Engineered tRNAs and methods of use

Abstract

Generally, the present disclosure relates to a tRNA that encodes one amino acid but is covalently linked to a different amino acid.The tRNA can correct missense mutations by providing different amino acids during protein synthesis.Such tRNAs can be used to correct pathogenic missense mutations.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 354,364, filed June 22, 2022, and U.S. Provisional Patent Application No. 63 / 438,236, filed January 10, 2023, each of which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under Grant No. GM105386 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] I. Sequence Listing This application contains a Sequence Listing which has been submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy, created on June 21, 2023, is named ARCD.P0781WO-Sequence Listing.txt, and is 172,211 bytes in size.

[0004] II. Technical Field The present invention relates to the fields of molecular biology, genetic engineering, and medicine. [Background technology]

[0005] III. Background Mutations in protein-coding regions of DNA can result in changes in amino acid identity in the protein product (missense) or the introduction of a premature stop codon (nonsense). Several patents and companies have recently embarked on the treatment of nonsense diseases using engineered tRNAs that read through the stop codon, thus bypassing the nonsense mutation (PMID: 30778053, 33567469). Similar principles can be applied to missense mutations.

[0006] The concept underlying MC-tRNAs has previously been described as missense suppressor tRNAs, which are related to the more commonly discussed nonsense suppressor tRNAs. These altered tRNAs have been shown to be functional in bacteria and yeast (ISBM:978-3-642-75178-3, PMID:2502189, PMID:30007351) and to occur at low levels in the human population (PMID:30643023), although their expression is unknown. In yeast, such tRNAs have been described through selection experiments to restore function to mutant proteins, suggesting that such an idea could be applied to disease (PMID:32476470). A fundamental obstacle to the implementation of this idea has to do with the proofreading of tRNAs by their cognate aminoacyl synthetases. The fidelity of the genetic code is ensured by the accurate charging of a set of isoaccepting tRNAs by their cognate synthetases; deviations from this guarantee proteome-wide mistranslation with both detrimental and adaptive effects. To ensure accurate charging, synthetases structurally inspect many "identity elements" of tRNAs to ensure accuracy. With the exception of type II tRNAs, tRNA-Ser and tRNA-Leu, the anticodon of tRNAs is considered the identity element for proofreading by synthetases (PMIDs: 4879401, 8128220). Therefore, it is reasonable to expect that mutation of the anticodon of a tRNA, like MC-tRNA, would result in a tRNA that is rejected by its cognate synthetases and is incapable of translation (PMIDs 11698642, 28660466, 9801296). Recently, different isodecoders, such as tRNA-Arg, have been shown to have different efficiencies and nonsense suppression (PMID: 30778053). This suggests that these tRNAs can tolerate mutations to the anticodon loop and remain translationally competent, indicating some level of successful aminoacylation. However, it is unclear whether these tRNAs can be engineered to translate non-stop codons.Furthermore, neither the level nor the identity of aminoacylation of these tRNAs is clear.

[0007] MC-tRNAs, which naturally deliver arginine to treat genetic diseases resulting from missense mutations of Arg residues, are of particular interest. Among known pathogenic single-nucleotide mutations, mutations of the Arg codon to several other amino acids have been found to be the most prevalent. Summary of the Invention

[0008] Generally, the present disclosure relates to the use of tRNAs that encode one amino acid but result in a different amino acid during protein synthesis to correct missense mutations. Such tRNAs can be used to correct missense mutations, including those that cause or contribute to disease.

[0009] Disclosed herein are tRNA molecules covalently linked to a first amino acid, the tRNA molecule comprising an anticodon loop sequence capable of hybridizing to an mRNA sequence encoding a second amino acid, the second amino acid being different from the first amino acid.

[0010] The first amino acid can be any amino acid, including any of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In some aspects, the first amino acid is Ala. In some aspects, the first amino acid is Arg. In some aspects, the first amino acid is Asn. In some aspects, the first amino acid is Asp. In some aspects, the first amino acid is Cys. In some aspects, the first amino acid is Glu. In some aspects, the first amino acid is Gln. In some aspects, the first amino acid is Gly. In some aspects, the first amino acid is His. In some aspects, the first amino acid is Ile. In some aspects, the first amino acid is Leu. In some aspects, the first amino acid is Lys. In some aspects, the first amino acid is Met. In some aspects, the first amino acid is Phe. In some aspects, the first amino acid is Pro. In some aspects, the first amino acid is Ser. In some aspects, the first amino acid is Thr. In some aspects, the first amino acid is Trp. In some aspects, the first amino acid is Tyr. In some aspects, the first amino acid is Val.

[0011] In some aspects, the first amino acid is not Ala. In some aspects, the first amino acid is not Arg. In some aspects, the first amino acid is not Asn. In some aspects, the first amino acid is not Asp. In some aspects, the first amino acid is not Cys. In some aspects, the first amino acid is not Glu. In some aspects, the first amino acid is not Gln. In some aspects, the first amino acid is not Gly. In some aspects, the first amino acid is not His. In some aspects, the first amino acid is not Ile. In some aspects, the first amino acid is not Leu. In some aspects, the first amino acid is not Lys. In some aspects, the first amino acid is not Met. In some aspects, the first amino acid is not Phe. In some aspects, the first amino acid is not Pro. In some aspects, the first amino acid is not Ser. In some aspects, the first amino acid is not Thr. In some aspects, the first amino acid is not Trp. In some aspects, the first amino acid is not Tyr. In some aspects, the first amino acid is not Val.

[0012] The second amino acid can be any amino acid, including any of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In some aspects, the second amino acid is Ala. In some aspects, the second amino acid is Arg. In some aspects, the second amino acid is Asn. In some aspects, the second amino acid is Asp. In some aspects, the second amino acid is Cys. In some aspects, the second amino acid is Glu. In some aspects, the second amino acid is Gln. In some aspects, the second amino acid is Gly. In some aspects, the second amino acid is His. In some aspects, the second amino acid is Ile. In some aspects, the second amino acid is Leu. In some aspects, the second amino acid is Lys. In some aspects, the second amino acid is Met. In some aspects, the second amino acid is Phe. In some aspects, the second amino acid is Pro. In some aspects, the second amino acid is Ser. In some aspects, the second amino acid is Thr. In some aspects, the second amino acid is Trp. In some aspects, the second amino acid is Tyr. In some aspects, the second amino acid is Val.

[0013] In some aspects, the second amino acid is not Ala. In some aspects, the second amino acid is not Arg. In some aspects, the second amino acid is not Asn. In some aspects, the second amino acid is not Asp. In some aspects, the second amino acid is not Cys. In some aspects, the second amino acid is not Glu. In some aspects, the second amino acid is not Gln. In some aspects, the second amino acid is not Gly. In some aspects, the second amino acid is not His. In some aspects, the second amino acid is not Ile. In some aspects, the second amino acid is not Leu. In some aspects, the second amino acid is not Lys. In some aspects, the second amino acid is not Met. In some aspects, the second amino acid is not Phe. In some aspects, the second amino acid is not Pro. In some aspects, the second amino acid is not Ser. In some aspects, the second amino acid is not Thr. In some aspects, the second amino acid is not Trp. In some aspects, the second amino acid is not Tyr. In some aspects, the second amino acid is not Val.

[0014] In some aspects, the second amino acid is glutamine, histidine, tryptophan, or cysteine.

[0015] The anticodon loop can comprise any anticodon sequence. In some aspects, the anticodon loop sequence is UUU. In some aspects, the anticodon loop sequence is UUC. In some aspects, the anticodon loop sequence is UUA. In some aspects, the anticodon loop sequence is UUG. In some aspects, the anticodon loop sequence is CUU. In some aspects, the anticodon loop sequence is CUC. In some aspects, the anticodon loop sequence is CUA. In some aspects, the anticodon loop sequence is CUG. In some aspects, the anticodon loop sequence is AUU. In some aspects, the anticodon loop sequence is AUC. In some aspects, the anticodon loop sequence is AUA. In some aspects, the anticodon loop sequence is AUG. In some aspects, the anticodon loop sequence is GUU. In some aspects, the anticodon loop sequence is GUC. In some aspects, the anticodon loop sequence is GUA. In some aspects, the anticodon loop sequence is GUG. In some aspects, the anticodon loop sequence is UCU. In some aspects, the anticodon loop sequence is UCC. In some aspects, the anticodon loop sequence is UCA. In some aspects, the anticodon loop sequence is UCG. In some aspects, the anticodon loop sequence is CCU. In some aspects, the anticodon loop sequence is CCC. In some aspects, the anticodon loop sequence is CCA. In some aspects, the anticodon loop sequence is CCG. In some aspects, the anticodon loop sequence is ACU. In some aspects, the anticodon loop sequence is ACC. In some aspects, the anticodon loop sequence is ACA. In some aspects, the anticodon loop sequence is ACG. In some aspects, the anticodon loop sequence is GCU. In some aspects, the anticodon loop sequence is GCC. In some aspects, the anticodon loop sequence is GCA. In some aspects, the anticodon loop sequence is GCG. In some aspects, the anticodon loop sequence is UAU.In some aspects, the anticodon loop sequence is UAC. In some aspects, the anticodon loop sequence is UAA. In some aspects, the anticodon loop sequence is UAG. In some aspects, the anticodon loop sequence is CAU. In some aspects, the anticodon loop sequence is CAC. In some aspects, the anticodon loop sequence is CAA. In some aspects, the anticodon loop sequence is CAG. In some aspects, the anticodon loop sequence is AAU. In some aspects, the anticodon loop sequence is AAC. In some aspects, the anticodon loop sequence is AAA. In some aspects, the anticodon loop sequence is AAG. In some aspects, the anticodon loop sequence is GAU. In some aspects, the anticodon loop sequence is GAC. In some aspects, the anticodon loop sequence is GAA. In some aspects, the anticodon loop sequence is GAG. In some aspects, the anticodon loop sequence is UGU. In some aspects, the anticodon loop sequence is UGC. In some aspects, the anticodon loop sequence is UGA. In some aspects, the anticodon loop sequence is UGG. In some aspects, the anticodon loop sequence is CGU. In some aspects, the anticodon loop sequence is CGC. In some aspects, the anticodon loop sequence is CGA. In some aspects, the anticodon loop sequence is CGG. In some aspects, the anticodon loop sequence is AGU. In some aspects, the anticodon loop sequence is AGC. In some aspects, the anticodon loop sequence is AGA. In some aspects, the anticodon loop sequence is AGG. In some aspects, the anticodon loop sequence is GGU. In some aspects, the anticodon loop sequence is GGC. In some aspects, the anticodon loop sequence is GGA. In some aspects, the anticodon loop sequence is GGG.

