Methods and compositions for treating premature termination codon-mediated disorder

Abstract

To provide improved compositions and methods for treating diseases mediated by premature termination codons, including Dravet syndrome.SOLUTION: The invention relates generally to modified tRNAs and the use of modified tRNAs to express in a mammalian cell a functional gene product encoded by a gene containing a premature termination codon and / or to treat a disease mediated by a premature termination codon, e.g., Dravet syndrome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 929,428, filed November 1, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates generally to methods and compositions for expressing gene products encoded by genes containing premature stop codons and / or for treating disorders mediated by premature stop codons. [Background technology]

[0003] background Protein synthesis is driven by a genetic code that contains 61 three-base pair codons that code for amino acids incorporated into the protein being synthesized and three three-base pair codons that terminate protein synthesis (called stop or termination codons). When a nucleic acid sequence encoding a protein is mutated to contain a premature termination codon instead of the codon for the next amino acid, the resulting protein is prematurely terminated and is often nonfunctional or less functional than the full-length or truncated protein. Such mutations, called nonsense mutations, are often associated with or are the causative factors for many different genetic diseases.

[0004] Many disorders are associated with or caused by nonsense mutations, including epilepsies such as 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, and SYNGAP. 1-related disorders include intellectual disability, pyridoxine-dependent epilepsy, familial infantile myoclonic epilepsy (FIME), myoclonic-astatic epilepsy, X-linked intellectual disability, partial epilepsy and recurrent ataxia, febrile seizures, autosomal dominant partial epilepsy with auditory symptoms (ADPEAF), PNPO deficiency, progressive myoclonic epilepsy, action myoclonus-renal failure (AMRF), CDKL5 deficiency disorders, and benign familial infantile spasms (BFIS).

[0005] For example, Dravet syndrome is a rare and devastating form of intractable epilepsy that begins in infancy. Initially, patients experience prolonged seizures. During the second year of life, additional seizure types begin to occur, typically accompanied by developmental delays, likely due to recurrent cerebral hypoxia. This results in inadequate 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 in the SCN1A gene, for example, that result in a premature stop codon, leading to a non-truncated protein or the absence or reduction of functional protein. The SCN1A gene normally encodes the neuronal voltage-gated sodium channel α subunit, Na(V)1.1. In mouse models, loss-of-function mutations in SCN1A have been observed to result in reduced sodium flux and impaired excitability of hippocampal GABAergic interneurons.

[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 an amino acid into a gene product encoded by a gene at a position where a truncated gene product would otherwise result from a premature termination codon (PTC) in the gene in mammalian cells. The present invention is further based, in part, on the discovery that tRNAs that enable the incorporation of an amino acid into a gene product encoded by a gene at a position where a truncated gene product would otherwise result from a PTC in the gene, such as the tRNAs described herein, can be used to treat a disease in a subject mediated by a PTC in a gene.

[0009] Thus, in one aspect, the present invention provides a tRNA comprising a nucleotide sequence set forth 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 includes one or more natural nucleotide modifications selected from, e.g., 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-described tRNAs. In certain embodiments, the expression vector comprises one, two, three, four, or more than four 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 a nucleotide sequence set forth in Table 4. In certain embodiments, the nucleotide sequence set forth in Table 4 is selected from SEQ ID NOs: 869 to 888. In certain embodiments, the nucleotide sequence set forth in Table 4 is operably linked to a nucleotide sequence encoding a tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in Table 4 is located 5' to the nucleotide sequence encoding the tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in Table 4 is located immediately 5' to (i.e., adjacent to) the nucleotide sequence encoding the tRNA.

[0012] In another aspect, the present invention provides expression vectors comprising 1, 2, 3, 4, or more than 4 copies of a nucleotide sequence encoding a tRNA set forth in Table 3. 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 set forth in Table 3, further comprising a nucleotide sequence set forth 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 set forth in Table 4 is selected from SEQ ID NOs: 869-888. In certain embodiments, the nucleotide sequence set forth in Table 4 is operably linked to a nucleotide sequence encoding a tRNA. In certain embodiments, within the expression vector, the nucleotide sequence set forth in Table 4 is 5' to the nucleotide sequence encoding the tRNA. In certain embodiments, within the expression vector, the nucleotide sequence set forth in Table 4 is immediately 5' to (i.e., adjacent to) the nucleotide sequence encoding the tRNA.

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

[0015] In another aspect, the present invention provides a pharmaceutical composition, comprising any of the above-mentioned tRNA or any of the above-mentioned expression vectors and a pharmaceutically acceptable excipient.In certain embodiments, tRNA or expression vector is not conjugated or bound to another moiety, for example, carrier particle, for example, aminolipid particle.In certain embodiments, the composition does not comprise nanoparticles and / or aminolipid delivery compounds.

[0016] In another aspect, the present invention provides a method for expressing in a mammalian cell a functional gene product encoded by a gene containing a premature stop codon, the method comprising introducing into the cell an effective amount of either the above-described tRNA or expression vector, thereby allowing an amino acid to be incorporated into the gene product at a position where the premature stop codon would otherwise result in a truncated gene product. In certain embodiments of any of the above methods, the gene is selected from the genes set forth in Table 5 or Table 6. In certain embodiments, the gene is the SCN1A gene.

[0017] In another aspect, the present invention provides a method for expressing in a mammalian cell a functional gene product encoded by a gene containing a premature stop codon, the method comprising introducing into the cell an effective amount of a tRNA set forth 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 allowing an amino acid to be incorporated into the gene product at a position where a truncated gene product would otherwise result due to the premature stop codon, wherein the gene is a gene set forth in Table 5. In a specific embodiment, the gene is the SCN1A gene.

[0018] In certain embodiments of any of the above methods, the cell contains less truncated gene product than a cell without said tRNA. In certain embodiments, the cell contains a greater amount of functional gene product than a cell without said tRNA.

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

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

[0021] In certain embodiments of any of the above methods, wherein the gene is the SCN1A gene, the SCN1A gene product produced by the tRNA is a functional SCN1A gene product. In certain embodiments, the functional SCN1A gene product has greater activity than a truncated SCN1A gene product. In certain embodiments, the functional SCN1A gene product is a Na v 1.1 Protein. In certain embodiments, the functional SCN1A gene product comprises the amino acid sequence of any one of SEQ ID NOs:863-868.

[0022] In certain embodiments of any of the above methods, the cell is a human cell. In certain embodiments, the cell is a central nervous system cell, e.g., a neuron. In certain embodiments, the tRNA is aminoacylated intracellularly.

[0023] In another aspect, the present invention provides a method for treating a premature stop codon-mediated disorder in a subject in need thereof, wherein the subject has a gene with a premature stop codon, the method comprising administering to the subject an effective amount of any of the above-mentioned tRNAs or any of the above-mentioned 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 a premature stop codon-mediated disorder in a subject in need thereof, wherein the subject has a gene with a premature stop codon, the method comprising administering to the subject an effective amount of a tRNA set forth 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 set forth in Table 5.

[0025] In certain embodiments of any of the above-mentioned methods of treatment, the disorder is epilepsy, such as Dravet syndrome. In certain embodiments, the subject is a human. In certain embodiments, the method further comprises administering to the subject an effective amount of another drug, such as DIACOMIT® (stiripentol), EPIODOLEX® (cannabidiol), a 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] 1001. A tRNA of the present invention, comprising a nucleotide sequence selected from SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186. [The present invention 1003] The tRNA of the present invention 1001 or 1002, which comprises a naturally occurring nucleotide modification. [The present invention 1004] The tRNA of any of claims 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 any one of the tRNAs of the present inventions 1001 to 1004. [The present invention 1006] 1005. An expression vector of the present invention comprising 1, 2, 3, 4, or more than 4 copies of a nucleotide sequence encoding said tRNA. [The present invention 1007] The expression vector of the invention 1005 or 1006 further comprising a nucleotide sequence set forth in Table 4. [The present invention 1008] 1007. An expression vector of the present invention, comprising a nucleotide sequence selected from SEQ ID NOs: 869 to 888. [The present invention 1009] An expression vector comprising 1, 2, 3, 4, or more than 4 copies of a nucleotide sequence encoding a tRNA shown in Table 3. [The present invention 1010] An expression vector comprising a nucleotide sequence encoding a tRNA shown in Table 3, and further comprising a 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 1005 to 1012 of the present invention, 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 the tRNA of any one of the present inventions 1001 to 1004 or the expression vector of any one of the present inventions 1005 to 1015, and a pharmaceutically acceptable excipient. [The present invention 1017] 1016. The pharmaceutical composition of claim 1016, wherein said tRNA or expression vector is not conjugated or linked to another moiety. [The present invention 1018] 1017. The pharmaceutical composition of claim 1017, wherein said tRNA or expression vector is not conjugated or bound to a carrier particle. [The present invention 1019] 1008. The pharmaceutical composition of the present invention, wherein the carrier particles are amino lipid particles. [The present invention 1020] Any of the pharmaceutical compositions of 1015 to 1019, which does not contain nanoparticles. [The present invention 1021] Any of the pharmaceutical compositions of claims 1015 to 1020, which does not contain an aminolipid delivery compound. [The present invention 1022] 1. A method for expressing in mammalian cells a functional gene product encoded by a gene containing a premature stop codon, comprising: introducing into said cells an effective amount of a tRNA of any one of inventions 1001 to 1004 or an expression vector of any one of inventions 1005 to 1015, thereby allowing an amino acid to be incorporated into the gene product at a position where a truncated gene product would otherwise result due to a premature stop codon; A method comprising: [The present invention 1023] 1023. The method of claim 1022, wherein said cell contains less truncated gene product than a cell without said tRNA. [The present invention 1024] 1024. The method of any one of claims 1022 to 1023, wherein said cells contain a functional gene product in an amount greater than a cell not having said tRNA. [The present invention 1025] The method of any one of claims 1022 to 1024, wherein the gene is a gene shown in Table 5 or Table 6. [The present invention 1026] The method of claim 1025, wherein said gene is a gene shown in Table 5. [The present invention 1027] 1. A method for expressing in mammalian cells a functional gene product encoded by a gene containing a premature stop codon, comprising: introducing into the cell an effective amount of a tRNA set forth in Table 3 or an expression vector comprising a nucleotide sequence encoding said tRNA, thereby allowing an amino acid to be incorporated into the gene product at a position where a truncated gene product would otherwise result due to a premature stop codon; Including, The gene is a gene shown in Table 5. method. [The present invention 1028] 1027. The method of claim 1027, wherein 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. [The present invention 1029] The method of any one of claims 1022 to 1028, wherein the gene is the SCN1A gene. [The present invention 1030] 1. A method for increasing the activity of a voltage-gated sodium channel encoded by an SCN1A gene containing a premature stop codon in a cell, comprising: introducing into said cells an effective amount of a tRNA of any one of inventions 1001 to 1004 or an expression vector of any one of inventions 1005 to 1015, thereby allowing an amino acid to be incorporated into the SCN1A gene product at a position where a truncated SCN1A gene product would otherwise result from a premature stop codon; A method comprising: [The present invention 1031] 1. A method for increasing the activity of a voltage-gated sodium channel encoded by an SCN1A gene containing a premature stop codon in a cell, comprising: introducing into the cell an effective amount of a tRNA set forth in Table 3 or an expression vector comprising a nucleotide sequence encoding said tRNA, thereby allowing the incorporation of an amino acid into the SCN1A gene product at a position where a truncated SCN1A gene product would otherwise result due to a premature stop codon; A method comprising: [The present invention 1032] 1031. The method of claim 1031, wherein 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. [The present invention 1033] The method of any of claims 1029 to 1032, wherein the SCN1A gene product produced by the tRNA is a functional SCN1A gene product. [The present invention 1034] The method of claim 1033, wherein the functional SCN1A gene product has greater activity than a truncated SCN1A gene product. [This invention 1035] The functional SCN1A gene product is v 1.1 The method of claim 1033 or 1034, wherein the protein is a protein. [The present invention 1036] The method of any of claims 1033 to 1035, wherein the functional SCN1A gene product comprises any one of SEQ ID NOs: 863 to 868. [This invention 1037] The method of claim 1036, wherein the functional SCN1A gene product comprises SEQ ID NO:863 or SEQ ID NO:864. [The present invention 1038] The method of any one of claims 1022 to 1037, wherein the cells are human cells. [This invention 1039] The method of any of claims 1022 to 1038, wherein the tRNA is aminoacylated intracellularly. [The present invention 1040] 1. A method of treating a premature stop codon mediated disorder in a subject in need thereof, comprising: the subject has a gene with a premature stop codon, The method comprises: administering to the subject an effective amount of the tRNA of any one of claims 1001 to 1004 of the present invention or the expression vector of any one of claims 1005 to 1015 of the present invention, thereby treating the disorder in the subject. A method comprising: [The present invention 1041] The method of claim 1040, wherein said disorder is a disorder shown in Table 5 or Table 6. [The present invention 1042] The method of claim 1041, wherein the disorder is a disorder shown in Table 5. [This invention 1043] 1. A method of treating a premature stop codon mediated disorder in a subject in need thereof, comprising: the subject has a gene with a premature stop codon, The method comprises: administering to the subject an effective amount of a tRNA set forth in Table 3 or an expression vector comprising a nucleotide sequence encoding the tRNA, thereby treating the disorder in the subject. Including, the disorder is a disorder shown in Table 5 method. [This invention 1044] The method of claim 1043, wherein 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. [This invention 1045] The method of any one of claims 1040 to 1044, wherein the disorder is Dravet syndrome. [The present invention 1046] The method of claim 1045, further comprising administering to said subject stiripentol, cannabidiol, a ketogenic diet, clobazam, topiramate, fenfluramine, or valproic acid. [This invention 1047] The method of any one of claims 1045 to 1046, wherein the gene is SCN1A. [This invention 1048] The method of any one of claims 1040 to 1047, wherein the subject is a human. [This invention 1049] Any of methods 1029-1039 or 1047, 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. [The present invention 1050] 1049. The method of claim 1049, 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 explanation of the drawings]

