEQ ID NOs: 2389 and 2501; SEQ ID NOs: 2390 and 2502; SEQ ID NOs: 2391 and 2503; SEQ ID NOs: 2392 and 2504; SEQ ID NOs: 2393 and 2505; SEQ ID NOs: 2394 and 2506; SEQ ID NOs: 2395 and 2507; SEQ ID NOs: 2396 and 2508; SEQ ID NOs: 2397 and 2509; SEQ ID NOs: 2398 and 2510; SEQ ID NOs: 2399 and 2511; SEQ ID NOs: 2400 and 2512; SEQ ID NOs: 2401 and 2513; SEQ ID NOs: 2402 and 2514; SEQ ID NOs: 2403 and 2515; SEQ ID NOs: 2404 and 2516; SEQ ID NOs: 2405 and 2517; SEQ ID NOs: 2406 and 2518; SEQ ID NOs: 2407 and 2519; SEQ ID NOs: 2408 and 2520; SEQ ID NOs: 2409 and 2521; SEQ ID NOs: 2410 and 2522; SEQ ID NOs: 2411 and 2523; SEQ ID NOs: 2412 and 2524; SEQ ID NOs: 2413 and 2525; SEQ ID NOs: 2414 and 2526; SEQ ID NOs: 2415 and 2527; SEQ ID NOs: 2416 and 2528; SEQ ID NOs: 2417 and 2529; SEQ ID NOs: 2418 and 2530; SEQ ID NOs: 2419 and 2531; SEQ ID NOs: 2420 and 2532; SEQ ID NOs: 2421 and 2533; SEQ ID NOs: 2422 and 2534; SEQ ID NOs: 2423 and 2535; SEQ ID NOs: 2424 and 2536; SEQ ID NOs: 2425 and 2537; SEQ ID NOs: 2426 and 2538; SEQ ID NOs: 2427 and 2539; SEQ ID NOs: 2428 and 2540; SEQ ID NOs: 2429 and 2541; SEQ ID NO: 2720 and 2840; SEQ ID NO: 2721 and 2841; SEQ ID NO: 2722 and 2842; SEQ ID NO: 2723 and 2843; SEQ ID NO: 2724 and 2844; SEQ ID NO: 2725 and 2845; SEQ ID NO: 2726 and 2846; SEQ ID NO: 2727 and 2847; SEQ ID NO: 2728 and 2848; SEQ ID NO: 2729 and 2849; SEQ ID NO: 2730 and 2850; SEQ ID NO: 2731 and 2851; SEQ ID NO: 2732 and 2852; SEQ ID NO: 2733 and 2853; SEQ ID NO: 2734 and 2854; SEQ ID NO: 2735 and 2855; SEQ ID NO: 2736 and 2856; SEQ ID NO: 2737 and 2857; SEQ ID NO: 2738 and 2858; SEQ ID NO: 2739 and 2859; SEQ ID NO: 2740 and 2860; SEQ ID NO: 2741 and 2861; SEQ ID NO: 2742 and 2862; SEQ ID NO: 2743 and 2863; SEQ ID NO: 2744 and 2864; SEQ ID NO: 2745 and 2865; SEQ ID NO: 2746 and 2866; SEQ ID NO: 2747 and 2867; SEQ ID NO: 2748 and 2868; SEQ ID NO: 2749 and 2869; SEQ ID NO: 2750 and 2870; SEQ ID NO: 2751 and 2871; SEQ ID NO: 2752 and 2872; SEQ ID NO: 2753 and 2873; SEQ ID NO: 2754 and 2874; SEQ ID NO: 2755 and 2875; SEQ ID NO: 2756 and 2876; SEQ ID NO: 2757 and 2877; SEQ ID NO: 2758 and 2878; SEQ ID NO: 2759 and 2879; SEQ ID NO: 2760 and 2880; SEQ ID NO: 2761 and 2881; SEQ ID NO: 2762 and 2882; SEQ ID NO: 2763 and 2883; SEQ ID NO: 2764 and 2884; SEQ ID NO: 2765 and 2885; SEQ ID NO: 2766 and 2886; SEQ ID NO: 2767 and 2887; SEQ ID NO: 2768 and 2888; SEQ ID NO: 2769 and 2889; SEQ ID NO: 2770 and 2890; SEQ ID NO: 2771 and 2891; SEQ ID NO: 2772 and 2892; SEQ ID NO: 2773 and 2893; SEQ ID NO: 2774 and 2894; SEQ ID NO: 2775 and 2895; SEQ ID NO: 2776 and 2896; SEQ ID NO: 2777 and 2897; SEQ ID NO: 2778 and 2898; SEQ ID NO: 2779 and 2899; SEQ ID NO: 2960 and 3340; SEQ ID NO: 2961 and 3341; SEQ ID NO: 2962 and 3342; SEQ ID NO: 2963 and 3343; SEQ ID NO: 2964 and 3344; SEQ ID NO: 2965 and 3345; SEQ ID NO: 2966 and 3346; SEQ ID NO: 2967 and 3347; SEQ ID NO: 2968 and 3348; SEQ ID NO: 2969 and 3349; SEQ ID NO: 2970 and 3350; SEQ ID NO: 2971 and 3351; SEQ ID NO: 2972 and 3352; SEQ ID NO: 2973 and 3353; SEQ ID NO: 2974 and 3354; SEQ ID NO: 2975 and 3355; SEQ ID NO: 2976 and 3356; SEQ ID NO: 2977 and 3357; SEQ ID NO: 2978 and 3358; SEQ ID NO: 2979 and 3359; SEQ ID NO: 2980 and 3360; SEQ ID NO: 2981 and 3361; SEQ ID NO: 2982 and 3362; SEQ ID NO: 2983 and 3363; SEQ ID NO: 2984 and 3364; SEQ ID NO: 2985 and 3365; SEQ ID NO: 2986 and 3366; SEQ ID NO: 2987 and 3367; SEQ ID NO: 2988 and 3368; SEQ ID NO: 2989 and 3369; SEQ ID NO: 2990 and 3370; SEQ ID NO: 2991 and 3371 ; SEQ ID NO: 2992 and 3372; SEQ ID NO: 2993 and 3373; SEQ ID NO: 2994 and 3374; SEQ ID NO: 2995 and 3375; SEQ ID NO: 2996 and 3376; SEQ ID NO: 2997 and 3377; SEQ ID NO: 2998 and 3378; SEQ ID NO: 2999 and 3379; SEQ ID NO: 3000 and 3380; SEQ ID NO: 3001 and 3381; SEQ ID NO: 3002 and 3382; SEQ ID NO: 3003 and 3383; SEQ ID NO: 3004 and 3384; SEQ ID NO: 3005 and 3385; SEQ ID NO: 3006 and 3386; SEQ ID NO: 3007 and 3387; SEQ ID NO: 3008 and 3388; SEQ ID NO: 3009 and 3389; SEQ ID NO: 3010 and 3390; SEQ ID NO: 3011 and 3391; SEQ ID NO: 3012 and 3392; SEQ ID NO: 3013 and 3393; SEQ ID NO: 3014 and 3394; SEQ ID NO: 3015 and 3395; SEQ ID NO: 3016 and 3396; SEQ ID NO: 3017 and 3397; SEQ ID NO: 3018 and 3398; SEQ ID NO: 3019 and 3399; SEQ ID NO: 3020 and 3400; SEQ ID NO: 3021 and 3401; SEQ ID NO: 3022 and 3402; SEQ ID NO: 3023 and 3403; SEQ ID NO: 3024 and 3404; SEQ ID NO: 3025 and 3405; SEQ ID NO: 3026 and 3406; SEQ ID NO: 3027 and 3407; SEQ ID NO: 3028 and 3408; SEQ ID NO: 3029 and 3409; SEQ ID NO: 3030 and 3410; SEQ ID NO: 3031 and 3411; SEQ ID NO: 3032 and 3412; SEQ ID NO: 3033 and 3413; SEQ ID NO: 3034 and 3414; SEQ ID NO: 3035 and 3415; SEQ ID NO: 3036 and 3416; SEQ ID NO: 3037 and 3417; SEQ ID NO: 3038 and 3418; SEQ ID NO: 3039 and 3419; SEQ ID NO: 3040 and 3420; SEQ ID NO: 3041 and 3421; SEQ ID NO: 3042 and 3422; SEQ ID NO: 3043 and 3423; SEQ ID NO: 3044 and 3424; SEQ ID NO: 3045 and 3425; SEQ ID NO: 3046 and 3426; SEQ ID NO: 3047 and 3427; SEQ ID NO: 3048 and 3428; SEQ ID NO: 3049 and 3429; SEQ ID NO: 3050 and 3430; SEQ ID NO: 3051 and 3431; SEQ ID NO: 3052 