Engineered tRNA and methods of use thereof

Abstract

In general, the present disclosure relates to tRNAs encoding an amino acid but covalently linked to a different amino acid. The tRNA can correct missense mutations during protein synthesis by providing different amino acids. Such tRNAs can be used to correct pathogenic missense mutations.

Description

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

[0003] This invention was made with government support awarded by the National Institutes of Health under Grant No. GM105386. The government has certain rights in this invention.

[0004] I. Sequence Listing

[0005] This application contains a sequence listing submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on June 21, 2023, named ARCD.P0781WO-Sequence Listing.txt, and is 172211 bytes in size. II. Technical field

[0006] The present invention relates to the fields of molecular biology, genetic engineering and medicine. III. Background Technology

[0007] Mutations in the protein coding regions of DNA may result in changes in the identity of the amino acids in the protein product (missense) or the introduction of premature stop codons (nonsense). Several patents and companies have recently begun to investigate the use of engineered tRNAs that can read through stop codons to treat nonsense mutation diseases, thereby bypassing nonsense mutations (PMID: 30778053, PMID: 33567469). Similar principles can be applied to missense mutations.

[0008] The basic concept of MC-tRNA has been described as missense suppressor tRNA, related to the more commonly discussed [nonsense] suppressor tRNA. These altered tRNAs have been shown to be functional in bacteria and yeast (ISBM: 978-3-642-75178-3, PMID: 2502189, PMID: 30007351), and are present at low levels in human populations (PMID: 30643023), although their expression is not known. In yeast, this tRNA has been described to restore function of mutant proteins by selection experiments, and it has been shown that this idea can be applied to disease (PMID: 32476470). The fundamental obstacle to the realization of this concept is related to the proofreading of the tRNA by its cognate aminoacyl synthetase. The fidelity of the genetic code is ensured by a set of isoacceptor tRNAs that accurately load their cognate synthetases, and deviations from this can lead to proteome-wide mistranslation with deleterious and adaptive effects. To ensure accurate loading, the synthetases structurally check the tRNA's many "identity elements" to ensure accuracy. In addition to type II tRNA, tRNA-Ser and tRNA-Leu, the anticodon of tRNA is considered to be an identity element for synthetase proofreading (PMID: 4879401, PMID: 8128220). Therefore, it is reasonable to expect that mutations in tRNA anticodons, as in MC-tRNA, will lead to the exclusion of the associated synthetase and produce tRNAs that cannot be translated (PMID11698642, PMID28660466 and PMID9801296). Recently, studies have shown that different iso-decoding tRNAs such as tRNA-Arg have different potencies and nonsense suppression (PMID: 30778053). This indicates that these tRNAs can tolerate mutations in the anticodon loop and maintain translation ability, indicating that aminoacylation is successful to some extent. However, it is not clear whether these tRNAs can be engineered to translate non-stop codons. In addition, the level or identity of aminoacylation of these tRNAs is unclear.

[0009] Of particular interest is that MC-tRNA can naturally deliver arginine to treat genetic diseases caused by missense mutations in Arg residues. Studies have found that among the known pathogenic single-base mutations, mutations in Arg codons are the most common compared to several other amino acids. Summary of the invention

[0010] In general, the present disclosure relates to tRNAs that use tRNAs that encode one amino acid but provide another 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.

[0011] Disclosed herein are tRNA molecules covalently linked to a first amino acid, wherein the tRNA molecule comprises an anticodon loop sequence capable of hybridizing to an mRNA sequence encoding a second amino acid different from the first amino acid.

[0012] The first amino acid can be any amino acid, including any one 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.

[0013] 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.

[0014] The second amino acid can be any amino acid, including any one 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.

[0015] 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.

[0016] In some aspects, the second amino acid is glutamine, histidine, tryptophan, or cysteine.

[0017] 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.

[0018] 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.

[0019] The tRNA can comprise an anticodon loop sequence and an amino acid not encoded by the anticodon loop. In some aspects, the amino acid not encoded by the anticodon loop is Arg. In some aspects, the anticodon loop sequence does not encode Arg.

[0020] In some aspects, the anticodon loop sequence is UGC, GCA, GUC, UUC, GAA, UCC, GUG, GAU, UUU, UAG, UAA, CAU, GUU, UGG, UUG, UCG, UGA, GCU, UGU, UAC, GUA, AGC, CGC, CUG, CUC, CCC, GCC, AAU, UAU, AAG, CAA, CAG, CUU, CGG, AGA, CGA, AGU, CGU, CCA, AAC, CAC or AGG. In some aspects, the anticodon loop sequence is GUG, CUG, UUG, AGG, CGG, UGG, AAG, CAG, UAG, GCA, CCA, GCU, GCC, CCC, UCC, CUU, UUU, CGU, UGU, UAU or CAU. In some aspects, the anticodon loop sequence is CUG, UUG, GUG, CCA or GCA from 5' end to 3' end. In some aspects, the anticodon loop sequence is not ACG, CCG, CCU, UCG, GCG or UCU from the 5' end to the 3' end. In some aspects, the mRNA sequence is not CGU, CGC, CGA, CGG, AGA or AGG from the 5' end to the 3' end. In some aspects, the mRNA sequence from 5' to 3' end is GCU, GCC, GCA, GCG, AAU, AAC, GAU, GAC, UGU, UGC, CAA, CAG, GAA, GAG, GGU, GGC, GGA, GGG, CAU, CAC, AUU, AUC, AUA, CUU, CUC, CUA, CUG, UUA, UUG, AAA, AAG, AUG, UUU, UUC, CCU, CCC, CCA, CCG, UCU, UCC, UCA, UCG, AGU, AGC, ACU, ACC, ACA, ACG, UGG, UAU, UAC, GUU, GUC, GUA or GUG.

[0021] In some aspects, the anticodon loop sequence is UGC, GCA, GUC, UUC, GAA, UCC, GUG, GAU, UUU, UAG, UAA, CAU, GUU, UGG, UUG, UCG, UGA, GCU, UGU, UAC, GUA, AGC, CGC, ACG, CCG, CCU, UCU, CUG, CUC, CCC, GCC, AAU, UAU, AAG, CAA, CAG, CUU, CGG, AGU, CGU, CCA, AAC, CAC or AGG. In some aspects, the codon loop sequence from the 5' end to the 3' end is GAA, CAA, UAA, AGG, CGG, UGG, AGU, CGU, UGU, AGC, CGC, UGC, GUA, GCA, ACG, GUU, AAU, GCC, CCU or UCU. In some aspects, the anticodon loop sequence is not AGA, CGA, GCU, UGA, ACU or GGA from the 5' end to the 3' end. In some aspects, the mRNA sequence is not UCU, UCC, UCA, UCG, AGU or AGC from the 5' end to the 3' end. In some aspects, the mRNA sequence is GCU, GCC, GCA, GCG, CGU, CGC, CGA, CGG, AGA, AGG, AAU, AAC, GAU, GAC, UGU, UGC, CAA, CAG, GAA, GAG, GGU, GGC, GGA, GGG, CAU, CAC, AUU, AUC, AUA, CUU, CUC, CUA, CUG, UUA, UUG, AAA, AAG, AUG, UUU, UUC, CCU, CCC, CCA, CCG, ACU, ACC, ACA, ACG, UGG, UAU, UAC, GUU, GUC, GUA or GUG from 5' to 3'. In some aspects, the mRNA sequence is not UAA, UGA or UAG.

[0022] The tRNA molecule may comprise a sequence having substitutions, deletions or additions relative to a mammalian tRNA molecule. Disclosed are tRNA molecules comprising a sequence having at most one, at most two, at most three, at most four or at most five substitutions relative to a mammalian tRNA molecule. In some aspects, the mammalian tRNA molecule is a human tRNA molecule.

[0023] Also disclosed is a nucleic acid encoding a tRNA sequence. The nucleic acid may comprise an expression vector. The nucleic acid may comprise a plasmid. The nucleic acid may be used to synthesize the tRNA sequence. This synthesis may occur in a cell. The cell may be a bacterial cell, an insect cell, a yeast cell, a vertebrate cell, or any other cell capable of expressing the tRNA. The synthesis may be performed by in vitro transcription.

[0024] A nucleic acid having a sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 144 is disclosed.

[0025] Also disclosed are tRNAs or nucleic acids encoding tRNAs, comprising one or more changes to the tRNAs disclosed herein. The changes may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more changes to the tRNAs. Also disclosed are tRNAs or nucleic acids encoding tRNAs, comprising one or more changes to the tRNAs of any one of SEQ ID NO: 1 to SEQ ID NO: 134. The changes may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more changes to the tRNAs. The changes may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more changes to the tRNAs of any one of SEQ ID NO: 1 to SEQ ID NO: 134.

[0026] 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 with any tRNA 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 with any tRNA 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 with any tRNA in SEQ ID NO. 1 to SEQ ID NO. 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 with any tRNA in SEQ ID NO. 1 to SEQ ID NO. 134, or any range derivable therein.

[0027] Also disclosed are vectors comprising any nucleic acid herein. The vector can be any vector, including any vector capable of delivering the nucleic acid to a cell of interest. 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 derivatives thereof.

[0028] Also disclosed are cells comprising any tRNA molecule disclosed herein. Also disclosed are cells comprising any nucleic acid disclosed herein. Also disclosed are cells comprising any vector disclosed herein. The cells may be cells for cell therapy such as autologous stem cell therapy.

[0029] Disclosed herein are methods for making tRNA disclosed herein. In some aspects, the methods include contacting a nucleic acid comprising an anticodon loop with an amino acid that is not naturally encoded by the anticodon loop. In some aspects, the nucleic acid is altered from a native tRNA sequence so that an aminoacyl-tRNA synthetase attaches an amino acid that is not normally encoded by the tRNA to the tRNA.

[0030] Disclosed herein is a method for using one or more than one tRNA disclosed herein to change the protein produced by a gene. Also disclosed is a method for generating a wild-type protein from a gene with a missense mutation. Also disclosed is a method for correcting missense mutations in the translation process of mRNA. Also disclosed is a method for producing genetically engineered proteins. Also disclosed is a method for introducing point mutations from mRNA to proteins. The method may include 1, 2, 3, 4, 5 or more than 5 steps, including any of the following steps: translating mRNA in the presence of one or more than one tRNA disclosed herein, administering an effective amount of one or more than one tRNA disclosed herein to a cell, administering an effective amount of one or more than one nucleic acid disclosed herein to a cell, administering an effective amount of one or more than one vector disclosed herein to a cell, and detecting proteins from cells. The administration may include any means of introducing tRNA, nucleic acid and / or vector into a cell, including by transfection, electroporation or transduction. The cell may be any cell, such as a mammalian cell. The cell may be a human cell. The cell may be a cell containing a missense mutation.