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

[0029] (Figure 1) Schematic diagram of a transcript (e.g., SCN1A transcript) containing a premature stop codon (PTC) that results in a truncated protein product (e.g., in a subject with Dravet syndrome). The native stop codon is indicated by a shaded circle, and the premature stop codon is indicated by an unshaded circle. Expression of a suppressor tRNA (e.g., anticodon-modified arginine tRNA) charged with its cognate amino acid (AA) allows read-through of the PTC and promotes expression of the full-length protein. (Figure 2A) Consensus tRNA secondary structure. Residue numbers are based on the tRNA numbering system described in Steinberg et al. (1993) NUCLEIC ACIDS RES. 21:3011-15. (Figure 2B) A table showing the modification profiles of tRNA sequences derived from the cytosol of specific eukaryotic organisms. The ratios in the table indicate the frequency of occurrence of the listed nucleotide at the numbered position shown in Figure 2A. Abbreviations for modified residues are defined in Motorin et al. (2005) "Transfer RNA Modification," ENCYCLOPEDIA OF LIFE SCIENCES, John Wiley & Sons, Inc. (Figure 3) Schematic diagram of the dual-fluorescence reporter construct containing three copies of red fluorescent protein (tdTomato), TEV protease, a 51-bp linker region containing PTC + / - 8 flanking codons, and three copies of green fluorescent protein (EGFP). Expression is driven by the promoter for elongation factor EF-1α, located upstream of the first copy of tdTomato. tdTomato, all copies of EGFP, and TEV protease are separated from each other by a TEV protease cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln-(Gly / Ser) (SEQ ID NO:902)). (Figure 4) Arg expression in the Flp-In-293 cell line stably expressing a dual fluorescent reporter with a targeted S-PTC linker region (SEQ ID NO: 29) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome. TCA 1 is a graph showing the read-through activity of suppressor tRNAs. TCACells were transfected with suppressor tRNAs (SEQ ID NOs: 1-25 and 35), and read-through activity was measured by flow cytometry in two independent experiments 24 h after transfection. "Parent" refers to the original Flp-In-293 cell line lacking the fluorescent reporter; "No PTC" refers to the Flp-In-293 cell line stably expressing the dual fluorescent reporter with a PTC-depleted version of the target S-PTC linker region (SEQ ID NO: 194); "EV" (empty vector) refers to cells transfected with an expression construct containing no tRNA; "TCG" refers to cells transfected with an expression construct containing wild-type Arg-tRNA with a TCG anticodon. Read-through activity is expressed as the percentage of surviving cells expressing both tdTomato and EGFP above background ("% double positive"). Error bars represent the standard deviation of the data. (Figure 5) Arg expression in Flp-In-3T3 cell lines stably expressing a dual fluorescent reporter with the R1407X-PTC linker region (SEQ ID NO: 30) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome. TCA 1 is a graph showing the read-through activity of suppressor tRNAs. TCACells were transfected with suppressor tRNAs (SEQ ID NOs: 1-25), and read-through activity was measured by flow cytometry in three independent experiments 24 h after transfection. "Parent" indicates the original 3T3 cell line lacking the fluorescent reporter; "No PTC" indicates a 3T3 cell line stably expressing a dual fluorescent reporter with a PTC-depleted version of the R1407X-PTC linker region (SEQ ID NO: 195); "Mock" indicates mock-transfected cells; "EV" (empty vector) indicates cells transfected with an expression construct containing neither tRNA nor EGFP reporter; "TCG" indicates cells transfected with an expression construct containing wild-type Arg-tRNA with a TCG anticodon. Read-through activity is expressed as the percentage of surviving cells expressing both tdTomato and EGFP above background ("% double positive"). Error bars represent the standard deviation of the data. (Figure 6) Arg expression in Flp-In-3T3 cells transiently expressing a dual fluorescent reporter with the target N-PTC linker region (SEQ ID NO: 28) derived from a clinically relevant SCN1A nonsense mutation associated with Dravet syndrome. TCA 1 is a graph showing the read-through activity of suppressor tRNAs. TCA Cells were co-transfected with suppressor tRNAs (SEQ ID NOs: 1-25 and 35), and read-through activity was measured by flow cytometry. "Mock" indicates mock-transfected cells; "No PTC" indicates cells transfected with a dual-fluorescence reporter containing a version of the target N-PTC linker region (SEQ ID NO: 193) lacking the PTC; "EV" (empty vector) indicates cells co-transfected with an expression construct containing no tRNA; and "TCG" indicates cells co-transfected with an expression construct containing wild-type Arg-tRNA with a TCG anticodon. Read-through activity was measured by flow cytometry and is expressed as the percentage of viable cells expressing both tdTomato and EGFP above background ("% double positive"). (Figure 7) The indicated Arg in Neuro-2a (N2a) and Flp-In-293 (293) cells. TCA 1 is a graph showing the read-through activity of suppressor tRNA (as measured by the percentage of positive GFP cells). TCA Cells were co-transfected with an expression construct containing a suppressor tRNA (SEQ ID NOs: 1-22). "EGFP" indicates cells transfected with a version of the EGFP reporter lacking the 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. Read-through activity is expressed as the percentage of viable cells expressing EGFP above background ("GFP+%"); all values ​​are normalized to cells expressing EGFP lacking the PTC. (Figure 8A) Diagram of the experimental approach to measure suppressor tRNA activity using a construct containing an EGFP reporter along with a PTC and suppressor tRNA. The native stop codon is indicated by a shaded circle, and the premature stop codon is indicated by an unshaded circle. Standard Arg-tRNA (containing an anticodon linked to CGA) cannot read through the PTC within EGFP, resulting in a non-functional truncated EGFP protein. Suppressor tRNA (containing an anticodon linked to UGA) allows readthrough of the PTC within EGFP, resulting in a full-length, functional EGFP protein. (FIG. 8B) Schematic diagram of an exemplary reporter construct containing EGFP together with a PTC and four copies of a suppressor tRNA. (FIG. 9) EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated Arg TCAFigure 1 shows fluorescent images of Neuro-2a cells transfected with expression constructs containing suppressor tRNAs 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 from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32). Images were taken approximately 24 hours after transfection. GFP controls, from left to right, are: 1) wild-type EGFP alone; 2) a single copy of Arg TCA 1) wild-type EGFP on an expression construct containing suppressor tRNA#001 (SEQ ID NO: 11), 2) the EGFP-R96X-TGA reporter alone, and 3) EGFP-R96X-TGA on an expression construct containing four copies of Arg-tRNA with an unmodified TCG anticodon. (FIG. 10) EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated Arg TCA Figure 1 shows fluorescent images of Flp-In-293 cells transfected with expression constructs containing suppressor tRNAs. The suppressor tRNAs were 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 from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32). Images were taken approximately 48 hours after transfection. GFP controls, from left to right, are: 1) wild-type EGFP alone; 2) a single copy of Arg TCA1) wild-type EGFP on an expression construct containing suppressor tRNA#001 (SEQ ID NO:11), 2) EGFP-R96X-TGA reporter alone, and 3) EGFP-R96X-TGA on an expression construct containing four copies of Arg-tRNA with an unmodified TCG anticodon. (Figure 11) EGFP-R96X-TGA reporter (SEQ ID NO: 177) and Arg TCA Fluorescence measured by flow cytometry in Neuro-2a and Flp-In-293 cells transfected with expression constructs containing suppressor tRNA#001 (SEQ ID NO:11). The expression constructs contained one (1x), two (2x), or four (4x) copies of the suppressor tRNA in the context of either (i) a U6 promoter ("U6") containing 19 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:33) and 46 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:34), or (ii) 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 200 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:27) ("flanking"). "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. Analysis was performed approximately 48 hours after transfection. Data are presented as histograms showing the frequency distribution of data relative to the fluorescence intensity in viable cells expressing EGFP above background. (FIG. 12) EGFP-R96X-TGA reporter (SEQ ID NO:177) and the indicated copy numbers of Arg-TCG-1-1 in the context of either (i) a U6 promoter ("U6") containing 19 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:33) and 46 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:34), or (ii) a 200 bp upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 200 bp downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:27) ("flanking"). TCA Figure 1 shows the percentage of EGFP-positive cells and the average EGFP intensity among all viable cells, as measured by flow cytometry, in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#001 (SEQ ID NO:11). "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. Analysis was performed approximately 48 hours after transfection. These plots summarize the data from Neuro-2a cells in Figure 11. (Figure 13) EGFP-R96X-TGA reporter (SEQ ID NO:31) and Arg TCAFigure 1 shows fluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#001 (SEQ ID NO:11). The expression constructs contained one (1x), two (2x), three (3x), or four (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contains 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32) ("flanking"). "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 approximately 48 hours after transfection. Data are presented as histograms showing the frequency distribution of data versus fluorescence intensity in all viable cells ("G1 gate") and viable cells expressing EGFP above background ("GFP+ gate"). (Figure 14) EGFP-R96X-TGA reporter (SEQ ID NO:31) and Arg TCAFigure 1 shows fluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#113 (SEQ ID NO:16). The expression constructs contained one (1x), two (2x), three (3x), or four (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contained 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32) ("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. Analysis was performed approximately 48 hours after transfection. Data are presented as histograms showing the frequency distribution of data versus 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 TCAFigure 1 shows fluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#115 (SEQ ID NO:18). The expression constructs contained one (1x), two (2x), three (3x), or four (4x) copies of the suppressor tRNA. Each copy of the suppressor tRNA also contained 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32) ("flanking"). "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 approximately 48 hours after transfection. Data are presented as histograms showing the frequency distribution of data versus fluorescence intensity in all viable cells ("G1 gate") and viable cells expressing EGFP above background ("GFP+ gate"). (Figure 16) The percentage of EGFP-positive cells among all viable cells ("% GFP+ cells") and the average EGFP intensity in EGFP-positive cells ("GFP+ gate") measured by flow cytometry in Neuro-2a cells transfected with expression constructs containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated copy numbers of the indicated suppressor tRNAs. The suppressor tRNAs were 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 the PTC, and "CAG:EGFP-PTC" indicates cells transfected with the EGFP-R96X-TGA reporter alone. These plots summarize the data from Figures 13-15. (Figure 17) EGFP-R96X-TGA reporter (SEQ ID NO:31) and the indicated Arg with the indicated copy number and flanking sequences. TCA The percentage of EGFP-positive cells, as measured by flow cytometry, in Neuro-2a cells transfected with expression constructs containing suppressor tRNAs TCA-001 (SEQ ID NO: 11), TCA-113 (SEQ ID NO: 16), and TCA-115 (SEQ ID NO: 18) is shown. "U6" indicates the U6 promoter including 19 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:33) and 46 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:34); "flanking (±200 bp)" indicates 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 200 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:27); and "flanking (+200 / -100 bp)" indicates 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 104 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:32) ("flanking"). "Mock" indicates mock-transfected cells, "Empty Vector" indicates cells transfected with an expression construct containing neither tRNA nor EGFP reporter, "4xTCG" indicates cells transfected with an expression construct containing the EGFP-R96X-TGA reporter and four copies of wild-type Arg-tRNA with a TCG anticodon, and "CAG:EGFP" indicates cells transfected with a version of the EGFP reporter lacking the PTC. All data are normalized to the positive control (CAG:EGFP). The plot shows the percentage of viable cells expressing GFP above background. (Figure 18) Dmd derived from a clinically relevant DMD nonsense mutation associated with Duchenne muscular dystrophy mdx-Gln in two independently derived Flp-In-293 cell lines (#2 and #10) stably expressing a dual fluorescent reporter with a PTC linker region (SEQ ID NO: 192). TTA 1 is a graph showing the read-through activity of suppressor tRNAs. TTA Cells were transfected with suppressor tRNAs (SEQ ID NOs: 36-48), and read-through activity was measured by flow cytometry 24 hours after transfection. "Parent" refers to the original Flp-In-293 cell line without the fluorescent reporter, and "No PTC" refers to a version of Dmd lacking the PTC. mdx Figure 1 shows the Flp-In-293 cell line stably expressing a dual fluorescent reporter with a -PTC linker region (SEQ ID NO:191), and "TTG" indicates cells transfected with an expression construct containing wild-type Gln-tRNA with a TTG anticodon. Read-through activity was measured by flow cytometry and is expressed as the percentage of viable cells expressing both tdTomato and EGFP above baseline ("% dual+ cells"). (FIG. 19) The indicated Gln in Neuro-2a cells co-transfected with an expression construct containing the EGFP-Q69X-TAA reporter (SEQ ID NO:175). TTA This graph shows the read-through activity of suppressor tRNAs (SEQ ID NOs: 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 a tRNA nor an EGFP reporter, and "TTG" indicates cells co-transfected with the EGFP-Q69X-TAA reporter and an expression construct containing a wild-type Gln-tRNA with a TTG anticodon. Read-through activity was measured by flow cytometry and is expressed as the percentage of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. (FIG. 20) The indicated Gln in Neuro-2a cells co-transfected with an expression construct containing the EGFP-Q69X-TAG reporter (SEQ ID NO:176). CTA This graph shows the read-through 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 the PTC, "EV" (empty vector) indicates cells transfected with an expression construct containing neither a tRNA nor an EGFP reporter, and "TTG" indicates cells co-transfected with the EGFP-Q69X-TAG reporter and an expression construct containing a wild-type Gln-tRNA with a TTG anticodon. Read-through activity was measured by flow cytometry and is expressed as the percentage of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. (FIG. 21) Neuro-2a cells approximately 24 hours after transfection with an expression construct containing the EGFP-R96X-TGA reporter ("GFP-PTC"), showing (i) the indicated copy number of Arg TCA Fluorescence images of Neuro-2a cells are shown that either contained suppressor tRNA#115 (SEQ ID NO:18) or were treated with (ii) the indicated concentrations of ataluren, (iii) the indicated concentrations of gentamicin, or (iv) the indicated concentrations of G418. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated concentrations of the indicated drugs were added. Controls in the left column are expression constructs containing wild-type EGFP ("WT-GFP") and the indicated drugs or the indicated copy numbers of Arg TCA Suppressor tRNA was transfected. (FIG. 22) A graph showing the percentage of GFP-positive cells measured by flow cytometry approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-R96X-TGA reporter ("GFP-PTC") and (i) the indicated copy number of Arg TCA Suppressor tRNA#115 (SEQ ID NO:18) was included, or cells were treated with either (ii) ataluren at the indicated concentration, (iii) gentamicin at the indicated concentration, or (iv) G418 at the indicated concentration. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentration. A reporter containing wild-type EGFP without a PTC ("WT-GFP") was used as a control. "Mock" indicates mock-transfected cells. "4XTCG" indicates cells transfected with an expression construct containing four copies of wild-type Arg-tRNA with a TCG anticodon and the EGFP-R96X-TGA reporter. The plot shows the percentage of surviving cells expressing EGFP above background. The percentage of cells expressing GFP ranged from 0.7 to 1.6% in the negative control, 0.9 to 4.9% in ataluren-treated cells, 1.2 to 6.5% in gentamicin-treated cells, and 6.7% to 26.7% in G418-treated cells. TCA In cells expressing the suppressor, it ranged from 73.0 to 76.2%. (FIG. 23) A graph showing cell viability from FIG. 22, measured by flow cytometry approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-R96X-TGA reporter ("GFP-PTC") and (i) the indicated copy number of Arg TCACells were either transfected with an expression construct containing suppressor tRNA#115 (SEQ ID NO:18) or treated with (ii) at the indicated concentrations of ataluren, (iii) at the indicated concentrations of gentamicin, or (iv) at the indicated concentrations of G418. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentrations. 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 four copies of wild-type Arg-tRNA with a TCG anticodon and the EGFP-R96X-TGA reporter. Cell viability was assessed by flow cytometry using 7-aminoactinomycin D (7-AAD), a membrane-impermeable dye that is normally excluded from viable cells. (Figure 24) Three clinically relevant Gln(Q)-to-TAG PTC mutations in SCN1A associated with Dravet syndrome (W1397 * , S1505 * , and Q1810 * Figure 1 shows the indicated Gln in Flp-In-293 cells transiently co-transfected with the indicated dual fluorescent reporter constructs containing the linker region (SEQ ID NOs: 889, 891, and 893) derived from CTAThis graph shows the read-through activity of suppressor tRNAs (SEQ ID NOs: 178-190). "Mock" indicates mock-transfected cells; "No PTC" indicates cells transfected with versions of the dual fluorescent reporter construct lacking the PTC (SEQ ID NOs: 890, 892, and 894); "EV" (empty vector) indicates cells transfected with an expression construct containing neither a tRNA nor a fluorescent reporter; and "TTG" indicates cells cotransfected with the indicated dual fluorescent reporter construct and an expression construct containing a wild-type Gln-tRNA with a TTG anticodon. Read-through activity was measured by flow cytometry approximately 24 hours after transfection and is expressed as the percentage of viable cells expressing both tdTomato and EGFP above baseline ("% dual+ cells"). (Figure 25) Clinically relevant PTC mutation in SCN1A associated with Dravet syndrome (W1397 * The indicated Gln in the Flp-In-293 cell line integrated with a dual fluorescent reporter construct containing the linker region (SEQ ID NO: 889) derived from CTA This graph shows the read-through activity of suppressor tRNAs (SEQ ID NOs: 178-190). "Mock" indicates mock-transfected cells, "RFP-EGFP" indicates the Flp-In-293 cell line containing a PTC-deficient version of the dual fluorescent reporter construct (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 a TTG anticodon. Read-through activity was measured by flow cytometry approximately 24 hours after transfection and is expressed as the percentage of cells expressing both tdTomato and EGFP above background ("% dual+ cells"). (Figure 26A) (i) 200 bp upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 26) and 104 bp downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 32) ("flanking 300"); (ii) 20 bp upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 895) and 17 bp downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 896) ("flanking 20"); (iii) 10 bp upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 897) and 17 bp downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: (iv) a single copy of the EGFP-R96X-TGA reporter (SEQ ID NO: 31) and a single copy of Arg in the context of either 0 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 and 17 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO: 896) ("flanking 0"). TCA Figure 1 shows fluorescence measured by flow cytometry in Neuro-2a cells transfected with an expression construct containing suppressor tRNA#115 (SEQ ID NO: 18). 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 PTC. Read-through activity was measured by flow cytometry approximately 24 hours after transfection. Data are presented as histograms showing the frequency distribution of data relative to the fluorescence intensity in cells expressing EGFP above background. (Figure 26B) Shows the percentage of EGFP-positive cells among all viable cells ("GFP+%") and the average EGFP intensity among viable cells expressing EGFP above background ("Average GFP signal") for the cells shown in Figure 26A. (Figure 27) Schematic diagram of the construct used to test the effect of 5' leader sequences on the readthrough of premature termination codons (PTCs) by suppressor tRNAs. The construct contained (i) a 100 bp 5' leader sequence derived from genomic DNA located upstream of a tRNA gene that is highly expressed in HEK293 cells, and (ii) a single copy of the 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 ("Stop"). (Figure 28) The indicated Arg expression levels were measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection. TCA 1 is a graph showing the read-through activity of suppressor tRNA expression constructs (i) EGFP-R96X-TGA reporter (SEQ ID NO:31) and (ii) Arg TCA Cells were co-transfected with a construct 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 a tRNA nor an EGFP reporter; "26 / 27" indicates cells co-transfected with ArgTCA suppressor tRNA#115 (SEQ ID NO:18) in the context of 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 200 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:27). The plot shows the percentage of viable cells expressing EGFP above background. The percentage of cells expressing EGFP was 0.4% in the empty vector control compared to 0.4% in the ArgTCA suppressor. TCA It ranged from 17.2% to 33.7% in cells expressing the suppressor. (Figure 29) The indicated Arg expression levels were measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection. TCA 1 is a graph showing the read-through activity of suppressor tRNA expression constructs (i) EGFP-R96X-TGA reporter (SEQ ID NO:31) and (ii) Arg TCA Cells were co-transfected with a construct 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 a tRNA nor an EGFP reporter; "26 / 27" indicates cells co-transfected with ArgTCA suppressor tRNA#115 (SEQ ID NO:18) in the context of 200 bp of upstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:26) and 200 bp of downstream flanking genomic DNA from tRNA-Arg-TCG-1-1 (SEQ ID NO:27). The plot shows the average EGFP intensity in cells expressing EGFP above background. The average EGFP intensity was 602 in the empty vector control compared to 602 in the Arg TCG-1-1 control. TCA In cells expressing the suppressor, it ranged from 1990 to 4319. (Figure 30) The indicated Gln ATP levels were measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection. TTA 1 is a graph showing the read-through activity of suppressor tRNA expression constructs (i) EGFP-Q69X-TAA reporter (SEQ ID NO:175) and (ii) Gln in the context of the indicated 100 bp upstream genomic DNA leader sequence (SEQ ID NO:869-888). 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 a tRNA nor an EGFP reporter; "173 / 174" indicates cells co-transfected with the Gln-TTG-1-1 gene in the context of 200 bp of upstream flanking genomic DNA from tRNA-Gln-TTG-1-1 (SEQ ID NO:173) and 200 bp of downstream flanking genomic DNA from tRNA-Gln-TTG-1-1 (SEQ ID NO:174). TTA Cells co-transfected with suppressor tRNA#163 (SEQ ID NO:45) are shown. The plot shows the percentage of viable cells expressing EGFP above background. The percentage of cells expressing EGFP was 0.3% in the empty vector control compared to 0.3% in the Gln TTA It ranged from 21.4% to 35.7% in cells expressing the suppressor. (Figure 31) The indicated Gln ATP levels were measured by flow cytometry in Flp-In-293 cells approximately 24 hours after transfection. TTA 1 is a graph showing the read-through activity of suppressor tRNA expression constructs (i) EGFP-Q69X-TAA reporter (SEQ ID NO:175) and (ii) Gln in the context of the indicated 100 bp upstream genomic DNA leader sequence (SEQ ID NO:869-888). 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 a tRNA nor an EGFP reporter; "173 / 174" indicates cells co-transfected with the Gln-TTG-1-1 gene in the context of 200 bp of upstream flanking genomic DNA from tRNA-Gln-TTG-1-1 (SEQ ID NO:173) and 200 bp of downstream flanking genomic DNA from tRNA-Gln-TTG-1-1 (SEQ ID NO:174).TTA 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 is 387 in the Gln versus 387 in the empty vector control. TTA In cells expressing the suppressor, it ranged from 1702 to 3822. (FIG. 32) A table summarizing the results of FIGS. 28-31, where values ​​are normalized to cells transfected with wild-type GFP that do not have PTC. (FIG. 33) is a graph showing the percentage of EGFP-positive cells measured by flow cytometry in three independent experiments approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-Q69X-TAA reporter ("EGFP-PTC") (SEQ ID NO: 175) and (i) the indicated number of copies of Gln TTASuppressor tRNA#002 (SEQ ID NO:36) was included, or cells were treated with either (ii) ataluren at the indicated concentration, (iii) gentamicin at the indicated concentration, or (iv) G418 at the indicated concentration. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentration. A reporter containing wild-type EGFP without a PTC ("EGFP") was used as a control. "Mock" indicates mock-transfected cells, "No tRNA" indicates cells transfected with the indicated EGFP expression construct alone, and "4X-Gln-TTG" indicates cells transfected with an expression construct containing four copies of wild-type Gln-tRNA with a TTG anticodon and the EGFP-Q69X-TAA reporter. The plot shows the percentage of viable cells expressing GFP above background. Error bars represent the standard deviation of the data. The percentage 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. TTA In cells expressing the suppressor, it ranged from 64.7 to 67.3%. (FIG. 34) A graph showing cell viability from FIG. 33, measured by flow cytometry approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-Q69X-TAA reporter ("EGFP-PTC") (SEQ ID NO: 175) and (i) the indicated number of copies of Gln TTASuppressor tRNA#002 (SEQ ID NO:36) was included, or cells were treated with either (ii) ataluren at the indicated concentration, (iii) gentamicin at the indicated concentration, or (iv) G418 at the indicated concentration. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentration. A reporter containing wild-type EGFP without a PTC ("EGFP") was used as a control. "Mock" indicates mock-transfected cells, "No tRNA" indicates cells transfected with the indicated EGFP expression construct alone, and "4X-Gln-TTG" indicates cells transfected with an expression construct containing four 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 normally excluded from viable cells. (Figure 35) is a graph showing the percentage of EGFP-positive cells measured by flow cytometry in three independent experiments approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-Q69X-TAG reporter ("EGFP-PTC") (SEQ ID NO: 176) and (i) the indicated number of copies of Gln CTASuppressor tRNA#196 (SEQ ID NO:178) was included, or cells were treated with either (ii) ataluren at the indicated concentration, (iii) gentamicin at the indicated concentration, or (iv) G418 at the indicated concentration. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentration. A reporter containing wild-type EGFP without a PTC ("EGFP") was used as a control. "Mock" indicates mock-transfected cells, "No tRNA" indicates cells transfected with the indicated EGFP expression construct alone, and "4X-Gln-TTG" indicates cells transfected with an expression construct containing four copies of wild-type Gln-tRNA with a TTG anticodon and the EGFP-Q69X-TAA reporter. The plot shows the percentage of viable cells expressing GFP above background. The percentage 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. CTA In cells expressing the suppressor, it ranged from 73.1 to 78.2%. (Figure 36) A graph showing cell viability from Figure 35, measured by flow cytometry approximately 48 hours after transfection. Neuro-2a cells were transfected with an expression construct containing the EGFP-Q69X-TAG reporter ("EGFP-PTC") (SEQ ID NO: 176) and (i) the indicated number of copies of Gln CTASuppressor tRNA#196 (SEQ ID NO:178) was included, or cells were treated with either (ii) ataluren at the indicated concentration, (iii) gentamicin at the indicated concentration, or (iv) G418 at the indicated concentration. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drug was added at the indicated concentration. A reporter containing wild-type EGFP without a PTC ("EGFP") was used as a control. "Mock" indicates mock-transfected cells, "No tRNA" indicates cells transfected with the indicated EGFP expression construct alone, and "4X-Gln-TTG" indicates cells transfected with an expression construct containing four 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 normally excluded from viable cells. (FIG. 37A) Western blot showing rescue of full-length SCN1A protein expression by suppressor tRNA. For Flp-In-293 cells, Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag peptide were used. Mouse SCN1A ("SCN1a (R1407 * )") and (i) an expression construct containing Arg TCACells were either cotransfected with an expression construct containing suppressor tRNA#115 (SEQ ID NO:18) ("Arg>TGA#115") or treated with (ii) ataluren at the indicated concentrations, (iii) gentamicin at the indicated concentrations, or (iv) G418 at the indicated concentrations. "SCN1a (wt)" 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. Molecular weights based on protein molecular weight markers are shown to the left of the gel. (Figure 37B) Quantification of the Western blot shown in Figure 37A. ImageJ was used to measure the intensity of the band corresponding to the size of the full-length SCN1A protein, and all intensity values ​​were normalized to the wild-type SCN1A protein ("SCN1a (wt)" lane). TCA Cells cotransfected with an expression vector containing the 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 tRNAs: (i) Arginine-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag peptide; Mouse SCN1A ("SCN1a (R1407 * )") and (ii) an expression construct containing #104 Arg TCA Suppressor tRNA (SEQ ID NO:6) ("Sup#104"), #106 Arg TCA suppressor tRNA (SEQ ID NO:8) ("Sup#106"), or Arg TCAFlp-In-293 cells were co-transfected with either suppressor tRNA#115 (SEQ ID NO: 18) ("Sup#115"). "SCN1a (wt)" 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 monoclonal anti-FLAG M2 antibody. Molecular weights based on protein molecular weight markers are shown on the left side of the gel. (Figure 38B) Quantification of the Western blot shown in Figure 38A. ImageJ was used to measure the intensity of the band corresponding to the size of the full-length SCN1A protein, and all intensity values ​​were normalized to the wild-type SCN1A protein ("SCN1a (wt)" lane). TCA Cells cotransfected with an expression vector containing a 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. Flp-In-293 cells were cultured in 6-well cell culture plates and transfected with (i) arginine-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag peptide. Mouse SCN1A ("SCN1a (R1407 * )") and (ii) an expression construct containing the indicated concentrations of Arg TCA The cells were co-transfected with suppressor tRNA#115 (SEQ ID NO:18) (“Arg>TGA#115”). 1x was 400 ng per well of Arg TCA suppressor tRNA construct, 0.3x, 133 ng Arg per well TCAsuppressor tRNA construct, 0.1x is 40 ng Arg per well TCA suppressor tRNA construct, 0.03x, 13 ng Arg per well TCA The suppressor tRNA construct is indicated. "SCN1a (wt)" 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. Molecular weights based on protein molecular weight markers are indicated on the left side of the gel. (Figure 39B) Quantification of the Western blot shown in Figure 39A. ImageJ was used to measure the intensity of the band corresponding to the size of the full-length SCN1A protein, and all intensity values ​​were normalized to the wild-type SCN1A protein ("SCN1a (wt)" lane). (Figure 40) Schematic diagram of the constructs packaged into AAV-PHP.eB capsids. Construct 262 contains wild-type EGFP driven by the EF1a promoter. Construct 269 contains EGFP-R96X-TGA (SEQ ID NO:177) driven by the EF1a promoter and two copies of Arg in the context of 55 bp of upstream flanking genomic DNA from tRNA-Tyr-GTA-5-1 (SEQ ID NO:900). TCA suppressor tRNA#115 (SEQ ID NO:18) ("TCA-115"). Both constructs contain 5' and 3' ITR sequences from AAV2, which provide cis-acting elements for AAV replication and packaging. (Figure 41) This figure shows the read-through activity of AAV-delivered suppressor tRNA. 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, which is required for reliable transduction by AAV-PHP.eB. Cells were transduced at an MOI of 1E5 vg / cell. Where indicated, cells were also transfected with an expression construct containing the EGFP-R96X-TGA reporter (SEQ ID NO:31) and an expression construct containing the ArgTCA suppressor tRNA#115 (SEQ ID NO:18). 72 hours after transduction, EGFP signals were quantified by immunofluorescence. Live-cell images were captured using an EVOS FL Auto 2 imaging system. CellProfiler software was used to segment and extract the nuclear-incorporated EGFP intensity in each image. These values ​​were averaged across all nuclei in each condition. From each of these averages, the background average, which was 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) Fluorescence measured by flow cytometry in Neuro-2a cells used in ribosome footprint profiling analysis. (i) EGFP-R96X-TGA reporter (SEQ ID NO: 177) and Arg TCA An expression construct containing suppressor tRNA#001 (SEQ ID NO:11) ("Arg TCA Cells were transfected with either (ii) an expression construct containing a version of the EGFP reporter lacking the PTC ("WT-EGFP"). (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) EGFP-R96X-TGA reporter (SEQ ID NO:177) and Arg TCA An expression construct containing suppressor tRNA#001 (SEQ ID NO:11) and TCA Cells were transfected with either (ii) an expression construct containing a version of the EGFP reporter lacking the PTC ("WT-EGFP"). Approximately 48 hours after transfection, cells were lysed and subjected to ribosome footprint profiling. Adapters were excised from the raw reads using Trimmomatic, followed by non-coding RNA removal by alignment to the Ensembl mouse mm10 ncRNA reference using bowtie2. The remaining reads were aligned to the UCSC mm10 mouse reference assembly, again using bowtie2. Multiple-mapped reads were discarded. The final set of aligned reads was quantified using the RiboProfiling package in R and custom Python scripts. Python was used to generate plots examining 3' UTR occupancy and fold change in each gene with 20 or more uniquely mapped reads, and their distribution in genes with each native stop codon was compared using a two-sample Kolmogorov-Smirnov test. DETAILED DESCRIPTION OF THE INVENTION