and 3432; SEQ ID NO: 3053 and 3433; SEQ ID NO: 3054 and 3434; SEQ ID NO: 3055 and 3435; SEQ ID NO: 3056 and 3436; SEQ ID NO: 3057 and 3437; SEQ ID NO: 3058 and 3438; SEQ ID NO: 3059 and 3439; SEQ ID NO: 3060 and 3440; SEQ ID NO: 3061 and 3441; SEQ ID NO: 3062 and 3442; SEQ ID NO: 3063 and 3443; SEQ ID NO: 3064 and 3444; SEQ ID NO: 3065 and 3445; SEQ ID NO: 3066 and 3446; SEQ ID NO: 3067 and 3447; SEQ ID NO: 3068 and 3448; SEQ ID NO: 3069 and 3449; SEQ ID NO: 3070 and 3450; SEQ ID NO: 3071 and 3451; SEQ ID NO: 3072 and 3452; SEQ ID NO: 3073 and 3453; SEQ ID NO: 3074 and 3454; SEQ ID NO: 3075 and 3455; SEQ ID NO: 3076 and 3456; SEQ ID NO: 3077 and 3457; SEQ ID NO: 3078 and 3458; SEQ ID NO: 3079 and 3459; SEQ ID NO: 3080 and 3460; SEQ ID NO: 3081 and 3461; SEQ ID NO: 3082 and 3462; SEQ ID NO: 3083 and 3463; SEQ ID NO: 3084 and 3464; SEQ ID NO: 3085 and 3465; SEQ ID NO: 3086 and 3466; SEQ ID NO: 3087 and 3467; SEQ ID NO: 3088 and 3468; SEQ ID NO: 3089 and 3469; SEQ ID NO: 3090 and 3470; SEQ ID NO: 3091 and 3471; SEQ ID NO: 3092 and 3472; SEQ ID NO: 3093 and 3473; SEQ ID NO: 3094 and 3474; SEQ ID NO: 3095 and 3475; SEQ ID NO: 3096 and 3476; SEQ ID NO: 3097 and 3477; SEQ ID NO: 3098 and 3478; SEQ ID NO: 3099 and 3479; SEQ ID NO: 3100 and 3480; SEQ ID NO: 3101 and 3481; SEQ ID NO: 3102 and 3482; SEQ ID NO: 3103 and 3483; SEQ ID NO: 3104 and 3484; SEQ ID NO: 3105 and 3485; SEQ ID NO: 3106 and 3486; SEQ ID NO: 3107 and 3487; SEQ ID NO: 3108 and 3488; SEQ ID NO: 3109 and 3489; SEQ ID NO: 3110 and 3490; SEQ ID NO: 3111 and 3491; SEQ ID NO: 3112 and 3492; SEQ ID NO: 3113 and 3493; SEQ ID NO: 3114 and 3494; SEQ ID NO: 3115 and 3495; SEQ ID NO: 3116 and 3496; SEQ ID NO: 3117 and 3497; SEQ ID NO: 3118 and 3498; SEQ ID NO: 3119 and 3499; SEQ ID NO: 3120 and 3500; SEQ ID NO: 3121 and 3501; SEQ ID NO: 3122 and 3502; SEQ ID NO: 3123 and 3503; SEQ ID NO: 3124 and 3504; SEQ ID NO: 3125 and 3505; SEQ ID NO: 3126 and 3506; SEQ ID NO: 3127 and 3507; SEQ ID NO: 3128 and 3508; SEQ ID NO: 3129 and 3509; SEQ ID NO: 3130 and 3510; SEQ ID NO: 3131 and 3511; SEQ ID NO: 3132 and 3512; SEQ ID NO: 3133 and 3513; SEQ ID NO: 3134 and 3514; SEQ ID NO: 3135 and 3515; SEQ ID NO: 3136 and 3516; SEQ ID NO: 3137 and 3517; SEQ ID NO: 3138 and 3518; SEQ ID NO: 3139 and 3519; SEQ ID NO: 3140 and 3520; SEQ ID NO: 3141 and 3521; SEQ ID NO: 3142 and 3522; SEQ ID NO: 3143 and 3523; SEQ ID NO: 3144 and 3524; SEQ ID NO: 3145 and 3525; SEQ ID NO: 3146 and 3526; SEQ ID NO: 3147 and 3527; SEQ ID NO: 3148 and 3528; and SEQ ID NO: 3149 and 3529.

15. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of human solute carrier family 6 member 19 (SLC6A19) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the nucleotide sequences of nucleotides 3 to 25, nucleotides 8 to 30, nucleotides 13 to 35, nucleotides 38 to 60, nucleotides 43 to 65, nucleotides 48 to 70, nucleotides 93 to 115, nucleotides 98 to 120, nucleotides 103 to 125, nucleotides 108 to 130, nucleotides 111 to 133, nucleotides 113 to 135, nucleotides 118 to 140, nucleotides 214 to 236, nucleotides 219 to 241, nucleotides 244 to 266, nucleotides 249 to 271, nucleotides 252 to 274, nucleotides 252 to 274, nucleotides 253 to 275, nucleotides 254 to 276, nucleotides 257 to 286, nucleotides 259 to 281, nucleotides 264 to 286, nucleotides 289 to 311, nucleotides 294 to 316, nucleotides 339 to 361, nucleotides 397 to 419, nucleotides 460 to 480, nucleotides 461 to 481, nucleotides 481 to 503, nucleotides 486 to 508, nucleotides 491 to 513, nucleotides 557 to 579, nucleotides 561 to 583, nucleotides 562 to 584, nucleotides 564 to 586, nucleotides 566 to 586, nucleotides 567 to 589, nucleotides 616 to 638, nucleotides 621 to 643, nucleotides 626 to 648, nucleotides 670 to 692, nucleotides 671 to 691, nucleotides 672 to 694, nucleotides 674 to 694, nucleotides 675 to 697, nucleotides 680 to 702, nucleotides 685 to 707, nucleotides 690 to 712, nucleotides 695 to 717, nucleotides 700 to 722, nucleotides 705 to 727, nucleotides 710 to 732, nucleotides 715 to 737, nucleotides 720 to 742, nucleotides 725 to 747, nucleotides 730 to 752, nucleotides 735 to 757, nucleotides 740 to 762, nucleotides 745 to 767, nucleotides 750 to 772, nucleotides 751 to 776, nucleotides 755 to 777, nucleotides 760 to 782, nucleotides 765 to 787, nucleotides 770 to 792, nucleotides 775 to 797, nucleotides 798 to 823, nucleotides 800 to 822, nucleotides 825 to 847, nucleotides 890 to 912, nucleotides 895 to 917, nucleotides 900 to 922, nucleotides 905 to 927, nucleotides 910 to 932, nucleotides 911 to 937, nucleotides 915 to 937, nucleotides 972 to 994, nucleotides 973 to 995, nucleotides 977 to 1000, nucleotides 977 to 999, nucleotides 978 to 1000, nucleotides 983 to 1005, nucleotides 1035 to 1057, nucleotides 1058 to 1080, nucleotides 1059 to 1081, nucleotides 1060 to 1082, nucleotides 1063 to 1082, nucleotides 1065 to 1087, nucleotides 1068 to 1087, nucleotides 1070 to 1094, nucleotides 1070 to 1092, nucleotides 1074 to 1096, nucleotides 1075 to 1097, nucleotides 1077 to 1096, nucleotides 1080 to 1102, nucleotides 1085 to 1107, nucleotides 1086 to 1106, nucleotides 1090 to 1112, nucleotides 1095 to 1117, nucleotides 1100 to 1122, nucleotides 1105 to 1127, nucleotides 1110 to 1132, nucleotides 1115 to 1137, nucleotides 1115 to 1137, nucleotides 1117 to 1139, nucleotides 1177 to 1199, nucleotides 1182 to 1204, nucleotides 1187 to 1209, nucleotides 1191 to 1214, nucleotides 1192 to 1214, nucleotides 1195 to 1217, nucleotides 1234 to 1256, nucleotides 1239 to 1261, nucleotides 1242 to 1264, nucleotides 1244 to 1266, nucleotides 1247 to 1268, nucleotides 1249 to 1271, nucleotides 1251 to 1270, nucleotides 1254 to 1276, nucleotides 1259 to 1281, nucleotides 1263 to 1282, nucleotides 1264 to 1286, nucleotides 1266 to 1285, nucleotides 1269 to 1291, nucleotides 1326 to 1348, nucleotides 1331 to 1353, nucleotides 1334 to 1361, nucleotides 1334 to 1356, nucleotides 1334 to 1356, nucleotides 1335 to 1357, nucleotides 1336 to 1358, nucleotides 1337 to 1359, nucleotides 1337 to 1359, nucleotides 1338 to 1360, nucleotides 1340 to 1362, nucleotides 1341 to 1363, nucleotides 1346 to 1368, nucleotides 1351 to 1373, nucleotides 1353 to 1372, nucleotides 1377 to 1399, nucleotides 1382 to 1404, nucleotides 1383 to 1403, nucleotides 1387 to 1409, nucleotides 1392 to 1414, nucleotides 1397 to 1419, nucleotides 1402 to 1424, nucleotides 1407 