WO2025080866A1 - Trna therapeutics for hemophilia - Google Patents

Abstract

The present invention is related at least in part to tRNA therapeutics that can be used for suppressing nonsense mutations and/or restoring functional proteins associated with monogenic, liver-based bleeding disorders, such as hemophilia, and related methods and compositions. In an embodiment, the cells that are targeted are endothelial cells.

Description

TRNA THERAPEUTICS FOR HEMOPHILIA

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119 of United States provisional application 63/589,073, filed October 10, 2023; United States provisional application 63/554,319, filed February 16, 2024; United States provisional application 63/644,365, filed May 8, 2024; United States provisional application 63/665,557, filed June 28, 2024; and United States provisional application 63/688,829, filed August 29, 2024, the entire contents of each which are incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (H101570057WO00-SEQ-JAV.xml; Size: 618,259 bytes; and Date of Creation: October 10, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

DNA molecules carry genetic information in the form of the sequence of the nucleotide bases that make up the DNA polymer. Only four nucleotide bases are utilized in DNA: adenine, guanine, cytosine, and thymine. This information, in the form of codons of three contiguous bases is transcribed into messenger RNA (mRNA), and then translated by transfer RNA (tRNA) and ribosomes to form proteins. Four nucleotide bases are utilized in RNA: adenine, guanine, cytosine, and uracil. The genetic code is the relation between a triplet codon and a particular amino acid. Sixty-four possible codon triplets form the genetic code, where three stop (also called “terminating” or “nonsense”) codons provide a signal to the translation machinery (cellular ribosomes) to stop protein production at the particular codon. The other sixty-one codon triplets (also called “sense codons”) correspond to one of the 20 standard amino acids.

DNA is translated by ribosomes, causing each amino acid to be linked together one by one to form polypeptides, according to the genetic instructions specifically provided by the DNA. Transfer RNAs translate mRNA into a protein on a ribosome. Each tRNA contains an “anticodon” region that hybridizes with a complementary codon on the mRNA. A tRNA that carries its designated amino acid is called a “charged” tRNA. If the tRNA is one of the 61 amino acid-associated tRNAs (or “sense tRNAs”), it will normally attach its amino acid to the growing peptide. The structural gene of tRNA is about 72-90 nucleotides long and folds into a cloverleaf structure.

SUMMARY OF THE INVENTION tRNAs can be engineered such that they recognize a specific codon and can carry an alternative amino acid for polypeptide production. These tRNAs can be used to target a nonsense mutation, such as one associated with a disease or disorder as provided herein, such as a monogenic, liver-based bleeding disorder, such as hemophilia (e.g., hemophilia A or B, such as severe hemophilia A or B). These tRNAs can also be used to target a nonsense mutation, such as one associated with cells of the liver, such as endothelial cells of the liver, such as hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells (LSECs). These tRNAs can also be used to target a nonsense mutation, such as one associated with endothelial cells, such as HUVECs and LSECs.

In one embodiment of any one of the tRNAs provided herein, the acceptor stem is specific for any amino acid to rescue a stop codon. In some embodiments, the stop codon is a premature stop codon in a mutant gene of the subject that has a monogenic, liver-based disease, such as hemophilia, such as a gene encoding a blood clotting factor, such as FVIII or FIX.

Preferably, in one embodiment of any one of the tRNAs provided herein, the amino acid restores protein function and, thus, may be the wild- type amino acid. In one embodiment of any one of the tRNAs provided herein, the amino acid is the cognate amino acid.

In another aspect, an oligonucleotide that encodes any one or more of the tRNAs provided herein is provided. In one embodiment of any one or more of the oligonucleotides provided herein, the oligonucleotide has a total length of less than 150 nucleotides. In one embodiment of any one or more of the oligonucleotides provided herein, the oligonucleotide is DNA. In one embodiment of any one or more of the oligonucleotides provided herein, the oligonucleotide is RNA.

In another aspect, an expression cassette comprising a promoter and a nucleic acid encoding any one or more of the tRNAs or any one or more of the oligonucleotides provided herein is provided. In one embodiment of any one of the expression cassettes provided herein, the promoter of the expression cassette is any one of the promoters provided herein. In one embodiment of any one of the expression cassettes provided herein, the expression cassette further comprises a terminator. In one embodiment of any one of the expression cassettes provided herein, the promoter of the expression cassette is any one of the terminators provided herein.

In another aspect, a vector comprising any one or more of the oligonucleotides or expression cassettes provided herein is provided. In one embodiment, the vector is a plasmid vector. In one embodiment, the plasmid vector is a nanoplasmid vector.

In another aspect, a composition comprising any one or more of the tRNAs, any one or more of the oligonucleotides, any one or more of the expression cassettes, or any one or more of the vectors provided herein, and a pharmaceutically acceptable carrier is provided.

In another aspect, a cell comprising any one or more of the tRNAs, any one or more of the oligonucleotides, any one or more of the expression cassettes or any one or more of the vectors provided herein is provided.

In another aspect, a method of rescuing a stop codon or premature termination codon (e.g., any one of the stop codons or premature termination codons described herein) and/or restoring protein function in cells of the liver, such as endothelial cells of the liver, such as hepatocytes, Kupffer cells, or LSECs, comprising contacting the cells with any one or more of the tRNAs, any one or more of the oligonucleotides, any one or more of the expression cassettes, any one or more of the vectors, or any one or more of the compositions provided herein is provided. In one embodiment, the contacting is in vitro. In another embodiment, the contacting is in vivo, such as by administration or delivery to a subject.

In another aspect, a method of treating a monogenic, liver-based bleeding disorder, such as hemophilia (e.g., hemophilia A or B, such as severe hemophilia A or B), comprising delivering or administering to a subject, or contacting cells associated with the disorder of the subject with, any one or more of the tRNAs, any one or more of the oligonucleotides, any one or more of the expression cassettes, any one or more of the vectors, or any one or more of the compositions provided herein is provided.

In one embodiment of any one of the methods provided herein, the amount of the tRNA(s), oligonucleotide(s), expression cassette(s), vector(s), or composition(s) is effective to rescue the stop codon, restore protein function and/or treat the monogenic, liver-based disorder. In one embodiment of any one of the methods or compositions provided herein, the liver cells are any one of the liver cells provided herein.

In one embodiment of any one of the methods or compositions provided herein, the cells are endothelial cells provided herein.

In one embodiment of any one of the methods or compositions provided herein, the hemophilia is hemophilia A or B.

In one embodiment of any one of the methods or compositions provided herein, the protein is encoded by a gene for a blood clotting factor, such as FVIII (Factor VIII, also referred to as F8) or FIX.

In one embodiment of any one of the methods or compositions provided herein, the nonsense mutation or stop codon or premature termination codon for rescue is one in a gene for a blood clotting factor, such as FVIII or FIX.

In one embodiment of any one of the methods or compositions provided herein, the tRNA is specific for TGA. In one embodiment of any one of the methods or compositions provided herein, the tRNA is specific for TAA. In one embodiment of any one of the methods or compositions provided herein, the tRNA is specific for TAG. In one embodiment of any one of the methods or compositions provided herein, the tRNA is specific for TGA and delivers an arginine. In one embodiment of any one of the methods or compositions provided herein, the tRNA is any one of the specific tRNAs provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 demonstrates that how, for example, an arginine suppressor tRNA that recognizes the UGA stop codon and inserts the amino acid arginine at that site can restore the normal sequence of the protein being made.

Figure 2 shows the general four-arm structure of tRNAs comprising a T-arm, a D arm, an anticodon arm, and an acceptor stem (or arm). These regions may also be referred to as ‘loops’ throughout.

Figure 3 shows minimal natural termination codon (NTC) read through by candidate ACE-tRNAs across a range of relevant doses.

Figure 4 shows high premature termination codon (PTC) correction by candidate ACE- tRNAs in a reporter construct with a PTC. Figure 5 shows levels of PTC correction and NTC readthrough in cells treated with 2 ng/mL, 10 ng/mL, 50 ng/mL, or 250 ng/mL of candidate ACE-tRNAs relative to untreated cells.

Figure 6 shows amino acid charge profile of two native Arg-tRNAs (CCT-2 and TCT- 1) and an ACE Arg-tRNA (delivered as a DNA nanoplasmid or a RNA nanoplasmid).

Figure 7 shows modification-induced mutations of two native Arg-tRNAs (CCT-2 and TCT-1) and an ACE Arg-tRNA (delivered as a DNA nanoplasmid or a RNA plasmid).

Figure 8 shows a schematic of a method used to evaluate biodistribution of nanoplasmid DNA after systemic administration.

Figure 9 shows biodistribution of AF647-labelled nanoplasmid DNA administered systemically by intravenous injection.

Figure 10 shows flow cytometry analysis of liver cells after systemic administration of AF647-labelled nanoplasmid DNA.

Figure 11 shows the median fluorescence intensity of hepatocytes and liver sinusoidal endothelial cells after systemic administration of AF647-labelled nanoplasmid DNA.

Figure 12 shows DNA FISH analysis of liver tissue after systemic administration of a control (vehicle) and AF647 -labelled nanoplasmid DNA (npDNA).

Figure 13 shows a schematic of a method used to evaluate PTC rescue in HEK293T cells transfected with a Gaussia luciferase reporter system.

Figure 14 shows recovery of F8 protein activity in a Gaussia Luciferase reporter system 48 hours after administration of ACE-tRNA nanoplasmids.

Figure 15 show a schematic of a method used to evaluate F8 activity after PTC rescue in HEK293T cells transfected with a Gaussia luciferase reporter system.

Figure 16 shows recovery of F8 48 hours after administration of ACE-tRNA nanoplasmids determined by FLAG tag quantitation.

Figure 17 shows recovery of F8 activity by a chromogenic coagulation assay 48 hours after administration of ACE-tRNA nanoplasmids.

Figure 18 shows in vivo and in vitro uptake of AF647-nanoplasmid DNA.

Figure 19 shows a schematic of a method to evaluate immunogenicity of nanoplasmid DNA in mice.

Figure 20 shows serum IL-6 levels 4 hours after administration of nanoplasmid DNA encapsulated in candidate lipid nanoparticles. Figure 21 shows serum TNF-a levels 4 hours after administration of nanoplasmid DNA encapsulated in candidate lipid nanoparticles.

Figure 22 shows serum INF-a levels 4 hours after administration of nanoplasmid DNA encapsulated in candidate lipid nanoparticles.

Figure 23 shows serum INF-y levels 4 hours after administration of nanoplasmid DNA encapsulated in candidate lipid nanoparticles.

Figure 24 shows a schematic of the method used to evaluate the dependence of cytokine release on the STING pathway.

Figure 25 shows serum IL-6 levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 26 shows serum TNF-a levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 27 shows serum INF-a levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 28 shows serum INF-y levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 29 shows serum MCP-1 levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 30 shows serum IP- 10 levels 4 hours after administration of nanoplasmid DNA encapsulated in a candidate lipid nanoparticle (LP-01) to wild-type mice versus STING knockout mice.

Figure 31 shows serum INF-a levels in human PBMCs after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 32 shows serum TNF-a levels in human PBMCs after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 33 shows serum IP- 10 levels in human PBMCs after treatment with a linear DNA construct or a nanoplasmid DNA construct. Figure 34 shows serum MCP-1 levels in human PBMCs after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 35 shows a schematic of a method used to evaluate immunogenicity of a linear DNA constructs and nanoplasmid DNA constructs in C57BL/6 mice.

Figure 36 shows IL-6 levels in mice sera after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 37 shows TNF-a levels in mice sera after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 38 shows INF-a levels in mice sera after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 39 shows INF-y levels in mice sera after treatment with a linear DNA construct or a nanoplasmid DNA construct.

Figure 40 shows an exemplary multi-patch nanoplasmid.

Figure 41 shows an exemplary multi-patch nanoplasmid.

Figure 42 shows a schematic of a method used to evaluate a multi-patch system to simultaneously target TGA, TAG, and TAA premature stop codons in HEK293T cells transfected with a nanoluciferase reporter system comprising a TGA, TAG, or TAA premature stop codon.

Figure 43 shows recovery of luciferase signal in HEK293T cells transfected with a nanoluciferase reporter system comprising a TGA, TAG, or TAA premature stop codon using a multi-patch ACE-tRNA.

Figure 44 shows a schematic of the method used to compare a multi-patch system with a single-patch system in HEK293T cells transfected with a F8 gene comprising a R427X mutation.

Figure 45 shows recovery of F8 48 hours after administration of ACE-tRNA nanoplasmids determined by FLAG tag quantitation.

Figure 46 shows recovery of F8 activity by a chromogenic coagulation assay 48 hours after administration of ACE-tRNA nanoplasmids.

Figure 47 shows a schematic of the method used to evaluate nanoplasmid DNA expression (using EGFP) in mice.

Figure 48 shows a schematic of a method used to evaluate in vivo PTC rescue of nanoluciferase using tRNA. Figure 49 shows bioluminescence signal in the liver 48 hour after administration of a negative control (PBC), a LNP containing mRNA encoding wild-type nanoluciferase, a LNP containing a mRNA encoding a nanoluciferase with a PTC, a LNP containing a mRNA encoding a nanoluciferase with a PTC and a LNP containing a tRNA, and a LNP containing a mRNA encoding a nanoluciferase with a PTC and free tRNA.

Figure 50 shows a schematic of a method used to evaluate in vivo PTC rescue of nanoluciferase using nanoplasmid DNA.

Figure 51 shows bioluminescence signal in the liver 48 hours after administration of a negative control (PBS), a LNP containing mRNA encoding a nanoluciferase with a PTC and LNP containing the nanoplasmid DNA, and a LNP containing mRNA encoding a wild-type nanoluciferase.

Figure 52 shows serum cytokine levels 4 and 24 hours post-administration of nanoplasmid DNA encapsulated in a LNP.

Figure 53 shows serum cytokine levels 4 and 24 hours post-administration of nanoplasmid DNA encapsulated in a LNP.

Figure 54 shows serum AST and ALT levels 4 and 24 hours post-administration of nanoplasmid DNA encapsulated in a LNP.

Figure 55 shows serum AST and ALT levels 4 and 24 hours post-administration of nanoplasmid NA encapsulated in a LNP.

Figure 56 shows a cartoon of a nonsense mutation resulting in a truncated protein and disease condition.