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

[0031] Thus, in one aspect, the present invention provides a tRNA (eg, an isolated tRNA) comprising a nucleotide sequence set forth in Table 2.

[0032] In another aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding a tRNA, for example, as shown in Tables 1 to 3. In certain embodiments, the expression vector comprises one, two, three, four, or more than four 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 a nucleotide sequence shown in Table 4.

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

[0034] In another aspect, the present invention provides a method for expressing in mammalian cells a functional gene product encoded by a gene containing a premature stop codon, the method comprising the step of introducing into a cell an effective amount of a tRNA (e.g., including SEQ ID NOs: 6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 shown in Tables 1-3 below) or an expression vector comprising a nucleotide sequence encoding the tRNA, thereby allowing an amino acid to be incorporated into the gene product at a position where a truncated gene product would otherwise result due to the premature stop codon.

[0035] In certain embodiments of any of the above methods, the cells contain fewer truncated gene products than cells without tRNA. For example, in certain embodiments, the cells contain less than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the truncated gene products compared to cells without tRNA. In certain embodiments, the cells contain about 5% to about 80%, about 5% to about 60%, about 5% to about 40%, about 5% to about 20%, about 5% to about 10%, about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60%, or about 60% to about 80% of the truncated gene product compared to cells without 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 cells, tissues, or subjects 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 cells, tissues, or subjects that do 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 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 250%, about 20% to about 300%, about 20% to about 350%, about 20% to about 400%, about 20% to about 450%, about 20% to about 500%, about 20% to about 550%, about 20% to about 600%, about 20% to about 650%, about 20% to about 650%, about 20% to about 700%, about 20% to about 750%, about 20% to about 850%, about 20% to about 900%, about 20% to about 950%, about 20% to about 950%, about 20% to about 1000%, about 20% to about 1500%, about 20% to about 1600%, about 20% to about 1700%, about 20% to about 1800%, about 20% to about 1900%, about 20% to about 1900%, about 20% to about 25 ... 0% to approximately 60%, approximately 20% to approximately 40%, approximately 40% to approximately 200%, approximately 40% to approximately 180%, approximately 40% to approximately 160%, approximately 40% to approximately 140%, approximately 40% to approximately 120%, approximately 40% to approximately 100%, approximately 40% to approximately 80%, approximately 40% to approximately 60%, approximately 60% to approximately 200%, approximately 60% to approximately 180%, approximately 60% to approximately 160%, approximately 60% to approximately 14 0%, approx. 60% to approx. 120%, approx. 60% to approx. 100%, approx. 60% to approx. 80%, approx. 80% to approx. 200%, approx. 80% to approx. 180%, approx. 80% to approx. 160%, approx. 80% to approx. 140%, approx. 80% to approx. 120%, approx. 80% to approx. 100%, approx. 100% to approx. 200%, approx. 100% to approx. 180%, approx. 100% to approx. 160%, approx. 100% to approx. The amount or expression of a 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 allows an amino acid to be incorporated at a position in a gene product corresponding to a premature stop codon (i.e., the tRNA allows readthrough of the premature stop codon), but the tRNA does not allow a substantial amount of an amino acid to be incorporated at a position in a gene product corresponding to a native stop codon (i.e., the tRNA does not allow readthrough of the native stop codon). For example, in certain embodiments, the disclosed tRNA does not increase readthrough of the native stop codon (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 contacted with the tRNA. Readthrough of the native stop codon can be measured by any method known in the art, for example, by 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 in a cell a functional SCN1A gene product encoded by an SCN1A gene containing a premature stop codon, the method comprising the step of introducing into a cell an effective amount of a tRNA (e.g., including SEQ ID NOs: 6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 shown in Tables 1-3 below) or an expression vector containing a nucleotide sequence encoding the tRNA, thereby allowing an amino acid to be incorporated into the SCN1A gene product at a position where a truncated SCN1A gene product would otherwise result from the premature stop codon.

[0040] In another aspect, the present invention provides a method for increasing the activity of a voltage-gated sodium channel encoded by an SCN1A gene containing a premature stop codon in a cell, the method comprising the step of introducing into a cell an effective amount of a tRNA (e.g., including SEQ ID NOs: 6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187 shown in Tables 1-3 below) or an expression vector containing a nucleotide sequence encoding the tRNA, thereby allowing an amino acid to be incorporated into the SCN1A gene product at a position where a truncated SCN1A gene product would otherwise result from the premature stop codon.

[0041] In another aspect, the present invention provides a method of treating a premature stop codon-mediated disorder in a subject in need thereof, wherein the subject has a gene with a premature stop codon, the method comprising administering to the subject an effective amount of a tRNA (e.g., comprising SEQ ID NOs: 6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187, as shown in Tables 1-3 below) or an expression vector comprising a nucleotide sequence encoding the tRNA, thereby treating the disorder in the subject. In certain embodiments, the disorder is selected from a disorder 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, wherein the subject has an SCN1A gene with a premature stop codon, the method comprising the step of administering to the subject an effective amount of a tRNA (e.g., comprising SEQ ID NOs: 6-9, 11, 16-22, 35-40, 44, 45, 178-182, 186, or 187, as shown in Tables 1-3 below) or an expression vector comprising 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. tRNAs are typically approximately 70–100 nucleotides long, and active tRNAs contain a 3' CCA sequence, which can be transcribed into tRNA during synthesis or added later during 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 to form an aminoacyl-tRNA (aa-tRNA). While the amino acid can spontaneously migrate from the 2'-hydroxyl group to the 3'-hydroxyl group or vice versa, 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 three-base sequence known as the anticodon. When this anticodon sequence hybridizes or base pairs with a complementary three-base codon sequence in the ribosome-bound messenger RNA (mRNA), the aa-tRNA binds to the ribosome and incorporates that amino acid into the polypeptide chain being synthesized by that ribosome. Translation of the genetic code is accomplished by tRNAs, as every tRNA that base pairs with a particular codon is aminoacylated with a single, specific amino acid. Each of the 61 non-terminating codons in the mRNA directs 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. Therefore, tRNAs derived from bacterial tRNAs, non-mammalian eukaryotic tRNAs, or mammalian (e.g., human) tRNAs can be useful in practicing the present invention. Nucleotide sequences encoding naturally occurring human tRNAs are known and generally available to those skilled 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 the site of a mutation, e.g., a PTC, within a protein-coding gene. The use of the term suppressor is based on the fact that, under certain circumstances, the modified tRNA "suppresses" the phenotypic effect of the coding mutation. Suppressor tRNAs typically contain a mutation (modification) either in the anticodon to alter codon specificity or at some position that alters the aminoacylation properties of the tRNA.

[0046] In certain embodiments, a tRNA (e.g., a suppressor tRNA) contains a modified anticodon region, where the modified anticodon hybridizes to a codon that differs 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 into the gene product at a position where the premature stop codon would otherwise result in a truncated gene product.

[0047] In certain embodiments, the tRNA comprises 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 comprises 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, tRNA can be aminoacylated or can be aminoacylated with any natural amino acid.For example, 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, tRNA can be aminoacylated with serine, leucine, glutamine, or arginine.In certain embodiments, tRNA can be aminoacylated with glutamine or arginine.In certain embodiments, tRNA can be aminoacylated with arginine.

[0049] In certain embodiments, the tRNA (i) comprises an anticodon that hybridizes to a codon set forth in Table 1, and (ii) is aminoacylated or capable of being aminoacylated with an amino acid set forth in Table 1.

[0050] [Table 1] TIFF2025148444000005.tif122137

[0051] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence set forth 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 a nucleotide sequence set forth 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 in which a 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 also comprises, consists essentially of, or consists of the same nucleotide sequence that includes uracil (U) in place of one or more thymines (T), or uracil (U) in place of all thymines (T). Similarly, it is understood that in each instance in which a tRNA comprises, consists essentially of, or consists of a nucleotide sequence that includes one or more uracils (U), it is also contemplated that the tRNA also comprises, consists essentially of, or consists of a nucleotide sequence that includes thymine (T) in place of one or more uracils (U), or thymine (T) in place of all uracils (U).

[0052] [Table 2] TIFF2025148444000007.tif234165TIFF2025148444000008.tif227151TIFF2025148444000009.t if227151TIFF2025148444000010.tif227151TIFF2025148444000011.tif227151TIFF20251484440 00012.tif227151TIFF2025148444000013.tif227151TIFF2025148444000014.tif227151TIFF202 5148444000015.tif227151TIFF2025148444000016.tif227151TIFF2025148444000017.tif107151

[0053] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence set forth 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 a nucleotide sequence set forth 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] TIFF2025148444000019.tif227152TIFF2025148444000020.tif227152TIFF2025148444000021.tif227152TIFF2025148444000022.tif227152TIFF2025148444000023.tif227152TIFF2025148444000024.tif227152TIFF2025148444000025.tif227152TIFF2025148444000026.tif227152TIFF2025148444000027.tif227152TIFF2025148444000028.tif227152TIFF2025148444000029.tif227152TIFF2025148444000030.tif227152TIFF2025148444000031.tif227152TIFF2025148444000032.tif227152TIFF2025148444000033.tif227152TIFF2025148444000034.tif227152TIFF2025148444000035.tif227152TIFF2025148444000036.tif227152TIFF2025148444000037.tif227152TIFF2025148444000038.tif220152TIFF2025148444000039.tif227152TIFF2025148444000040.tif220152TIFF2025148444000041.tif227152TIFF2025148444000042.tif220152TIFF2025148444000043.tif227152TIFF2025148444000044.tif227152TIFF2025148444000045.tif227152TIFF2025148444000046.tif227152TIFF2025148444000047.tif227152TIFF2025148444000048.tif227152TIFF2025148444000049.tif227152TIFF2025148444000050.tif227152TIFF2025148444000051.tif227152TIFF2025148444000052.tif227152TIFF2025148444000053.tif221152TIFF2025148444000054.tif161152.

[0055] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence set forth 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 of 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, a tRNA can contain one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) compared to a reference tRNA sequence (e.g., a tRNA disclosed herein). In certain embodiments, a tRNA can comprise, consist of, or consist essentially of a single mutation or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 mutations. It is contemplated that a tRNA can comprise, consist of, or consist essentially of 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 within the skill of 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 algorithm 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 searches. For a discussion of basic issues in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6:119-129. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the entire length of the sequences being compared. Search parameters for histogram, description, alignment, expectation (i.e., the statistical significance threshold for reporting matches to database sequences), cutoff, matrix, and filter are at 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). Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generate word hits every wink positions along the query); and gapw=16 (sets the window width over which gapped alignments are generated).Equivalent Blastp parameter settings would be Q=9; R=2; wink=1; and gapw=32. Searches can also be performed using NCBI (National Center for Biotechnology Information) BLAST advanced options parameters (e.g., -G, gap widening cost [integer]: default = 5 for nucleotides / 11 for proteins; -E, gap extension cost [integer]: default = 2 for nucleotides / 1 for proteins; -q, nucleotide mismatch penalty [integer]: default = -3; -r, nucleotide match reward [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, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments can also be used (default parameters may include, for example, a Blosum62 matrix and gap opening penalty = 10 and gap extension penalty = 0.1). Bestfit comparisons between sequences, available in the GCG package version 10.0, use DNA parameters of GAP = 50 (gap creation penalty) and LEN = 3 (gap extension penalty); the equivalent settings for protein comparisons are GAP = 8 and LEN = 2.