[0016] In some aspects, the anticodon loop sequence is not UUU. In some aspects, the anticodon loop sequence is not UUC. In some aspects, the anticodon loop sequence is not UUA. In some aspects, the anticodon loop sequence is not UUG. In some aspects, the anticodon loop sequence is not CUU. In some aspects, the anticodon loop sequence is not CUC. In some aspects, the anticodon loop sequence is not CUA. In some aspects, the anticodon loop sequence is not CUG. In some aspects, the anticodon loop sequence is not AUU. In some aspects, the anticodon loop sequence is not AUC. In some aspects, the anticodon loop sequence is not AUA. In some aspects, the anticodon loop sequence is not AUG. In some aspects, the anticodon loop sequence is not GUU. In some aspects, the anticodon loop sequence is not GUC. In some aspects, the anticodon loop sequence is not GUA. In some aspects, the anticodon loop sequence is not GUG. In some aspects, the anticodon loop sequence is not UCU. In some aspects, the anticodon loop sequence is not UCC. In some aspects, the anticodon loop sequence is not UCA. In some aspects, the anticodon loop sequence is not UCG. In some aspects, the anticodon loop sequence is not CCU. In some aspects, the anticodon loop sequence is not CCC. In some aspects, the anticodon loop sequence is not CCA. In some aspects, the anticodon loop sequence is not CCG. In some aspects, the anticodon loop sequence is not ACU. In some aspects, the anticodon loop sequence is not ACC. In some aspects, the anticodon loop sequence is not ACA. In some aspects, the anticodon loop sequence is not ACG. In some aspects, the anticodon loop sequence is not GCU. In some aspects, the anticodon loop sequence is not GCC. In some aspects, the anticodon loop sequence is not GCA. In some aspects, the anticodon loop sequence is not GCG. In some aspects, the anticodon loop sequence is not UAU.In some aspects, the anticodon loop sequence is not UAC. In some aspects, the anticodon loop sequence is not UAA. In some aspects, the anticodon loop sequence is not UAG. In some aspects, the anticodon loop sequence is not CAU. In some aspects, the anticodon loop sequence is not CAC. In some aspects, the anticodon loop sequence is not CAA. In some aspects, the anticodon loop sequence is not CAG. In some aspects, the anticodon loop sequence is not AAU. In some aspects, the anticodon loop sequence is not AAC. In some aspects, the anticodon loop sequence is not AAA. In some aspects, the anticodon loop sequence is not AAG. In some aspects, the anticodon loop sequence is not GAU. In some aspects, the anticodon loop sequence is not GAC. In some aspects, the anticodon loop sequence is not GAA. In some aspects, the anticodon loop sequence is not GAG. In some aspects, the anticodon loop sequence is not UGU. In some aspects, the anticodon loop sequence is not UGC. In some aspects, the anticodon loop sequence is not UGA. In some aspects, the anticodon loop sequence is not UGG. In some aspects, the anticodon loop sequence is not CGU. In some aspects, the anticodon loop sequence is not CGC. In some aspects, the anticodon loop sequence is not CGA. In some aspects, the anticodon loop sequence is not CGG. In some aspects, the anticodon loop sequence is not AGU. In some aspects, the anticodon loop sequence is not AGC. In some aspects, the anticodon loop sequence is not AGA. In some aspects, the anticodon loop sequence is not AGG. In some aspects, the anticodon loop sequence is not GGU. In some aspects, the anticodon loop sequence is not GGC. In some aspects, the anticodon loop sequence is not GGA. In some aspects, the anticodon loop sequence is not GGG.

[0017] The tRNA can include an anticodon loop sequence and an amino acid not encoded by the anticodon loop. In certain aspects, the amino acid not encoded by the anticodon loop is Arg. In certain aspects, the anticodon loop sequence does not encode Arg.

[0018] In certain aspects, the anticodon loop sequence is TIFF2025525377000001.tif26160. In certain aspects, the anticodon loop sequence is TIFF2025525377000002.tif11131. In certain aspects, the anticodon loop sequence is CUG, UUG, GUG, CCA, or GCA from 5' to 3'. In some aspects, the anticodon loop sequence is not ACG, CCG, CCU, UCG, GCG, or UCU from 5' to 3'. In some aspects, the mRNA sequence is not CGU, CGC, CGA, CGG, AGA, or AGG from 5' to 3'. In some aspects, the mRNA sequence is not CGU, CGC, CGA, CGG, AGA, or AGG from 5' to 3'. The file is TIFF2025525377000003.tif33160.

[0019] In certain aspects, the anticodon loop sequence is TIFF2025525377000004.tif26160. In certain aspects, the anticodon loop sequence is From 5' to 3' TIFF2025525377000005.tif11135. In certain aspects, the anticodon loop sequence is not AGA, CGA, GCU, UGA, ACU, or GGA from 5' to 3'. In some aspects, the mRNA sequence is not UCU, UCC, UCA, UCG, AGU, or AGC from 5' to 3'. TIFF2025525377000006.tif33160. In some aspects, the mRNA sequence is not UAA, UGA, or UAG.

[0020] The tRNA molecule can contain a sequence with substitutions, deletions, or additions relative to a mammalian tRNA molecule. Disclosed are tRNA molecules containing a sequence with up to one, two, three, four, or five substitutions relative to a mammalian tRNA molecule. In certain aspects, the mammalian tRNA molecule is a human tRNA molecule.

[0021] Nucleic acids encoding tRNA sequences are also disclosed. The nucleic acids can comprise expression vectors. The nucleic acids can comprise plasmids. The nucleic acids can be used to synthesize tRNA sequences. Such synthesis can occur in cells. The cells can be bacterial cells, insect cells, yeast cells, vertebrate cells, or any other cells that can express tRNA. Synthesis can occur by in vitro transcription.

[0022] Nucleic acids having the sequence of any one of SEQ ID NOs: 1-144 are disclosed.

[0023] Also disclosed are tRNAs or nucleic acids encoding tRNAs that contain one or more modifications to the tRNAs disclosed herein. The modifications can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications to the tRNA. Also disclosed are tRNAs or nucleic acids encoding tRNAs that contain one or more modifications to the tRNA of any one of SEQ ID NOs. 1-134. The modifications can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications to the tRNA. The modifications can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications to the tRNA of any one of SEQ ID NOs. 1-134.

[0024] Also disclosed are tRNAs having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any range derivable therein, sequence identity to any of the tRNAs disclosed herein. Also disclosed are nucleic acids encoding tRNAs having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any range derivable therein, sequence identity to any of the tRNAs disclosed herein. Also disclosed are tRNAs having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any range derivable therein, sequence identity to a tRNA of any of SEQ ID NOs. 1-134. Also disclosed are nucleic acids encoding tRNAs having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any range derivable therein, sequence identity to a tRNA of any of SEQ ID NOs. 1-134.

[0025] Also disclosed is a vector comprising any of the nucleic acids herein.The vector can be any vector, including any vector that can deliver nucleic acid to target cells.In some aspects, the vector is a virus.In some aspects, the vector is an adenovirus, a retrovirus, a lentivirus, or an adeno-associated virus (AAV), including any derivative thereof.

[0026] Also disclosed are cells comprising any of the tRNA molecules disclosed herein.Also disclosed are cells comprising any of the nucleic acids disclosed herein.Also disclosed are cells comprising any of the vectors disclosed herein.The cells can be cells used for cell therapy, for example, autologous stem cell therapy.

[0027] The present specification discloses a method for producing the tRNA disclosed herein.In certain aspects, the method comprises contacting the nucleic acid comprising anticodon loop with the amino acid that is not naturally encoded by anticodon loop.In certain aspects, the nucleic acid is modified from natural tRNA sequence so that aminoacyl-tRNA synthetase can bind the amino acid that is not normally encoded by tRNA.

[0028] Disclosed herein are methods for modifying proteins produced by genes using one or more of the tRNAs disclosed herein. Also disclosed are methods for generating wild-type proteins from genes with missense mutations. Also disclosed are methods for correcting missense mutations during mRNA translation. Also disclosed are methods for generating engineered proteins. Also disclosed are methods for introducing point mutations in proteins from mRNA. The methods can include one, two, three, four, five, or more steps, including any of the following: translating mRNA in the presence of one or more of the tRNAs disclosed herein, administering to a cell an effective amount of one or more of the tRNAs disclosed herein, administering to a cell an effective amount of one or more of the nucleic acids disclosed herein, administering to a cell an effective amount of one or more of the vectors disclosed herein, and detecting the protein from the cell. Administration can include any means of introducing the tRNA, nucleic acid, and / or vector into a cell, including by transfection, electroporation, or transduction. The cell can be any cell, for example, a mammalian cell. The cell can be a human cell. The cell may be a cell containing a missense mutation.

[0029] Disclosed herein are methods for treating or preventing a disease, such as a genetic disease, in a subject. Also disclosed are methods for treating or preventing limb-girdle disease. Also disclosed are methods for restoring CAPN3 function in a cell. Also disclosed are methods for reversing the effects of CAPN3 loss of function in a cell. Also disclosed are methods for restoring CAPN3 function in a subject. Also disclosed are methods for reversing the effects of CAPN3 loss of function in a subject. Any of the methods can include one, two, three, or more steps, including any of the following: administering to a subject a therapeutically effective amount of one or more tRNAs disclosed herein, administering to a subject a therapeutically effective amount of one or more nucleic acids disclosed herein, and administering to a subject a therapeutically effective amount of one or more vectors disclosed herein. The subject may have a genetic disease. The subject may have cancer. Diseases, including genetic diseases or cancer, can be characterized, caused, or accelerated by single nucleotide variations (SNVs). SNVs can result in missense mutations in genes. Genes disclosed herein that may have missense mutations include ABCD1, CAPN3, GLA, GBA, GALC, ARSA, SGSH, HGSNAT, IDS, OTC, DHCR7, or HEXA. Methods for correcting missense mutations in genes are disclosed herein. The gene may be ABCD1, CAPN3, GLA, GBA, GALC, ARSA, SGSH, HGSNAT, IDS, OTC, DHCR7, or HEXA. The gene may be any gene in Table 2. The SNV in the gene may be any SNV disclosed in Table 2. In some aspects, the SNV is recessive. In some aspects, the genetic disease is a recessive disease. The disease may be adrenoleukodystrophy, Fabry disease, Gaucher disease type I, metachromatic leukodystrophy, mucopolysaccharidosis, ornithine transcarbamylase deficiency, Smith-Lemli-Opitz syndrome, Tay-Sachs disease, Niemann-Pick disease, or very long-chain acyl-CoA dehydrogenase deficiency.

[0030] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0031] The use of the word "a" or "an" in conjunction with the term "comprising" can mean "one," but this is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Any term used in the singular also includes the plural and vice versa.

[0032] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination with or excluding each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspects.

[0033] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), "characterized by" (and any form of including, such as "characterized as"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude further, unrecited elements or method steps.

[0034] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this specification. The phrase "consisting of" excludes any unspecified element, step, or ingredient. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term "comprising" can also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0035] It is contemplated that any aspect discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0036] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as "use of" any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0037] The use of one or more sequences or compositions can be used according to any of the methods described herein.Other aspects and embodiments are discussed throughout this application.Any embodiment or aspect discussed in one aspect of this disclosure also applies to other aspects of this disclosure, and vice versa.

[0038] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention can be applied to any other embodiment or aspect of the invention. Moreover, any composition of the invention can be used in any method of the invention, and any method of the invention can be used to produce or utilize any composition of the invention. Aspects of an embodiment described in an example are also aspects that can be implemented in the context of aspects discussed elsewhere in a different example or elsewhere in this application, e.g., in the Summary of the Invention, Detailed Description, Claims, and Brief Description of the Drawings.