Figure 57 shows a schematic of an exemplary anticodon-engineered (ACE) tRNA design to rescue Arg-TGA PTCs.

Figure 58 is a schematic of a reporter assay in which HEK293T cells were stably transfected with a nanoluciferase reporter gene with a TGA premature stop codon.

Figure 59A shows three replicates of fold-change of luminescence activity that is normalized to untreated control after treatment with various doses of the ACE-tRNA.

Figure 59B shows six replicates of fold-change of luminescence activity, normalized to untreated control, after treatment with various doses of ACE-tRNA.

Figure 60 shows protein restoration following treatment with ACE-tRNA, but not lipid nanoparticles encapsulating nanoplasmid DNA for native tRNA (control). Figure 61A shows dose-dependent uptake of nanoplasmid DNA, expression of ACE- tRNA, and protein restoration (as measured by nanoluciferase activity).

Figure 61B shows dose-dependent uptake of nanoplasmid DNA in a cell-based assay.

Figure 61C shows dose-dependent expression of ACE-tRNA in a cell-based assay.

Figure 62A is a schematic overview of the experimental design to evaluate codonspecific restoration of full-length protein by ACE-tRNA.

Figure 62B shows codon- specific suppression of three different PTCs using different ACE-tRNAs.

Figure 63 shows a schematic overview of a reporter assay with B-domain deleted FVIII. HEK293T cells were transfected with a B-domain deleted FVIII gene with a TGA premature stop codon.

Figure 64 shows western blot analysis of cell lysates after treatment with ACE-tRNA, showing restoration of B-domain deleted FVIII.

Figure 65 shows fold change FLAG expression after treatment with ACE-tRNA, relative to untreated.

Figure 66 shows fold change in B-domain deleted FVIII activity after treatment with ACE-tRNA, relative to untreated.

Figure 67 shows a schematic overview of a reporter assay with full-length FVIII. COS- 7 cells were transfected with full-length FVIII gene with a TGA premature stop codon.

Figure 68 shows dose-dependent FVIII coagulation activity in the supernatant of cells treated with ACE-tRNA.

Figure 69 shows a schematic overview of an in vivo assay in which AlexFluor647- labeled nanoplasmid DNA was administered intravenously and tissues were collected 4 hours after administration.

Figure 70 shows biodistribution in tissues measured by fluorescence intensity after intravenous injection of vehicle, 0.1 mg/kg ACE-tRNA, or 1 mg/kg ACE-tRNA.

Figure 71 shows cellular distribution in liver measured by flow cytometry after intravenous injection of vehicle, 0.1 mg/kg ACE-tRNA, or 1 mg/kg ACE-tRNA.

Figure 72 shows a schematic overview of an in vivo assay in which LumA mice were injected intravenously with vehicle (control), LNP encapsulating nanoplasmid DNA encoding native tRNA (Native tRNA npDNA), or LNP encapsulating nanoplasmid DNA encoding Ace tRNA (ACE-tRNA npDNA). Figure 73 shows IVIS imaging of firefly luciferase in whole animals three days postinjection.

Figure 74 shows a schematic overview of a high throughput assay to evaluate npDNA uptake, ACE-tRNA expression, and PTC rescue activity. Analyses include quantitative polymerase chain reaction (qPCR), reverse transcription quantitative PCR (RT-qPCR), and nanoluciferase (Nluc) assay.

Figure 75 shows npDNA uptake (left panel), ACE-tRNA expression (middle panel), and nanoluciferase activity (right panel) in cells treated with 1 pg/ml npDNA assayed over 48 hours.

Figure 76 shows npDNA uptake and ACE-tRNA expression in cells treated with 1 pg/ml npDNA assayed over 48 hours.

Figure 77 shows ACE-tRNA expression and nanoluciferase activity in cells treated with 1 pg/ml npDNA assayed over 48 hours.

Figure 78 shows npDNA uptake (left panel), ACE-tRNA expression (middle panel), and nanoluciferase activity (right panel) in cells treated with 0.3 pg/ml npDNA assayed over 48 hours.

Figure 79 shows npDNA uptake and ACE-tRNA expression in cells treated with 0.3 pg/ml npDNA assayed over 48 hours.

Figure 80 shows ACE-tRNA expression and nanoluciferase activity in cells treated with 0.3 pg/ml npDNA assayed over 48 hours.

Figure 81 shows a western blot of full-length Factor VIII rescue in cell lysates after treatment with 1 pg/ml npDNA.

Figure 82 is a schematic overview of an assay to evaluate four cell lines for their ability to produce full-length Factor VIII. HEK293T, COS-7, CHO, and BHK cells were transiently transfected with a full-length Factor VIII with an R-TGA PTC and treated with npDNA. Factor VIII activity was assayed 48 hours after npDNA treatment by FLAG tag quantitation.

Figure 83 shows rescue of Factor VIII activity, relative to wild-type Factor VIII activity, in CHO, BHK, COS-7, and HEK293T (pink) cells (untreated) or transfected with full- length Factor VIII (WT), full-length Factor VIII comprising an R-TGA PTC (PTC), or full- length Factor VIII comprising an R-TGA PTC and administered 0.25 pg/ml, 0.5 pg/ml, or 1 pg/ml npDNA. Figure 84 shows a schematic overview of an assay to evaluate location dependency of PTC rescue in nanoluciferase. Constructs were designed to have a PTC at R13, R45, K77, R114, or R143 in nanoluciferase. Rescue of luciferase activity was evaluated 48 hours postadministration of npDNA.

Figure 85 shows a schematic overview of an assay to evaluate location dependency of PTC rescue in full-length Factor VIII. Constructs were designed to have a PTC at R15, R427, R814, R1215, R1715, R2116, or R2228 in full-length Factor VIII. Rescue of full-length Factor VIII activity was measured by FLAG tag quantitation 48 post-administration of npDNA.

Figure 86 shows rescue of nanoluciferase activity in constructs comprising a PTC at R13 (circle), R45 (square), K77 (up triangle), R114 (down triangle), or R143 (diamond) 48 hours after administration of npDNA.

Figure 87 shows rescue of full-length Factor VIII activity in constructs comprising a PTC at R15, R427, R814, R1215, R1715, R2116, or R2228, 48 hours after administration of npDNA.

Figure 88 shows four strategies for biodistribution analysis: 1) labeling using AF647,

2) DNA in situ hybridization (DNA ISH), using nanoplasmid specific oligonucleotide probes;

3) EGFP expression using nanoplasmids expressing EGFP, and 4) TdTomato Expression using plasmids expressing Cre.

Figure 89 shows DNA in situ hybridization of mouse liver sections following intravenous administration of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA. Red arrows point indicate npDNA uptake in liver sinusoidal endothelial cells.

Figure 90 plots the optical density of npDNA in mouse liver following intravenous administration of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to mouse liver obtained from a naive mouse.

Figure 91 is a schematic overview of an assay to evaluate the effect of dexamethasone pre-treatment on cytokine production levels following npDNA administration. Dexamethasone (10 mg/kg) is administered to CD1 mice 3 hours prior to intravenous administration of 1 mg/kg npDNA. Serum is collected 4, 24, and 72 hours post-npDNA administration and evaluated for cytokine and liver enzyme levels.

Figure 92 shows serum IL-6 levels in CD1 mice administered vehicle, or 4 hours, 24 hours, or 72 hours post-npDNA administration with dexamethasone pre-treatment (w Dex) or without dexamethasone pre-treatment (wo Dex). Figure 93 shows serum IFN-a levels in CD1 mice administered vehicle, or 4 hours, 24 hours, or 72 hours post-npDNA administration with dexamethasone pre-treatment (w Dex) or without dexamethasone pre-treatment (wo Dex).

Figure 94 shows serum IFN-y levels in CD1 mice administered vehicle, or 4 hours, 24 hours, or 72 hours post-npDNA administration with dexamethasone pre-treatment (w Dex) or without dexamethasone pre-treatment (wo Dex).

Figure 95 shows serum alanine aminotransferase (ALT) levels in CD1 mice administered vehicle, or 4 hours, 24 hours, or 72 hours post-npDNA administration with dexamethasone pre-treatment (w Dex) or without dexamethasone pre-treatment (wo Dex).

Figure 96 shows a schematic of the method used to evaluate the dependence of cytokine release on the STING pathway.

Figure 97 shows serum alanine aminotransferase (ALT) levels in C57BL/6 STING knockout (KO) mice administered vehicle or 1 mg/kg npDNA (red) and in C57BL/6 wild-type mice administered 1 mg/kg npDNA with or without dexamethasone pre-treatment (black).

Figure 98 shows serum aspartate aminotransferase (AST) levels in C57BL/6 STING knockout (KO) mice administered vehicle or 1 mg/kg npDNA (red) and in C57BL/6 wild-type mice administered 1 mg/kg npDNA with or without dexamethasone pre-treatment.

Figure 99 shows a schematic overview of an assay to evaluate ACE-tRNA expression in mouse liver. CD1 mice are pre-treated with 10 mg/kg dexamethasone 3 hours prior to intravenous administration of npDNA at 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg. 72 hours post-npDNA administration, mouse livers are collected and evaluated by qPCR or RT- qPCR.

Figure 100 shows copy number of npDNA per mg of liver tissue in dexamethasone pre-treated mice measured 72 hours post-administration of vehicle, 0.1 mg/kg npDNA, 0.25 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA.

Figure 101 shows copy number of ACE-tRNA per mg of liver tissue in dexamethasone pre-treated mice measured 72 hours post-administration of vehicle, 0.1 mg/kg npDNA, 0.25 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA.

Figure 102 shows a schematic overview of an assay to evaluate the durability of npDNA and ACE-tRNA expression in mice. CD1 mice are optionally pre-treated with 10 mg/kg dexamethasone 3 hours prior to intravenous administration of 1 mg/kg npDNA. Mouse liver is collected on days 1, 3, 7, 14, and 30 and evaluated for npDNA and ACE-tRNA expression.

Figure 103 shows npDNA copies (relative to vehicle) and ACE-tRNA expression (relative to vehicle) in mouse evaluated on days 1, 3, 7, 14, and 30 post-npDNA administration.

Figure 104 is a schematic of an assay to evaluate immune stimulation in PBMCs derived from mouse, non-human primate, or human. M<I> = macrophages; NK = natural killer cells; DC = dendritic cell.

Figure 105 is an overview of immune stimulation in mouse, non-human primate (NHP), and human PBMCs. Dark = responsive; Light = not responsive.

Figure 106 shows IP- 10 levels measured in human, non-human primate (NHP), and mouse PBMC supernatant after treatment with varying npDNA doses, relative to untreated PMBC supernatant.

Figure 107 shows the maximum fold change in IP- 10 levels measured in human, non- human primate (NHP), and mouse PBMC supernatant after treatment with npDNA relative to untreated PBMC supernatant.

Figure 108 is a schematic overview of a non-human primate study. Arrows indicate npDNA dosing events; dexamethasone pre-treatment 3 hours prior to administration of npDNA; necropsy events; brackets designate sampling windows.

Figure 109 shows change in body weight 1 day prior to or days 1, 2, and 3 after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP.

Figure 110 shows change in body temperature 1 day prior to or days 1, 2, and 3 after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP.

Figure 111 shows serum TNF-a levels measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP.

Figure 112 shows serum INF-a levels measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP. Figure 113 shows serum AST levels measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP. Light grey shaded region indicates moderate increase in serum AST levels (up to 5-fold) relative to vehicle-treated NHP; dark grey shaded region indicates severe increase in serum AST levels (up to 10-fold) relative to vehicle-treated NHP.

Figure 114 shows serum ALT levels measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP. Light grey shaded region indicates moderate increase in serum ALT levels (up to 5-fold) relative to vehicle-treated NHP; dark grey shaded region indicates severe increase in serum ALT levels (up to 10-fold) relative to vehicle-treated NHP.

Figure 115 shows white blood cell counts measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP. Light grey shaded region indicates moderate increase in white blood cell counts (up to 5-fold) relative to vehicle-treated NHP; dark grey shaded region indicates severe increase in white blood cell counts (up to 10-fold) relative to vehicle-treated NHP.

Figure 116 shows neutrophil counts measured 24 hours prior to or 6, 24, and 724 hours after administration of a first or second dose of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to naive NHP. Light grey shaded region indicates moderate increase in neutrophil counts (up to 5-fold) relative to vehicle-treated NHP; dark grey shaded region indicates severe increase in neutrophil cell counts (up to 10-fold) relative to vehicle- treated NHP.

Figure 117 shows npDNA copies per mL plasma in naive NHP, compared to vehicle- treated NHP or NHP treated with 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA. npDNA copies were measured 5 minutes, 30 minutes, 2 hours, 6 hours, 24 hours, and 72 hours post first and second dose.

Figure 118 shows npDNA copies per mg of liver, spleen, lung, colon, or kidney tissue obtained from naive NHP, vehicle-treated NHP, or NHP treated with 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA. Figure 119 shows relative ACE-tRNA expression (compared to vehicle) in NHP liver and spleen obtained from naive NHP, vehicle-treated NHP, or NHP treated with 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA.

Figure 120 shows DNA in situ hybridization of NHP liver sections following intravenous administration of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA. Arrows point indicate npDNA uptake in liver sinusoidal endothelial cells.

Figure 121 plots the optical density of npDNA in NHP liver following intravenous administration of vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA compared to NHP liver obtained from a naive NHP.

Figure 122 shows a schematic overview of an assay evaluating the effects of acute steroid treatment on cytokine production in non-human primates. 2 mg/kg dexamethasone is intramuscularly injected 24 hours and 2 hours prior to intravenous infusion of 0.5 mg/kg npDNA. Dexamethasone is additionally injected 2 hours post-npDNA administration. NHP sera is collected prior to the first dexamethasone injection, and at 6, 24, and 72 hours post- npDNA administration.

Figure 123 shows serum IL-6 levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 124 shows serum TNF-a levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 125 shows serum IFN-y levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 126 shows serum IP- 10 levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration. Figure 127 shows serum MCP-1 levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 128 shows serum ALT levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 129 shows serum AST levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 130 shows serum creatine kinase levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 131 shows serum C-reactive protein levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 132 shows fasting plasma glucose levels from vehicle-treated NHP, NHP treated with 0.5 mg/kg without dexamethasone treatment, and NHP treated with 0.5 mg/kg with dexamethasone treatment measured 24 hours prior to npDNA administration, immediately prior to npDNA expression (marked by arrow), and 1 and 3 days post-npDNA administration.