[0059] It is assumed that tRNA can 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 adding specific ligand or antigenic, fluorescent, affinity, reactive, spectral, or other probe moiety; tRNA containing one or more ribose moieties that are methylated or otherwise modified; aa-tRNA that is aminoacylated with amino acids other than the 20 natural amino acids, including unnatural amino acids that function as reagent carriers, specific ligands, or antigenic, fluorescent, reactive, affinity, spectral, or other probes; or any combination of these compositions.Additionally, a single tRNA scaffold was developed by 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; According to 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 comprises a naturally occurring nucleotide modification. 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 Figure 2B. In certain embodiments, the tRNA contains 2'-O-methylguanosine or G at position 0; pseudouridine or U 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 at position 9. N2-methylguanosine or G at position 10; N4-acetylcytidine or C at position 12; pseudouridine, U, 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, U, or modified U at position 20a; D, pseudouridine, or U at position 20b; pseudouridine or U at position 25; pseudouridine, U, N2,N2-dimethylguanosine, N2-methylguanosine, G, or modified G at position 26; pseudouridine, U, N2,N2-dimethylguanosine, or G at position 27; pseudouridine or U at position 28; pseudouridine or U at position 30; pseudouridine or U at position 31; 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, U, 2'-O-methylcytidine, 3-methylcytidine, C, or modified C at position 32;Inosine at position 34, 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, keuosine, mannosyl-keuosine, galactosyl-keuosine pseudouridine or U at position 35; pseudouridine, U, or modified U at position 36; 1-methylinosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-threonylcarbamoyladenosine, A, modified A, 1-methylguanosine, peroxywybutosine, wybutosine, G, or modified G at position 37; pseudouridine, U, 5-methylcytidine, C, or modified C at position 38; 1-methylpseudouridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, U, 2'-O-methylguanosine, or G at position 39; pseudouridine, U, 5-methylcytidine, or C at position 40; 2'-O-methyluridine, U, or modified U at position 44; pseudouridine or U at position e11; pseudouridine or U at position e12; pseudouridine or U at position e14; 3-methylcytidine or C at position e2; 7-methylguanosine or G at position 46; D, U, or modified U at position 47; D, U, 5-methylcytidine, C, or modified C; A, modified A, 5-methylcytidine, C, or modified C at position 49; pseudouridine, U, 5-methylcytidine, or C at position 50; 5,2'-O-dimethyluridine, 5-methyluridine, pseudouridine, or U at position 54; pseudouridine or U at position 55; 1-methyladenosine, A, or modified A at position 58; 2'-O-ribosyladenosine (phosphate), A, 2'-O-ribosylguanosine (phosphate), G, or modified G at position 64; pseudouridine or U at position 65;The tRNAs contain 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. 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 comprises 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. Methods for Producing tRNA It is contemplated that tRNA molecules (e.g., suppressor tRNAs) useful in the practice of the present invention 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 a tRNA can be synthesized chemically or by recombinant DNA methods. For example, a tRNA sequence can be synthesized or cloned from a library by conventional hybridization or polymerase chain reaction (PCR) techniques using appropriate synthetic nucleic acid primers. The resulting tRNA-encoding DNA molecule can be ligated with other appropriate nucleotide sequences, including, for example, expression control sequences, to produce a conventional gene expression construct (i.e., an expression vector) encoding the tRNA. The production of a defined gene construct is within the skill of the art. A nucleic acid encoding a desired tRNA can be incorporated (ligated) into an expression vector, such as the expression vectors described in the following section, and the expression vector can be introduced into a host cell via 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, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and bone marrow cells. Transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the 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 before introduction into a cell 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 Vectors The tRNA of interest can be expressed in a cell of interest by incorporating the gene encoding the tRNA of interest into a suitable expression vector.As used herein, "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence to be expressed.An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be provided by host cells or in vitro expression systems.Expression vectors include all those known in the art that incorporate the recombinant polynucleotide of interest, such as cosmids, (e.g., naked or contained in liposomes) plasmids, retrotransposons (e.g., piggyback, sleeping beauty), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0066] In certain embodiments, the expression vector is a viral vector. The term "virus" is used herein to refer to an obligate intracellular parasite that does not have a protein synthesis or energy generation mechanism. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, Epstein-Barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolar virus 40 (SV40) vectors), poxvirus vectors, and pseudovirus vectors.

[0067] Viruses can be RNA viruses (having a genome composed of RNA) or DNA viruses (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA viral vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfar viruses, herpes viruses (e.g., herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papilloma viruses (e.g., HPV), polyoma viruses (e.g., simian vacuolar virus 40 (SV40)), and pox viruses (e.g., vaccinia viruses, cowpox viruses, smallpox viruses, fowlpox viruses, sheeppox viruses, myxoma viruses). In certain embodiments, the viral vector is an RNA viral vector. Exemplary RNA viruses include bunyaviruses (e.g., hantaviruses), coronaviruses, flaviviruses (e.g., yellow fever virus, West Nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)), and toroviruses.

[0068] In certain embodiments, the expression vector comprises 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. Functionally linked nucleotide sequences are typically contiguous. However, because enhancers usually function even when located several kilobases away from the promoter, and intron sequences can vary in length, some polynucleotide elements may be operably linked but not directly adjacent, and may function in trans from different alleles or even chromosomes.

[0069] The tRNA gene preferably has a strong promoter that is active in a variety of cell types. The promoter for eukaryotic tRNA genes is typically present within the structural sequence encoding the tRNA molecule itself. Elements that regulate transcriptional activity are present within the 5' upstream region, but the length of an 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, SV40 promoters, human cytomegalovirus (CMV) promoters, U6 promoters, or any other promoter (e.g., cellular promoters, 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, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of an appropriate promoter will be apparent to one skilled in the art from the teachings contained herein.

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

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

[0073] In certain embodiments, the expression vector comprises a nucleotide sequence set forth 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 a nucleotide sequence set forth in Table 4. In certain embodiments, within the expression vector, the nucleotide sequence set forth in Table 4 is operably linked to a nucleotide sequence encoding a tRNA. In certain embodiments, within the expression vector, the nucleotide sequence set forth in Table 4 is 5' or 3' (e.g., immediately 5' or immediately 3') to the nucleotide sequence encoding the 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 an expression vector described in Example 8 or Example 9 herein.

[0075] [Table 4] TIFF2025148444000056.tif226155TIFF2025148444000057.tif220155TIFF2025148444000058.tif54155

[0076] Adeno-associated virus (AAV) vectors In certain embodiments, the expression vector is an adeno-associated virus (AAV) vector.AAV is a small, non-enveloped, icosahedral virus belonging to the Dependoparvovirus genus and the Parvoviridae family.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 show different tissue tropism.

[0077] AAV includes numerous serologically distinguishable types, including serotypes AAV-1 to AAV-12 and over 100 serotypes derived from non-human primates (see, for example, 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, and 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; and 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 comprises a sequence disclosed in U.S. Patent No. 7,198,951, such as, but not limited to, 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 macaques, such as rh.8, rh.10, rh.39, rh.43, and rh.74, are also contemplated in the present invention. In addition to native 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), which contain signal sequences that guide 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 encoding the rep and cap genes. The two rep promoters, along with differential splicing of a single AAV intron, 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), which 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, part or all of the 4.3 kb internal genome can be replaced with foreign DNA, for example, an expression cassette for an exogenous gene of interest. Thus, in certain embodiments, the AAV vector comprises a genome containing an expression cassette for an exogenous gene flanked by the 5' ITR and 3' ITR. The ITRs can be derived from the same serotype as the capsid or can be derivatives thereof. Alternatively, the ITRs can be of a different serotype from the capsid, thereby generating a pseudotype AAV. In certain embodiments, the ITRs are derived from AAV-2. In certain embodiments, the ITRs are derived from AAV-5. At least one of the ITRs can be modified to mutate or remove a terminal resolution site, thereby generating a self-complementary AAV vector.

[0080] To produce an AAV vector, the rep and cap proteins can be provided in trans, for example, on a plasmid. A host cell line permissive for 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 adenovirus 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 producing 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). Many cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells (see, e.g., 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 Publication Nos. WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types using one plasmid containing an expression cassette flanked by ITRs and one or more additional plasmids providing additional AAV and helper virus genes.

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

[0082] AAV vectors can be 4.7 kb in size, or have single-stranded genomes larger or smaller than 4.7 kb, including oversized genomes as large as 5.2 kb or as small as 3.0 kb. Thus, if the exogenous gene of interest to be expressed from an AAV vector is small, the AAV genome can include a stuffer sequence. Furthermore, the vector genome can be substantially self-complementary, thereby enabling rapid expression in cells. In certain embodiments, the genome of a self-complementary AAV vector comprises, from 5' to 3', a 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 without a functional terminal resolution site; a second nucleic acid sequence complementary or substantially complementary to the first nucleic acid sequence; and a 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 #AAV502A-1), and pAAVK-EF1α-MCS1-CMV-MCS2 (System Bio catalog #AAV502A-1). 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), pAA These vectors include VS1-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 suit therapeutic use. For example, an exogenous gene of interest can be inserted into the multiple cloning site and the selectable marker (e.g., a gene encoding a puro or 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. Pat. 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, for example, the central nervous system, in a subject, for example, a human subject.Exemplary AAV vectors that can target the nervous system include AAV9 variants AAV-PHP.B (see, for example, Deverman et al. (2016) NAT. BIOTECHNOL. 34(2):204-209), AAV-AS (see, for example, Choudhury et al. (2016) MOL. THER. 24:726-35), and AAV-PHP.eB (see, for example, Chan et al. (2017) NAT. NEUROSCI. 20:1172-79).Another exemplary AAV-based strategy for targeting the nervous system is 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 vectors In certain embodiments, viral vectors can be retroviral vectors.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 for mediating retroviral-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 virus's structural genes are removed and replaced with a gene of interest. Often, 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 comprises, from 5' to 3': a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and / or enhancer, an exogenous gene of interest, and a 3' LTR. If no exogenous promoter is provided, gene expression is driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression. For lentivirus production, the structural genes can be provided on a separate vector, thereby rendering the resulting virions replication-deficient. 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. Tat can also be eliminated from the packaging system by using a retroviral vector containing a chimeric 5' LTR in which the U3 region of the 5' LTR has been replaced with a heterologous regulatory element.

[0088] Genes can be incorporated into the proviral backbone in a variety of common ways. The most straightforward construct is one in which the structural genes of the retrovirus are replaced by a single gene that is transcribed under the control of the viral regulatory sequences in the LTR. Retroviral vectors that can introduce two or more genes into target cells have also been constructed. Usually, 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] Thus, the new gene is flanked by 5' and 3' LTRs, respectively, which function to promote transcription and polyadenylation of virion RNA. The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, which in their native sequence are directly repeated and contain the U3, R, and U5 regions. LTRs usually provide essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain many regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region is adjacent to the U3 and U5 regions. In certain embodiments, the R region contains a transactivation response (TAR) gene element that interacts with a transactivator (tat) gene element to enhance viral replication. This element is not required in embodiments in which 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. Modifications to the 3' LTR are often made 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 refers to a replication-deficient retroviral vector in which the U3 region of the 3' LTR has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the U3 region of the 3' LTR is used as a template for the U3 region of the 5' LTR during viral replication, and therefore, viral transcripts cannot be generated without the U3 enhancer-promoter. In further embodiments, the 3' LTR is modified so that the U5 region is replaced, for example, with an ideal polyadenylation sequence. It should be noted that modifications to the LTRs, such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs, are contemplated as 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 the transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, the promoters of 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). Typical promoters can drive high-level transcription in a Tat-independent manner. This replacement reduces the possibility of recombination that generates replicative viruses, since the complete U3 sequence is no longer present in the virus 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 term "packaging signal" or "packaging sequence" refers to a sequence located within the retroviral genome that is required for encapsidation of the retroviral RNA strand during viral particle formation (see, for example, Clever et al., 1995 J. VIROLOGY, 69(4):2101-09). The packaging signal may be a minimal packaging signal (also referred to as the Psi [Ψ] sequence) required for encapsidation of the viral genome.

[0093] In certain embodiments, the retroviral vector (e.g., 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, the central initiation of the positive-strand DNA in the cPPT and the central termination in the CTS form a triple-stranded DNA structure: a central DNA flap. Without wishing to be bound by any theory, the DNA flap may act as a cis-acting determinant for the nuclear import of the lentiviral genome and / or increase viral titer. In certain embodiments, the retroviral vector backbone comprises one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in certain embodiments, the transfer plasmid comprises a FLAP element. In one embodiment, a 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 comprises an export element. In one embodiment, a retroviral vector comprises one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) 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, the RNA export element is located within the 3' UTR of a gene and can be inserted as one or multiple copies.

[0095] In certain embodiments, retroviral vectors (e.g., lentiviral vectors) further comprise posttranscriptional regulatory elements. Various posttranscriptional regulatory elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; see Zufferey et al., (1999) J. VIROL., 73:2886); the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (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. Posttranscriptional regulatory elements are usually located at the 3' end of the 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 posttranscriptional regulatory element sequence. In certain embodiments, the vectors of the present invention lack or do not contain post-transcriptional regulatory elements, such as a WPRE or HPRE, because in some instances these elements increase the risk of cell transformation and / or do not substantially or significantly increase the amount of mRNA transcripts or increase mRNA stability. Thus, in certain embodiments, the vectors of the present invention lack or do not contain a WPRE or HPRE as an additional safety measure.

[0096] The elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes.Transcription termination signals are usually found downstream of polyadenylation signals.Therefore, in certain embodiments, retroviral vectors (e.g., lentiviral vectors) further comprise polyadenylation signals.The term "polyadenylation signal" or "polyadenylation sequence" as used herein refers to a DNA sequence that directs both the termination and polyadenylation of newly formed RNA transcripts by RNA polymerase H.Efficient polyadenylation of recombinant transcripts is desirable because transcripts that lack polyadenylation signals are unstable and rapidly degraded. 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), the bovine growth hormone polyadenylation sequence (BGHpA), the 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 comprises an insulator element. The insulator element can contribute to protecting the retroviral expression sequence, e.g., a therapeutic gene, from integration site effects (i.e., position effects; see, e.g., Burgess-Beusse et al., (2002) PROC. NATL. ACAD. SCI., USA, 99:16433; and Zhan et al., 2001, HUM. GENET., 109:471), which can be mediated by cis-acting elements present in genomic DNA and can result in unregulated expression of the transferred sequence. In certain embodiments, the retroviral vector comprises an insulator element in one or both 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 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™ (Thermo Fisher), pLenti6.2 / V5-DEST™ (Thermo Fisher), 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 Purified These vectors include 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), and pInducer10-mir-RUP-PheS (Addgene plasmid #44011). These vectors can be modified to make them suitable for therapeutic use. For example, the selectable marker (e.g., puro, EGFP, or mCherry) can be removed or replaced with a second exogenous gene of interest.Further examples of lentiviral vectors are described 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, Nos. 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, and PCT Publication No. WO2017 / 091786.

[0099] Adenovirus vectors In certain embodiments, the viral vector may be an adenovirus 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 genus Adenoviridae, including, but not limited to, the human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. Typically, adenovirus vectors are generated by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome to allow the insertion of non-native nucleic acid sequences into the adenovirus, for example, for gene transfer.

[0100] Human adenovirus can be used as a source of adenoviral genome for adenoviral 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 serogroups (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 for producing non-group C adenovirus vectors, and methods for 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 Publication Nos. WO1997 / 012986 and WO1998 / 053087.

[0101] Non-human adenoviruses (e.g., ape, monkey, bird, dog, sheep, or bovine adenoviruses) can be used to generate adenovirus vectors (i.e., as the source of the adenovirus genome for adenovirus vectors). For example, adenovirus vectors can be based on simian adenoviruses, including those from both New World and Old World monkeys (see, for example, Virus Taxonomy: VHIth Report of the International Committee on Taxonomy of Viruses (2005)). Phylogenetic analysis of adenoviruses that infect primates is disclosed, for example, in Roy et al. (2009) PLOS PATHOG. 5(7):e1000503. Gorilla adenovirus can be used as the source of the adenovirus genome for adenovirus vectors. Gorilla adenovirus and adenoviral vectors are described, for example, in PCT Publication Nos. WO2013 / 052799, WO2013 / 052811, and WO2013 / 052832. Adenoviral vectors can also contain subunit combinations and thus can be "chimeric" adenoviral vectors.

[0102] Adenoviral vectors can be replication-competent, conditionally replication-competent, or replication-deficient. Replication-competent adenoviral vectors can replicate in typical host cells, i.e., cells that can typically be infected by adenovirus. Conditionally replicating adenoviral vectors are adenoviral vectors engineered to replicate under defined conditions. For example, replication-essential gene functions, such as those encoded by adenoviral early regions, can be operably linked to inducible, repressible, or tissue-specific transcription 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 adenoviral vectors that require the complementation of one or more gene functions or regions of the adenoviral genome required for replication, for example, as a result of the deficiency of one or more replication-essential gene functions or regions, which prevents the adenoviral vector from replicating in typical host cells, particularly human cells, that are infected by the adenoviral vector.

[0103] Preferably, the adenoviral vector is replication-deficient, and replication-deficient adenoviral vectors require complementation of at least one replication-essential gene function in one or more regions of the adenoviral genome for propagation (e.g., to form adenoviral vector particles). The adenoviral vector can be defective with respect to only the early region of the adenoviral genome (i.e., E1-E4 regions), only the late region of the adenoviral genome (i.e., L1-L5 regions), one or more replication-essential gene functions in both the early and late regions of the adenoviral genome, or all adenoviral genes (i.e., high-capacity adeno vectors (HC-Ad)). See, e.g., 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 adenoviral vectors are disclosed in U.S. Pat. Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, 6,482,616, and 7,195,896, and PCT Publication Nos. 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 the gene functions that are not present in the replication-deficient adenoviral vector but are required for viral propagation at an appropriate level to produce high-titer viral vector stocks.Such complementing cell lines are known and include but are not limited to 293 cells (e.g., as described in Graham et al. (1977) J. GEN. VIROL. 36: 59-72), PER.C6 cells (e.g., as described in PCT Publication No. WO1997 / 000326 and U.S. Patent No. 5,994,128 and U.S. Patent No. 6,033,908), and 293-ORF6 cells (e.g., as described 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 (see, for example, U.S. Patent Publication No. 2008 / 0233650) that are designed to propagate adenoviral vectors encoding transgenes whose expression inhibits viral growth in host cells. Additional suitable complementing cells are described, for example, in U.S. Patent 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. Patent Nos. 6,225,289 and 6,514,943 and PCT Publication No. WO2000 / 034444.

[0105] Additional exemplary adenoviral vectors and / or methods of generating or propagating adenoviral vectors are described in U.S. Patent 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 adenoviral vector systems include the ViraPower™ Adenoviral Expression System available from Thermo Fisher Scientific, the AdEasy™ Adenoviral Vector System available from Agilent Technologies, and the Adeno-X™ Expression System 3 available from Takara Bio USA, Inc.

[0107] Viral vector production 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 appropriate conditions that allow the production of infectious viral particles. Nucleic acids encoding viral genes and / or tRNAs can be incorporated into plasmids and introduced into host cells via conventional transfection or transformation techniques. Exemplary host cells suitable for producing 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 may 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 may include plaque purification, centrifugation, e.g., cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography, or filtration steps.

[0109] IV. Pharmaceutical Compositions For therapeutic use, the tRNA and / or expression vector is preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals, within the scope of sound medical judgment, at a reasonable benefit / risk ratio, and without undue toxicity, irritation, allergic response, or other problem or complication.

[0110] The term "pharmaceutically acceptable carrier," as used herein, refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues, at a reasonable benefit / risk ratio, without undue toxicity, irritation, allergic response, or other problems or complications. Pharmaceutically acceptable carriers include any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain 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 and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0111] In certain embodiments, pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, 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 amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; 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); colors, flavors, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions ( for example, 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., pluronic, PEG, sorbitan esters, polysorbates such as 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 may contain nanoparticles, e.g., 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%) nanoparticles or aminolipid delivery compounds, e.g., as described in U.S. Patent Publication No. 2017 / 0354672. In certain embodiments, the tRNA or expression vector introduced into the cell or administered to the subject is not conjugated or associated with another moiety, e.g., a carrier particle, e.g., an aminolipid particle. As used herein, the term "conjugate," when used in reference to two or more moieties, means that the moieties are physically bound or connected to one another, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. Typically, the moieties are attached either by one or more covalent bonds or by mechanisms involving specific binding. Alternatively, a sufficient number of weak interactions may provide sufficient stability for the moieties to remain physically associated.