[0039] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. (Figure 1) Figures 1A-1D. Distribution of pathogenic missense mutations in human diseases. A) Pie charts illustrating the distribution of known SNVs among all SNVs (left) and among pathogenic SNVs (right) in the ClinVar database. B) A few missense SNVs are common in the population and have been reported multiple times to ClinVar, while most SNVs are infrequent and reported only a few times. C) Among pathogenic missense mutations, not all amino acids give rise to pathogenic SNVs equally. D) At the codon level, the Arg codon gives rise to multiple pathogenic SNVs. Codon-level mutations with a maximum number of submitters for any individual SNV of 10 or more are labeled. (Figure 2-1) Figures 2A-2F. Arginine iso-decoder tRNAs can be engineered into the read-through mutant codon to restore function to the reporter protein. A) Illustration of a tandem fluorescent reporter for arginine mistranslation. In GFP, Arg96 is required for rapid fluorophore maturation; mutation of Arg96 results in non-fluorescent GFP and the native RFP loading control. Expression of a mutator MC-tRNA to decode mutant position 96 and deliver Arg restores GFP fluorescence. B) The construct in A was repeated for a full set of potential SNVs that could change the Arg codon to Cys, Trp, Gin, or His. C) (Top) (Left) Flow cytometry shows that GFP and RFP fluorescence scale together in positive control cells. (Right) The GFP-to-RFP ratio is near 1 because the reporter has a tandem protein. (Bottom) For GFP* constructs in which Arg96 is mutated as in A and B, GFP fluorescence does not increase with RFP. (Left) Representative data from the Arg96His construct. (Right) GFP / RFP ratio traces for all mutant constructs confirming loss of fluorescence. D) Flow cytometry showing restoration of GFP fluorescence compared to RFP fluorescence loading control. GFP fluorescence is restored when tRNA, the appropriate tRNA, is expressed. E) Annexin staining (right) indicates early cell death, which increases with tRNA expression levels. F) DAPI staining indicates late cell death. Cells expressing low levels of MC-tRNA are not toxic. (Figure 2-2) See the description of Figure 2-1. (Figure 3A) Figures 3A-3C. Mass spectrometry analysis shows wild-type sequence reinstatement for disease-associated peptides. A) Schematic of peptide expression constructs. Because the Arg>His_G>A SNV is associated with many SNVs from many diseases and many peptides, we expressed constructs with tandem disease-associated peptides separated by a Lys-C protease site and a C-terminal affinity purification tag. Similar constructs were made for each Arg-associated SNV. B) Representative LC-MS-MS data for peptide XYZ, associated with Xyz syndrome. C) Summary of peptide reinstatement for many disease-associated peptides with expression of MC-tRNAArg. (Figure 3B) See legend to Figure 3A. (Figure 3C) See legend to Figure 3A. (Figure 4A-4F). MC-tRNA can be applied to other isoacceptors besides arginine. A) Schematic of a fluorescent reporter for monitoring serine delivery at position 65 of GFP. Position 65 is mutated to ArgCCG, which disrupts GFP chromophore formation and results in non-fluorescent cells. Delivery of serine to position 65 by MC-tRNA restores fluorescence. B) Control transfections were performed using plasmids expressing no tRNA but GFP only, RFP only, or tandem GFP-RFP. Cells were filtered for RFP expression above the black line for further analysis. (Left) Fluorescence for the GFP and RFP channels is shown for cells 24 h posttransfection, representing a single replicate experiment. (Right) Histograms of the GFP-to-RFP ratio are shown for RFP- and GFP-RFP-expressing cells. C) HEK293T cells were transfected with a plasmid carrying a tandem GFP*-RFP construct in which Ser65 from A was mutated to Arg(CCG) and a tRNA construct. Fluorescence was monitored by flow cytometry at 24, 48, and 72 hours posttransfection. GFP and RFP fluorescence for a single replicate experiment at 24 hours are shown. D) Histograms of the GFP-to-RFP ratio for the constructs in C are shown. Expression of tRNASer(CGG) in several different backbones all restores GFP fluorescence. Expression of tRNASer(CGA) does not restore fluorescence because it does not decode ArgCCG. E) The GFP-to-RFP ratio is used as a surrogate for tRNA expression and compared with Annexin V staining for cell death. An increase in the GFP-RFP ratio does not correlate with increased cell death. F) Annexin fluorescence was used to determine cells as live or dead using the threshold indicated by the dashed line in E. The percentage of dead cells was calculated for each expression plasmid at 24 and 48 hours for three replicate experiments. (Figure 4B) See legend to Figure 4A. (Figure 4C) See legend to Figure 4A. (Figure 4D) See legend to Figure 4A. (Figure 4E) See legend to Figure 4A. (Figure 4F) See legend to Figure 4A. (Figure 5A) Figures 5A-5C. Stable lines are viable and recover GFP. A) Cells expressing the WT GFP-RFP construct are polyclonal. Two populations are visible, one with high levels of GFP fluorescence. Only cells with low DAPI live / dead staining are shown. B) Cells expressing a GFP-RFP construct carrying the R96C mutation, which abolishes GFP fluorescence. This population has low GFP fluorescence. Only cells with low DAPI live / dead staining are shown. C) Cells expressing the Arg>Cys repair mc-tRNA are viable but proliferate slowly. Compared to B, cells with low DAPI live / dead staining and GFP fluorescence indicating low levels of repair of the GFP mutation R96C are shown here. (Figure 5B) See legend to Figure 5A. (Figure 5C) See legend to Figure 5A. (Figure 6A) Figures 6A-6C. Stable lines are viable and recover GFP. A) Cells expressing the WT GFP-RFP construct are polyclonal. Two populations are visible, one with high levels of GFP fluorescence. Only cells with low DAPI live / dead staining are shown. B) Cells expressing a GFP-RFP construct carrying the R96Q mutation, which abolishes GFP fluorescence. This population has low GFP fluorescence. Only cells with low DAPI live / dead staining are shown. C) Cells expressing the Arg>Gln repair mc-tRNA are viable but proliferate slowly. Compared to B, cells with low DAPI live / dead staining and GFP fluorescence indicating low levels of repair of the GFP mutation R96Q are shown here. (Figure 6B) See legend to Figure 6A. (Figure 6C) See legend to Figure 6A. (Figure 7A) Figures 7A-7E. MC-tRNA-expressing cells show minimal disruption to native gene expression. mRNA sequencing was performed on stable cell lines and compared to positive, negative, and null controls. In all figures, the Y-axis is transcripts per million (TPM). A) Stable cell lines show negligible induction of the heat shock response compared to wild-type cells. A positive control of heat-shocked cells is included for comparison. B) Genes related to apoptosis were measured in stable cell lines and compared to untransfected and control stressed cells. Minimal induction of apoptotic pathways was observed. C) Genes related to the general stress response further demonstrate minimal changes in gene expression and, therefore, minimal disruption to cellular physiology. D) To understand whether there is an immunogenic effect on mc-tRNA expression in this setting, genes related to the innate immune response were analyzed. Minimal changes in the expression of these genes were observed. E) Expression of the glycolytic enzyme GAPDH was used as a control since no changes in gene expression for this pathway are expected. These data confirm that global measures of gene expression behave as expected. (Figure 7B) See legend to Figure 7A. (Figure 7C) See legend to Figure 7A. (Figure 7D) See legend to Figure 7A. (Figure 7E) See legend to Figure 7A. (Figure 8A) Figures 8A-8E. Validation of a GFP-based mistranslation reporter. (A) Flow cytometry analysis of GFP expression in cells overexpressing WT and mutant GFP-mCherry fusion proteins. All single live cells are shown. Gates were set based on single-color controls. (B) Density curves of GFP expression in cells overexpressing WT and mutant GFP-mCherry fusion proteins. (C)-(E) GFP signal normalized to mCherry for cells overexpressing GFP-mCherry fusion proteins with point mutations in GFP65 (C), GFP96 (D), and other GFP point mutations (E). Each dot represents one biological replicate. The mean and standard deviation for each sample are shown as solid black dots and vertical lines, respectively. (Figure 8B) See legend to Figure 8A. (Figure 8C) See legend to Figure 8A. (Figure 8D) See legend to Figure 8A. (Figure 8E) See legend to Figure 8A. Figure 9A-9C. Validation of other fluorescent protein (FP)-based mistranslation reporters. (A) Flow cytometry analysis of mPlum expression in cells overexpressing WT and R96C eGFP-mPlum fusion proteins. All single live cells are shown. Gates were set based on single-color controls. (B) Density curves of mPlum expression in cells overexpressing WT and R96C eGFP-mPlum fusion proteins. (C) FP signal normalized to eGFP for cells overexpressing each FP-based mistranslation reporter. Each dot represents one biological replicate. The mean and standard deviation for each sample are shown as solid black dots and vertical lines, respectively. (Figure 10) Overview of all quantified mistranslation reporters. Each well was labeled with a fluorescent protein (FP) and the position of the mutation that acts as the mistranslation reporter. When the WT amino acid (aa) is mutated to a mutant amino acid (Mut aa) at this position, the FP reporter signal is reduced by more than 2-fold. Previously reported FP reporters are colored gray. (Figure 11A) Figures 11A-11G. Figure 3: mctRNA Ser-tRNA Arg(CCG) Results. (A) Ser-tRNA Arg(CCG) Sequence of the expression cassette. The tRNA sequence is highlighted in bold, and the anticodon is underlined. 200 bp of endogenous sequence upstream and downstream of the tRNA-Ser-CGA-1-1 gene was maintained to allow proper transcription and processing of the mctRNA. (B) Density curves of GFP signal normalized to mCherry in cells overexpressing i) WT GFP, ii) GFP(S65R), and iii) GFP(S65R) together with mctRNA (n=3). (C) Ser-tRNA Arg(CCG) Ser-tRNA normalized to endogenous tRNA-Ser-CGA-1-1 in cells overexpressing GFP(S65R) with or without Arg(CCG) (D) Ser-tRNA expression levels (n=3). Arg(CCG) Endogenous tRNA-Ser-CGA-1-1 and Ser-tRNA in cells overexpressing GFP(S65R) together with Arg(CCG) (E) Charging levels of endogenous tRNA-Ser-CGA-1-1 and Ser-tRNA Arg(CCG) Average mutation rate at each nucleotide position (n=3). (F) mctRNA Ser-tRNA Arg(CCG) Differential gene expression in cells expressing GFP(S65R)-mCherry with vs. without. (G) Biological process gene ontology enrichment analysis for significantly up- or down-regulated genes (p>0.05, absolute fold change (FC)>2). Vertical solid lines indicate p=0.05. (Figure 11B) See legend to Figure 11A. (Figure 11C) See legend to Figure 11A. (Figure 11D) See legend to Figure 11A. (Figure 11E) See legend to Figure 11A. (Figure 11F) See legend to Figure 11A. (Figure 11G) See legend to Figure 11A. (Figure 12A) Figures 12A-12H. mctRNA Arg-tRNA Cys / His / Gln / Trp(***) (A) mctRNA Arg-tRNAs of 19 different endogenous Arg tRNA isodecoder backbones Gln(CUG) (B) GFP signal compared to WT GFP in cells overexpressing GFP(R96Q) with Arg-tRNA. WT GFP and GFP(R96Q) are shown for comparison. Cys / His / Gln / Trp(***) Sequence of the expression cassette. The tRNA sequence is highlighted in bold, and the anticodon region is underlined. To generate the homologous mctRNA, the anticodon region was mutated to Cys, His, Gln, and Trp anticodons. 200 bp of endogenous sequence upstream and downstream of tRNA-Arg-CCT-4-1 was maintained to allow proper transcription and processing of the mctRNA. (C) Density curves of GFP signal normalized to mCherry for i) WT GFP, ii) the GFP R96 mutant, and iii) cells overexpressing the GFP R96 mutant with the corresponding mctRNA. (D) IP-MS quantification of the mctRNA-modified peptide SAMPEGYVQER in cells overexpressing the GFP R96 mutant with and without the corresponding mctRNA (n = 2). (E) MS spectrum of the mctRNA-modified peptide SAMPEGYVQER in cells overexpressing the GFP R96 mutant with the corresponding mctRNA. (F) Expression levels of Arg mctRNA normalized to endogenous tRNA-Arg-CCT-4-1 in cells overexpressing GFP R96 mutants with or without the corresponding mctRNA (n=3). (G) Charging levels of endogenous tRNA-Arg-CCT-4-1 and Arg mctRNA in cells overexpressing GFP R96 mutants with the corresponding mctRNA (n=3). (H) Average mutation rates at each nucleotide position of endogenous tRNA-Ser-CGA-1-1 and Arg mctRNA (n=3). (Figure 12B) See legend to Figure 12A. (Figure 12C) See legend to Figure 12A. (Figure 12D) See legend to Figure 12A. (Figure 12E) See legend to Figure 12A. (Figure 12F) See legend to Figure 12A. (Figure 12G) See legend to Figure 12A. (Figure 12H) See legend to Figure 12A. (Figure 13A) Figures 13A-13E. Cellular response to Arg mctRNA. (A) Differential gene expression in cells expressing GFP mutant-mCherry and the corresponding mctRNA versus cells expressing only GFP mutant-mCherry. (B)-(E) Biological Process Gene Ontology (GO) analysis for genes significantly up- or down-regulated with Arg mctRNA expression (p>0.05, absolute fold change (FC)>2). Vertical solid lines indicate p=0.05. b-c. The top five GO terms with the highest fold enrichment for up- or down-regulated genes are displayed. (Figure 13B) See legend to Figure 13A. (Figure 13C) See legend to Figure 13A. (Figure 13D) See legend to Figure 13A. (Figure 13E) See legend to Figure 13A. Figure 14A-14C. Arg-tRNA Gln(CUG) rescued mutant LGMD2A-associated protein CAPN3. (A) Schematic diagram of the domains and mutation sites of calpain 3 protein. (B) Arg-tRNA Gln(CUG) Western blot of cells transfected with or without mock construct, WT CAPN3, catalytically inactive mutant C129S, and deletion mutant R490Q. In the figure, full-length (FL) CAPN3 (94 kDa), two bands of autolysis products TIFF2025525377000007.tif5128, and GAPDH (37 KDa) are marked. GAPDH is a loading control. (C) Quantification of relative Western blot band intensities (n=4 biological replicates). DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description of the Invention Many genetic diseases are caused by mutations in the protein-coding region of DNA that result in changes in amino acid identity (missense) in the protein product. The resulting mutant proteins may be biologically inactive and therefore may result in significant functional defects leading to disease. Specific missense-correcting tRNAs (MC-tRNAs) for protein biosynthesis in cells are disclosed. The MC-tRNAs have a covalently attached (aminoacylated or charged) amino acid that does not match the anticodon sequence for reading the codon for the charged amino acid. These engineered MC-tRNAs can restore the original protein sequence during translation, thereby generating functional proteins and altering disease outcomes. Specific Arg or Ser mutations are inserted into each MC-tRNA. Arg and MC-tRNA Ser Disclosed are MC-tRNAs that can be modified into functional proteins by Arg Also disclosed is the restoration of wild-type sequence of disease-related peptides by MC-tRNA.Also disclosed is the MC-tRNA that naturally delivers arginine to treat genetic diseases caused by missense mutation of Arg residue.In certain aspects, single base mutations that lead to disease are corrected, including the mutation of Arg to a different amino acid, which is the most common mutation that leads to disease.