Figure 133 is a schematic of a phase 1 dose escalation study.

Figure 134 is a flowchart illustrating the dose escalation scheme for a phase 1 study.

Figure 135 is a schematic of a chromogenic substrate assay that can be utilized to measure Factor VIII activity in individuals on Hemlibra.

Figure 136 shows rescue of full-length Factor VIII activity with a PTC at R427, with or without Hemlibra, following treatment with vehicle (0) or 1 pg/mL npDNA (1).

Figure 137 shows rescue of full-length Factor VIII activity with a PTC at R814, with or without Hemlibra, following treatment with vehicle (0) or 1 pg/mL npDNA (1). Figure 138 shows results of a Factor VIII ELISA test demonstrating rescue of Factor VIII activity in COS-7 and HEK293T cells following administration of npDNA (+), compared to untreated cells, vehicle-treated cells (-), or cells transfected with wild-type full-length Factor VIII.

Figure 139 is a schematic overview of a cell-based assay to characterize rescue of FVIII-PTC (R427X). COS-7 cells were transfected with full-length FVIII gene with a TGA PTC.

Figure 140 shows FVIII protein measured in cell lysates.

Figure 141 shows FVIII coagulation activity by Coatest in the supernatant of cells treated with npDNA increases with increasing doses of npDNA.

Figure 142 shows FVIII coagulation activity (Coatest) from multiple cellular systems, CHO, BHK, and COS-7 cells.

Figure 143 shows dose-dependent uptake of nanoplasmid DNA in HUVEC cells.

Figure 144 shows dose-dependent expression of ACE-tRNA in HUVEC cells.

Figure 145 shows dose-dependent uptake of nanoplasmid DNA in LSEC cells.

Figure 146 shows dose-dependent expression of ACE-tRNA in LSEC cells.

Figure 147 is a schematic of the experimental design. LumA mice were given an intravenous (i.v.) administration of vehicle or npDNA (0.1 mg/kg).

Figure 148 is a representative IVIS imaging of firefly luciferase signal in liver on day 3.

Figure 149 shows quantification of luciferase signal in liver.

Figure 150 is a schematic overview of a study design to evaluate restoration of full- length mCherry protein in liver sinusoidal endothelial cells in vivo.

Figure 151 shows flow cytometry quantification of mCherry-positive liver cell types (Hepatocytes, Kupffer cells, and LSECs).

Figure 152 shows plasma persistence of nanoplasmid DNA up to 72 hours after IV administration of npDNA (1 mg/kg), measured by qPCR. N=5 to 9 per timepoint.

Figure 153 is a schematic overview of the experimental design to evaluate biodistribution of npDNA in mice.

Figure 154 shows the biodistribution of nanoplasmid DNA in tissues measured by qPCR. Figure 155 shows liver persistence of nanoplasmid DNA up to 3 months after IV administration, measured by qPCR. N=8 to 10 per timepoint.

Figure 156 shows liver persistence of nanoplasmid DNA at 1 month after IV administration in NHP, measured by qPCR.

DETAILED DESCRIPTION

Transfer RNA are decoders of DNA and RNA “blueprints” and help to make the proteins that form the structure of cells and tissues. These RNA molecules can be modified or engineered such that they can enable the systematic “recoding” of the genetic code. Provided herein are compositions and methods related to a technology based on site-directed changes in transfer RNA (tRNA) such that they can be used to alter amino acids of codons in polypeptide or protein production.

“Nonsense suppressors” are alleles of tRNA genes that contain an altered anticodon, such that instead of triggering a “stop” signal, they insert an amino acid in response to a termination codon. For example, an ochre mutation results in the creation of a UAA codon in an mRNA. An ochre suppressor gene produces tRNA with an AUU anticodon that inserts an amino acid at the UAA site, which permits the continued translation of the mRNA despite the presence of a codon that would normally trigger a stop in translation. The tRNAs provided herein can be engineered to target any one or more of unwanted stop codons, such as any one or more of the stop codons provided herein, in any of the genes associated with a disease or disorder as provided herein. The disease or disorder may be a “liver-based disease”, which term refers to any disease where disease occurrence, pathology and/or symptoms involve, originate from, or occur in liver cells or liver tissue and/or any disease where delivery and/or uptake of the compositions provided herein to liver cells or tissue can result in treatment of the disease, inhibition of disease progression, and/or alleviation of one or more symptoms of the disease. The disease or disorder may be an “endothelial cell-based disease”, which term refers to any disease where disease occurrence, pathology and/or symptoms involve, originate from, or occur in endothelial cells and/or any disease where delivery and/or uptake of the compositions provided herein to endothelial cells can result in treatment of the disease, inhibition of disease progression, and/or alleviation of one or more symptoms of the disease. Preferably, in an embodiment, the disease or disorder is both liver-based and endothelial cellbased. In an embodiment, the disease or disorder is preferably monogenic. “Monogenic” as used herein refers to an inherited condition caused by a mutation in a single gene. The term is meant to encompass any disease or disorder where it is related to or controlled by a single gene. In an embodiment, the disease or disorder is preferably a bleeding disorder. As used herein, “bleeding disorder” refers to a group of conditions that result when the blood cannot clot properly.

In some embodiments, the bleeding disorder is hemophilia (e.g., Hemophilia A or Hemophilia B). In some embodiments, the bleeding disorder is hemophilia A. Hemophilia A is a rare, congenital, recessive X-linked disorder caused by the lack of or deficiency of clotting factor VIII (FS). Exemplary F8 mRNA sequences may be found, for example, in NCBI Accession Nos.: NM_000132.4 and NM_019863.3. The severity of the disease depends on the reduction of levels of F8, which are determined by the type of mutation in the gene encoding F8. Approximately 20% of severe hemophilia A cases in the United States are caused by nonsense mutations that result in premature termination codons (PTCs) in the F8 gene result. Of these nonsense mutations, approximately 20% result in a TAA (stop) PTC and approximately 20% result in a TAG (stop) PTC. The majority of nonsense mutations (>50%) were found to be a result of an Arg to TGA (stop) nonsense mutation. These nonsense mutations are heterogeneously dispersed across F8 gene locations (e.g., R15X, R427X, R814X, R1215X, R1715X, R2116X, and R2228X). Any one of the methods or compositions provided herein may be used to target any one of the foregoing nonsense mutations in any one of the cells provided herein, such as liver cells, such as hepatocytes, Kupffer cells or LSECs.

As used herein, the amino acid coded for by its anticodon is the amino acid to which the anticodon is specific. As used herein, when referring to the amino acid specificity of the acceptor stem or arm, “specific for” refers to the amino acid that is or can be carried, delivered or provided by the acceptor stem or arm of the tRNA.

By altering anticodon loops of tRNA (the part that binds to RNA messages), tRNA sequences have been identified that possess the ability to switch protein codon meanings. In effect, switching the genetic meaning of the aforementioned codons. This therapeutic approach takes advantage of “code switching.” Code- switching by administering codon- selective amino acid conversion allows for protein modification.

It has been found that a tRNA can be changed through molecular editing of the anticodon sequence within the tRNA. This approach allows for reprogramming a codon, such as a rare codon to be substituted with an amino acid, such as a cognate amino acid. The small size of these tRNA molecules makes them amenable to ready expression, for example, a tRNA + the promoter is only -300 bp or less. A further advantage of the present invention is that it provides facile expression and cell delivery because the entire system can be compact. Briefly, an oligonucleotide can be synthesized that comprises the structural component of a tRNA gene functional in cells, such as in human cells. The sequence of this oligonucleotide is designed based upon the known sequence with substitutions made in the anticodon region of the tRNA causing the specific tRNA to recognize a codon, such as a stop codon, but deliver an alternative amino acid, such as the cognate amino acid. tRNAs have a general four-arm structure comprising a T-arm, a D-arm, an anticodon arm, and an acceptor stem or arm (Figure 2). The T-arm is made up of a “T-stem” and a “T<pc loop.” Any one of the tRNAs provided herein can comprise this four-arm structure. The tRNAs are approximately 100 nucleotides in length, in some embodiments, and can be readily introduced into cells.

In certain embodiments, the tRNA is encoded in an expression cassette. Because of the internal promoter sequences of tRNA encoding sequences, the tRNA sequence need not be included in a separate transcription unit, although one may be provided. Thus, the present invention also provides an expression cassette comprising a sequence encoding a tRNA as provided herein. In certain embodiments, the expression cassette further contains a promoter. In certain embodiments, the promoter is a regulatable promoter. In certain embodiments, the promoter is a constitutive promoter. The promoter to drive expression of the sequence encoding the tRNA to be delivered can be any desired promoter, selected by known considerations, such as the level of expression of a nucleic acid functionally linked to the promoter and the cell type in which the vector is to be used. Promoters can be an exogenous or an endogenous promoter. In certain embodiments, the promoter is a liver- specific promoter. In an embodiment of any one of the nucleic acids provided herein, the promoter may be any one of the promoters provided herein.

“Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate cell, which may include a promoter operably linked to the nucleotide sequence of interest that may be operably linked to termination signals. The expression cassette including the nucleotide sequence of interest may be chimeric. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of a regulatable promoter. The expression cassette may be or contained in a vector. In an embodiment of any one of the expression cassettes or vectors provided herein, the promoter may be any one of the promoters provided herein.

“Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The present invention provides a method of administering or delivering a nucleic acid to a cell, such as a liver cell, such as a hepatocyte, Kupffer or LSEC. Administration to the cell can be accomplished by any means, including simply contacting the cell. “Contacting” or “placing in contact” includes anything that allows for a physical and/or chemical interaction. The contact with the cells can be for any desired length of time. The cells can include any desired cell in humans as well as other large (non-rodent) mammals, such as primates, horse, sheep, goat, pig, and dog. Any one of the subjects provided herein can be a human or other mammal. The term “mammal” includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep.

Suitable methods for the administration or delivery or introduction into a subject are also provided or otherwise understood in the art. In one embodiment, pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the nucleic acid of interest, e.g., an amount sufficient to rescue a nonsense mutation, stop codon or premature termination codon, restore protein expression or function, to treat a disease or disorder as provided herein, or to reduce or ameliorate symptoms of a disease or disorder as provided herein, or an amount sufficient to confer a desired benefit.

The tRNAs can be delivered in an effective amount, and, preferably in an embodiment, into a cell with tRNA synthetase, such as endogenous tRNA synthetase. A tRNA synthetase is considered to be “endogenous” to a cell if it is present in the cell into which a tRNA is introduced according to the present invention. As will be the apparent to those of ordinary skill in the art, a tRNA synthetase may be considered to be endogenous for these purposes whether it is naturally found in cells of the relevant type, or whether the particular cell at issue has been engineered or otherwise manipulated by the hand of man to contain or express it.

The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings of this specification, an effective amount of the tRNAs provided may be empirically determined. Administration can be effected in one dose, continuously or intermittently, throughout the course of treatment. Methods of determining the most effective means and dosages of administration may vary with the composition of the therapy, target cells, such as liver cells, and the subject being treated, etc. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

Vehicles including water, aqueous saline, artificial CSF, or other known substances can be employed with the subject invention. To prepare a formulation, the purified composition can be isolated. The composition may then be adjusted to an appropriate concentration and packaged for use.

In some aspects of the present disclosure, compositions comprising the nucleic acid molecules, tRNAs, cassettes or vectors are provided. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, a pharmaceutical composition may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0 and water. Any one of the compositions provided herein can be placed in contact with, administered to or introduced into a cell with genetic transfer methods, such as transfection. The exogenous genetic material (e.g., encoding a tRNA) can be introduced into a cell in vivo by genetic transfer methods, such as transfection. Various expression vectors (z.e., vehicles for facilitating delivery of exogenous genetic material into a target cell) are known to one of ordinary skill in the art. As used herein, “transfection of cells” refers to the acquisition by a cell of new genetic material by incorporation. Thus, transfection refers to the insertion of nucleic acid into a cell using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art or are otherwise described herein.

As used herein, “exogenous genetic material” refers to a nucleic acid or an oligonucleotide, either natural or synthetic, that is not naturally found in the cells; or if it is naturally found in the cells, it is not transcribed or expressed at biologically significant levels by the cells. Thus, “exogenous genetic material” includes, for example, a non-naturally occurring nucleic acid that can be transcribed into a tRNA.

Typically, the exogenous genetic material includes the heterologous gene together with a promoter to control transcription of the new gene. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (e.g., enhancers) required to obtain the desired gene transcription activity. The exogenous genetic material may introduced into the cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence.

In addition to at least one promoter and at least one heterologous nucleic acid, the expression vector may include a selection gene, for example, green fluorescent protein (GFP), for facilitating selection of cells that have been transfected with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the tRNA, the other vector containing a selection gene. The selection of a suitable promoter, enhancer, selection gene and/or signal sequence is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.

In an embodiment of any one of the compositions or methods provided herein, the nucleic acid is DNA that encodes for the at least one tRNA.

In an embodiment of any one of the compositions or methods provided herein, the DNA is in a closed form, such as in a plasmid, nanoplasmid or minicircle. In an embodiment of any one of the compositions or methods provided herein, the DNA is a linear DNA, such as a DNA thread. In an embodiment of any one of the compositions or methods provided herein, the expression cassette in any one of the formats described herein further comprises a promoter as provided herein.

In an embodiment of any one of the compositions or methods provided herein, promoters (or leader sequences) may be between (35-105 bp in size). The promoters may be any known promoters, including native tRNA leader sequences, which sequences may be ~ 50- 60 bp in size. In an embodiment of any one of the compositions or methods provided herein, the promoter (or leader sequences) may be reduced- sequence or re-configured promoters. Example promoter or leader sequences that may be comprised in any one of the nucleic acids, expression cassettes (or constructs) or vectors provided herein include, but are not limited to, any one of the following sequences. Accordingly, in an embodiment, any one of the expression cassettes provided herein may comprise any one of the promoter or leader sequences provided in Table 1. In one aspect, such nucleic acids, expression cassettes or vectors are provided herein.