[0113] In certain embodiments, pharmaceutical compositions may contain sustained- or controlled-delivery formulations. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bioerodible 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 may be prepared by any of a variety of methods known in the art.

[0114] Pharmaceutical compositions containing the tRNA and / or expression vector disclosed herein may be provided in dosage unit form and may be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include 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 may be prepared by methods known in the pharmaceutical arts. See, e.g., Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and isotonicity adjusting 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 preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., 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). tRNA can be modified to prevent rapid degradation of tRNA by endo- and exonucleases in vivo, or can be delivered using a drug delivery system. tRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. tRNA molecules can also be conjugated or otherwise linked to aptamers. 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 promote binding of (negatively charged) tRNA molecules and enhance interaction with negatively charged cell membranes to enable efficient uptake of tRNA by cells. Cationic lipids, dendrimers, or polymers can be either bound to RNA, such as tRNA, or induced to form vesicles or micelles that surround RNA (see, for example, Kim et al. (2008) JOURNAL OF CONTROLLED RELEASE 129(2):107-116). Methods for producing and administering cationic RNA complexes are well within the capabilities of those skilled 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, e.g., 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 polyamidoamines (Tomalia et al. (2007) BIOCHEM. SOC. TRANS. 35:61-67; Yoo et al. (1999) PHARM. RES. 16:1799-1804). In certain embodiments, tRNA is complexed with cyclodextrin for systemic administration. Methods and pharmaceutical compositions for administering RNA and cyclodextrin can be found in U.S. Patent No. 7,427,605.

[0117] Pharmaceutical preparations are preferably sterile.Sterilization can be achieved by any suitable method, for example, by filtration through a sterile filtration membrane.When the composition is lyophilized, sterilization by filtration can be carried out before or after lyophilization and reconstitution.

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

[0119] Generally, a therapeutically effective amount of an active ingredient, e.g., a tRNA and / or expression vector, ranges from 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg. In certain embodiments, a therapeutically effective amount of a viral expression vector ranges from 10 2 ~10 15 Plaque-forming units (pfu), e.g., 10 2 ~10 10 , 10 2 ~10 5 , 10 5 ~10 15 , 10 5 ~10 10 , or 10 10 ~10 15 The range of plaque-forming units (PFU) is used. The amount administered may depend on variables such as the type and severity of the disease or condition being treated, the patient's overall health, 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 to rapidly achieve the desired blood or tissue level. Alternatively, the initial dose may be lower than the 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 conducted at 0.5 mg / kg to 20 mg / kg. The dosing frequency may vary depending on factors such as the route of administration, dosage, serum half-life, and the disease being treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks. A preferred route of administration is parenteral, e.g., 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, tRNA or expression vector is not conjugated or combined with another part, for example, carrier particle, for example, aminolipid particle.In certain embodiments, tRNA or expression vector is introduced into cell or administered to subject in a dosage form that does not contain nanoparticles.In certain embodiments, tRNA or expression vector is introduced into cell or administered to subject in a dosage form that does not contain aminolipid delivery compound, for example, as described in US 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 subjects.As used herein, the term "PTC-mediated disorders" refers to disorders that are mediated by, enhanced by, worsened by, or otherwise accelerated by, or associated with, intragenic PTC.

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

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

[0124] [Table 5] TIFF2025148444000060.tif188128

[0125] In certain embodiments, the premature stop codon-mediated disorder is a disorder listed in Table 6 below, and / or the gene having a premature stop 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 impairment in the subject.For example, in certain embodiments, the method reduces the seizure frequency in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, for example, for a period of 1 day, 1 week, or 1 month.In certain embodiments, the method reduces the seizure severity by 50%, for example, for a period of 1 day, 1 week, or 1 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 codons within the SCN1A gene are 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.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.127 8C>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, or a combination of mutations selected from the group consisting of: 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 c.58G>T, c.575G>A, c.664C>T, c.962C>G, c.1095dupT, c.1129C>T, c.1315C>T, c.1348C>T, c.13 66G>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.26 It is caused by a mutation or combination of mutations selected from 35delC, 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 in the SCN1A gene are 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.In certain embodiments, the premature stop codon in the SCN1A gene is caused by a mutation selected from c.4573C>T, c.5656C>T, and c.5734C>T. In certain embodiments, the premature stop codon in 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 in the SCN1A gene is caused by a mutation shown in Table 7, or a combination of mutations shown in Table 7.

[0130] [Table 7] TIFF2025148444000063.tif22678TIFF2025148444000064.tif131128

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

[0132] In another aspect, the present invention provides a method of treating Dravet syndrome in a subject in need thereof, wherein the subject has 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 the suppressor class shown in the same row in Table 7 as the mutation, or an expression vector comprising a nucleotide sequence encoding the tRNA. As used in Table 7, the term "suppressor class" (e.g., Arg>TGA) refers to 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, a subject has an SCN1A gene with 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 an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs:6-9, 11, 16-18, 19-22, and 35. In certain embodiments, a subject has an SCN1A gene with 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 an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs:36-40, 44, and 45. 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 an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs:178-182, 186, and 187.

[0134] In certain embodiments, the gene is the SCN1A gene, and the SCN1A gene product produced by the tRNA is a functional SCN1A gene product. In certain embodiments, the functional SCN1A gene product has greater activity, e.g., greater voltage-gated sodium channel activity, than a truncated SCN1A gene product. In certain embodiments, the method increases 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 that does not have the tRNA. In certain embodiments, the method includes increasing 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 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 240%, about 20% to about 250%, about 20% to about 300%, about 20% to about 320%, about 20% to about 350%, about 20% to about 360%, about 20% to about 370%, about 20% to about 380%, about 20% to about 400%, about 20% to about 450%, about 20% to about 460%, about 20% to about 470%, about 20% to about 480%, about 20% to about 500%, about 20% to about 510%, about 20% to about 520%, about 20% to about 530%, about 20% to about 540%, about 20% to about 550%, about 20% to about 560%, about 20% to about 570%, about 20% to about 580%, about 20% to about 590%, about 20% to about 600%, about 20% to about 610%, about 20% to about 620%, about 20% to about 630%, about 20% to about 640%, about 20% to about 650%, about 20% to about Approximately 80%, approximately 20% to approximately 60%, approximately 20% to approximately 40%, approximately 40% to approximately 200%, approximately 40% to approximately 180%, approximately 40% to approximately 160%, approximately 40% to approximately 140%, approximately 40% to approximately 120%, approximately 40% to approximately 100%, approximately 40% to approximately 80%, approximately 40% to approximately 60%, approximately 60% to approximately 200%, approximately 60% to approximately 180%, approximately 60% to approximately 160%, approximately 60 % to approximately 140%, approximately 60% to approximately 120%, approximately 60% to approximately 100%, approximately 60% to approximately 80%, approximately 80% to approximately 200%, approximately 80% to approximately 180%, approximately 80% to approximately 160%, approximately 80% to approximately 140%, approximately 80% to approximately 120%, approximately 80% to approximately 100%, approximately 100% to approximately 200%, approximately 100% to approximately 180%, approximately 100% to approximately 160%, approximately 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%.Voltage-gated sodium channel activity can be measured by any method known in the art, for example, as 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 Na v 1.1 Proteins. In certain embodiments, the functional SCN1A gene product comprises, consists essentially of, or consists of the amino acid sequence 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: TIFF2025148444000065.tif95149TIFF2025148444000066.tif223149TIFF2025148444000067.tif223149 TIFF2025148444000068.tif229149TIFF2025148444000069.tif223149TIFF2025148444000070.tif177149

[0136] The term "effective amount," as used herein, refers to an amount of an active agent (e.g., a tRNA or expression vector according to the invention or a second active agent in a combination therapy) sufficient to exert a beneficial or desired effect. An 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" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., halting its progression; and (b) palliating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to organisms 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.), and more preferably include 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 is suffering from a disease, so that the effects of the treatments on the patient overlap at some point. In certain embodiments, the delivery of one treatment is occurring even when the delivery of a second treatment begins, so 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 they are administered in combination. 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., less of the second treatment is used to achieve the same effect, or the second treatment reduces symptoms to a greater extent, or the same situation occurs with the first treatment. In certain embodiments, delivery is such that the reduction of symptoms or other parameters related to disorder is greater than that observed when one treatment is delivered without the other.The effect of two treatments can be partially additive, totally additive, or more than additive.Delivery can be such that the effect of the first treatment 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), a 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), a ketogenic diet, ONFI® (clobazam), TOPAMAX® (topiramate), fenfluramine, or valproic acid.

[0140] Throughout the detailed description where compositions are described as having, including, or comprising particular components, or where processes and methods are described as having, including, or comprising particular steps, it is further assumed that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are 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 stated to be included in and / or selected from a list of elements or components mentioned, it is to be understood that the element or component can be any one of the elements or components mentioned, or that the element or component can be selected from a group consisting of two or more of the elements or components mentioned.

[0142] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether expressly or impliedly herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, where reference is made to a particular compound, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, although embodiments are described and illustrated herein to allow for clear and concise examples to be described and illustrated, it is intended and will be understood that the embodiments can be combined or separated in various ways without departing from the present teachings and the invention. For example, it will be understood that all features described and illustrated herein may be applicable to all aspects of the invention described and illustrated herein.

[0143] The phrase "at least one of" should be understood to include each individual referenced object following the phrase and various combinations of two or more of the referenced objects, unless otherwise understood from context and usage. The phrase "and / or" in the context of three or more referenced objects should be understood to have the same meaning, unless otherwise understood from context.

[0144] The use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," and "containing," including grammatical equivalents thereof, should be understood to be generally open-ended and open-ended, e.g., not excluding additional, unstated elements or steps, unless specifically stated otherwise or understood from the context.

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

[0146] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0147] The use of any and all examples or exemplary language herein, such as "for example" or "including," is intended merely to more fully describe the invention and does not impose limitations on the scope of the invention unless otherwise recited in the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [Example]

[0148] The following examples are illustrative only 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 a premature termination codon (PTC).

[0150] Suppressor tRNAs were generated from endogenous mouse arginine-tRNAs by converting their normal anticodons to TCA anticodons that recognize the TGA stop codon (Arg TCA Five of the endogenous arginine-tRNAs contained introns that had to be removed by splicing to generate the mature tRNA; the corresponding Arg tRNAs with and without these intron sequences wereTCA Suppressor tRNAs were constructed and the suppressor tRNA sequences are shown in Table 8.

[0151] [Table 8] TIFF2025148444000072.tif220154TIFF2025148444000073.tif226154TIFF2025148444000074.tif67154

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

[0153] This Arg TCAThe suppressors were tested for PTC readthrough activity by flow cytometry in cell lines containing dual fluorescent readthrough reporters. These reporters contain three copies of red fluorescent protein (tdTomato), TEV protease, a linker region containing a PTC, and three copies of 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 (thus, only red fluorescence would be detected). PTC readthrough activity as a result of the suppressor tRNA would allow translation to proceed through the PTC within the linker region, resulting in the expression of both tdTomato and EGFP (thus, both red and green fluorescence would be detected). Therefore, readthrough can be assessed by quantifying the proportion of viable cells expressing both red and green fluorescent reporters above background (% double positives).

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

[0155] The linker region derived from the SCN1A transcript of subject N is as follows, and the reporter containing this linker region is referred to as subject N-PTC reporter. The corresponding linker with the wild-type Arg codon instead of TIFF2025148444000075.tif4147PTC was used as a control and had the following sequence: TIFF2025148444000076.tif4150

[0156] The linker region derived from the SCN1A transcript of the subject S is as follows, and the reporter containing this linker region is referred to as the subject S-PTC reporter. The corresponding linker with the wild-type Arg codon instead of TIFF2025148444000077.tif4147PTC was used as a control and had the following sequence: TIFF2025148444000078.tif4150

[0157] An additional 51-base pair linker region was obtained from a mouse model of Dravet syndrome, which is 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 the reporter containing this linker region is referred to as the R1407X-PTC reporter. The corresponding linker with the wild-type Arg codon instead of TIFF2025148444000079.tif4147PTC was used as a control and had the following sequence: TIFF2025148444000080.tif4150

[0158] Arg TCA Suppressors are designed to (i) stably express the S-PTC reporter of interest and contain Arg TCA (ii) a human Flp-In-293 cell line transiently transfected with a plasmid encoding the suppressor (results shown in Fig. 4 ), (iii) a cell line stably expressing the R1407X-PTC reporter and expressing Arg TCA (iii) a murine Flp-In-3T3 cell line transiently transfected with a plasmid encoding the suppressor (results shown in Figure 5 ), and (iv) a plasmid encoding the N-PTC reporter of interest and Arg TCA Multiple assay configurations were tested, including Flp-In-293 cells transiently cotransfected with a plasmid encoding a suppressor tRNA (results shown in Figure 6 ). Transfections were performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol.

[0159] An additional reporter construct was generated containing EGFP with a nuclear localization signal (NLS) and an arginine-to-TGA mutation (R96X) within the EGFP open reading frame that abolishes fluorescence in the absence of PTC readthrough. An outline of the experimental approach is shown in Figure 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: TIFF2025148444000081.tif196149

[0160] Arg TCA HEK293 cells and mouse Neuro-2a cells (a neural crest-derived cell line widely used to study neuronal differentiation) transiently co-transfected with a plasmid encoding a suppressor tRNA and a plasmid encoding a CAG:NLS-EGFP (R96X-TGA) reporter were used to express Arg. TCA The activity of the suppressors was evaluated. Transfections were performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. Cotransfections were performed using equal amounts of the indicated suppressor plasmid and the 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 Figure 7.

[0161] Generally, Arg TCAThe relative readthrough activity of suppressor tRNAs remained consistent across multiple assay formats. The following suppressors reliably demonstrated readthrough 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] Taken together, these results indicate that the described suppressor tRNAs are able to promote the expression of transcripts containing premature stop codons associated with disorders such as Dravet syndrome, such as the SCN1A transcript.

[0163] Example 2 This example describes the effect of expression vector characteristics on readthrough of premature termination codons (PTCs) by arginine aminoacylated suppressor tRNAs.

[0164] Arg TCA Expression constructs containing both the suppressor tRNA and the EGFP (R96X-TGA) reporter on the same plasmid were generated in a pGL4 vector backbone. These constructs contained one, two, three, or four copies of the Arg (described in Example 1 and shown in Table 8) TCASuppressor tRNAs 113 (SEQ ID NO: 16), 115 (SEQ ID NO: 18), and 001 (SEQ ID NO: 11) were included. Each copy of the tRNA sequence was expressed in the context of either (i) 200 bp upstream genomic flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 26) and 200 bp downstream genomic flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 27); (ii) 200 bp upstream genomic flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 26) and 104 bp downstream genomic flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 32); or (iii) an upstream U6 promoter containing 19 bp upstream flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 33) and 46 bp downstream flanking sequence from tRNA-Arg-TCG-1-1 (SEQ ID NO: 34). An outline of the experimental approach is shown in Figure 8A, and an outline of an exemplary reporter construct containing four copies of the suppressor tRNA is shown in Figure 8B. The reporter constructs were 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, the sequence of which is as follows: TIFF2025148444000082.tif208149

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

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

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

[0168] C57BL / 6J mice have a spontaneous C-to-T mutation in 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). TCA Suppressor 120 contains a T at position 51. Arg TCA A modified suppressor tRNA containing a T to C substitution at position 51 of suppressor 120 was generated (Arg TCA Suppressor 179, which has the nucleotide sequence of SEQ ID NO: 35). TCA Suppressors 120 and 179 were tested by transfection into the Flp-In-293 cell line, which stably expresses the S-PTC reporter of interest, and by cotransfection into Flp-In-3T3 cells with a plasmid encoding the N-PTC reporter of interest. Transfections were performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. The results are shown in Figures 4 and 6. Arg TCASuppressor 120 is nonfunctional, whereas Arg containing only a single substitution TCA Suppressor 179 exhibited PTC readthrough activity.

[0169] Example 4 This example describes a glutamine aminoacylated suppressor tRNA that promotes readthrough of a premature termination codon (PTC).

[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 (The suppressor tRNA sequences are referred to as suppressor tRNAs.) The suppressor tRNA sequences are shown in Table 9.

[0171] [Table 9] TIFF2025148444000084.tif213154TIFF2025148444000085.tif213154TIFF2025148444000086.tif80154

[0172] In this example, all mature tRNA sequences were expressed in the context of upstream and downstream genomic flanking sequences (±200 bp) from the highly expressed glutamine-tRNA, tRNA-Gln-TTG-1-1, i.e., tRNA sequences were expressed with 5' flanking sequences of SEQ ID NO: 173 and 3' flanking sequences of SEQ ID NO: 174. All mature tRNA sequences, including 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 independently derived Flp-In-293 cell lines containing an integrated fluorescent readthrough reporter. The results are shown in Figure 18. The reporter contained three copies of red fluorescent protein (tdTomato), TEV protease, a linker region containing a PTC, and three copies of green fluorescent protein (EGFP). A schematic 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 (and thus only red fluorescence would be detected). PTC readthrough activity as a result of the suppressor tRNA would allow translation to proceed through the PTC within the linker region, resulting in the expression of both tdTomato and EGFP (and thus both red and green fluorescence would be detected). Therefore, readthrough can be assessed using flow cytometry by quantifying the proportion of viable cells expressing both red and green fluorescent reporters above background (% double positive). The linker is mouse Dmd mdx It was obtained from the transcript and had the following sequence: The corresponding linker with the wild-type Gln codon instead of TIFF2025148444000087.tif4149PTC was used as a control and had the following sequence: TIFF2025148444000088.tif4150

[0174] An additional reporter construct containing EGFP with a nuclear localization signal (NLS) and a glutamine-to-TAA mutation (Q69X) that abolishes fluorescence in the absence of PTC readthrough was generated in the pGL4 vector backbone. EGFP expression was driven by the CMV early enhancer / chicken β-actin (CAG) promoter. This reporter construct is designated CAG:NLS-EGFP (Q69X-TAA) and its sequence is as follows: TIFF2025148444000089.tif195149

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

[0176] An additional reporter construct containing EGFP with a nuclear localization signal (NLS) and a glutamine-to-TAG mutation (Q69X) that abolishes fluorescence in the absence of PTC readthrough was generated in the pGL4 vector backbone. 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: TIFF2025148444000090.tif194149

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

[0178] Taken together, these results indicate that the described suppressor tRNAs are able to promote the expression of transcripts containing premature stop codons associated with the disorder.

[0179] Example 5 This example describes the read-through activity of the disclosed suppressor tRNAs and small molecule nonsense suppression therapy.

[0180] The disclosed suppressor tRNAs were tested together with the nonsense suppressors translaruna (ataluren), gentamicin, and G418 (geneticin). PTC readthrough activity was assessed 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), in which (i) the indicated copy number of Arg was expressed on the same construct. TCA Neuro-2a cells were measured by either including a suppressor tRNA or by treating them with (ii) ataluren, (iii) gentamicin, or (iv) G418. Transfections were 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 were added at the indicated concentrations. PTC readthrough activity was measured as the percentage of EGFP-positive cells. A reporter containing wild-type EGFP without PTC was used as a control. Cell viability in the above treatment sets was assessed 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 are shown in Figures 21–23. Collectively, the results demonstrate that Arg TCA The results show that suppressor tRNA#115 (SEQ ID NO:18) produces much more readthrough than either of the nonsense suppressors. Furthermore, unlike either of the nonsense suppressors, the results show that Arg TCA This indicates that treatment with suppressor tRNA does not result in a decrease in cell viability.