[0042] Specific missense-corrected tRNAs (MC-tRNAs) for protein biosynthesis in cells xxx(yyy), where xxx is the amino acid attached to the 3' end and yyy is the anticodon sequence. MC-tRNAs have a covalently attached (charged) amino acid that does not match the anticodon sequence for reading the codon for the charged amino acid. The use of engineered MC-tRNAs can correct genetic diseases resulting from missense mutations, such as adrenoleukodystrophy, Sanfilippo syndrome (MPS-III-A), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), among others. In certain aspects, this approach is well suited to recessive diseases where restoring the activity of a small amount of native protein can correct the phenotype. Certain aspects focus on rare metabolic diseases with infantile onset and poor prognosis.

[0043] Certain aspects relate to identifying unequal distribution of specific types of mutations, with Arg to Cys, Trp, Gln, and His mutations being the most prevalent, accounting for 9% of all pathogenic SNVs. Aspects are disclosed relating to correcting genetic diseases by co-translational missense correction (CoMED) of genetic diseases.

[0044] I. Nucleotide Acquisition A.Synthesis Nucleic acid molecules, including the tRNA or the nucleic acid that encodes the tRNA described herein, can be produced by nucleic acid synthesis.tRNA or the nucleic acid that encodes the tRNA can be synthesized using any method known in the art, for example, phosphoramidite synthesis and / or solid-phase synthesis.tRNA or the nucleic acid that encodes the tRNA can be synthesized.

[0045] B. Expression Nucleic acid molecules, including any tRNA or tRNA-encoding nucleic acid described herein, can be produced by an expression vector. The expression vector used herein can include sequences for maintaining a plasmid or virus, and for cloning and expressing an exogenous nucleotide sequence. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, a replication origin, a transcription termination sequence, and a selection marker element. Such sequences and their use are well known in the art.

[0046] 1. Expression system There are many expression systems that contain at least some or all of the above-mentioned expression vectors. Prokaryotic and / or eukaryotic systems can be used in conjunction with the aspect of producing nucleic acid sequences. Commercially and widely available systems include, but are not limited to, bacterial, mammalian, yeast, and insect cell systems. Those skilled in the art can use an appropriate expression system to express the vector to produce the nucleic acid sequence.

[0047] 2. Gene transfer method Suitable methods for nucleic acid delivery to result in expression of the compositions are expected to include virtually any method by which nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue, or organism, as described herein or as would be known to one of skill in the art. Such methods include, but are not limited to, injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), e.g., by microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, each of which is incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, each of which is incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by particle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); or by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include gene transfer by viral transduction, e.g., lentiviral or retroviral transduction.

[0048] 3.Host cells In another aspect, the use of a host cell into which a recombinant expression vector has been introduced is contemplated.Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.Some vectors can use control sequences that allow them to replicate and / or express in both prokaryotic and eukaryotic cells.Those skilled in the art will understand the conditions for incubating host cells to maintain and allow vector replication.Techniques and conditions that may allow large-scale production of vectors and production of nucleic acids encoded by vectors are also understood and known.

[0049] It is known that for the stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of cells can integrate foreign DNA into their genome.In order to identify and select these integrants, a selectable marker (for example, for antibiotic resistance) is generally introduced into host cells together with the gene of interest.The cells that are stably transfected with introduced nucleic acid can be identified by drug selection (for example, the cells that have integrated the selectable marker gene survive, while other cells die), among other methods known in the art.

[0050] II. Pharmaceutical Compositions In certain aspects, compositions or agents for use in the methods disclosed herein, including, for example, tRNA, nucleic acids encoding tRNA, vectors containing tRNA, or cells containing tRNA, are suitably contained in a pharmaceutically acceptable carrier. The carrier can be non-toxic and biocompatible and can be selected so as not to adversely affect the biological activity of the agent. In some aspects of the present disclosure, the agents can be formulated into preparations for local delivery (i.e., to specific parts of the body, such as the brain, nervous tissue, or other tissues) or systemic delivery in solid, semi-solid, gel, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, allowing for oral, parenteral, or surgical administration. Certain aspects of the present disclosure also contemplate local administration of the composition, such as by coating a medical device.

[0051] Suitable carriers for parenteral delivery by injection, infusion, or irrigation, and for topical delivery include distilled water, physiological phosphate-buffered saline, normal Ringer's solution or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol.In addition, sterile, fixed oils can be used as solvents or suspension media.For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides.In addition, fatty acids such as oleic acid are used to prepare injection solutions.Carriers and agents can be formulated into solutions, suspensions, polymerizable or non-polymerizable gels, pastes, or ointments.

[0052] A carrier can also include a delivery vehicle to sustain (i.e., extend, delay, or modulate) the delivery of an agent or to enhance the delivery, uptake, stability, or pharmacokinetics of a therapeutic agent. Such delivery vehicles can include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles.

[0053] The solution of pharmaceutical composition can be prepared in water suitably mixed with surfactant, for example, hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, their mixture, and in oil.Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0054] In certain aspects, pharmaceutical compositions are conveniently administered in the form of injectable compositions, either as liquid solutions or suspensions, and solid forms suitable for dissolving or suspending in liquid before injection can also be prepared.These preparations can also be emulsified.Typical compositions for such purposes include a pharmaceutically acceptable carrier.For example, the composition can contain up to 10 mg, 25 mg, 50 mg, or up to about 100 mg of human serum albumin per milliliter of phosphate-buffered saline.Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, such as salts, preservatives, buffers, etc.

[0055] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antibacterial agents, antifungal agents, antioxidants, chelating agents, and inert gases. The pH and precise concentration of various components of the pharmaceutical composition are adjusted according to well-known parameters.

[0056] Further formulations are suitable for oral administration. Oral formulations contain typical excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0057] In further aspects, pharmaceutical compositions can include classical pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects can be via any common route, so long as the target tissue is available via that route. This can include oral, nasal, buccal, rectal, vaginal, or topical. Alternatively, administration can be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions will typically be administered as pharmaceutically acceptable compositions containing physiologically acceptable carriers, buffers, or other excipients. For the treatment of pulmonary conditions, aerosol delivery can be used. The volume of the aerosol can be, for example, between about 0.01 ml and 0.5 ml.

[0058] The effective amount of the pharmaceutical composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the pharmaceutical composition calculated to produce the desired response described above in conjunction with its administration, i.e., an appropriate route and treatment regimen.

[0059] A. Protein The nucleotide and protein, polypeptide and peptide sequences of various genes have been disclosed previously and can be found in recognized computerized databases.Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / World Wide Web) and The Universal Protein Resource (UniProt; uniprot.org World Wide Web).The coding region of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0060] B. Other agents It is contemplated that other agents can be used in combination with certain aspects.These additional agents include those that act in combination with and / or synergistically with tRNA, the nucleic acid encoding tRNA, the vector containing tRNA, or the cell containing tRNA as described herein.The additional agent can include those that reduce the symptoms of the disorders disclosed herein, or those that reduce the side effects associated with the therapeutic compositions disclosed herein.

[0061] III. Sequence and Single-Base Variation

[0062] Table 1. Nucleic acids according to aspects disclosed herein <h2 style=";text-align:left;direction:ltr">TIFF2025525377000008.tif222159TIFF2025525377000009.tif244159TIFF2025525377000010.tif237159TIFF2025525377000011.tif24 4159TIFF2025525377000012.tif237159TIFF2025525377000013.tif244159TIFF2025525377000014.tif237159TIFF2025525377000015.t if244159TIFF2025525377000016.tif237159TIFF2025525377000017.tif244159TIFF2025525377000018.tif242159TIFF2025525377000019.tif242159TIFF2025525377000020.tif242159TIFF2025525377000021.tif242159TIFF2025525377000022.tif242159TIFF20255253770 00023.tif242159TIFF2025525377000024.tif235159TIFF2025525377000025.tif242159TIFF2025525377000026.tif242159TIFF2025525377000027.tif242159TIFF2025525377000028.tif242159TIFF2025525377000029.tif242159TIFF2025525377000030.tif242159TIFF202 5525377000031.tif242159TIFF2025525377000032.tif242159TIFF2025525377000033.tif242159TIFF2025525377000034.tif242159TIFF2025525377000035.tif242159TIFF2025525377000036.tif243159TIFF2025525377000037.tif243159TIFF2025525377000038.tif118159<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0063] <h2 style=";text-align:left;direction:ltr"> Table 2. Single-nucleotide mutations in genetic disorders characterized by missense mutations TIFF2025525377000039.tif110159TIFF2025525377000040.tif243159TIFF2025525377000041.tif243159TIFF2025525377000042.tif118159

[0064] Table 3. Single-nucleotide mutations in genetic disorders characterized by missense or nonsense mutations TIFF2025525377000043.tif141170 [Example]

[0065] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0066] Example 1: Results Distribution analysis of pathogenic missense mutations We searched the single-nucleotide variants (SNVs) reported in the ClinVar database (ref.) to obtain detailed information about the types of pathogenic SNVs in CDS. Missense mutations accounted for 92% of all analyzed CDS SNVs, and the remainder represented nonsense mutations among all SNVs. However, many missense mutations are not disease-associated. Among CDS SNVs annotated as "pathogenic," "possibly pathogenic," and "pathogenic / possibly pathogenic," missense mutations accounted for 58% (Figure 1A). Of the approximately 42,500 total missense mutations, most had only one submitter, suggesting rare allele frequencies, and the majority were reported less than 10 times, whereas approximately 100 specific pathogenic SNVs were reported more than 20 times (Figure 1B). These results indicate that pathogenic mutations are unevenly distributed throughout the human genome and that there are specific hotspot mutations that recur at unusually high frequencies.