Table 1

Any one of the expression cassettes as provided herein can include a heterologous gene (encoding a tRNA), preferably, together with a promoter to control transcription. The heterologous gene may be introduced into a nucleic acid immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence.

The nucleic acids, expression cassettes or vectors encoding the tRNA(s) as provided herein can be generated synthetically.

Nucleotide sequences encoding several hundred human tRNAs are known and generally available to those of skill in the art through sources such as GenBank. The structure of tRNAs is highly conserved, and tRNAs can be functional across species. Thus, bacterial or other eukaryotic tRNA sequences are also potential sources for the tRNAs of the invention. The determination of whether a particular tRNA is functional as desired, such as in a desired mammalian cell, can be ascertained as described herein or through other experimentation that will be apparent to one of ordinary skill in the art with the benefit of the teachings provided herein. In an embodiment of any one of the compositions or methods provided herein, the tRNA sequences may be any of the sequences provided in PCT/US2018/059065, W02019/090154, W02019/090169, and Lueck et al., Nature Communications 10, 822, 2019, the sequences of which are incorporated herein by reference. In an embodiment of any one of the compositions or methods provided herein, the tRNA sequence is an arginine suppressor tRNA. In an embodiment of any one of the compositions or methods provided herein, each of the tRNA sequences of the nucleic acid, expression cassette or vector may be 70-90 nt in length.

In an embodiment, any one of the nucleic acids, expression cassettes or vectors provided herein may comprise at least one, at least two or at least three heterologous nucleic acid sequences encoding any one of the tRNAs of Table 2.

Table 2

Optionally, the nucleic acids, expression cassettes or vectors provided herein may further include additional sequences (e.g., 3’ tRNA tail or trailer sequences). Such sequences may be between 2-20 bp in length. They may include natural sequences or engineered variants with 3-10 consecutive “T” residues. In an embodiment, any one of the nucleic acids, expression cassettes or vectors provided herein may comprise any one of the sequences of

Table 3.

Table 3

Minimal random or restriction-enzyme site spacer sequences of 0 to 80 bp may also be included in any one of the expression cassettes provided herein. Example restriction enzyme sequences that could be introduced in part, in whole, and/or as part of a joining series include, but are not limited to, those of Table 4. In an embodiment, any one of the nucleci acids, expression cassettes or vectors provided herein may comprise any one of the sequences of Table 4.

Table 4

In an embodiment, any one of the nucleci acids, expression cassettes or vectors comprises any one of the sequences of Table 5.

Table 5

In some embodiments, the present disclosure provides compositions that comprise any one of the nucleic acids, expression cassettes or vectors disclosed herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the tRNA is delivered into the nucleus of a cell.

The present invention includes compositions and methods for rescuing a stop codon or premature termination codon (e.g., any one of the stop codons or premature termination codons described herein) and/or restoring protein function in cells of the liver, such as endothelial cells of the liver, such as hepatocytes, Kupffer cells, or LSECs through the use of the tRNAs, or nucleic acids that encode them, as provided herein. The present invention in another embodiment includes compositions and methods for treating a monogenic, liver-based bleeding disorder, such as hemophilia (e.g., hemophilia A or B, such as severe hemophilia A or B) through administration or delivery of the tRNAs, or nucleic acids that encode them, as provided herein. In an embodiment, the tRNA comprises an anticodon specific for TGA and delivers an arginine.

In some embodiments, a tRNA, or nucleic acid that encodes it, is uncharged with an amino acid when administered to a subject to delivered to a cell, and can become charged in the subject or in the cell. Certain embodiments of the present disclosure provide a method of administering or delivery to a subject. A subject may be a mammal. In certain embodiments, the mammal is human. Certain embodiments of the present disclosure provide a use of a tRNA, nucleic acid, expression cassette, vector or composition as described herein to prepare a medicament useful for rescuing a stop codon or premature termination codon (e.g., any one of the stop codons or premature termination codons described herein) and/or restoring protein function in cells of the liver, such as endothelial cells of the liver, such as hepatocytes, Kupffer cells, or LSECs. Certain embodiments of the present disclosure provide a use of a tRNA, nucleic acid, expression cassette, vector or composition as described herein to prepare a medicament useful for treating a monogenic, liver-based bleeding disorder, such as hemophilia (e.g., hemophilia A or B, such as severe hemophilia A or B) in a subject, such as a mammal, such as a human.

The present disclosure also provides a cell containing a tRNA, oligonucleotide, expression cassette, or vector described herein. The cell may be mammalian, such as human. According to one aspect, a cell expression system is provided. The expression system comprises a cell and an expression cassette as provided herein. Expression cassettes include, but are not limited to, plasmids, viral vectors, and other vehicles for delivering heterologous genetic material to cells. The cell expression system can be formed in vivo.

According to yet another aspect, a method for treating a subject in vivo is provided. The method includes introducing any one of the tRNAs, nucleic acids, expression cassettes, vector, or compositions provided herein to a subject in vivo. The subject may be mammalian, such as human.

The terms “treat” and “treatment” refer to both therapeutic treatment and measures that can alleviate symptoms or provide some benefit to a subject, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition, disease or disorder.

The phrase “therapeutically effective amount” means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. The agents of the invention can be administered so as to result in a reduction in at least one symptom associated with a disease or disorder as provided herein. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal. Such factors can be readily determined by the clinician employing animal models or other test systems that are known to the art.

Administration of a tRNA, nucleic acid, expression cassette, vector, or composition in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is therapeutic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

One or more suitable unit dosage forms having the tRNA, nucleic acid, expression cassette, vector, or composition of the invention may be formulated and can be administered by a variety of routes. When the agents of the invention are prepared for administration, they may be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. Pharmaceutical formulations containing the agents of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. The agents of the invention can also be formulated as solutions appropriate for administration. The pharmaceutical formulations of the agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

Thus, the agent may be formulated for administration and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in a suitable vehicle, e.g., sterile, pyrogen-free water, before use. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0 and water.

Any of the compositions provided herein can be placed in contact with, administered to or introduced into a cell, with genetic transfer methods, such as transfection or transduction. Thus, any of the compositions provided herein can be included with or in a gene delivery vehicle. The gene delivery vehicle can be any delivery vehicle known in the art and can include naked nucleic acid that is facilitated by a receptor and/or lipid mediated transfection, as well as any of a number of vectors. Vectors include but are not limited to eukaryotic vectors, prokaryotic vectors (such as for example bacterial vectors) and viral vectors including, but not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentivirus vectors (human and other including porcine), Herpes virus vectors, Epstein-Barr viral vectors, SV40 virus vectors, pox virus vectors, and pseudotyped viral vectors.

The compositions provided herein can be contacted with cells or delivered or administered to a subject within a particle, such as a nanoparticle. A particle, such as a nanoparticle, can be, but is not limited to, lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles where the majority of the material that makes up their structure are lipids), virus-like particles (i.e., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), and/or particles with a combination of nanomaterials. The particles may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like.

In some embodiments, particles, such as nanoparticles, may comprise one or more lipids. In some embodiments, particles, such as nanoparticles, may comprise liposomes. In some embodiments, particles, such as nanoparticles, may comprise a lipid bilayer. In some embodiments, particles, such as nanoparticles, may comprise a lipid monolayer. In some embodiments, particles, such as nanoparticles, may comprise a micelle.

In some embodiments, the synthetic carrier comprises a lipid nanoparticle (LNP), a liposome, a polymeric nanoparticle (NP), a polymersome, electrostatic complexes, an exosome, a polymer/lipid based nanocarrier, and/or a protein-based nanocarrier.

The compositions provided herein can be contacted with cells or delivered or administered to a subject with an extracellular vesicle or exosome. Generally, exosomes are nano-sized extracellular vesicles (EVs) (30-150 nm in diameter) which can be formed and released by many mammalian cells. The EVs or exosomes can be loaded with an agent of interest, for example by pre-treatment of cells with the agent and then isolation of loaded EVs or exosomes.

EVs or exosomes can be derived from human embryonic kidney cells, bone marrow stem cells, immature dendritic cells, red blood cells as well as from milk. EVs or exosomes can be isolated and purified with a number of different techniques. Such methods include, but are not limited to ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), precipitation with polymers, and separation by affinity-based methods, such as immunomagnetic -based isolation.

In an embodiment of any one of the compositions or methods provided herein, a nucleic acid sequence encoding a tRNA is in a closed-end form, such as in a plasmid, nanoplasmid or minicircle. In an embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encoding a tRNA is in the form of a linear DNA, such as a DNA thread. In an embodiment of any one of the compositions or methods provided herein, the DNA is in any one of the formats provided herein, such as in a plasmid, a nanoplasmid, a minicircle, a DNA fragment, linear DNA, a DNA thread, or a closed-end DNA thread.

The term “minicircle”, as used herein, refers to small circular DNA fragments that are largely or completely free of non-essential prokaryotic elements. Minicircles include circular forms of DNA without prokaryotic elements and/or in which prokaryotic elements have been removed. Minicircles can be from a parental plasmid where bacterial DNA sequences have been excised. The minicircle may be in the form of any suitable recombinant plasmid that comprises a heterologous nucleic acid sequence to be delivered to a target cell, either in vitro or in vivo. The preparation of minicircles have been described in the art (e.g., in Nehlsen et al., Gene Ther. Mol. Biol. 10: 233-244, 2006; and Kay et al., Nature Biotechnology. 28: 1287- 1289, 2010). The preparation can, for example, follow a two-step procedure: (i) production of a ‘parental plasmid’ (bacterial plasmid with eukaryotic inserts); and (ii) induction of a sitespecific recombinase at the end of this process. These steps can be followed by the excision of prokaryotic vector parts via recombinase-target sequences and recovery by capillary gel electrophoresis.

As a nonlimiting example, a minicircle may be produced as follows. An expression cassette, which comprises the polynucleotide coding sequence along with regulatory elements for its expression, is flanked by attachment sites for a recombinase. A sequence encoding the recombinase is located outside of the expression cassette and includes elements for inducible expression (such as, for example, an inducible promoter). Upon induction of recombinase expression, the vector DNA is recombined, resulting in two distinct circular DNA molecules. One of the circular DNA molecules is relatively small, forming a minicircle that comprises the expression cassette for the polynucleotide; this minicircle DNA vector is devoid of any bacterial DNA sequences. The second circular DNA sequence contains the remaining vector sequence, including the bacterial sequences and the sequence encoding the recombinase. The minicircle DNA containing the polynucleotide sequence can then be separately isolated and purified. In some embodiments, a minicircle DNA vector may be produced using plasmids similar to pBAD.([).C31.hFIX and pBAD.([).C31.RHB. See, e.g., Chen et al. (2003) Mol. Ther. 8:495-500, or as otherwise provided herein.

Examples of recombinases that may be used for creating a minicircle include, but are not limited to, Streptomyces bacteriophage (|)31 integrase, Cre recombinase, and the integrase/DNA topoisomerase IV complex. Each of these recombinases catalyzes recombination between distinct sites. For example, (|)31 integrase catalyzes recombination between corresponding attP and attB sites, Cre recombinase catalyzes recombination between loxP sites, and the integrase/DNA topoisomerase IV complex catalyzes recombination between bacteriophage attP and attB sites.

Published US Application 20170342424 also describes a system making use of a parent plasmid which is exposed to an enzyme which causes recombination at recombination sites, thereby forming a (i) minicircle including the polynucleotide sequence and (ii) miniplasmid comprising the remainder of the parent plasmid. One recombination site is modified at the 5’ end such that its reaction with the enzyme is less efficient than the wild type site, and the other recombination site is modified at the 3’ end such that its reaction with the enzyme is less efficient than the wild type site, and the other recombination site is modified at the 3’ end such that its reaction with the enzyme is less efficient than the wild type site, both modified sites being located in the minicircle after recombination.

Removal of prokaryotic sequences ideally should be efficient, using the smallest possible excision site, while creating supercoiled DNA minicircles which consist solely of gene expression elements under appropriate — preferably mammalian — control regions. Some techniques for minicircle production use bacterial phage lambda ( ) integrase mediated recombination to produce minicircle DNA. See, for example, Darquet, et al. 1997 Gene Ther 4(12): 1341-9; Darquet et al. 1999 Gene Ther 6(2): 209-18; and Kreiss, et al. 1998 Appl Micbiol Biotechnol 49(5):560-7).

Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). For example, a MC-EasyTM (Cat # MN920A-1, SBI System Biosciences) Minicircle DNA production kit can be used to obtain high-quality minicircle DNA. Information on minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 21 8, 1526-1535 and Hou et al., Molecular Therapy — Methods & Clinical Development, Article number: 14062 (2015) doi:10.1038/mtm.2014.62. More information on Minicircles is provided in Chen Z Y, He C Y, Ehrhardt A, Kay M A. Mol Ther. 2003 September; 8(3):495-500 and Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Gracey Maniar L E, Maniar J M, Chen Z Y, Lu J, Fire A Z, Kay M A. Mol Ther. 2013 January; 21(1):131.

In some embodiments, the closed-end form is a supercoiled helix. DNA supercoiling refers to the amount of twist in a particular DNA strand. Supercoiled DNA can be positively supercoiled DNA or negatively supercoiled DNA. As used herein, “supercoiled DNA” refers to a DNA molecule, or fragment of a DNA molecule, wherein one or both DNA strands comprise increased twisting compared to the amount of twisting in a reference state known as “relaxed B-form” DNA. In a “relaxed” double-helical segment of DNA, the two strands twist around the helical axis once every 10.4-10.5 base pairs of sequence. A given DNA strand may be “positively supercoiled” or “negatively supercoiled” (i.e., more or less tightly wound). Supercoiling creates twist strain in the DNA strand. The amount of a strand’s supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code (which strongly affects DNA metabolism and possibly gene expression). Certain enzymes (e.g., topoisomerases) are capable of increasing or decreasing the amount of twisting (e.g., supercoiling) in a DNA strand in order to facilitate functions such as DNA replication and transcription. If a DNA segment under twist strain is closed into a circle by joining its two ends, and then allowed to move freely, it may form a supercoiled structure. Examples of supercoiled structures of circular DNA molecules include, but are not limited to a figure-eight structure, a plectonemic structure, or a toroidal structure.

In some embodiments, the DNA thread is any one of the DNA threads as described in PCT/US2024/020795, which DNA threads and methods of their use and production are incorporated herein by reference in their entirety. The term “DNA thread”, as used herein, refers to small DNA fragments that are largely or completely free of non-essential prokaryotic elements. In some embodiments, the nucleic acid molecule encoding the tRNA is in a closed- end, open-ended or both DNA thread.

