Methods and compositions for treating immature termination codon-mediated disorders

Abstract

The present invention relates generally to modified tRNAs and their use for expressing functional gene products in mammalian cells that are encoded by genes containing premature stop codons and / or for treating diseases mediated by premature stop codons, such as Dravet syndrome.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 929,428, filed on November 1, 2019, and for all purposes, the entire disclosure thereof is incorporated herein by reference in its entirety.

[0002] Field of the Invention The present invention generally relates to methods and compositions for expressing gene products encoded by genes containing premature termination codons and / or for treating disorders mediated by premature termination codons.

Background Art

[0003] Background Protein synthesis is governed by the genetic code, which includes 61 three - base - pair codons that encode the amino acids incorporated into the synthesized protein and three three - base - pair codons (referred to as stop or termination codons) that terminate protein synthesis. When the nucleic acid sequence encoding a protein mutates to contain a premature termination codon instead of the codon for the next amino acid, the resulting protein ends at an immature stage, which is often non - functional or less functional than the non - truncated or full - length protein. Such mutations, called nonsense mutations, are often associated with, or causative factors for, many different genetic diseases.

[0004] Many disorders are associated with or caused by nonsense mutations. These include epilepsy, e.g. Dravet syndrome, genetic epilepsy with febrile seizures (GEFS), benign familial infantile epilepsy (BFIE), early infantile epileptic encephalopathy (EIEE), Lennox-Gastaut syndrome, Rett syndrome, PPM-X syndrome, Ohtahara syndrome, episodic ataxia, hemiplegic migraine, idiopathic generalized epilepsy, FOXG1 syndrome, familial focal epilepsy with variable foci (FFEVF), childhood-onset epileptic encephalopathy, SYNGAP, and others. 1Associated conditions include intellectual disability, pyridoxine-dependent epilepsy, familial infantile myoclonic epilepsy (FIME), myoclonic-astatic epilepsy, X-linked intellectual disability, partial and recurrent ataxias, febrile seizures, autosomal dominant partial epilepsy with auditory symptoms (ADPEAF), PNPO deficiency, progressive myoclonic epilepsy, action myoclonus-renal failure (AMRF), CDKL5 deficiency disorder, and benign familial infantile spasms (BFIS).

[0005] As an example, Dravet syndrome is a rare and devastating form of intractable epilepsy that begins in infancy. Initially, patients experience prolonged seizures. In the second year, additional types of seizures begin to occur, typically accompanied by developmental decline, likely due to recurrent cerebral hypoxia. This results in poor development of language and motor skills. Mutations in the SCN1A (encoding the voltage-gated sodium channel α subunit Nav1.1), SCN1B (encoding the voltage-gated sodium channel β1 subunit), SCN2A (encoding Nav1.2), SCN3A (encoding Nav1.3), SCN9A (encoding Nav1.7), GABRG2 (encoding the gamma-aminobutyric acid receptor γ2 subunit), GABRD (encoding the gamma-aminobutyric acid receptor Δ subunit) and / or PCDH19 (encoding protocadherin-19) genes have been associated with Dravet syndrome.

[0006] Dravet syndrome can be caused by nonsense mutations within the SCN1A gene that, for example, result in premature stop codons and the absence or reduction of non-truncated or functional proteins. The SCN1A gene normally encodes Na(V)1.1, a voltage-gated sodium channel α subunit of neurons. In mouse models, loss-of-function mutations within SCN1A have been observed to result in a decrease in sodium flux and a failure of excitability of GABAergic interneurons in the hippocampus.

[0007] Despite efforts to date, there remains a need in the art for improved compositions and methods for treating diseases mediated by premature stop codons, including Dravet syndrome. SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, on the discovery of tRNAs (e.g., suppressor tRNAs) that enable the incorporation of amino acids at positions in a gene product encoded by a gene where a shortened gene product would otherwise result from a premature termination codon (PTC) within that gene in mammalian cells. The present invention is further based, in part, on the discovery that a tRNA that enables the incorporation of amino acids at positions in a gene product encoded by a gene where a shortened gene product would otherwise result from a PTC within that gene, e.g., a tRNA described herein, can be used to treat a disease mediated by a PTC within a gene in a subject.

[0009] Accordingly, in one aspect, the present invention provides a tRNA comprising the nucleotide sequence shown in Table 2. In certain embodiments, the tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186.

[0010] In certain embodiments, the tRNA comprises one or more natural nucleotide modifications selected from, for example, 5-methyluridine, 5-carbamoylmethyluridine, 5-carbamoyl-methyl-2-O-methyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine.

[0011] In another aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding any of the above tRNAs. In certain embodiments, the expression vector comprises the nucleotide sequence encoding the tRNA in a copy number of 1, 2, 3, 4, or more than 4. In certain embodiments, the expression vector comprises a nucleotide sequence corresponding to a genomic DNA sequence adjacent to a wild-type tRNA gene. For example, in certain embodiments, the expression vector comprises the nucleotide sequence shown in Table 4. In certain embodiments, the nucleotide sequence shown in Table 4 is selected from SEQ ID NOs: 869-888. In certain embodiments, the nucleotide sequence shown in Table 4 is operably linked to the nucleotide sequence encoding the tRNA. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is located on the 5' side of the nucleotide sequence encoding the tRNA. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is immediately adjacent to the 5' side of the nucleotide sequence encoding the tRNA (i.e., adjacent).

[0012] In another aspect, the present invention provides an expression vector comprising the nucleotide sequence encoding the tRNA shown in Table 3 in a copy number of 1, 2, 3, 4, or more than 4. In certain embodiments, the tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187.

[0013] In another aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding a tRNA shown in Table 3 and further comprising a nucleotide sequence shown in Table 4. In certain embodiments, the tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187. In certain embodiments, the nucleotide sequence shown in Table 4 is selected from SEQ ID NOs: 869-888. In certain embodiments, the nucleotide sequence shown in Table 4 is operably linked to the nucleotide sequence encoding the tRNA. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is located on the 5' side of the nucleotide sequence encoding the tRNA. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is immediately adjacent to the 5' side of the nucleotide sequence encoding the tRNA (i.e., adjacent).

[0014] In certain embodiments of any of the above expression vectors, the expression vector is a viral vector, such as a DNA viral vector, such as an adeno-associated virus (AAV) vector.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising any of the above tRNAs or any of the above expression vectors and a pharmaceutically acceptable excipient. In certain embodiments, the tRNA or expression vector is not conjugated or bound to another moiety, such as a carrier particle, such as an amino lipid particle. In certain embodiments, the composition does not contain nanoparticles and / or amino lipid delivery compounds.

[0016] In another aspect, the present invention provides a method for expressing a functional gene product encoded by a gene containing a premature termination codon in mammalian cells, the method comprising the step of introducing into the cells either an effective amount of said tRNA or an expression vector, thereby enabling an amino acid to be incorporated at a position in the gene product where, in the absence thereof, a truncated gene product would result from the premature termination codon. In any particular embodiment of the above method, the gene is selected from the genes shown in Table 5 or Table 6. In a particular embodiment, the gene is the SCN1A gene.

[0017] In another aspect, the present invention provides a method for expressing a functional gene product encoded by a gene containing a premature termination codon in mammalian cells, the method comprising the step of introducing into the cells an effective amount of the tRNA shown in Table 3 (for example, a tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187) or an expression vector containing a nucleotide sequence encoding the tRNA, thereby enabling an amino acid to be incorporated at a position in the gene product where, in the absence thereof, a truncated gene product would result from the premature termination codon, wherein the gene is the gene shown in Table 5. In a particular embodiment, the gene is the SCN1A gene.

[0018] In any particular embodiment of the above method, the cells contain fewer truncated gene products than cells that do not have said tRNA. In a particular embodiment, the cells contain a greater amount of the functional gene product than cells that do not have said tRNA.

[0019] In another aspect, the present invention provides a method for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature termination codon in a cell, the method comprising the step of introducing into the cell an effective amount of any one of the above tRNAs or any one of the above expression vectors, whereby an amino acid can be incorporated into a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise be produced due to the premature termination codon.

[0020] In another aspect, the present invention provides a method for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature termination codon in a cell, the method comprising the step of introducing into the cell an effective amount of a tRNA shown in Table 3 (for example, a tRNA containing a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187) or an expression vector containing a nucleotide sequence encoding the tRNA, whereby an amino acid can be incorporated into a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise be produced due to the premature termination codon.

[0021] In a specific embodiment of any of the above methods where the gene is the SCN1A gene, the SCN1A gene product generated by the tRNA is a functional SCN1A gene product. In a specific embodiment, the functional SCN1A gene product has higher activity than the truncated SCN1A gene product. In a specific embodiment, the functional SCN1A gene product is the Na v 1.1 protein. In a specific embodiment, the functional SCN1A gene product contains any one of the amino acid sequences of SEQ ID NOs: 863-868.

[0022] In a specific embodiment of any of the above methods, the cell is a human cell. In a specific embodiment, the cell is a central nervous system cell, such as a neuron. In a specific embodiment, the tRNA is aminoacylated intracellularly.

[0023] In another aspect, the present invention provides a method of treating an immature termination codon-mediated disorder in a subject in need thereof, the subject having a gene with an immature termination codon, the method comprising administering to the subject an effective amount of any of the above tRNAs or any of the above expression vectors, thereby treating the disorder in the subject. In certain embodiments, the disorder is selected from the disorders shown in Table 5 or Table 6.

[0024] In another aspect, the present invention provides a method of treating an immature termination codon-mediated disorder in a subject in need thereof, the subject having a gene with an immature termination codon, the method comprising administering to the subject an effective amount of a tRNA shown in Table 3 (e.g., a tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187) or an expression vector comprising a nucleotide sequence encoding the tRNA, thereby treating the disorder in the subject, wherein the disorder is a disorder shown in Table 5.

[0025] In certain embodiments of any of the above treatment methods, the disorder is epilepsy, such as Dravet syndrome. In certain embodiments, the subject is human. In certain embodiments, the method further comprises administering to the subject an effective amount of another agent, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), the ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), fenfluramine, or valproic acid.

[0026] In certain embodiments of any of the above methods wherein the gene is the SCN1A gene, the premature stop codon in the SCN1A gene is caused by a mutation or combination of mutations selected from c.664C>T, c.1129C>T, c.1492A>T, c.1624C>T, c.1738C>T, c.1837C>T, c.2134C>T, c.2593C>T, c.3637C>T, c.3733C>T, c.3985C>T, c.4573C>T, c.5656C>T, and c.5734C>T. In certain embodiments, the premature stop codon is caused by a mutation selected from c.1738C>T and c.3985C>T.

[0027] [The present invention 1001] A tRNA comprising the nucleotide sequence shown in Table 2. [The present invention 1002] The tRNA of the present invention 1001, comprising a nucleotide sequence selected from SEQ ID NOs: 19 to 21, 37, 39, 40, 44, 179, 181, 182, and 186. [The present invention 1003] The tRNA of the present invention 1001 or 1002, comprising naturally occurring nucleotide modifications. [The present invention 1004] The tRNA of any one of the present inventions 1001 to 1003, comprising one or more nucleotide modifications selected from 5-methyluridine, 5-carbamoylmethyluridine, 5-carbamoyl-methyl-2-O-methyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine. [The present invention 1005] An expression vector comprising a nucleotide sequence encoding the tRNA of any one of the present inventions 1001 to 1004. [The present invention 1006] The expression vector of the present invention 1005, comprising the nucleotide sequence encoding the tRNA in a copy number of 1, 2, 3, 4, or more than 4. [The present invention 1007] The expression vector of the present invention 1005 or 1006, further comprising the nucleotide sequence shown in Table 4. [The present invention 1008] The expression vector of the present invention 1007, comprising a nucleotide sequence selected from SEQ ID NOs: 869 to 888. [The present invention 1009] An expression vector comprising the nucleotide sequence encoding the tRNA shown in Table 3 in a copy number of 1, 2, 3, 4, or more than 4. [The present invention 1010] An expression vector comprising the nucleotide sequence encoding the tRNA shown in Table 3, and further comprising the nucleotide sequence shown in Table 4. [The present invention 1011] The expression vector of the present invention 1009 or 1010, wherein the tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 6 to 9, 11, 16 to 18, 22, 35, 36, 38, 45, 178, 180, and 187. [The present invention 1012] The expression vector of the present invention 1010 or 1011, comprising a nucleotide sequence selected from SEQ ID NOs: 869 to 888. [The present invention 1013] The expression vector of any one of the present inventions 1005 to 1012, which is a viral vector. [The present invention 1014] The expression vector of the present invention 1013, wherein the viral vector is a DNA viral vector. [The present invention 1015] The expression vector of the present invention 1013 or 1014, wherein the viral vector is an adeno-associated virus (AAV) vector. [The present invention 1016] A pharmaceutical composition comprising any one of the tRNAs of the present invention 1001 to 1004 or any one of the expression vectors of the present invention 1005 to 1015 and a pharmaceutically acceptable excipient. [The present invention 1017] The pharmaceutical composition of the present invention 1016, wherein the tRNA or the expression vector is not conjugated or bound to another moiety. [The present invention 1018] The pharmaceutical composition of the present invention 1017, wherein the tRNA or the expression vector is not conjugated or bound to carrier particles. [The present invention 1019] The pharmaceutical composition of the present invention 1008, wherein the carrier particles are amino lipid particles. [The present invention 1020] The pharmaceutical composition according to any one of the present invention 1015 to 1019, which does not contain nanoparticles. [The present invention 1021] The pharmaceutical composition according to any one of the present invention 1015 to 1020, which does not contain an amino lipid delivery compound. [The present invention 1022] A method for expressing a functional gene product encoded by a gene containing a premature stop codon in mammalian cells, comprising the step of introducing an effective amount of any one of the tRNAs of the present invention 1001 to 1004 or any one of the expression vectors of the present invention 1005 to 1015 into the cells, thereby enabling an amino acid to be incorporated at a position in the gene product where a truncated gene product would otherwise occur due to the premature stop codon. The method comprising the above step. [The present invention 1023] The method of the present invention 1022, wherein the cells contain fewer truncated gene products than cells without the tRNA. [The present invention 1024] The method of the present invention 1022 or 1023, wherein the cells contain a greater amount of the functional gene product than cells without the tRNA. [The present invention 1025] The method according to any one of the present invention 1022 to 1024, wherein the gene is the gene shown in Table 5 or Table 6. [The present invention 1026] The method of the present invention 1025, wherein the gene is the gene shown in Table 5. [The present invention 1027] A method for expressing a functional gene product encoded by a gene containing a premature stop codon in mammalian cells, A step of introducing into the cell an expression vector containing an effective amount of the tRNA shown in Table 3 or a nucleotide sequence encoding the tRNA, thereby enabling an amino acid to be incorporated at a position in the gene product where a truncated gene product would otherwise be produced due to a premature stop codon comprising wherein the gene is the gene shown in Table 5 a method [Inventive concept 1028] The method according to Inventive concept 1027, wherein the tRNA comprises a nucleotide sequence selected from SEQ ID NO: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187 [Inventive concept 1029] The method according to any one of Inventive concepts 1022-1028, wherein the gene is the SCN1A gene [Inventive concept 1030] A method for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature stop codon in a cell, comprising a step of introducing into the cell an effective amount of any one of the tRNAs of Inventive concepts 1001-1004 or any one of the expression vectors of Inventive concepts 1005-1015, thereby enabling an amino acid to be incorporated at a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise be produced due to a premature stop codon a method [Inventive concept 1031] A method for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature stop codon in a cell, comprising a step of introducing into the cell an effective amount of the tRNA shown in Table 3 or an expression vector containing a nucleotide sequence encoding the tRNA, thereby enabling an amino acid to be incorporated at a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise be produced due to a premature stop codon a method [Inventive concept 1032] The method according to Inventive concept 1031, wherein the tRNA comprises a nucleotide sequence selected from SEQ ID NO: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187 [Inventive concept 1033] The method according to any one of Inventive concepts 1029-1032, wherein the SCN1A gene product generated by the tRNA is a functional SCN1A gene product [Inventive concept 1034] The method according to Inventive concept 1033, wherein the functional SCN1A gene product has higher activity than the truncated SCN1A gene product [The present invention 1035] The method of the present invention 1033 or 1034, wherein the functional SCN1A gene product is the Na v 1.1 protein. [The present invention 1036] The method according to any one of the present inventions 1033 to 1035, wherein the functional SCN1A gene product comprises any one of SEQ ID NOs: 863 to 868. [The present invention 1037] The method of the present invention 1036, wherein the functional SCN1A gene product comprises SEQ ID NO: 863 or SEQ ID NO: 864. [The present invention 1038] The method according to any one of the present inventions 1022 to 1037, wherein the cell is a human cell. [The present invention 1039] The method according to any one of the present inventions 1022 to 1038, wherein the tRNA is aminoacylated intracellularly. [The present invention 1040] A method for treating premature termination codon-mediated disorders in a subject in need thereof, wherein the subject has a gene having a premature termination codon, and the method comprises administering to the subject an effective amount of any one of the tRNAs of the present inventions 1001 to 1004 or any one of the expression vectors of the present inventions 1005 to 1015, thereby treating the disorder in the subject, step. [The present invention 1041] The method of the present invention 1040, wherein the disorder is the disorder shown in Table 5 or Table 6. [The present invention 1042] The method of the present invention 1041, wherein the disorder is the disorder shown in Table 5. [The present invention 1043] A method for treating premature termination codon-mediated disorders in a subject in need thereof, wherein the subject has a gene having a premature termination codon, and the method comprises administering to the subject an effective amount of the tRNA shown in Table 3 or an expression vector comprising the nucleotide sequence encoding the tRNA, thereby treating the disorder in the subject, comprising, wherein the disorder is the disorder shown in Table 5, method. [The present invention 1044] The method of the present invention 1043, wherein the tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 6 to 9, 11, 16 to 18, 22, 35, 36, 38, 45, 178, 180, and 187. [The present invention 1045] The method according to any one of the present inventions 1040 to 1044, wherein the disorder is Dravet syndrome. [The present invention 1046] The method of the present invention 1045, further comprising administering to the subject stiripentol, cannabidiol, ketogenic diet, clobazam, topiramate, fenfluramine, or valproic acid. [The present invention 1047] The method of the 1045th or 1046th aspect of the present invention, wherein the gene is SCN1A. [1048th aspect of the present invention] The method according to any one of aspects 1040 to 1047 of the present invention, wherein the subject is a human. [1049th aspect of the present invention] The method according to any one of aspects 1029 to 1039 or 1047 of the present invention, wherein the premature stop codon in the SCN1A gene is caused by a mutation selected from c.664C>T, c.1129C>T, c.1492A>T, c.1624C>T, c.1738C>T, c.1837C>T, c.2134C>T, c.2593C>T, c.3637C>T, c.3733C>T, c.3985C>T, c.4573C>T, c.5656C>T, and c.5734C>T. [1050th aspect of the present invention] The method of the 1049th aspect of the present invention, wherein the premature stop codon in the SCN1A gene is caused by a mutation selected from c.1738C>T and c.3985C>T. These and other aspects and features of the present invention are set forth in the following detailed description and claims.

Brief Description of the Drawings

[0028] The present invention can be more fully understood by reference to the following drawings.

[0029] (FIG. 1) Schematic diagram of a transcript (e.g., an SCN1A transcript) containing a premature termination codon (PTC) that results in a truncated protein product (e.g., a protein product in a subject with Dravet syndrome). The native stop codon is indicated by a shaded circle, and the premature termination codon is indicated by an unshaded circle. Expression of a suppressor tRNA (e.g., an anticodon-modified arginine tRNA) charged with its cognate amino acid (A.A.) enables read-through of the PTC and promotes expression of the full-length protein. (FIG. 2A) It is a common tRNA secondary structure. The residue numbers are based on the tRNA numbering system described in Steinberg et al. (1993) NUCLEIC ACIDS RES. 21:3011-15. (FIG. 2B) It is a table showing the modification profile of tRNA sequences derived from the cytoplasm of specific eukaryotes. The ratios in the table indicate the occurrence frequencies of the nucleotides listed at the positions of the numbers shown in FIG. 2A. The abbreviations of the modified residues are defined in Motorin et al. (2005) “Transfer RNA Modification,” ENCYCLOPEDIA OF LIFE SCIENCES, John Wily & Sons, Inc. (FIG. 3) It is a schematic diagram of a dual-fluorescent reporter construct containing 3 copies of red fluorescent protein (tdTomato), TEV protease, a 51-bp linker region containing PTC+ / -8 flanking codons, and 3 copies of green fluorescent protein (EGFP). Expression is driven by the promoter of elongation factor EF-1α located upstream of the first copy of tdTomato. All copies of tdTomato, EGFP, and TEV protease are separated from each other by the TEV protease cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln-(Gly / Ser) (SEQ ID NO:902) ) (FIG. 4) Arg in a Flp-In-293 cell line stably expressing a dual-fluorescent reporter with a target S-PTC linker region (SEQ ID NO: 29) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome TCA It is a graph showing the readthrough activity of the suppressor tRNA. The indicated Arg TCASuppressor tRNAs (SEQ ID NOs: 1-25 and 35) were transfected into cells, and readthrough activity was measured by flow cytometry in two independent experiments 24 hours after transfection. "Parent" refers to the original Flp-In-293 cell line without a fluorescent reporter, "PTC-free" refers to a Flp-In-293 cell line stably expressing a dual fluorescent reporter with a version of the target S-PTC linker region (SEQ ID NO: 194) lacking the PTC, "EV" (empty vector) refers to cells transfected with an expression construct not containing tRNA, and "TCG" refers to cells transfected with an expression construct containing wild-type Arg-tRNA with a TCG anticodon. Readthrough activity is expressed as the ratio of viable cells expressing both tdTomato and EGFP above background ("double positive %"). Error bars represent the standard deviation of the data. (Figure 5) Arg in a Flp-In-3T3 cell line stably expressing a dual fluorescent reporter with an R1407X-PTC linker region (SEQ ID NO: 30) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome TCA Graph showing the readthrough activity of suppressor tRNAs. The indicated Arg TCASuppressor tRNA (SEQ ID NO: 1 - 25) was transfected into cells, and readthrough activity was measured by flow cytometry in three independent experiments 24 hours after transfection. "Parent" refers to the original 3T3 cell line without a fluorescent reporter. "PTC - free" refers to a 3T3 cell line stably expressing a dual - fluorescent reporter with a version of the R1407X - PTC linker region (SEQ ID NO: 195) lacking the PTC. "Mock" refers to mock - transfected cells. "EV" (empty vector) refers to cells transfected with an expression construct containing neither tRNA nor the EGFP reporter. "TCG" refers to cells transfected with an expression construct containing wild - type Arg - tRNA with a TCG anticodon. Readthrough activity is represented as the ratio of viable cells expressing both tdTomato and EGFP above background ("double - positive %"). Error bars represent the standard deviation of the data. (Figure 6) Arg in Flp - In - 3T3 cells transiently expressing a dual - fluorescent reporter with the subject N - PTC linker region (SEQ ID NO: 28) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome TCA Graph showing the readthrough activity of suppressor tRNA. The indicated Arg TCA Suppressor tRNA (SEQ ID NO: 1 - 25 and 35) was co - transfected into cells, and readthrough activity was measured by flow cytometry. "Mock" refers to mock - transfected cells. "PTC - free" refers to cells transfected with a dual - fluorescent reporter with a version of the subject N - PTC linker region (SEQ ID NO: 193) lacking the PTC. "EV" (empty vector) refers to cells co - transfected with an expression construct without tRNA. "TCG" refers to cells co - transfected with an expression construct containing wild - type Arg - tRNA with a TCG anticodon. Readthrough activity was measured by flow cytometry and is represented as the ratio of viable cells expressing both tdTomato and EGFP above background ("double - positive %"). (FIG. 7) Graph showing the readthrough activity of the indicated Arg TCA suppressor tRNAs (measured by the percentage of positive GFP cells) in Neuro-2a (N2a) and Flp-In-293 (293) cells. An expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) was co-transfected into cells with an expression construct containing the indicated Arg TCA suppressor tRNAs (SEQ ID NOs: 1-22). "EGFP" indicates cells transfected with a version of the EGFP reporter lacking a PTC, and "EGFP-PTC" indicates cells transfected with the EGFP-R96X-TGA reporter alone. EGFP expression was analyzed by flow cytometry approximately 24 hours after transfection in 293 cells and approximately 48 hours after transfection in N2a cells. Readthrough activity is represented as the ratio of viable cells expressing EGFP above background ("GFP+%"), and all values are normalized to cells expressing EGFP lacking a PTC. (FIG. 8A) Schematic of the experimental approach for measuring suppressor tRNA activity using a construct containing an EGFP reporter with a PTC and suppressor tRNAs. Native stop codons are indicated by shaded circles, and premature stop codons are indicated by unshaded circles. Standard Arg-tRNA (containing an anticodon that binds to CGA) cannot read through the PTC in EGFP, resulting in a non-functional truncated EGFP protein. Suppressor tRNA (containing an anticodon that binds to UGA) enables readthrough of the PTC in EGFP, resulting in a full-length functional EGFP protein. (FIG. 8B) Schematic of an exemplary reporter construct containing EGFP with a PTC and 4 copies of a suppressor tRNA. (FIG. 9) EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated number of copies of the indicated Arg TCAFluorescent images of Neuro-2a cells transfected with an expression construct containing suppressor tRNA are shown. The suppressor tRNAs are TCA-001 (SEQ ID NO:11), TCA-113 (SEQ ID NO:16), and TCA-115 (SEQ ID NO:18). Each copy of the suppressor tRNA also contains 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and 104 bp of downstream flanking genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1. The images were taken approximately 24 hours after transfection. The GFP controls are, from left to right, 1) wild-type EGFP alone, 2) wild-type EGFP on an expression construct containing a single copy of Arg TCA suppressor tRNA #001 (SEQ ID NO:11), 3) EGFP-R96X-TGA reporter alone, and 4) EGFP-R96X-TGA on an expression construct containing four copies of Arg-tRNA with an unmodified TCG anticodon. (Figure 10) Fluorescent images of Flp-In-293 cells transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated copy number of the indicated Arg TCA suppressor tRNA are shown. The suppressor tRNAs are TCA-001 (SEQ ID NO:11), TCA-113 (SEQ ID NO:16), and TCA-115 (SEQ ID NO:18). Each copy of the suppressor tRNA also contained 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and 104 bp of downstream flanking genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1. The images were taken approximately 48 hours after transfection. The GFP controls are, from left to right, 1) wild-type EGFP alone, 2) a single copy of Arg TCAWild-type EGFP on an expression construct containing suppressor tRNA#001 (SEQ ID NO:11), 3) EGFP-R96X-TGA reporter alone, and 4) EGFP-R96X-TGA on an expression construct containing 4 copies of Arg-tRNA with an unmodified TCG anticodon. (Figure 11) Fluorescence measured by flow cytometry in Neuro-2a and Flp-In-293 cells transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:177) and Arg TCA suppressor tRNA#001 (SEQ ID NO:11). The expression constructs contained 1 (1x), 2 (2x) or 4 (4x) copies of the suppressor tRNA under the context of either (i) a U6 promoter ("U6") containing 19 bp of upstream flanking genomic DNA (SEQ ID NO:33) from tRNA-Arg-TCG-1-1 and 46 bp of downstream flanking genomic DNA (SEQ ID NO:34) from tRNA-Arg-TCG-1-1, or (ii) 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and 200 bp of downstream flanking genomic DNA (SEQ ID NO:27) from tRNA-Arg-TCG-1-1 ("flanking"). "Empty vector" indicates cells transfected with an expression construct that contains neither tRNA nor the EGFP reporter, and "EF1a:EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC. Analyses were performed at approximately 48 hours post-transfection. Data are presented as a histogram showing the frequency distribution of data for fluorescence intensity in viable cells expressing EGFP above background. (Figure 12) Under the context of either (i) a U6 promoter ("U6") containing 19 bp of upstream flanking genomic DNA (SEQ ID NO:33) derived from tRNA-Arg-TCG-1-1 and 46 bp of downstream flanking genomic DNA (SEQ ID NO:34) derived from tRNA-Arg-TCG-1-1, or (ii) 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) and 200 bp of downstream flanking genomic DNA (SEQ ID NO:27) derived from tRNA-Arg-TCG-1-1 ("flanking"), the EGFP-R96X-TGA reporter (SEQ ID NO:177) and the indicated copy number of Arg TCA The percentage of EGFP-positive cells and the mean EGFP intensity in all viable cells measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing the suppressor tRNA #001 (SEQ ID NO:11) are shown. "Empty vector" indicates cells transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "EF1a:EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC. The analysis was performed approximately 48 hours after transfection. These plots summarize the data from the Neuro-2a cells in Figure 11. (Figure 13) The EGFP-R96X-TGA reporter (SEQ ID NO:31) and Arg TCAFluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#001 (SEQ ID NO:11) is shown. The expression construct contained 1 (1x), 2 (2x), 3 (3x) or 4 (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contained 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and 104 bp of downstream flanking genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1 (the "flank"). "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC, and "mock" indicates cells transfected with the EGFP-R96X-TGA reporter alone. Analysis was performed at approximately 48 hours after transfection. Data are presented as a histogram showing the frequency distribution of data for fluorescence intensity in all live cells ("G1 gate") and live cells expressing EGFP above background ("GFP+ gate"). (FIG. 14) EGFP-R96X-TGA reporter (SEQ ID NO:31) and Arg TCAFluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#113 (SEQ ID NO:16) is shown. The expression construct contained 1 (1x), 2 (2x), 3 (3x) or 4 (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contained 200 bp of upstream genomic DNA (SEQ ID NO:26) and 104 bp of downstream genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1 (the "flanking"). "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC, and "mock" indicates cells transfected with the EGFP-PTC reporter alone. Analyses were performed at approximately 48 hours post-transfection. Data are presented as histograms showing the frequency distribution of data for fluorescence intensity in all viable cells ("G1 gate") and viable cells expressing EGFP above background ("GFP+ gate"). (Figure 15) EGFP-R96X-TGA reporter (SEQ ID NO:31) and Arg TCAFluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#115 (SEQ ID NO:18) is shown. The expression construct contained 1 (1x), 2 (2x), 3 (3x), or 4 (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contained 200 bp of upstream flanking genomic DNA (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1 (the "flanking"). "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking a PTC, and "Mock" indicates cells transfected with the EGFP-R96X-TGA reporter alone. Analysis was performed at approximately 48 hours after transfection. Data are presented as histograms showing the frequency distribution of data for fluorescence intensity in all viable cells ("G1 gate") and viable cells expressing EGFP above background ("GFP+ gate"). (Figure 16) Ratio of EGFP-positive cells ("GFP+ cell %") and mean EGFP intensity in EGFP-positive cells ("GFP+ gate") measured by flow cytometry in all viable Neuro-2a cells transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated copy number of the indicated suppressor tRNA are shown. The suppressor tRNAs are TCA-001 (SEQ ID NO:11), TCA-113 (SEQ ID NO:16), and TCA-115 (SEQ ID NO:18). "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking a PTC, and "CAG:EGFP-PTC" indicates cells transfected with the EGFP-R96X-TGA reporter alone. These plots summarize data from Figures 13-15. (Figure 17) Ratio of EGFP-positive cells measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated suppressor tRNAs with the indicated copy numbers and flanking sequences. The suppressor tRNAs are TCA-001 (SEQ ID NO: 11), TCA-113 (SEQ ID NO: 16), and TCA-115 (SEQ ID NO:18). "U6" indicates the U6 promoter containing the 19 bp upstream flanking genomic DNA (SEQ ID NO:33) from tRNA-Arg-TCG-1-1 and the 46 bp downstream flanking genomic DNA (SEQ ID NO:34) from tRNA-Arg-TCG-1-1, "flanking (±200bp)" indicates the 200 bp upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and the 200 bp downstream flanking genomic DNA (SEQ ID NO:27) from tRNA-Arg-TCG-1-1, "flanking (+200 / -100bp)" indicates the 200 bp upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and the 104 bp downstream flanking genomic DNA (SEQ ID NO:32) from tRNA-Arg-TCG-1-1 ("flanking"). "Mock" indicates mock-transfected cells, "empty vector" indicates cells transfected with an expression construct containing neither tRNA nor the EGFP reporter, "4xTCG" indicates cells transfected with an expression construct containing the EGFP-R96X-TGA reporter and 4 copies of wild-type Arg-tRNA with the TCG anticodon, and "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking a PTC. All data are normalized to the positive control (CAG:EGFP). The plot shows the ratio of viable cells expressing GFP above background. TCA (Figure 18) Dmd derived from clinically relevant DMD nonsense mutations associated with Duchenne muscular dystrophy mdx ​- In two separately obtained Flp-In-293 cell lines (#2 and #10) that stably express a dual-fluorescent reporter with a PTC linker region (SEQ ID NO:192), Gln TTA Graph showing the readthrough activity of the suppressor tRNA. The Gln shown TTA The suppressor tRNA (SEQ ID NO:36 - 48) was transfected into cells, and the readthrough activity was measured by flow cytometry 24 hours after transfection. "Parent" indicates the original Flp-In-293 cell line without the fluorescent reporter, and "PTC-free" indicates a version of Dmd lacking the PTC mdx - A Flp-In-293 cell line that stably expresses a dual-fluorescent reporter with a PTC linker region (SEQ ID NO:191) is shown. "TTG" indicates cells transfected with an expression construct containing wild-type Gln-tRNA with a TTG anticodon. The readthrough activity was measured by flow cytometry and is represented as the ratio of viable cells expressing both tdTomato and EGFP above baseline ("double + cell %"). (Figure 19) In Neuro-2a cells co-transfected with an expression construct containing the EGFP-Q69X-TAA reporter (SEQ ID NO:175), the Gln shown TTA Graph showing the readthrough activity of the suppressor tRNA (SEQ ID NO:36 - 48). "Mock" indicates mock-transfected cells, "EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC, "EV" (empty vector) indicates cells transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "TTG" indicates cells co-transfected with the EGFP-Q69X-TAA reporter and an expression construct containing wild-type Gln-tRNA with a TTG anticodon. The readthrough activity was measured by flow cytometry and is represented as the ratio of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. (Figure 20) In Neuro-2a cells co-transfected with an expression construct containing the EGFP-Q69X-TAG reporter (SEQ ID NO: 176), the indicated Gln CTA Graph showing the readthrough activity of suppressor tRNAs (SEQ ID NOs: 78-90). "Mock" indicates mock-transfected cells, "EGFP" indicates cells transfected with a version of the EGFP reporter lacking a PTC, "EV" (empty vector) indicates cells transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "TTG" indicates cells co-transfected with the EGFP-Q69X-TAG reporter and an expression construct containing wild-type Gln-tRNA with a TTG anticodon. Readthrough activity was measured by flow cytometry and is represented as the percentage of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. (Figure 21) Neuro-2a cells approximately 24 hours after transfection with an expression construct containing the EGFP-R96X-TGA reporter ("GFP-PTC"), (i) either containing the indicated copy number of Arg TCA on the same construct, including suppressor tRNA #115 (SEQ ID NO: 18), or (ii) treated with the indicated concentration of ataluren, (iii) treated with the indicated concentration of gentamicin, or (iv) treated with the indicated concentration of G418. Fluorescent images of Neuro-2a cells that underwent any one of these are shown. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug at the indicated concentration was added. The controls in the left column were transfected with an expression construct containing wild-type EGFP ("WT-GFP") and the indicated drug or the indicated copy number of Arg TCA suppressor tRNA. (Figure 22) It is a graph showing the ratio of GFP-positive cells measured by flow cytometry approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-R96X-TGA reporter (「GFP-PTC」) was transfected, and (i) on the same construct, the indicated copy number of Arg TCA suppressor tRNA#115 (SEQ ID NO:18) was included, or (ii) treated with the indicated concentration of ataluren, (iii) treated with the indicated concentration of gentamicin, or (iv) treated with the indicated concentration of G418. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, and at this time, the indicated drug at the indicated concentration was added. A reporter containing wild-type EGFP without PTC (「WT-GFP」) was used as a control. 「Mock」 indicates mock-transfected cells. 「4XTCG」 indicates cells transfected with an expression construct containing 4 copies of wild-type Arg-tRNA with the TCG anticodon and the EGFP-R96X-TGA reporter. The plot shows the ratio of viable cells expressing EGFP above background. The ratio of cells expressing GFP was 0.7 - 1.6% in the negative control, 0.9 - 4.9% in ataluren-treated cells, 1.2 - 6.5% in gentamicin-treated cells, and 6.7% - 26.7% in G418-treated cells, compared to TCA 73.0 - 76.2% in cells expressing the Arg suppressor. (Figure 23) It is a graph showing the cell viability from Figure 22 measured by flow cytometry approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-R96X-TGA reporter (「GFP-PTC」) was transfected, and (i) on the same construct, the indicated copy number of Arg TCAEither (i) transfect an expression construct containing suppressor tRNA#115 (SEQ ID NO:18), (ii) treat with ataluren at the indicated concentrations, (iii) treat with gentamicin at the indicated concentrations, or (iv) treat with G418 at the indicated concentrations. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drugs at the indicated concentrations were added. A reporter containing wild-type GFP without a PTC (“WT-GFP”) was used as a control. “Mock” indicates mock-transfected cells. “4XTCG” indicates cells transfected with an expression construct containing 4 copies of wild-type Arg-tRNA with a TCG anticodon and an EGFP-R96X-TGA reporter. Cell viability was assessed by flow cytometry using 7-aminoactinomycin D (7-AAD), a membrane-impermeable dye that is excluded from normally viable cells. (Figure 24) In Flp-In-293 cells transiently co-transfected with the indicated dual-fluorescent reporter constructs containing linker regions (SEQ ID NO:889, 891, and 893) derived from three clinically relevant Gln(Q)-to-TAG PTC mutations (W1397 * , S1505 * , and Q1810 * ) associated with Dravet syndrome, the indicated Gln CTAGraph showing the readthrough activity of suppressor tRNA (SEQ ID NO: 178 - 190). "Mock" indicates mock-transfected cells, "PTC-free" indicates cells transfected with a version of the dual-fluorescent reporter construct lacking the PTC (SEQ ID NO: 890, 892, and 894), "EV" (empty vector) indicates cells transfected with an expression construct containing neither tRNA nor the fluorescent reporter, and "TTG" indicates cells co-transfected with the indicated dual-fluorescent reporter construct and an expression construct containing wild-type Gln-tRNA with the TTG anticodon. Readthrough activity was measured by flow cytometry approximately 24 hours after transfection and is represented as the percentage of viable cells expressing both tdTomato and EGFP above baseline ("double + cell %"). (Figure 25) In the Flp-In-293 cell line incorporating a dual-fluorescent reporter construct containing the linker region (SEQ ID NO: 889) derived from the clinically relevant PTC mutation (W1397 * ) in SCN1A associated with Dravet syndrome CTA Graph showing the readthrough activity of suppressor tRNA (SEQ ID NO: 178 - 190). "Mock" indicates mock-transfected cells, "RFP-EGFP" indicates the Flp-In-293 cell line incorporating a version of the dual-fluorescent reporter construct lacking the PTC (SEQ ID NO: 890), "EV" (empty vector) indicates cells transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "TTG" indicates cells transfected with wild-type Gln-tRNA with the TTG anticodon. Readthrough activity was measured by flow cytometry approximately 24 hours after transfection and is represented as the percentage of cells expressing both tdTomato and EGFP above background ("double + cell %"). (FIG. 26A) (i) 200 bp upstream adjacent genomic DNA (SEQ ID NO: 26) derived from tRNA-Arg-TCG-1-1 and 104 bp downstream adjacent genomic DNA (SEQ ID NO: 32) derived from tRNA-Arg-TCG-1-1 (“adjacent 300”), (ii) 20 bp upstream adjacent genomic DNA (SEQ ID NO: 895) derived from tRNA-Arg-TCG-1-1 and 17 bp downstream adjacent genomic DNA (SEQ ID NO: 896) derived from tRNA-Arg-TCG-1-1 (“adjacent 20”), (iii) 10 bp upstream adjacent genomic DNA (SEQ ID NO: 897) derived from tRNA-Arg-TCG-1-1 and 17 bp downstream adjacent genomic DNA (SEQ ID NO: 896) derived from tRNA-Arg-TCG-1-1 (“adjacent 10”), or (iv) 0 bp upstream adjacent genomic DNA derived from tRNA-Arg-TCG-1-1 and 17 bp downstream adjacent genomic DNA (SEQ ID NO: 896) derived from tRNA-Arg-TCG-1-1 (“adjacent 0”), under the context of either, the EGFP-R96X-TGA reporter (SEQ ID NO: 31) and a single copy of Arg TCA Suppressor tRNA #115 (SEQ ID NO: 18) are shown for Neuro-2a cells transfected with the expression construct containing them, as measured by flow cytometry. Details of the expression vectors are shown in Table 11. “GFP-PTC” indicates cells transfected with the EGFP-R96X-TGA reporter alone (SEQ ID NO: 31), and “GFP” indicates cells transfected with a version of the EGFP reporter lacking the PTC. Readthrough activity was measured by flow cytometry at approximately 24 hours after transfection. The data are presented as a histogram showing the frequency distribution of the data relative to the fluorescence intensity in cells expressing EGFP above background. (FIG. 26B) Shown are the ratio of EGFP-positive cells (“GFP+%”) in all viable cells and the mean EGFP intensity in viable cells expressing EGFP above background (“mean GFP signal”) for the cells shown in FIG. 26A. (Figure 27) Schematic diagram of the constructs used to test the effect of the 5’ leader sequence on readthrough of premature termination codons (PTCs) by suppressor tRNAs. The constructs contain (i) a 100 bp 5’ leader sequence derived from genomic DNA located upstream of a tRNA gene highly expressed in HEK293 cells, (ii) a single copy of either Arg TCA suppressor tRNA#115 (SEQ ID NO:18) or Gln TTA suppressor tRNA#163 (SEQ ID NO:45), and (iii) an RNA polymerase III termination signal (“Terminator”). (Figure 28) Graph showing the readthrough activity of the indicated Arg TCA suppressor tRNA expression constructs as measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection. Under the context of the indicated 100 bp upstream genomic DNA leader sequence (SEQ ID NO:869 - 888), cells were co-transfected with (i) the EGFP-R96X-TGA reporter (SEQ ID NO:31) and (ii) a construct containing Arg TCA suppressor tRNA#115 (SEQ ID NO:18). “WT-EGFP” indicates cells transfected with a reporter containing wild-type GFP without a PTC, “EV” (empty vector) indicates cells co-transfected with an expression construct containing neither tRNA nor the EGFP reporter, and “26 / 27” indicates cells co-transfected with ArgTCA suppressor tRNA#115 (SEQ ID NO:18) under the context of 200 bp upstream flanking genomic DNA (SEQ ID NO:26) from tRNA-Arg-TCG-1-1 and 200 bp downstream flanking genomic DNA (SEQ ID NO:27) from tRNA-Arg-TCG-1-1. The plot shows the percentage of viable cells expressing EGFP above background. The percentage of cells expressing EGFP ranged from 17.2% to 33.7% in cells expressing the Arg TCA suppressor compared to 0.4% in the empty vector control. (Figure 29) Arg measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection TCA Graph showing the readthrough activity of the indicated Arg suppressor tRNA expression constructs. Under the context of the indicated 100 bp upstream genomic DNA leader sequences (SEQ ID NOs: 869 - 888), (i) the EGFP-R96X-TGA reporter (SEQ ID NO: 31) and (ii) Arg TCA Cells were co-transfected with constructs containing suppressor tRNA#115 (SEQ ID NO: 18). "WT-EGFP" indicates cells transfected with a reporter containing wild-type GFP without a PTC, "EV" (empty vector) indicates cells co-transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "26 / 27" indicates cells co-transfected with ArgTCA suppressor tRNA#115 (SEQ ID NO: 18) under the context of 200 bp upstream adjacent genomic DNA (SEQ ID NO: 26) from tRNA-Arg-TCG-1-1 and 200 bp downstream adjacent genomic DNA (SEQ ID NO: 27) from tRNA-Arg-TCG-1-1. The plot shows the mean EGFP intensity in cells expressing EGFP above background. The mean EGFP intensity ranged from 1990 - 4319 in cells expressing the Arg suppressor compared to 602 in the empty vector control TCA suppressor. (Figure 30) Gln measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection TTA Graph showing the readthrough activity of the indicated Gln suppressor tRNA expression constructs. Under the context of the indicated 100 bp upstream genomic DNA leader sequences (SEQ ID NOs: 869 - 888), (i) the EGFP-Q69X-TAA reporter (SEQ ID NO: 175) and (ii) Gln TTACells were co-transfected with a construct containing suppressor tRNA#163 (SEQ ID NO:45). "WT-EGFP" indicates cells transfected with a reporter containing wild-type GFP without a PTC, "EV" (empty vector) indicates cells co-transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "173 / 174" indicates Gln under the context of 200 bp upstream flanking genomic DNA (SEQ ID NO:173) from tRNA-Gln-TTG-1-1 and 200 bp downstream flanking genomic DNA (SEQ ID NO:174) from tRNA-Gln-TTG-1-1 TTA Shows cells co-transfected with suppressor tRNA#163 (SEQ ID NO:45). The plot shows the proportion of viable cells expressing EGFP above background. The proportion of cells expressing EGFP was Gln relative to 0.3% in the empty vector control TTA In the range of 21.4% - 35.7% in cells expressing the suppressor (Figure 31) Gln shown, measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection TTA Graph showing the readthrough activity of the suppressor tRNA expression construct. Under the context of the indicated 100 bp upstream genomic DNA leader sequences (SEQ ID NO:869 - 888), (i) EGFP-Q69X-TAA reporter (SEQ ID NO:175) and (ii) Gln TTA Cells were co-transfected with a construct containing suppressor tRNA#163 (SEQ ID NO:45). "WT-EGFP" indicates cells transfected with a reporter containing wild-type GFP without a PTC, "EV" (empty vector) indicates cells co-transfected with an expression construct containing neither tRNA nor the EGFP reporter, and "173 / 174" indicates Gln under the context of 200 bp upstream flanking genomic DNA (SEQ ID NO:173) from tRNA-Gln-TTG-1-1 and 200 bp downstream flanking genomic DNA (SEQ ID NO:174) from tRNA-Gln-TTG-1-1TTA Cells co-transfected with suppressor tRNA#163 (SEQ ID NO:45) are shown. The plot shows the average EGFP intensity in cells expressing EGFP above background. The average EGFP intensity was 1702 - 3822 in cells expressing the suppressor, compared to 387 in the empty vector control, for Gln TTA It was in the range of 1702 - 3822 in cells expressing the suppressor. (Figure 32) A table summarizing the results of Figures 28 - 31, with values normalized to cells transfected with wild-type GFP without a PTC. (Figure 33) A graph showing the percentage of EGFP-positive cells measured by flow cytometry in three independent experiments approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-Q69X-TAA reporter ("EGFP-PTC") (SEQ ID NO:175) was transfected, and (i) on the same construct, the indicated copy number of Gln TTAEither include suppressor tRNA#002 (SEQ ID NO:36), or (ii) treat with ataluren at the indicated concentration, (iii) treat with gentamicin at the indicated concentration, or (iv) treat with G418 at the indicated concentration. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, and at this point, the indicated drug at the indicated concentration was added. A reporter containing wild-type EGFP without a PTC (“EGFP”) was used as a control. “Mock” indicates mock-transfected cells, “tRNAless” indicates cells transfected with the indicated EGFP expression construct alone, and “4X-Gln-TTG” indicates cells transfected with an expression construct containing 4 copies of wild-type Gln-tRNA with the TTG anticodon and the EGFP-Q69X-TAA reporter. The plot shows the ratio of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. The ratio of cells expressing GFP was 0.2 - 0.7% in the negative control, 0.4 - 0.6% in ataluren-treated cells, 0.4% in gentamicin-treated cells, and 1.7% - 4.5% in G418-treated cells, compared to 64.7 - 67.3% in cells expressing the Gln TTA suppressor. (Figure 34) A graph showing the cell viability from Figure 33, measured by flow cytometry approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-Q69X-TAA reporter (“EGFP-PTC”) (SEQ ID NO:175) was transfected, and (i) on the same construct, the indicated copy number of Gln TTAEither (i) include suppressor tRNA#002 (SEQ ID NO:36), or (ii) treat with ataluren at the indicated concentration, (iii) treat with gentamicin at the indicated concentration, or (iv) treat with G418 at the indicated concentration. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug at the indicated concentration was added. A reporter containing wild-type EGFP without a PTC (“EGFP”) was used as a control. “Mock” indicates mock-transfected cells, “tRNA minus” indicates cells transfected with the indicated EGFP expression construct alone, and “4X-Gln-TTG” indicates cells transfected with an expression construct containing 4 copies of wild-type Gln-tRNA with a TTG anticodon and the EGFP-Q69X-TAA reporter. Cell viability was assessed by flow cytometry using 7-aminoactinomycin D (7-AAD), a membrane-impermeable dye that is excluded from normally viable cells. (Figure 35) Graph showing the ratio of EGFP-positive cells measured by flow cytometry in three independent experiments approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-Q69X-TAG reporter (“EGFP-PTC”) (SEQ ID NO:176) was transfected, and (i) on the same construct, the indicated copy number of Gln CTAEither include suppressor tRNA#196 (SEQ ID NO:178), or (ii) treat with ataluren at the indicated concentration, (iii) treat with gentamicin at the indicated concentration, or (iv) treat with G418 at the indicated concentration. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, and at this point, the indicated drug at the indicated concentration was added. A reporter containing wild-type EGFP without a PTC (「EGFP」) was used as a control. 「Mock」 indicates mock-transfected cells, 「tRNA minus」 indicates cells transfected with the indicated EGFP expression construct alone, and 「4X-Gln-TTG」 indicates cells transfected with an expression construct containing 4 copies of wild-type Gln-tRNA with a TTG anticodon and the EGFP-Q69X-TAA reporter. The plot shows the ratio of viable cells expressing GFP above background. The ratio of cells expressing GFP was 0.5 - 0.8% in the negative control, 0.5 - 0.7% in ataluren-treated cells, 0.5 - 1.1% in gentamicin-treated cells, and 13.6 - 20.6% in G418-treated cells, compared to 73.1 - 78.2% in cells expressing the suppressor. CTA It was in the range of 73.1 - 78.2% in cells expressing the suppressor. (Figure 36) A graph showing the cell viability from Figure 35, measured by flow cytometry approximately 48 hours after transfection. For Neuro-2a cells, an expression construct containing the EGFP-Q69X-TAG reporter (「EGFP-PTC」)(SEQ ID NO:176) was transfected, and (i) on the same construct, the indicated copy number of Gln CTAEither (i) include suppressor tRNA#196 (SEQ ID NO:178), or (ii) treat with ataluren at the indicated concentration, or (iii) treat with gentamicin at the indicated concentration, or (iv) treat with G418 at the indicated concentration. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, and at this point, the indicated drug at the indicated concentration was added. A reporter containing wild-type EGFP without a PTC (“EGFP”) was used as a control. “Mock” indicates mock-transfected cells, “tRNA minus” indicates cells transfected with the indicated EGFP expression construct alone, and “4X-Gln-TTG” indicates cells transfected with an expression construct containing 4 copies of wild-type Gln-tRNA with a TTG anticodon and the EGFP-Q69X-TAA reporter. Cell viability was evaluated by flow cytometry using 7-aminoactinomycin D (7-AAD), a membrane-impermeable dye that is excluded from normally viable cells. (FIG. 37A) Western blot showing rescue of full-length SCN1A protein expression by suppressor tRNA. For Flp-In-293 cells, an expression construct containing Arg(R)-to-TGA PTC (R1407X) and a 3xFLAG tag peptide at the C-terminus TIFF0007712270000001.tif4128 (SEQ ID NO:901) of mouse SCN1A ( “SCN1a (R1407 * )”) was transfected, and (i) Arg TCAEither co-transfect an expression construct containing suppressor tRNA#115 (SEQ ID NO:18) (“Arg>TGA#115”), or (ii) treat with ataluren at the indicated concentrations, or (iii) treat with gentamicin at the indicated concentrations, or (iv) treat with G418 at the indicated concentrations. “SCN1a (w.t.)” refers to cells transfected with an expression construct containing wild-type mouse SCN1A and a C-terminal 3xFLAG tag peptide (SEQ ID NO: 898). Proteins were isolated 24 hours after transfection and SCN1A was detected using a monoclonal anti-FLAG M2 antibody. The molecular weights based on protein molecular weight markers are shown on the left side of the gel. (FIG. 37B) Quantification of the Western blot shown in FIG. 37A. The intensity of the band corresponding to the size of the full-length SCN1A protein was measured using ImageJ, and all intensity values were normalized to the wild-type SCN1A protein (“SCN1a (w.t.)” lane). Expression vectors containing mutant SCN1A and Arg TCA Cells co-transfected with an expression vector containing suppressor tRNA expressed more than 70% of the full-length SCN1A expressed by cells transfected with an expression construct containing wild-type SCN1A. (FIG. 38A) Western blot showing rescue of full-length SCN1A protein expression by suppressor tRNA. (i) Arginine-to-TGA PTC (R1407X) and a 3xFLAG tag peptide at the C-terminus TIFF0007712270000002.tif4128 (SEQ ID NO:901) of mouse SCN1A ( “SCN1a (R1407 * )”), and (ii) #104 Arg TCA suppressor tRNA (SEQ ID NO:6) (“Sup#104”), #106 Arg TCA suppressor tRNA (SEQ ID NO:8) (“Sup#106”), or Arg TCAEither suppressor tRNA#115 (SEQ ID NO;18) (“Sup#115”) was co-transfected into Flp-In-293 cells. “SCN1a (w.t.)” indicates cells transfected with an expression construct containing wild-type mouse SCN1A and a C-terminal 3xFLAG tag peptide (SEQ ID NO: 898). Proteins were isolated 24 hours after transfection, and SCN1A was detected using a monoclonal anti-FLAG M2 antibody. The molecular weight based on the protein molecular weight marker is shown on the left side of the gel. (FIG. 38B) Quantification of the Western blot shown in FIG. 38A. The intensity of the band corresponding to the size of the full-length SCN1A protein was measured using ImageJ, and all intensity values were normalized to the wild-type SCN1A protein (“SCN1a (w.t.)” lane). Expression vectors containing mutant SCN1A and Arg TCA Cells co-transfected with an expression vector containing suppressor tRNA expressed more than 30% (Sup#104), more than 60% (Sup#106), or more than 70% (Sup#115) of the full-length SCN1A expressed by cells transfected with an expression construct containing wild-type SCN1A. (FIG. 39A) Western blot showing rescue of full-length SCN1A protein expression by suppressor tRNA. For Flp-In-293 cells, cells were cultured in a 6-well cell culture plate, and (i) an expression construct containing arginine-to-TGA PTC (R1407X) and a 3xFLAG tag peptide TIFF0007712270000003.tif4128 (SEQ ID NO:901) at the C-terminus and mouse SCN1A ( “SCN1a (R1407 * )”) and (ii) Arg at the indicated concentrations TCA were co-transfected with suppressor tRNA#115 (SEQ ID NO:18) (“Arg>TGA#115”). 1x indicates 400 ng of Arg suppressor tRNA construct per well, and 0.3x indicates 133 ng of Arg suppressor tRNA construct per well. TCA per well. TCASuppressor tRNA constructs are shown, 0.1x being 40 ng of Arg per well TCA Suppressor tRNA constructs are shown, 0.03x being 13 ng of Arg per well TCA Suppressor tRNA constructs are shown. "SCN1a (w.t.)" indicates cells transfected with an expression construct containing wild-type mouse SCN1A and a C-terminal 3xFLAG tag peptide (SEQ ID NO: 898). Proteins were isolated 24 hours after transfection and SCN1A was detected using a monoclonal anti-FLAG M2 antibody. The molecular weights based on protein molecular weight markers are shown on the left side of the gel. (Figure 39B) Quantification of the Western blot shown in Figure 39A. The intensity of the band corresponding to the size of the full-length SCN1A protein was measured using ImageJ, and all intensity values were normalized to the wild-type SCN1A protein ("SCN1a (w.t.)" lane). (Figure 40) Schematic diagram of constructs packaged in the AAV-PHP.eB capsid. Construct 262 contains wild-type EGFP driven by the EF1a promoter. Construct 269 contains EGFP-R96X-TGA (SEQ ID NO: 177) and 2 copies of Arg driven by the EF1a promoter under the context of 55 bp upstream flanking genomic DNA (SEQ ID NO: 900) from tRNA-Tyr-GTA-5-1 TCA containing suppressor tRNA #115 (SEQ ID NO: 18) ("TCA-115"). Both constructs contain AAV2-derived 5' and 3' ITR sequences that provide cis-acting elements for AAV replication and packaging. (Figure 41) Shows the readthrough activity of suppressor tRNA delivered by AAV. AAV-PHP.eB containing the construct shown in Figure 40 was produced by Vigene Biosciences. 48 hours prior to AAV transduction, 293 cells were pre-transfected with an expression construct containing the LY6A gene required for reliable transduction by AAV-PHP.eB. Cells were transduced at an MOI of 1E5 vg / cell. When indicated, cells were also transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and an expression construct containing ArgTCA suppressor tRNA#115 (SEQ ID NO:18). 72 hours after transduction, the EGFP signal was quantified by immunofluorescence. Live cell images were taken with an EVOS FL Auto 2 imaging system. Using CellProfiler software, the EGFP intensity incorporated into the nucleus was segmented and extracted in each image. These values were averaged among all nuclei for each condition. From each of these averages, the background average, which is the intensity of incorporated EGFP in the negative control condition, was subtracted and all values were normalized. The plot shows the normalized GFP intensity %, and all values are normalized to cells transduced with AAV-PHP.eB containing construct 262. (Figure 42) Shows fluorescence measured by flow cytometry in Neuro-2a cells used in ribosome footprint profiling analysis. (i) An expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:177) and Arg TCA suppressor tRNA#001 (SEQ ID NO:11) on the same construct (「Arg TCA EGFP-PTC」) or (ii) an expression construct containing a version of the EGFP reporter lacking the PTC (「WT-EGFP」) was transfected into the cells. (Figure 43) shows the fold change in 3' UTR read density distribution (determined by ribosome profiling) between the two Neuro-2a cell populations shown in Figure 42. (i) An expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:177) and the Arg TCA suppressor tRNA #001 (SEQ ID NO:11) on the same construct (「Arg TCA EGFP-PTC」) or (ii) an expression construct containing a version of the EGFP reporter lacking the PTC (「WT-EGFP」) was transfected into the cells. Cells were lysed approximately 48 hours after transfection and subjected to ribosome footprint profiling. Adapters were removed from the raw reads using Trimmomatic, and non-coding RNAs were removed by aligning to the Ensembl mouse mm10 ncRNA reference using bowtie2. The remaining reads were aligned to the UCSC mm10 mouse reference assembly using bowtie2 again. Multiply mapped reads were discarded. The final set of aligned reads obtained was quantified using the RiboProfiling package with R and custom Python scripts. Using Python, plots were created to test the 3' UTR occupancy and fold change in each gene with 20 or more uniquely mapped reads, and the distributions in genes with each native stop codon were compared using the two-sample Kolmogorov-Smirnov test. **DETAILED DESCRIPTION**

