Suppressor trna construct with spacer sequences for treating stop-codon-associated diseases

Abstract

The present disclosure provides an expression insert for expressing a suppressor tRNA, which contains two or more copies of a nucleic acid encoding a suppressor tRNA, a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer contains a nucleotide sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a promoter operably linked to each nucleic acid encoding the suppressor tRNA. The present disclosure also provides expression vectors containing the expression insert, cells containing the expression vector, compositions containing the suppressor tRNA, and methods of treating stop-codon-associated genetic diseases and restoring translation to nucleotide sequences comprising nonsense mutations.

Description

SUPPRESSOR tRNA CONSTRUCT WITH SPACER SEQUENCES FOR TREATING STOP-CODON-ASSOCIATED DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 749,064, filed January 24, 2025, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0002] The Sequence Listing XML file, entitled SequenceListing.xml, created on January 23, 2026, having a size of 11,604 bytes, which is submitted herewith, is incorporated herein by reference in its entirety.FIELD OF INVENTION

[0003] The present disclosure relates to genetic constructs, methods, and compositions for expressing suppressor transfer RNA (tRNA) molecules, and more particularly to expression inserts comprising multiple copies of suppressor tRNA sequences with novel spacer elements for suppressing premature stop codons and treating stop-codon-associated genetic diseases (e.g., a disease caused or mediated by a premature stop codon).BACKGROUND

[0004] Protein synthesis occurs when genetic instructions contained in messenger RNA (mRNA) are translated at ribosomes. During translation, ribosomes connect amino acids presented by transfer RNAs (tRNAs) to form complex polypeptides. Each tRNA contains an anticodon region that hybridizes with mRNA and is aminoacylated to contain a corresponding amino acid for incorporation into the growing polypeptide chain. Each tRNA is encoded by a gene of about 72-90 nucleotides and folds into a characteristic cloverleaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenic split promoters that become a part of the mature tRNA coding sequence (Sharp S. J., Schaack J., Coolen L., Burke D. J. and Soil D., “Structure and transcription of eukaryotic tRNAgenes”, Crit. Rev. Biochem, 19:107-144 (1985); GeiduschekE. O., and Tocchini-Valentini, “Transcription by RNA polymerase III”, Annu. Rev. Biochem. 57:873-914 (1988)).

[0005] The three stop codons in the genetic code are UAG (amber), UAA (ochre), and UGA (opal). When a stop codon is read during translation, it is interpreted as a stop signal that terminates protein production. Premature stop codons, also known as nonsense mutations, arise when a nucleotide substitution introduces a stop codon within the coding sequence of a gene. This results in premature termination of translation, leading to truncated and often nonfunctional proteins. Such mutations are associated with a range of genetic disorders, including cystic fibrosis, Duchenne muscular dystrophy, and certain types of cancer.

[0006] The process of aging is closely related to the accurate reading, writing, and copying of DNA. Genomic instability, which results in DNA mutations, can reduce the amount of mRNA in cells. This reduction can lead to malfunction of proteins that regulate processes such as DNA repair, epigenetic regulation of protein expression, and tumor suppression. These suppressors play a crucial role in preventing the development of cancer. Mutations in nucleotides that result in nonsense or stop mutations represent a common pathway leading to such events. Nonsense mutations at arginine CGA codons resulting in the stop codon UGA, caused by hydrolytic deamination of 5-methylcytosine at CpG sites, occur frequently in tumor suppressor genes (Fig. 1). The numerous pathologies that result from accumulated nonsense mutations have solidified genomic instability as a core hallmark of aging.

[0007] Different strategies have been attempted to mitigate the effects of nonsense mutations. Nonsense-mediated mRNA decay (NMD) is a conserved pathway for the surveillance and degradation of abnormal mRNAs identified based on premature termination codons. Drugs that block NMD can activate premature termination codon readthrough, as is the case for aminoglycosides such as G418 and NB-124, or PTC-124 (Ataluren). However, this approach has drawbacks including low efficiency, incorporation of near-cognate amino acids at premature termination codons, and readthrough at natural termination codons, resulting in aberrant protein products and small therapeutic windows.

[0008] Pseudouridylation represents another approach that can be tailored to a specific disease-causing premature termination codon. Unlike aminoglycosides, pseudouridylation raises less concern about global natural termination codon readthrough, mitigating potentialoff-target activity. However, like aminoglycosides, pseudouridylation promotes the misincorporation of near-cognate amino acids.

[0009] Nonsense mutations can also be suppressed by a mutation in the anticodon sequence of a tRNA molecule so that it recognizes the stop codon instead. Such suppressor tRNAs work to suppress the effect of a nonsense mutation by reinserting an amino acid despite the presence of the premature stop codon (see Fig. 2). While suppressor tRNAs occur naturally due to mutations, they can be detrimental because they may cause partial loss of translation capability for a given amino acid, which can be lethal in insects and mammals. In addition, naturally occurring suppressor tRNAs may bind with both nonsense and normal stop codons, generating extended versions of proteins whose genes were not mutated.

[0010] Accordingly, there remains interest in developing improved genetic constructs, methods, and compositions for expressing suppressor tRNAs and treating disorders mediated by premature stop codons.SUMMARY

[0011] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0012] In one embodiment, an expression insert for expressing a suppressor tRNA is provided. In this embodiment, the expression insert comprises two or more copies of a nucleic acid encoding a suppressor tRNA, a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a promoter operably linked to each nucleic acid encoding the suppressor tRNA.

[0013] In another embodiment, a method of treating a stop-codon-associated genetic disease in a subject in need thereof is provided. In this embodiment, the method comprises administering to the subject a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA. The expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA. Thespacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. A promoter is operably linked to each nucleic acid encoding the suppressor tRNA.