[0031] 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-band disease. Also disclosed are methods for restoring CAPN3 function in a cell. Also disclosed are methods for reversing the effects of a loss of CAPN3 function in a cell. Also disclosed are methods for restoring CAPN3 function in a subject. Also disclosed are methods for reversing the effects of a loss of CAPN3 function in a subject. Any of the methods may include 1, 2, 3, or more than 3 steps, including any of the following steps: 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 one or more vectors disclosed herein. The subject may have a genetic disease. The subject may have cancer. The disease, including a genetic disease or cancer, may be characterized by, caused by, or promoted by a single nucleotide variation (SNV). SNVs may result in missense mutations in a gene. 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 carbamoyltransferase deficiency, S-Lewis-O'Neal syndrome, Tay-Sachs disease, Niemann-Pick disease, or very long chain acyl-CoA dehydrogenase deficiency.

[0032] Throughout this application, the term "about" is used according to its ordinary and customary meaning in the fields of cell biology 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.

[0033] When used with the terms "including" or "comprising" without a quantifier in front of an element, it can mean "one / an", but is also consistent with the meaning of "one or more / one or more", "at least one / at least one" and "one or more than one / one or more than one".

[0034] As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined or exclusive of 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 can be specifically excluded from one or more than one aspect.

[0035] The words "comprising," "including," "having," "characterized by," "containing," and the like are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0036] The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any of the ingredients 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 subject matter to the specified materials or steps, and those materials or steps that do not materially affect the basic and novel characteristics thereof. It is contemplated that embodiments and aspects described in the context of the terms "comprising" or "including" may also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0037] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to implement the methods of the invention.

[0038] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in the language of a "use" claim, such as "use" of any compound, composition, or agent discussed herein to achieve or carry out the therapeutic, diagnostic, or physiological purpose or effect.

[0039] The purposes of one or more than one sequence or composition can be applied based on any method described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed about one aspect of the present disclosure is also applicable to other aspects of the present disclosure, and vice versa.

[0040] It is particularly contemplated that any limitation discussed with respect to one embodiment or aspect of the present invention may be applied to any other embodiment or aspect of the present invention. In addition, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to produce or use any composition of the present invention. Aspects of the embodiments described in the examples are also aspects that may be implemented elsewhere in the various examples or elsewhere in the present application in the context of aspects discussed in the Summary of the Invention, the Detailed Description of the Invention, the Claims, and the Description of the Figures.

[0041] Other purposes, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating specific aspects of the present invention, are given only as illustrations, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description provided herein.

[0043] Figure 1A to Figure 1D. Distribution of pathogenic missense mutations in human diseases. A) Pie charts show the distribution of known SNVs among all SNVs (left) and pathogenic SNVs (right) in the ClinVar database. B) A few missense SNVs are common in the population and reported to ClinVar many times, while most SNVs are less frequent and reported less often. C) Among pathogenic missense mutations, not all amino acids produce pathogenic SNVs. D) At the codon level, the Arg codon produces multiple pathogenic SNVs. Mutations at the codon level are marked, where the maximum number of submitters for any single SNV is greater than 10 or equal to 10.

[0044] Figure 2A to Figure 2F. Arginine isoencoding tRNAs can be engineered to read mutant codons and restore reporter protein function. A) Illustration of a tandem fluorescent reporter gene for reporting arginine mistranslation. In GFP, Arg96 is required for rapid maturation of the fluorophore; mutations in Arg96 result in non-fluorescent GFP and a natural RFP internal reference. Expression of mutagenic MC-tRNA to decode the 96th position of the mutation and deliver Arg restores GFP fluorescence. B) Repeat the construct in A, except that the Arg codon can be converted to Cys, Trp, Gln, or His, thereby obtaining a full set of potential SNVs. C) (Top) (Left) Flow cytometry shows that GFP and RFP fluorescence appear together in positive control cells. (Right) Because the reporter gene has a tandem protein, the ratio of GFP to RFP is close to 1. (Bottom) For GFP* constructs with mutations in Arg96 as shown in A and B, GFP fluorescence does not increase as RFP increases. (Left) Representative data from Arg96His constructs. (Right) Traces of the GFP / RFP ratio for all mutant constructs confirm the loss of fluorescence. D) Flow cytometry shows the restoration of GFP fluorescence relative to the RFP fluorescence internal control. When the appropriate tRNA is expressed, GFP fluorescence is restored. E) Annexin staining (right) shows early cell death; cell death increases with increasing tRNA expression levels. F) DAPI staining shows late cell death. Cells with low MC-tRNA expression levels are not affected by toxic effects.

[0045] Figure 3A to Figure 3C .Mass spectrometry indicates wild-type sequence recovery of disease-associated peptides. A) Diagram of peptide expression constructs. Since Arg>His_G>A SNVs are associated with many SNVs from multiple diseases and multiple peptides, the inventors expressed constructs with tandem disease-associated peptides separated by a Lys-C protease site and a C-terminal affinity purification tag. Similar constructs were constructed for each Arg-associated SNV. B) Representative LC-MS-MS data for peptide XYZ associated with Xyz syndrome. C) Summary of peptide recovery for many disease-associated peptides expressing MC-tRNAArg.

[0046] Fig. 4A to Fig. 4F.MC-tRNA can be applied outside of arginine isoacceptors. A) A diagram of a fluorescent reporter gene for monitoring the delivery of serine at position 65 of GFP. Position 65 mutates to ArgCCG, which destroys the formation of the GFP chromophore, resulting in cells without fluorescence. Fluorescence can be restored by delivering serine to position 65 by MC-tRNA. B) Control transfections were performed using a plasmid that does not express tRNA, as well as a plasmid that expresses only GFP, a plasmid that expresses only RFP, or a plasmid that expresses tandem GFP-RFP. Cells expressing RFP above the black line were screened out for further analysis. (Left) Fluorescence of the GFP channel and the RFP channel of cells 24 hours after transfection are shown, showing a single repeat. (Right) A histogram of the GFP to RFP ratio is a cell expressing RFP and a cell expressing GFP-RFP. C) Plasmids and tRNA constructs with a tandem GFP*-RFP construct mutated from A to Arg(CCG) at Ser65 were transfected into HEK293T cells. Fluorescence was monitored by flow cytometry 24 hours, 48 ​​hours and 72 hours after transfection. GFP fluorescence and RFP fluorescence of a single repeat at 24 hours are shown. D) A histogram of the GFP to RFP ratio of the construct in C is shown. The expression of tRNASer (CGG) with several different skeletons can restore GFP fluorescence. The expression of tRNASer (CGA) did not restore fluorescence because it could not decode ArgCCG. E) The ratio of GFP to RFP is used as a representative of tRNA expression and is compared with Annexin V staining for detecting cell death. The increase in the GFP-RFP ratio is not related to the increase in cell death. F) Annexin fluorescence is used to determine whether the cell is a live cell or a dead cell by the threshold value shown by the dotted line in E. At 24 hours and 48 hours, the ratio of dead cells in 3 replicates of each expression plasmid is calculated.

[0047] Figures 5A to 5C .Stable cell lines are viable and have restored GFP. A) Cells expressing the WT GFP-RFP construct are polyclonal. Two populations of cells are visible, one of which has high levels of GFP fluorescence. Only cells with low staining levels of the DAPI live-dead stain are shown. B) Cells expressing a GFP-RFP construct in which GFP has the mutation R96C, which eliminates fluorescence. This population has lower GFP fluorescence. Only cells with low DAPI staining levels are shown. C) Cells expressing the Arg>Cys restored mc-tRNA are viable but grow slowly. Shown here are cells with low staining levels of the DAPI live-dead stain, and the GFP fluorescence indicates lower levels of restoration of the GFP mutation R96C compared to B.

[0048] Figures 6A to 6C.Stable cell lines are viable and have restored GFP. A) Cells expressing the WT GFP-RFP construct are polyclonal. Two populations of cells are visible, one of which has high levels of GFP fluorescence. Only cells with low staining levels of the DAPI live-dead stain are shown. B) Cells expressing a GFP-RFP construct in which GFP has the mutation R96Q, which eliminates fluorescence. This population has lower GFP fluorescence. Only cells with low DAPI staining levels are shown. C) Cells expressing the Arg>Gln restored mc-tRNA are viable but grow slowly. Shown here are cells with low staining levels of the DAPI live-dead stain, and the GFP fluorescence indicates lower levels of restoration of the GFP mutation R96Q compared to B.

[0049] 7A to 7E .Cells expressing mc-tRNAs show very little disruption to native gene expression. Stable cell lines were subjected to mRNA sequencing and compared to positive, negative, and blank controls. In all graphs, the Y-axis represents transcripts per million (TPM). A) Stable cell lines show negligible induction in response to heat shock compared to wild-type cells. A positive control of heat shocked cells was also included for comparison. B) Genes associated with apoptosis were examined in stable cell lines compared to untransfected cells and control stressed cells. Very low induction of apoptotic pathways was observed. C) Genes associated with general stress responses also showed very little changes in gene expression, so disruption to cell physiology was very minimal. D) Genes associated with the innate immune response were analyzed to see if expression of the mc-tRNA in this setting had an immunogenic effect. Very low changes in expression of these genes were observed. E) Expression of the glycolytic enzyme GAPDH was used as a control, as it was not expected that gene expression in this pathway would change. These data confirm that the overall test results for gene expression were as expected.

[0050] Fig. 8A to Fig. 8E. Validation of mistranslation reporter gene based on GFP. (A) Flow cytometry analysis of GFP expression in cells overexpressing WT GFP-mCherry fusion protein and mutant GFP-mCherry fusion protein. All living single cells are shown. Gating is set according to monochrome control. (B) Density curve of GFP expression in cells overexpressing WT GFP-mCherry fusion protein and mutant GFP-mCherry fusion protein. (C) to (E) For cells overexpressing GFP-mCherry fusion protein and point mutations at GFP65 (C), GFP96 (D) and other GFP point mutations (E), GFP signal is standardized with mCherry. Each point represents a biological replicate. The mean and standard deviation of each sample are shown as solid black dots and vertical lines, respectively.