[0030] Detailed Description The present invention is based in part on the discovery of a tRNA (e.g., a suppressor tRNA) that enables the incorporation of an amino acid at a position in a gene product encoded by a gene in mammalian cells where a shortened gene product would otherwise result from a premature termination codon (PTC) within that gene. The present invention is further based in part on the discovery that a tRNA that enables the incorporation of an amino acid at a position in a gene product encoded by a gene where a shortened gene product would otherwise result from a PTC within that gene can be used to treat a disease mediated by a PTC within a gene in a subject.

[0031] Accordingly, in one aspect, the present invention provides a tRNA (e.g., an isolated tRNA) comprising the nucleotide sequence shown in Table 2.

[0032] In another aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding a tRNA, such as those shown in Tables 1-3. In certain embodiments, the expression vector comprises 1, 2, 3, 4 or more than 4 copies of the nucleotide sequence encoding the tRNA. In certain embodiments, the expression vector comprises a nucleotide sequence corresponding to a genomic DNA sequence adjacent to a wild-type tRNA gene. For example, in certain embodiments, the expression vector comprises the nucleotide sequence shown in Table 4.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising any of the above tRNAs or any of the above expression vectors and a pharmaceutically acceptable excipient.

[0034] In another aspect, the present invention provides a method for expressing a functional gene product encoded by a gene containing a premature stop codon in mammalian cells, the method comprising the step of introducing into the cells an expression vector containing an effective amount of a tRNA (e.g., including SEQ ID NO:6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 as shown in Tables 1-3 below) or a nucleotide sequence encoding the tRNA, whereby amino acids are incorporated at positions in the gene product where, in the absence thereof, a truncated gene product would otherwise result from the premature stop codon.

[0035] In certain embodiments of any of the above methods, the cells contain fewer truncated gene products than cells without the tRNA. For example, in certain embodiments, the cells contain about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or less than about 90% fewer truncated gene products compared to cells without the tRNA. In certain embodiments, the cells contain about 5% - about 80%, about 5% - about 60%, about 5% - about 40%, about 5% - about 20%, about 5% - about 10%, about 10% - about 80%, about 10% - about 60%, about 10% - about 40%, about 10% - about 20%, about 20% - about 80%, about 20% - about 60%, about 20% - about 40%, about 40% - about 80%, about 40% - about 60%, or about 60% - about 80% fewer truncated gene products compared to cells without the tRNA. In certain embodiments, no detectable truncated gene product is present in the cells. The amount or expression of the truncated gene product can be measured by any method known in the art, such as Western blot or ELISA.

[0036] In certain embodiments, the cells contain a greater amount of functional gene product than cells that do not have tRNA. For example, in certain embodiments, the method increases the amount of functional gene product in a cell, tissue, or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, or about 500% compared to a cell, tissue, or subject that does not have tRNA. In certain embodiments, the method increases the amount of functional gene product in a cell, tissue, or subject by about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200% compared to a cell, tissue, or subject that does not have tRNA. The amount or expression of the functional gene product can be measured by any method known in the art, such as Western blot or ELISA.