to 1429, nucleotides 1412 to 1434, nucleotides 1417 to 1439, nucleotides 1456 to 1478, nucleotides 1458 to 1477, nucleotides 1481 to 1503, nucleotides 1486 to 1508, nucleotides 1491 to 1513, nucleotides 1496 to 1518, nucleotides 1521 to 1543, nucleotides 1568 to 1590, nucleotides 1569 to 1591, nucleotides 1574 to 1596, nucleotides 1578 to 1621, nucleotides 1579 to 1601, nucleotides 1583 to 1605, nucleotides 1583 to 1605, nucleotides 1584 to 1606, nucleotides 1584 to 1606, nucleotides 1585 to 1607, nucleotides 1585 to 1607, nucleotides 1587 to 1609, nucleotides 1588 to 1610, nucleotides 1589 to 1611, nucleotides 1594 to 1616, nucleotides 1595 to 1617, nucleotides 1599 to 1621, nucleotides 1601 to 1620, nucleotides 1602 to 1624, nucleotides 1642 to 1664, nucleotides 1749 to 1775, nucleotides 1750 to 1772, nucleotides 1755 to 1777, nucleotides 1757 to 1779, nucleotides 1758 to 1780, nucleotides 1760 to 1782, nucleotides 1761 to 1792, nucleotides 1764 to 1783, nucleotides 1765 to 1787, nucleotides 1770 to 1792, nucleotides 1809 to 1831, nucleotides 1810 to 1829, nucleotides 1812 to 1832, nucleotides 1814 to 1836, nucleotides 1852 to 1881, nucleotides 1853 to 1875, nucleotides 1854 to 1876, nucleotides 1858 to 1880, nucleotides 2168 to 2188, nucleotides 2333 to 2353, nucleotides 2454 to 2474, nucleotides 2507 to 2527, nucleotides 2593 to 2613, nucleotides 3244 to 3264, nucleotides 3288 to 3308, nucleotides 3304 to 3324, nucleotides 3332 to 3352, nucleotides 3333 to 3353, nucleotides 3501 to 3523, nucleotides 3595 to 3615, nucleotides 3595 to 3617, nucleotides 3643 to 3671, nucleotides 3646 to 3668, nucleotides 3646 to 3668, nucleotides 3647 to 3669, nucleotides 3648 to 3668, nucleotides 3648 to 3670, nucleotides 3648 to 3670, nucleotides 3649 to 3671, nucleotides 3651 to 3670, nucleotides 3651 to 3673, nucleotides 3651 to 3673, nucleotides 3652 to 3671, nucleotides 3712 to 3733, nucleotides 3713 to 3735, nucleotides 3763 to 3786, nucleotides 3763 to 3785, nucleotides 3764 to 3786, nucleotides 3791 to 3811, nucleotides 3796 to 3816, nucleotides 3801 to 3829, nucleotides 3801 to 3820, nucleotides 3802 to 3821, nucleotides 3804 to 3826, nucleotides 3809 to 3831, nucleotides 3810 to 3832, nucleotides 3812 to 3833, nucleotides 3812 to 3834, nucleotides 3813 to 3835, nucleotides 3814 to 3833, nucleotides 3814 to 3836, nucleotides 3814 to 3836, nucleotides 3839 to 3859, nucleotides 3840 to 3862, nucleotides 3902 to 3924, nucleotides 3965 to 3987, nucleotides 3995 to 4015, nucleotides 3998 to 4020, nucleotides 4100 to 4124, nucleotides 4102 to 4124, nucleotides 4141 to 4163, nucleotides 4141 to 4163, nucleotides 4142 to 4164, nucleotides 4143 to 4165, nucleotides 4144 to 4166, nucleotides 4146 to 4168, nucleotides 4150 to 4172, nucleotides 4151 to 4173, nucleotides 4192 to 4214, nucleotides 4197 to 4219, nucleotides 4201 to 4223, nucleotides 4265 to 4287, nucleotides 4270 to 4292, nucleotides 4318 to 4338, nucleotides 4319 to 4341, nucleotides 4329 to 4349, nucleotides 4348 to 4386, nucleotides 4349 to 4371, nucleotides 4350 to 4369, nucleotides 4350 to 4372, nucleotides 4350 to 4372, nucleotides 4351 to 4370, nucleotides 4352 to 4371, nucleotides 4352 to 4374, nucleotides 4353 to 4372, nucleotides 4353 to4375, nucleotides 4354 to 4376, nucleotides 4359 to 4381, nucleotides 4361 to 4383, nucleotides 4362 to 4384, nucleotides 4362 to 4384, nucleotides 4364 to 4386, nucleotides 4365 to 4387, nucleotides 4395 to 4415, nucleotides 4396 to 4416, nucleotides 4409 to 4431, nucleotides 4410 to 4432, nucleotides 4412 to 4434, nucleotides 4637 to 4657, nucleotides 4638 to 4658, nucleotides 4640 to 4660, nucleotides 5103 to 5123, nucleotides 5124 to 5144, nucleotides 5126 to 5146, or nucleotides 5127 to 5147of SEQ ID NO: 1, and the antisense strand comprises at least 19 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