[0014] In yet another embodiment, a method of restoring translation to a nucleotide sequence comprising a nonsense mutation in a cell is provided. In this embodiment, the method comprises introducing into the cell a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA. The expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA. The spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. A promoter is operably linked to each nucleic acid encoding the suppressor tRNA. The suppressor tRNA hybridizes to a premature stop codon in the nucleotide sequence and permits incorporation of an amino acid at a position corresponding to the premature stop codon.

[0015] In yet another embodiment, an expression vector is provided. In this embodiment, the expression vector comprises the expression insert comprising two or more copies of a nucleic acid encoding a suppressor tRNA, a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a promoter operably linked to each nucleic acid encoding the suppressor tRNA.

[0016] In yet another embodiment, a cell is provided. In this embodiment, the cell comprises an expression vector comprising an expression insert comprising two or more copies of a nucleic acid encoding a suppressor tRNA, a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a promoter operably linked to each nucleic acid encoding the suppressor tRNA.

[0017] In yet another embodiment, a composition is provided. In this embodiment, the composition comprises a suppressor tRNA encoded by an expression insert comprising two or more copies of a nucleic acid encoding a suppressor tRNA, a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and a promoter operably linked to each nucleic acid encoding the suppressor tRNA, and a pharmaceutically acceptable carrier.

[0018] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.BRIEF DESCRIPTION OF FIGURES

[0019] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below. Nonlimiting and non-exhaustive examples are described with reference to the following figures.

[0020] Fig. 1 depicts a chemical reaction diagram illustrating the mechanism of CGA to UGA mutation by deamination of 5-methylcytosine, according to aspects of the present disclosure.

[0021] Fig. 2 depicts a diagram illustrating the mechanism of suppression of a nonsense amber (UAG) codon using a suppressor tRNA, according to an embodiment.

[0022] Fig. 3 depicts a two-dimensional cloverleaf structure representation of a suppressor tRNAArg / Opmolecule, according to aspects of the present disclosure.

[0023] Fig. 4A depicts a Western blot analysis and a bar graph showing relative p53 expression levels in cells treated with different compounds based on the experiment in Example 1.

[0024] Fig. 4B depicts the results of genome-wide ribosomal profiling using calu6 cells as described in Example 1.

[0025] Fig. 5 depicts a Western blot analysis showing protein expression results based on the experiment in Example 2.

[0026] Fig. 6A depicts microscopy images showing fluorescence intensity data from an AAV9 transduction experiment in Calu-6 cells based on the experiment in Example 3.

[0027] Fig. 6B depicts a bar graph showing fluorescence intensity data from the AAV9 transduction experiment of Fig. 6A.

[0028] Fig. 7 depicts a Western blot analysis showing protein expression levels across multiple sample lanes based on the experiment in Example 3.

[0029] Fig. 8 depicts a line graph showing mouse weight change during a toxicology experiment comparing a Control group and a Vec3 group over a 14-day study period based on the experiment in Example 4.

[0030] Fig. 9 depicts a series of line graphs showing changes in white blood cell parameters including white blood cell counts, neutrophils, monocytes, eosinophils, lymphocytes, and basophils comparing PBS vehicle control and Vec3 treatment groups based on the experiment in Example 4.

[0031] Fig. 10 depicts a series of bar graphs showing mean relative expression levels of Vec3 in various tissues including brain, heart, lung, pancreas, liver, and kidney compared to vehicle control based on the experiment in Example 4.DETAILED DESCRIPTION

[0032] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in enzymology, biochemistry, cellular biology, molecular biology, genetics, and medicine.

[0033] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0034] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on thescope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified. Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the present specification and viewing the present drawings that various modifications and variations can be made thereto.

[0035] The present disclosure relates to genetic constructs, methods, and compositions for expressing suppressor transfer RNA (tRNA) molecules and for treating genetic diseases associated with premature stop codons. In some cases, the genetic constructs described herein comprise expression inserts containing two or more copies of a nucleic acid encoding a suppressor tRNA, wherein spacer sequences are positioned between respective copies of the nucleic acid encoding the suppressor tRNA. In some cases, a promoter may be operably linked to each nucleic acid encoding a suppressor tRNA within the expression insert.

[0036] Suppressor tRNAs are tRNA molecules that recognize and hybridize to nonsense stop codons, such as UAG (amber), UAA (ochre), and UGA (opal) codons, during translation. When a suppressor tRNA hybridizes to a premature stop codon in a messenger RNA (mRNA) transcript, the suppressor tRNA may permit incorporation of an amino acid at the position corresponding to the premature stop codon, thereby allowing translation to continue and enabling production of a full-length protein rather than a truncated protein product.

[0037] Premature stop codons, also referred to as nonsense mutations, arise when a nucleotide substitution introduces a stop codon within the coding sequence of a gene. Such mutations result in premature termination of translation, leading to truncated and often nonfunctional proteins. Nonsense mutations are associated with a range of genetic disorders and diseases, and the constructs and methods described herein may provide therapeutic approaches for addressing these conditions.

[0038] In contrast to small-molecule read-through agents such as aminoglycosides (e.g., G418) or ataluren, the suppressor tRNA constructs described herein may selectively suppress premature stop codons with minimal read-through of native termination codons, thereby reducing off-target protein extension. Aminoglycoside drugs such as G418 may promoteread-through at both premature and normal stop codons, which may result in non-specific effects including the production of aberrantly extended proteins from genes that do not contain nonsense mutations. In some cases, ataluren may fail to achieve rescue of protein expression in cells harboring certain nonsense mutations. The suppressor tRNA constructs of the present disclosure may provide improved specificity by recognizing and hybridizing to premature stop codons while exhibiting reduced activity at normal termination codons. In some aspects, this selectivity may result from the anticodon-mediated recognition mechanism of the suppressor tRNA, which may differ from the mechanisms employed by small-molecule read-through agents. The improved specificity of the suppressor tRNA constructs may reduce the potential for off-target effects associated with global termination codon read-through.