[0037] In certain embodiments, the tRNA enables an amino acid to be incorporated at a position in the gene product corresponding to a premature stop codon (i.e., the tRNA enables readthrough of the premature stop codon), but the tRNA does not enable a substantial amount of amino acid to be incorporated at a position in the gene product corresponding to a native stop codon (i.e., the tRNA does not enable readthrough of the native stop codon). For example, in certain embodiments, the disclosed tRNA does not increase readthrough of native stop codons (or all native stop codons) in a cell, tissue, or subject, or increases readthrough by less than about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% compared to a cell, tissue, or subject not in contact with the tRNA. Readthrough of native stop codons can be measured by any method known in the art, such as ribosome profiling as described in Example 13 herein.

[0038] In certain embodiments of any of the above methods, the gene is selected from the genes shown in Table 5 or Table 6. In certain embodiments, the gene is the SCN1A gene.

[0039] In another aspect, the present invention provides a method for expressing a functional SCN1A gene product encoded by an SCN1A gene containing a premature stop codon in a cell, the method comprising introducing into the cell an expression vector comprising an effective amount of a tRNA (e.g., including SEQ ID NO:6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 as shown in Tables 1-3 below) or a nucleotide sequence encoding the tRNA, thereby enabling an amino acid to be incorporated at a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise result due to the premature stop codon.

[0040] In another aspect, the present invention provides a method for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature termination codon in a cell, the method comprising the step of introducing into the cell an expression vector comprising an effective amount of a tRNA (e.g., including SEQ ID NO:6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 as shown in Tables 1-3 below) or a nucleotide sequence encoding the tRNA, thereby enabling the incorporation of an amino acid at a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise result from the premature termination codon.

[0041] In another aspect, the present invention provides a method for treating a premature termination codon-mediated disorder in a subject in need thereof, the subject having a gene with a premature termination codon, the method comprising the step of administering to the subject an expression vector comprising an effective amount of a tRNA (e.g., including SEQ ID NO:6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 as shown in Tables 1-3 below) or a nucleotide sequence encoding the tRNA, thereby treating the disorder in the subject. In certain embodiments, the disorder is selected from the disorders shown in Table 5 or Table 6.

[0042] In another aspect, the present invention provides a method for treating Dravet syndrome in a subject in need thereof, the subject having an SCN1A gene with a premature termination codon, the method comprising the step of administering to the subject an expression vector comprising an effective amount of a tRNA (e.g., including SEQ ID NO:6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 as shown in Tables 1-3 below) or a nucleotide sequence encoding the tRNA, thereby treating Dravet syndrome in the subject.

[0043] I. tRNA and suppressor tRNA During protein synthesis, transfer RNA (tRNA) delivers amino acids to the ribosome for incorporation into the growing protein (polypeptide) chain. tRNA is typically about 70 - 100 nucleotides in length, and active tRNA contains a 3' CCA sequence, which can be transcribed into the tRNA during its synthesis or added during subsequent post-transcriptional processing. During aminoacylation, the amino acid added to the tRNA molecule is covalently attached to the 2' or 3' hydroxyl group of the 3' terminal ribose, forming aminoacyl-tRNA (aa-tRNA). The amino acid can move spontaneously from the 2'-hydroxyl group to the 3'-hydroxyl group or vice versa, but it is understood to be incorporated into the growing protein chain on the ribosome from the 3'-OH position. The loop at the other end of the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon. When this anticodon sequence hybridizes or forms base pairs with a complementary three-base codon sequence in messenger RNA (mRNA) bound to the ribosome, the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptide chain being synthesized by that ribosome. Since all tRNAs that form base pairs with a particular codon are aminoacylated with a single specific amino acid, the genetic code is translated by tRNAs. Each of the 61 non-stop codons in mRNA guides the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing polypeptide chain being synthesized by the ribosome.

[0044] tRNAs are generally highly conserved and often function across species. Thus, tRNAs from bacterial, non-mammalian eukaryotic, or mammalian (e.g., human) sources may be useful in practicing the present invention. Nucleotide sequences encoding naturally occurring human tRNAs are known and generally available to those of skill in the art through sources such as Genbank. See also Sprinzl et al. (2005) NUCLEIC ACIDS RES. 33: D139-40; Buckland et al. (1996) GENOMICS 35(1):164-71; Schimmel et al. (Eds.) (1979) “Transfer-RNA: Structure, Properties, and Recognition,” Cold Spring Harbor Laboratory; Agris (1983) "The Modified Nucleosides of Transfer RNA, II,” Alan R. Liss Inc. tRNAs are generally highly conserved and often function across species.

[0045] A suppressor tRNA is a modified tRNA that inserts the appropriate amino acid at a mutant site within a protein-coding gene, e.g., a PTC. The use of this term under suppressor is based on the fact that, in certain circumstances, this modified tRNA "suppresses" the phenotypic effect of the coding mutation. Suppressor tRNAs typically contain a mutation (modification) either within the anticodon to change codon specificity or at several positions that alter the aminoacylation properties of the tRNA.

[0046] In certain embodiments, a tRNA (e.g., a suppressor tRNA) contains a modified anticodon region, and the modified anticodon hybridizes to a codon that is different from the corresponding naturally occurring anticodon. In certain embodiments, the modified anticodon hybridizes to a stop codon, e.g., a PTC, such that the tRNA incorporates an amino acid into the gene product rather than terminating protein synthesis. In certain embodiments, the modified anticodon hybridizes to a premature stop codon, such that the tRNA incorporates an amino acid at a position in the gene product where, without the tRNA, a truncated gene product would result due to the premature stop codon.

[0047] In certain embodiments, the tRNA contains an anticodon that hybridizes to a codon selected from UAG (i.e., the “amber” stop codon), UGA (i.e., the “opal” stop codon), and UAA (i.e., the “ochre” stop codon). In certain embodiments, the anticodon hybridizes to a codon selected from UGA to UAA. In certain embodiments, the anticodon hybridizes to UGA. In certain embodiments, the tRNA contains an anticodon that hybridizes to a non-standard stop codon, e.g., a 4-nucleotide codon (see, e.g., Moore et al. (2000) J. Mol. Biol. 298:195, and Hohsaka et al. (1999) J. Am. Chem. Soc. 121:12194).

[0048] In certain embodiments, the tRNA is or can be aminoacylated with any natural amino acid. For example, the tRNA can be aminoacylated with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the tRNA can be aminoacylated with serine, leucine, glutamine, or arginine. In certain embodiments, the tRNA can be aminoacylated with glutamine or arginine. In certain embodiments, the tRNA can be aminoacylated with arginine.

[0049] In certain embodiments, the tRNA comprises an anticodon that hybridizes to the codons shown in Table 1 and is or can be aminoacylated with the amino acids shown in Table 1.

[0050]

Table 1

[0051] In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence shown in Table 2. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence shown in Table 2. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence selected from SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186. Throughout the detailed description, in each instance where the tRNA comprises, consists essentially of, or consists of a nucleotide sequence that includes one or more thymines (T), it is also contemplated that the tRNA comprises, consists essentially of, or consists of the same nucleotide sequence with uracil (U) substituted for one or more thymines (T), or uracil (U) substituted for all thymines (T). Similarly, in each instance where the tRNA comprises, consists essentially of, or consists of a nucleotide sequence that includes one or more uracils (U), it is understood that it is also contemplated that the tRNA comprises, consists essentially of, or consists of a nucleotide sequence with thymine (T) substituted for one or more uracils (U), or thymine (T) substituted for all uracils (U).

[0052] [Table 2] TIFF0007712270000007.tif234165TIFF0007712270000008.tif227151TIFF0007712270000009.tif227151TIFF0007712270000010.tif227151TIFF0007712270000011.tif227151TIFF0007712270000012.tif227151TIFF0007712270000013.tif227151TIFF0007712270000014.tif227151TIFF0007712270000015.tif227151TIFF0007712270000016.tif227151TIFF0007712270000017.tif107151

[0053] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence shown in Table 3. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence shown in Table 3. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187.

[0054]

Table 3

[0055] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence shown in any one of Tables 8-10. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence in any one of Tables 8-10.

[0056] In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:6. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:7. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:8. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:9. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:11. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:16. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:17. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:18. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:19. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:20. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:21. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:22. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:35. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:36. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:37.In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:38. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:39. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:40. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:44. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:45. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:178. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:179. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:180. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:181. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:182. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:186. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:187.

[0057] In certain embodiments, the tRNA can include one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) as compared to a reference tRNA sequence (e.g., a tRNA disclosed herein). In certain embodiments, the tRNA can consist of, consist essentially of, or include a combination of a single mutation, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 mutations. It is contemplated that the tRNA can consist of, consist essentially of, or include 1 to 15, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 15, 2 to 10, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 15, 3 to 10, 3 to 7, 3 to 6, 3 to 5, or 3 to 4 mutations.

[0058] Sequence identity can be determined in a variety of ways that are well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithms employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402) is designed for sequence similarity searching. For a discussion of basic matters in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6:119-129. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. Search parameters regarding histograms, descriptions, alignments, expect values (i.e., thresholds of statistical significance for reporting matches against database sequences), cutoffs, matrices and filters are those of the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919). The four blastn parameters can be adjusted as follows: Q = 10 (gap formation penalty); R = 10 (gap extension penalty); wink = 1 (generate word hits every wink-th position along the query); and gapw = 16 (set the window width for generating alignments that include gaps).Equal Blastp parameter settings can be Q = 9; R = 2; wink = 1; and gapw = 32. Searches can also be performed using NCBI (National Center for Biotechnology Information) BLAST advanced option parameters (e.g., -G, cost to open a gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost to extend a gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, drop-off (X) for bit-unit blast extension: default = 20 for blastn / 7 for others; -X, X drop-off value (bit-unit) for alignments containing gaps: default = 15 for all programs, not applicable to blastn; and -Z, final X drop-off value (bit-unit) for alignments containing gaps: 50 for blastn, 25 for others). ClustalW for pairwise protein alignment can also be used (default parameters can include, for example, the Blosum62 matrix and gap open penalty = 10 and gap extension penalty = 0.1). Bestfit comparison between sequences available in the GCG package version 10.0 uses DNA parameters of GAP = 50 (gap formation penalty) and LEN = 3 (gap extension penalty), and the equivalent settings for protein comparison are GAP = 8 and LEN = 2.

[0059] It is contemplated that the tRNA may contain one or more modifications. Exemplary modified tRNAs include acylated tRNA; alkylated tRNA; tRNA containing one or more bases other than adenine, cytosine, guanine, or uracil; tRNA covalently modified by the addition of a specific ligand or an antigenic, fluorescent, affinity, reactive, spectral, or other probe moiety; tRNA containing one or more ribose moieties that have been methylated or otherwise modified; aa-tRNA aminoacylated with an amino acid other than one of the 20 natural amino acids, including a reagent carrier, a specific ligand, or an unnatural amino acid that functions as an antigenic, fluorescent, reactive, affinity, spectral, or other probe; or any combination of these compositions.Exemplary modified tRNA molecules are described in Soll et al. (1995) “tRNA: Structure, Biosynthesis, and Function,” ASM Press; El Yacoubi et al. (2012) ANNU. REV. GENET. 46:69-95; Grosjean et al. (1998) “Modification and Editing of RNA.” ASM Press; Hendrickson et al. (2004) ANNU. REV. BIOCHEM. 73:147-176, 2004; Ibba et al. (2000) ANNU. REV. BIOCHEM. 69:617-650; Johnson et al. (1995) COLD SPRING HARBOR SYMP. QUANT. BIOL. 60:71-82; Johnson et al. (1982) J. MOL. BIOL. 156:113-140; Crowley et al. (1994) CELL 78:61-71; Beier et al. (2001) NUCLEIC ACIDS RES. 29:4767-4782; Torres et al. (2014) TRENDS MOL. MED. 20:306-314; Bjork et al. (1987) ANNU. REV. BIOCHEM. 56:263-287; Schaffrath et al. (2017) RNA BIOL. 14(9):1209-1222; and Johansson et al. (2008) MOL. CELL. BIOL. 28(10):3301-12.

[0060] In certain embodiments, the tRNA includes naturally occurring nucleotide modifications. Naturally occurring tRNAs contain a wide variety of post-transcriptionally modified nucleotides, including, for example, one or more of the residues described in Machnicka et al. (2014) RNA BIOLOGY 11(12): 1619-1629 and shown in FIG. 2B. In certain embodiments, the tRNA is 2'-O-methylguanosine or G at position 0; pseudouridine or Ψ at position 1; 2'-O-methyladenosine, A, 2'-O-methyluridine, U, 2'-O-methylcytidine, C, 2'-O-methylguanosine, or G at position 4; N2-methylguanosine or G at position 6; N2-methylguanosine or G at position 7; 1-methyladenosine, A, 1-methylguanosine, G, or modified G at position 9; N2-methylguanosine or G at position 10; N4-acetylcytidine or C at position 12; pseudouridine, Ψ, 2'-O-methylcytidine, or C at position 13; 1-methyladenosine, A, or modified A at position 14; dihydrouridine (D) or U at position 16; D or U at position 17; 2'-O-methylguanosine or G at position 18; 3-(3-amino-3-carboxypropyl)uridine, D, or U at position 20; 3-(3-amino-3-carboxypropyl)uridine, D, pseudouridine, Ψ, or modified U at position 20a; D, pseudouridine, or Ψ at position 20b; pseudouridine or Ψ at position 25; pseudouridine, Ψ, N2,N2-dimethylguanosine, N2-methylguanosine, G, or modified G at position 26; pseudouridine, Ψ, N2,N2-dimethylguanosine, or G at position 27; pseudouridine or Ψ at position 28; pseudouridine or Ψ at position 30; pseudouridine or Ψ at position 31; 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, Ψ, 2'-O-methylcytidine, 3-methylcytidine, C, or modified C at position 32;34 - inosine, A, 2 - thiouridine, 2'-O - methyluridine, 5 - (carboxyhydroxymethyl)uridine methyl ester, 5 - carbamoylmethyluridine, 5 - carboxymethylaminomethyl - 2'-O - methyluridine, 5 - methoxycarbonylmethyl - 2 - thiouridine, 5 - methoxycarbonylmethyluridine, pseudouridine, U, modified U, 2'-O - methylcytidine, 5 - formyl - 2'-O - methylcytidine, 5 - methylcytidine, C, modified C, queuosine, mannosyl - queuosine, galactosyl - queuosine, 2'-O - methylguanosine, or G; at position 35, pseudouridine or U; at position 36, pseudouridine, U, or modified U; at position 37, 1 - methylinosine, 2 - methylthio - N6 - threonylcarbamoyladenosine, N6 - isopentenyladenosine, N6 - methyl - N6 - threonylcarbamoyladenosine, N6 - threonylcarbamoyladenosine, A, modified A, 1 - methylguanosine, peroxisome wobutosine, wobutosine, G, or modified G; at position 38, pseudouridine, U, 5 - methylcytidine, C, or modified C; at position 39, 1 - methylpseudouridine, 2'-O - methylpseudouridine, 2'-O - methyluridine, pseudouridine, U, 2'-O - methylguanosine, or G; at position 40, pseudouridine, U, 5 - methylcytidine, or C; at position 44, 2'-O - methyluridine, U, or modified U; at position e11, pseudouridine or U; at position e12, pseudouridine or U; at position e14, pseudouridine or U; at position e2, 3 - methylcytidine or C; at position 46, 7 - methylguanosine or G; at position 47, D, U, or modified U; at position 48, D, U, 5 - methylcytidine, C, or modified C; at position 49, A, modified A, 5 - methylcytidine, C, or modified C; at position 50, pseudouridine, U, 5 - methylcytidine, or C; at position 54, 5,2'-O - dimethyluridine, 5 - methyluridine, pseudouridine, or U; at position 55, pseudouridine or U; at position 58, 1 - methyladenosine, A, or modified A; at position 64, 2'-O - ribosyladenosine (phosphate), A, 2'-O - ribosylguanosine (phosphate), G, or modified G; at position 65, pseudouridine or U;It contains one or more residues selected from the group consisting of pseudouridine, U, N2-methylguanosine, or G at position 67; pseudouridine or U at position 68; and pseudouridine, U, 5-methylcytidine, or C at position 72. A, C, G, and U represent unmodified adenine, cytosine, guanine, and uracil, respectively. The residue numbers are based on the tRNA numbering system described in Steinberg et al., (1993) NUCLEIC ACIDS RES. 21:3011-15.;

[0061] In certain embodiments, the tRNA contains one or more nucleotide modifications selected from 5-methyluridine, 5-carbamoylmethyluridine, 5-carbamoyl-methyl-2-O-methyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine.;

[0062] II. Method for producing tRNA It is contemplated that tRNA molecules useful in the practice of the present invention (e.g., suppressor tRNAs) can be produced by methods known in the art, including extracellular production by synthetic chemical methods, intracellular production by recombinant DNA methods, or purification from natural sources.;

[0063] For example, a DNA molecule encoding tRNA can be synthesized chemically or by recombinant DNA methods. For example, the sequence of tRNA can be synthesized or cloned from a library by conventional hybridization techniques or polymerase chain reaction (PCR) techniques using appropriate synthetic nucleic acid primers. The resulting DNA molecule encoding tRNA can be ligated with other appropriate nucleotide sequences, such as expression control sequences, to produce a conventional gene expression construct (i.e., expression vector) encoding tRNA. The production of defined gene constructs is within the common general knowledge in the art. The nucleic acid encoding the desired tRNA can be incorporated (ligated) into an expression vector, such as the expression vectors described in the following sections, and this expression vector can be introduced into a host cell through conventional transfection or transformation techniques. Exemplary host cells are Escherichia coli (E. coli) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and bone marrow cells. The transformed host cells can be grown under conditions in which the host cell can express the gene encoding tRNA. Specific expression and purification conditions will vary depending on the expression system used.

[0064] Alternatively, tRNA can be chemically synthesized or purified from natural sources by methods known in the art. If the tRNA is aminoacylated prior to introduction into cells or administration to a subject, the tRNA can be aminoacylated with the desired amino acid by any method known in the art, including chemical or enzymatic aminoacylation.

[0065] III. Expression vector The tRNA of interest can be expressed in the cells of interest by incorporating the gene encoding the tRNA of interest into an appropriate expression vector. As used herein, "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate a recombinant polynucleotide of interest, such as cosmids, plasmids (e.g., naked or contained within liposomes), retrotransposons (e.g., piggyback, sleeping beauty), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0066] In certain embodiments, the expression vector is a viral vector. As used herein, the term "virus" refers to an obligate intracellular parasite that has no protein synthesis or energy generation mechanism. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Epstein-Barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotyped virus vectors.

[0067] The virus can be an RNA virus (having a genome composed of RNA) or a DNA virus (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvovirus (e.g., adeno-associated virus), adenovirus, asfarvirus, herpesvirus (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papillomavirus (e.g., HPV), polyomavirus (e.g., simian vacuolating virus 40 (SV40)), and poxvirus (e.g., vaccinia virus, cowpox virus, variola virus, fowlpox virus, goatpox virus, myxoma virus). In certain embodiments, the viral vector is an RNA viral vector. Exemplary RNA viruses include bunyavirus (e.g., hantavirus), coronavirus, flavivirus (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis virus (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza virus (e.g., influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, norovirus (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retrovirus (e.g., human immunodeficiency virus-1 (HIV-1)), and torovirus.

[0068] In certain embodiments, the expression vector includes a regulatory sequence or promoter operably linked to a nucleotide sequence encoding a tRNA. The term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, enhancers usually function even when located several kilobases away from a promoter, and intron sequences can vary in length, so some polynucleotide elements are operably linked but not directly adjacent and can function in trans even from different alleles or chromosomes.

[0069] The tRNA gene preferably has a strong promoter that is active in various cell types. Promoters for eukaryotic tRNA genes are typically present within the structural sequence itself that encodes the tRNA molecule. Elements that regulate transcriptional activity are present within the 5' upstream region, but the length of the active transcription unit can be much shorter than 500 base pairs.

[0070] Additional exemplary promoters that can be used include, but are not limited to, retroviral LTRs, the SV40 promoter, the human cytomegalovirus (CMV) promoter, the U6 promoter, or any other promoter (e.g., eukaryotic cellular promoters, including but not limited to histone, pol III, and β-actin promoters). Other viral promoters that can be used include, but are not limited to, the adenovirus promoter, the TK promoter, and the B19 parvovirus promoter. The selection of an appropriate promoter will be apparent to those skilled in the art from the teachings contained herein.

[0071] In certain embodiments, the expression vector comprises a tRNA coding sequence encoding a tRNA that comprises, consists essentially of, or consists of the nucleotide sequence shown in Table 2 or Table 3. In certain embodiments, the expression vector comprises a tRNA coding sequence encoding a tRNA that comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in Table 2 or Table 3.

[0072] In certain embodiments, in addition to the tRNA coding sequence, the expression vector comprises a nucleotide sequence corresponding to a genomic DNA sequence adjacent to the wild-type tRNA gene (i.e., a DNA sequence that is from the same genome as the wild-type tRNA gene and is located 5' or 3' to the wild-type tRNA gene within that genome, e.g., a DNA sequence that is immediately 5' or 3' to the wild-type tRNA gene within that genome). In certain embodiments, in addition to the tRNA coding sequence, the expression vector comprises a nucleotide sequence corresponding to an exogenous promoter.