In some embodiments of any one of the compositions or methods provided herein, DNA threads of the present disclosure are less than 500, 475, 450, 425, 400, 375, 350, 325, 300 or 275 base pairs in length. In some embodiments of any one of the compositions or methods provided herein, DNA threads of the present disclosure are less than ~ 200-300 base pairs in length. In some embodiments of any one of the compositions or methods provided herein, DNA threads of the present disclosure are less than 270 base pairs in length. In an embodiment of any one of the compositions or methods provided herein, the DNA thread is less than 265 bps, less than 260 bps, less than 255 bps, less than 250 bps, less than 245 bps, less than 240 bps, less than 235 bps, less than 230 bps, less than 225 bps, less than 220 bps, less than 215 bps, less than 210 bps, less than 205 bps, less than 200 bps, less than 195 bps, less than 190 bps, less than 185 bps, less than 180 bps, less than 175 bps, less than 170 bps, less than 165 bps, less than 160 bps, less than 155 bps, or less than 150 bps, less than 145 bps, or less than 140 bps in length.

In any one of the foregoing embodiments, the DNA thread is greater than 100 bps, greater than 105 bps, greater than 110 bps, greater than 115 bps, greater than 120 bps, greater than 125 bps, greater 130 bps, greater than 135 bps, greater than 140 bps, greater than 145 bps, greater than 150 bps, greater than 155 bps, greater than 160 bps, greater than 165 bps, greater than 170 bps, greater than 175 bps, greater than 180 bps, greater than 185 bps, greater than 190 bps, greater than 195 bps, or greater than 200 bps in length. In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 100 bps, less than 400 bps but greater than 100 bps, less than 350 bps but greater than 100 bps, less than 300 bps but greater than 100 bps, less than 275 bps but greater than 100 bps, less than 250 bps but greater than 100 bps, less than 225 bps but greater than 100 bps, less than 200 bps but greater than 100 bps, less than 195 bps but greater than 100 bps, less than 190 bps but greater than 100 bps, less than 185 bps but greater than 100 bps, less than 180 bps but greater than 100 bps, less than 175 bps but greater than 100 bps, less than 170 bps but greater than 100 bps, less than 165 bps but greater than 100 bps, less than 160 bps but greater than 100 bps, less than 155 bps but greater than 100 bps, less than 150 bps but greater than 100 bps, less than 145 bps but greater than 100 bps, or less than 140 bps but greater than 100 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 110 bps, less than 400 bps but greater than 110 bps, less than 350 bps but greater than 110 bps, less than 300 bps but greater than 110 bps, less than 275 bps but greater than 110 bps, less than 250 bps but greater than 110 bps, less than 225 bps but greater than 110 bps, less than 200 bps but greater than 110 bps, less than 195 bps but greater than 110 bps, less than 190 bps but greater than 110 bps, less than 185 bps but greater than 110 bps, less than 180 bps but greater than 110 bps, less than 175 bps but greater than 110 bps, less than 170 bps but greater than 110 bps, less than 165 bps but greater than 110 bps, less than 160 bps but greater than 110 bps, less than 155 bps but greater than 110 bps, less than 150 bps but greater than 110 bps, less than 145 bps but greater than 110 bps, or less than 140 bps but greater than 110 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 120 bps, less than 400 bps but greater than 120 bps, less than 350 bps but greater than 120 bps, less than 300 bps but greater than 120 bps, less than 275 bps but greater than 120 bps, less than 250 bps but greater than 120 bps, less than 225 bps but greater than 120 bps, less than 200 bps but greater than 120 bps, less than 195 bps but greater than 120 bps, less than 190 bps but greater than 120 bps, less than 185 bps but greater than 120 bps, less than 180 bps but greater than 120 bps, less than 175 bps but greater than 120 bps, less than 170 bps but greater than 120 bps, less than 165 bps but greater than 120 bps, less than 160 bps but greater than 120 bps, less than 155 bps but greater than 120 bps, less than 150 bps but greater than 120 bps, less than 145 bps but greater than 120 bps, or less than 140 bps but greater than 120 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 130 bps, less than 400 bps but greater than 130 bps, less than 350 bps but greater than 130 bps, less than 300 bps but greater than 130 bps, less than 275 bps but greater than 130 bps, less than 250 bps but greater than 130 bps, less than 225 bps but greater than 130 bps, less than 200 bps but greater than 130 bps, less than 195 bps but greater than 130 bps, less than 190 bps but greater than 130 bps, less than 185 bps but greater than 130 bps, less than 180 bps but greater than 130 bps, less than 175 bps but greater than 130 bps, less than 170 bps but greater than 130 bps, less than 165 bps but greater than 130 bps, less than 160 bps but greater than 130 bps, less than 155 bps but greater than 130 bps, less than 150 bps but greater than 130 bps, less than 145 bps but greater than 130 bps, or less than 140 bps but greater than 130 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 140 bps, less than 400 bps but greater than 140 bps, less than 350 bps but greater than 140 bps, less than 300 bps but greater than 140 bps, less than 275 bps but greater than 140 bps, less than 250 bps but greater than 140 bps, less than 225 bps but greater than 140 bps, less than 200 bps but greater than 140 bps, less than 195 bps but greater than 140 bps, less than 190 bps but greater than 140 bps, less than 185 bps but greater than 140 bps, less than 180 bps but greater than 140 bps, less than 175 bps but greater than 140 bps, less than 170 bps but greater than 140 bps, less than 165 bps but greater than 140 bps, less than 160 bps but greater than 140 bps, less than 155 bps but greater than 140 bps, or less than 150 bps but greater than 140 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 150 bps, less than 400 bps but greater than 150 bps, less than 350 bps but greater than 150 bps, less than 300 bps but greater than 150 bps, less than 275 bps but greater than 150 bps, less than 250 bps but greater than 150 bps, less than 225 bps but greater than 150 bps, less than 200 bps but greater than 150 bps, less than 195 bps but greater than 150 bps, less than 190 bps but greater than 150 bps, less than 185 bps but greater than 150 bps, less than 180 bps but greater than 150 bps, less than 175 bps but greater than 150 bps, less than 170 bps but greater than 150 bps, less than 165 bps but greater than 150 bps, or less than 160 bps but greater than 150 bps. In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 160 bps, less than 400 bps but greater than 160 bps, less than 350 bps but greater than 160 bps, less than 300 bps but greater than 160 bps, less than 275 bps but greater than 160 bps, less than 250 bps but greater than 160 bps, less than 225 bps but greater than 160 bps, less than 200 bps but greater than 160 bps, less than 195 bps but greater than 160 bps, less than 190 bps but greater than 160 bps, less than 185 bps but greater than 160 bps, less than 180 bps but greater than 160 bps, less than 175 bps but greater than 160 bps, or less than 170 bps but greater than 160 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 170 bps, less than 400 bps but greater than 170 bps, less than 350 bps but greater than 170 bps, less than 300 bps but greater than 170 bps, less than 275 bps but greater than 170 bps, less than 250 bps but greater than 170 bps, less than 225 bps but greater than 170 bps, less than 200 bps but greater than 170 bps, less than 195 bps but greater than 170 bps, less than 190 bps but greater than 170 bps, less than 185 bps but greater than 170 bps, or less than 180 bps but greater than 170 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 180 bps, less than 400 bps but greater than 180 bps, less than 350 bps but greater than 180 bps, less than 300 bps but greater than 180 bps, less than 275 bps but greater than 180 bps, less than 250 bps but greater than 180 bps, less than 225 bps but greater than 180 bps, less than 200 bps but greater than 180 bps, less than 195 bps but greater than 180 bps, or less than 190 bps but greater than 180 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 190 bps, less than 400 bps but greater than 190 bps, less than 350 bps but greater than 190 bps, less than 300 bps but greater than 190 bps, less than 275 bps but greater than 190 bps, less than 250 bps but greater than 190 bps, less than 225 bps but greater than 190 bps, or less than 200 bps but greater than 190 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 200 bps, less than 400 bps but greater than 200 bps, less than 350 bps but greater than 200 bps, less than 300 bps but greater than 200 bps, less than 275 bps but greater than 200 bps, less than 250 bps but greater than 200 bps, or less than 225 bps but greater than 200 bps. In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 210 bps, less than 400 bps but greater than 210 bps, less than 350 bps but greater than 210 bps, less than 300 bps but greater than 210 bps, less than 275 bps but greater than 210 bps, less than 250 bps but greater than 210 bps, or less than 225 bps but greater than 210 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 220 bps, less than 400 bps but greater than 220 bps, less than 350 bps but greater than 220 bps, less than 300 bps but greater than 220 bps, less than 275 bps but greater than 220 bps, or less than 250 bps but greater than 220 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 230 bps, less than 400 bps but greater than 230 bps, less than 350 bps but greater than 230 bps, less than 300 bps but greater than 230 bps, less than 275 bps but greater than 230 bps, or less than 250 bps but greater than 230 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 240 bps, less than 400 bps but greater than 240 bps, less than 350 bps but greater than 240 bps, less than 300 bps but greater than 240 bps, less than 275 bps but greater than 240 bps, or less than 250 bps but greater than 240 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 250 bps, less than 400 bps but greater than 250 bps, less than 350 bps but greater than 250 bps, less than 300 bps but greater than 250 bps, or less than 275 bps but greater than 250 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 260 bps, less than 400 bps but greater than 260 bps, less than 350 bps but greater than 260 bps, less than 300 bps but greater than 260 bps, or less than 275 bps but greater than 260 bps.

In some embodiments of any one of the compositions or methods provided herein, the DNA thread is less than 450 bps but greater than 270 bps, less than 400 bps but greater than 270 bps, less than 350 bps but greater than 270 bps, less than 300 bps but greater than 270 bps, less than 290 bps but greater than 270 bps, or less than 280 bps but greater than 270 bps.

It should be appreciated that the DNA thread can be any size that is compatible with the tRNA to be delivered and/or the desired effects, such as those provided herein. As a nonlimiting example, a closed-end DNA thread may be produced as follows. A circular DNA plasmid, which can comprise the polynucleotide coding sequence along with regulatory elements for its expression and at least one protelomerase resolution site (telRL), is produced. In some embodiments, the circular DNA plasmid is processed with protelomerase TelN, acting on a telRL site, to convert a circular plasmid DNA into linear covalently closed DNA construct in a single-step enzyme reaction. In some embodiments, the circular DNA plasmid can contain two or more telRL sites, which result in multiple linear closed-end DNA constructs after processing with pro telomerase. In some embodiments, a closed-end DNA thread may be produced from linear open-end DNA construct comprising at least two telRL sites. Processing a linear DNA construct comprising at least two telRL sites with protelomerase can result in at least one closed-end DNA thread.

Cell-free production of closed-end DNA threads has been described in WO 2010/086626, WO 2012/017210, and WO 2021/252354, which production steps are hereby incorporated by reference. The preparation can, for example, follow a two-step procedure: (i) production of a ‘parental plasmid’ (bacterial plasmid with eukaryotic inserts) or linear DNA construct containing a protelomerase resolution site; and (ii) induction of a protelomerase enzymatic reaction and recovery of the desired sequence by gel electrophoresis.

In an embodiment of any one of the compositions or methods provided herein, the expression cassette is comprised in a nanoplasmid. The term “nanoplasmid”, as used herein, refers to circular DNA fragments that are largely or completely free of non-essential prokaryotic elements. Nanoplasmids include circular forms of DNA without prokaryotic elements and/or in which prokaryotic elements have been removed. Nanoplasmids can be from a parental, larger plasmid where bacterial DNA sequences have been excised.

In an embodiment of any one of the compositions or methods provided herein nanoplasmids may comprise a nucleic acid, expression cassette or vector provided herein. Accordingly, the nanoplasmid may comprise a promoter, such as any one of the promoters provided herein, and a nucleic acid encoding at least one, two, three or four transfer RNAs (tRNAs), such as any one or one combination of tRNAs as provided herein. In some embodiments, the nanoplasmid is less than 3000 bp, less than 2900 bp, less than 2800 bp, less than 2750 bp, less than 2600 bp, less than 2500 bp, less than 2400 bp, less than 2300 bp, less than 2200 bp, less than 2100 bp, less than 2000 bp, less than 1900 bp, less than 1800 bp, less than 1750 bp, less than 1700 bp, less than 1600 bp, less than 1500 bp, less than 1400 bp, less than 1300 bp, less than 1250 bp, less than 1200 bp or less than 1100 bp. In any one of the foregoing embodiments, the nanoplasmid is greater than 1000 bp, greater than 1100 bp, greater than 1200 bp, greater than 1250 bp, greater than 1200 bp, greater than 1100 bp in size or greater than 1000 bp in size. In some embodiments, the nanoplasmid is less than 2000 bp but greater than 1000 bp, greater than 1100 bp, greater than 1200 bp, greater than 1300 bp, greater than 1400 bp or greater than 1500 bp in size. It should be appreciated that the nanoplasmid can be any size that is compatible with the tRNA(s) to be delivered.