[0051] Fig. 9A to Fig. 9C. other verification of mistranslation reporter genes based on fluorescent protein (FP). (A) Flow cytometry analysis of mPlum expression in cells overexpressing WT eGFP-mPlum fusion protein and R96C eGFP-mPlum fusion protein. All living single cells are shown. Gate control is set according to single color control. (B) Density curve of mPlum expression in cells overexpressing WT eGFP-mPlum fusion protein and R96C eGFP-mPlum fusion protein. (C) For cells overexpressing corresponding mistranslation reporter genes based on FP, FP signal is standardized with eGFP. Each point represents a biological repeat. The mean value and standard deviation of each sample are shown as solid black dots and vertical lines, respectively.

[0052] Fig.10 Summary of all quantified mistranslation reporters. Each well is labeled with a fluorescent protein (FP) and the mutation position of the mistranslation reporter. When the WT amino acid at this position (WT aa) is mutated to the mutant amino acid (Mut aa), the FP reporter signal decreases by more than 2-fold. Previously reported FP reporters are shown in gray.

[0053] Figure 11A to Figure 11G. Figure 3: mctRNA Ser-tRNA Arg(CCG) (A) Ser-tRNA Arg(CCG) Sequences of expression components. The tRNA sequence is highlighted in bold and the anticodon is underlined. The 200 bp endogenous sequence upstream of the tRNA-Ser-CGA-1-1 gene and the 200 bp endogenous sequence downstream are retained to enable correct transcription and processing of mctRNA. (B) Density curves of GFP signals in i) cells overexpressing WT GFP, ii) cells overexpressing GFP(S65R), and iii) cells overexpressing GFP(S65R) and mctRNA, which are normalized with mCherry (n=3). (C) Containing or not containing Ser-tRNA Arg (CCG) Ser-tRNA in cells overexpressing GFP(S65R) Arg(CCG) The expression level of Ser-tRNA was normalized by endogenous tRNA-Ser-CGA-1-1 (n=3). (D) Overexpression of GFP (S65R) and Ser-tRNA Arg(CCG) Endogenous tRNA-Ser-CGA-1-1 and Ser-tRNA in cells Arg(CCG) (E) In the presence of endogenous tRNA-Ser-CGA-1-1 and Ser-tRNA Arg(CCG)The average mutation rate at each nucleotide position (n = 3). (F) Arg(CCG) Ser-tRNA without mctRNA Arg(CCG) (G) Gene ontology enrichment analysis of significantly up-regulated or down-regulated genes (p>0.05, absolute fold change (FC)>2). Solid vertical lines mark p=0.05.

[0054] Figure 12A to Fig.12H .mctRNA Arg-tRNA Cys / His / Gln / Trp(***) (A) mctRNA Arg-tRNA encoding the tRNA backbone in cells overexpressing GFP(R96Q) and 19 different endogenous Arg tRNA isoforms Gln(CUG) GFP signal relative to WT GFP in cells. WT GFP and GFP(R96Q) are shown for comparison. (B) Arg-tRNA Cys / His / Gln / Trp(***) Sequence of expression component. tRNA sequence is highlighted in bold, and anticodon region is underlined. Anticodon region is mutated to anticodon of Cys, His, Gln and Trp to generate associated mctRNA. 200bp endogenous sequence upstream of tRNA-Arg-CCT-4-1 and 200bp endogenous sequence downstream are retained to enable correct transcription and processing of mctRNA. (C) Signal density curve of GFP in i) cells overexpressing WT GFP, ii) cells overexpressing GFP R96 mutant, and iii) cells overexpressing GFP R96 mutant and corresponding mctRNA, which is normalized with mCherry. D) IP-MS quantification (n=2) of peptide SAMPEGYVQER corrected by mctRNA in cells overexpressing GFP R96 mutant with or without corresponding mctRNA. (E) MS spectrum of peptide SAMPEGYVQER corrected by mctRNA in cells overexpressing GFP R96 mutant containing corresponding mctRNA. (F) Expression levels of Arg mctRNA in cells overexpressing GFP R96 mutants with or without the corresponding mctRNA, normalized with endogenous tRNA-Arg-CCT-4-1 (n=3). (G) Loading levels of endogenous tRNA-Arg-CCT-4-1 and Arg mctRNA in cells overexpressing GFP R96 mutants and 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).

[0055] Fig.13A Figure 13E. Cellular response to Arg mctRNA. (A) Differential gene expression in cells expressing GFP mutant-mCherry and corresponding mctRNA and in cells expressing only GFP mutant-mCherry. (B) to (E) Gene ontology (GO) enrichment analysis of biological processes for genes significantly upregulated or downregulated in Arg mctRNA expression (p>0.05, absolute fold change (FC)>2). Solid vertical lines mark p=0.05. Figures b to c show the top 5 GO entries with the highest enrichment folds among upregulated and downregulated genes.

[0056] Figures 14A to 14C. Arg-tRNA Gln(CUG) Restored mutations in the LGMD2A-associated protein CAPN3. (A) Schematic representation of the Calpain 3 protein domains and mutation sites. (B) Transfection of mock constructs, WT CAPN3, the catalytically inactive mutation C129S, and tRNAs with or without Arg-tRNA Gln(CUG) Western blot of cells with defective mutation R490Q. Full-length (FL) CAPN3 (94 KDa), two autolysate bands (① about 55 KDa, ② about 65 KDa) and GAPDH (37 KDa) are marked in the figure. GAPDH is an internal reference. (C) Quantification of relative Western blot band intensities (biological replicates n=4). DETAILED DESCRIPTION

[0057] Many genetic diseases are caused by mutations in the protein coding regions of DNA, which result in altered amino acid identities (missense) in the protein product. The resulting mutant protein may be biologically inactive, leading to loss of key functions and thus causing disease. Disclosed is a specific missense-correcting tRNA ( m issense c The MC-tRNA has a covalently linked (aminoacylated or charged) amino acid that does not match the anticodon sequence of the codon used to read the charged amino acid. These engineered MC-tRNAs can restore the original protein sequence during translation, thereby generating functional proteins and altering disease outcomes. Arg and MC-tRNA Ser MC-tRNA that corrects a specific Arg mutation or Ser mutation into a functional protein. Also disclosed is a method for ArgRestoring the wild-type sequence of disease-associated peptides. Also disclosed are MC-tRNAs that deliver arginine in a natural setting to treat genetic diseases caused by missense mutations of Arg residues. Certain aspects can correct single base mutations that cause disease, including mutations in which Arg mutates to different amino acids, which is the most common mutation that causes disease.

[0058] Disclosed are specific missense correction tRNAs (MC-tRNAs) for protein biosynthesis in cells xxx (yyy), where xxx = the amino acid attached to the 3' end and yyy = the anticodon sequence). The MC-tRNA has a covalently linked (charged) amino acid that does not match the anticodon sequence of the codon used to read the charged amino acid. Genetic diseases caused by missense mutations such as adrenoleukodystrophy, mucopolysaccharidosis type III (MPS-III-A), very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), etc. can be corrected using engineered MC-tRNA. In some aspects, this approach is well suited for recessive diseases where restoring a small amount of native protein activity can correct the phenotype. Some aspects focus on rare metabolic diseases that develop in infancy and have a poor prognosis.

[0059] Certain aspects relate to the identification of uneven distribution of specific types of mutations, and that Arg to Cys, Arg to Trp, Arg to Gln, and Arg to His mutations are the most common, accounting for 9% of all pathogenic SNVs. Aspects related to correcting genetic diseases by co-translational missense correction of genetic diseases (CoMED) are disclosed.

[0060] I. Obtaining Nucleotides

[0061] A. Synthesis

[0062] Nucleic acid molecules, including tRNAs described herein or nucleic acids encoding the tRNAs, can be generated by nucleic acid synthesis. tRNAs or nucleic acids encoding the tRNAs can be synthesized using any method known in the art, such as phosphoramidite synthesis and / or solid phase synthesis. tRNAs or nucleic acids encoding the tRNAs can be synthesized.

[0063] B. Expression

[0064] Nucleic acid molecules, including any tRNA described herein or nucleic acids encoding the tRNA, can be generated by expression vectors. The expression vectors used herein may contain sequences for plasmid maintenance or viral maintenance and sequences for cloning and expressing exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and generally include one or more of the following operably linked nucleotide sequences: a promoter, one or more than one enhancer sequence, an origin of replication, a transcription termination sequence, and a selective marker element. Such sequences and methods of using them are well known in the art.

[0065] 1. Expression system

[0066] There are many expression systems that contain at least some or all of the above expression vectors. Prokaryotic and / or eukaryotic systems can be used to generate nucleic acid sequences. Widely commercially available systems include, but are not limited to, bacterial cell systems, mammalian cell systems, yeast cell systems, and insect cell systems. Those skilled in the art can utilize appropriate expression systems to express vectors to generate nucleic acid sequences.

[0067] 2. Gene transfer method

[0068] As described herein or known to those of ordinary skill in the art, methods suitable for delivery of nucleic acids to achieve expression of the composition are contemplated to include virtually any method of introducing nucleic acid (e.g., DNA, including viral and non-viral vectors) into a cell, tissue, or organism. Such methods include, but are not limited to, direct delivery of DNA, such as by 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, all of which are incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, which is incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, which is incorporated herein by reference); by 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 microprojectile bombardment (PCT Applications WO 94 / 09699 and WO 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, all of which are incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, all of which are incorporated herein by reference); tumefaciens-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, both of which are incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, both of which are incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral transduction or retroviral transduction.

[0069] 3. Host cells

[0070] On the other hand, it is contemplated to use host cells into which recombinant expression vectors have been introduced. The vector DNA can be introduced into prokaryotes or eukaryotic cells by conventional transformation techniques or transfection techniques. Some vectors may employ control sequences that enable replication and / or expression in prokaryotes and eukaryotic cells. Those skilled in the art will appreciate the conditions under which host cells are incubated to maintain them and allow vector replication. It is also understood and known that techniques and conditions that allow large-scale production of vectors and production of nucleic acids encoded by the vectors.