[0073] In certain embodiments, the expression vector comprises the nucleotide sequence shown in Table 4. In certain embodiments, the expression vector comprises a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence shown in Table 4. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is operably linked to a nucleotide sequence encoding a tRNA. In certain embodiments, within the expression vector, the nucleotide sequence shown in Table 4 is located 5' or 3' (e.g., immediately 5' or immediately 3') to the nucleotide sequence encoding a tRNA. In certain embodiments, the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 869 - 888, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 869 - 888.

[0074] In certain embodiments, the expression vector is the expression vector described in Example 8 or Example 9 herein.

[0075]

Table 4

[0076] Adeno-associated virus (AAV) vector In certain embodiments, the expression vector is an adeno-associated virus (AAV) vector. AAV is a small, non-enveloped icosahedral virus of the genus Dependoparvovirus and the family Parvoviridae. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV can infect both dividing and quiescent cells of various tissue types, and different AAV serotypes exhibit different tissue tropisms.

[0077] AAV includes a number of serologically distinguishable types, including serotypes AAV-1 to AAV-12 and over 100 serotypes derived from non-human primates (see, e.g., Srivastava (2008) J. CELL BIOCHEM., 105(1): 17-24, and Gao et al. (2004) J. VIROL., 78(12), 6381-6388). The serotype of the AAV vector used in the present invention can be selected by those skilled in the art based on delivery efficiency, tissue tropism, and immunogenicity. For example, AAV-1, AAV-2, AAV-4, AAV-5, AAV-8, and AAV-9 can be used for delivery to the central nervous system; AAV-1, AAV-8, and AAV-9 can be used for delivery to the heart; AAV-2 can be used for delivery to the kidney; AAV-7, AAV-8, and AAV-9 can be used for delivery to the liver; AAV-4, AAV-5, AAV-6, AAV-9 can be used for delivery to the lung; AAV-8 can be used for delivery to the pancreas, AAV-2, AAV-5, and AAV-8 can be used for delivery to photoreceptor cells; AAV-1, AAV-2, AAV-4, AAV-5, and AAV-8 can be used for delivery to the retinal pigment epithelium; AAV-1, AAV-6, AAV-7, AAV-8, and AAV-9 can be used for delivery to skeletal muscle. In certain embodiments, the AAV capsid protein includes the sequences disclosed in U.S. Patent No. 7,198,951, e.g., without limitation, AAV-9 (SEQ ID NOs: 1-3 of U.S. Patent No. 7,198,951), AAV-2 (SEQ ID NO: 4 of U.S. Patent No. 7,198,951), AAV-1 (SEQ ID NO: 5 of U.S. Patent No. 7,198,951), AAV-3 (SEQ ID NO: 6 of U.S. Patent No. 7,198,951), and AAV-8 (SEQ ID NO: 7 of U.S. Patent No. 7,198,951). AAV serotypes identified from rhesus monkeys, such as rh.8, rh.10, rh.39, rh.43, and rh.74 are also contemplated in the present invention. In addition to natural AAV serotypes, modified AAV capsids have been developed to improve delivery efficiency, tissue tropism, and immunogenicity.Exemplary native and modified AAV capsids are disclosed in U.S. Patent Nos. 7,906,111, 9,493,788, and 7,198,951, and PCT Publication No. WO2017189964A2.

[0078] The wild-type AAV genome contains two 145-nucleotide inverted terminal repeats (ITRs) that contain signal sequences that direct AAV replication, genome encapsidation, and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive the expression of two open reading frames that encode the rep and cap genes. Along with differential splicing of a single AAV intron, two rep promoters generate four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. The rep proteins are responsible for genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3) that are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0079] Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome can be replaced with foreign DNA, such as an expression cassette for an exogenous gene of interest. Thus, in certain embodiments, an AAV vector comprises a genome that includes an expression cassette for an exogenous gene flanked by 5' and 3' ITRs. The ITRs can be from the same serotype as the capsid or a derivative thereof. Alternatively, the ITRs can be from a different serotype than the capsid, thereby generating a pseudotyped AAV. In certain embodiments, the ITR is from AAV-2. In certain embodiments, the ITR is from AAV-5. At least one of the ITRs can be modified to mutate or remove the terminal resolution site, thereby generating a self-complementary AAV vector.

[0080] To generate an AAV vector, the rep and cap proteins can be provided in trans, for example, on a plasmid. Host cell lines that permit AAV replication must express the rep and cap genes, an expression cassette flanked by ITRs, and helper functions provided by a helper virus, such as the adenoviral genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011). Methods for generating and purifying AAV vectors have been described in detail (see, for example, Mueller et al., (2012) CURRENT PROTOCOLS IN MICROBIOLOGY, 14D.1.1-14D.1.21, Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors). A number of cell types, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells, are suitable for the production of AAV vectors (see, for example, U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, and 8,163,543, U.S. Patent Publication No. 20020081721, and PCT Publications WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types by one plasmid containing an expression cassette flanked by ITRs, and one or more additional plasmids that provide additional AAV and helper virus genes.

[0081] In the present invention, AAV of any serotype can be used. Similarly, it is envisioned that any adenovirus type can be used and that those skilled in the art will be able to identify AAV and adenovirus types suitable for the production of their desired recombinant AAV vector (rAAV). AAV particles can be purified, for example, by affinity chromatography, iodixanol gradient, or CsCl gradient.

[0082] The AAV vector can be 4.7 kb in size, or have a single-stranded genome larger or smaller than 4.7 kb, including a large oversize genome of 5.2 kb or a small one of 3.0 kb. Thus, when the exogenous gene of interest to be expressed from the AAV vector is small, the AAV genome can contain a stuffer sequence. Furthermore, the vector genome can be substantially self-complementary, thereby allowing for rapid expression in cells. In certain embodiments, the genome of the self-complementary AAV vector comprises, from 5' to 3': 5' ITR; a first nucleic acid sequence comprising a promoter and / or enhancer operably linked to the coding sequence of the gene of interest; a modified ITR having no functional terminal resolution site; a second nucleic acid sequence complementary or substantially complementary to the first nucleic acid sequence; and 3' ITR. AAV containing all types of genomes is suitable for use in the methods of the present invention.

[0083] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EF1α-MCS (System Bio catalog #AAV502A-1), pAAVK-EF1α-MCS1-CMV-MCS2 (System Bio catalog #AAV503A-1), pAAV-ZsGreen1 (Clontech catalog #6231), pAAV-MCS2 (Addgene plasmid #46954), AAV-Stuffer (Addgene plasmid #106248), pAAVscCBPIGpluc (Addgene plasmid #35645), AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid #68375), pAAV-RAM-d2TTA::TRE-MCS-WPRE-pA (Addgene plasmid #63931), pAAV-UbC (Addgene plasmid #62806), pAAVS1-P-MCS (Addgene plasmid #80488), pAAV-Gateway (Addgene plasmid #32671), pAAV-Puro_siKD (Addgene plasmid #86695), pAAVS1-Nst-MCS (Addgene plasmid #80487), pAAVS1-Nst-CAG-DEST (Addgene plasmid #80489), pAAVS1-P-CAG-DEST (Addgene plasmid #80490), pAAVf-EnhCB-lacZnls (Addgene plasmid #35642), and pAAVS1-shRNA (Addgene plasmid #82697). These vectors can be modified to be suitable for therapeutic use. For example, an exogenous gene of interest can be inserted into the multiple cloning site, and a selectable marker (e.g., a gene encoding puro or a fluorescent protein) can be removed or replaced with another (same or different) exogenous gene of interest.Further examples of AAV vectors are disclosed in U.S. Patent Nos. 5,871,982, 6,270,996, 7,238,526, 6,943,019, 6,953,690, 9,150,882, and 8,298,818, U.S. Patent Publication No. 2009 / 0087413, and PCT Publication Nos. WO2017075335A1, WO2017075338A2, and WO2017201258A1.

[0084] In certain embodiments, the expression vector is an AAV vector that can target the nervous system, such as the central nervous system, in a subject, such as a human subject. Exemplary AAV vectors that can target the nervous system include AAV-PHP.B, which is an AAV9 variant (see, e.g., Deverman et al. (2016) NAT. BIOTECHNOL. 34(2):204-209), AAV-AS (see, e.g., Choudhury et al. (2016) MOL. THER. 24:726-35), and AAV-PHP.eB (see, e.g., Chan et al. (2017) NAT. NEUROSCI. 20:1172-79). Further exemplary AAV-based strategies for targeting the nervous system are described in Bedrook et al. (2018) ANNU REV NEUROSCI. 41:323-348. In certain embodiments, the AAV vector is an AAV-PHP.eB vector.

[0085] Lentiviral vector In certain embodiments, the viral vector can be a retroviral vector. Examples of retroviral vectors include Moloney murine leukemia virus vectors, spleen necrosis virus vectors, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Retroviral vectors are useful as agents that mediate retrovirus-mediated gene transfer into eukaryotic organisms.

[0086] In certain embodiments, the retroviral vector is a lentiviral vector. Exemplary lentiviral vectors include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis-encephalitis virus (CAEV).

[0087] Retroviral vectors are typically constructed such that most of the sequences encoding the viral structural genes are removed and replaced with a gene of interest. In many cases, the structural genes (e.g., gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques known in the art. Thus, a minimal retroviral vector contains, from 5' to 3': a 5' long terminal repeat (LTR), a packaging signal, any exogenous promoter and / or enhancer, an exogenous gene of interest, and a 3' LTR. In the absence of an exogenous promoter, gene expression is driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. For the production of lentiviruses, the structural genes can be provided on a separate vector, thereby rendering the virions produced replication-defective. Specifically, for lentiviruses, the packaging system can include a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, and a third separate vector encoding the envelope protein Env (usually VSV-G due to its broad infectivity). To improve the safety of the packaging system, the packaging vector can be split to express Rev from one vector and Gag and Pol from another vector. By using a retroviral vector containing a chimeric 5' LTR in which the U3 region of the 5' LTR is replaced with a heterologous regulatory element, Tat can also be removed from the packaging system.

[0088] Genes can be incorporated into the proviral backbone by a variety of common methods. The most straightforward construct is one in which the retroviral structural genes are replaced by a single gene transcribed under the control of viral regulatory sequences within the LTR. Retroviral vectors have also been constructed that can introduce two or more genes into target cells. Typically, in such vectors, one gene is under the regulatory control of the viral LTR, and the second gene is either expressed from a spliced message or under the control of its own internal promoter.

[0089] Accordingly, the new gene is flanked by the 5' and 3' LTRs, respectively, which function to promote transcription and polyadenylation of the virion RNA. The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the ends of retroviral DNA that are tandem repeats in their native sequences and contain the U3, R, and U5 regions. The LTRs generally provide functions essential for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) as well as viral replication. The LTRs contain many regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for replication and integration of the viral genome. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region flanks the U3 and U5 regions. In certain embodiments, the R region contains a trans-activation response (TAR) gene element that interacts with the trans-activator (tat) gene element to enhance viral replication. This element is not required in embodiments where the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0090] In certain embodiments, the retroviral vector comprises a modified 5' LTR and / or 3' LTR. Modification of the 3' LTR is often done to improve the safety of lentiviral or retroviral systems by conferring replication deficiency to the virus. In certain embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector represents a replication-deficient retroviral vector in which the U3 region of the 3' LTR is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because during viral replication, the U3 region of the 3' LTR is used as a template for the U3 region of the 5' LTR, and thus viral transcripts cannot be generated without the U3 enhancer-promoter. In further embodiments, the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal polyadenylation sequence. Note that modifications to the LTRs, such as to the 3' LTR, 5' LTR, or both 3' and 5' LTRs, are envisioned to be useful in the practice of the present invention.

[0091] In certain embodiments, the U3 region of the 5' LTR is replaced with a heterologous promoter that drives transcription of the viral genome upon production of viral particles. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters can drive high levels of transcription in a Tat-independent manner. This replacement reduces the likelihood of recombination that generates replication-competent virus because the complete U3 sequence is no longer present in the viral production system.

[0092] Adjacent to the 5' LTR are sequences required for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site). As used herein, the terms "packaging signal" or "packaging sequence" refer to sequences located within the retroviral genome that are required for encapsidation of the retroviral RNA strand during viral particle formation (see, e.g., Clever et al., 1995 J. VIROLOGY, 69(4):2101-09). The packaging signal can be the minimal packaging signal required for encapsidation of the viral genome (also referred to as the psi [Ψ] sequence).

[0093] In certain embodiments, a retroviral vector (e.g., a lentiviral vector) further comprises a FLAP. As used herein, the term "FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al. (2000) CELL, 101:173. During reverse transcription, initiation at the center of the plus-strand DNA in the cPPT and termination at the center in the CTS form a triple-stranded DNA structure: the central DNA flap. Without wishing to be bound by any theory, the DNA flap may act as a cis-acting determinant for nuclear import of the lentiviral genome and / or may increase the viral titer. In certain embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest within the vector. For example, in certain embodiments, the transfer plasmid comprises a FLAP element. In one embodiment, the vector of the invention comprises a FLAP element isolated from HIV-1.

[0094] In certain embodiments, a retroviral vector (e.g., a lentiviral vector) further includes an export element. In one embodiment, a retroviral vector includes one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the cell nucleus to the cytoplasm. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) Rev-responsive element (RRE) (see, e.g., Cullen et al., (1991) J. VIROL. 65: 1053; and Cullen et al., (1991) CELL 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, an RNA export element is located within the 3' UTR of a gene and can be inserted as one or more copies.

[0095] In certain embodiments, a retroviral vector (e.g., a lentiviral vector) further includes a post-transcriptional regulatory element. A variety of post-transcriptional regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; see Zufferey et al., (1999) J. VIROL., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., MOL. CELL. BIOL., 5:3864); and others (Liu et al., (1995), GENES DEV., 9:1766) can increase the expression of heterologous nucleic acids. Post-transcriptional regulatory elements are typically located at the 3' end of a heterologous nucleic acid sequence. This configuration synthesizes an mRNA transcript whose 5' portion contains the heterologous nucleic acid coding sequence and whose 3' portion contains the post-transcriptional regulatory element sequence. In certain embodiments, the vectors of the invention lack or do not include a post-transcriptional regulatory element, such as WPRE or HPRE, in some instances because these elements increase the risk of cell transformation and / or do not substantially or significantly increase the amount of the mRNA transcript or increase the stability of the mRNA. Thus, in certain embodiments, the vectors of the invention lack or do not include WPRE or HPRE as an additional safety measure.

[0096] Elements that drive efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are typically found downstream of polyadenylation signals. Thus, in certain embodiments, retroviral vectors (e.g., lentiviral vectors) further include a polyadenylation signal. As used herein, the term "polyadenylation signal" or "polyadenylation sequence" means a DNA sequence that drives both the termination and polyadenylation of an RNA transcript newly formed by RNA polymerase H. Since transcripts lacking a polyadenylation signal are unstable and rapidly degraded, efficient polyadenylation of recombinant transcripts is desirable. Specific examples of polyadenylation signals that can be used in the vectors of the present invention include ideal polyadenylation sequences (e.g., AATAAA, ATTAAA AGTAAA), bovine growth hormone polyadenylation sequence (BGHpA), rabbit β-globin polyadenylation sequence (rβgpA), or other suitable heterologous or endogenous polyadenylation sequences known in the art.

[0097] In certain embodiments, the retroviral vector further includes an insulator element. The insulator element can be mediated by a cis-acting element present in genomic DNA and can contribute to the protection of the retroviral expression sequence, such as a therapeutic gene, from the integration site effect (i.e., position effect; see, for example, Burgess-Beusse et al., (2002) PROC. NATL. ACAD. SCI., USA, 99:16433; and Zhan et al., 2001, HUM. GENET., 109:471), which can result in unregulated expression of the introduced sequence. In certain embodiments, the retroviral vector includes an insulator element in one or both of the LTRs or elsewhere within the region of the vector that integrates into the cellular genome. Insulators suitable for use in the present invention include, but are not limited to, the chicken β-globin insulator (see Chung et al., (1993). CELL 74:505; Chung et al., (1997) PROC. NATL. ACAD. SCI., USA 94:575; and Bell et al., 1999. CELL 98:387). Examples of insulator elements include, but are not limited to, insulators derived from the β-globin locus, such as chicken HS4.

[0098] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech catalog #631984), pLVX-EF1alpha-IRES-mCherry (Clontech catalog #631987), pLVX-Puro (Clontech catalog #632159), pLVX-IRES-Puro (Clontech catalog #632186), pLenti6 / V5-DEST (trademark) (Thermo Fisher), pLenti6.2 / V5-DEST (trademark) (Thermo Fisher), pLKO.1 (Addgene plasmid #10878), pLKO.3G (Addgene plasmid #14748), pSico (Addgene plasmid #11578), pLJM1-EGFP (Addgene plasmid #19319), FUGW (Addgene plasmid #14883), pLVTHM (Addgene plasmid #12247), pLVUT-tTR-KRAB (Addgene plasmid #11651), pLL3.7 (Addgene plasmid #11795), pLB (Addgene plasmid #11619), pWPXL (Addgene plasmid #12257), pWPI (Addgene plasmid #12254), EF.CMV.RFP (Addgene plasmid #17619), pLenti CMV Puro DEST (Addgene plasmid #17452), pLenti-puro (Addgene plasmid #39481), pULTRA (Addgene plasmid #24129), pLX301 (Addgene plasmid #25895), pHIV-EGFP (Addgene plasmid #21373), pLV-mCherry (Addgene plasmid #36084), pLionII (Addgene plasmid #1730), pInducer10-mir-RUP-PheS (Addgene plasmid #44011). These vectors can be modified to be suitable for therapeutic use. For example, a selectable marker (e.g., puromycin, EGFP, or mCherry) can be removed or replaced with a second exogenous gene of interest.Further examples of lentiviral vectors are disclosed in U.S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, as well as PCT Publication No. WO2017 / 091786.

[0099] Adenoviral vector In certain embodiments, the viral vector can be an adenoviral vector. Adenoviruses are medium-sized (90 - 100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The term "adenovirus" refers to any virus of the family Adenoviridae, including but not limited to human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgroups. Typically, an adenoviral vector is generated by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus to allow insertion of a non-native nucleic acid sequence, e.g., for gene transfer.

[0100] Human adenoviruses can be used as a source of the adenovirus genome for adenovirus vectors. For example, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22 - 30, 32, 33, 36 - 39, and 42 - 48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), unclassified serogroup (e.g., serotypes 49 and 51), or any other adenovirus serogroup or serotype. Adenovirus serotypes 1 - 51 are available from the American Type Culture Collection (ATCC, Manassas, Virginia). Non-group C adenovirus vectors, methods of making non-group C adenovirus vectors, and methods of using non-group C adenovirus vectors are disclosed, for example, in U.S. Patent Nos. 5,801,030, 5,837,511, and 5,849,561, and PCT Publications WO1997 / 012986 and WO1998 / 053087.

[0101] Non-human adenoviruses (e.g., simian, monkey, avian, canine, ovine, or bovine adenoviruses) can be used to generate adenoviral vectors (i.e., as a source of the adenovirus genome for an adenoviral vector). For example, an adenoviral vector can be based on simian adenoviruses, including both New World and Old World monkeys (see, e.g., Virus Taxonomy: VHIth Report of the International Committee on Taxonomy of Viruses (2005)). Phylogenetic analyses of adenoviruses infecting primates are disclosed, for example, in Roy et al. (2009) PLOS PATHOG. 5(7):e1000503. Gorilla adenoviruses can be used as a source of the adenovirus genome for an adenoviral vector. Gorilla adenoviruses and adenoviral vectors are described, for example, in PCT Publications WO2013 / 052799, WO2013 / 052811, and WO2013 / 052832. An adenoviral vector can also include combinations of sub-types and thus can be a “chimeric” adenoviral vector.

[0102] Adenoviral vectors can be replicable, conditionally replicable, or replication-deficient. Replicable adenoviral vectors can replicate in a typical host cell, i.e., a cell that can typically be infected by adenovirus. Conditionally replicating adenoviral vectors are adenoviral vectors that have been engineered to replicate under defined conditions. For example, gene functions essential for replication, such as those encoded by the adenovirus early regions, can be operably linked to inducible, repressible, or tissue-specific transcriptional control sequences, such as promoters. Conditionally replicating adenoviral vectors are further described in U.S. Patent No. 5,998,205. Replication-deficient adenoviral vectors are, for example, adenoviral vectors that, as a result of the deletion of one or more gene functions or regions essential for replication, require complementation of one or more gene functions or regions of the adenoviral genome required for replication, such that the adenoviral vector does not replicate in a typical host cell that it infects, particularly human cells.