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

[0182] Suppressor tRNAs are generated from endogenous mouse tRNAs by converting the normal anticodon into an anticodon that recognizes a premature termination codon (PTC). The suppressor tRNA sequences are shown in Table 10.

[0183] [Table 10] TIFF2025148444000092.tif199132TIFF2025148444000093.tif212132TIFF2025148444000094.tif226132 TIFF2025148444000095.tif220132TIFF2025148444000096.tif220132TIFF2025148444000097.tif220132 TIFF2025148444000098.tif206132TIFF2025148444000099.tif213132TIFF2025148444000100.tif213132 TIFF2025148444000101.tif213132TIFF2025148444000102.tif213132TIFF2025148444000103.tif213132 TIFF2025148444000104.tif213132TIFF2025148444000105.tif213132TIFF2025148444000106.tif220132 TIFF2025148444000107.tif220132TIFF2025148444000108.tif226132TIFF2025148444000109.tif213132 TIFF2025148444000110.tif213132TIFF2025148444000111.tif213132TIFF2025148444000112.tif226132 TIFF2025148444000113.tif220132TIFF2025148444000114.tif220132TIFF2025148444000115.tif100132

[0184] Suppressor tRNAs are tested for PTC readthrough activity by flow cytometry in cell lines containing dual fluorescent readthrough reporters. These reporters contain three copies of red fluorescent protein (tdTomato), TEV protease, a linker region containing a PTC, and three copies of green fluorescent protein (EGFP). An overview of the reporter construct is shown in Figure 3. In the absence of any PTC readthrough resulting from the suppressor tRNA, translation will terminate at the PTC within the linker region, and only tdTomato will be expressed (thus, only red fluorescence will be detected). PTC readthrough activity resulting from the suppressor tRNA will allow translation to proceed through the PTC within the linker region, resulting in the expression of both tdTomato and EGFP (thus, both red and green fluorescence will be detected). Therefore, readthrough can be assessed by quantifying the proportion of viable cells expressing both red and green fluorescent reporters above background (% double positives).

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

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

[0187] Gln CTAThe suppressor tRNA was 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 cotransfected with an expression construct containing a dual fluorescent readthrough reporter. These reporters included three copies of red fluorescent protein (tdTomato), TEV protease, a linker region containing a PTC, and three copies of green fluorescent protein (EGFP). A schematic of the reporter constructs is shown in Figure 3. In the absence of any PTC readthrough resulting from the suppressor tRNA, translation would terminate through the PTC in the linker region, and only tdTomato would be expressed (and thus only red fluorescence would be detected). PTC readthrough activity resulting from the suppressor tRNA would allow translation to proceed through the PTC in the linker region, resulting in the expression of both tdTomato and EGFP (and thus both red and green fluorescence would be detected). Therefore, readthrough was assessed by quantifying the proportion of viable cells expressing both red and green fluorescent reporters above background (% double positive) using flow cytometry. To screen for suppressor tRNAs that exhibit readthrough activity in PTCs associated with Dravet syndrome, we generated PTCs from the SCN1A transcripts of three patients with Gln(Q)-to-TAG nonsense mutations in SCN1A: patient 3 (W1397X), patient 4 (S1505X), and patient 5 (Q1810X). Linker regions containing eight adjacent codons on either side of the PTC were generated.

[0188] The linker region derived from the SCN1A transcript of patient 3 is as follows, and the reporter containing this linker region is referred to as the patient 3-Gln-TAG (W1397X) reporter. The corresponding linker with the wild-type Trp(W) codon instead of TIFF2025148444000116.tif4149PTC was used as a control and had the following sequence: TIFF2025148444000117.tif4150

[0189] The linker region derived from the SCN1A transcript of patient 4 is as follows, and the reporter containing this linker region is referred to as the patient 4-Gln-TAG (S1505X) reporter. The corresponding linker with wild-type Ser (S) instead of TIFF2025148444000118.tif4150PTC was used as a control and had the following sequence: TIFF2025148444000119.tif4150

[0190] The linker region derived from the SCN1A transcript of patient 5 is as follows, and the reporter containing this linker region is referred to as the patient 5-Gln-TAG (Q1810X) reporter. The corresponding linker with the wild-type Gln codon instead of TIFF2025148444000120.tif4150PTC was used as a control and had the following sequence: TIFF2025148444000121.tif4150

[0191] Gln CTA The suppressor tRNAs (SEQ ID NOs:178-190) were expressed in (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) in cells stably expressing the patient 3-Gln-TAG (W1397X) reporter and expressing Gln CTA PTC read-through activity was tested by flow cytometry in multiple assay configurations, including the human Flp-In-293 cell line transiently transfected with a plasmid encoding a suppressor tRNA (results shown in Figure 25). Transfections were performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol.

[0192] Taken together, these results indicate that the described suppressor tRNAs are able to promote the expression of transcripts containing premature stop codons associated with disorders such as Dravet syndrome, such as the SCN1A transcript.

[0193] Example 8 This example describes the effect of nucleotide sequences adjacent to a suppressor tRNA on the readthrough of a premature termination codon (PTC) by the suppressor tRNA.

[0194] EGFP-R96X-TGA reporter (described in Example 1, SEQ ID NO: 31) and a single copy of Arg TCA Expression vectors were generated that encoded suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18, as described in Example 1). The expression vectors were derived from genomic DNA in each case 5' and 3' to the mouse tRNA-Arg-TCG-1-1 gene, but contained different lengths of sequences immediately 5' and 3' to the tRNA coding sequence. Details of the expression vectors are shown in Table 11.

[0195] [Table 11]

[0196] The expression vectors in Table 11 were tested for PTC read-through activity in Neuro-2a cells by flow cytometry. Transfection was performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. The results are shown in Figures 26A and 26B, and show that the Arg TCA Although suppressor tRNA#115 exhibited activity even with a random 5' leader sequence, this indicates that 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 nucleotide sequences adjacent to a suppressor tRNA on the readthrough of a premature termination codon (PTC) by the suppressor tRNA.

[0198] A library of expression vectors was generated containing nucleotide sequences encoding (ii) TCA-115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, 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) in combination with (i) one of 20 unique 100-nt leader sequences (the sequence immediately 5' to the tRNA coding sequence) derived from human genomic DNA that is immediately 5' to the endogenous tRNA gene. A schematic diagram showing the expression vector construct design for the library is shown in Figure 27. The 20 unique 100 nt leader sequences include those derived from the most abundant tRNAs in human HEK293 cells and include 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:889), Arg-TCG-5-1 (SEQ ID NO:889), Arg-TCG-6-1 (SEQ ID NO:889 ...TCG-5-1 (SEQ ID NO:889), Arg-TCG-6-1 (SEQ ID NO:889), Arg-TCG-5-1 (SEQ ID NO:871), Lys-TTT-6-1 (SEQ ID NO:889), Asn-GTT-1-1 (SEQ ID NO:880), Arg-CCG-2-1 (SEQ ID NO:877), Ala-AGC-4-1 (SEQ ID NO:889), Arg-TCG-5-1 (SEQ ID NO:889), Arg-TC The proteins contained leader sequences derived from the Leu-TAA-1-1 (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 suppressor tRNA#163 (SEQ ID NO:45) combination was TCA Constructs include EGFP-R96X-TGA reporter (SEQ ID NO: 31) or Gln TTA The constructs were tested for PTC readthrough activity by flow cytometry in cell lines cotransfected with the EGFP-Q69X-TAA reporter (SEQ ID NO:175). The results, shown in Figures 28-32, demonstrate that (i) the activity of suppressor tRNAs is affected by leader sequences and (ii) suppressor tRNAs (including different classes of suppressor tRNAs) exhibited high readthrough activity when combined with the identified leader sequences.

[0200] Example 10 This example describes the read-through activity of certain suppressor tRNAs and small molecule nonsense suppression therapies disclosed herein.

[0201] The suppressor tRNAs were tested together with the nonsense suppressors translaruna (ataluren), gentamicin, and G418 (geneticin). PTC readthrough activity was measured in Neuro-2a cells approximately 48 hours after transfection with an expression construct containing a CAG:NLS-EGFP (Q69X-TAA) reporter (SEQ ID NO: 175, described in Example 4), either (i) containing the indicated copy number of Gln suppressor tRNAs (#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) on the same construct, or (ii) treated with ataluren, (iii) treated with gentamicin, or (iv) treated with G418. Transfections were performed using Lipofectamine 3000 Transfection Reagent according to the manufacturer's protocol. In all experimental conditions, cell culture medium was replaced with fresh medium approximately 6 hours after transfection, at which point the indicated drugs were added at the indicated concentrations. PTC read-through activity was measured as the percentage of EGFP-positive cells as determined by flow cytometry. A reporter containing wild-type EGFP without PTC was used as a control. Cell viability in cells subjected to the above treatment sets was assessed 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. Results were analyzed using Gln TTA Suppressor tRNA#002 (SEQ ID NO:36) is shown in Figures 33-34, and Gln CTASuppressor tRNA#196 (SEQ ID NO:178) is shown in Figures 35-36. Collectively, the results demonstrate that this suppressor tRNA results in greater readthrough than any of the nonsense suppressors. Furthermore, the results demonstrate that, unlike any of the nonsense suppressors, treatment with the 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 specific suppressor tRNAs disclosed herein.

[0203] Flp-In-293 cells were transfected with (i) an expression construct containing mouse SCN1A with Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag peptide (DYKDHD-G-DYKDHD-I-DYKDDDDK) (SEQ ID NO:901), and (ii) Arg TCA An expression construct containing suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18) was either co-transfected 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 NOs:898 and 899 are as follows: TIFF2025148444000123.tif42149TIFF2025148444000124.tif226149TIFF2025148444000125.tif226149TIFF20251484440 00126.tif227152TIFF2025148444000127.tif226149TIFF2025148444000128.tif226149TIFF2025148444000129.tif57149

[0204] SCN1A was detected by Western blot as follows. 24 hours after transfection, proteins were isolated in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific #89900) containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific #87786) according to the manufacturer's protocol. 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 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 a primary anti-FLAG M2 antibody (Sigma, F1804-200UG, 1:1000) in TBST overnight at 4°C, washed three times with TBST, and incubated with a goat anti-mouse IgG (H+L) secondary antibody, HRP secondary antibody (Thermo Fisher Scientific #31431, 1:30,000) for 1 hour at room temperature. The blot was developed by applying SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific #34094), and signals were detected using an iBright imaging system. The results are shown in Figure 37. TCA This shows that suppressor tRNA#115 was able to rescue full-length SCN1A protein expression, whereas small molecule drugs were not.

[0205] Also, (i) an expression construct containing mouse SCN1A with an Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag (SEQ ID NO:901), and (ii) an Arg TCA 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 The suppressor tRNA #115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18) was co-transfected into Flp-In-293 cells. SCN1A expression was measured by Western blot using anti-FLAG antibody as described above in this example. The results are shown in Figure 38 and demonstrate that each suppressor tRNA tested rescued full-length SCN1A protein expression.

[0206] We also investigated the effect of (i) an expression construct containing mouse SCN1A with an Arg(R)-to-TGA PTC (R1407X) and a C-terminal 3xFLAG tag (SEQ ID NO:901) 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 plates) of Arg(R)-to-TGA PTC (R1407X). TCA Flp-In-293 cells were co-transfected with an expression construct containing suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18). SCN1A expression was measured by Western blot using anti-FLAG antibody as described above in this example. The results are shown in Figure 39 and demonstrate that tRNA#115 was able to rescue full-length SCN1A protein expression over a wide dose range.

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

[0208] The constructs packaged into AAV-PHP.eB capsids 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) driven by the EF1a promoter and two copies of Arg in the context of 55 bp of upstream flanking genomic DNA from tRNA-Tyr-GTA-5-1 (SEQ ID NO:900). TCA Construct 262 contains suppressor tRNA#115 (tRNA-Arg-TCT-2-1-TCA-SUP_intronless, SEQ ID NO:18, described in Example 1). Both constructs contain 5' and 3' ITR sequences from AAV2, which provide cis-acting elements for AAV replication and packaging. Construct 262 and AAV-PHP.eB containing construct 269 were produced 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 reliable 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 cells were allowed to recover for an additional 24 hours before viral transduction. Cells were then transduced at an MOI of 1E5 vg / cell. The results are shown in Figure 41. AAV-delivered Arg TCASuppressor tRNA#115 achieved approximately 13.2% PTC readthrough based on GFP intensity. Suppressor tRNAs exhibit comparable readthrough activity when delivered by AAV or transient transfection.

[0210] Example 13 This example describes ribosome profiling experiments that show that the disclosed suppressor tRNAs do not cause readthrough of significant amounts 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 compared with (ii) cells transfected with an expression construct lacking the suppressor tRNA. Ribosomes typically terminate translation when they encounter a stop codon. Therefore, if suppressor tRNAs increase readthrough of native stop codons, this would be indicated by an increased density of ribosomes found in the 3' UTR of mRNAs in cells expressing the suppressor tRNA, particularly in 3' UTR mRNAs containing the native stop codon recognized by the expressed suppressor tRNA.

[0212] Neuro-2a cells were incubated with (i) the EGFP-R96X-TGA reporter (SEQ ID NO:177) and Arg TCACells were transfected with either (i) an expression construct containing suppressor tRNA#001 (SEQ ID NO:11) or (ii) an expression construct lacking the suppressor tRNA and containing a wild-type version of the EGFP reporter. EGFP expression is shown in Figure 42. Approximately 48 hours after transfection, cells were subjected to ribosome footprint profiling as follows: Cells were lysed in lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl, 100 mM KCl, 1% Triton X-100, 1 mM DTT, 50 μg / mL Emetine (Sigma #324693), and 500 U / mL RNAsin (Promega #N2615)), and the cell lysate was sheared 10 times using a 25-gauge needle, followed by centrifugation 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, followed by the addition of 5 μL of SuperAse-IN (Thermo Fisher Scientific #AM2694) to stop the reaction. The MNase-treated extract was applied to a 15-45% sucrose gradient and separated by density for 2:26 hours at 41,000 rpm in a SW 41Ti swinging bucket rotor (Beckman Coulter #331362) at 4°C. After fractionation and collection of the monosome-containing fraction, ribosomal-protected mRNA fragments were precipitated from the sucrose with 1.25 mL of 95% ethanol overnight at -20°C. 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 ranging in size from 26 to 34 nt were excised from the gel and isolated to generate a ribosome-protected fragment library.After 3' linker ligation, rRNA removal using Ribo-Zero reagent from 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). Barcoded cDNA libraries were extracted from the gel and sequenced in single-read runs on a NextSeq 550 sequencing system. After sequencing, adapters were excised from the raw reads using Trimmomatic, and non-coding RNA was removed by aligning them to the Ensembl mouse mm10 ncRNA reference using bowtie2. The remaining reads were aligned to the UCSC mm10 mouse reference assembly, again using bowtie2. Duplicate-mapped reads were discarded. The resulting final set of aligned reads was quantified using the RiboProfiling package in R and custom Python scripts. Using Python, plots were generated examining 3' UTR occupancy and fold change in each gene with 20 or more uniquely mapped reads, and the distribution of genes with each native stop codon was compared using a two-sample Kolmogorov-Smirnov test. The results are shown in Figure 43.

[0213] Taken together, these results indicate that the suppressor tRNA can promote the expression of transcripts containing premature stop codons, such as EGFP-R96X-TGA, without causing readthrough of significant amounts of off-target native stop codons in cells expressing it.

[0214] INCORPORATION BY REFERENCE The entire disclosure of each of the patent and scientific literature cited herein is incorporated by reference for all purposes.

[0215] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments, therefore, should be considered in all respects as illustrative and not limiting on the invention described herein. The scope of the present invention is therefore indicated by the appended claims, rather than the above detailed description, and all changes made within the spirit and range of equivalents of the claimed invention are intended to be embraced therein.