[0071] For stable transfection of mammalian cells, it is known that only a small portion of cells can integrate foreign DNA into their genomes, depending on the expression vector and transfection technique used. In order to identify and select these integrants, selective markers (e.g., resistance to antibiotics) are usually co-introduced into host cells with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selective marker gene can survive, while other cells die) and other methods known in the art.

[0072] II. Pharmaceutical Compositions

[0073] In some aspects, compositions or agents, including compositions or agents used in the methods disclosed herein, such as tRNA, nucleic acids encoding tRNA, vectors comprising tRNA, or cells comprising tRNA, are suitably contained in a pharmaceutically acceptable carrier. The carrier can be nontoxic, biocompatible, and selected to not adversely affect the biological activity of the agent. The medicament in some aspects of the present disclosure can be formulated into a preparation for local delivery (i.e., delivery to a specific location of the body, such as the brain, neural tissue, or other tissues) or systemic delivery, which is in the form of a solid, semisolid, gel, liquid, or gas, such as a tablet, capsule, powder, granule, ointment, solution, storage agent, inhalant, and injection, which can be administered orally, parenterally, or surgically. Some aspects of the present disclosure also contemplate local application of compositions by coating medical devices, etc.

[0074] Suitable carriers for parenteral delivery and topical delivery include distilled water, physiological phosphate buffered saline, normal Ringer's solution or lactated Ringer's solution, dextrose solution, Hank's solution or propylene glycol. In addition, sterile fixed oils can be used as solvents or suspension media. Any biocompatible oil including synthetic monoglycerides or diglycerides can be used for this. In addition, fatty acids such as oleic acid can also be used to prepare injections. The carrier and the medicament can be mixed into a liquid, a suspension, a polymerizable or non-polymerizable gel, a paste or an ointment.

[0075] The carrier can also include a delivery vehicle to maintain (i.e., extend, delay or regulate) the delivery of one or more than one medicament or enhance the delivery, uptake, stability or pharmacokinetics of one or more than one therapeutic agent. As non-limiting examples, such a delivery vehicle can include microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer hydrogels or copolymer hydrogels and polymer micelles.

[0076] Solutions of pharmaceutical compositions can be prepared in water with suitable mixing with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0077] In certain aspects, the pharmaceutical composition is advantageously administered in the form of an injectable composition that is a liquid solution or suspension; it can also be prepared in solid form, which is suitable for dissolution or suspension in a liquid prior to injection. These preparations can also be emulsified. Typical compositions for this purpose include a pharmaceutically acceptable carrier. For example, the composition can contain 10 mg or less, 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, including salts, preservatives, buffers, etc.

[0078] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, such as ethyloleate. Aqueous carriers include water, alcohol / water solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include liquids and nutritional supplements. Preservatives include antimicrobials, antifungals, antioxidants, chelating agents, and inert gases. The pH and exact concentrations of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

[0079] Other formulations are suitable for oral administration. Oral formulations include typical excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. The composition is in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation or powder.

[0080] In other aspects, the pharmaceutical composition may include a classical pharmaceutical preparation. According to certain aspects, the administration of the pharmaceutical composition may be carried out by any common route, as long as the target tissue can be reached by the route. This may include oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, it may be administered by in situ injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection. Such compositions are usually administered in the form of a pharmaceutically acceptable composition, which includes a physiologically acceptable carrier, buffer or other excipients. For the treatment of pulmonary disorders, an aerosol delivery method may be used. The volume of the aerosol may, for example, be from about 0.01 ml to about 0.5 ml.

[0081] The effective amount of the pharmaceutical composition is determined according to the intended purpose. The term "unit dose" or "dose" refers to physically discrete units suitable for use in subjects, each unit containing a predetermined amount of the pharmaceutical composition calculated to produce the above-mentioned desired response associated with its administration, i.e., appropriate route and treatment regimen.

[0082] A. Protein

[0083] The nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed 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 (www.ncbi.nlm.nih.gov / ) and the Global Protein Resource Database (UniProt; www.uniprot.org / ). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those of ordinary skill in the art.

[0084] B. Other preparations

[0085] It is contemplated that other agents may be used in conjunction with certain aspects. These additional agents include preparations that combine and / or synergize with the tRNA, nucleic acids encoding tRNA, vectors comprising tRNA, or cells comprising tRNA as described herein. Additional agents may include agents that alleviate the symptoms of the diseases disclosed herein, or may include agents that alleviate the side effects associated with the therapeutic compositions disclosed herein.

[0086] III. Sequence and Single Nucleotide Variations

[0087] Table 1: Nucleic acids for use in aspects disclosed herein

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Table 2 – Single nucleotide variants in genetic diseases characterized by missense mutations

[0120]

[0121]

[0122]

[0123]

[0124] Table 3: Single nucleotide variants in genetic diseases characterized by missense or nonsense mutations.

[0125]

[0126]

[0127] Example

[0128] The following examples are used to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes of practice of the present invention. However, it will be appreciated by those skilled in the art that, without departing from the spirit and scope of the present invention, many changes may be made to the disclosed specific embodiments and the same or similar results may be obtained.

[0129] Example 1: Results

[0130] Distribution analysis of pathogenic missense mutations

[0131] The inventors searched the ClinVar database (reference) for reported single nucleotide variations (SNVs) to obtain detailed information on the types of pathogenic SNVs in CDS. Among the SNVs of all analyzed CDS, missense mutations accounted for 92%, and the rest were nonsense mutations; however, many missense mutations were not associated with disease. Among the SNVs of CDS annotated as "pathogenic", "likely pathogenic" and "pathogenic / likely pathogenic", missense mutations accounted for 58% (Figure 1A). Of the total of approximately 42,500 missense mutations, most missense mutations had only one submitter, indicating that the allele frequency was rare and the vast majority of them were reported less than 10 times; however, about 100 specific pathogenic SNVs were reported more than 20 times (Figure 1B). This result shows that pathogenic mutations are unevenly distributed in the human genome, and there are specific hotspot mutations that recur at abnormally high frequencies.

[0132] Dividing pathogenic missense mutations into single amino acids showed that the highest proportion of mutations of Arg to another amino acid was >6700 (16% of all pathogenic missense SNVs; 9% of pathogenic SNVs including nonsense mutations), followed by mutations of Gly to another amino acid (Figure 1C). The frequencies of pathogenic mutations of the remaining 18 amino acids were similar and more than 2-fold lower than those of Arg mutations or Gly mutations. In addition, the analysis of the types of pathogenic mutations combined with the number of reported disease occurrences (Figure 1B) showed that the top four common pathogenic SNVs were indeed represented by four Arg codon mutations: Arg codon mutation to Cys, Arg codon mutation to Trp, Arg codon mutation to Gln, and Arg codon mutation to His (Figure 1D). These results indicate that mutations of Arg to just four amino acids represent the vast majority of pathogenic single-base mutations.

[0133] In order to determine the Arg In view of the fact that mutations of Arg codon to Cys, Arg codon to Trp, Arg codon to Gln and Arg codon to His can treat specific diseases, the inventors searched for diseases with the following criteria in ClinVar:

[0134] (i) The disease is recessively inherited.

[0135] (ii) The mutation is in the coding sequence.

[0136] (iii) The mutation is associated with a known rare metabolic disorder.

[0137] (iv) Each SNV is submitted by 4 or more submitters.

[0138] Applying these criteria, we identified 12 major diseases that are associated with at least 3 of the 4 Arg codon mutations (Table 1). Combining estimates of disease incidence and SNV frequency, we estimated that using only MC-tRNA Arg (GTG), which can treat about 55 new patients in the United States each year. Similarly, MC-tRNA Arg (GCA), MC-tRNA Arg (CCA) and MC-tRNA Arg (TTG) can treat approximately 49, approximately 44, and approximately 51 new patients per year, respectively. In total, the four MC-tRNAs for this selected rare disease Arg The construct could treat about 200 new patients in the U.S. each year.

[0139] Using MC-tRNA Arg Generating functional proteins from DNA mutations

[0140] The inventors designed a reporter protein to test the use of MC-tRNA Arg The feasibility of generating functional proteins from genes containing Arg mutations at the DNA level during translation in the absence of MC-tRNA Arg In the absence of a functionally defective protein (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 the seven codons present in the most frequent pathogenic Arg mutations (generating GFP*), which include Arg to Cys (CGC / CGT to TGC / TGT), Arg to Trp (CGG to TGG), Arg to Gln (CGA / CGG to CAA / CAG), and Arg to His (CGC / CGT to CAC / CAT) (Figure 2B, see below). This mutation extends the maturation time of GFP from hours to months (PMID: 14523232, PMID: 16331981, PMID: 18470931), so that the resulting GFP is virtually non-fluorescent on the time scale of laboratory experiments.

[0141]

[0142] MC-tRNA Arg The design of is more complicated because many tRNA entities can play this role. The inventors chose to test 20 different tRNAs in the reference human genome (Genomic tRNA Database, http: / / gtmadb.ucsc.edu / ) ArgThe design will be each tRNA Arg The anticodon sequence of the human tRNA was changed to the anticodon sequence of the human tRNA that reads the Cys / Trp / Gln / His sequence (Table 2). Cys has a single anticodon sequence GCA, which reads TGC and TGT codons with a GT wobble. Arg (GCA) can read TGC and TGT mutations in GFP constructs with Arg96 mutated to Cys. Natural tRNA Arg (TCT) sequence consists of 6 tRNA Arg (TCT) genes represent these genes, which contain introns that may affect MC-tRNA Arg Therefore, the inventors also used another 6 intron-containing constructs to replace the MC-tRNA corresponding to each anticodon. Arg The total number of MC-tRNA increased to 26. Arg For example, Trp (CCA) and His (GTG) have a single natural anticodon sequence, so MC-tRNA Arg (CCA) and MC-tRNA Arg (GTG) is designed to have the same Arg The inventors added these two anticodons to the design, thus creating MC-tRNA. Arg (TTG) and MC-tRNA Arg (CTG). Each backbone was paired with its endogenous promoter and terminator by including 200 bp upstream and downstream of the genomic context for plasmid-based expression.