16. The dsRNA agent of any one of claims 11-15, wherein: the double stranded region is 17-30 nucleotide pairs in length; the double stranded region is 17-25 nucleotide pairs in length; the double stranded region is 17-23 nucleotide pairs in length, each strand is independently no more than 30 nucleotides in length; the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length; the region of complementarity is at least 17 nucleotides in length, optionally wherein the region of complementarity is between 19 and 23 nucleotides in length, optionally wherein the region of complementarity is 19 nucleotides in length; and/or at least one strand comprises a 3’ overhang of at least 2 nucleotides.

17. An exon skipping antisense oligonucleotide comprising any of SEQ ID NOs: 2542-2719.

18. The exon skipping antisense oligonucleotide of claim 17 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.

19. The exon skipping antisense oligonucleotide of claim 18, wherein the modified internucleoside linkage comprises a phosphorothioate linkage.

20. The exon skipping antisense oligonucleotide of claim 19, wherein the at least one modified internucleoside linkage, sugar moiety, or nucleobase comprises a modification selected from the group consisting of a deoxy-nucleoside, a 3 ’-terminal deoxy -thymine (dT) nucleoside, a 2 -0- methyl (2'-0Me) modified nucleoside, a 2'-fluoro (2'-F ) modified nucleoside, a 2'-deoxy-modified nucleoside, a locked nucleotide (LNA), an unlocked nucleoside, a conformationally restricted nucleoside, a constrained ethyl nucleoside, an abasic nucleoside, a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), a 2’-amino-modified nucleoside, a 2’-O-allyl-modified nucleoside, 2’-C- alkyl-modified nucleoside, 2’-hydroxly-modified nucleoside, a 2’-methoxyethyl (2 -MOE) modified nucleoside, a 2’-O-alkylmodified nucleoside, a morpholino nucleoside, a phosphorami date, a non-natural base comprising nucleoside, a tetrahydropyran modified nucleoside, a 1,5-anhydrohexitol modified nucleoside, a cyclohexenyl modified nucleoside, a nucleotide comprising a 5 -phosphorothioate group, a nucleotide comprising a 5'- methylphosphonate group, a nucleotide comprising a 5’ phosphate or 5’ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleoside comprising adenosine-glycol nucleic acid (GNA), a nucleoside comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2’deoxythymidine-.3’phosphate, a nucleotide comprising 2 ’-deoxyguanosine-3 ’-phosphate, and a terminal nucleoside linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof, optionally wherein the exon skipping antisense oligonucleotide comprises one or more 2 ’-methoxy ethyl (2-MOE) modified nucleosides, optionally wherein all nucleosides of the exon skipping antisense oligonucleotide are 2’ -methoxy ethyl (2 -MOE) modified nucleosides.

21. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims, wherein the antisense compound, dsRNA agent, or exon skipping antisense oligonucleotide comprises at least one modified nucleotide.

22. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 21, wherein substantially all of the nucleotides of the sense strand of the dsRNA agent; or substantially all of the nucleotides of the antisense strand of the antisense compound, exon skipping antisense oligonucleotide or the dsRNA agent comprise a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the dsRNA agent comprise a modification.

23. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 21 or claim 22, wherein all of the nucleotides of the sense strand of the dsRNA agent comprise a modification; all of the nucleotides of the antisense strand of the antisense compound, exon skipping antisense oligonucleotide or the dsRNA agent comprise a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand of the dsRNA agent comprise a modification.

24. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 21-23, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3 ’-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2 -deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C- alkyl-modified nucleotide, 2’-hydroxly-modified nucleotide, a 2’ -methoxyethyl modified nucleotide, a 2’-O-alkyl-modified nucleotide, a morpholino nucleotide (e.g., a phosphorodiamidate morpholino nucleic acid (PMO)), a phosphorami date, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a phosphoryl guanidine-based backbone, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5’-phosphate, a nucleotide comprising a 5’-phosphate mimic, a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

25. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 21-24, wherein the modifications on the nucleotides are selected from the group consisting of:

LNA, HNA, CeNA, 2 -methoxy ethyl, 2 -O-alkyl, 2'-O-allyl, 2'-C- allyl, 2 '-fluoro, 2'- deoxy, 2’ -hydroxyl, and glycol; and combinations thereof; a C7-modified deaza-adenine, a C7-modified deaza-guanosine, a C5-modified cytosine, a C5-modified uridine, Nl-methyl-pseudouridine (ml\|/), 1-ethyl-pseudouridine (ely), 5-methoxy- uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (\|/), 5-methoxymethyl uridine, 5- methylthio uridine, 1 -methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, or any combination thereof; a phosphorothioate (PS) modification, a phosphoryl guanidine (PN) modification, a borano-phosphate modification, an alkyl phosphonate nucleic acid (phNA), a peptide nucleic acid (PNA), or any combination thereof; a deoxyribonucleic acid (DNA), optionally wherein the DNA is or comprises a DNA analog, optionally wherein the DNA analog comprises one or more morpholino subunits linked together by phosphorus-containing linkage(s), optionally wherein the DNA analog is or comprises a phosphorodiamidate morpholino nucleic acid (PMO), optionally wherein the PMO comprises about 12-40 nucleotides; a peptide nucleic acid (PNA) modification; and/or one or more modification to a 5’ end of the antisense compound or dsRNA agent, optionally wherein the modification to the 5' end is a 5’ amino modification.

26. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 21-25, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fhioro modified nucleotide, a 2'-deoxy- modified nucleotide, a glycol modified nucleotide (GNA), and a vinyl-phosphonate nucleotide; and combinations thereof.

27. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 21-26, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, and a 2'-fluoro modified nucleotide; and combinations thereof.

28. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims comprising one or more deoxy-nucleotides, optionally wherein a heteroduplex oligonucleotide (HDO) comprises the antisense compound or the exon skipping antisense oligonucleotide and/or the dsRNA agent is a HDO.

29. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims, directly or indirectly conjugated to a targeting moiety.

30. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 29, wherein the antisense compound, exon skipping antisense oligonucleotide or dsRNA agent and the targeting moiety are indirectly conjugated by way of a linker.

31. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 29 or claim 30, wherein the targeting moiety specifically binds a cell surface factor, optionally a kidney cell surface factor.

32. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 31, wherein the cell surface factor is internalized when bound by the targeting moiety.

33. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 31 or claim 32, wherein the kidney cell surface factor is a receptor, optionally megalin or cubilin.

24. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 29-33, wherein the targeting moiety is chosen from: a polypeptide, an aminoglycoside, an endogenous ligand (e.g., a ligand disclosed in Table 1 or Table 2), a xenobiotic, an antibody or a fragment thereof, an aptamer, a small molecule, or any combination thereof.

35. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 34, wherein the targeting moiety is or comprises an endogenous ligand, e.g., a ligand disclosed in Table 1 or Table 2.

36. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 29-35, wherein the targeting moiety is or comprises a vitamin, optionally a vitamin provided in Table 1, optionally wherein the vitamin is or comprises vitamin B12.

37. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 36, wherein the targeting moiety is or comprises a polypeptide.

38. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 37, wherein the polypeptide is chosen from: a fragment of receptor associated protein (RAP), a peptide derived from a radiopharmaceutical conjugate such as ocreotide, ocreotate, exendin, minigastrin, and/or neurotensin; or any combination thereof.

39. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 38, wherein the polypeptide comprises a RAP fragment, or a variant thereof, optionally wherein the RAP fragment comprises a polypeptide comprising residues 219-323 of RAP.

40. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 37, wherein the polypeptide is or comprises a peptide derived from a radiopharmaceutical conjugate such as ocreotide, ocreotate, exendin, minigastrin, and/or neurotensin.

41. The ASO, dsRNA agent, or exon skipping anti sense oligonucleotide of any one of claims 37-40, wherein the polypeptide is or comprises a knotted peptide.

42. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 29 or claim 30, wherein the targeting moiety is or comprises an aminoglycoside, optionally wherein the aminoglycoside is chosen from one or more, or all of: streptomycin, neomycin, kanamycin, paromomycin, gentamicin, G-418 (geneticin) ELX-202, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekacin, isepamicin, framycetin, paromomycin, apramycin, fradiomycin, arbekacin, plazomicin, or a derivative, or a fragment, or a variant thereof.

43. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 29 or claim 30, wherein the targeting moiety is or comprises a xenobiotic, optionally wherein the xenobiotic is or comprises polymixin, aprotinin, trichosanthin, or any combination thereof.

44. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 29 or claim 30, wherein the targeting moiety is or comprises an antibody or a fragment thereof, optionally wherein the antibody or fragment thereof selectively binds Megalin, Cubilin, or both.

45. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 44, wherein the antibody or fragment thereof specifically binds Megalin.

46. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of claim 44, wherein the antibody or fragment thereof specifically binds Cubilin.

47. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 43-46, wherein the antibody or fragment thereof is a bispecific antibody or a multi-specific antibody.

48. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 42-46, wherein the antibody comprises one or more modifications of an Fc domain, e.g., an Fc variant.

49. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims, wherein the antisense compound or dsRNA agent is characterized in that when delivered to a cell expressing the target, reduced expression and/or activity of the target is observed as compared to a cell which has not been delivered the antisense compound, dsRNA agent, or exon skipping antisense oligonucleotide or to a cell which does not express the target.

50. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 30-49, wherein the linker is a cleavable linker, optionally wherein the linker becomes cleaved when exposed to a cell-internal environment.

51. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims, wherein the antisense compound, dsRNA agent, or exon skipping antisense oligonucleotide comprises one or more extended nucleic acid ("exNA") modifications, optionally wherein the one or more exNA modification(s) is/are positioned at or near a 3'-end of the nucleic acid.

52. The ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims, wherein the antisense compound, dsRNA agent, or exon skipping antisense oligonucleotide comprises one or more phosphoryl guanidine-containing backbone ("PN backbone") and/or mesyl phosphoramidate modifications.

53. A pharmaceutical composition comprising the ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of the preceding claims and a pharmaceutically acceptable penetration enhancer, carrier, or diluent, optionally wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, intramuscular, parenteral, or oral delivery

54. A method for the prevention, amelioration, or treatment of a kidney disease or condition in a subject comprising administration of the ASO, dsRNA, exon skipping antisense oligonucleotide, or pharmaceutical composition of any one of the preceding claims to the subject in need of such intervention.

55. A method for the prevention, amelioration, or treatment of a kidney disease or condition in a subject comprising administration of an inhibitory ASO, exon skipping antisense oligonucleotide, or dsRNA to the subject in need of such intervention, wherein the inhibitory' antisense compound, exon skipping antisense oligonucleotide, or dsRNA targets the human SLC6A19 m RN A of SEQ ID NO: 1 .

56. The method of claim 54 or claim 55, wherein the kidney disease or condition is a glomerular disorder, a renal tubular disorder, Chronic Kidney Disease, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof.

57. The method of claim 55, wherein the kidney disease or condition is phenylketonuria (PKU).

58. The method of claim 55, wherein the kidney disease or condition is a urea cycle disorder.

59. A method for reducing SI..C6 A19 mRNA or B°AT1 polypeptide levels in a cell, the method comprising contacting the cell with an ASO, dsRNA agent, or exon skipping antisense oligonucleotide of any one of claims 2-52 in an amount sufficient to reduce SLC6A19 mRNA or B°AT1 polypeptide levels in the cell.

60. The method of ciaim 59, wherein the cel 1 is chosen from: immune cells; nervous system cells; muscle cells; small intestine cells; colon cells; adipocytes; kidney cells; liver cells; lung cells; splenic cells; stomach cells; esophagus cells; bladder cells; pancreas cells; thyroid cells; salivary gland cells; adrenal gland cells; pituitary gland cells; breast cells; skin cells; ovary cells; uterus cells; placenta cells; prostate cells; parathyroid cells; cells of the inner ear; or testis cells, or any combination thereof, optionally wherein the cell is a kidney cell, optionally wherein the kidney cell is a proximal tubular epithelial cell and/or a podocyte.

61. The method of claim 59 or claim 60, wherein the cell is a mammalian cell, optionally a human cell, optionally a human cell of a subject, optionally a human cell of a subject in situ.

62. Use of an ASO, dsRNA, exon skipping antisense oligonucleotide, or pharmaceutical composition of any one of claims 2-53 in the manufacture of a medicament for the treatment, prevention or amelioration of a kidney disease or condition, wherein the medicament reduces the expression of a nucleic acid molecule encoding B°AT1.

63. The use of claim 62, wherein the kidney disease or condition comprises a glomerular disorder, a renal tubular disorder, Chronic Kidney Disease, other renal disorders, an inborn error of metabolism, a systemic metabolic disorder, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the inner ear, a neurological disorder, or a viral infection, or any combination thereof.

64. The use of claim 62, wherein the kidney disease or condition is PKU.

65. The use of claim 62, wherein the kidney disease or condition is a urea cycle disorder.

66. A kit comprising the ASO, dsRNA, exon skipping antisense oligonucleotide, or pharmaceutical composition of any one of claims 2-53, and instructions for its use.

67. A vial comprising the ASO, dsRNA, exon skipping antisense oligonucleotide, or pharmaceutical composition of any one of claims 2-53.

68. A syringe comprising the ASO, dsRNA, exon skipping antisense oligonucleotide, or pharmaceutical composition of any one of claims 2-53.