[0216] Sequence information SEQUENCE LISTING <110> TEVARD BIOSCIENCES, INC. <120> METHODS AND COMPOSITIONS FOR TREATING A PREMATURE TERMINATION CODON-MEDIATED DISORDER <150> US 62 / 929,428 <151> 2019-11-01 <160> 902 <170> PatentIn version 3.5 <210> 1 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 gggccagtgg cgcaatggat aacgcgtctg acttcagatc agaagattcc aggttcgact 60 cctggctggc tcg 73 <210> 2 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 gggccagtgg cgcaatggat aacgcgtctg acttcagatc agaagattgt aggttcgact 60 cctacctggc tcg 73 <210> 3 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 gggccagtgg cgcaatggat aacgcgtctg acttcagatc agaagattct aggttcgact 60 cctggctggc tcg 73 <210> 4 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 ggccgcgtgg cctaatggat aaggcgtctg atttcagatc agaagattga gggttcgagt 60 cccttcgtgg tcg 73 <210> 5 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 ggccgcgtgg cctaatggat aaggcgtctg atttcagatc agaagattgg gggttcgagt 60 cccttcgtgg tcg 73 <210> 6 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 gacccagtgg cctaatggat aaggcatcag ccttcagagc tggggattgt gggttcgagt 60 cccatctggg tcg 73 <210> 7 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 gccccagtgg cctaatggat aaggcactgg ccttcaaagc cagggattgt gggttcgagt 60 cccacctggg gta 73 <210> 8 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 gccccagtgg cctaatggat aaggcactgg ccttcaaagc cagggattgt gggttcgagt 60 cccacctggg gtg 73 <210> 9 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 9 gccccggtgg cctaatggat aaggcattgg ccttcaaagc cagggattgt gggttcgagt 60 cccacccggg gta 73 <210> 10 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 gccccagtgg cctaatggat aaggcattgg ccttcaaagc cagggattgt gggttcgagt 60 cccatctggg gtg 73 <210> 11 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 ggccgcgtgg cctaatggat aaggcgtctg acttcagatc agaagattgc aggttcgagt 60 cctgccgcgg tcg 73 <210> 12 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 12 gaccgcgtgg cctaatggat aaggcgtctg acttcagatc agaagattga gggttcgagt 60 cccttcgtgg tcg 73 <210> 13 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 13 gaccacgtgg cctaatggat aaggcgtctg acttcagatc agaagattga gggttcgaat 60 cccttcgtgg ttg 73 <210> 14 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 gaccacgtgg cctaacggat aaggcgtctg acttcagatc agaagattga gggttcgaat 60 cccttcgtgg tta 73 <210> 15 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 15 ggctctgtgg cgcaatggat agcgcattgg acttcaagtg acgagaaagc gattcaaagg 60 ttgtgggttc gaatcccacc agagtcg 87 <210> 16 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 ggctctgtgg cgcaatggat agcgcattgg acttcaaatt caaaggttgt gggttcgaat 60 cccaccagag tcg 73 <210> 17 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 ggctccgtgg cgcaatggat agcgcattgg acttcaagag gctgaaggca ttcaaaggtt 60 ccgggttcga gtcccggcgg agtcg 85 <210> 18 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 ggctccgtgg cgcaatggat agcgcattgg acttcaaatt caaaggttcc gggttcgagt 60 cccggcggag tcg 73 <210> 19 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 19 ggctctgtgg cgcaatggat agcgcattgg acttcaagca tgattgagag attcaaaggt 60 tgcgggttcg agtcccgcca gagtcg 86 <210> 20 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 ggctctgtgg cgcaatggat agcgcattgg acttcaaatt caaaggttgc gggttcgagt 60 cccgccagag tcg 73 <210> 21 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 ggctctgtgg cgcaatggat agcgcattgg acttcaagac aaatggaggc attcaaaggt 60 tgtgggttcg agtcccacca gagtcg 86 <210> 22 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 ggctctgtgg cgcaatggat agcgcattgg acttcaaatt caaaggttgt gggttcgagt 60 cccaccagag tcg 73 <210> 23 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 23 gtctctgtgg cgcaatggac gagcgcgctg gacttcaaat ccagaggttc tgggttcgag 60 tcccggcaga gatg 74 <210> 24 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 24 ggctctgtgg agcaatggat agcacattgg acttcaagca tgaccgagag attcaaaggt 60 tgcgggttcg agtcccacca gagttg 86 <210> 25 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 ggctctgtgg agcaatggat agcacattgg acttcaaatt caaaggttgc gggttcgagt 60 cccaccagag ttg 73 <210> 26 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 26 ctacccagag gcaggcggga gactcccccg agcgtccaat aagagcgccg ccaatggagc 60 cgcccgcccg cgggggtgca gagggacttc cgggtgaggt cctccgctac ttccctcccc 120 acggaaaaga tagaccagtc tgacgcgagc ctgaaggcgg ctacacgctt taagctaagt 180 aaaggcacct tctcgctggc 200 <210> 27 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 27 acttgtatgt tgtttttatc tgtcagtttg ttaatcccaa gattcccttt ggaaataaag 60 cgaaattgac cgtagtggtt atgaccaact tctagtctaa acttaattct tggaactcaa 120 ggatctgagc aaacaactgt cagggtgaca cattgcttaa acggtgacag cggtcgagag 180 ccttgtcccg gatggagagt 200 <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 28 ctgagacctc taagagcctt atcttgattt gaagggatga gggtggttgt g 51 <210> 29 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 29 acaagccttt tcagctttag agggtgagca aaggatgtgg gatctgagaa c 51 <210> 30 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 30 ctaatagaaa gaaatgagac cgcctgatgg aaaaatgtga aagtaaactt t 51 <210> 31 <211> 1854 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 31 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 360 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 420 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 480 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 540 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 600 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 660 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg 720 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg 780 ggagcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc 840 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac 900 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc 960 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt 1020 ctcatcattt tggcaaagaa ttgcggccca acggtaccgg atccaccggc cgccaccatg 1080 ggaagcccaa agaagaagcg taaggtaatg gtgagcaagg gcgaggagct gttcaccggg 1140 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 1200 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1260 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 1320 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1380 ggctacgtcc aggagtgaac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 1440 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1500 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1560 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1620 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1680 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1740 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1800 ctcggcatgg acgagctgta caagggaagc cccaagaaaa agcggaaggt gtaa 1854 <210> 32 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 32 acttgtatgt tgtttttatc tgtcagtttg ttaatcccaa gattcccttt ggaaataaag 60 cgaaattgac cgtagtggtt atgaccaact tctagtctaa actt 104 <210> 33 <211> 269 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 33 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgggcggagg aaggcacctt ctcgctggc 269 <210> 34 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 34 acttgtatgt tgtttttatc tgtcagtttg ttaatcccaa gattcc 46 <210> 35 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 35 gtctctgtgg cgcaatggac gagcgcgctg gacttcaaat ccagaggttc cgggttcgag 60 tcccggcaga gatg 74 <210> 36 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 36 ggtcccatgg tgtaatggtt agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 37 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 37 ggttccatgg tgtaatggtt agcactctgg actttaaatc cagcgacccg agttcaaatc 60 tcggtgggac ct 72 <210> 38 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 38 ggttccatgg tgtaatggtt agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtggaac ct 72 <210> 39 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 39 ggttccatgg tgtaatggtt agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 40 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 40 ggttccatgg tgtaatggtg agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 41 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 41 ggttccatgg tgtaatggct agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtgggat tt 72 <210> 42 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 42 ggttccatgg tgtaatggtt agcactctgg actttaaatc cagccataca agttcaaatc 60 tcagtggaac ct 72 <210> 43 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 43 ggttccttgg tgtaagatga gcactctgga ttttaaatcc agcgatcaga gttcaaatct 60 cggtgggacc t 71 <210> 44 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 44 ggtcccatgg tgtaatggtt agcactctgg actttaaatc cagcaatctg agttcaaatc 60 tcggtgggac ct 72 <210> 45 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 45 ggccccatgg tgtaatggtt agcactctgg actttaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 46 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 46 ggtctcatgg tgtaatggtt agcacactgg actttaagtc cagcaatccg agttcgagtc 60 ttggtgagac ca 72 <210> 47 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 47 ggacccatgg tgtaatggtt agcactctgg actttaaatc cagcaatcca agttcaaatc 60 tcggtgggac ct 72 <210> 48 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 48 gtttccatgg tgtaatggtt ggcactctgg actttaaatc cagcaatcca agttcaagtc 60 tctgtgggac ct 72 <210> 49 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 49 gtcaggatgg ccgagtggtc taaggcgcca gactctagct atggcttcct cgctctgagg 60 gttctggtct cccctggagg cgtgggttcg aatcccactt ctgaca 106 <210> 50 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 50 gtcaggatgg ccgagtggtc taaggcgcca gactctagct tagcttccct gtctggggat 60 tctggtctcc gtatggaggc gtgggttcga atcccacttc tgaca 105 <210> 51 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 51 gtcaggatgg ccgagtggtc taaggcgcca gactctaggt gacaagcctt acctacgggt 60 gttctggtct ccgaatggag gcgtgggttc gaatcccact tctgaca 107 <210> 52 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 52 gtcaggatgg ccgagtggtc taaggcgcca gactctagcg ttcgcttcct ctactgaggg 60 ttctggtctc cgtgtggagg cgtgggttcg aatcccactt ctgaca 106 <210> 53 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 53 gtcaggatgg ccgagtggtc taaggcgcca gacttcagct atggcttcct cgctctgagg 60 gttctggtct cccctggagg cgtgggttcg aatcccactt ctgaca 106 <210> 54 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 54 gtcaggatgg ccgagtggtc taaggcgcca gacttcagct tagcttccct gtctggggat 60 tctggtctcc gtatggaggc gtgggttcga atcccacttc tgaca 105 <210> 55 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 55 gtcaggatgg ccgagtggtc taaggcgcca gacttcaggt gacaagcctt acctacgggt 60 gttctggtct ccgaatggag gcgtgggttc gaatcccact tctgaca 107 <210> 56 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 56 gtcaggatgg ccgagtggtc taaggcgcca gacttcagcg ttcgcttcct ctactgaggg 60 ttctggtctc cgtgtggagg cgtgggttcg aatcccactt ctgaca 106 <210> 57 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 57 gtcaggatgg ccgagtggtc taaggcgcca gactttagct atggcttcct cgctctgagg 60 gttctggtct cccctggagg cgtgggttcg aatcccactt ctgaca 106 <210> 58 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 58 gtcaggatgg ccgagtggtc taaggcgcca gactttagct tagcttccct gtctggggat 60 tctggtctcc gtatggaggc gtgggttcga atcccacttc tgaca 105 <210> 59 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 59 gtcaggatgg ccgagtggtc taaggcgcca gactttaggt gacaagcctt acctacgggt 60 gttctggtct ccgaatggag gcgtgggttc gaatcccact tctgaca 107 <210> 60 <211> 106 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 60 gtcaggatgg ccgagtggtc taaggcgcca gactttagcg ttcgcttcct ctactgaggg 60 ttctggtctc cgtgtggagg cgtgggttcg aatcccactt ctgaca 106 <210> 61 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 61 ccttcgatag ctcagttggt agagcggagg actctagagt tactagaata gtgatcctta 60 ggtcgctggt tcgaatccgg ctcgaagga 89 <210> 62 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 62 ccttcgatag ctcagttggt agagcggagg actctagtca gtacaatatg gtaatcctta 60 ggtcgctggt tcgattccgg ctcgaagga 89 <210> 63 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 63 ccttcgatag ctcagctggt agagcggagg actctaggct tgtggctgtg gacatcctta 60 ggtcgctggt tcgattccgg ctcgaagga 89 <210> 64 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 64 ccttcgatag ctcagctggt agagcggagg actctagcta actccccgtt agaagacatc 60 cttaggtcgc tggttcgact ccggctcgaa gga 93 <210> 65 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 65 ctttcgatag ttcagttggt agagcggagg actctagagt attaacgttg gtgatcctta 60 ggtcgctggt tcgagtccgg ctcgaagga 89 <210> 66 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 66 ccttcgatag ctcagttggt agagcggagg actttagagt tactagaata gtgatcctta 60 ggtcgctggt tcgaatccgg ctcgaagga 89 <210> 67 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 67 ccttcgatag ctcagttggt agagcggagg actttagtca gtacaatatg gtaatcctta 60 ggtcgctggt tcgattccgg ctcgaagga 89 <210> 68 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 68 ccttcgatag ctcagctggt agagcggagg actttaggct tgtggctgtg gacatcctta 60 ggtcgctggt tcgattccgg ctcgaagga 89 <210> 69 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 69 ccttcgatag ctcagctggt agagcggagg actttagcta actccccgtt agaagacatc 60 cttaggtcgc tggttcgact ccggctcgaa gga 93 <210> 70 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 70 ctttcgatag ttcagttggt agagcggagg actttagagt attaacgttg gtgatcctta 60 ggtcgctggt tcgagtccgg ctcgaagga 89 <210> 71 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 71 gggggtatag ctcagtggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 72 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 cagatgcccc ct 72 <210> 73 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 73 gggggtatag ctcaggggta gagtatttgg cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 74 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 74 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtcctt ggttcaaatc 60 caggtgtccc ct 72 <210> 75 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 75 gggggtatag ctcagaggta gagcatttga cttcagatca agagatctct ggttcaaatc 60 caggtgcccc ct 72 <210> 76 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 76 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct agttcaaatc 60 caggtgcccc ct 72 <210> 77 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 77 ggtggtatag ctcaggggta gagcatttga cttcagatca agagatccct ggttcgaatc 60 caggtgcccc ct 72 <210> 78 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 78 gggggtataa ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 79 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 79 tggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 80 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 80 gggggtatag ctcagaggaa gagcatttga cttcagatca agaggtccct gattcaaatc 60 caggtgcccc ct 72 <210> 81 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 81 gggggtaaag ctcaggggta gagcatttga cttcagatta agaggtccct ggttcaaatc 60 caggtacccc ct 72 <210> 82 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 82 gggggtatag ctcagtggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 cgggtgcccc ct 72 <210> 83 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 83 ggggttatag ctcaggtgta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 84 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 84 gggggtatag ctcaggggta gagcatttga cttcagatca cgaggtccct ggttcaaatc 60 gaggtgcccc ct 72 <210> 85 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 85 gggggtatag ctcaggggtg gagcatttga cttcagatca aggggtccct gtttcaaatc 60 caggtgcccc ct 72 <210> 86 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 86 gggggtatag ctcagtggta gagcatttga cttcagatca agaggtcccc ggttcaaatc 60 cgggtgcccc ct 72 <210> 87 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 87 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 88 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 88 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 cgggtgcccc ct 72 <210> 89 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 89 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtccct ggttcaaatc 60 caggtacccc ct 72 <210> 90 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 90 gggggtatag ctcaggggta gagcatttga cttcagatca agaggtcccc ggttcaaatc 60 cgggtgcccc ct 72 <210> 91 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 91 gggggcatag ctcaggggta gagcatttga cttcagatca agaggtcccc ggttcaaatc 60 cgggtgctcc ct 72 <210> 92 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 92 gggggtatag ctcaggggta gagcatttga cttcagatta agaggtccct ggttcaaatc 60 caggtgcccc ct 72 <210> 93 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 93 tccctggtgg tctagtggtt aggattcggc gctctaaccg ccgcggcccg ggttcgattc 60 ccggtcaggg aa 72 <210> 94 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 94 tccctggtgg tctagtggtt aggatttggc gctctaaccg ccgcggcctg ggttcgattc 60 ccggtcaggg aa 72 <210> 95 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 95 tccctggtgg tctagtggtt aggctttggt gctctaacct ccatggccca ggtttgattc 60 ctggtcaggg aa 72 <210> 96 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 96 tccctggtgg tctagtggtt aggattcggc gctttaaccg ccgcggcccg ggttcgattc 60 ccggtcaggg aa 72 <210> 97 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 97 tccctggtgg tctagtggtt aggatttggc gctttaaccg ccgcggcctg ggttcgattc 60 ccggtcaggg aa 72 <210> 98 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 98 tccctggtgg tctagtggtt aggctttggt gctttaacct ccatggccca ggtttgattc 60 ctggtcaggg aa 72 <210> 99 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 99 tcccacatgg tctagcggtt aggattcctg gttctaaccc aggcggcccg ggttcgactc 60 ccggtgtggg aa 72 <210> 100 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 100 tcccatatgg tctagcggtt aggattcctg gttctaaccc aggcggcccg ggttcgactc 60 ccggtatggg aa 72 <210> 101 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 101 tccctggtgg tctagtggct aggattcggc gctctaaccg ccgcggcccg ggttcgattc 60 ccggtcaggg aa 72 <210> 102 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 102 tcccacatgg tctagcggtt aggattcctg gttttaaccc aggcggcccg ggttcgactc 60 ccggtgtggg aa 72 <210> 103 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 103 tcccatatgg tctagcggtt aggattcctg gttttaaccc aggcggcccg ggttcgactc 60 ccggtatggg aa 72 <210> 104 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 104 tccctggtgg tctagtggct aggattcggc gctttaaccg ccgcggcccg ggttcgattc 60 ccggtcaggg aa 72 <210> 105 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 105 gtttccgtag tgtagtggtt agcgcgttcg ccttcaaaag cgaaaggtcc ccggttcgaa 60 accgggcgga aaca 74 <210> 106 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 106 gcgccgctgg tgtagtggta tcatgcaaga tttcaattct tgcgacccgg gttcgattcc 60 cgggcggcgc a 71 <210> 107 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 107 gcattggtag ttcaatggta gaattctcgc cttcaacgcg ggtgacccgg gttcgattcc 60 cggccaatgc a 71 <210> 108 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 108 gcattggtgg ttcaatggta gaattctcgc cttcaacgcg ggtgacccgg gttcgattcc 60 cggccaatgc a 71 <210> 109 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 109 gcattggtgg ttcaatggta gaattctcgc cttcaactcg ggtgacccgg gttcgattcc 60 cggccaatgc a 71 <210> 110 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 110 gcatgggtgg ttcagtggta gaattctcgc cttcaacgcg ggaggcccgg gttcgattcc 60 cggcccatgc a 71 <210> 111 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 111 gcattggtgg ttcagtggta gaattctcgc cttcaacgcg ggaggcccgg gttcgattcc 60 cggccaatgc a 71 <210> 112 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 112 gcattggtgg ttcagtggta gaattctcgc cttcaacgcg ggaggcccgg gtttgattcc 60 cggccaatgc a 71 <210> 113 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 113 gcattggtgg ttcagtggta gaattctcgc cttcaacgcg ggaggcccgg gttcggttcc 60 cggccaatgc a 71 <210> 114 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 114 gcgttggtgg tatagtggtg agcatagctg ccttcaaagc agttgacccg ggttcgattc 60 ccggccaacg ca 72 <210> 115 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 115 ggtagcgtgg ccgagcggtc taaggcgctg gattctagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 116 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 116 ggtagtgtgg ccgagcggtc taaggcgctg gattctagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 117 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 117 ggtagtgtgg ccgagcggtc taaggcgctg gattctagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 118 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 118 ggtagcgtgg ccgagcggtc taaggcgctg gatttcagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 119 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 119 ggtagtgtgg ccgagcggtc taaggcgctg gatttcagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 120 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 120 ggtagtgtgg ccgagcggtc taaggcgctg gatttcagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 121 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 121 ggtagcgtgg ccgagcggtc taaggcgctg gattttagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 122 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 122 ggtagtgtgg ccgagcggtc taaggcgctg gattttagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 123 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 123 ggtagtgtgg ccgagcggtc taaggcgctg gattttagct ccagtctctt cgggggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 124 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 124 gtcaggatgg ccgagtggtc taaggcgcca gactctagtt ctggtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 125 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 125 gtcaggatgg ccgagtggtc taaggcgcca gactctagtt ctggtctccg tatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 126 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 126 gtcaggatgg ccgagtggtc taaggcgcca gactctagtt ctggtctccg aatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 127 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 127 gtcaggatgg ccgagtggtc taaggcgcca gactctagtt ctggtctccg tgtggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 128 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 128 gtcaggatgg ccgagtggtc taaggcgcca gacttcagtt ctggtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 129 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 129 gtcaggatgg ccgagtggtc taaggcgcca gacttcagtt ctggtctccg tatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 130 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 130 gtcaggatgg ccgagtggtc taaggcgcca gacttcagtt ctggtctccg aatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 131 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 131 gtcaggatgg ccgagtggtc taaggcgcca gacttcagtt ctggtctccg tgtggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 132 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 132 gtcaggatgg ccgagtggtc taaggcgcca gactttagtt ctggtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 133 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 133 gtcaggatgg ccgagtggtc taaggcgcca gactttagtt ctggtctccg tatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 134 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 134 gtcaggatgg ccgagtggtc taaggcgcca gactttagtt ctggtctccg aatggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 135 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 135 gtcaggatgg ccgagtggtc taaggcgcca gactttagtt ctggtctccg tgtggaggcg 60 tgggttcgaa tcccacttct gaca 84 <210> 136 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 136 gtcaggatgg ccgagcggtc taaggcgctg cgttctagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 137 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 137 gtcaggatgg ccgagcggtc taaggcgctg cgttctagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 138 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 138 gtcaggatgg ccgagtggtc taaggagctg tgttctagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 139 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 139 gtcaggatgg ccgagcagtc taaggcactg cgttctagtc gcagtctccc ctggaggcgt 60 ggattcgaat cccactcctg aca 83 <210> 140 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 140 gtcaggatgg ccgagcggtc taaggcgctg cgtttcagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 141 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 141 gtcaggatgg ccgagcggtc taaggcgctg cgtttcagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 142 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 142 gtcaggatgg ccgagtggtc taaggagctg tgtttcagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 143 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 143 gtcaggatgg ccgagcagtc taaggcactg cgtttcagtc gcagtctccc ctggaggcgt 60 ggattcgaat cccactcctg aca 83 <210> 144 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 144 gtcaggatgg ccgagcggtc taaggcgctg cgttttagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 145 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 145 gtcaggatgg ccgagcggtc taaggcgctg cgttttagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccacttctg aca 83 <210> 146 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 146 gtcaggatgg ccgagtggtc taaggagctg tgttttagtc gcagtctccc ctggaggcgt 60 gggttcgaat cccactcctg aca 83 <210> 147 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 147 gtcaggatgg ccgagcagtc taaggcactg cgttttagtc gcagtctccc ctggaggcgt 60 ggattcgaat cccactcctg aca 83 <210> 148 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 148 accagaatgg ccgagtggtt aaggcgttgg actctagatc caatggattt atatccgcgt 60 gggttcgaac cccacttctg gta 83 <210> 149 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 149 accaggatgg ccgagtggtt aaggcgttgg actctagatc caatggacat atgtctgcgt 60 gggttcgaac cccactcctg gta 83 <210> 150 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 150 actgggatgg ctgagtggtt aaggcgttgg actctagatc caatgggcgg ttgcctgcgt 60 gggttcgaac cccactccca gta 83 <210> 151 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 151 gatgggatgg ctgagaggtt aaggctttgg actctagatc caatgggcag atgcctgcgt 60 gggtttgaac cccactccca ata 83 <210> 152 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 152 accagaatgg ccgagtggtt aaggcgttgg acttcagatc caatggattt atatccgcgt 60 gggttcgaac cccacttctg gta 83 <210> 153 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 153 accaggatgg ccgagtggtt aaggcgttgg acttcagatc caatggacat atgtctgcgt 60 gggttcgaac cccactcctg gta 83 <210> 154 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 154 actgggatgg ctgagtggtt aaggcgttgg acttcagatc caatgggcgg ttgcctgcgt 60 gggttcgaac cccactccca gta 83 <210> 155 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 155 gatgggatgg ctgagaggtt aaggctttgg acttcagatc caatgggcag atgcctgcgt 60 gggtttgaac cccactccca ata 83 <210> 156 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 156 accagaatgg ccgagtggtt aaggcgttgg actttagatc caatggattt atatccgcgt 60 gggttcgaac cccacttctg gta 83 <210> 157 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 157 accaggatgg ccgagtggtt aaggcgttgg actttagatc caatggacat atgtctgcgt 60 gggttcgaac cccactcctg gta 83 <210> 158 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 158 actgggatgg ctgagtggtt aaggcgttgg actttagatc caatgggcgg ttgcctgcgt 60 gggttcgaac cccactccca gta 83 <210> 159 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 159 gatgggatgg ctgagaggtt aaggctttgg actttagatc caatgggcag atgcctgcgt 60 gggtttgaac cccactccca ata 83 <210> 160 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 160 ggtagcgtgg ccgagcggtc taaggcgctg gattctagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 161 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 161 ggtagtgtgg ccgagcggtc taaggcgctg gattctagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 162 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 162 ggtagcgtgg ccgagtggtc taaggcgctg gattctagct ccagtcattt cgatggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 163 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 163 ggtagcgtgg ccgagcggtc taaggcgctg gatttcagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 164 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 164 ggtagtgtgg ccgagcggtc taaggcgctg gatttcagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 165 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 165 ggtagcgtgg ccgagtggtc taaggcgctg gatttcagct ccagtcattt cgatggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 166 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 166 ggtagcgtgg ccgagcggtc taaggcgctg gattttagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 167 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 167 ggtagtgtgg ccgagcggtc taaggcgctg gattttagct ccagtctctt cggaggcgtg 60 ggttcgaatc ccaccactgc ca 82 <210> 168 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 168 ggtagcgtgg ccgagtggtc taaggcgctg gattttagct ccagtcattt cgatggcgtg 60 ggttcgaatc ccaccgctgc ca 82 <210> 169 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 169 gcccagctag ctcagttggt agagcgtggg actctaaatc ctagggtcgt gggttcgaac 60 cccacgttgg gcg 73 <210> 170 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 170 gcccagctag ctcagtctgt agagcatgag actctaagtc tcagggtcat gggttggagc 60 cccatgttgt gca 73 <210> 171 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 171 gcctagctag ttcagtcggt agagcatgag actctaaatc tcaggttcat gagtttgagc 60 cccatgttgg tttggca 77 <210> 172 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 172 ccccggctag ctcagtcagt agagcttgag aatctaaatc tcagggtcgt gggttggagc 60 cccacgttgg gcg 73 <210> 173 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 173 gatcaccgga agaggtgaca gacacctcgg ggcccatgaa cgtttggaat tcgtaaggac 60 atgagaatct cggtggttcc gtgtctgccc gccatcgcgg ccaccggcca cgggcccaag 120 ccaagtgtag cgaagcttag aaaaggttgc ccaacgtcat gtggcttgag aaggctgccg 180 ggcgccttaa gccgccagca 200 <210> 174 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 174 cactgaacct ttttttggcc ttagaatccc tgttttgggg cctgcaggaa gtagcaacca 60 acccgagcct ccgcagggaa tgcactgacc tgtagaatgg acgttcagct tccctccctg 120 tgtctcaaca cgattacatt tcaggaacag cctgggctgg gaggcactgc gcacgcgcgc 180 cgagtcgggc ggaaaaataa 200 <210> 175 <211> 1854 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 175 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 360 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 420 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 480 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 540 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 600 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 660 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg 720 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg 780 ggagcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc 840 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac 900 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc 960 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt 1020 ctcatcattt tggcaaagaa ttgcggccca acggtaccgg atccaccggc cgccaccatg 1080 ggaagcccaa agaagaagcg taaggtaatg gtgagcaagg gcgaggagct gttcaccggg 1140 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 1200 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1260 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgtaatgc 1320 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1380 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 1440 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1500 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1560 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1620 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1680 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1740 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1800 ctcggcatgg acgagctgta caagggaagc cccaagaaaa agcggaaggt gtaa 1854 <210> 176 <211> 1854 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 176 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 360 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 420 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 480 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 540 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg 600 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac 660 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg 720 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg 780 ggagcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc 840 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac 900 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc 960 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt 1020 ctcatcattt tggcaaagaa ttgcggccca acggtaccgg atccaccggc cgccaccatg 1080 ggaagcccaa agaagaagcg taaggtaatg gtgagcaagg gcgaggagct gttcaccggg 1140 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 1200 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1260 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgtagtgc 1320 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1380 ggctacgtcc aggagcgcac catcttcttc areacgacg gcaactacaa gacccgcgcc 1440 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1500 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1560 tatatcatgg ccgacaagca gaagacggc atcaaggtga acttcaagat ccgccacaac 1620 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1680 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1740 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1800 ctcggcatgg acgagctgta caaggaagc cccaagaaaa agcggaaggt gtaa 1854 <210> 177 <211> 1981 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 177 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180 ctagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360 gagagttcgt ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgtggcctgg 420 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540 caagatagtc ttgtaaatgc gggccaagat cagcacactg gtatttcggt ttttggggcc 600 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660 gcgcggccac cgagaatcgg acgggggtag tctcaagctg cccggcctgc tctggtgcct 720 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagcac aaaatggagg 840 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960 tagttctcca gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc caccggccgc 1200 caccatggga agcccaaaga agaagcgtaa ggtaatggtg agcaagggcg aggagctgtt 1260 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1320 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1380 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 1440 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1500 gcccgaaggc tacgtccagg agtgaaccat cttcttcaag gacgacggca actacaagac 1560 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1620 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1680 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 1740 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 1800 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 1860 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 1920 gatcactctc ggcatggacg agctgtacaa gggaagcccc aagaaaaagc ggaaggtgta 1980 a 1981 <210> 178 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 178 ggtcccatgg tgtaatggtt agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 179 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 179 ggttccatgg tgtaatggtt agcactctgg actctaaatc cagcgacccg agttcaaatc 60 tcggtgggac ct 72 <210> 180 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 180 ggttccatgg tgtaatggtt agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtggaac ct 72 <210> 181 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 181 ggttccatgg tgtaatggtt agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 182 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 182 ggttccatgg tgtaatggtg agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 183 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 183 ggttccatgg tgtaatggct agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtgggat tt 72 <210> 184 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 184 ggttccatgg tgtaatggtt agcactctgg actctaaatc cagccataca agttcaaatc 60 tcagtggaac ct 72 <210> 185 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 185 ggttccttgg tgtaagatga gcactctgga ttctaaatcc agcgatcaga gttcaaatct 60 cggtgggacc t 71 <210> 186 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 186 ggtcccatgg tgtaatggtt agcactctgg actctaaatc cagcaatctg agttcaaatc 60 tcggtgggac ct 72 <210> 187 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 187 ggccccatgg tgtaatggtt agcactctgg actctaaatc cagcgatccg agttcaaatc 60 tcggtgggac ct 72 <210> 188 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 188 ggtctcatgg tgtaatggtt agcacactgg actctaagtc cagcaatccg agttcgagtc 60 ttggtgagac ca 72 <210> 189 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 189 ggacccatgg tgtaatggtt agcactctgg actctaaatc cagcaatcca agttcaaatc 60 tcggtgggac ct 72 <210> 190 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 190 gtttccatgg tgtaatggtt ggcactctgg actctaaatc cagcaatcca agttcaagtc 60 tctgtgggac ct 72 <210> 191 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 191 ctgcaaagtt ctttgaaaga gcaacaaaat ggcttcaact atctgagtga c 51 <210> 192 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 192 ctgcaaagtt ctttgaaaga gcaataaaat ggcttcaact atctgagtga c 51 <210> 193 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 193 ctgagacctc taagagcctt atctcgattt gaagggatga gggtggttgt g 51 <210> 194 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 194 acaagccttt tcagctttag agggcgagca aaggatgtgg gatctgagaa c 51 <210> 195 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 195 ctaatagaaa gaaatgagac cgcccggtgg aaaaatgtga aagtaaactt t 51 <210> 196 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 196 gcccggctag ctcagtcggt agagcatggg actctaaatc ccagggtcgt gggttcgagc 60 cccacgttgg gcg 73 <210> 197 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 197 gcccggctag ctcagtcggt agagcatgag actctaaatc tcagggtcgt gggttcgagc 60 cccacgttgg gcg 73 <210> 198 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 198 gcccagctag ctcagtctgt agagcatgag actctaaatc tcagggtcgt gagttcgagc 60 cccacgttgg gtg 73 <210> 199 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 199 gcccagatag ctcagtgggt agagcatgag actctaaatc tcagggtcat gggttcatgc 60 cccatgttgg gta 73 <210> 200 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 200 gtcctgctgg ctcagtcggt acagcatggg actctaaatc ccagggtcgt gggttcgagc 60 tccacgttgg gta 73 <210> 201 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 201 gcctggctag ctcagtccat agagcatggg actctaaatc ccagggtcat gggttcgagc 60 cccatattag gca 73 <210> 202 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 202 gcccagctag cttagttggt agagcatgag actctaaatc tcagagtcat gggttcaggc 60 ctcatgtttg gca 73 <210> 203 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 203 aacctggcta ggtcagttgg tagatcatga gactctaaat ctcagggtca tgggttcaag 60 ccccatgttg gttt 74 <210> 204 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 204 gcccagctag ctcagttggt agagcgtggg actttaaatc ctagggtcgt gggttcgaac 60 cccacgttgg gcg 73 <210> 205 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 205 gcccagctag ctcagtctgt agagcatgag actttaagtc tcagggtcat gggttggagc 60 cccatgttgt gca 73 <210> 206 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 206 gcctagctag ttcagtcggt agagcatgag actttaaatc tcaggttcat gagtttgagc 60 cccatgttgg tttggca 77 <210> 207 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 207 ccccggctag ctcagtcagt agagcttgag aatttaaatc tcagggtcgt gggttggagc 60 cccacgttgg gcg 73 <210> 208 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 208 gcccggctag ctcagtcggt agagcatggg actttaaatc ccagggtcgt gggttcgagc 60 cccacgttgg gcg 73 <210> 209 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 209 gcccggctag ctcagtcggt agagcatgag actttaaatc tcagggtcgt gggttcgagc 60 cccacgttgg gcg 73 <210> 210 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 210 gcccagctag ctcagtctgt agagcatgag actttaaatc tcagggtcgt gagttcgagc 60 cccacgttgg gtg 73 <210> 211 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 211 gcccagatag ctcagtgggt agagcatgag actttaaatc tcagggtcat gggttcatgc 60 cccatgttgg gta 73 <210> 212 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 212 gtcctgctgg ctcagtcggt acagcatggg actttaaatc ccagggtcgt gggttcgagc 60 tccacgttgg gta 73 <210> 213 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 213 gcctggctag ctcagtccat agagcatggg actttaaatc ccagggtcat gggttcgagc 60 cccatattag gca 73 <210> 214 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 214 gcccagctag cttagttggt agagcatgag actttaaatc tcagagtcat gggttcaggc 60 ctcatgtttg gca 73 <210> 215 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 215 aacctggcta ggtcagttgg tagatcatga gactttaaat ctcagggtca tgggttcaag 60 ccccatgttg gttt 74 <210> 216 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 216 gcccggatag ctcagtcggt agagcatcag actctaaatc tgagggtcca gggttcaagt 60 ccctgttcgg gcg 73 <210> 217 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 217 gcctggatag ctcagtcggt agagcatcag actctaaatc tgagggtcca gggttcaagt 60 ccctgttcag gcg 73 <210> 218 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 218 gcctggatag ctcaattggt agagcatcag actctaaatc tgagggttca gggttcaagt 60 ccctgttcag gcg 73 <210> 219 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 219 gcccagccag ctcagtaggt agagtatgag actctaaatc tcagggtggt gggttcgagc 60 cccatgttgg ggg 73 <210> 220 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 220 tgtggtgtag ctcagtcggt agagcatcag actctaaatc tgagggtcca gggttcaggt 60 ccctgttcgg gtgccaaaa 79 <210> 221 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 221 gcccggatag ctcagtcggt agagcatcag actttaaatc tgagggtcca gggttcaagt 60 ccctgttcgg gcg 73 <210> 222 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 222 gcctggatag ctcagtcggt agagcatcag actttaaatc tgagggtcca gggttcaagt 60 ccctgttcag gcg 73 <210> 223 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 223 gcctggatag ctcaattggt agagcatcag actttaaatc tgagggttca gggttcaagt 60 ccctgttcag gcg 73 <210> 224 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 224 gcccagccag ctcagtaggt agagtatgag actttaaatc tcagggtggt gggttcgagc 60 cccatgttgg ggg 73 <210> 225 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 225 tgtggtgtag ctcagtcggt agagcatcag actttaaatc tgagggtcca gggttcaggt 60 ccctgttcgg gtgccaaaa 79 <210> 226 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 226 gtagtcgtgg ccgagtggtt aaggcgatgg actctaaatc cattggggtt tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 227 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 227 gtagtcgtgg ccgagtggtt aaggcgatgg actctaaatc cattggggtc tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 228 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 228 gtagtcgtgg ccaagtgagt aaggcaatgg actctaaatc cattggggtc tcccagcaca 60 ggttcaaatc ctgctgacta tg 82 <210> 229 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 229 gtagtcgtgg ccgagtggtt aaggcgatgg acttcaaatc cattggggtt tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 230 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 230 gtagtcgtgg ccgagtggtt aaggcgatgg acttcaaatc cattggggtc tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 231 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 231 gtagtcgtgg ccaagtgagt aaggcaatgg acttcaaatc cattggggtc tcccagcaca 60 ggttcaaatc ctgctgacta tg 82 <210> 232 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 232 gtagtcgtgg ccgagtggtt aaggcgatgg actttaaatc cattggggtt tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 233 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 233 gtagtcgtgg ccgagtggtt aaggcgatgg actttaaatc cattggggtc tccccgcgca 60 ggttcgaatc ctgccgacta cg 82 <210> 234 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 234 gtagtcgtgg ccaagtgagt aaggcaatgg actttaaatc cattggggtc tcccagcaca 60 ggttcaaatc ctgctgacta tg 82 <210> 235 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 235 gctgtgatgg ccgagtggtt aaggcgttgg actctaaatc caatggggtc tccccgcgca 60 ggttcgaatc ctgctcacag cg 82 <210> 236 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 236 gtcacggtgg ccgagtggtt aaggcgttgg actctaaatc caatggggtt tccccgcaca 60 ggttcgaatc ctgttcgtga cg 82 <210> 237 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 237 gctgtgatgg ccgagtggtt aaggcgttgg actctaaatc caatgggttc ttcccgcgca 60 ggttcaaatc ctgctcacag cg 82 <210> 238 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 238 gctgtgatgg ccgagtggtt aaggcgttgg acttcaaatc caatggggtc tccccgcgca 60 ggttcgaatc ctgctcacag cg 82 <210> 239 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 239 gtcacggtgg ccgagtggtt aaggcgttgg acttcaaatc caatggggtt tccccgcaca 60 ggttcgaatc ctgttcgtga cg 82 <210> 240 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 240 gctgtgatgg ccgagtggtt aaggcgttgg acttcaaatc caatgggttc ttcccgcgca 60 ggttcaaatc ctgctcacag cg 82 <210> 241 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 241 gctgtgatgg ccgagtggtt aaggcgttgg actttaaatc caatggggtc tccccgcgca 60 ggttcgaatc ctgctcacag cg 82 <210> 242 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 242 gtcacggtgg ccgagtggtt aaggcgttgg actttaaatc caatggggtt tccccgcaca 60 ggttcgaatc ctgttcgtga cg 82 <210> 243 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 243 gctgtgatgg ccgagtggtt aaggcgttgg actttaaatc caatgggttc ttcccgcgca 60 ggttcaaatc ctgctcacag cg 82 <210> 244 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 244 gacgaggt...