A nonlimiting example of a nanoplasmid encoding a tRNA described herein comprises a nucleotide sequence as shown below:

CATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAG ATTTCCTTCAGGTTTATATAAGGAGCCATAAGAAGAATTCCTTCATGGGTGCCCCA GTGGCCTAATGGATAAGGCACTGGCCTTCAAAGCCAGGGATTGTGGGTTCGAGTC CCACCTGGGGTGGTGCTTTTTTCCATTGAGGCTATGTACTCTGATATTTAGCATGTC GCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTT ATATAAGGAGCCATAAGAAGAATTCCTTCATGGGTGCCCCAGTGGCCTAATGGAT AAGGCACTGGCCTTCAAAGCCAGGGATTGTGGGTTCGAGTCCCACCTGGGGTGGT GCTTTTTTCCAAATCTGTAGTCAAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAATCTGTAAGTCACTG ATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGA TTTCCTTCAGGTTTATATAAGGAGCCATAAGAAGAATTCCTTCATGGGTGCCCCAG TGGCCTAATGGATAAGGCACTGGCCTTCAAAGCCAGGGATTGTGGGTTCGAGTCC CACCTGGGGTGGTGCTTTTTTCTGATTGACCAATGTACTCTGATATTTAGCATGTCG CTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTA TATAAGGAGCCATAAGAAGAATTCCTTCATGGGTGCCCCAGTGGCCTAATGGATA AGGCACTGGCCTTCAAAGCCAGGGATTGTGGGTTCGAGTCCCACCTGGGGTGGTG_{CTTTTTTCATTACTCTGGATTGTACTCAGGTGTGGAAAGTCCCCAGGCTCCCCAGC} AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGCCCTACGTGC TGCCTCGCATGGCCCGGCTGACCTCTTGACCCCTCTGGGGCTAGCTGGCTTGTTGT CCACAACCATTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCTTAT AAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTC AAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACATTAGCCATGAGAG CTTAGTACATTAGCCATGAGGGTTTAGTTCATTAAACATGAGAGCTTAGTACATTA AACATGAGAGCTTAGTACATTAAACATGAGAGCTTAGTACATACTATCAACAGGT TGAACTGCTGATCTGTACAGTAGAATTGGTAAAGAGAGTTGTGTAAAATATTGAG TTCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGAC AAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGTAC (SEQ ID NO: 682)

Any one of the nucleic acids, expression cassettes or vectors may comprise the sequence shown directly above.

In an embodiment of any one of the compositions or methods provided herein, an oligonucleotide encoding any one of the tRNAs is provided. In an embodiment of any one of the compositions or methods provided herein, an oligonucleotide described herein further comprises a promoter, such as any one of the promoters provided herein. Such an oligonucleotide may be in any one of the formats provided herein or comprised in a vector.

In some embodiments, an oligonucleotide as provided herein comprises a nucleic acid sequence capable of directing expression of a particular nucleotide sequence, such as any one of the sequences provided herein, in an appropriate cell, such as any one of the cells provided herein, which may include any one of the promoters provided herein operably linked to any one of the nucleotide sequences of interest provided herein that may also be operably linked to any one of the termination signals or terminators provided herein. An oligonucleotide as provided herein may be in a recombinant form useful for heterologous expression.

In some embodiments of any one of the compositions or methods provided, the tRNAs, nucleic acids, expression cassettes or vectors are not found in nature. In some embodiments of the foregoing, the tRNA, or sequence that encodes it, is engineered or modified from that found in nature. In some embodiments of the foregoing, the tRNAs, nucleic acids, expression cassettes or vectors is recombinant.

Electroporation may also be used for delivery or administration.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

Example 1. Engineered transfer RNAs for suppression of PTCs.

Approximately 10-15% of all inherited genetic disease is driven by nonsense mutations that form premature termination codons (PTCs), leading to truncated proteins. These truncated proteins can be rescued by tRNAs as provided herein, and in some embodiments are known as anticodon engineered transfer RNAs (ACE-tRNAs). Candidate ACE-tRNAs were first evaluated for their potential to induce global readthrough of natural termination codons (NTCs). Across a range of relevant doses, candidate ACE-tRNAs were found to have minimal NTC readthrough (Figure 3). In contrast, candidate ACE-tRNAs were found to significantly correct PTCs when delivered in both a DNA nanoplasmid and a RNA nanoplasmid, as evidenced by recovery of luminescence in a reporter construct with a PTC (Figure 4). Across a range of doses (2-250 ng/mL), PTC correction was found to be between 15-300-fold more in treated cells versus untreated cells while NTC readthrough was found to be less than ~15x-fold in treated cells relative to untreated cells (Figure 5).

Further, ACE-tRNAs were found to largely behave as native tRNAs within cells. ACE- tRNAs were evaluated for their ability to be charged with the correct cognate amino acid in vitro. Amino acid charge profiling of two native Arg-tRNAs (CCT-2 and (TCT-2)) and an ACE Arg-tRNA (delivered as a DNA plasmid or a RNA plasmid) showed that ACE-tRNAs were charged with the correct amino acid (arginine) at a similar level as the two control native tRNAs (Figure 6). Additionally, the modification profile of the native tRNAs and ACE-tRNAs were found to be nearly identical (Figure 7).

These results suggest that ACE-tRNAs can effectively suppress PTCs and faithfully encode their cognate amino acid.

Example 2. ACE-tRNAs for PTC suppression of genes associated with hemophilia A.

Hemophilia A is a rare congenital, recessive X-linked disorder caused by the lack of or deficiency of clotting factor VIII (F8) and occurs in approximately 13 out of every 100,000 male births (representing about 43,000 patients in the United States), The severity of the disease depends on the reduction of levels of F8, which are determined by the type of mutation in the gene encoding F8. Multiple mutations have been described, and evaluation of their correlation with severity of disease have shown that 97% of PTCs result in severe disease (defined as <1 lU/dL or <1% of WT levels). Replacement of F8 is known to restore normal clotting and has previously been demonstrated by gene replacement with AAV therapy. However, AAV therapy can result in liver enzyme elevation and the need for P- Glucosylceramide (GC) treatment. Additionally, AAV-driven expression has been shown in long-term follow up studies to wane over time and all patients treated with AAV therapy were found to have developed anti- AAV anti-drug antibodies. Since PTCs drive a significant portion of severe cases, ACE-tRNA therapy represents a promising candidate because unlike with gene therapy approaches, the protein (F8) cannot be overexpressed and lead to thrombotic events because F8 expression remains under the control of its endogenous promoter.

Exemplary ACE-tRNA drug products were developed and in some embodiments can be in a nanoplasmid format and/or are delivered using lipid nanoparticles. Nanoplasmids are advantageous because they use a non-bacterial backbone that is compact in size, durable, and have lower immunogenicity. Further, nanoplasmids have been shown to be durable, scalable for manufacturing, and are able to encode multiple tRNAs. Similarly, lipid nanoparticles have lower immunogenicity and are scalable for manufacturing. Lipid nanoparticles are also biodegradable.

Thus, exemplary nanoplasmid DNA systems were developed and evaluated for their biodistribution after systemic administration (Figure 8). Briefly, AF647-labeled nanoplasmid DNA encoding an ACE-tRNA were encapsulated into lipid nanoparticles and injected intravenously into mice. Four hours after injection, fluorescence measurement was evaluated in nine tissues and quantified by flow cytometry. Fluorescence intensity measurements showed that the nanoparticle was distributed primarily to the liver and spleen after intravenous administration in a dose-dependent manner (Figure 9). Flow cytometry analysis of the liver cells demonstrated that 98% of liver endothelial cells were positive for nanoplasmid DNA (Figure 10). Further, evaluation of the median fluorescence intensity (MFI) demonstrated that on a per-cell basis, liver sinusoidal endothelial cells take up more copies of plasmid than hepatocytes (Figure 11). The cellular biodistribution was confirmed by DNA FISH (Figure 12).

Additionally, the ability of ACE-tRNAs to rescue expression of F8 protein and activity was evaluated in a Gaussia Luciferase reporter system (Figure 13). HEK293T cells were transfected with a reporter plasmid having a mutation at amino acid position 427 in F8 (R427X), resulting in a premature stop codon. Cells treated with ACE-tRNA nanoplasmids were found to have over 3-fold increase in luminescence as compared to untreated cells (Figure 14). Further, restoration of full-length protein and subsequent luciferase activity was seen only in cells treated with ACE-tRNA and not in untreated cells. The transfected HEK293T cells were also evaluated for FLAG expression and F8 functional activity (Figure 15). The recovery of F8 was validated by FLAG-tag quantitation (Figure 16), and activity of the recovered F8 was validated by a chromogenic coagulation assay (Figure 17).

The uptake of AF647 -nanoplasmid DNA was compared to the in vitro studies of Example 1 to evaluate the predicted levels of natural termination codon read through. A 1 mg/kg dose (20 pg) of ACE-tRNA nanoplasmid DNA in mice was determined to be approximately equivalent to a 50 ng/mL in vitro dose (Figure 18), which showed minimal NTC read through and high PTC rescue (Figure 3 and Figure 5).

Example 3. Cytokine production after administration of ACE-tRNA compositions.

The immunogenicity of the lipid nanoparticle was evaluated in mice. Briefly, C57BL/6 wild-type mice were injected intravenously with 2 pg or 20 pg nanoplasmid DNA encapsulated in candidate lipid nanoparticles. Four hours after administration, serum was collected for cytokine analysis (Figure 19). The results showed reduced serum cytokines when the candidate lipid nanoparticle was used (Figures 20-23).

To evaluate the dependence of cytokine release on the STING pathway, C57BL/6 wildtype mice and C57BL/6 STING knockout mice were injected intravenously with 2 pg or 20 pg of a composition comprising nanoplasmid DNA encapsulated in a lipid nanoparticle. Four hours after administration, serum was collected and analyzed for cytokine levels (Figure 24). The results showed a significant reduction in IL-6, TNF-a, INF-a, and INF-y serum levels in STING knockout mice compared to wild-type (Figures 25-30), suggesting that the STING pathway is the major contributor of cytokine induction by nanoplasmids encapsulated by the candidate lipid nanoparticles.

Example 4. Comparison of small linear DNA versus nanoplasmid DNA in in vitro human PBMCs.

A small linear DNA construct comprising 1 copy of R-TGA was compared to a nanoplasmid DNA construct comprising 4 copies of R-TGA. Human PBMCs were incubated overnight with lipid nanoparticles comprising one of the two constructs to evaluate their immunogenicity. The results showed that the linear DNA construct had low to no immunogenicity compared to the nanoplasmid DNA (Figures 31-34). This result was replicated in a mouse study. Briefly, C57BL/6 mice were intravenously administered lipid nanoparticles comprising either the linear DNA construct or the nanoplasmid DNA construct. Four hours after administration, serum was collected for cytokine analysis (Figure 35). Similar to the results in human PBMCs, the linear DNA construct showed almost no immunogenicity compared to the nanoplasmid DNA construct in mice (Figures 36-39).

Example 5. Multi Patch ACE-tRNA.

The ability to rescue three PTCs simultaneously was evaluated in a multi patch system (Figures 40 and 41, see also Table 6). Briefly, a nanoplasmid comprising R-TGA, Q-TAA, and Q-TAG (ACE-tRNA Multi) was evaluated against a nanoplasmid comprising a copy of the native ArgTGA (CCT-2-1), a copy of the native GlnTAG (CTG-3-1), and a copy of the native GlnTAA (TTG-1-1) (Native-tRNA Multi) in HEK392T cells transfected with a nanoluciferase reporter system comprising a TGA, TAG, or TAA premature stop codon (Figure 42). After 48 hours of incubation, the cells were evaluated for luciferase activity. The results demonstrated that the multi patch system (ACE-tRNA Multi) led to luciferase signal in all three cell lines, while single copy ACE-tRNA plasmids were only able to rescue the cell line containing the cognate PTC (Figure 43).

Furthermore, the multi patch system was comparable to the single patch system (nanoplasmid comprising four copies of R-TGA) in rescuing F8 activity. This was demonstrated in HEK293T cells transfected with a F8 gene comprising a R427X sequence (Figure 44). Briefly, cells were transiently transfected with plasmids encoding for expression of wild-type or BDD F8 R427X sequence. After 24 hours, cells were transfected with 1 pg/mL of a single-patch nanoplasmid construct or a multi-patch nanoplasmid construct for 48 hours. FLAG expression was seen in wild- type cells and only in PTC expressing cells that were treated with ACE-tRNA (Figure 45). Additionally, no signal over background was detected in cells containing the PTC without treatment. F8 protein activity was also only seen in wild-type cells or in PTC expressing cells treated with ACE-tRNA (Figure 46).

Table 6.

Example 6. EGFP Expression by nanoplasmid DNA in LSEC.

An EGFP nanoplasmid was encapsulated in a lipid nanoparticle formulation and evaluated for expression after intravenous injection in mice (Figure 47). Three days after administration, flow cytometry analysis was performed on liver cells. Nanoplasmid DNA expression (measured by EGFP) was confirmed in liver sinusoidal endothelial cells and CD45+ cells.

Example 7. PTC rescue in liver after intravenous administration of tRNA or nanoplasmid DNA.

In vitro PTC rescue after intravenous administration of tRNA or nanoplasmid DNA was evaluated using IVIS imaging in mouse liver 49 hours after injection. To evaluate in vitro PTC rescue with tRNA, mice were injected with either PBC, a LNP containing wild-type nanoluciferase mRNA, a lipid nanoparticle containing nanoluciferase mRNA with a PTC, a lipid nanoparticle containing nanoluciferase mRNA with a PTC with a tRNA encapsulated in a lipid nanoparticle, or a lipid nanoparticle containing nanoluciferase mRNA with a PTC and a tRNA (Figure 48). The results show administration of a tRNA encapsulated in a LNP resulted in a 6-fold increase in signal, and administration of the free tRNA resulted in a 193-fold increase in signal (Figure 49), demonstrating in vivo PTC rescue. Similarly, in vivo PTC rescue was found in the liver when mice were administered a nanoplasmid DNA (Figure 50 and Figure 51).

Example 8. Evaluating safety of LNP formulations.

Lipid nanoparticles were produced and evaluated for their immunogenic properties. Mice were immunized with 2 pg, 10 pg, or 20 pg of nanoplasmid DNA encapsulated in lipid nanoparticles, and serum was collected at 4 hours and 24 hours post-administration. Cytokine analysis of sera revealed a dose-dependent and transient induction of cytokines by the nanoplasmid DNA. All cytokines were resolved after 24 hours, and no significant difference was found between the LNPs (Figures 52-53).

Liver inflammation was also evaluated by testing alanine aminotransferase (ALT) and aspartate transferase (AST) levels in wild-type mice. Overall, the level of AST and ALT was found to be higher than usual due in part from hemolysis for many serum samples. No significant induction of AST or ALT was found using some LNPs (Figure 54). However, other LNPs induced AST and ALT 24 hours post-injection in a dose-dependent manner (Figure 55).

Example 8. Nonsense mutation rescue with anticodon-engineered tRNA for the treatment of severe hemophilia A.

Hemophilia A is driven by mutations in the Factor VIII F8) gene and affect approximately 13 in every 100,000 male births. Severe hemophilia A poses a significant burden to the healthcare system. Approximately 20% of severe hemophilia A cases in the United States are caused by nonsense mutations that result in premature termination codons (PTCs) in the F8 gene result (Figure 56). Of these nonsense mutations, approximately 20% result in a TAA (stop) PTC and approximately 20% result in a TAG (stop) PTC. The majority of nonsense mutations (>50%) were found to be a result of an Arg to TGA (stop) nonsense mutation (Figure 57).