[0143] The experimental test included the following steps. First, GFP*-RFP and a single MC-tRNA in the same plasmid were Arg Transfected into human HEK293T cell cultures. The inventors also included a positive control of a wild-type GFP-RFP construct and two negative controls, one of which contained only the GFP*-RFP construct and the other contained GFP*-RFP and a tRNA that reads only the Arg codon. Arg(TCG). After 24 hours, 48 ​​hours and 72 hours, the green fluorescence level and red fluorescence level in the transfected cells were detected by flow cytometry. The positive control showed high levels of fluorescence for GFP and RFP along the diagonal (Figure 2C). All negative controls for GFP*-RFP (Figure 2C, at the bottom). MC-tRNA Arg The presence of tRNA will produce a large amount of functional GFP protein. The actual intensity of green fluorescence depends on the MC-tRNA used. Arg type and GFP* mutation in DNA (Figure 2D). These results indicate that MC-tRNA Arg The strategy works in human cells, generating functional proteins at the translation level from genetic mutations at the DNA level.

[0144] MC-tRNA Arg In principle, many non-Arg codons will be read, resulting in a large number of mistranslations. However, mistranslation is not inherently fatal. In fact, cells can tolerate high levels of mistranslation and even naturally regulate the level and type of mistranslation (reference). Nevertheless, the inventors conducted additional experiments to examine the expression of MC-tRNA Arg 2E). Both annexin V stainings indicated cell death. The inventors observed similar levels of cell death in cells expressing MC-tRNA and control cells, indicating very little toxic effect. The inventors found that although some MC-tRNA Arg showed strong toxicity, leading to massive cell death, but other MC-tRNA Arg showed weaker toxicity, suggesting that specific MC-tRNA can be obtained Arg To minimize toxicity derived from mistranslation.

[0145] In order to prove that MC-tRNA Arg Indeed, MC-tRNA was expressed in cells, and the inventors performed high-throughput tRNA sequencing on several transfected cells that showed the highest levels of GFP fluorescence (Figure 2F). Arg Can be distinguished from endogenous tRNA by its anticodon sequence. Arg The anticodons of tRNA are ICG (GCG in sequencing), TCG, CTC, and TTC, while MC-tRNA Arg The anticodons of MC-tRNA are GCA, CCA, GTG, TTG and CTG. Arg The expression level is quite high, accounting for 1% of the endogenous tRNA in the experiment. Arg from X% to Y%.

[0146] MC-tRNA Arg Restoration of Arg residues in disease-associated peptides

[0147] In order to prove that MC-tRNA Arg To ensure that non-Arg codons are read and Arg is incorporated into translation, the inventors performed mass spectrometry analysis on reporter protein constructs containing natural peptide sequences in the disease context (Figure 3A, Table 3). A single polypeptide reporter construct contains a fusion block of a 21 amino acid peptide sequence centered on the pathogenic mutation (disease-associated peptide), each separated by a Gly-Lys-Gly sequence that provides a Lys-C protease cleavage site. Each reporter gene also contains a triple Flag tag near the C-terminus to facilitate purification by immunoprecipitation. The inventors designed a total of 4 constructs, each of which contained a single type of Arg mutation to the same amino acid. For example, all disease-causing Arg to His mutations are present in the same reporter construct, so all missense correction events from His to Arg can be corrected with MC-tRNA Arg (GTG) was tested. A similar strategy was applied to MC-tRNA Arg (GCA), MC-tRNA Arg (CCA), MC-tRNA Arg (TTG or CTG) reporter constructs were tested for Arg to Cys, Arg to Trp, and Arg to Gln mutations.

[0148] Our mass spectrometry results showed that in the absence of MC-tRNA Arg In this case, the cell naturally produces the disease-associated peptide. Arg In the presence of , endogenous tRNA was still used to produce disease-associated peptides, and missense correction peptides corresponding to the correction of X mutation to Arg during translation were also detected (Figure 3B). The results of each X mutation to Arg correction peptide can be semi-quantitatively compared with the disease-associated peptide ( Figure 3C ). As expected, in most cases, MC-tRNA Arg showed significant levels of correct peptide production. Arg (CTG) can only read the CAG codon of Gln, but not the CAA codon of Gln, so it is expected that MC-tRNA Arg (CTG) has no corrective effect on the Arg to Gln (CAA) mutation.

[0149] Missense correction also occurs at MC-tRNA Ser

[0150] To investigate missense correction of amino acids other than Arg, the inventors designed similar constructs to test MC-tRNA SerThis construct was also made of fused GFP and RFP, except that GFP contained a mutation of the Arg codon from Ser65 to CGG, which resulted in GFP not fluorescing ( FIG. 4A ). The inventors also constructed several constructs containing GFP (Ser65 mutated to CGG)-RFP (GFP*-RFP) and MC-tRNA Ser Plasmid construct of (CCG). MC-tRNA Ser (CCG)1 and MC-tRNA Ser (CCG)2 has tRNA Ser (AGA) and tRNA Ser (CGA) isoenzyme decoding tRNA tRNA main body sequence. MC-tRNA Ser (CCG)2+ is derived from MC-tRNA Ser (CCG)2 has the same tRNA main sequence, but the variable loop is extended from 3 nucleotides to 6 nucleotides. Finally, the inventors also constructed a tRNA containing GFP*-RFP and tRNA Ser (CGA) negative control construct.

[0151] At 24 hours and 48 hours after transfection, the inventors used flow cytometry to detect the fluorescence levels of GFP and RFP in HEK293T cells transfected with these plasmids. Cells were gated by RFP fluorescence to verify whether the transfection was successful (Figure 4B). As expected, the negative control of the GFP*-RFP plasmid without any additional tRNA showed only red fluorescence (red). The positive control of the wild-type GFP-RFP plasmid showed higher green and red fluorescence (green). All three containing MC-tRNA Ser The plasmids containing (CCG) (rows 2, 3, and 4) all showed restored green fluorescence, with levels within several folds of that of the positive control GFP-RFP plasmid (Figure 4C). Ser The negative control (CGA) (fifth row) showed no green fluorescence recovery, which supports the MC-tRNA Ser of positive results.

[0152] Although all three MC-tRNA Ser (CCG) all restored comparable levels of GFP fluorescence, but the quantitative differences between these constructs highlight the importance of using different tRNA body sequences for missense correction. Ser Compared with (CCG)1, MC-tRNA Ser (CCG)2 had a narrower distribution and a higher percentage of cells with high GFP fluorescence levels, indicating that MC-tRNA Ser (CCG)2 is higher than MC-tRNASer (CCG)1 is a better missense correction factor (Figure 4D). Ser Extension of the variable loop in (CCG)2+ and MC-tRNA Ser (CCG)2 showed no substantial differences compared to the parent sequence, although there are literature reports indicating that extended variable loops can lead to a 3-fold increase in stop codon repression levels (PMID: 20026070). It remains to be seen whether other changes between the tRNA main sequence and the sequence in the reference human genome will produce more effective missense correction factors. The inventors further explored whether the expression of MC-tRNA would lead to cell death. It has been previously reported that this type of extensive mistranslation may be cytotoxic. However, using Annexin V staining to detect cell death, the inventors found that the cell death rate of cells expressing MC-tRNA was not increased compared to control cells (Figure 4E, Figure 4F). The inventors used the ratio of GFP to RFP expression in cells expressing MC-tRNA as a representative of tRNA expression levels in living cells. The inventors did not find a correlation between the GFP to RFP ratio and Annexin V staining (Figure 4E).

[0153] Finally, the inventors reviewed the literature and identified 18 fluorescent proteins that can be used to study missense correction in the same way as Arg and Ser (Table 4). The use of these proteins can be used to perform high-throughput screening of effective MC-tRNAs for each amino acid mutation that causes human disease.

[0154] Example 2: Discussion

[0155] Potential of CoMED strategies for the treatment of genetic diseases: In this work, the inventors describe a strategy to use missense correction tRNAs to treat genetic mutations that are pathogenic or likely to be pathogenic in thousands of reported human diseases. The basic concept relies on the use of MC-tRNAs that charge codons for one amino acid but read a different amino acid when translated. The co-translational missense correction process results in a functional protein, whereas the same protein translated according to the genetic code is nonfunctional. The inventors demonstrate that MC-tRNA Arg and MC-tRNA Ser The respective missense mutations were robustly corrected in human cell cultures, providing proof of principle for the concept of co-translational missense correction (CoMED) of genetic diseases.

[0156] In some ways, the strategy is well suited for treating genetic recessive diseases, in which a genetic mutation produces little or no functional protein, so using MC-tRNA could produce useful amounts of the protein to treat the disease.

[0157] Missense correction and toxicity: The main concern for introducing MC-tRNA into cells is toxicity, because MC-tRNA will also misread other codons in other proteins, thereby increasing the degree of mistranslation. In the central dogma of molecular biology, the typical fidelity range for replication is 10 -8 Up to 10 -9 , for transcription is 10 -5 Up to 10 -6 , for the translation of 10 -3 Up to 10 -4 . Not only does translation have a minimum level of fidelity, but cells can also actively change the fidelity of translation in response to environmental conditions to expand their proteome diversity to better respond and adapt (reference). It is also clear that a large amount of mistranslation can lead to high levels of proteotoxic stress and cell death. The toxic effects may depend on many factors, such as the cell type (neuron, liver, kidney, etc.), the amount of MC-tRNA expressed desired for disease treatment, the length of MC-tRNA exposure, and the specific type of MC-tRNA.

[0158] The MC-tRNA backbone is not limited to tRNAs in the reference human genome: in this work, the inventors used only tRNA backbones from the reference human genome, which was derived from mixed DNA from about 100 individuals. However, there is a large library of other tRNA sequences in the human population (PMID: 30643023), so the number of naturally available tRNA backbones can be easily exploited for additional testing of MC-tRNA efficacy. In addition, tRNA sequences of non-human origin, such as mouse / rat, yeast and even bacterial tRNAs can also be tested, as long as they can be loaded by the corresponding human aminoacyl-tRNA synthetase, they can be functional MC-tRNAs, such as non-human MC-tRNA Arg Can be charged by human arginyl-tRNA synthetase.

[0159] Modifications of tRNA may be required to fine-tune the efficacy of MC-tRNA: the innate immune system recognizes unmodified tRNA as foreign, while certain modified tRNAs do not trigger an immune response (reference). Therefore, to achieve the highest efficacy while minimizing the immune response, MC-tRNA can contain specific modifications. tRNA modifications are also very effective in regulating co-translational efficiency and decoding selectivity. Human cytoplasmic tRNA contains an average of 13 modifications per molecule, but not all modifications are required for maximum efficiency. For example, pseudouridine alone has been shown to be very effective in minimizing the immune response of mRNA vaccines for COVID-19. Various modifications and their combinations can be tested to obtain the optimal MC-tRNA construct with the highest efficacy.