[0103] Preferably, the adenovirus vector is replication-deficient, and a replication-deficient adenovirus vector requires complementation of at least one gene function essential for replication of one or more regions of the adenovirus genome (e.g., for forming adenovirus vector particles) for propagation. The adenovirus vector can be deficient with respect to only the early regions of the adenovirus genome (i.e., the E1-E4 regions), only the late regions of the adenovirus genome (i.e., the L1-L5 regions), one or more gene functions essential for replication of both the early and late regions of the adenovirus genome, or all adenovirus genes (i.e., high-capacity adenovectors (HC-Ad)). See, for example, Morsy et al. (1998) PROC. NATL. ACAD. SCI. USA 95: 965-976, Chen et al. (1997) PROC. NATL. ACAD. SCI. USA 94: 1645-1650, and Kochanek et al. (1999) HUM. GENE THER. 10(15):2451-9. Examples of replication-deficient adenovirus vectors are disclosed in U.S. Patent Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, 6,482,616, and 7,195,896, and PCT Publications WO1994 / 028152, WO1995 / 002697, WO1995 / 016772, WO1995 / 034671, WO1996 / 022378, WO1997 / 012986, WO1997 / 021826, and WO2003 / 022311.

[0104] The replication-deficient adenoviral vector of the present invention can be produced in a complementing cell line that provides at an appropriate level the gene functions that are not present in the replication-deficient adenoviral vector but are required for viral growth to generate a high-titer viral vector stock. Such complementing cell lines are known and include, but are not limited to, 293 cells (described, for example, in Graham et al. (1977) J. GEN. VIROL. 36: 59-72), PER.C6 cells (described, for example, in PCT Publication No. WO1997 / 000326 and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described, for example, in PCT Publication No. WO1995 / 034671 and Brough et al. (1997) J. VIROL. 71: 9206-9213). Other suitable complementing cell lines for producing the replication-deficient adenoviral vector of the present invention include complementing cell lines created to propagate an adenoviral vector encoding a transgene whose expression inhibits viral growth in the host cell (see, for example, U.S. Patent Publication No. 2008 / 0233650). Further suitable complementing cells are described, for example, in U.S. Pat. Nos. 6,677,156 and 6,682,929, and PCT Publication No. WO2003 / 020879. Formulations for adenoviral vector-containing compositions are further described, for example, in U.S. Pat. Nos. 6,225,289 and 6,514,943, and PCT Publication No. WO2000 / 034444.

[0105] Further exemplary adenoviral vectors and / or methods of generating or propagating adenoviral vectors are described in U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, 6,083,716, 6,113,913, 6,303,362, 7,067,310, and 9,073,980.

[0106] Commercially available adenovirus vector systems include the ViraPower™ adenovirus expression system available from Thermo Fisher Scientific, the AdEasy™ adenovirus vector system available from Agilent Technologies, and the Adeno-X™ expression system 3 available from Takara Bio USA, Inc.

[0107] Production of viral vectors Methods for producing viral vectors are known in the art. Typically, the virus of interest is produced in a suitable host cell using conventional techniques including culturing transfected or infected host cells under conditions that permit the production of infectious viral particles. Nucleic acids encoding viral genes and / or tRNAs can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells suitable for the production of the disclosed viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions will vary depending on the virus and production system used.

[0108] In certain embodiments, producer cells can be administered directly to a subject, while in other embodiments, after production, infectious viral particles are recovered from the culture and optionally purified. Typical purification steps can include plaque purification, centrifugation, e.g., cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatography steps, e.g., ion exchange chromatography or filtration steps.

[0109] IV. Pharmaceutical composition For therapeutic use, the tRNA and / or expression vector is preferably combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, under a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic response, or other problems or complications, suitable for use in contact with human and animal tissues. It represents compounds, substances, compositions, and / or dosage forms.

[0110] As used herein, the term "pharmaceutically acceptable carrier" means buffers, carriers, and excipients suitable for use in contact with human and animal tissues under a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic response, or other problems or complications. Pharmaceutically acceptable carriers include any standard pharmaceutical carrier, such as an aqueous phosphate buffered saline solution, water, an emulsion (e.g., an oil / water or water / oil emulsion), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0111] In certain embodiments, the pharmaceutical composition may contain formulation materials for changing, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition.In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine or lysine); antibacterial substances; antioxidants (e.g., ascorbic acid, sodium sulfite or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose or dextrin); proteins (e.g., serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); isotonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0112] In certain embodiments, the pharmaceutical composition can contain nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29). In certain embodiments, the composition does not contain (or is substantially free of, e.g., the composition contains less than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% thereof) nanoparticles or amino lipid delivery compounds, such as those described in U.S. Patent Publication No. 2017 / 0354672. In certain embodiments, the tRNA or expression vector introduced into cells or administered to a subject is not conjugated or otherwise bound to another moiety, such as a carrier particle, e.g., an amino lipid particle. As used herein, the term "conjugate," when used with respect to two or more moieties, means that these moieties are physically bonded or connected to each other either directly or via one or more additional moieties functioning as a linker, such that the structure forms a structure that is sufficiently stable to maintain the physical bond between these moieties under the conditions in which the structure is used, e.g., physiological conditions. Typically, these moieties are added either by one or more covalent bonds or by a mechanism involving specific binding. Alternatively, a sufficient number of weak interactions can provide sufficient stability for the moieties to maintain a physical bond.

[0113] In certain embodiments, the pharmaceutical composition may contain a sustained or controlled release formulation. Techniques for incorporating sustained or controlled release means, such as liposome carriers, biodegradable microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained release preparations may include, for example, porous polymer microparticles in the form of shaped articles, such as films or microcapsules, or semipermeable polymer matrices. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes which can be prepared by any of a variety of methods known in the art.

[0114] The pharmaceutical compositions containing the tRNA and / or expression vectors disclosed herein can be provided in unit dosage form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the tRNA and / or expression vector is administered intrathecally. In certain embodiments, the tRNA and / or expression vector is administered by injection. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, for example, Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and isotonicity regulating agents such as sodium chloride or dextrose.

[0115] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be protected against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, polyethylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0116] Generally, any method for delivering nucleic acid molecules can be adapted for use with tRNA (see, for example, Akhtar et al. (1992) TRENDS CELL. BIOL. 2(5):139-144 and PCT Publication No. WO94 / 02595). The tRNA can be modified to prevent rapid degradation of the tRNA by endo- and exonucleases in vivo, or can be delivered using a drug delivery system. The tRNA molecule can be modified by chemical conjugation to a lipophilic group, such as cholesterol, to enhance cell uptake and prevent degradation. The tRNA molecule can also be conjugated or otherwise bound to an aptamer. The tRNA can also be delivered using a drug delivery system, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems enhance the interaction with the negatively charged cell membrane to facilitate binding of the (negatively charged) tRNA molecule and enable efficient uptake of the tRNA by the cell. Cationic lipids, dendrimers, or polymers can either bind to RNA, such as tRNA, or be induced to form vesicles or micelles that surround the RNA (see, for example, Kim et al. (2008) JOURNAL OF CONTROLLED RELEASE 129(2):107-116). Methods for making and administering cationic RNA complexes are well within the capabilities of those of ordinary skill in the art (see, for example, Sorensen et al. (2003) J. MOL. BIOL 327:761-766; Verma et al. (2003) CLIN. CANCER RES. 9:1291-1300; Arnold et al. (2007) J. HYPERTENS. 25:197-205).Some non-limiting examples of drug delivery systems useful for systemic delivery of RNA, such as tRNA, include DOTAP (Sorensen et al. (2003) supra; Verma et al. (2003), supra), oligofectamine, solid-phase nucleic acid lipid particles (Zimmermann et al. (2006) NATURE 441:111-114), cardiolipin (Chien et al. (2005) CANCER GENE THER. 12:321-328; Pal et al. (2005) INT J. ONCOL. 26:1087-1091), polyethyleneimine (Bonnet et al. (2008) PHARM. RES. 25(12):2972-82; Aigner (2006) J. BIOMED. BIOTECHNOL. 71659), Arg-Gly-Asp (RGD) peptide (Liu (2006) MOL. PHARM. 3:472-487), and polyamidoamine (Tomalia et al. (2007) BIOCHEM. SOC. TRANS. 35:61-67; Yoo et al. (1999) PHARM. RES. 16:1799-1804). In certain embodiments, the tRNA forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNA and cyclodextrin can be found in U.S. Patent No. 7,427,605.

[0117] The pharmaceutical formulation is preferably sterile. Sterility can be achieved by any suitable method, such as filtration through a sterile filtration membrane. If the composition is lyophilized, filtration sterilization can be performed before or after lyophilization and reconstitution.

[0118] The compositions described herein can be administered locally or systemically. Administration will typically be parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in an even more preferred embodiment, intravenously. Preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions.

[0119] Generally, the therapeutically effective amount of the active ingredient, such as tRNA and / or the expression vector, is in the range of 0.1 mg / kg to 100 mg / kg, such as 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg. In certain embodiments, the therapeutically effective amount of the viral expression vector is 10 2 ~10 15 plaque forming units (pfu), such as 10 2 ~10 10 、10 2 ~10 5 、10 5 ~10 15 、10 5 ~10 10 、or 10 10 ~10 15 pfu. The amount administered may depend on variables such as the type and degree of the disease or condition being treated, the overall health of the patient, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The initial dose may be increased above the upper limit level in order to rapidly achieve the desired blood level or tissue level. Alternatively, the initial dose may be less than its optimal amount and the daily dose may be gradually increased during the course of treatment. The human dose may be optimized in a conventional Phase I dose escalation study designed, for example, to be carried out at 0.5 mg / kg to 20 mg / kg. The dosing frequency may vary depending on factors such as the route of administration, the dose, the serum half-life, and the disease being treated. Exemplary dosing frequencies are once a day, once a week, and once every two weeks. A preferred route of administration is parenteral, such as intravenous infusion. In certain embodiments, the polypeptide and / or multimeric protein is lyophilized and then reconstituted with buffered saline at the time of administration.

[0120] In certain embodiments, the tRNA or expression vector is not conjugated or bound to another moiety, such as a carrier particle, such as an amino lipid particle. In certain embodiments, the tRNA or expression vector is introduced into cells or administered to a subject in a dosage form lacking nanoparticles. In certain embodiments, the tRNA or expression vector is introduced into cells or administered to a subject in a dosage form lacking an amino lipid delivery compound, such as described in U.S. Patent Publication No. 2017 / 0354672.

[0121] V. Therapeutic use The compositions and methods disclosed herein can be used to treat premature termination codon (PTC)-mediated disorders in a subject. As used herein, the term "PTC-mediated disorder" refers to a disorder mediated by, enhanced by, exacerbated by, or otherwise promoted by a PTC in a gene, or associated with a PTC in a gene.

[0122] The present invention provides a method of treating a PTC-mediated disorder in a subject in need thereof. The method includes administering to the subject, in either case alone or in combination with another therapeutic agent, an effective amount of a tRNA and / or an expression vector, such as a tRNA and / or an expression vector disclosed herein, for treating a PTC-mediated disorder in the subject.

[0123] In certain embodiments, the premature termination codon-mediated disorder is a disorder listed in Table 5 below, and / or the gene having the premature termination codon is a gene listed in the corresponding row of Table 5 below.

[0124]

Table 5

[0125] In certain embodiments, the premature termination codon-mediated disorder is a disorder listed in Table 6 below, and / or the gene having a premature termination codon is a gene listed in the corresponding row of Table 6 below.

[0126] [Table 6]

[0127] In certain embodiments, the PTC-mediated disorder is epilepsy (e.g., Dravet syndrome), and the method reduces the seizure frequency, seizure severity, and / or cognitive dysfunction in a subject. For example, in certain embodiments, the method reduces the seizure frequency in a subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% over a period of, for example, one day, one week, or one month. In certain embodiments, the method reduces the seizure severity by 50% over a period of, for example, one day, one week, or one month.

[0128] In certain embodiments, the PTC-mediated disorder is dystonia and / or the gene having a premature stop codon is SCN1A. In certain embodiments, the premature stop codon within the SCN1A gene is c.5745C>G, c.5713G>T, c.5701C>T, c.5677C>T, c.5641C>T, c.5629C>T, c.5623C>T, c.5503A>T, c.5473G>T, c.5437G>T, c.5428C>T, c.5403G>A, c.5402G>A, c.5383G>T, c.5371G>T, c.5049T>G, c.4921G>T, c.4900C>T, c.4873C>T, c.4779del, c.4778G>A, c.4774G>T, c.4761T>G, c.4648G>T, c.4540C>T, c.4516A>T, c.4514C>A, c.4508T>G, c.4488C>G, c.4471G>T, c.4300A>T, c.4269G>A, c.4268G>A, c.4233T>A, c.4222G>T, c.4191G>A, c.4190G>A, c.4186C>T, c.4159A>T, c.4155C>A, c.3964del, c.3952C>T, c.3825G>A, c.3824G>A, c.3819G>A, c.3818G>A, c.3795T>A, c.3789T>G, c.3779G>A, c.3750C>G, c.3724G>T, c.3700C>T, c.3697C>T, c.3657dup, c.3624G>A, c.3604C>T, c.3582G>A, c.3578G>A, c.3574C>T, c.3463C>T, c.3454del, c.3424G>T, c.3422C>A, c.3406G>T, c.3328G>T, c.3273C>A, c.3262G>T, c.3073C>T, c.3060T>A, c.2844T>A, c.2749C>T, c.2695C>T, c.2645T>A, c.2560C>T, c.2551C>T, c.2546C>A, c.2462G>A, c.2298del, c.2228G>A, c.2181G>A, c.2180G>A, c.2101C>T, c.2038A>T, c.1958T>A, c.1837C>T, c.1834C>T, c.1804G>T, c.1795G>T, c.1738C>T, c.1702C>T, c.1660C>T, c.Resulting from a mutation, or combination of mutations, selected from 1624C>T, c.1516C>T, c.1378C>T, c.1363C>T, c.1354A>T, c.1348C>T, c.1345G>T, c.1344dup, c.1306G>T, c.1278C>A, c.1278C>G, c.1151G>A, c.1129C>T, c.1118T>A, c.942del, c.751del, c.644T>A, c.327C>G, c.249C>A, c.121A>T, c.4846_4850dup, c.4787_4788del, c.4578_4612dup, c.4211_4212del, c.4125_4130delinsATAATCATACTGATTGCCTAAAACTAAT, c.3690_3693del, c.3338_3339del, c.1247_1248insGTAGA, c.825_826insGTATA, and c.278_279dup. In certain embodiments, the premature stop codons within the SCN1A gene are caused by a mutation, or combination of mutations, selected from c.58G>T, c.575G>A, c.664C>T, c.962C>G, c.1095dupT, c.1129C>T, c.1315C>T, c.1348C>T, c.1366G>T, c.1492A>T, c.1537G>T, c.1624C>T, c.1738C>T, c.1804G>T, c.1837C>T, c.2134C>T, c.2370T>A, c.2495G>A, c.2593C>T, c.2635delC, c.2904C>A, c.3295G>T, c.3311C>A, c.3452C>G, c.3637C>T, c.3656G>A, c.3733C>T, c.3783C>A, c.3829C>T, c.3985C>T, c.4359T>G, c.4547C>A, c.4573C>T, c.4721C>G, c.4954G>T, c.5641G>T, c.5656C>T, and c.5734C>T. In certain embodiments, the premature stop codons within the SCN1A gene are caused by a mutation, or combination of mutations, selected from c.664C>T, c.1129C>T, c.1492A>T, c.1624C>T, c.1738C>T, c.1837C>T, c.2134C>T, c.2593C>T, c.3637C>T, c.3733C>T, c.3985C>T, c.It is caused by a mutation selected from 4573C>T, c.5656C>T, and c.5734C>T. In certain embodiments, the premature stop codon within the SCN1A gene is caused by a mutation selected from c.1738C>T and c.3985C>T.

[0129] In certain embodiments, the premature stop codon within the SCN1A gene is caused by the mutations shown in Table 7, or combinations of the mutations shown in Table 7.

[0130]

Table 7

[0131] Additional exemplary mutations, including exemplary mutations that result in a premature stop codon within a gene, such as the SCN1A gene, can be found in ClinVar (available at world wide web ncbi.nlm.nih.gov / clinvar / ), “A catalog of SCN1A variants” Lossin et al. (2009) BRAIN DEV. 2009 31(2):114-30, SCN1A Registry (available at world wide web scn1a.net / scn1a-registry / ), SCN1A Mutation Database (available at world wide web gzneurosci.com / scn1adatabase), and Leiden Open Variation Database (LOVD v.3.0; available at world wide web databases.lovd.nl / shared / genes / SCN1A). Unless otherwise indicated, all SCN1A mutations described herein are with respect to SCN1a isoform 1 (NCBI reference sequence NM_001165963, SEQ ID NO:863).

[0132] In another aspect, the present invention provides a method for treating Dravet syndrome in a subject in need thereof, the subject having an SCN1A gene with a mutation shown in a row of Table 7, the method comprising administering to the subject an effective amount of a suppressor tRNA of a suppressor class shown in the same row of Table 7 as the mutation, or an expression vector comprising a nucleotide sequence encoding the tRNA. The "suppressor class" (e.g., Arg>TGA) used in Table 7 represents the type of endogenous tRNA from which the suppressor tRNA is derived (e.g., arginine tRNA) and the stop codon recognized by the suppressor tRNA (e.g., TGA). Exemplary Arg>TGA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 19-22, and 35. Exemplary Gln>TAA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 36-40, 44, and 45. Exemplary Gln>TAG suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 178-182, 186, and 187.

[0133] For example, in certain embodiments, the subject has an SCN1A gene having a premature stop codon selected from c.664C>T, c.3637C>T, c.3733C>T, c.2134C>T, and c.1837C>T, and the method comprises administering to the subject a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 6-9, 11, 16-18, 19-22, and 35 in an effective amount. In certain embodiments, the subject has an SCN1A gene having a premature stop codon selected from c.3607C>T, c.2782C>T, c.3829C>T, and c.2893C>T, and the method comprises administering to the subject a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 36-40, 44, and 45 in an effective amount. In certain embodiments, the subject has an SCN1A gene having a premature stop codon selected from c.3106C>T, c.3496C>T, c.5662C>T, c.5461C>T, and c.3730C>T, and the method comprises administering to the subject a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 178-182, 186, and 187 in an effective amount.

[0134] In certain embodiments where the gene is the SCN1A gene, the SCN1A gene product generated by tRNA is a functional SCN1A gene product. In certain embodiments, the functional SCN1A gene product has higher activity, e.g., higher voltage-gated sodium channel activity, than the truncated SCN1A gene product. In certain embodiments, the method increases the voltage-gated sodium channel activity in a cell, tissue, or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% compared to a cell, tissue, or subject without tRNA. In certain embodiments, the method increases the voltage-gated sodium channel activity in a cell, tissue, or subject by about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200% compared to a cell, tissue, or subject without tRNA.Voltage-gated sodium channel activity can be measured by any method known in the art, such as those described in Kalume et al. (2007) J. NEUROSCI. 27(41):11065-74, Yu et al. (2007) NAT. NEUROSCI. 9(9): 1142-9, and Han et al. (2012) NATURE 489(7416): 385-390.

[0135] In certain embodiments, the functional SCN1A gene product is the Na v 1.1 protein. In certain embodiments, the functional SCN1A gene product comprises, consists essentially of, or consists of any one of the following amino acid sequences (each corresponding to a different isoform of SCN1A), or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the following amino acid sequences: TIFF0007712270000065.tif95149TIFF0007712270000066.tif223149TIFF0007712270000067.tif223149TIFF0007712270000068.tif229149TIFF0007712270000069.tif223149TIFF0007712270000070.tif177149

[0136] As used herein, the term "effective amount" refers to the amount of an active agent (e.g., a tRNA or expression vector according to the invention or a second active agent in combination therapy) sufficient to exert a beneficial or desired effect. The effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0137] As used herein, "treat", "treating" and "treatment" mean treating a disease in a subject, such as a human. This includes (a) suppressing the disease, i.e., stopping its progression; and (b) alleviating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), more preferably humans.

[0138] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or treatment modalities. The term "administered in combination" as used herein is understood to mean that two (or more) different treatments are delivered to a subject while the subject has a disease such that the effects of the treatments on the subject overlap at some point during the treatment of the subject. In certain embodiments, the delivery of one treatment is carried out even when the delivery of a second treatment has begun such that there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either example, the treatments are more effective because the administrations are combined. For example, the second treatment is more effective than would be seen if the second treatment were administered in the absence of the first treatment, e.g., an equivalent effect is seen with a lesser amount of the second treatment, or the second treatment results in a greater reduction of symptoms, or a similar situation is seen for the first treatment. In certain embodiments, the delivery is such that the reduction in symptoms, or other parameters associated with the disorder, is greater than would be observed if one treatment were delivered in the absence of the other. The effects of the two treatments can be partially additive, wholly additive, or more than additive. The delivery can be such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered.

[0139] In certain embodiments, the methods or compositions described herein are administered in combination with one or more additional therapeutic agents, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), the ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), fenfluramine, or valproic acid. For example, during the treatment of Dravet syndrome, the methods or compositions described herein are administered in combination with one or more additional therapeutic agents, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), the ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), fenfluramine, or valproic acid.