Claims

1. A tRNA encoded by a sequence comprising a nucleotide sequence selected from any one of SEQ ID NOs: 39, 181, 40, 179, 182, and 186.

2. a) contain 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 of claim 1.

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

4. a) comprising 1, 2, 3, 4, or more than 4 copies of a nucleotide sequence encoding said tRNA; b) comprising a nucleotide sequence selected from any one of SEQ ID NOs: 869 to 888; and / or c) comprising the nucleotide sequence shown in Table 4; The expression vector of claim 3.

5. 4. The expression vector of 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 viral (AAV) vector; The expression vector of claim 5.

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

8. a) the tRNA or expression vector is not conjugated or attached to another moiety or carrier particle, and / or b) the pharmaceutical composition does not comprise nanoparticles and / or does not comprise an aminolipid delivery compound; 8. The pharmaceutical composition of claim 7.

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

10. a) the cells contain fewer truncated gene products than cells without the tRNA; b) the cells contain a greater amount of a functional gene product than cells without the tRNA; c) the gene is a 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 termination codon is the SCN1A gene; 10. The pharmaceutical composition of claim 9.

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

12. a) the SCN1A gene product produced by said tRNA is a functional SCN1A gene product comprising any one of SEQ ID NOs: 863-868; and / or b) a 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; 11. The pharmaceutical composition of claim 10.

13. a) the functional SCN1A gene product has greater activity than the truncated SCN1A gene product; and / or b) Functional SCN1A gene product is v 1.1 Protein, 13. The pharmaceutical composition of claim 12.

14. 10. The pharmaceutical composition of claim 9, wherein the cells are human cells.

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

16. a) the gene is a gene shown in Table 5 or Table 6, and / or b) the premature stop codon-mediated disorder is Duchenne muscular dystrophy or Dravet syndrome and / or a disorder shown in Table 5 or Table 6; 16. The pharmaceutical composition of claim 15.

17. 17. The pharmaceutical composition of claim 16, further comprising stiripentol, cannabidiol, a ketogenic diet, clobazam, topiramate, fenfluramine, or valproic acid.

18. 16. The pharmaceutical composition of claim 15, wherein the subject is a human.

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