[0039] The expression inserts described herein may comprise spacer sequences positioned between copies of the nucleic acid encoding the suppressor tRNA. In some cases, the spacer sequences may be positioned between a termination sequence of one copy of the nucleic acid encoding the suppressor tRNA and a promoter operably linked to a subsequent copy of the nucleic acid encoding the suppressor tRNA. The spacer sequences may facilitate expression of multiple copies of the suppressor tRNA from a single expression insert.

[0040] The expression inserts may be incorporated into expression vectors, including viral vectors and plasmid vectors, for delivery to cells. In some cases, the expression vectors may be administered to a subject as part of a pharmaceutical composition for treating a stopcodon-associated genetic disease. The suppressor tRNA molecules encoded by the expression inserts may hybridize to premature stop codons and restore translation of nucleotide sequences comprising nonsense mutations.

[0041] Referring to FIG. 1, a mechanism by which CGA to UGA mutations arise through deamination of 5-methylcytosine is depicted. Cytosine may undergo methylation to form 5-methylcytosine, wherein a methyl group is attached to the carbon at position 5 of the pyrimidine ring. The 5-methylcytosine may then undergo deamination, wherein the amino group is replaced with a carbonyl oxygen, resulting in thymine. This chemical transformation at CpG sites may cause a CGA codon, which encodes arginine, to mutate to a UGA codon, which is an opal stop codon. Such mutations occur frequently in tumor suppressor genes and may contribute to various cancers and age-related diseases.

[0042] Three stop codons exist in the genetic code: UAG (amber), UAA (ochre), and UGA (opal). During translation, when a ribosome encounters a stop codon in an mRNAtranscript, the stop codon is interpreted as a signal to terminate protein production. When a stop codon arises prematurely within the coding sequence of a gene due to a nonsense mutation, translation terminates at the position of the premature stop codon rather than at the natural termination codon. This premature termination results in production of truncated proteins that are often nonfunctional or have altered function compared to the corresponding full-length proteins.

[0043] With reference to FIG. 2, a mechanism of suppression of a nonsense amber (UAG) codon using a suppressor tRNA is illustrated. As shown in FIG. 2, an mRNA carrying a nonsense mutation contains a premature stop codon (UAG) within the gene region. During translation of the mutated mRNA without suppression, a short defective protein is produced due to premature termination at the nonsense codon.

[0044] As further shown in FIG. 2, a wild-type glutamine-tRNA comprises a wild-type anticodon (GUC) that recognizes glutamine codons. The wild-type glutamine-tRNA may undergo a mutation to produce a mutated glutamine-tRNA with an altered anticodon (AUC). The altered anticodon (AUC) of the mutated glutamine-tRNA is complementary to the UAG nonsense codon rather than to the normal glutamine codon.

[0045] With continued reference to FIG. 2, when the mutated glutamine-tRNA with the AUC anticodon is present during translation, the mutated glutamine-tRNA may bind to the UAG nonsense codon on the mRNA. This binding permits translation to continue through the premature stop codon. The mutated glutamine-tRNA, being aminoacylated with glutamine, permits glutamine to be incorporated into the growing polypeptide chain at the position corresponding to the nonsense codon. As a result, a full-length protein with glutamine inserted at the position of the nonsense codon may be produced rather than a truncated protein product.

[0046] Referring to FIG. 3, a two-dimensional cloverleaf structure representation of a suppressor tRNA molecule of SEQ ID NO: 6 of the present invention is depicted. The suppressor tRNA molecule shown in FIG. 3 is a tRNAArg / Opsuppressor that recognizes and hybridizes to UGA opal stop codons. The cloverleaf structure of the suppressor tRNA comprises four distinct stem-loop regions arranged in a characteristic pattern.

[0047] As shown in FIG. 3, the suppressor tRNA comprises an acceptor stem positioned at the top of the structure, wherein the 5' and 3' ends of the tRNA molecule are positioned adjacent to each other. The 3' end terminates in a single-stranded region that forms theacceptor stem where amino acids attach during aminoacylation. The acceptor stem extends downward and is formed by base pairing between nucleotides from the 5' and 3' ends of the molecule.

[0048] With continued reference to FIG. 3, a D-loop extends from the central junction of the suppressor tRNA molecule and projects to one side of the structure. The D-loop contains several unpaired nucleotides in a loop region. On the opposite side of the structure, a T-loop extends outward and similarly contains unpaired nucleotides within a loop region. The D-loop and T-loop contribute to the three-dimensional folding of the tRNA molecule.

[0049] As further shown in FIG. 3, an anti-codon loop extends from the bottom of the suppressor tRNA structure. The anti-codon loop contains a three-nucleotide anticodon sequence that recognizes and hybridizes to the UGA opal stop codon during translation. The nucleotide bases are represented throughout the structure, with base pairs indicated between complementary nucleotides in the stem regions. The overall arrangement of the suppressor tRNA demonstrates the folded configuration that permits the tRNA to function in protein synthesis.

[0050] In some cases, at least one copy of a nucleic acid encoding a suppressor tRNA may encode a modified or engineered suppressor tRNA. The modified or engineered suppressor tRNA may comprise an anticodon that hybridizes to a nonsense stop codon and may be capable of being aminoacylated with an amino acid. When the suppressor tRNA is expressed in a cell and aminoacylated with the amino acid, the suppressor tRNA may hybridize to the nonsense stop codon and may permit the amino acid to be incorporated into a gene product at a position that would otherwise result in a truncated gene product caused by the nonsense stop codon.

[0051] In some cases, the nonsense stop codon may be a UGA stop codon. In such cases, the suppressor tRNA may be aminoacylated or may be capable of being aminoacylated with arginine. The suppressor tRNA that recognizes UGA stop codons and is aminoacylated with arginine may be referred to as a tRNAArg / Opsuppressor. When the tRNAArg / Opsuppressor encounters a premature UGA stop codon during translation, the tRNAArg / Opsuppressor may permit arginine to be incorporated into the growing polypeptide chain at the position corresponding to the premature stop codon.