[0160] Combining 200 tRNAs to Treat All Single Nucleotide Missense Mutations: In summary, the inventors describe a CoMED strategy for using MC-tRNAs to treat human genetic diseases, with the first focus on using 4 to 5 MC-tRNAs for approximately 200 potential new patients per year in the U.S. Arg Targeting Arg mutations to Cys / Trp / His / Gln mutations. In principle, a collection of about 200 MC-tRNAs can treat all single nucleotide missense mutations that cause human diseases.

[0161] Example 3: Materials and Methods

[0162] MC-tRNA Ser experiment

[0163] MC-tRNA containing promoter Ser Construct:

[0164] The tRNA sequences and DNA blocks used to express MC-tRNA are shown in Table 1. These blocks were cloned into a mammalian expression plasmid that also expressed a tandem GFP*-RFP polypeptide reporter gene.

[0165] Transfection protocol:

[0166] HEK293T cells are grown to confluence. Cells are trypsinized and washed. Depending on the [number] of cells, they are mixed with [ng of plasmid] and [lipofectamine] for transfection. After [incubation], cells are washed and incubated at [temperature] for [time].

[0167] Staining protocol:

[0168] When indicated, cells were stained with DAPI or Annexin-V AlexaFluor 647 or both as follows: [DAPI]. [Annexin-V]

[0169] Flow cytometry protocol:

[0170] At 24, 48, and 72 hours post-transfection, cells were trypsinized with [Trypsen reagent] in 96-well plates. Cells were pelleted, supernatant discarded, washed with PBS, and resuspended in 200uL PBS. Cells were then analyzed on [instrument] using [laser]. Instrument default voltages were used. Forward and side scatter parameters were used to gate live cells, and 10,000 events in this gate were recorded. Subsequent analysis was done with custom scripts in R using the FlowCore and FlowVisCRAN packages.

[0171] ClinVar calculation

[0172] ClinVar data were downloaded from (https: / / ftp.ncbi.nlm.nih.gov / pub / clinvar / tab_delimited / ) (PubMed PMID: 31777943) on January 4, 2022. The analysis was done using custom scripts in R.

[0173] Comparison of missense SNVs and nonsense SNVs among all SNVs

[0174] The mutation summary dataset from ClinVar was filtered by Assembly = "hg38", type = "single nucleotide variant". The name field was parsed to obtain the WT and SNV amino acid identities as well as the WT and SNV nucleotides. If the SNV amino acid was "Ter", indicating terminator, it was marked as a nonsense mutation, otherwise the entry was marked as a missense mutation. The number of missense and nonsense SNVs reported was summarized.

[0175] Comparison of pathogenic SNVs in amino acids

[0176] The above parsed SNV set was further screened according to the ClinicalSignificance field, accepting "pathogenic", "likely pathogenic" and "pathogenic / likely pathogenic". The screened set was used as the set of pathogenic SNVs. The SNVs were grouped according to the wild-type amino acid, and the number of SNV entries in each group was added up for comparison.

[0177] Comparison of the number of submitters for each SNV

[0178] In the pathogenic SNV set selected above, the NumberSubmitters field is used to make a histogram to reflect the frequency of reporting of single SNVs from the population.

[0179] Comparison of the number of missense pathogenic SNVs and each SNV submitter

[0180] The PhenotypelDS field is parsed to identify the Orphanet ID associated with each SNV. The SNVs are then grouped based on their mutation type, i.e., a combination of WT amino acid, WT nucleotide, SNV amino acid, and SNV nucleotide. This identification information represents codon-level information, although each combination can be matched to a small set of possible codons. Using these groups, the NumberSubmitter for each SNV is added to derive "total_reports". In addition, a list of unique PhenotypelDS entries is counted to track the "number_diseases" associated with each SNV type. The number of submitters for each SNV is also tracked. For plotting, only the names of SNV types where at least one single SNV has more than 10 submitters are shown.

[0181] Example 4: Detailed information on the CAPN3 gene associated with the disease LGMD2A and missense mutations:

[0182] The disease limb-girdle muscular dystrophy type 2A (LGMD2A) is associated with mutations in the gene Cablain3, CAPN3. Typically, this disease is thought to be genetically recessive. The inventors speculated that restoring the enzymatic function of a small amount of CAPN3 would be sufficient to improve symptoms in LGMD2A patients, so tRNA therapy would be a good choice. This disease was chosen as a good candidate for commercial development for the following 6 reasons: 1) Since CAPN3 is an enzyme and the disease is genetically recessive, tRNA therapy is likely to be effective; 2) There is evidence that gene therapy has safety issues due to off-target cardiac delivery of the CAPN3 gene; 3) The disease is non-lethal, so there is an adult patient population that can participate in clinical trials without waiting for diagnosis; 4) The number of patients, although small, may be sufficient to support treatment development, estimated at 1 in 100,000 newborns in the United States; 5) The affected tissue is skeletal muscle, which has multiple feasible delivery options; 6) There are convenient assays to detect gene function, such as autoprotein hydrolysis.

[0183] The disease and SNVs appear in ClinVar, so the inventors analyzed known pathogenic SNVs and possible pathogenic SNVs associated with the disease. The inventors found that 65.2% of the SNVs were missense mutations, compared to 34.8% nonsense mutations (there are mutations that affect splicing or frameshift, which are excluded from this analysis). Among the missense mutations, Arg>Gln accounted for 24% of the reported cases; Arg>Trp accounted for 12%; Arg>Cys accounted for 4%; Arg>His accounted for 3%, a trend consistent with the analysis presented in this article, indicating that mutations from Arg account for the majority of pathogenic missense mutations in all ClinVar diseases. Although each disease is unique, the distribution of SNVs in LGMD2A in the human population follows the same trend. Although Arg>Gln mutations are the largest single mutation type, it includes many different SNVs, all of which should be treatable with mc-tRNA. The Supplementary Table illustrates the exact SNVs of CAPN3 reported in Clinvar, as well as statistics for the composite score of Arg>Gln mutations included therein, and the score of missense mutations compared to nonsense mutations.

[0184] To test whether mc-tRNA can truly restore CAPN3 function, the inventors purchased plasmids expressing mutant CAPN3 that are predicted to be dysfunctional in their well-documented autoproteolytic activity compared to wild-type. These CAPN3 mutants will be expressed in HEK293 cells and co-expressed with mc-tRNA to repair their corresponding mutations. The inventors selected CAPN3 mutations that have either been biochemically validated in the literature or confirmed to be present in LGMD2A patients by the Coalition to Cure Calpain 3. The inventors performed control western blot experiments using HEK293 cells expressing CAPN3 or mutant CAPN3, but not expressing mc-tRNA constructs.

[0185] Example 5: Generation of stable cell lines expressing GFP.RFP, or mutant GFP.RFP, or mutant GFP.RFP and corrective tRNA

[0186] Stable cell line generation

[0187] HEK293 cells were purchased from ATCC and cultured using standard sterile mammalian cell culture procedures. More specifically, HEK293 cells were maintained in culture in 10 cm polystyrene cell culture treated dishes in DMEM supplemented with 10% FBS, penicillin, and streptomycin at 37°C in a humidified atmosphere containing 5% CO2. To generate stable cell lines expressing the protein and tRNA of interest, HEK293 cells were split and subcultured in 6-well cell culture treated plates, transfected with plasmids expressing the desired protein / tRNA of interest and a selectable marker, and then screened using antibiotic-based selection. The goal of this project was to study mutation correction and stress in cells constitutively expressing tRNAs engineered to recognize and correct specific missense mutations (mc-tRNA).

[0188] For transfection, healthy HEK293 cells were first seeded into 6-well cell culture treated plates in DMEM supplemented with 10% FBS only at a density of 900,000 cells / well. The cells were allowed to adhere for 24 hours. After 24 hours, the cells were transfected using lipofectamine 2000 and the plasmid of interest. More specifically, for each well, 5ul of lipofectamine 2000 reagent was mixed with 2ug of the plasmid of interest in 500ul of OptiMEM and incubated at room temperature for 20 minutes. The mixed lipofectamine / plasmid transfection reagent was then added dropwise to the corresponding wells and mixed by shaking the wells clockwise and counterclockwise 20 times. The transfected cells were incubated for 4 hours at 37°C in a humidified atmosphere containing 5% CO2. After 4 hours, the medium was removed from each well and replaced with new DMEM supplemented with 10% FBS, penicillin and streptomycin. All transfected plasmids contained an ampicillin resistance component and a puromycin resistance component.

[0189] The following plasmids were transfected:

[0190] -p2- Positive control plasmid expressing eGFP.mCherry fusion protein from CMV promoter. Both fluorescent proteins are functional.

[0191] -p100- Negative control expressing an eGFP.mCherry fusion protein containing a mutant eGFP. The mutant eGFP has an amino acid change from R to Q at position 97 (R97Q). The mutant is described herein as eGFP(R97Q).mCherry. The codon for Q at this position is CAG. This mutation renders eGFP non-fluorescent, but the RFP protein still fluoresces.

[0192] -p108- Test plasmid expressing eGFP(R97Q).mCherry and mc-tRNA that recognizes the mutant CAG codon but delivers arginine. This tRNA has previously been shown to correct the mutation in eGFP in transient transfection-based experiments.

[0193] -p401- Negative control expressing an eGFP.mCherry fusion protein containing a mutant eGFP. The mutant eGFP has an amino acid change from R to C at position 97 (R97C). The mutant is described herein as eGFP(R97C).mCherry. The codon for C at this position is TGC. The mutation of the eGFP greatly reduces its fluorescence, but the RFP protein still has full fluorescence.

[0194] -p404 - a test plasmid expressing eGFP(R97C), mCherry and mc-tRNA that recognizes the mutant TGC codon but delivers arginine. This tRNA has previously been shown to correct mutations in eGFP in transient transfection-based experiments.

[0195] A total of 2x6 well plates of cells were transfected. The first plate was transfected with plasmid p2, plasmid p100, plasmid p103 and plasmid p108. The second plate was transfected with plasmid p2, p401 and p404. Additional untransfected wells were retained to provide screening controls.