[0067] Breaking down pathogenic missense mutations into individual amino acids shows that Arg mutations to other amino acids account for the highest proportion (>6700) (16% of all pathogenic missense SNVs; 9% of pathogenic SNVs, including nonsense mutations), followed by Gly mutations to other amino acids (Figure 1C). Pathogenic mutations to the other 18 amino acids are similarly frequent, more than twofold less frequent than Arg or Gly mutations. Furthermore, combining the type of pathogenic mutation with the reported number of disease occurrences (Figure 1B) shows that the top four most frequent pathogenic SNVs are indeed represented by four Arg codon mutations to Cys, Trp, Gln, and His (Figure 1D). These results indicate that Arg mutations to only four amino acids represent the highest group of pathogenic single-nucleotide mutations.

[0068] MC-tRNA for Cys, Trp, Gln, and Arg codon mutations to His Arg In order to identify specific diseases that can be treated with (i) The disease is genetically recessive (ii) the mutation is in a coding sequence (iii) the mutation is associated with a known rare metabolic disorder (iv) Each SNV was submitted by four or more submitters. ClinVar was searched for diseases using the following criteria.

[0069] Applying these criteria, we identified 12 major diseases associated with at least three of the four Arg codon mutations (Table 1). Combining disease incidence estimates and SNV frequencies, we identified MC-tRNA mutations. Arg It is estimated that approximately 55 new patients in the United States per year could be treated using MC-tRNA (GTG) alone. Arg (GCA), MC-tRNA Arg (CCA), and MC-tRNA Arg (TTG) can treat approximately 49, 44, and 51 new patients per year, respectively. Arg The construct could treat approximately 200 new patients per year in the United States for this select group of rare diseases.

[0070] MC-tRNA for generating functional proteins from DNA mutations Arg Use of The present inventors have developed MC-tRNA Arg To generate functional proteins during translation from genes containing Arg mutations at the DNA level, which are thought to generate functionally defective proteins in the absence of MC-tRNA ArgTo test the feasibility of using a reporter protein, we designed one (reference). This dual fluorescent protein reporter contains both green fluorescent protein (GFP) and red fluorescent protein (RFP) in a single polypeptide (Figure 2A). The Arg96 residue in GFP is mutated to one of seven codons present in the most frequent pathogenic Arg mutations (generating GFP*), including Arg to Cys (CGC / CGT to TGC / TGT), Trp (CGG to TGG), Gln (CGA / CGG to CAA / CAG), and His (CGC / CGT to CAC / CAT) (Figure 2B; see below). This mutation extends the maturation time of GFP from hours to months (PMIDs: 14523232, 16331981, 18470931), making GFP virtually non-fluorescent on the timescale of laboratory experiments. TIFF2025525377000044.tif25160

[0071] MC-tRNA Arg The design of tRNA bodies is more complex because many tRNA bodies may fulfill this role. We have identified 20 different tRNA bodies in the reference human genome (Genome tRNA Database, http: / / gtrnadb.ucsc.edu / ). Arg The sequence to be tested is selected. Arg The anticodon sequence of each of the above is changed to that of a human tRNA that reads a Cys / Trp / Gln / His sequence (Table 2). For example, human tRNA Cys has a single anticodon sequence of GCA that reads both TGC and TGT codons through a GT wobble. Arg (GCA) is expected to read both the TGC and TGT mutants in the GFP Arg96-to-Cys construct. Arg The (TCT) sequence is MC-tRNA Arg Six tRNAs containing introns that may affect the decoding efficiency of Arg(TCT) gene. Therefore, we included six additional constructs containing introns, and for each anticodon, MC-tRNA Arg Increase the total number of MC-tRNA to 26. Arg Following the example of (GCA), there is a single native anticodon sequence for Trp (CCA) and His (GTG), and therefore MC-tRNA Arg (CCA) and MC-tRNA Arg The design of (GTG) involves the use of MC-tRNA Arg There are two native anticodon sequences for Gln (TTG, CTG), and TTG can read both Gln codons, CAA and CAG, whereas CTG can only read CAG. Arg (TTG) and MC-tRNA Arg Both anticodons were included in the design to create (CTG) backbones. Each backbone is paired with its endogenous promoter and terminator, capturing 200 bp upstream and downstream genomic context for plasmid-based expression.

[0072] The experimental test included the following steps: First, GFP*-RFP and one single MC-tRNA present in the same plasmid were transfected with GFP*-RFP and one single MC-tRNA. Arg is transfected into HEK293T human cell cultures. One positive control of the wild-type GFP-RFP construct and two negative controls, namely, one GFP*-RFP only construct and one GFP*-RFP+tRNA that reads only the Arg codon, are included. Arg After 24, 48, and 72 hours, both the green and red fluorescence levels of the transfected cells are measured by flow cytometry. The positive control shows high fluorescence for both GFP and RFP along the diagonal (Figure 2C). All negative controls are GFP*-RFP (Figure 2C, bottom). MC-tRNA ArgThe presence of MC-tRNA generates substantial amounts of functional GFP protein, and the actual magnitude of green fluorescence depends on the MC-tRNA used. Arg Type of GFP and its DNA * These results suggest that the MC-tRNA is highly dependent on both the MC-tRNA and the MC-tRNA mutations (Figure 2D). Arg We demonstrate that the strategy works in human cells, producing functional proteins at the translational level from genetic mutations at the DNA level.

[0073] MC-tRNA Arg In principle, MC-tRNA can read many non-Arg codons, causing substantial mistranslation. However, mistranslation is not inherently lethal; in fact, cells can tolerate high levels of mistranslation and even naturally regulate the level and type of mistranslation (references). Nevertheless, we conducted further experiments to demonstrate that MC-tRNA Arg The potential toxic effects of expressing MC-tRNA were investigated (Figure 2E). Both Annexin V stainings indicate cell death. Similar levels of cell death were observed in MC-tRNA-expressing cells and controls, indicating minimal toxic effects. We found that MC-tRNA, which exhibits strong toxic effects leading to substantial cell death, was not associated with MC-tRNA expression. Arg However, MC-tRNA exhibits mild toxicity. Arg We also found that certain MC-tRNAs were used to minimize toxicity from mistranslation. Arg This shows that it is possible to obtain

[0074] MC-tRNA Arg To demonstrate that MC-tRNA is indeed expressed in cells, we performed high-throughput tRNA sequencing of several transfected cells that exhibited the highest levels of GFP fluorescence (Figure 2F). Arg can be distinguished from endogenous tRNAs by their anticodon sequences. Arg has anticodons ICG (GCG in sequencing), TCG, CTC, and TTC, but MC-tRNA ArgMC-tRNA has the anticodons GCA, CCA, GTG, TTG, and CTG. Arg is expressed at a significant level and is expressed in the presence of endogenous tRNAs in experiments. Arg It constitutes X to Y% of the total.

[0075] MC-tRNA Arg restores Arg residues in disease-associated peptides MC-tRNA Arg To demonstrate that MC-tRNA actually reads non-Arg codons and incorporates Arg during translation, we performed mass spectrometry analysis of reporter protein constructs containing natural peptide sequences in disease contexts (Figure 3A, Table 3). The single-polypeptide reporter constructs contain fusion blocks of 21-amino acid peptide sequences centered around the pathogenic mutation (disease-associated peptide), with each block separated by a Gly-Lys-Gly sequence that provides a Lys-C protease cleavage site. Each reporter also contains a triple Flag tag near the C-terminus to facilitate purification by immunoprecipitation. We designed a total of four constructs, each consisting of a single type of Arg mutation for the same amino acid. For example, all of the pathogenic Arg-to-His mutations were present in the same reporter construct, resulting in the expression of MC-tRNA. Arg (GTG) can be used to test all missense correction events from His to Arg. A similar strategy can be used to test all missense correction events from His to Arg, respectively. Arg (GCA), MC-tRNA Arg (CCA), MC-tRNA Arg (TTG or CTG) and applied to Arg to Cys, Arg to Trp, and Arg to Gln reporter constructs.

[0076] Mass spectrometry results showed that cells Arg It is shown that the corresponding MC-tRNA naturally produces disease-associated peptides in the absence of ArgIn the presence of MC-tRNAArg, disease-associated peptides are still produced using endogenous tRNAs, and at the same time, missense-corrected peptides corresponding to the X-to-Arg correction during translation are also detected (Figure 3B). This result can be semiquantitatively compared for each X-to-Arg-corrected peptide and the disease-associated peptide (Figure 3C). As expected, in most cases, MC-tRNAArg shows significant levels of corrected peptide production. Arg Since (CTG) can only read CAG and cannot read the CAA codon of Gln, MC-tRNA with the mutation Arg to Gln(CAA) Arg A non-corrective effect of (CTG) is expected.

[0077] Missense correction is MC-tRNA Ser But it also occurs To study missense correction for amino acids other than Arg, MC-tRNA Ser A similar construct was designed to test missense correction by MC-tRNA. This construct was also made with fused GFP and RFP, except that GFP contained a Ser65 to CGG to Arg codon mutation that rendered GFP non-fluorescent (Figure 4A). GFP (Ser65 to CGG)-RFP (GFP*-RFP) and MC-tRNA Ser Several plasmid constructs containing (CCG) MC-tRNA were also generated. Ser (CCG)1 and MC-tRNA Ser (CCG)2 is tRNA Ser (AGA) and tRNA Ser (CGA) tRNA body sequence derived from isodecoder. Ser (CCG)2+ is MC-tRNA Ser It is derived from the same tRNA body sequence as (CCG)2, but the variable loop is expanded from 3 to 6 nucleotides. Finally, GFP*-RFP and tRNA Ser A negative control construct containing (CGA) was also generated.

[0078] The GFP and RFP fluorescence levels of HEK293T cells transfected with these plasmids were examined using flow cytometry 24 and 48 hours after transfection. Cells were gated by RFP fluorescence to verify successful transfection (Figure 4B). As expected, the negative control of the GFP*-RFP plasmid without any additional tRNA shows only red fluorescence (red). The positive control of the wild-type GFP-RFP plasmid shows high green and red fluorescence (green). MC-tRNA Ser All three plasmids (columns 2, 3, and 4) containing (CCG) show restoration of green fluorescence at levels within a few fold of the positive control GFP-RFP plasmid (Figure 4C). Ser The negative control (column 5) containing (CGA) showed the restoration of green fluorescence, which is due to the MC-tRNA Ser This reinforces the positive results of

[0079] All three MC-tRNAs Ser Although (CCG) restored significant levels of GFP fluorescence, the quantitative difference between these constructs highlights the importance of using different tRNA body sequences for missense correction. Ser (CCG)2 is MC-tRNA Ser Compared with (CCG)1, the distribution of cells with high levels of GFP fluorescence was narrower and the proportion was higher, which indicates that MC-tRNA Ser (CCG)2 is MC-tRNA Ser This suggests that MC-tRNA is a better missense corrector than (CCG)1 (Figure 4D). Although literature reports suggest that expansion of the variable loop can lead to a three-fold higher level of stop codon suppression (PMID: 20026070), MC-tRNA Ser The expansion of the variable loop of (CCG)2+ SerThe tRNA body sequences showed no substantial differences from the parent sequence of (CCG)2. It remains to be seen whether further alteration of the tRNA body sequence from the reference human genome sequence will generate more efficient missense correctors. We further explored whether expression of MC-tRNA leads to cell death. It has previously been reported that this type of widespread mistranslation can be cytotoxic. However, when using Annexin V staining to measure cell death, we found no increase in cell death in MC-tRNA-expressing cells compared with control cells (Figure 4E, Figure 4F). We used the GFP-to-RFP expression ratio in MC-tRNA-expressing cells as a proxy for tRNA expression levels among live cells. No correlation was found between the GFP-to-RFP ratio and Annexin V staining (Figure 4E).