[0052] In some cases, the suppressor tRNA may comprise a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some cases, the suppressor tRNA maycomprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6. Variants of the suppressor tRNA sequence may retain the ability to hybridize to an opal (UGA) nonsense stop codon and may retain the capability of being aminoacylated with arginine such that the tRNA, when expressed in a cell and aminoacylated with arginine, hybridizes to a premature stop codon and permits arginine to be incorporated into a gene product at a position that would otherwise result in a truncated gene product caused by the premature stop codon.

[0053] The suppressor tRNA molecules described herein may be produced by various methods. In some cases, tRNA molecules may be produced by extracellular production using synthetic chemical methods. In some cases, tRNA molecules may be produced by intracellular production using recombinant DNA methods. In some cases, tRNA molecules may be produced by purification from natural sources.

[0054] In some cases, the suppressor tRNA may be aminoacylated with a desired amino acid prior to introduction into a cell or administration to a subject. The aminoacylation may be performed by chemical aminoacylation methods or by enzymatic aminoacylation methods. Aminoacylation is the enzymatic or chemical attachment of an amino acid to a corresponding tRNA, and aminoacylation represents a step for ensuring proper translation. For therapeutic purposes, modified tRNAs designed to recognize nonsense mutations may be aminoacylated with appropriate amino acids, permitting incorporation of the amino acids into a growing polypeptide chain at positions corresponding to nonsense stop codons.

[0055] In some embodiments, the suppressor tRNA is aminoacylated with an amino acid selected from arginine, glutamine, tryptophan, tyrosine, lysine, or another amino acid that preserves structural or functional integrity of the translated protein. The selection of the amino acid for aminoacylation may depend on the specific nonsense mutation being targeted and the amino acid that was originally encoded at the position prior to the mutation. In some cases, the amino acid selected for aminoacylation may be the same amino acid that was encoded by the wild-type codon before the nonsense mutation occurred. In other cases, the amino acid selected for aminoacylation may be a different amino acid that maintains similar physicochemical properties, such as charge, hydrophobicity, or size, to the original amino acid. The aminoacylation of the suppressor tRNA with an appropriate amino acid may permitrestoration of protein function when the suppressor tRNA hybridizes to the premature stop codon and permits incorporation of the amino acid into the growing polypeptide chain.

[0056] While arginine-aminoacylated suppressor tRNAs are exemplified herein, a person of ordinary skill in the art would understand that suppressor tRNAs aminoacylated with other amino acids capable of restoring structural or functional integrity of a target protein may be designed using the same principles, including anticodon selection and compatibility with endogenous aminoacyl-tRNA synthetases.Definitions

[0057] As used herein, the term "suppressor tRNA" refers to a transfer RNA molecule that comprises an anticodon sequence capable of hybridizing to a nonsense stop codon, such as UAG (amber), UAA (ochre), or UGA (opal), during translation. A suppressor tRNA is aminoacylated or is capable of being aminoacylated with an amino acid such that, when the suppressor tRNA hybridizes to a premature stop codon in a messenger RNA transcript, the amino acid is incorporated into the growing polypeptide chain at the position corresponding to the premature stop codon. Suppressor tRNAs permit translation to continue through premature stop codons, thereby enabling production of full-length proteins rather than truncated protein products. The suppressor rRNA of the present invention includes engineered, synthetic, and variant tRNAs meeting functional criteria set forth herein.

[0058] As used herein, the term "spacer" refers to a nucleotide sequence positioned between two copies of a nucleic acid encoding a suppressor tRNA within an expression insert. In an embodiment, the spacer is positioned between a termination sequence of a first copy of the nucleic acid encoding the suppressor tRNA and a promoter operably linked to a second copy of the nucleic acid encoding the suppressor tRNA. The spacer facilitates expression of multiple copies of the suppressor tRNA from a single expression insert. In an embodiment, the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. The spacer is not limited to transcriptional function. The spacer may function as a transcriptional insulator, a structural separator, or both; however, no particular mechanism of action is required provided that expression of adjacent suppressor tRNA copies is maintained.

[0059] As used herein, the term "minimal read-through" refers to a characteristic of a suppressor tRNA or suppressor tRNA construct wherein the suppressor tRNA exhibits reduced activity at native termination codons compared to premature stop codons. Thus, the term “minimal read-through” herein refers to a level of read-through at native termination codons that is substantially lower than that observed with aminoglycoside-based read-through agents under comparable conditions. A suppressor tRNA exhibiting minimal read-through hybridizes to premature stop codons arising from nonsense mutations while exhibiting reduced or negligible hybridization to normal termination codons at the 3' ends of coding sequences. Minimal read-through reduces the production of aberrantly extended proteins from genes that do not contain nonsense mutations.

[0060] As used herein, the term "stop-codon-associated genetic disease" or "stop-codon-associated genetic disorder" refers to a disease or disorder that is mediated, enhanced, facilitated by, or associated with a premature stop codon in a gene. Stop-codon-associated genetic diseases include diseases wherein a nonsense mutation introduces a premature stop codon within the coding sequence of a gene, resulting in production of a truncated and often nonfunctional protein. Stop-codon-associated genetic diseases germline, somatic, mosaic, and tissue-restricted mutations. Examples of stop-codon-associated genetic diseases include, but are not limited to, P-thalassemia, Choroideremia, Cystic Fibrosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Hurler Syndrome, Marfan Syndrome, Spinal Muscular Atrophy, and cancers associated with nonsense mutations in tumor suppressor genes such as p53, as well as other stop-codon-associated genetic diseases discussed elsewhere herein.