Lipid nanoparticles with nanoplasmid DNA were evaluated for their ability to read through PTCs and restore F8 function. First, a reporter assay was developed in which HEK293T cells were stably transfected with a nanoluciferase reporter gene with a TGA premature stop codon (e.g., Argl3-TGA), which results in a truncated NanoLuciferase incapable of generating a bioluminescence signal (Figure 58). The cells were then treated with either a lipid nanoparticle (LNP) encapsulating a nanoplasmid DNA encoding anticodon- engineered (ACE)-tRNA or a native tRNA, and luciferase activity was measured 48 hours after transfection. The dose range of the ACE-tRNA was between 0.007813 pg/mL and 2 pg/mL. Treatment with three doses of the ACE-tRNA showed read-through of the PTC and restoration of functional NanoLuciferase in a dose-dependent manner (Figure 59A and Figure 59B). In contrast, protein restoration was not observed following treatment with the LNP encapsulating nanoplasmid DNA encoding native tRNA (Figure 60). Additional experiments showed dosedependent uptake of nanoplasmid DNA, expression of ACE-tRNA, and restoration of nanoluciferase activity using ACE-tRNA (Figures 61A-61C). These responses were observed to be well-correlated with the dose-dependent restoration of full-length protein.

Next, three different reporter cell lines expressing nanoluciferase mRNA with R-TGA (Argl3-TGA), Q-TAG (Glnl4-TAG), or Q-TAA (Gln22-TAA) premature stop codons were generated and treated with three different ACE-tRNAs (Figure 62A). Each reporter cell line was transfected with the three ACE-tRNAs and treated with lipofectamine to enable uptake of plasmid DNA encoding ACE-tRNA at dose levels of 0.7 pg/mL of 2 pg/.mL, and luciferase activity was measured 48 hours after transfection. The results showed codon-specific suppression of each of the three PTCs by the appropriate ACE-tRNA (Figure 62B).

Based on these results, a reporter assay was developed in which HEK293T cells were transfected with a B-domain deleted F8 gene with a TGA premature stop codon. These cells were transfected with a lipid nanoparticle (LNP) encapsulating a nanoplasmid DNA encoding anticodon-engineered (ACE)-tRNA, and F8 activity and FLAG expression were measured 48 hours post-transfection (Figure 63). Western blot analysis showed restoration of B-domain deleted F8 in cell lysates after treatment with ACE-tRNA (Figure 64). Relative to untreated cells, treatment with ACE-tRNA showed over 5-fold increase in FLAG expression (Figure 65). F8 coagulation activity was also significantly increased after treatment with ACE-tRNA, with over 60-fold increase in F8 activity compared to untreated cells (Figure 66).

These results were replicated in an in vitro study in which COS-7 cells were transfected with a full-length F8 gene having a TGA premature stop codon. These cells were then treated with ACE-tRNA, and F8 activity was measured 48 hours after incubation (Figure 67). The results showed that ACE-tRNA was capable of restoring functional full-length F8 in vitro, and that there was a dose-dependent coagulation response in the supernatant of cells treated with ACE-tRNA (Figure 68 and Figure 81). The biodistribution of the LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA was evaluated by intravenous administration of labeled nanoplasmid DNA to mice. AF647 fluorescent-labeled npDNA was administered to WT C57BL/6 mice (n=3 per group) as a single dose of 0.1 mg/kg or 1 mg/kg via IV injection. Animals were sacrificed 4 hours after treatment (when sufficient tissue penetration was expected) and a panel of tissues (heart, lung, liver, spleen, pancreas, kidney, colon, bone marrow, and plasma) were collected. (Figure 69). Tissues were homogenized and total AF647 fluorescence was measured. As expected for ENP formulations, fluorescence analysis showed ENPs encapsulating nanoplasmid DNA encoding ACE-tRNA were primarily sequestered in the liver, followed by the spleen and plasma (Figure 70). Minimal signal above background was detected in the remaining organs evaluated, heart, lung, pancreas, kidney, colon, and bone marrow. Since LSECs are the disease-relevant target cells for npDNA delivery as they represent the primary location for FVIII protein expression, distribution of labeled nanoplasmid DNA in the liver was further examined at a cellular level using flow cytometry after digesting the liver and producing single-cell suspensions. Approximately 25% and 95% of endothelial cells were positive for labeled npDNA at 0.1 and 1 mg/kg dose, respectively. Cellular distribution of the LNPs in the liver was evaluated by flow cytometry, demonstrating that the LNPs were primarily delivered to endothelial and Kupffer cells of the liver (Figure 71). These data confirm delivery and cellular uptake of the npDNA to the target cell type (LSECs) for treatment of hemophilia A.

The tissue distribution of npDNA was further evaluated in mice using a qPCR method to detect npDNA in tissue samples. npDNA was administered to WT C57BL/6 mice (n=3 per group) at a single 1 mg/kg dose via IV injection. Animals were sacrificed 4 hours after treatment and a panel of tissues (heart, lung, liver, spleen, pancreas, kidney, and colon) were collected (Figure 153). Total DNA was isolated from all collected tissues and the amount of nanoplasmid DNA was quantified by qPCR. The majority of nanoplasmid DNA was detected in the liver and spleen (Figure 154).

Finally, an in vivo study was conducted to evaluate restoration of full-length protein by suppression of a nonsense mutation using ACE-tRNA nanoplasmid DNA. LumA mice were injected intravenously with vehicle (control), LNP encapsulating nanoplasmid DNA encoding native tRNA (Native tRNA npDNA), or LNP encapsulating nanoplasmid DNA encoding ACE-tRNA (ACE-tRNA npDNA) (Figure 72). IVIS imaging performed 72 hours post- injection confirmed suppression of the PTC and restoration of the full-length protein, as evidenced by recovery of luciferase signal (Figure 73).

Example 9. Time-dependent npDNA uptake, ACE-tRNA expression, and PTC rescue.

A high throughput assay was developed in which HEK293T cells were seeded in a 96 well plate and dosed with either 1 pg/mL or 0.3 pg/mL of LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA). The wells were quantified at multiple time points between 0 and 50 hours post-dose, and evaluated for npDNA uptake via qPCR, tRNA expression via RT-qPCR, and PTC rescue by a nanoluciferase assay (Figure 74). npDNA uptake was found to be both dose- and time-dependent, with cells showing increasing npDNA uptake in the first 10 hours post-dose (Figure 75 and Figure 78). ACE- tRNA expression was found to broadly follow npDNA uptake, with the greatest ACE-tRNA expression values measured following peak npDNA uptake (Figure 76 and Figure 79). ACE- tRNA expression was between 200 and 520 fold greater than npDNA, as measured by copies of ACE-tRNA or npDNA per well. Similarly, nanoluciferase activity peaked a few hours after peak ACE-tRNA activity was measured (Figure 77 and Figure 80). These results demonstrate the time- and dose-dependent activity of an npDNA for PTC rescue.

Example 10. Rescue of full-length Factor VIII-PTC in multiple cell lines.

To demonstrate that LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA) were capable of rescuing full-length Factor VIII-PTC in multiple cell lines, HEK293T cells were transfected with a B-domain deleted Factor VIII with an Arg-TGA PTC and COS-7, Chinese Hamster Ovary (CHO), and Baby Hamster Kidney (BHK) cells were transfected with a full-length factor VIII with an Arg-TGA PTC were dosed with 0.25 pg/mL, 0.5 pg/mL, or 1 pg/mL of npDNA and evaluated for Factor VIII activity 48 hours post-dose (Figure 82). Factor VIII activity 48 hours post-dose was compared to untreated cells, cells transfected with full-length Factor VIII, and cells transfected with Factor VIII comprising an R-TGA PTC (Figure 83). Full-length Factor VIII activity was restored in all four cell lines, suggested that all four cell lines are viable options for producing full-length Factor VIII.

Example 11. PTC rescue is location agnostic. Since nonsense mutations are heterogeneously dispersed across F8 gene locations in patients with hemophilia A, full-length PTC restoration activity of LNPs encapsulating ACE- tRNA (npDNA) was evaluated for various PTC locations on the F8 mRNA. To evaluate whether PTC rescue is location agnostic, nanoluciferase constructs comprising an R-TGA or K-TGA PTC at R13, R45, K77, R114, or R143 and full-length factor VIII constructs comprising an R-TGA PTC at R15, R427, R814, R1215, R1715, R2116, or R2228 were evaluated for PTC rescue 48 hours post-dose with various concentrations of LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA) (Figure 84 and Figure 85). HEK293T cells transfected with a nanoluciferase PTC construct demonstrated that PTC rescue was dose-dependent and location agnostic, with all constructs showing similar luminescence signal over background at each concentration of npDNA evaluated (Figure 86). Similarly, COS-7 cells transfected with a full-length Factor VIII PTC construct demonstrated PTC rescue 48 hours post-dose with 1 pg/ml npDNA in a location agnostic manner (Figure 87). These results demonstrate that npDNA constructs are capable of rescuing TGA PTCs across genes of interest.

Example 12. Biodistribution of npDNA.

To evaluate biodistribution of LNPs encapsulating nanoplasmid DNA encoding ACE- tRNA (npDNA) in mice, multiple strategies were developed (Figure 88). First, the biodistribution results from Example 8 were verified in non-human primates (NHPs). As expected, the main target organ for npDNAs are the liver, followed by the spleen. Within the liver, liver sinusoidal endothelial cells (LSECs) were found to be the main target, followed by hepatocytes and Kupffer cells (Figure 89 and Figure 90). This is due to the fact that the liver sinusoidal endothelium is fenestrated, which results in enrichment of -100 nm LNPs in non- human primate LSECs (Table 7).

Table 7. Average fenestrae size in various species.

Example 13. Control of cytokine production in mice following npDNA administration. As demonstrated in Example 3, transient cytokine and ALT production in mice was observed following administration of LNPs encapsulating nanoplasmid DNA encoding ACE- tRNA (npDNA). To evaluate the effects of a dexamethasone pre-treatment on cytokine production, CD1 mice were intravenously administered 10 mg/kg dexamethasone 3 hours prior to the administration of 1 mg/kg npDNA. Serum was collected 4 hours, 24 hours, and 72 hours post-npDNA administration and evaluated for cytokines (IL-6, INF-a, INF-y) and alanine aminotransferase (ALT) levels (Figure 91). The results showed a significant reduction in IL-6, INF-a, and INF-y serum levels in mice pre-treated with dexamethasone compared to mice that were not pre-treated with dexamethasone (Figures 92-94). Additionally, dexamethasone pretreatment was found to ameliorate liver enzyme release as measured by ALT (Figure 95).

To further investigate liver enzyme release in mice, serum liver enzyme (ALT and aspartate aminotransferase (ALT)) levels 24 hours post-dose with 1 mg/kg npDNA were measured in C57BL/6 wild-type mice and compared to levels in C57BL/6 STING knockout mice. (Figure 96). The results demonstrated that both ALT and AST levels were reduced in STING knockout mice, as compared to wild-type mice, suggesting that liver enzyme elevation is driven by cytokine release. Further, ALT and AST levels in wild-type mice pre-treated with dexamethasone were similar to levels in STING knockout mice suggested that the observed liver enzyme elevation in mice is mild (Figure 97 and Figure 98).

To evaluate the effect of dexamethasone pre-treatment on ACE-tRNA biodistribution in mouse liver, CD1 mice were pre-treated with dexamethasone as described above. Three hours after pre-treatment, mice were administered 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg of npDNA. Mouse livers were collected 72 hours post-npDNA administration and evaluated for npDNA and ACE-tRNA copy numbers (Figure 99). The results showed that npDNA and ACE-tRNA levels remain dose-dependent in liver following pre-treatment with dexamethasone (Figure 100 and Figure 101).

Further, npDNA and ACE-tRNA levels were found to be durable. CD1 mice were optionally pre-treated with dexamethasone as described above and injected with 1 mg/kg npDNA. Livers were collected on day 1, day 3, day 7, day 14, and day 30 post-npDNA administration (Figure 102). ACE-tRNA expression was found to be detectable in vivo 1 month after a single injection (Figure 103).

Example 14. npDNA administration in non-human primates (NHPs). An in vitro assay with mouse, NHP, and human PBMCs were stimulated with LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA) and evaluated for cytokine production in supernatant (Figure 104). Mouse PBMCs were found to be more sensitive to npDNA-mediated stimulation than both NHP and human PBMCs (Figure 105). This was further verified by supernatant IP- 10 measurements, which showed higher IP- 10 production by mouse PBMCs than both NHP and human PBMCs (Figure 106 and Figure 107).

Thus, a first NHP study was developed to evaluate PTC rescue. NHPs were administered vehicle, 0.1 mg/kg npDNA, 0.5 mg/kg npDNA, or 1 mg/kg npDNA on day 0 and serum was collected from days 0 to 3. For NHPs receiving vehicle or 0.1 mg/kg npDNA, an additional dose was administered on day 7 followed by necropsy on day 10. Serum sampling was performed between days 7 and 10. For NHPs receiving 0.5 mg/kg npDNA or 1 mg/kg npDNA, an additional dose was administered on day 36, with a dexamethasone pre-treatment as described above, followed by necropsy on day 39. Serum sampling was performed between days 36 and 39 (Figure 108). The following readouts were performed during the course of the study: body weight, body temperature, food intake, clinical observation, serum cytokines, clinical chemistry (hematology, coagulation, complement), plasma PK of nanoplasmid DNA, histopathology in tissues (liver, spleen, lung, kidney, colon, heart), npDNA quantification in tissues, and ACE-tRNA quantification in liver and spleen.

Overall, npDNA was found to be well-tolerated in NHPs, with no significant difference observed in body weight and body temperature with or without npDNA treatment (Figure 109 and Figure 110). Additionally, while a transient and dose-dependent increase of cytokines (TNF-a, INF-a, IE-6, IP- 10, MCP-1, INF-y), liver enzymes (AST, AFT), white blood cells, and neutrophils were observed, all were mostly resolved 24-72 hours after npDNA administration (Figures 111-116). No significant changes were observed in other cell types. These results demonstrate that there are no significant safety concerns with npDNA administration in NHPs.