[0196] Transfected cells were grown and expressed at 37°C in a humidified atmosphere containing 5% CO2 for 48 hours. After 48 hours, cells were screened using 0.5ug / ml puromycin. Cells were maintained in a medium containing 0.5ug / ml puromycin for 2 weeks, with the medium replaced every 2 to 3 days. After 2 weeks of screening, cells that had stably integrated the plasmid into their genome were still alive and growing. These stable cells were expanded into 10cm cell culture dishes and maintained at 37°C in a humidified atmosphere containing 5% CO2 in DMEM supplemented with 10% FBS, penicillin, streptomycin and 0.3ug / ml puromycin.

[0197] The following stable cell lines were generated:

[0198] - Cell line 03-00 - HEK293 cells transfected with p2, expressing WT eGFP.mCherry, polyclonal, resistant to at least 0.5ug / ml of puromycin

[0199] - Cell line 04-00 - HEK293 cells transfected with p401, expressing eGFP(R97C).mCherry, polyclonal, resistant to at least 0.5ug / ml of puromycin

[0200] - Cell line 05-00 - HEK293 cells transfected with p404, expressing eGFP(R97C).mCherry and mc-tRNAArg-tRNA-CysGCA, polyclonal, resistant to at least 0.5ug / ml puromycin

[0201] Resistant

[0202] - Cell line 06-00 - HEK293 cells transfected with p2, expressing WT eGFP.mCherry, polyclonal, resistant to at least 0.5ug / ml of puromycin

[0203] - Cell line 07-00 - HEK293 cells transfected with p100, expressing eGFP(R97Q).mCherry, polyclonal, resistant to at least 0.5ug / ml of puromycin

[0204] - Cell line 08-00 - HEK293 cells transfected with p108, expressing eGFP(R97Q), mCherry and mc-tRNAArg-tRNA-GlnCUG, polyclonal, resistant to at least 0.5ug / ml puromycin

[0205] Resistant

[0206] *Note: The naming convention for cell lines is as follows: cell line number-passage number. For example: 06-03 represents cell line 6, passaged 3 times.

[0207] It is noteworthy that the stable cell line 05 transfected with plasmid p404 grew very slowly compared to the other cell lines. The inventors believe that this is due to the large repressive effect of the mc-tRNA, which restored 10% of the eGFP fluorescence in the transient transfection-based experiment. In addition, the positive control cell lines (transfected with p2) in both culture plates survived the screening, thus generating two polyclonal positive control cell lines (03-00 and 06-00).

[0208] Fluorescence recovery

[0209] Fluorescence recovery was examined using flow cytometry. Stable cell lines were prepared for flow cytometry as described below. Stable cells were seeded into 6-well plates at a density of 300,000 cells / well and allowed to grow for 24 hours. The culture medium was then removed and each well was washed with 1ml 1xPBS. The PBS was then removed and 500ul 0.25% Trypsin:EDTA was added to each well and incubated at room temperature for 5 minutes. After the incubation was complete, 500ul DMEM supplemented with 10% FBS was added to each well. The cells were then resuspended with a pipette, transferred to a 1.7ml centrifuge tube, and centrifuged at 5000xg for 5 minutes to precipitate. The supernatant was removed from the precipitated cells, the cell pellet was carefully stirred, and the cells were resuspended in 500ul lxPBS. The cells were then stained with DAPI to identify dead and live cells. DAPI staining was performed by adding 10ul DAPI (10ug / ml stock solution) to 500ul of resuspended cells and incubating at room temperature for 15 minutes. After incubation, add another 500ul of 1xPBS to resuspend the cells. Keep the cells on ice until use.

[0210] Fluorescence was measured using an Attune flow cytometer. GFP and DAPI signals were detected in all stable cell lines. GFP signal was used to measure missense repression of mc-tRNA. DAPI signal was used to measure cell death caused by mc-tRNA expression.

[0211] Next-generation sequencing

[0212] 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.

[0213] Example 6: Other cell culture data

[0214] Mammalian cell culture

[0215] HEK293 cells were authenticated and tested negative for mycoplasma. HEK293 cells were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle's (DMEM) high glucose medium (Cytiva Catalog No. SH30022.01) supplemented with 10% heat-inactivated fetal bovine serum and 100 U / ml penicillin-streptomycin.

[0216] Plasmids

[0217] The plasmid was synthesized and verified by GenScript, inc. The fluorescent protein-based mistranslation reporter gene is driven by the CMV promoter. The MctRNA expression component contains the endogenous sequence of 200 bp upstream and 200 bp downstream of the tRNA gene. The exact sequence is as follows:

[0218] (1)Ser-tRNA Arg(CCG) (SEQ ID NO: 140):

[0219]

[0220] (2) Arg-tRNA Cys(GCA) (SEQ ID NO: 141):

[0221]

[0222] (3) Arg-tRNA His(GUG) (SEQ ID NO: 142):

[0223]

[0224] (4) Arg-tRNA Gln(CUG) (SEQ ID NO: 143):

[0225]

[0226] (5)Arg-tRNA Trp(CCA) (SEQ ID NO: 144):

[0227]

[0228] Transfection

[0229] The cells were plated in 96-well or 6-well plates one day before transfection, and the cell confluence reached 50% to 70% on the day of transfection. TM Plasmid Miniprep / Midiprep System (Promega Catalog No. A1222 / A2492) and ethanol precipitation were used to prepare plasmids for transfection. TM 3000 (Invitrogen TM Catalog number L3000015) and transfection was performed according to the manufacturer's protocol.

[0230] Flow cytometry

[0231] Flow cytometry was performed 48 hours after transfection.TM Catalog number 25200056) HEK293 cells were digested from the plate and resuspended in 1xPBS, 5mMEDTA, 25mM HEPES pH 7.0, 1% FBS and 100ng / ml DAPI pre-cooled on ice before sorting. The flow cytometer was NovoCyte Penteon5-30. Fluorescent protein (FP) signals were detected using the following relevant detection channels: GFP, B525; mCherry, Y615; dsRed / Zoan2rfp, Y586; mPlum, Y667. For each flow cytometry run, wild-type HEK293 cells without any staining were used as background controls, and HEK293 cells overexpressing a single FP were used as gating references. The collected flow cytometry data were analyzed using custom R scripts.

[0232] Immunoprecipitation

[0233] HEK293 cells were collected 48 hours after transfection and washed twice with 1xPBS. 3-7 million cells were then incubated in 500 μl lysis buffer (1xPBS, 1% IGEPAL (NP-40), 0.1% SDS, 0.5% sodium deoxycholate by weight / volume, 1x protease inhibitor cocktail (Nacalai Cat. No. 25955)) in a rotator at 4°C for 15 minutes, followed by centrifugation at 1000g, 4°C for 5 minutes to pellet cell debris. GFP-mCherry fusion protein was immunoprecipitated (IP) by GFP monoclonal antibody (Invitrogen Cat. No. MA515256), and CAPN3 was immunoprecipitated (IP) by CAPN3 monoclonal antibody ( Catalog No. 67366-1-Ig) for immunoprecipitation. TMM-280 goat anti-mouse IgG magnetic beads (Cat. No. 11202D) were incubated at 4°C for 4 hours and then added to the cell lysate. The ratio of antibody to magnetic beads was 4 μg to 50 μl, and the antibody in the cell lysate was diluted at a ratio of 1 μg in 50 μl. The antibody-coupled magnetic beads were washed 3 times with 1 ml of lysis buffer, and then the magnetic beads were added to the cell lysate. The mixture was incubated overnight at 4°C. Subsequently, the magnetic beads were washed 3 times with 1 ml of high salt wash buffer (50 mM Tris-HCl pH7.4, 1 M NaCl, 1 mM EDTA, 1% IGEPAL (NP-40), 0.1% SDS, 0.5 weight / volume% sodium deoxycholate, 1x protease inhibitor cocktail), and the magnetic beads were washed 3 times with 1 ml of low salt wash buffer (20 mM Tris-HCl pH7.4, 10 mM MgCl2, 0.2% Tween-20, 1x protease inhibitor cocktail). The samples were analyzed by mixing with non-reducing 1x NuPAGE LDS sample buffer (Invitrogen TM The immunoprecipitated proteins were eluted from the beads and directly loaded onto 4% to 12% NuPAGE gels (Invitrogen TM NuPAGE gels were stained with Coomassie G-250 stain (Cat. #1610786), and the desired bands were cut and stored at 4°C.

[0234] Mass spectrometry sample preparation

[0235] Protein bands were extracted from SDS-PAGE gels and cut into 1 mm × 1 mm cubes. In-gel digestion was performed according to published protocols with some modifications. 1. First, the gel fragments were dehydrated with acetonitrile and the solution was removed after 5 minutes. Then, the gel fragments were covered with a sufficient volume of a solution of 5 mM TCEP in 40 mM ammonium bicarbonate containing 25% acetonitrile. The mixture was incubated at 37°C for 5 minutes to fully reduce the disulfide bonds in the protein. The gel fragments were dehydrated again with acetonitrile and another solution of 5 mM TCEP in 40 mM ammonium bicarbonate containing 25% acetonitrile was added. This step was intended to further reduce the disulfide bonds and was incubated at 65°C for 15 minutes with gentle stirring. After cooling to room temperature, the gel fragments were treated with a solution of 40 mM iodoacetamide in 40 mM ammonium bicarbonate containing 25% acetonitrile for alkylation. The gel fragments were incubated in the dark at room temperature for 15 minutes. Subsequently, the gel fragments were washed with 40 mM ammonium bicarbonate containing 25% acetonitrile for 5 minutes and then dehydrated with acetonitrile. This washing step was repeated again. The gel pieces were then swollen in a digestion buffer containing trypsin / Lys-C (8 ng / pL) in 40 mM ammonium bicarbonate and 0.5 mM CaCl2 and placed on ice for 1 hour. The gel pieces were placed on ice and excess trypsin solution was removed. The samples were digested at 37°C for 12 hours. To extract the peptides, 0.2% formic acid in 60% acetonitrile was used twice and 0.15% formic acid in 60% acetonitrile was used once, with incubation times of 10 minutes and 30 minutes, respectively. Finally, the peptides were dried in vacuo.