[0061] As used herein, the term "pharmaceutically acceptable carrier" refers to buffers, carriers, excipients, solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and other materials that are compatible with pharmaceutical administration and are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions (including oil / water and water / oil emulsions), and various types of wetting agents. In an embodiment, the pharmaceutically acceptable carrier is a polymeric nanoparticle, a liposome, or a micelle.

[0062] As used herein, the term "therapeutically effective amount" refers to an amount of an active agent, such as a suppressor tRNA or an expression vector encoding a suppressortRNA, sufficient to effect beneficial or desired results in a subject. A therapeutically effective amount is administered in one or more administrations, applications, or dosages and is not limited to a particular formulation or administration route. The therapeutically effective amount varies depending on factors such as the type and extent of disease or indication to be treated, the overall health of the subject, the in vivo potency of the active agent, the pharmaceutical formulation, and the route of administration.

[0063] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Subjects include mammals, such as murines, simians, equines, bovines, porcines, canines, felines, and humans. In an embodiment, the subject is a human.

[0064] As used herein, the term "subject in need thereof or "patient in need thereof' refers to a subject having, suspected of having, or at risk of developing a stop-codon-associated genetic disease or disorder. In an embodiment, a subject in need thereof is a subject having a nonsense mutation in a gene associated with a genetic disease.

[0065] As used herein, the terms "treat," "treating," and "treatment" refer to the treatment of a disease or disorder in a subject. Treatment includes inhibiting the disease or disorder, such as arresting development of the disease or disorder, and relieving the disease or disorder, such as causing regression of the disease state. Treatment encompasses administration of a composition to a subject to achieve a therapeutic effect.

[0066] As used herein, the term "in combination" refers to administration of two or more different treatments to a subject during the course of the subject's affliction with a disorder, such that the effects of the treatments on the subject overlap at a point in time. In an embodiment, the delivery of one treatment is still occurring when the delivery of a second treatment begins, such that there is overlap in terms of administration.

[0067] As used herein, the term "simultaneous" or "concurrent delivery" refers to administration of two or more treatments wherein the delivery of one treatment is still occurring when the delivery of another treatment begins. Simultaneous or concurrent delivery results in overlap of the treatments in terms of administration timing.

[0068] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is operably linked when the nucleic acid sequence is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a gene if the promoter affects thetranscription of the gene. Operably linked nucleotide sequences are typically contiguous; however, enhancers function when separated from the promoter by several kilobases, and intronic sequences are of variable lengths, such that some polynucleotide elements are operably linked but not directly flanked.

[0069] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression, and other elements for expression are supplied by a host cell or in an in vitro expression system. Expression vectors include cosmids, plasmids (naked or contained in liposomes), retrotransposons, and viruses (such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0070] As used herein, the term "expression insert" refers to a nucleic acid sequence comprising one or more copies of a nucleic acid encoding a suppressor tRNA, wherein a promoter is operably linked to each nucleic acid encoding the suppressor tRNA. In an embodiment, the expression insert comprises two or more copies of the nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA.

[0071] As used herein, the term "termination sequence" refers to a nucleotide sequence that directs termination of transcription. In an embodiment, the termination sequence is a hexa-thymine sequence that permits transcription termination of small RNA transcribed by RNA polymerase III.

[0072] As used herein, the term "repeating unit" refers to a nucleic acid sequence comprising a promoter, a nucleic acid encoding a suppressor tRNA, and a termination sequence. In an embodiment, the expression insert comprises two or more repeating units, wherein a spacer is positioned between successive repeating units. For example, a repeating unit may comprise, in a 5' to 3' orientation, a U6 promoter operably linked to a nucleic acid encoding a tRNAArg / Opsuppressor followed by a hexa-thymine termination sequence. In some cases, the nucleic acid encoding the suppressor tRNA within the repeating unit may include flanking sequences derived from genomic DNA sequences that naturally flank wildtype tRNA genes.

[0073] As used herein, the term "sequence identity" refers to the percentage of nucleotides or amino acids that are identical between two sequences when the sequences arealigned for comparison using standard sequence alignment algorithms. Sequence identity is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences, dividing by the total number of positions in the reference sequence, and multiplying by 100.

[0074] As used herein, the term "nonsense mutation" refers to a point mutation in a nucleotide sequence that results in a premature stop codon within the coding sequence of a gene. Nonsense mutations cause premature termination of translation, resulting in truncated protein products.

[0075] As used herein, the term "premature stop codon" refers to a stop codon (UAG, UAA, or UGA) that occurs within the coding sequence of a gene as a result of a nonsense mutation, rather than at the natural termination position at the end of the coding sequence.

[0076] As used herein, the term "native termination codon" or "normal termination codon" refers to a stop codon that occurs at the natural termination position at the 3' end of a coding sequence and signals the end of translation under normal conditions.

[0077] As used herein, the term "aminoacylation" refers to the enzymatic or chemical attachment of an amino acid to a corresponding tRNA molecule. Aminoacylation is performed by aminoacyl-tRNA synthetases in enzymatic aminoacylation or by chemical methods in chemical aminoacylation.

[0078] As used herein, the term "viral vector" refers to a virus or virus-derived construct used to deliver genetic material into cells. Viral vectors include adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and adenoviral vectors.

[0079] As used herein, the term "adeno-associated viral vector" or "AAV vector" refers to a vector derived from adeno-associated virus, a small, non-enveloped virus of the genus Dependoparvovirus and family Parvovirus. AAV vectors are capable of infecting both dividing and quiescent cells of several tissue types.Expression Insert Components and Structure

[0080] An expression insert for expressing a suppressor tRNA comprises two or more copies of a nucleic acid encoding a suppressor tRNA. A spacer is positioned between respective copies of the nucleic acid encoding the suppressor tRNA. A promoter is operably linked to each nucleic acid encoding a suppressor tRNA within the expression insert.

[0081] The spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In an embodiment, the spacer comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of any of SEQ ID NOs: 1-5.