[0140] Throughout the detailed description where a composition is described as having, including, or comprising a particular component, or a process and method is described as having, including, or comprising a particular step, it is contemplated that there are also compositions of the invention that consist essentially of, or consist of, the recited components, and processes and methods according to the invention that consist essentially of, or consist of, the recited processing steps.

[0141] In instances where an element or component is said to be included in and / or selected from a recited list of elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0142] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the invention, whether explicitly or implicitly set forth herein. For example, if a reference is made to a particular compound, that compound can be used in various aspects of the compositions of the invention and / or in the methods of the invention, unless the context indicates otherwise. In other words, in this application, aspects are described and presented so as to be able to describe and depict clear and concise examples, but it is intended and will be understood that aspects can be variously combined or separated without departing from the present teachings and the invention. For example, it will be understood that all features described and presented herein can be applicable to all aspects of the invention described and presented herein.

[0143] The expression "at least one of" should be understood to include each individual one of the objects recited after the expression and various combinations of two or more of the recited objects, unless the context and usage indicate otherwise. The expression "and / or" in relation to three or more recited objects should also be understood to have the same meaning, unless the context indicates otherwise.

[0144] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", "containing", including their grammatical equivalents, is generally open-ended and non-limiting, unless specifically stated otherwise or unless the context indicates otherwise, i.e., it should be understood not to exclude additional unrecited elements or steps.

[0145] When the term "about" is used before a numerical value, the present invention includes the specific numerical value itself as well, unless specifically stated otherwise. As used herein, the term "about" represents a variation of ±10% from the indicated value, unless otherwise indicated or inferred.

[0146] It should be understood that the order of steps or the order in which a particular act is performed is not important as long as the present invention is feasible. Further, two or more steps or acts may be performed simultaneously.

[0147] Any and all examples or exemplary language herein, such as "for example" or "including", are intended only to more fully illustrate the present invention and do not impose a limitation on the scope of the present invention unless otherwise recited in the claims. No language in this specification should be construed as indicating any element not recited in the claims as essential to the practice of the invention.

Examples

[0148] The following examples are merely examples and are not intended to limit the scope or content of the present invention in any way.

[0149] Example 1 This example describes an arginine aminoacylated suppressor tRNA that promotes readthrough of premature termination codons (PTCs).

[0150] Suppressor tRNAs were generated by converting the normal anticodons of endogenous mouse arginine-tRNAs to a TCA anticodon that recognizes the TGA stop codon (Arg TCA suppressor tRNA). Five of the endogenous arginine-tRNAs contained introns that had to be removed by splicing to generate mature tRNAs; corresponding Arg with and without these intron sequencesTCA A suppressor tRNA was prepared. The suppressor tRNA sequence is shown in Table 8.

[0151] [Table 8] TIFF0007712270000072.tif220154TIFF0007712270000073.tif226154TIFF0007712270000074.tif67154

[0152] In this example, all mature tRNA sequences (predicted by GtRNAdb; http: / / gtrnadb.ucsc.edu) were expressed under the context of upstream and downstream genomic flanking sequences (±200 bp) derived from the highly expressed arginine tRNA, tRNA-Arg-TCG-1-1, that is, the tRNA sequences were expressed together with the 5'-flanking sequence of SEQ ID NO:26 and the 3'-flanking sequence of SEQ ID NO:27. All mature tRNA sequences containing the upstream and downstream genomic flanking sequences were generated within the pGL4 vector backbone.

[0153] This Arg TCAThe suppressor was tested for PTC readthrough activity by flow cytometry in cell lines containing the dual-fluorescent readthrough reporter. These reporters contain 3 copies of the red fluorescent protein (tdTomato), TEV protease, a linker region containing the PTC, and 3 copies of the green fluorescent protein (EGFP). An overview of the reporter construct is shown in Figure 3. In the absence of any PTC readthrough as a result of the suppressor tRNA, translation would terminate at the PTC in the linker region and only tdTomato would be expressed (thus, only red fluorescence would be detected). PTC readthrough activity as a result of the suppressor tRNA would allow translation to proceed past the PTC in the linker region and express both tdTomato and EGFP (thus, both red and green fluorescence would be detected). Thus, readthrough can be evaluated by quantifying the proportion of viable cells (double positive %) that express both red and green fluorescent reporters above background.

[0154] To screen for suppressor tRNAs that exhibit readthrough activity at PTCs associated with Dravet syndrome, linkers containing PTCs and eight adjacent codons on either side of the PTC were generated from the SCN1A transcripts of two patients, subject N and subject S, who have nonsense mutations within the SCN1A gene.

[0155] The linker region derived from the SCN1A transcript of subject N is as follows, and the reporter containing this linker region is designated the subject N-PTC reporter. TIFF0007712270000075.tif4147The corresponding linker with a wild-type Arg codon instead of the PTC was used as a control and had the following sequence. TIFF0007712270000076.tif4150

[0156] The linker region derived from the SCN1A transcript of subject S is as follows, and the reporter containing this linker region is designated the subject S-PTC reporter. TIFF0007712270000077.tif4147Instead of the PTC, the corresponding linker with a wild-type Arg codon was used as a control, which had the following sequence. TIFF0007712270000078.tif4150

[0157] An additional 51-base pair linker region was obtained from a mouse model of Dravet syndrome caused by the R1407X nonsense mutation in SCN1A (Ogiwara et al., 2007, Neurobiology of Disease). The linker region from the SCN1A R1407X transcript is as follows, and a reporter containing this linker region is designated as the R1407X-PTC reporter. TIFF0007712270000079.tif4147Instead of the PTC, the corresponding linker with a wild-type Arg codon was used as a control, which had the following sequence. TIFF0007712270000080.tif4150

[0158] Arg TCA The suppressor was (i) a human Flp-In-293 cell line that stably expressed the target S-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 4), (ii) a mouse Flp-In-3T3 cell line that stably expressed the R1407X-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 5), and (iii) tested in multiple assay settings including Flp-In-293 cells that were transiently co-transfected with a plasmid encoding the target N-PTC reporter and a plasmid encoding the suppressor tRNA (the results are shown in Figure 6). Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. TCA The suppressor was (i) a human Flp-In-293 cell line that stably expressed the target S-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 4), (ii) a mouse Flp-In-3T3 cell line that stably expressed the R1407X-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 5), and (iii) tested in multiple assay settings including Flp-In-293 cells that were transiently co-transfected with a plasmid encoding the target N-PTC reporter and a plasmid encoding the suppressor tRNA (the results are shown in Figure 6). Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. TCA The suppressor was (i) a human Flp-In-293 cell line that stably expressed the target S-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 4), (ii) a mouse Flp-In-3T3 cell line that stably expressed the R1407X-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 5), and (iii) tested in multiple assay settings including Flp-In-293 cells that were transiently co-transfected with a plasmid encoding the target N-PTC reporter and a plasmid encoding the suppressor tRNA (the results are shown in Figure 6). Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. TCA The suppressor was (i) a human Flp-In-293 cell line that stably expressed the target S-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 4), (ii) a mouse Flp-In-3T3 cell line that stably expressed the R1407X-PTC reporter and was transiently transfected with a plasmid encoding the suppressor (the results are shown in Figure 5), and (iii) tested in multiple assay settings including Flp-In-293 cells that were transiently co-transfected with a plasmid encoding the target N-PTC reporter and a plasmid encoding the suppressor tRNA (the results are shown in Figure 6). Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol.

[0159] We generated an additional reporter construct that contains EGFP with a nuclear localization signal (NLS) and an arginine-to-TGA mutation (R96X) that ablates fluorescence in the absence of PTC readthrough within the EGFP open reading frame. A schematic of the experimental approach is shown in Fig. 8A. EGFP expression was driven by the CMV early enhancer / chicken β-actin (CAG) promoter. This reporter construct is designated CAG:NLS-EGFP (R96X-TGA), and its sequence is as follows. TIFF0007712270000081.tif196149

[0160] Arg TCA In HEK293 cells and mouse Neuro-2a cells (a neural crest-derived cell line widely used to study neural differentiation) transiently co-transfected with a plasmid encoding a suppressor tRNA and a plasmid encoding the CAG:NLS-EGFP (R96X-TGA) reporter, Arg TCA suppressor activity was evaluated. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. Co-transfection was performed using equal amounts of the indicated suppressor plasmid and CAG:NLS-EGFP (R96X-TGA) reporter plasmid. EGFP expression was analyzed by flow cytometry approximately 24 hours after transfection in 293 cells and approximately 48 hours after transfection in Neuro-2a cells. The results are shown in Fig. 7.

[0161] Generally, Arg TCAThe relative read-through activity of suppressor tRNAs was maintained consistent among multiple assay formats. The following suppressors reliably showed read-through activity above baseline: TCA-001 (SEQ ID NO:11), TCA-89 (SEQ ID NO:1), TCA-90 (SEQ ID NO:2), TCA-105 (SEQ ID NO:7), TCA-106 (SEQ ID NO:8), TCA-107 (SEQ ID NO:9), TCA-113 (SEQ ID NO:16), TCA-114 (SEQ ID NO:17), TCA-115 (SEQ ID NO:18), TCA-116 (SEQ ID NO:19), TCA-117 (SEQ ID NO:20), TCA-118 (SEQ ID NO:21), and TCA-119 (SEQ ID NO:22).

[0162] Collectively, these results indicate that the described suppressor tRNAs can promote the expression of transcripts containing premature termination codons (PTCs) associated with disorders such as Dravet syndrome, e.g., SCN1A transcripts.

[0163] Example 2 This example describes the effect of an expression vector's characteristics on the read-through of premature termination codons (PTCs) by arginine aminoacylated suppressor tRNAs.

[0164] Arg TCA Expression constructs containing both suppressor tRNA and the EGFP (R96X-TGA) reporter on the same plasmid were made within the pGL4 vector backbone. These constructs had 1, 2, 3, or 4 copies of Arg (described in Example 1 and shown in Table 8) TCAIt included suppressor tRNAs 113 (SEQ ID NO:16), 115 (SEQ ID NO:18), and 001 (SEQ ID NO:11). Each copy of the tRNA sequence was expressed under the context of either (i) the 200 bp upstream genomic flanking sequence (SEQ ID NO: 26) from tRNA-Arg-TCG-1-1 and the 200 bp downstream genomic flanking sequence (SEQ ID NO: 27) from tRNA-Arg-TCG-1-1, (ii) the 200 bp upstream genomic flanking sequence (SEQ ID NO: 26) from tRNA-Arg-TCG-1-1 and the 104 bp downstream genomic flanking sequence (SEQ ID NO: 32) from tRNA-Arg-TCG-1-1, or (iii) the 19 bp upstream flanking genomic sequence (SEQ ID NO: 33) from tRNA-Arg-TCG-1-1 and the 46 bp downstream flanking genomic sequence (SEQ ID NO: 34) from tRNA-Arg-TCG-1-1. The schematic of the experimental approach is shown in Figure 8A, and the overview of an exemplary reporter construct containing 4 copies of the suppressor tRNA is shown in Figure 8B. The reporter construct was either the CAG:NLS-EGFP (R96X-TGA) reporter construct (described in Example 1) or the EF1a:NLS-EGFP (R96X-TGA) reporter construct in which the CAG promoter was replaced with the elongation factor 1 alpha (EF1a) promoter (this sequence is as follows). TIFF0007712270000082.tif208149

[0165] These constructs were transfected into Neuro-2a cells or HEK293 (FlpIn-293) cells, and readthrough was assayed by fluorescence imaging or flow cytometry at approximately 24 or 48 hours after transfection. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol.

[0166] The results are shown in FIGS. 9-10 (fluorescent images) and FIGS. 11-17 (quantification of fluorescent signals measured by flow cytometry). For suppressors 001 and 113, improved readthrough was seen with increasing copy number. Overall, the results indicate that an increase in the copy number of the suppressor tRNA module in the reporter construct often leads to an improvement in readthrough activity. Furthermore, although the U6-containing constructs showed PTC readthrough activity, it was generally not as active as that seen in equivalent suppressor tRNAs expressed under the context of the adjacent genome.

[0167] Example 3 This example describes the design of a functional arginine aminoacylated suppressor tRNA that promotes readthrough of premature termination codons (PTCs) in transcripts.

[0168] C57BL / 6J mice have a naturally occurring C-to-T mutation at the T loop (position 51) of Arg-TCT-5-1, which has been shown to affect pre-tRNA processing and function (Ryuta Ishimura et al., Science, 2014). (Arg described in Example 1 and shown in Table 8) TCA Suppressor 120 contains a T at position 51. Arg TCA A modified suppressor tRNA containing a substitution of T to C at position 51 of suppressor 120 was generated (Arg TCA designated suppressor 179 and having the nucleotide sequence of SEQ ID NO:35). Arg TCA Suppressors 120 and 179 were tested by transfection into a Flp-In-293 cell line stably expressing the target S-PTC reporter and co-transfection with a plasmid encoding the target N-PTC reporter into Flp-In-3T3 cells. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. The results are shown in FIGS. 4 and 6. Arg TCASuppressor 120 is non-functional, whereas Arg contains only a single substitution TCA Suppressor 179 showed PTC readthrough activity.

[0169] Example 4 This example describes glutamine aminoacylated suppressor tRNAs that promote readthrough of premature termination codons (PTCs).

[0170] Suppressor tRNAs were generated from endogenous mouse glutamine-tRNAs by converting their normal anticodons to TTA or CTA anticodons (Gln TTA or Gln CTA collectively referred to as suppressor tRNAs). The suppressor tRNA sequences are shown in Table 9.

[0171] [Table 9] TIFF0007712270000084.tif213154TIFF0007712270000085.tif213154TIFF0007712270000086.tif80154

[0172] In this example, all mature tRNA sequences were expressed under the context of upstream and downstream genomic flanking sequences (±200 bp) derived from the highly expressed glutamine-tRNA, tRNA-Gln-TTG-1-1, i.e., the tRNA sequences were expressed together with the 5' flanking sequence of SEQ ID NO:173 and the 3' flanking sequence of SEQ ID NO:174. All mature tRNA sequences containing the upstream and downstream genomic flanking sequences were generated within the pGL4 vector backbone.

[0173] Gln TTAThe suppressor was tested for PTC readthrough activity by flow cytometry in two separately obtained Flp-In-293 cell lines containing the integrated fluorescent readthrough reporter. The results are shown in Figure 18. The reporter contained 3 copies of the red fluorescent protein (tdTomato), TEV protease, a linker region containing the PTC, and 3 copies of the green fluorescent protein (EGFP). An overview of the reporter construct is shown in Figure 3. In the absence of any PTC readthrough as a result of the suppressor tRNA, translation would terminate at the PTC within the linker region and only tdTomato would be expressed (therefore, only red fluorescence would be detected). PTC readthrough activity as a result of the suppressor tRNA would allow translation to proceed past the PTC within the linker region and express both tdTomato and EGFP (therefore, both red and green fluorescence would be detected). Thus, readthrough can be evaluated by quantifying, using flow cytometry, the proportion of viable cells (double positive %) that express both red and green fluorescent reporters above background. The linker was obtained from the mouse Dmd mdx transcript and had the following sequence. TIFF0007712270000087.tif4149A corresponding linker with a wild-type Gln codon instead of the PTC was used as a control and had the following sequence. TIFF0007712270000088.tif4150

[0174] A further reporter construct containing EGFP with a nuclear localization signal (NLS) and a glutamine-to-TAA mutation (Q69X) that extinguishes fluorescence in the absence of PTC readthrough was generated within the pGL4 vector backbone. EGFP expression was driven by the CMV early enhancer / chicken β-actin (CAG) promoter. This reporter construct was designated CAG:NLS-EGFP (Q69X-TAA) and its sequence is as follows. TIFF0007712270000089.tif195149

[0175] Gln TTA In Neuro-2a cells transiently co-transfected with the plasmid encoding suppressor tRNA and the plasmid encoding CAG:NLS-EGFP (Q69X-TAA) reporter, Gln TTA suppressor activity was evaluated by flow cytometry. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. The results are shown in Figure 19.

[0176] A further reporter construct was generated within the pGL4 vector backbone that contained EGFP with a nuclear localization signal (NLS) and a glutamine-to-TAG mutation (Q69X) that extinguishes fluorescence in the absence of PTC readthrough. EGFP expression was driven by the CMV early enhancer / chicken β-actin (CAG) promoter. This reporter construct is designated CAG:NLS-EGFP (Q69X-TAG), and its sequence is as follows. TIFF0007712270000090.tif194149

[0177] Gln CTA In Neuro-2a cells transiently co-transfected with the plasmid encoding suppressor tRNA and the plasmid encoding CAG:NLS-EGFP (Q69X-TAG) reporter, Gln CTA suppressor activity was evaluated. The results are shown in Figure 20.

[0178] Collectively, these results indicate that the described suppressor tRNA can promote the expression of transcripts containing premature termination codons associated with disorders.

[0179] Example 5 This example describes the readthrough activity of the disclosed suppressor tRNA and small molecule nonsense suppression therapy.

[0180] The disclosed suppressor tRNAs were tested with the nonsense suppressor drug translarna (ataluren), gentamicin, and G418 (geneticin). The PTC readthrough activity was measured in Neuro-2a cells approximately 48 hours after transfection with an expression construct containing the CAG:NLS-EGFP (R96X-TGA) reporter (described in Example 1), where (i) on the same construct, the indicated copy number of Arg TCA suppressor tRNA was included, or (ii) the cells were treated with ataluren, (iii) the cells were treated with gentamicin, or (iv) the cells were treated with G418. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, and at this time, the indicated drugs at the indicated concentrations were added. The PTC readthrough activity was measured as the ratio of EGFP-positive cells. A reporter containing wild-type EGFP without a PTC was used as a control. The cell viability in the cells treated with the above treatment sets was evaluated by flow cytometry after staining with 7-aminoactinomycin D (7-AAD; Thermo Fisher Scientific #006993-50), a membrane-impermeable dye that is excluded from normally viable cells, according to the manufacturer's protocol. The results are shown in FIGS. 21-23. In summary, the results show that Arg TCA suppressor tRNA #115 (SEQ ID NO: 18) produces much more readthrough than any of the nonsense suppressor drugs. Furthermore, the results show that, unlike any of the nonsense suppressor drugs, Arg TCA treatment with suppressor tRNA is not associated with a decrease in cell viability.

[0181] Example 6 This example describes aminoacylated suppressor tRNAs that promote readthrough of premature termination codons (PTCs).

[0182] SuppressortRNAisproducedbyconvertingthenormalanticodonfromendogenousmousetRNAintoananticodonthatrecognizesaprematureterminationcodon(PTC).ThesuppressortRNAsequencesareshowninTable10.

[0183]

Table10

[0184] The suppressor tRNA is tested for PTC readthrough activity by flow cytometry in cell lines containing a dual-fluorescent readthrough reporter. These reporters contain 3 copies of the red fluorescent protein (tdTomato), TEV protease, a linker region containing the PTC, and 3 copies of the green fluorescent protein (EGFP). An overview of the reporter construct is shown in Figure 3. In the absence of any PTC readthrough as a result of the suppressor tRNA, translation will terminate at the PTC within the linker region and only tdTomato will be expressed (therefore, only red fluorescence will be detected). PTC readthrough activity as a result of the suppressor tRNA will allow translation to proceed past the PTC within the linker region and express both tdTomato and EGFP (therefore, both red and green fluorescence will be detected). Thus, readthrough can be evaluated by quantifying the proportion of viable cells (double positive %) that express both red and green fluorescent reporters above background.

[0185] Example 7 This example describes a glutamine aminoacylated suppressor tRNA that promotes readthrough of premature termination codons (PTCs).

[0186] In this example, all mature tRNA sequences were expressed under the context of the upstream and downstream genomic flanking sequences (±200 bp) derived from tRNA-Gln-TTG-1-1, a highly expressed glutamine tRNA, i.e., the tRNA sequences were expressed using the 5' flanking sequence of SEQ ID NO:173 and the 3' flanking sequence of SEQ ID NO:174. All mature tRNA sequences containing the upstream and downstream genomic flanking sequences were generated within the pGL4 vector backbone.

[0187] Gln CTASuppressor tRNAs were tested for PTC readthrough activity by flow cytometry in Flp-In-293 cells that either contained an integrated dual-fluorescent readthrough reporter or were transiently co-transfected with an expression construct containing a dual-fluorescent readthrough reporter. These reporters contained 3 copies of the red fluorescent protein (tdTomato), TEV protease, a linker region containing the PTC, and 3 copies of the green fluorescent protein (EGFP). An overview of the reporter construct is shown in Figure 3. In the absence of any PTC readthrough as a result of the suppressor tRNA, translation would terminate at the PTC within the linker region and only tdTomato would be expressed (therefore, only red fluorescence would be detected). PTC readthrough activity as a result of the suppressor tRNA would allow translation to proceed past the PTC within the linker region and express both tdTomato and EGFP (therefore, both red and green fluorescence would be detected). Thus, readthrough was evaluated by quantifying, using flow cytometry, the percentage of viable cells (double positive %) that expressed both red and green fluorescent reporters above background. To screen for suppressor tRNAs that exhibit readthrough activity at PTCs associated with Dravet syndrome, linkers were generated that contained PTCs from the SCN1A transcripts of three patients, Patient 3 (W1397X), Patient 4 (S1505X), and Patient 5 (Q1810X), who have Gln(Q)-to-TAG nonsense mutations in SCN1A, and eight adjacent codons on either side of the PTC.