Pharmacokinetic analysis of npDNA in plasma showed that npDNA is rapidly cleared from circulation and is largely undetectable by 72 hours post-npDNA administration (Figure 117). Additionally, no significant change was observed following the second dose suggesting that there was no formation of antidrug antibodies. Biodistribution analysis of npDNA showed that liver and spleen are the major targets for npDNA, followed by lung, colon, and kidney (Figure 118). This biodistribution pattern was consistent with npDNA biodistribution in mice. Further, expression of ACE-tRNA in liver and spleen was confirmed and found to be dosedependent (Figure 119). DNA in situ hybridization further confirmed that liver sinusoidal endothelial cells are the main targets of npDNA (Figure 120 and Figure 121).

Finally, it was shown that cytokines and liver enzymes in NHPs could be suppressed by acute steroid treatment. For this study, NHPs were administered 2 mg/kg dexamethasone by intramuscular injection 24 hours and 2 hours prior to intravenous infusion of 0.5 mg/kg npDNA. 2 hours post-npDNA administration, 2 mg/kg of dexamethasone was administered to via intramuscular injection. Serum was collected prior to the first dexamethasone injection (24 hours prior to npDNA administration), and at 6, 24, and 72 hours post-npDNA administration (Figure 122). All serum cytokine (IL-6, TNF-a, INF-y, IP-10, MCP-1) and liver enzyme (ALT, AST, creatine kinase, C-reactive protein high sensitive, fasting plasma glucose) levels were significantly reduced with acute dexamethasone treatment, as compared to without (Figures 123-132).

Example 15. npDNA administration in humans.

A phase 1 dose escalation study in adult males with severe Hemophilia A is evaluated. Males, 18 years or older, with severe Hemophilia A who have R-TGA PTC mutations and without history of inhibitors are enrolled in this study. Patients on emicizumab are also allowed to participate in this study. The primary objective of this study is to evaluate the safety and tolerability of LNPs encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA). Additional objectives include characterizing the pharmacokinetics of npDNA after a single IV administration, evaluating Eactor VIII activity levels, and evaluating the presence of R-TGA tRNA in circulation (Figure 133). The dose escalation scheme is shown in Figure 134. Within each cohort, dosing is performed as follows:

• The first subject receives a single dose of npDNA. If the dose is well-tolerated, and results in Factor VIII activity that is equal or more than a pre-determined Factor VIII level, then up to two additional subjects are administered a dose at the same dose level.

• If the dose is well-tolerated in the additional subjects but results in Factor VIII that is less than a pre-determined Factor VIII level, then the dose is escalated to the next dose level.

• If the dose is well-tolerated and results in Factor VIII activity > 70% WT, the dose escalation is stopped. At the highest dose selected, 10 additional patients are dosed at that selected dose. To measure Factor VIII, a chromogenic substrate assay suitable for measuring Factor VIII activity was developed. Briefly, plasma is treated with purified coagulation factors from bovine prior to measure chromogenic activity. Evaluation of Factor VIII PTC rescue is shown in Figure 136 and Figure 137). Recruitment of Hemlibra patients in clinical trials is supported. Finally, a preliminary Factor VIII ELISA test was developed and showed specific detection of both full- length and B-domain deleted Factor VIII (Figure 138).

Example 16. In vitro dose-dependent restoration of full-length FVIII protein in cell-based assays.

To demonstrate disease-relevant biological activity of ACE-tRNA, COS-7 cells were transiently transfected with plasmids that encode full-length FVIII-PTC (Arg427-TGA, R427X). Transfected cells were treated with LNP encapsulating nanoplasmid DNA encoding ACE-tRNA (npDNA). ACE-tRNA doses ranged between 0.004 pg/mL and 1 pg/mL. Cells were incubated with npDNA for 48 hours. At the completion of a 48-hour incubation period, supernatant and cell lysate samples were collected (Figure 139). FVIII protein levels in cell lysates were measured by western blotting, and FVIII activity was measured in supernatant cells with a standard Coatest chromogenic substrate (CS) coagulation assay. Treatment with npDNA restored full-length FVIII protein (Figure 140) and coagulation activity (Figure 141) in a dose-dependent manner.

The same experiment was completed in 2 additional cell lines, Chinese hamster ovary (CHO) cells and Baby hamster kidney (BHK) cells using a single dose of ACE-tRNA (0.5 pg/mL). Using the same Coatest CS coagulation assay, restoration of full-length FVIII protein in all 3 cell lines was observed, demonstrating that restoration of protein mediated by ACE- tRNA is consistent in cell lines across rodent and non-human primate (NHP) species (Figure 142).

Example 17. In vitro nanoplasmid DNA uptake and ACE-tRNA expression in human endothelial cells.

To demonstrate the uptake of nanoplasmid DNA and expression of ACE-tRNA in human endothelial cells considered relevant cell types for FVIII, two human endothelial cell lines (primary HUVEC and immortalized LSECs) were evaluated. Each cell type was treated with LNPs encoding ACE-tRNA at a dose range between 0.015625 |ag/mL and 4 jag/mL. Cells were incubated for 48 hours, followed by quantification of the amount of nanoplasmid DNA and ACE-tRNA expression by qPCR and RT-qPCR, respectively. A dose-dependent uptake of nanoplasmid DNA and expression of ACE-tRNA was observed in both types of endothelial cells (Figures 143-146).

Example 18. FVIII restoration in endothelial cells.

To demonstrate biological activity (restoration of full-length FVIII) of LNP encapsulating ACE-tRNA (npDNA) in disease-relevant cell types, HUVEC and LSECs will be evaluated by transiently transfecting plasmids that encode full-length F8 containing an Arg- TGA PTC (Arg27-TGA, R427X). Transfected cells will be treated with npDNA at a dose range between 0.0004 pg/mL and 1 pg/mL and incubated for 48 hours. At the completion of the 48-hour incubation period, supernatant and cell lysate samples will be collected for assays. FVIII protein levels in cell lysates will be measured by Western Blotting and/or ELISA, and FVIII activity will be measured in supernatant samples with a standard Coatest CS coagulation assay.

Example 19. In vivo pharmacology studies.

In vivo pharmacology studies have been conducted with surrogate animal model systems to demonstrate the pharmacological activity of npDNA (in vivo restoration of full- length surrogate protein).

A LumA transgenic mouse model which ubiquitously expresses a firefly luciferase mRNA with an Arg-TGA PTC (R387-TGA) was used to evaluate npDNA in vivo. In this model, the luciferase mRNA carriers the TGA PTC (UGA in mRNA) which upon translation results in a truncated, inactive form of the protein incapable of generating a luminescence signal. Mice in the two studies (n=4 per vehicle and n=8 per npDNA) were given an IV administration of vehicle or npDNA (0.1 mg/kg). All mice were pre-treated with dexamethasone (10 mg/kg) before dosing (Figure 147). After 3 days (sufficient time for expression of ACE-tRNA), all animals were subjected to imaging using an In Vivo Imaging System (IVIS) system. Expression of the restored firefly luciferase bioluminescence signal was detected in the liver of npDNA treated mice (Figure 148 and Figure 149) compared to the vehicle group where a luminescence signal was not detected. The restored signal observed in npDNA-treated mice confirms delivery to target tissues (liver) and the putative mechanism of action (correction of an Arg-TGA PTC in an mRNA derived from a chromosomal gene) in a surrogate test system with a reporter protein.

To demonstrate functional restoration of a full-length protein in LSECs within the liver, npDNA and LNP encapsulating mCherry reporter mRNA carrying the Arg-TGA PTC (Arg 18- TGA) sequential-administration studies were carried out in CD-I mice. Mice (N = 4 per group) were intravenously injected with either vehicle, an LNP encapsulating nanoplasmid DNA encoding native, non-engineered tRNA (1 mg/kg), or npDNA (1 mg/kg). All mice were pretreated with dexamethasone (10 mg/kg) 3 hours prior to dosing. 2 days post- injection, all mice receive a single IV administration of the LNP carrying mCherry Arg-TGA PTC mRNA (1 mg/kg) (Figure 150). As with the initial test article administration, all mice were pre-treated with the same dexamethasone treatment. One day after the mCherry mRNA injection, the mice were sacrificed and liver tissues were collected. mCherry expression was analyzed by flow cytometry after dissociating the liver into a single-cell suspension. Plow cytometry results of liver single-cell suspension samples from treated mice confirmed mCherry positive signal in LSECs, demonstrating biological activity (PTC rescue) of npDNA in target relevant cells in vivo (Figure 151).

These data provide a biologically relevant measure of npDNA-mediated rescue of Arg- TGA PTC in F8 mRNA in physiologically relevant target cells (LSECs). Average levels of circulating WT PVIII in healthy individuals are in the range of 100 lU/dL, produced by the full complement of LSECs. The results demonstrate that full-length protein was restored, and the dose was pharmacologically active. In the mice, approximately 25% of LSECs took up npDNA, and an equivalent human dose is expected to achieve circulating restored PVIII.

Example 20. Plasma persistence of npDNA in mice.

To evaluate the persistence of drug substance nanoplasmid DNA in mouse plasma, npDNA (1 mg/kg) was administered into WT CD-I mice (N = 5 to 9 per timepoint) intravenously, and plasma was collected at different time points (pre-dose, 5 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 72 h). The amount of nanoplasmid DNA was quantified by qPCR. Nanoplasmid DNA levels peaked at the earliest time tested (5 minutes) with a rapid decline by 1 hour and to minimally detectable levels (limit of quantification of qPCR assay is around 10 copies per pL of plasma) by 72 hours (Figure 152). Example 21. Liver persistence of npDNA in mice.

A study in CD-I mice was completed. npDNA was administered to WT CD-I mice (N = 8 to 10 per timepoint) as a single IV injection at 0.25 mg/kg and 1 mg/kg dose levels. All mice were pre-treated with dexamethasone (10 mg/kg) before dosing. Animals were sacrificed at pre-dose, 24 h, 72 h, 1 week, 2 weeks, 1 month, and 3 month time points and liver tissues were collected. Total DNA was isolated from liver and the amount of nanoplasmid DNA was quantified by qPCR. Nanoplasmid DNA peaked at the earliest time tested (24 hours) with gradual decrease over time (Figure 155).

Example 22. Liver persistence of npDNA in NHP.

A study in NHP was completed. npDNA was administered to NHP as a single IV injection at 1 mg/kg. Total DNA was isolated from liver and the amount of nanoplasmid DNA was quantified by qPCR. Nanoplasmid DNA was detectable at 1 month after dosing (Figure 156).

Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (z.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of suppressing or rescuing a stop codon in cells, comprising contacting the cells with a nucleic acid that encodes a transfer RNA (tRNA) comprising an acceptor stem and an anticodon, wherein the anticodon is specific for any one of the stop codons provided herein and, optionally, the tRNA can suppress or rescue the stop codon with any one of the amino acids provided herein, wherein the stop codon is in a gene associated with a liver-based bleeding disorder.

2. A method of restoring protein function or activity in cells, wherein the protein is of a gene associated with a liver-based bleeding disorder, comprising contacting cells with a nucleic acid that encodes a transfer RNA (tRNA) comprising an acceptor stem and an anticodon, wherein the anticodon is specific for any one of the stop codons provided herein and, optionally, the tRNA can suppress or rescue the stop codon with any one of the amino acids provided herein.

3. The method of claim 1 or 2, wherein the liver-based bleeding disorder is also endothelial cell-based.

4. The method of any one of claims 1-3, wherein the liver-based bleeding disorder is monogenic.

5. The method of claim 4, wherein the liver-based bleeding disorder is hemophilia A.

6. The method of any one of claims 1-5, wherein the gene is any one of the genes provided herein.

7. The method of any one of claims 1-5, wherein the stop codon is any one of the stop codons provided herein.

8. The method of any one of claims 2-7, wherein the protein is Factor VIII (F8) or Factor IX.

9. The method of any one of the preceding claims, wherein protein function or activity is restored partially or fully.

10. The method of claim 9, wherein protein function or activity is restored such that coagulation activity occurs or is increased as compared to in the absence of the contacting.

11. The method of any one of the preceding claims, wherein the stop codon is TGA.

12. The method of any one of the preceding claims, wherein the amino acid is arginine.

13. An oligonucleotide that encodes at least one, two, three or four of the tRNAs of any one of claims 1-12.

14. The oligonucleotide of claim 13, wherein the oligonucleotide is DNA.

15. The oligonucleotide of claim 13 or 14, wherein the sequence of the oligonucleotide comprises any one of the sequences provided herein.

16. An expression cassette comprising any one of the promoters provided herein and a nucleic acid encoding at least one tRNA of any one of claims 1-12 or the oligonucleotide of any one of claims 13-15, optionally, wherein the expression cassette is in a DNA thread format.

17. The expression cassette of claim 16, further comprising any one of the terminators provided herein.

18. A vector comprising the oligonucleotide of any one of claims 13-15 or the expression cassette of claim 16 or 17.

19. The vector of claim 18, wherein the vector is a plasmid vector, such as a nanoplasmid.

20. A composition comprising: the oligonucleotide of any one of claims 13-15, the expression cassette of claim 16 or 17, or the vector of claim 18 or 19, and a pharmaceutically acceptable carrier.

21. The composition of claim 20, wherein the pharmaceutically acceptable carrier is a particle, such as a nanoparticle.

22. The composition of claim 21, wherein the particle is a lipid nanoparticle.

23. The method of any one of claims 1-12, wherein the tRNA is encoded by a nucleic acid that comprises any one or more or any one combination of the sequences provided herein.

24. The method of claim 23, wherein the nucleic acid is or comprises the oligonucleotide of any one of claims 13-15, the expression cassette of claim 16 or 17, or the vector of claim 18 or 19, and, optionally, a pharmaceutical carrier.

25. The method of claim 24, wherein the pharmaceutically acceptable carrier is a particle, such as a nanoparticle.

26. The method of claim 25, wherein the particle is a lipid nanoparticle.

27. The method of any one of claims 1-12 and 23-26, wherein when there is more than one tRNA encoded, the sequences of the encoded tRNAs are in the same oligonucleotide, expression cassette, vector or composition.

28. The method, oligonucleotide, expression cassette, vector or composition of any one of the preceding claims, wherein at least three tRNAs that are each specific for a different stop codon are encoded.