[0082] The spacer is located between a termination sequence of a first copy of the nucleic acid encoding the suppressor tRNA positioned 5' to the spacer and the promoter positioned 3' to the spacer. The promoter positioned 3' to the spacer is operably linked to a second copy of the nucleic acid encoding the suppressor tRNA. In an embodiment, the termination sequence comprises a hexa-thymine sequence. The hexa-thymine sequence permits transcription termination of small RNA transcribed by pol III RNA polymerase.

[0083] In some embodiments, the nucleic acid encoding the suppressor tRNA excludes one or more native flanking sequences normally associated with endogenous tRNA genes. In such embodiments, the suppressor tRNA may consist essentially of the tRNA coding sequence required for folding, aminoacylation, and anticodon function. In other embodiments, the nucleic acid encoding the suppressor tRNA includes one or more flanking sequences derived from genomic DNA sequences that naturally flank wild-type tRNA genes. The inclusion or exclusion of flanking sequences may affect the expression level, processing, or stability of the suppressor tRNA in a cell Jean.

[0084] In an embodiment, the spacer has an additional 1 to 20 nucleotides. The distribution of the additional nucleotides is all at the 5' end, all at the 3' end, or split between the 5' and the 3' end. For example, where the additional nucleotides are split between the 5' and the 3' end, each side has 1 to 19 nucleotides such that the total of additional nucleotides at the 5' and 3' region totals no more than 20 nucleotides. In an embodiment, the spacer has at least 3, at least 5, at least 7, at least 9, at least 10, at least 12, at least 15, or at least 17 additional nucleotides at the 5' end. In an embodiment, the spacer has at least 3, at least 5, at least 7, at least 9, at least 10, at least 12, at least 15, or at least 17 additional nucleotides at the 3' end.

[0085] The expression insert comprises from 2 to 10 copies of the nucleic acid encoding the suppressor tRNA. In an embodiment, the expression insert contains 2-10 copies, 2-9copies, 2-8 copies, 2-7 copies, 2-6 copies, 2-5 copies, 2-4 copies, 2-3 copies, 3-10 copies, 3-9 copies, 3-8 copies, 3-7 copies, 3-6 copies, 3-5 copies, 3-4 copies, 4-10 copies, 4-9 copies, 4-8 copies, 4-7 copies, 4-6 copies, 4-5 copies, 5-10 copies, 5-9 copies, 5-8 copies, 5-7 copies, 5-6 copies, 6-10 copies, 6-9 copies, 6-8 copies, 6-7 copies, 7-10 copies, 7-9 copies, 7-8 copies, 8-10 copies, 8-9 copies, or 9-10 copies of the nucleic acid encoding the suppressor tRNA.Nucleic Acid Sequence Configurations

[0086] In an embodiment, each copy of the nucleic acid encoding the suppressor tRNA comprises an identical nucleotide sequence. When each copy of the nucleic acid encoding the suppressor tRNA comprises an identical nucleotide sequence, the expression insert produces multiple copies of the same suppressor tRNA molecule upon transcription. The identical nucleotide sequences encoding the suppressor tRNA permit consistent expression of a single suppressor tRNA species from the expression insert.

[0087] In an embodiment, at least two copies of the nucleic acid encoding the suppressor tRNA comprise different nucleotide sequences. When at least two copies of the nucleic acid encoding the suppressor tRNA comprise different nucleotide sequences, the expression insert produces two or more different suppressor tRNA molecules upon transcription. The different nucleotide sequences encoding the suppressor tRNA permit expression of multiple suppressor tRNA species from a single expression insert. The different suppressor tRNA species may recognize different nonsense stop codons or may be aminoacylated with different amino acids.

[0088] In an embodiment, the expression insert comprises an expression cassette that comprises a nucleotide sequence corresponding to genomic DNA sequences flanking a corresponding wild-type tRNA gene. The flanking sequences provide regulatory elements and sequence context that facilitate proper transcription and processing of the suppressor tRNA. The expression cassette comprises the nucleic acid encoding the suppressor tRNA together with the flanking sequences derived from genomic DNA sequences that naturally flank wild-type tRNA genes.

[0089] In an embodiment, the expression cassette comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 7. In an embodiment, the expression cassette comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7. The expression cassette of SEQ ID NO: 7 comprises the tRNAArg / Opsuppressor coding sequence embedded within flanking genomic DNA sequences.

[0090] In an embodiment, the expression cassette terminates with a termination sequence to permit transcription termination of small RNA transcribed by pol III RNA polymerase. In an embodiment, the termination sequence is a hexa-thymine sequence. The hexa-thymine sequence signals transcription termination for RNA polymerase III, which transcribes tRNA genes.Promoter Elements and Repeating Units

[0091] The promoter operably linked to each nucleic acid encoding the suppressor tRNA is selected from the group consisting of a retroviral LTR, a SV40 promoter, a human cytomegalovirus (CMV) promoter, a U6 promoter, an adenovirus promoter, a TK promoter, and a B19 parvovirus promoter. The selection of the promoter depends on the expression system employed and the desired level and pattern of suppressor tRNA expression.

[0092] In an embodiment, the retroviral LTR promoter comprises a long terminal repeat sequence derived from a retrovirus. The U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Heterologous promoters that replace the U3 region include viral simian virus 40 (SV40) promoters (early or late), cytomegalovirus (CMV) promoters (immediate early), Moloney murine leukemia virus (MoMLV) promoters, Rous sarcoma virus (RSV) promoters, and herpes simplex virus (HSV) thymidine kinase promoters. The replacement of the U3 region with a heterologous promoter reduces the possibility of recombination to generate replication-competent virus.

[0093] In an embodiment, the promoter is a U6 promoter. The U6 promoter is a pol III promoter that drives expression of small RNAs. The U6 promoter comprises a sequence comprising SEQ ID NO: 8 or a nucleotide sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8. The U6 promoter drives high levels of transcription of the suppressor tRNA in a variety of cell types.