[0080] Finally, we searched the literature and identified 18 fluorescent proteins that can be used to study missense correction in the same manner as Arg and Ser (Table 4). The use of these proteins will enable efficient, high-throughput screening of MC-tRNAs for all amino acid mutations that lead to human disease.

[0081] Example 2: Discussion Potential of the CoMED strategy for treating genetic diseases: In this study, we described a strategy that uses missense-correcting tRNAs to treat pathogenic or potentially pathogenic genetic mutations in thousands of reported human diseases. The underlying concept relies on the use of MC-tRNAs that are charged with one amino acid but read the codon for another amino acid in translation. The co-translational missense correction process generates a functional protein, whereas the same protein translated according to the genetic code would be non-functional. We demonstrated that the MC-tRNA Arg and MC-tRNA Ser We show that these genes function fully to correct their respective missense mutations in human cell culture, thus providing proof-of-principle for cotranslational missense correction (CoMED) of genetic diseases.

[0082] In certain aspects, this strategy is well suited to treating genetically recessive diseases, where the genetic mutation results in little or no functional protein, and the use of MC-tRNA can produce amounts of functional protein useful for disease treatment.

[0083] Missense correction and toxicity: A major consideration in introducing MC-tRNA into cells is toxicity, because MC-tRNA may misread other codons for other proteins, increasing the level of mistranslation. The typical range of fidelity in the central dogma of molecular biology is 10 for replication. -8 ~10 -9 , 10 for transcription -5 ~10 -6 , and 10 for translation -3 ~10 -4 Not only does translation have a minimum level of fidelity, but cells can actively alter translation fidelity in response to environmental conditions to expand their proteome diversity for better response and adaptation (references). It is also clear that large levels of mistranslation can lead to high levels of proteotoxic stress and cell death. The toxic effects likely depend on many factors, including the cell type (neuron, liver, kidney, etc.), the amount of MC-tRNA expression required for disease treatment, the length of MC-tRNA exposure, and the specific type of MC-tRNA.

[0084] The backbone of MC-tRNA is not limited to tRNAs in the reference human genome: in this study, we only used tRNA bodies derived from the reference human genome, which was derived from pooled DNA of approximately 100 individuals. However, since a large reservoir of other tRNA sequences exists in the human population (PMID: 30643023), a number of naturally available tRNA bodies can easily be used for further testing of the effectiveness of MC-tRNAs. Furthermore, tRNA sequences from non-human sources, such as mouse / rat, yeast, or even bacterial tRNAs, can also be tested, and these may be functional MC-tRNAs as long as they can be charged with the corresponding human aminoacyl-tRNA synthetases (e.g., non-human MC-tRNAs that can be charged by human arginyl-tRNA synthetases). Arg ).

[0085] Modifications of tRNA may be required to fine-tune the efficacy of MC-tRNA: While the innate immune system recognizes unmodified tRNA as foreign, certain modified tRNAs do not elicit an immune response (reference). Therefore, to achieve the highest efficacy while minimizing the immune response, MC-tRNAs can contain specific modifications. tRNA modifications are also highly effective in fine-tuning cotranslational efficiency and decoding selectivity. Human cytosolic tRNAs contain an average of 13 modifications per molecule, although not all of these are required for maximum efficiency. For example, pseudouridine alone has proven highly effective in minimizing immune responses in COVID-19 mRNA vaccines. Various modifications and their combinations can be tested to obtain the optimal MC-tRNA construct with the highest efficacy.

[0086] A collection of 200 tRNAs to treat all single-base missense mutations: In summary, we describe the CoMED strategy of using MC-tRNAs to treat human genetic diseases. The initial focus will be on the development of 4-5 MC-tRNAs targeting Cys / Trp / His / Gln-to-Arg mutations for approximately 200 potential new patients per year in the United States. Arg In principle, a collection of approximately 200 MC-tRNAs could treat all single-base missense mutations that cause human disease.

[0087] Example 3: Materials and Methods MC-tRNA Ser Experiment MC-tRNAser construct containing the promoter: Table 1 shows the tRNA sequences and DNA blocks used for expression of MC-tRNA. These blocks were cloned into mammalian expression plasmids that also express a tandem GFP*-RFP polypeptide reporter.

[0088] Transfection Protocol: HEK293T cells were grown to confluence. Cells were trypsinized and washed. [number] cells were transfected with [ng of plasmid] and [lipofectamine]. After [incubation], cells were washed and grown at [temperature] for [hours].

[0089] Staining Protocol: Where indicated, cells were stained with either DAPI or Annexin-V AlexaFluor 647 or both as follows: [DAPI], [Annexin-V].

[0090] Flow Cytometry Protocol: At 24, 48, and 72 hours post-transfection, cells were trypsinized in 96-well plates using Trypsin Reagent. Cells were pelleted, decanted, washed with PBS, and resuspended in 200 μL of PBS. Cells were then analyzed on the instrument using the laser. The instrument's default voltages were used. Forward and side scatter parameters were used to gate on live cells, and 10k events in this gate were recorded. Subsequent analysis was performed using custom scripts in R using the FlowCore and FlowVis CRAN packages.

[0091] ClinVar computation ClinVar data were downloaded on January 4, 2022 (https: / / ftp.ncbi.nlm.nih.gov / pub / clinvar / tab_delimited / ) (PubMed PMID: 31777943). Analysis was performed using custom scripts in R.

[0092] Comparison of missense vs. nonsense SNVs among all SNVs The ClinVar variant summary dataset was filtered for assembly="hg38" and type="single-nucleotide variant". The name field was parsed to obtain the WT and SNV amino acid identity as well as the WT and SNV nucleotides. If the SNV amino acid was "Ter", indicating a terminator, it was labeled as a nonsense mutation; otherwise, the entry was labeled as missense. The number of SNVs reported for both missense and nonsense was summed.

[0093] Comparison of pathogenic SNVs between amino acids The parsed set of SNVs from above was further filtered for the Clinical Significance field to accept "pathogenic," "possibly pathogenic," and "pathogenic / possibly pathogenic." This filtered set was used as the set of pathogenic SNVs. SNVs were grouped by wild-type amino acid, and the number of SNV entries in each group was summed and compared.

[0094] Comparison of the number of submitters per SNV In the filtered set of pathogenic SNVs derived above, the NumberSubmitters field was used to generate a histogram reflecting how frequently individual SNVs were reported from the population.

[0095] Comparison of the number of SNVs and submitters per SNV for missense pathogenic mutations The PhenotypeIDS field was parsed to identify the Orphanet ID associated with each SNV. SNVs were then grouped by their mutation type: WT amino acid, WT nucleotide, SNV amino acid, and SNV nucleotide combination. This identification information is a proxy for codon-level information, but it allows each combination to be matched to a small group of possible codons. Using these groups, the number of submitters for each SNV was summed to obtain "total_reports." Additionally, the list of unique PhenotypeIDS entries was matched to track the "number_diseases" associated with each SNV type. The number of submitters for each SNV was also tracked. For plotting, only names are displayed for SNV types for which at least one individual SNV had more than 10 submitters.

[0096] Example 4: Details of the CAPN3 gene and missense mutations associated with LGMD2A disease: Limb-girdle muscular dystrophy type 2A (LGMD2A) is associated with mutations in the gene Caplain 3 (CAPN3). This disease is generally considered to be genetically recessive. The inventors hypothesized that restoring a small amount of CAPN3 enzyme function would be sufficient to improve the symptoms of patients with LGMD2A, and therefore, tRNA therapy would be an excellent candidate. This disease was selected as an excellent candidate for commercial development for six reasons: 1) CAPN3 is an enzyme and the disease manifests as a genetic recessive, making tRNA therapy potentially effective; 2) there is evidence of safety concerns with gene therapy based on off-target cardiac delivery of the CAPN3 gene; 3) the disease is non-fatal, making it possible for a population of adult patients to enroll in clinical trials without waiting for a diagnosis; 4) the patient population, while small, is estimated at 1 in 100,000 births in the United States, potentially large enough to support therapeutic development; 5) the affected tissue is skeletal muscle, for which there are many viable delivery options; and 6) there are convenient assays to test gene function, such as autoproteolysis.

[0097] Because this disease and SNVs appear in ClinVar, we analyzed known pathogenic and potentially pathogenic SNVs associated with this disease. We found that 65.2% of SNVs were missense mutations compared to 34.8% of nonsense mutations (mutations affecting splicing or frameshifts exist and were excluded from this analysis). Among missense mutations, Arg>Gln accounts for 24% of reported cases; Arg>Trp accounts for 12%; Arg>Cys accounts for 4%; and Arg>His accounts for 3%. This trend is consistent with the analysis presented herein, which shows that Arg-based mutations account for the majority of pathogenic missense mutations across all ClinVar diseases. While every individual disease is unique, the SNV landscape in the human population for LGMD2A follows this same trend. Although Arg>Gln mutations account for the largest single type of mutation, this includes many different SNVs, all of which can be reasonably treated with mc-tRNA. A supplemental table is included that clearly shows the exact SNVs reported in Clinvar for CAPN3, as well as combined statistics for the rate of Arg>Gln mutations and the rate of missense versus nonsense mutations.

[0098] To test whether mc-tRNA can truly restore function to CAPN3, we purchased plasmids expressing mutant CAPN3s predicted to be functionally impaired with well-documented autoproteolytic activity compared to wild-type CAPN3. These CAPN3 mutants were expressed in HEK293 cells, and coexpressed with mc-tRNA to repair their corresponding mutations. We selected CAPN3 mutations that had either been biochemically verified in the literature or confirmed to be present in patients with LGMD2A by the Coalition to Cure Calpain 3. Control Western blots were performed using HEK293 cells expressing CAPN3 or mutant CAPN3 but not the mc-tRNA expression construct.

[0099] Example 5: Generation of stable cell lines expressing GFP.RFP or mutant GFP.RFP or mutant GFP.RFP and corrective tRNA Generation of stable cell lines HEK293 cells were purchased from ATCC and cultured using standard sterile mammalian cell culture techniques. Specifically, HEK293 cells were maintained in 10 cm polystyrene cell culture-treated dishes in DMEM supplemented with 10% FBS, penicillin, and streptomycin at 37°C in a humidified 5% CO atmosphere. To generate stable cell lines expressing proteins and tRNAs of interest, HEK293 cells were split into 6-well cell culture-treated plates, subcultured, transfected with plasmids expressing the desired protein / tRNA of interest and a selectable marker, and then selected using antibiotic-based selection. The goal of this project was to study mutation correction and stress in cells constitutively expressing a tRNA (mc-tRNA) engineered to recognize and correct specific missense mutations.

[0100] For transfection, healthy HEK293 cells were first seeded into 6-well cell culture-treated plates at a density of 900,000 cells / well in DMEM supplemented with 10% FBS only. The cells were allowed to adhere to the wells for 24 hours. After 24 hours, the cells were transfected using Lipofectamine 2000 and the plasmid of interest. More specifically, for each well, 5 μL of Lipofectamine 2000 reagent was combined with 2 μg of the plasmid of interest in 500 μL of OptiMEM and incubated at room temperature for 20 minutes. The combined Lipofectamine / plasmid transfection reagent was then added dropwise to the appropriate well and mixed by swirling the well clockwise and counterclockwise 20 times. The transfected cells were incubated at 37°C for 4 hours in a humidified 5% CO2 atmosphere. After 4 hours, the medium was removed from each well and replaced with fresh DMEM supplemented with 10% FBS, penicillin, and streptomycin. All transfected plasmids contain ampicillin and puromycin resistance cassettes.