[0236] LC-MS parameters

[0237] The samples were analyzed using an Exploris 480 mass spectrometer connected to an UltiMate 3000 liquid chromatography system (Thermo Scientific). The chromatography system used a MonoCap column from GL Sciences measuring 50 cm in length and 0.75 mm in inner diameter (Cat. No. 5020-10006). The flow rate was maintained at 500 nL / min and the temperature was maintained at 25°C. A gradient method was used over 75 minutes using mobile phase A (0.15% formic acid in water) and mobile phase B (0.15% formic acid in 100% acetonitrile). The gradient was as follows: 5% B for 5 minutes, followed by a transition from 5% B to 22% B in 46.5 minutes, from 22% B to 34% B in 7.5 minutes, and then a rapid transition from 30% B to 95% B in 1 minute. The composition was maintained at 95% B for 4 minutes. Full scan MS spectra from m / z 350 to 1650 were collected with a resolution of 120000 at m / z 200. The maximum injection time was set to 50 ms and the AGC target value was set to 3e6. The cycle time for data acquisition was set to 3 s, while the intensity threshold was set to 5e4. For the MS / MS scan, a resolution of 15000, maximum acquisition time was set to automatic, and the AGC target value was 4e4 was adopted. The isolation window of the Orbitrap unit was set to 1.6 m / z and the first mass was set to 110 m / z. The collision energy of HCD was set to 32. A dynamic exclusion duration of 10 s was implemented, and unassigned, 1, and charge states of 8 or above were excluded. The heated capillary temperature was set to 300 °C.

[0238] MS data analysis

[0239] The raw MS data were processed and searched by Proteome Discoverer (version 3.0.0.757; ThermoFisher Scientific) using the Sequest HT search engine. The precursor mass tolerance used was 10 ppm and the fragment mass tolerance was 0.02 Da. The data were filtered using a 1% FDR cutoff, which was estimated by the Target Decoy PSM Validator. Trypsin (all) was set as the enzyme in the search. The maximum mass cut was set to 3 and the peptide length was 4 to 30. Carbamidomethyl (+57.021 Da on C) was selected as a fixed modification, while oxidation (+15.995 Da on M), deamidation (+0.984 Da on N and Q), protein N-terminal Met deletion (-131.040 Da), acetylation (+42.011 Da on N-terminal) and protein N-terminal Met deletion + acetylation (-89.030 Da) were dynamic modifications. To determine mutational transitions, the signature peptide SAMPEGYVQER was used. Signals were normalized by the amount of peptide injected.

[0240] tRNA sequencing and data analysis

[0241] According to the previously published MSR-seq protocol 2 , tRNA sequencing libraries were constructed using approximately 1 μg of total RNA. The original 100bp paired-end sequencing reads were obtained from the Illumina NovaSeq platform. Data analysis also followed the MSR-seq data processing process with minor customization. Specifically, sequencing fragment 2 was processed and mapped to a selected reference, which included all human tRNA sequences and 5 mctRNA sequences. Given the sequence similarity between mctRNA and its related endogenous tRNA, only mapped sequencing fragments longer than 60 nt were used for abundance, load, and mutation analysis.

[0242] RNA sequencing and data analysis

[0243] RNA-seq experiments were performed in 3 independent replicates of HEK293 cells overexpressing a mistranslated reporter with or without the relevant mctRNA. HEK293 cells were sorted on a FACSAria Fusion 5-18 cell sorter for mCherry+, i.e., the population of cells that were successfully transfected 48 hours after transfection. TRIzol TMTotal RNA was extracted from sorted cells using the PCR amplification reagent (Invitrogen Cat. No. 15596026). 1 μg of total RNA from each 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 on the Novasek 6000 platform (Illumina), and approximately 120 million directional 100 bp paired-end (PE) sequencing fragments were obtained for each sample. The PE sequencing fragments were mapped using STAR 2.7.10b using the reference human genome GRCh38.p10. Sequencing fragments with unique mappings were filtered, and the number of sequencing fragments for each gene in all genes was counted using featureCounts 2.0.1. Differential gene analysis was performed using edgeR (version 3.40.2). For genes that were significantly up-regulated or down-regulated (p < 0.05 and absolute fold change > 2), gene ontology analysis was performed using clusterProfiler v4.6.2.

[0244] Western blotting

[0245] Total protein samples were extracted from HEK293 cells 48 hours after transfection. To prevent CAPN3 autolytic activity, 15 mM EDTA and 1x protease inhibitor cocktail were added to all buffers during sample preparation. In addition, samples were kept on ice until denaturation. One to two million transfected HEK293 cells were washed once with ice-cold 1x PBS and washed with 30 μl of CelLytic TM M buffer (Sigma-Aldrich Product No. C2978). Centrifuge the cell lysate at 17000g, 4°C for 15 minutes to precipitate the cell debris. Collect the supernatant and incubate at 70°C for 15 minutes after adding 10 μl of 4xNuPAGE LDS sample buffer containing 5% β-mercaptoethanol. Load the denatured protein sample onto a 4% to 12% NuPAGE gel for gel electrophoresis. Before transfer, place the NuPAGE gel, filter paper, and pre-soaked -P PVDF membrane (Sigma-Aldrich Product No. IPVH00010) was incubated in transfer buffer (25mM Tris-HCl, 192mM glycine, 10% methanol) for 10 minutes. The transfer was performed using the Trans-Blot Turbo transfer system using the following settings: constant 25V; limit 1A; 20 minutes. The membrane was first blocked in blocking buffer (5% nonfat dry milk (BIO-RAD Product No. 1706404) in 1xTBST buffer) for 1 hour at room temperature and then incubated at 4°C with the primary antibody, anti-CAPN3 ( No. 67366-1-Ig) and anti-GAPDH (Invitrogen Catalog No. MA5-15738) were incubated overnight. The membrane was then washed three times with 1xTBST for 10 minutes each. Next, the membrane was incubated with 1:10,000 dilution of 680RD goat anti-mouse IgG secondary antibody (LI- Catalog No. 926-68070) in blocking buffer for 1 hour at room temperature. The blot was then washed three times with 1xTBST for 10 minutes each and then washed in Amersham Typhoon TM IR short-pass imaging.

[0246] ***

[0247] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it is obvious to those skilled in the art that the steps of the methods and methods described herein or the order of steps of the methods can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it is obvious that certain chemically related and physiologically related reagents can replace the reagents described herein and achieve the same or similar results. All these similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined in the appended claims.

[0248] References

[0249] The following references, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0250] Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Anal Chem 68, 850-858 (1996).

[0251] Watkins,C.P.,Zhang,W.,Wylder,A.C.,Katanski,C.D.&Pan,T.A multiplexplatform for small RNA sequencing elucidates multifaceted tRNA stressresponse and translational regulation.Nat Commun 13,(2022).

Claims

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, the mRNA sequence encoding a second amino acid different from the first amino acid.

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

3. The tRNA molecule of claim 2, wherein the anticodon loop sequence is UGC, GCA, GUC, UUC, GAA, UCC, GUG, GAU, UUU, UAG, UAA, CAU, GUU, UGG, UUG, UCG, UGA, GCU, UGU, UAC, GUA, AGC, CGC, CUG, CUC, CCC, GCC, AAU, UAU, AAG, CAA, CAG, CUU, CGG, AGA, CGA, AGU, CGU, CCA, AAC, CAC or AGG.

4. The tRNA molecule of claim 3, wherein the anticodon loop sequence is GUG, CUG, UUG, AGG, CGG, UGG, AAG, CAG, UAG, GCA, CCA, GCU, GCC, CCC, UCC, CUU, UUU, CGU, UGU, UAU or CAU.

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

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

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

8. The tRNA molecule of any one of claims 2 to 6, wherein the mRNA sequence from 5' to 3' end is GCU, GCC, GCA, GCG, AAU, AAC, GAU, GAC, UGU, UGC, CAA, CAG, GAA, GAG, GGU, GGC, GGA, GGG, CAU, CAC, AUU, AUC, AUA, CUU, CUC, CUA, CUG, UUA, UUG, AAA, AAG, AUG, UUU, UUC, CCU, CCC, CCA, CCG, UCU, UCC, UCA, UCG, AGU, AGC, ACU, ACC, ACA, ACG, UGG, UAU, UAC, GUU, GUC, GUA or GUG.

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

10. The tRNA molecule of claim 9, wherein the anticodon loop sequence is UGC, GCA, GUC, UUC, GAA, UCC, GUG, GAU, UUU, UAG, UAA, CAU, GUU, UGG, UUG, UCG, UGA, GCU, UGU, UAC, GUA, AGC, CGC, ACG, CCG, CCU, UCU, CUG, CUC, CCC, GCC, AAU, UAU, AAG, CAA, CAG, CUU, CGG, AGU, CGU, CCA, AAC, CAC or AGG.

11. The tRNA molecule of claim 10, wherein the anticodon loop sequence from the 5' end to the 3' end is GAA, CAA, UAA, AGG, CGG, UGG, AGU, CGU, UGU, AGC, CGC, UGC, GUA, GCA, ACG, GUU, AAU, GCC, CCU or UCU.

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

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

14. The tRNA molecule of any one of claims 9 to 12, wherein the mRNA sequence from 5' to 3' end is GCU, GCC, GCA, GCG, CGU, CGC, CGA, CGG, AGA, AGG, AAU, AAC, GAU, GAC, UGU, UGC, CAA, CAG, GAA, GAG, GGU, GGC, GGA, GGG, CAU, CAC, AUU, AUC, AUA, CUU, CUC, CUA, CUG, UUA, UUG, AAA, AAG, AUG, UUU, UUC, CCU, CCC, CCA, CCG, ACU, ACC, ACA, ACG, UGG, UAU, UAC, GUU, GUC, GUA or GUG.

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

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

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

20. The tRNA molecule of claim 19, wherein the tRNA molecule comprises a sequence having up to three substitutions relative to a human tRNA molecule.

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

22. The nucleic acid according to claim 21, wherein the sequence is one of SEQ ID NO: 1 to SEQ ID NO:

144.

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

24. The vector of claim 23, wherein the vector is an AAV vector.

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

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

27. A method for altering a protein produced by a gene, the method comprising administering 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 to a cell.

28. A method for producing a wild-type protein from a gene having a missense mutation, the method comprising administering 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 to a cell.

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

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

31. A method for treating or preventing a genetic disease in a subject, the method 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. The method of claim 31, wherein the genetic disease is characterized by the presence of a single nucleotide variation (SNV) that results in a missense mutation in a gene.

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

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

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

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

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, Schizotypy-Lewis syndrome, Tay-Sachs disease, Niemann-Pick disease, or very long-chain acyl-CoA dehydrogenase deficiency.

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