[0094] The expression insert comprises a repeating unit. The repeating unit comprises the promoter, the nucleic acid encoding the suppressor tRNA, and a termination sequence. The repeating unit provides a modular structure that permits multiple copies of the suppressor tRNA to be expressed from a single expression insert. Each repeating unit contains the regulatory elements and coding sequence for expression of one copy of the suppressor tRNA.

[0095] In an embodiment, the repeating unit comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9. In an embodiment, the repeating unit comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 9. The repeating unit of SEQ ID NO: 9 comprises a U6 promoter, the tRNAArg / Opsuppressor coding sequence with flanking regions, and a termination sequence.

[0096] Where multiple copies of the repeating unit are present in the expression insert, a spacer is positioned between each successive copy of the repeating unit. In an embodiment, the spacer is the same between each successive copy of the repeating unit. In an embodiment, at least one spacer located between successive copies of the repeating unit is different from another spacer in the expression insert.

[0097] In an embodiment, the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the spacer is positioned after a final repeating unit of the expression insert. The spacer positioned after the final repeating unit provides a terminal sequence element that completes the expression insert structure.

[0098] In an embodiment, the expression insert comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 10. In an embodiment, the expression insert comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 10. The expression insert of SEQ ID NO: 10 comprises three copies of the repeating unit containing a U6 promoteroperably linked to the tRNAArg / Opsuppressor, wherein spacers are positioned between the first and second copies, between the second and third copies, and after the final copy.Expression Vectors

[0099] An expression vector comprises the expression insert described herein. The expression vector provides a vehicle for delivering the expression insert to a cell, wherein the expression insert directs expression of the suppressor tRNA molecules encoded by the nucleic acid sequences within the expression insert. The expression vector comprises sufficient cis-acting elements for expression, and other elements for expression are supplied by a host cell or by an in vitro expression system.

[0100] The expression vector includes cosmids, plasmids, retrotransposons, and viruses. In an embodiment, the expression vector is a plasmid. The plasmid is naked or is contained in liposomes. Plasmids contained in liposomes provide protection from degradation and facilitate cellular uptake of the plasmid. In an embodiment, the expression vector is a cosmid. Cosmids are plasmid vectors that contain cos sequences from bacteriophage lambda and permit packaging of large DNA inserts.

[0101] In an embodiment, the expression vector is a retrotransposon. Retrotransposons include piggyback and sleeping beauty transposon systems. The piggyback transposon system provides stable integration of the expression insert into a host cell genome. The sleeping beauty transposon system provides stable integration of the expression insert into a host cell genome through a cut-and-paste transposition mechanism.

[0102] In an embodiment, the expression vector is a viral vector. Viral vectors include lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses. Viral vectors provide efficient delivery of the expression insert to target cells and tissues. The selection of a viral vector depends on the target cell type, the desired duration of expression, and the therapeutic application.

[0103] In an embodiment, the expression vector further comprises a reporter gene. The reporter gene permits monitoring of expression from the expression vector. Reporter genes include genes encoding fluorescent proteins, such as enhanced green fluorescent protein (EGFP), and genes encoding enzymes that produce detectable products. The reporter gene isoperably linked to a promoter within the expression vector such that expression of the reporter gene indicates successful delivery and expression from the expression vector.Adeno-Associated Viral Vectors

[0104] In an embodiment, the expression vector is an adeno-associated viral (AAV) vector. AAV is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV is capable of infecting both dividing and quiescent cells of several tissue types, with different AAV serotypes exhibiting different tissue tropism.

[0105] In an embodiment, the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAV10. AAV includes numerous serologically distinguishable types including serotypes AAV-1 to AAV-12, as well as more than 100 serotypes from nonhuman primates. The serotype of the AAV vector is selected based on the efficiency of delivery, tissue tropism, and immunogenicity for the intended therapeutic application.

[0106] AAV-1 is used for delivery to the central nervous system, the heart, and skeletal muscle. AAV-2 is used for delivery to the central nervous system, the kidney, and photoreceptor cells. AAV-4 is used for delivery to the central nervous system, the lung, and photoreceptor cells. AAV-5 is used for delivery to the central nervous system, the lung, photoreceptor cells, and retinal pigment epithelium. AAV -6 is used for delivery to the lung and skeletal muscle. AAV-7 is used for delivery to the liver and skeletal muscle. AAV-8 is used for delivery to the central nervous system, the heart, the liver, the pancreas, photoreceptor cells, retinal pigment epithelium, and skeletal muscle. AAV-9 is used for delivery to the central nervous system, the heart, the liver, the lung, and skeletal muscle. AAV9 is a vector for clinical use due to the ability of AAV9 to efficiently transduce various tissues and organs, including the heart, liver, skeletal muscle, and central nervous system (CNS). AAV9 has higher transduction efficiency in the CNS than other AAV serotypes, making AAV9 an attractive vector for treating neurological disorders.

[0107] In an embodiment, the AAV capsid protein comprises a sequence as disclosed in U.S. Pat. No. 7,198,951. The AAV capsid protein sequences disclosed in U.S. Pat. No.7,198,951 include AAV-9 (SEQ IDNOs: 1-3 of U.S. Pat. No. 7,198,951), AAV-2 (SEQ ID NO: 4 of U.S. Pat. No. 7,198,951), AAV-1 (SEQ ID NO: 5 of U.S. Pat. No. 7,198,951),1AAV-3 (SEQ ID NO: 6 of U.S. Pat. No. 7,198,951), and AAV-8 (SEQ ID NO: 7 of U.S. Pat. No. 7,198,951). In an embodiment, AAV serotypes identified from rhesus monkeys are contemplated, including rh.8, rh.10, rh.39, rh.43, and rh.74. Modified AAV capsids have been developed for improving efficiency of delivery, tissue tropism, and immunogenicity.