[0101] The following plasmids were transfected: - p2 - a positive control plasmid expressing an eGFP.mCherry fusion protein from the CMV promoter. Both fluorescent proteins are functional. - p100- A negative control expressing an eGFP.mCherry fusion protein containing a mutant eGFP. This mutant eGFP has an amino acid change from R to Q at position 97 (R97Q). This mutant is referred to herein as eGFP(R97Q).mCherry. The codon for Q at this position is CAG. This mutation prevents eGFP from becoming fluorescent, but the RFP protein remains fluorescent. - p108- eGFP(R97Q). A test plasmid expressing mCherry and mc-tRNA that recognizes the mutant CAG codon but delivers arginine. This tRNA was previously shown to correct a mutation in eGFP in transient transfection-based experiments. - p401 - A negative control expressing an eGFP.mCherry fusion protein containing a mutant eGFP. This mutant eGFP has an amino acid change from R to C at position 97 (R97C). This mutant is referred to herein as eGFP(R97C).mCherry. The codon for C at this position is TGC. This mutation in eGFP greatly reduces its fluorescence, yet the RFP protein remains fully fluorescent. - p404- eGFP(R97C). A test plasmid expressing mCherry and mc-tRNA that recognizes the mutant TGC codon but delivers arginine. This tRNA was previously shown to correct a mutation in eGFP in transient transfection-based experiments.

[0102] A total of 2 x 6-well plates of cells were transfected. The first plate was transfected with plasmids p2, p100, p103, and p108. The second plate was transfected with plasmids p2, p401, and p404. Additional untransfected wells were maintained to provide selection controls.

[0103] Transfected cells were grown at 37°C in a humidified 5% CO2 atmosphere for 48 hours to allow expression of the transfected construct. After 48 hours, cells were selected using 0.5 μg / ml puromycin. Cells were maintained in medium containing 0.5 μg / ml puromycin for 2 weeks, with medium changes approximately every 2-3 days. After 2 weeks of selection, cells that had stably integrated the plasmid into their genome were still viable and proliferating. These stable cells were expanded into 10 cm cell culture dishes and maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin, and 0.3 μg / ml puromycin at 37°C in a humidified 5% CO2 atmosphere.

[0104] The following stable cell lines were generated: - Cell line 03-00 - HEK293 cells transfected with p2 that express WT eGFP.mCherry, are polyclonal, and are resistant to at least 0.5ug / ml puromycin. - Cell line 04-00 - HEK293 cells transfected with p401 that express eGFP(R97C).mCherry, are polyclonal, and are resistant to at least 0.5ug / ml of puromycin. - Cell line 05-00- HEK293 cells transfected with p404 expressing eGFP(R97C).mCherry and mc-tRNA Arg-tRNA-CysGCA, which are polyclonal and resistant to at least 0.5ug / ml of puromycin. - Cell line 06-00 - p2 transfected HEK293 cells expressing WT eGFP.mCherry, polyclonal, and resistant to at least 0.5ug / ml puromycin. - Cell line 07-00 - HEK293 cells transfected with p100 that express eGFP(R97Q).mCherry, are polyclonal, and are resistant to at least 0.5ug / ml of puromycin. - Cell line 08-00- HEK293 cells transfected with p108 expressing eGFP(R97Q).mCherry and mc-tRNA Arg-tRNA-GlnCUG, which are polyclonal and resistant to at least 0.5ug / ml of puromycin.

[0105] *Note: Cell line nomenclature is as follows: line number-division number. Example: 06-03 is cell line 6, division 3.

[0106] Notably, stable cell line 05 transfected with plasmid p404 grew very slowly compared to the other cell lines. We attribute this to the significant inhibitory effect of this mc-tRNA, which restored 10% of eGFP fluorescence in transient transfection-based experiments. Furthermore, the positive control lines (transfected with p2) on both plates survived selection, leading to the generation of two polyclonal positive control lines (03-00 and 06-00).

[0107] Fluorescence recovery Fluorescence recovery was examined using flow cytometry. Stable cell lines for flow cytometry were prepared as follows: Stable cells were seeded in 6-well dishes at 300,000 cells / well and allowed to grow for 24 hours. The medium was then removed, and each well was washed with 1 ml of 1x PBS. The PBS was then removed, and 500 μl of 0.25% trypsin:EDTA was added to each well and incubated at room temperature for 5 minutes. Once the incubation was complete, 500 μl of DMEM supplemented with 10% FBS was added to each well. The cells were then resuspended by pipetting, transferred to a 1.7 ml centrifuge tube, and pelleted by centrifugation at 5000 × g for 5 minutes. Carefully removing the supernatant from the pelleted cells without disturbing the cell pellet, the cells were resuspended in 500 μl of 1x PBS. The cells were then stained with DAPI to distinguish between dead and live cells. DAPI staining was performed by adding 10 μl of DAPI (10 μg / ml stock) to 500 μl of resuspended cells and incubating for 15 minutes at room temperature. After incubation, an additional 500 μl of 1×PBS was added to the resuspended cells. Cells were kept on ice until use.

[0108] Fluorescence was measured using an Attune flow cytometer. GFP and DAPI signals were measured from all stable cell lines. GFP signal was used as a measure of missense suppression by mc-tRNA. DAPI signal was used as a measure of cell death due to mc-tRNA expression.

[0109] Next-generation sequencing Total RNA was isolated from stable cell lines using a standard TRIzol:chloroform extraction procedure. mRNA was isolated and DNA libraries were constructed using the Illumina Stranded mRNA prep kit. Libraries were sequenced on an Illumina MiSeq system.

[0110] Example 6: Further cell culture data Mammalian cell culture HEK293 cells have proven reliable and tested negative for mycoplasma. HEK293 cells were cultured in Dulbecco's Modified Eagle's (DMEM) High Glucose Medium (Cytiva, catalog number SH30022.01) supplemented with 10% heat-inactivated fetal bovine serum and 100 U / ml penicillin-streptomycin at 37°C and 5% CO2.

[0111] Plasmid The plasmid was synthesized and verified by GenScript, Inc. The fluorescent protein-based mistranslation reporter was driven by a CMV promoter. The MctRNA expression cassette contained 200 bp of endogenous sequence upstream and downstream of the tRNA gene. The exact sequence is as follows: (1) Ser-tRNA Arg(CCG) Expression cassette for (SEQ ID NO: 140): TIFF2025525377000045.tif69160(2)Arg-tRNA Cys(GCA) Expression cassette for (SEQ ID NO: 141): TIFF2025525377000046.tif62159(3)Arg-tRNA His(GUG) Expression cassette for (SEQ ID NO: 142): TIFF2025525377000047.tif62159(4)Arg-tRNA Gln(CUG) Expression cassette for (SEQ ID NO: 143): TIFF2025525377000048.tif62159(5)Arg-tRNA Trp(CCA) Expression cassette for (SEQ ID NO: 144): As shown in TIFF2025525377000049.tif62159.

[0112] Transfection Cells were seeded into 96- or 6-well plates one day before transfection, and the cells reached 50–70% confluence on the day of transfection. Plasmids used for transfection were purified using PureYield (商標) Plasmids were prepared using the miniprep / midiprep system (Promega, Cat. No. A1222 / A2492) and precipitated with ethanol. Transfection was performed using Lipofectamine. (商標) 3000 (Invitrogen (商標) The procedure was carried out using a ELISA kit (catalog number L3000015) according to the manufacturer's protocol.

[0113] Flow cytometry Flow cytometry assays were performed 48 hours after transfection. (商標) HEK293 cells were detached from plates using a PBS buffer (Cat. No. 25200056) and resuspended in ice-cold 1x PBS, 5 mM EDTA, 25 mM HEPES pH 7.0, 1% FBS, and 100 ng / ml DAPI before sorting. The flow cytometer was a NovoCyte Penteon 5-30. Fluorescent protein (FP) signals were detected using the following cognate detection ion channels: GFP, B525; mCherry, Y615; dsRed / Zoan2rfp, Y586; and mPlum, Y667. For all flow cytometry runs, wild-type HEK293 cells without any staining were used as background controls, and HEK293 cells overexpressing a single FP were used as gating references. Collected flow cytometry data were analyzed using custom R scripts.

[0114] Immunoprecipitation HEK293 cells were harvested 48 hours posttransfection and washed twice with 1x PBS. Three to seven million cells were then incubated in 500 μl of lysis buffer (1x PBS, 1% IGEPAL (NP-40), 0.1% SDS, 0.5% w / v sodium deoxycholate, 1x protease inhibitor cocktail (Nacalai, Cat. No. 25955)) on a rotator for 15 minutes at 4°C, and then centrifuged at 1,000 g for 5 minutes at 4°C to pellet cell debris. The GFP-mCherry fusion protein was immunoprecipitated (IP) with a GFP monoclonal antibody (Invitrogen, Cat. No. MA515256), and CAPN3 was immunoprecipitated with a CAPN3 monoclonal antibody (Proteintech). (登録商標) IP was performed with pre-washed Dynabeads (Cat. No. 67366-1-Ig). (商標) The beads were incubated with M-280 sheep anti-mouse IgG beads (Cat. No. 11202D) for 4 hours at 4°C before being added to the cell lysate. The antibody-to-beads ratio was 4 μg to 50 μl, and the dilution of antibody in the cell lysate was 1 μg in 50 μl. The antibody-conjugated beads were washed three times with 1 ml of lysis buffer and added to the cell lysate. The mixture was incubated overnight at 4°C. The beads were then washed three times with 1 ml of high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% IGEPAL (NP-40), 0.1% SDS, 0.5% w / v sodium deoxycholate, 1x protease inhibitor cocktail) and three times with 1 ml of low-salt wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl, 0.2% Tween-20, 1x protease inhibitor cocktail). The beads were washed three times with non-reducing 1x NuPAGE LDS sample buffer (Invitrogen). (商標) The IPed proteins were eluted from the beads by incubating them at 70°C for 15 minutes in a 4-12% NuPAGE gel (Invitrogen, Cat. No. NP0007). (商標)The gel was directly loaded onto a NuPAGE gel (catalog no. NP0321BOX) and subjected to gel electrophoresis. The NuPAGE gel was stained with Coomassie G-250 stain (catalog no. 1610786), and the band of interest was excised and stored at 4°C.

[0115] Mass spectrometry sample preparation Protein spots were extracted from SDS-PAGE gels and cut into 1 mm x 1 mm cubes according to published protocols with some modifications. 1 The in-gel digestion process was performed according to

[14] . First, the gel pieces were dehydrated using acetonitrile, and the solution was removed after 5 minutes. The gel pieces were then covered with a sufficient amount of a solution containing 5 mM TCEP in 40 mM ammonium bicarbonate with 25% acetonitrile. The mixture was incubated at 37°C for 5 minutes to fully reduce disulfide bridges in the protein. The gel pieces were again dehydrated using acetonitrile, and then another solution of 5 mM TCEP in 40 mM ammonium bicarbonate with 25% acetonitrile was added to it. This step aimed to further reduce disulfide bridges, and the mixture was incubated at 65°C for 15 minutes with gentle stirring. After cooling to room temperature, the gel pieces were treated with 40 mM iodoacetamide in 40 mM ammonium bicarbonate with 25% acetonitrile for alkylation. The gel pieces were incubated in the dark at room temperature for 15 minutes. The gel pieces were then washed with 40 mM ammonium bicarbonate containing 25% acetonitrile for 5 minutes, followed by dehydration using acetonitrile. This washing step was repeated once more. The gel pieces were then swollen in a digestion buffer containing trypsin / Lys-C (8 ng / μL) in 40 mM ammonium bicarbonate and 0.5 mM CaCl2 using an ice-cold bath for 1 hour. Excess trypsin solution was removed from the gel pieces while they were kept on ice. The samples were digested for 12 hours at 37°C. To extract peptides, two changes of 60% acetonitrile in 0.2% formic acid and one change of 60% acetonitrile in 0.15% formic acid were used, with incubation times of 10 and 30 minutes, respectively. Finally, the peptides were dried under vacuum.