[0188] The linker region derived from the SCN1A transcript of Patient 3 is as follows and the reporter containing this linker region is designated the Patient 3-Gln-TAG (W1397X) reporter. TIFF0007712270000116.tif4149The corresponding linker with a wild-type Trp (W) codon instead of the PTC was used as a control and had the following sequence. TIFF0007712270000117.tif4150

[0189] The linker region derived from the SCN1A transcript of patient 4 is as follows, and a reporter containing this linker region is referred to as the patient 4-Gln-TAG (S1505X) reporter. TIFF0007712270000118.tif4150 The corresponding linker with wild-type Ser (S) instead of 4150PTC was used as a control, which had the following sequence. TIFF0007712270000119.tif4150

[0190] The linker region derived from the SCN1A transcript of patient 5 is as follows, and a reporter containing this linker region is referred to as the patient 5-Gln-TAG (Q1810X) reporter. TIFF0007712270000120.tif4150 The corresponding linker with wild-type Gln codon instead of 4150PTC was used as a control, which had the following sequence. TIFF0007712270000121.tif4150

[0191] Gln CTA The suppressor tRNA (SEQ ID NO: 178 - 190) was tested for PTC readthrough activity by flow cytometry in a plurality of assay settings including (i) human Flp-In-293 cells transiently co-transfected with the patient 3-Gln-TAG (W1397X) reporter, the patient 4-Gln-TAG (S1505X) reporter, or the patient 5-Gln-TAG (Q1810X) reporter (results shown in Figure 24), and (ii) human Flp-In-293 cell lines stably expressing the patient 3-Gln-TAG (W1397X) reporter and transiently transfected with a plasmid encoding the suppressor tRNA (results shown in Figure 25). Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. CTA

[0192] In summary, these results indicate that the described suppressor tRNA can promote the expression of transcripts containing premature termination codons associated with disorders such as Dravet syndrome, such as the SCN1A transcript.

[0193] Example 8 This example describes the effect of the nucleotide sequence adjacent to the suppressor tRNA on the read-through of premature termination codons (PTCs) by the suppressor tRNA.

[0194] EGFP-R96X-TGA reporter (SEQ ID NO:31 described in Example 1) and a single copy of Arg TCA An expression vector encoding suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP without intron, SEQ ID NO:18 described in Example 1) was constructed. This expression vector was obtained from genomic DNA on the 5' and 3' sides of the mouse tRNA-Arg-TCG-1-1 gene in each case and contained sequences on the immediate 5' and 3' sides of the tRNA coding sequence that differed in length. The details of the expression vector are shown in Table 11.

[0195]

Table 11

[0196] The expression vectors in Table 11 were tested for PTC read-through activity by flow cytometry in Neuro-2a cells. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. The results are shown in Figures 26A and 26B, and Arg TCA suppressor tRNA#115 showed activity even with a random 5’ leader sequence, indicating that the suppressor tRNA activity can be increased by using a 5' leader sequence derived from an endogenous tRNA gene.

[0197] Example 9 This example describes the effect of the nucleotide sequence adjacent to the suppressor tRNA on the read-through of premature termination codons (PTCs) by the suppressor tRNA.

[0198] (i) In combination with one of 20 unique 100-nt leader sequences (sequences immediately 5' to the tRNA coding sequence) derived from human genomic DNA immediately 5' to the endogenous tRNA gene, and (ii) an expression vector library containing a nucleotide sequence encoding TCA-115 (tRNA-Arg-TCT-2-1-TCA-SUP without intron, SEQ ID NO:18 as described in Example 1) or TTA-163 (tRNA-Gln-TTG-3-1-TTA-SUP, SEQ ID NO:45 as described in Example 4) was prepared. A schematic diagram showing the design of the expression vector constructs of the library is shown in Figure 27. The 20 unique 100-nt leader sequences contain sequences derived from the most abundant tRNAs in human HEK293 cells and include leader sequences derived from the Arg-TCT-1-1 (SEQ ID NO:875), Tyr-GTA-5-1 (SEQ ID NO:883), Ser-GCT-3-1 (SEQ ID NO:878), Arg-TCG-1-1 (SEQ ID NO:886), Arg-TCG-3-1 (SEQ ID NO:888), Ser-TGA-1-1 (SEQ ID NO:879), Arg-TCG-5-1 (SEQ ID NO:871), Lys-TTT-6-1 (SEQ ID NO:887), Asn-GTT-1-1 (SEQ ID NO:880), Arg-CCG-2-1 (SEQ ID NO:877), Ala-AGC-4-1 (SEQ ID NO:874), Leu-TAA-1-1 (SEQ ID NO:876), Ser-CGA-4-1 (SEQ ID NO:870), Ser-TGA-4-1 (SEQ ID NO:869), Ser-GCT-2-1 (SEQ ID NO:872), Arg-TCT-2-1 (SEQ ID NO:881), Thr-TGT-1-1 (SEQ ID NO:885), Ile-AAT-4-1 (SEQ ID NO:873), Val-CAC-2-1 (SEQ ID NO:884), or Asn-GTT-3-1 (SEQ ID NO:882) genes.

[0199] These leader sequences and Arg TCA suppressor tRNA #115 (SEQ ID NO:18) or GlnTTA The combination of suppressor tRNA#163 (SEQ ID NO:45) with Arg TCA For constructs, flow cytometry was used to test PTC readthrough activity in cell lines co-transfected with the EGFP-R96X-TGA reporter (SEQ ID NO:31) or Gln TTA For constructs, the EGFP-Q69X-TAA reporter (SEQ ID NO:175). The results are shown in FIGS. 28-32, showing that (i) the activity of the suppressor tRNA is affected by the leader sequence, and (ii) suppressor tRNAs (including different classes of suppressor tRNAs) show high readthrough activity when combined with the specified leader sequence.

[0200] Example 10 This example describes the readthrough activity of specific suppressor tRNAs and small molecule nonsense suppression therapies disclosed herein.

[0201] The suppressor tRNA was tested together with the nonsense suppressing drug Translarna (ataluren), gentamicin, and G418 (geneticin). The PTC readthrough activity was measured in Neuro-2a cells approximately 48 hours after transfection with an expression construct containing the CAG:NLS-EGFP (Q69X-TAA) reporter (SEQ ID NO:175 described in Example 4), in which (i) either the indicated copy number of Gln suppressor tRNA (#002, tRNA-Gln-TTG-1-1-TTA-SUP, SEQ ID NO:36, or #196, tRNA-Gln-TTG-1-1-CTA-SUP, SEQ ID NO:178, both described in Example 4) was included on the same construct, or (ii) treated with ataluren, (iii) treated with gentamicin, or (iv) treated with G418. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. In all experimental conditions, the cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drugs at the indicated concentrations were added. The PTC readthrough activity was measured as the ratio of EGFP-positive cells measured by flow cytometry. A reporter containing wild-type EGFP without a PTC was used as a control. The cell viability in the cells subjected to the above treatment set was evaluated by flow cytometry after staining with 7-aminoactinomycin D (7-AAD; Thermo Fisher Scientific #006993-50), a membrane-impermeable dye that is normally excluded from viable cells, according to the manufacturer's protocol. The results for Gln TTA For suppressor tRNA #002 (SEQ ID NO:36), see Figures 33-34, Gln CTASuppressor tRNA #196 (SEQ ID NO:178) is shown in FIGS. 35-36. In summary, the results show that this suppressor tRNA produces more readthrough than any of the nonsense suppressor drugs. Furthermore, the results show that, unlike any of the nonsense suppressor drugs, treatment with suppressor tRNA does not result in a decrease in cell viability.

[0202] Example 11 This example describes the rescue of full-length SCN1a protein expression by a specific suppressor tRNA disclosed herein.

[0203] For Flp-In-293 cells, (i) mouse SCN1A having Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag peptide (DYKDHD-G-DYKDHD-I-DYKDDDDK) (SEQ ID NO:901) was transfected with an expression construct containing to and (ii) either transfected with an expression construct containing Arg TCA suppressor tRNA #115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18) or treated with G418, gentamicin, or ataluren. An expression construct containing wild-type mouse SCN1A and a C-terminal 3xFLAG tag peptide (SEQ ID NO:898) was used as a control. SEQ ID NO:898 and 899 are as follows: TIFF0007712270000123.tif42149TIFF0007712270000124.tif226149TIFF0007712270000125.tif226149TIFF0007712270000126.tif227152TIFF0007712270000127.tif226149TIFF0007712270000128.tif226149TIFF0007712270000129.tif57149

[0204] SCN1A was detected by Western blot as follows. Twenty-four hours after transfection, proteins were isolated according to the manufacturer's protocol in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific #89900) containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific #87786). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific #23225). 30 μg of protein was separated on either a NuPAGE 4-12% Bis-Tris (Thermo Fisher Scientific #NP0322BOX) or NuPAGE 3-8%, Tris-Acetate (Thermo Fisher Scientific #EA0375BOX) protein gel at 150 V for 1.5 hours and then transferred to a PVDF membrane at 30 V overnight followed by 250 mA at 4°C for 30 minutes. The blot was blocked in SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific #37536) for 1 hour at room temperature, incubated with the primary anti-FLAG M2 antibody (Sigma, F1804-200UG, 1:1000) in TBST overnight at 4°C, washed 3 times with TBST, and then incubated with the HRP secondary antibody, a goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific #31431, 1:30,000) for 1 hour at room temperature. This blot was developed by applying SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific #34094) and the signal was detected by an iBright imaging system. The results are shown in Figure 37, Arg TCA Suppressor tRNA #115 was able to rescue full-length SCN1A protein expression, indicating that the small molecule drug was not able to do so.

[0205] Also, (i) mouse SCN1A having an Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag (SEQ ID NO:901) and an expression construct containing the same, and (ii) Arg to suppressor tRNA#104 (tRNA-Arg-CCG-3-1-TCA-SUP, SEQ ID NO:6), Arg TCA suppressor tRNA#106 (tRNA-Arg-CCT-2-1-TCA-SUP, SEQ ID NO:8) or Arg TCA suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP without intron, SEQ ID NO:18) were co-transfected into Flp-In-293 cells. SCN1A expression was measured by Western blot using an anti-FLAG antibody as described above in this example. The results are shown in FIG. 38, indicating that each of the tested suppressor tRNAs rescued full-length SCN1A protein expression. TCA

[0206] (SEQ ID NO:901) Also, (i) mouse SCN1A having an Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag to and an expression construct containing the same, and (ii) various doses (13 ng per well, 40 ng per well, 113 ng per well, or 400 ng per well; 6-well cell culture plate) of Arg suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP without intron, SEQ ID NO:18) were co-transfected into Flp-In-293 cells. SCN1A expression was measured by Western blot using an anti-FLAG antibody as described above in this example. The results are shown in FIG. 39, indicating that tRNA 115 was able to rescue full-length SCN1A protein expression over a wide dose range. TCA

[0207] Example 12 ​​ This example describes the read-through activity of the disclosed suppressor tRNA delivered by an adeno-associated virus (AAV) vector.

[0208] The constructs packaged in the AAV-PHP.eB capsid are shown in Figure 40. Construct 262 contains wild-type EGFP driven by the EF1a promoter. Construct 269 contains EGFP-R96X-TGA (SEQ ID NO:177) and 2 copies of Arg TCA suppressor tRNA #115 (tRNA-Arg-TCT-2-1-TCA-SUP without intron, SEQ ID NO:18, described in Example 1) under the context of 55 bp upstream flanking genomic DNA (SEQ ID NO:900) from tRNA-Tyr-GTA-5-1, driven by the EF1a promoter. Both constructs contain AAV2-derived 5' and 3' ITR sequences that provide the cis-acting elements for AAV replication and packaging. AAV-PHP.eB containing constructs 262 and 269 was generated by Vigene Biosciences.

[0209] Prior to AAV transduction, 293 cells (Agilent #240073) were pre-transfected with an expression construct containing the LY6A gene (CCDS ID 27540.1) driven by the CMV early enhancer / chicken β-actin (CAG) promoter, which is required for efficient transduction by AAV-PHP.eB. For pre-transfection, cells were transiently transfected with the LY6A expression construct using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. Approximately 24 hours after transfection, the medium was replaced with fresh medium and the cells were allowed to recover for an additional 24 hours prior to viral transduction. Cells were then transduced at an MOI of 1E5 vg / cell. The results are shown in Figure 41. Arg delivered by AAV TCASuppressor tRNA #115 achieved approximately 13.2% PTC readthrough based on GFP intensity. Suppressor tRNAs exhibit equivalent readthrough activity when delivered by AAV or transient transfection.

[0210] Example 13 This example describes ribosome profiling experiments showing that the disclosed suppressor tRNAs do not cause readthrough of a significant amount of off-target native stop codons.

[0211] To determine whether suppressor tRNAs cause readthrough of native stop codons, ribosome profiling was used to quantify (i) the number of ribosomes found in the 3' UTR of mRNAs from cells transfected with an expression construct containing the suppressor tRNA, in comparison to (ii) cells transfected with an expression construct lacking the suppressor tRNA. Ribosomes typically terminate translation when they encounter a stop codon. Thus, if suppressor tRNAs increase readthrough of native stop codons, this would be indicated by an increase in ribosome density found in the 3' UTR of mRNAs in cells expressing the suppressor tRNA, particularly in 3' UTR mRNAs containing native stop codons recognized by the expressed suppressor tRNA.

[0212] Neuro-2a cells were transfected with (i) an EGFP-R96X-TGA reporter (SEQ ID NO:177) and Arg on the same construct TCAEither an expression construct containing suppressor tRNA#001 (SEQ ID NO:11) or (ii) an expression construct lacking suppressor tRNA and containing the wild-type version of the EGFP reporter was transfected. The expression of EGFP is shown in Figure 42. Approximately 48 hours after transfection, the cells were subjected to ribosome footprint profiling as follows. The cells were lysed in lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 1 mM DTT, 50 μg / mL Emetine (Sigma #324693), and 500 U / mL RNAsin (Promega #N2615)), the cell lysate was sheared 10 times using a 25-gauge needle, and then centrifuged at 20,000 g for 10 minutes at 4°C. The supernatant was digested with micrococcal nuclease (MNase; 120 units / OD A260 lysate; New England Biolabs #M0247S) for 30 minutes at room temperature, and then the reaction was stopped by adding 5 μL of SuperAse-IN (Thermo Fisher Scientific #AM2694). The MNase-treated extract was applied to a 15-45% sucrose gradient and separated by density at 41,000 rpm for 2:26 hours at 4°C in a SW 41Ti swinging bucket rotor (Beckman Coulter #331362). After fractionation and collection of the monosome-containing fractions, the mRNA fragments protected by ribosomes were sedimented from sucrose at -20°C overnight using 1.25 mL of 95% ethanol. The mRNA fragments were resuspended in 10 mM Tris-HCl, pH 8.0 and separated on a 15% denaturing polyacrylamide gel (TBE-urea gel; Thermo Fisher Scientific #EC68852BOX). RNA fragments having a size in the range of 26-34 nt were excised from the gel and isolated to generate a ribosome-protected fragment library.After ligation of the 3' linker, rRNA depletion using the Ribo-Zero reagent of the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina #20020598), reverse transcription, circularization, and PCR amplification using index primers, the PCR products were separated on an 8% non-denaturing polyacrylamide gel (Thermo Fisher Scientific #EC62152BOX). The barcoded cDNA library was extracted from the gel and sequenced in single-read run mode using the NextSeq 550 sequencing system. After sequencing, adapters were excised from the raw reads using Trimmomatic, and then non-coding RNAs were removed by aligning them to the Ensembl mouse mm10 ncRNA reference using bowtie2. The remaining reads were also aligned to the UCSC mm10 mouse reference assembly using bowtie2. Duplicate-mapped reads were discarded. The final set of aligned reads obtained was quantified using the RiboProfiling package with R and custom Python scripts. Using Python, plots were created to examine the 3' UTR occupancy and fold change in each gene with 20 or more uniquely mapped reads, and the distributions of genes with each native stop codon were compared using the two-sample Kolmogorov–Smirnov test. The results are shown in Figure 43.

[0213] Collectively, these results indicate that suppressor tRNAs can promote the expression of transcripts containing premature termination codons, such as EGFP-R96X-TGA, without causing significant amounts of readthrough of off-target native stop codons in the cells that express them.

[0214] Incorporation by reference For all purposes, the entire disclosure of each patent and scientific document cited herein is incorporated by reference.

[0215] Equivalents The present invention can be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above aspects should be regarded as illustrative rather than restrictive of the invention described herein in every respect. Accordingly, the scope of the present invention is indicated by the appended claims rather than the above detailed description, and all changes made within the scope of the claimed invention and equivalents thereof are intended to be embraced by the present invention.

Claims

1. A tRNA encoded by an array comprising a nucleotide sequence selected from any one of SEQ ID NO: 39, 181, 40, 179, 182, and 186.

2. a) Comprising naturally occurring nucleotide modifications and / or b) Comprising one or more nucleotide modifications selected from 5-methyluridine, 5-carbamoylmethyluridine, 5-carbamoyl-methyl-2-O-methyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine, The tRNA according to claim 1.

3. An expression vector comprising a nucleotide sequence encoding the tRNA according to claim 1 or 2.

4. a) Comprising 1, 2, 3, 4, or more than 4 copies of the nucleotide sequence encoding the tRNA, b) Comprising a nucleotide sequence selected from any one of SEQ ID NO: 869 - 888, and / or c) Comprising the nucleotide sequences shown in Table 4, The expression vector according to claim 3.

5. The expression vector according to claim 3, which is a viral vector.

6. a) The viral vector is a DNA viral vector and / or b) The viral vector is an adeno-associated virus (AAV) vector, The expression vector according to claim 5.

7. A pharmaceutical composition comprising the tRNA according to claim 1 or 2 or the expression vector according to claim 3 and a pharmaceutically acceptable excipient.

8. a) The tRNA or expression vector is not conjugated or bound to another moiety or carrier particle, and / or b) The pharmaceutical composition does not contain nanoparticles and / or does not contain amino lipid delivery compounds, The pharmaceutical composition according to claim 7.

9. A pharmaceutical composition for expressing a functional gene product encoded by a gene containing a premature termination codon in mammalian cells, Comprising an effective amount of the tRNA according to claim 1 or 2 or the expression vector according to claim 3 and a pharmaceutically acceptable excipient, enabling an amino acid to be incorporated at a position in the gene product where a truncated gene product would otherwise occur due to the premature termination codon.

10. a) The cell contains fewer truncated gene products than the cell without the tRNA. b) The cell contains a greater amount of functional gene products than the cell without the tRNA. c) The gene is the gene shown in Table 5 or Table 6. d) The gene containing the premature stop codon is the dystrophin gene, and / or e) The gene containing the premature stop codon is the SCN1A gene. The pharmaceutical composition according to claim 9.

11. A pharmaceutical composition for increasing the voltage-gated sodium channel activity encoded by the SCN1A gene containing a premature stop codon in a cell, comprising an effective amount of the tRNA according to claim 1 or 2 or the expression vector according to claim 3 and a pharmaceutically acceptable excipient, and enabling an amino acid to be incorporated at a position in the SCN1A gene product where a truncated SCN1A gene product would otherwise occur due to the premature stop codon.

12. a) The SCN1A gene product generated by the tRNA is a functional SCN1A gene product comprising any one of SEQ ID NOs: 863 to 868, and / or b) The premature stop codon in the SCN1A gene is caused by a mutation selected from c.664C>T, c.1129C>T, c.1492A>T, c.1624C>T, c.1738C>T, c.1837C>T, c.2134C>T, c.2593C>T, c.3637C>T, c.3733C>T, c.3985C>T, c.4573C>T, c.5656C>T, and c.5734C>T. The pharmaceutical composition according to claim 10.

13. a) The functional SCN1A gene product has higher activity than the truncated SCN1A gene product, and / or The pharmaceutical composition according to claim 12. b) The functional SCN1A gene product is Na v 1.1 protein,

14. The pharmaceutical composition according to claim 9, wherein the cell is a human cell.

15. A pharmaceutical composition for treating a premature stop codon-mediated disorder in a subject in need thereof, comprising an effective amount of the tRNA according to claim 1 or 2 or the expression vector according to claim 3 and a pharmaceutically acceptable excipient.

16. a) The gene is the gene shown in Table 5 or Table 6, and / or ​ b) the immature termination codon-mediated disorder is Duchenne muscular dystrophy or the Drave syndrome and / or is a disorder shown in Table 5 or Table 6, The pharmaceutical composition according to claim 15.

17. The pharmaceutical composition according to claim 16, further comprising stiglentinol, cannabidiol, ketogenic diet, clobazam, topiramate, fenfluramine, or valproic acid.

18. The pharmaceutical composition according to claim 15, wherein the subject is a human.

Images