[0116] LC-MS parameters Samples were analyzed using an Exploris 480 mass spectrometer connected to an UltiMate 3000 liquid chromatography system (Thermo Scientific). The chromatography system utilized a 50 cm long, 0.75 mm internal diameter GL Sciences MonoCap column (catalog number 5020-10006). The flow rate was maintained at 500 nL / min, and the temperature was kept constant at 25°C. A 75-minute gradient method was used, including mobile phase A (0.15% formic acid in water) and mobile phase B (0.15% formic acid in 100% acetonitrile). The gradient proceeded as follows: 5% B for 5 minutes, followed by a transition from 5% to 22% B over 46.5 minutes, a transition from 22% to 34% B over 7.5 minutes, and a rapid transition from 30% to 95% B within 1 minute. The composition was maintained at 95% B for 4 minutes. Full-scan MS spectra ranging from 350 to 1650 m / z were collected at a resolution of 120,000 at m / z 200. The maximum injection time was set to 50 ms, and the AGC target value was set to 3e6. The data acquisition cycle time was set to 3 seconds, while the intensity threshold was set to 5e4. For MS / MS scans, a resolution of 15,000 was used, the maximum acquisition time was set to automatic, and the AGC target was set to 4e4. The isolation window in the Orbitrap cell was set to 1.6 m / z, and the first mass was set to 110 m / z. The collision energy for the HCD was set to 32. A 10-second dynamic exclusion period was performed to exclude unassigned, 1, and charge states greater than 8. The heated capillary temperature was set to 300 °C.

[0117] MS data analysis Raw MS data were processed and searched in Proteome Discoverer (version 3.0.0.757; Thermo Fisher Scientific) using the Sequest HT search engine. A precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da were used. A 1% FDR cutoff estimated by the Target Decoy PSM Validator was applied to filter the data. Trypsin (complete) was set as the enzyme in the search. The maximum mass cutoff was set to 3, and the peptide length ranged from 4 to 30. Carbamidomethyl (+57.021 Da at C) was selected as the fixed modification, while oxidation (+15.995 Da at M), deamidation (+0.984 Da at N and Q), loss of Met at the protein N-terminus (-131.040 Da), acetyl (+42.011 Da at N-terminus), and loss of Met + acetyl at the protein N-terminus (-89.030 Da) were selected as dynamic modifications. The proteotypic peptide, SAMPEGYVQER, was used to determine mutational conversion. Signals were normalized by the amount of injected peptide.

[0118] tRNA sequencing and data analysis Approximately 1 μg of total RNA was used to perform the previously published MSR-seq protocol. 2 A tRNA sequencing library was constructed according to [1]. Raw 100-bp paired-end sequencing reads were obtained from the Illumina NovaSeq platform. Data analysis also followed a slightly customized MSR-seq data processing pipeline. In particular, read 2 was processed and mapped against a curated reference containing all human tRNA sequences and five mctRNA sequences. Given the sequence similarity between mctRNA and its cognate endogenous tRNA, only mapped reads longer than 60 nt were used for abundance, charging, and mutation analysis.

[0119] RNA-seq and data analysis RNA-seq experiments were performed on three independent replicates from HEK293 cells overexpressing the mistranslation reporter with and without the cognate mctRNA. Forty-eight hours after transfection, HEK293 cells were sorted on a FACSAria Fusion 5-18 cell sorter for the mCherry+, i.e., successfully transfected, population. TRIzol (商標) Total RNA was extracted from sorted cells using a reagent (Invitrogen, catalog number 15596026). 1 μg of total RNA per sample was used as input for RNA-seq library construction. Total RNA samples were polyA-selected to enrich for mature mRNA species. All RNA libraries were multiplexed and sequenced on the Novaseq 6000 platform (Illumina), yielding approximately 120 million directional 100-bp paired-end (PE) reads for each sample. PE reads were mapped using STAR 2.7.10b using the reference human genome GRCh38.p10. Uniquely mapped reads were filtered, and reads per gene were counted for all genes using featureCounts 2.0.1. Differential gene analysis was performed using edgeR (version 3.40.2). Gene ontology analysis was performed using clusterProfiler v4.6.2 for significantly up- or down-regulated genes (p<0.05 and absolute fold change>2).

[0120] Western blot Total protein samples were extracted from HEK293 cells 48 hours after transfection. To prevent autolytic activity of CAPN3, 15 mM EDTA and 1x protease inhibitor cocktail were added to all buffers during sample preparation. Furthermore, samples were kept on ice until denaturation. One to two million transfected HEK293 cells were washed once with ice-cold 1x PBS and denatured in 30 μl of CelLytic. (商標)The cells were lysed in NuPAGE M buffer (Sigma-Aldrich, catalog no. C2978). The cell lysate was centrifuged at 17,000 g for 15 minutes at 4°C to pellet cell debris. The supernatant was collected and immediately incubated at 70°C for 15 minutes with 10 μl of 4x NuPAGE LDS sample buffer containing 5% β-mercaptoethanol. The denatured protein sample was loaded onto a 4-12% NuPAGE gel for gel electrophoresis. The NuPAGE gel, filter paper, and pre-wetted Immobilon®-P PVDF membrane (Sigma-Aldrich, catalog no. IPVH00010) were incubated in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 10% methanol) for 10 minutes before transfer. Membrane transfer was performed using a Trans-Blot Turbo transfer system using the following settings: constant voltage 25 V; limit voltage 1 A; 20 minutes. The membrane was first blocked in blocking buffer (5% non-fat dry milk (BIO-RAD, Cat. No. 1706404) in 1× TBST buffer) for 1 hour at room temperature, and then blocked with a 1:1000 dilution of primary antibody, anti-CAPN3 (Proteintech (登録商標) The membrane was incubated overnight at 4°C in blocking buffer with anti-GAPDH (Invitrogen, Cat. No. 67366-1-Ig) and anti-GAPDH (Invitrogen, Cat. No. MA5-15738). The membrane was then washed three times with 1x TBST for 10 minutes each. The membrane was then washed with a 1:10,000 dilution of IRDye. (登録商標) 680RD goat anti-mouse IgG secondary antibody (LI-COR (登録商標) The blotted membrane was then incubated with Amersham Typhoon Antibody (Cat. No. 926-68070) in blocking buffer for 1 hour at room temperature. The blotted membrane was then washed three times with 1x TBST for 10 minutes each time and then washed with Amersham Typhoon Antibody (Cat. No. 926-68070) in blocking buffer for 1 hour at room temperature. (商標) Imaging was performed in the IR short channel.

[0121] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0122] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025525377000050.tif34160

Claims

1. 1. A tRNA molecule covalently linked to a first amino acid, the tRNA molecule comprising an anticodon loop sequence capable of hybridizing to an mRNA sequence encoding a second amino acid, the second amino acid being different from the first amino acid.

2. 2. The tRNA molecule of claim 1, wherein the first amino acid is arginine.

3. The anticodon loop sequence 3. The tRNA molecule of claim 2, wherein

4. The anticodon loop sequence 4. The tRNA molecule of claim 3, wherein:

5. 5. The tRNA molecule of claim 4, wherein the anticodon loop sequence is CUG, UUG, GUG, CCA, or GCA from 5' to 3'.

6. 3. The tRNA molecule of claim 2, wherein the anticodon loop sequence is not ACG, CCG, CCU, UCG, GCG, or UCU from 5' to 3'.

7. 7. The tRNA molecule of any one of claims 2 to 6, wherein the mRNA sequence from 5' to 3' is not CGU, CGC, CGA, CGG, AGA, or AGG.

8. The mRNA sequence is The tRNA molecule of any one of claims 2 to 6, wherein

9. 2. The tRNA molecule of claim 1, wherein the first amino acid is serine.

10. The anticodon loop sequence 10. The tRNA molecule of claim 9, wherein

11. The anticodon loop sequence is 11. The tRNA molecule of claim 10, wherein

12. 10. The tRNA molecule of claim 9, wherein the anticodon loop sequence is not AGA, CGA, GCU, UGA, ACU, or GGA from 5' to 3'.

13. 13. The tRNA molecule of any one of claims 9 to 12, wherein the mRNA sequence is not UCU, UCC, UCA, UCG, AGU, or AGC from 5' to 3'.

14. The mRNA sequence is The tRNA molecule of any one of claims 9 to 12, wherein

15. 15. The tRNA molecule of any one of claims 1 to 14, wherein the first amino acid is alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine.

16. 15. The tRNA molecule of any one of claims 1 to 14, wherein the second amino acid is alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine.

17. 17. The tRNA molecule of any one of claims 1 to 16, wherein the second amino acid is glutamine, histidine, tryptophan, or cysteine.

18. 18. The tRNA molecule of any one of claims 1 to 17, wherein the mRNA sequence is not UAA, UGA, or UAG.

19. 19. The tRNA molecule of any one of claims 1 to 18, comprising a sequence with up to three substitutions relative to a mammalian tRNA molecule.

20. 20. The tRNA molecule of claim 19, comprising a sequence with up to three substitutions relative to a human tRNA molecule.

21. A nucleic acid comprising a sequence encoding the tRNA of any one of claims 1 to 20.

22. 22. The nucleic acid of claim 21, wherein the sequence is one of SEQ ID NOs: 1-144.

23. A vector comprising the nucleic acid of claim 21 or 22.

24. 24. The vector of claim 23, which is an AAV vector.

25. 25. A cell comprising a tRNA molecule according to any one of claims 1 to 20, a nucleic acid according to any one of claims 21 to 22, and / or a vector according to any one of claims 23 to 24.

26. 26. The cell of claim 25, wherein the nucleic acid and / or vector is stably expressed.

27. 25. A method for modifying a protein produced by a gene, comprising administering to a cell an effective amount of the tRNA of any one of claims 1 to 20, the nucleic acid of claim 21 or 22, or the vector of claim 23 or 24.

28. 25. A method for producing a wild-type protein from a gene having a missense mutation, the method comprising administering to a cell an effective amount of the tRNA of any one of claims 1 to 20, the nucleic acid of claim 21 or 22, or the vector of claim 23 or 24.

29. 29. The method of claim 27 or 28, wherein the cell is a mammalian cell.

30. 30. The method of claim 29, wherein the cell is a human cell.

31. 25. A method for treating or preventing a genetic disease in a subject, comprising administering to the subject a therapeutically effective amount of the tRNA of any one of claims 1 to 20, the nucleic acid of claim 21 or 22, or the vector of claim 23 or 24.

32. 32. The method of claim 31, wherein the genetic disease is characterized by the presence of a single nucleotide variant (SNV) that results in a missense mutation in the gene.

33. 33. The method of claim 32, wherein the gene is ABCD1, GLA, GBA, GALC, ARSA, SGSH, HGSNAT, IDS, OTC, DHCR7, or HEXA.

34. 34. The method of any one of claims 31 to 33, wherein said gene is a gene in Table 2.

35. 35. The method of any one of claims 31 to 34, wherein the SNV is an SNV in Table 2.

36. 36. The method of any one of claims 31 to 35, wherein the genetic disease is a recessive disease.

37. 37. The method of any one of claims 31 to 36, wherein the genetic disease is adrenoleukodystrophy, Fabry disease, Gaucher disease type I, metachromatic leukodystrophy, mucopolysaccharidosis, ornithine transcarbamylase deficiency, Smith-Lemli-Opitz syndrome, Tay-Sachs disease, Niemann-Pick disease, or very long-chain acyl-CoA dehydrogenase deficiency.

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