[0108] The wild-type AAV genome contains two 145 nucleotide inverted terminal repeats (ITRs), which contain signal sequences directing AAV replication, genome encapsidation, and integration. In addition to the ITRs, three AAV promoters, p5, pl 9, and p40, drive expression of two open reading frames encoding rep and cap genes. Two rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. Rep proteins are responsible for genomic replication. The Cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. The capsid proteins form the capsid of the AAV particle.

[0109] Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome is replaced with foreign DNA, such as an expression cassette for an exogenous gene of interest. In an embodiment, the AAV vector comprises a genome comprising an expression cassette for an exogenous gene flanked by a 5' ITR and a 3' ITR. The ITRs are derived from the same serotype as the capsid or a derivative thereof. In an embodiment, the ITRs are of a different serotype from the capsid, thereby generating a pseudotyped AAV. In an embodiment, the ITRs are derived from AAV-2. In an embodiment, the ITRs are derived from AAV-5.

[0110] In an embodiment, at least one of the ITRs is modified to mutate or delete the terminal resolution site, thereby allowing production of a self-complementary AAV vector. The rep and cap proteins are provided in trans, such as on a plasmid, to produce an AAV vector. A host cell line permissive of AAV replication expresses the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, such as adenoviral genes Ela, Elb55K, E2a, E4orf6, and VA.[OHl] In an embodiment, the AAV vector has single-stranded genomes that are 4.7 kb in size, or are larger or smaller than 4.7 kb, including oversized genomes that are as large as 5.2 kb, or as small as 3.0 kb. Where the exogenous gene of interest to be expressed from the AAV vector is small, the AAV genome comprises a stuffer sequence. In an embodiment, the AAV vector genome is substantially self-complementary, thereby allowing for rapidexpression in the cell. In an embodiment, the genome of a self-complementary AAV vector comprises from 5' to 3': a 5' ITR; a first nucleic acid sequence comprising a promoter and / or enhancer operably linked to a coding sequence of a gene of interest; a modified ITR that does not have a functional terminal resolution site; a second nucleic acid sequence complementary or substantially complementary to the first nucleic acid sequence; and a 3' ITR. AAV vectors containing genomes of all types are used in the methods described herein.

[0112] In an embodiment, the expression vector is an AAV vector capable of targeting the nervous system in a subject, such as a human subject. The AAV vector capable of targeting the nervous system includes AAV9 variants AAV-PHP.B, AAV-AS, and AAV-PHP.eB. AAV-PHP.B is an AAV9 variant that efficiently transduces the central nervous system. AAV-AS is an AAV9 variant that targets the central nervous system. AAV-PHP.eB is an AAV9 variant that has enhanced transduction efficiency in the central nervous system compared to other AAV serotypes.Retroviral and Lenti viral Vectors

[0113] In an embodiment, the viral vector is a retroviral vector. Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. The retroviral vector is selected from moloney murine leukemia virus vectors, spleen necrosis virus vectors, and vectors derived from retroviruses such as rous sarcoma virus, harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.

[0114] In an embodiment, the retroviral vector is a lentiviral vector. The lentiviral vector is derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), or caprine arthritis encephalitis virus (CAEV).

[0115] Retroviral vectors are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene or genes of interest. The structural genes (gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques. A minimum retroviral vector comprises from 5' to 3': a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and / or enhancer, an exogenous gene of interest, and a 3' LTR. If no exogenous promoter is provided, geneexpression is driven by the 5' LTR. The structural genes are provided in separate vectors for manufacture of the lentivirus, rendering the produced virions replication-defective.

[0116] With respect to lentivirus, the packaging system comprises a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, and a third, separate vector encoding the envelope protein Env. The envelope protein is VSV-G due to the wide infectivity of VSV-G. To improve the safety of the packaging system, the packaging vector is split, expressing Rev from one vector and Gag and Pol from another vector. Tat is eliminated from the packaging system by using a retroviral vector comprising a chimeric 5' LTR, wherein the U3 region of the 5' LTR is replaced with a heterologous regulatory element.

[0117] The new genes are flanked by 5' and 3' LTRs, which serve to promote transcription and polyadenylation of the virion RNAs, respectively. The term "long terminal repeat" or "LTR" refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs provide functions fundamental to the expression of retroviral genes (promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences for replication and integration of the viral genome. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In an embodiment, the R region comprises a trans-activation response (TAR) genetic element, which interacts with the transactivator (tat) genetic element to enhance viral replication. The TAR element is not required in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0118] In an embodiment, the retroviral vector comprises a modified 5' LTR and / or 3' LTR. Modifications of the 3' LTR are made to improve the safety of lentiviral or retroviral systems by rendering viruses replication-defective. In an embodiment, the retroviral vector is a self-inactivating (SIN) vector. A SIN retroviral vector refers to a replication-defective retroviral vector in which the 3' LTR U3 region has been modified by deletion or substitution to prevent viral transcription beyond the first round of viral replication. The 3' LTR U3 region is used as a template for the 5' LTR U3 region during viral replication, and the viral transcript is not made without the U3 enhancer-promoter. In an embodiment, the 3' LTR is modified such that the U5 region is replaced with an ideal polyadenylation sequence.

[0119] In an embodiment, the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles.Heterologous promoters include viral simian virus 40 (SV40) promoters (early or late), cytomegalovirus (CMV) promoters (immediate early), Moloney murine leukemia virus (MoMLV) promoters, Rous sarcoma virus (RS V) promoters, and herpes simplex virus (HS V) thymidine kinase promoters. The heterologous promoters drive high levels of transcription in a Tat-independent manner. The replacement reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system.

[0120] Adjacent the 5' LTR are sequences for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). The term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation. The packaging signal is a minimal packaging signal (also referred to as the psi [ ] sequence) for encapsidation of the viral genome.

[0121] In an embodiment, the retroviral vector further comprises a FLAP element. The term "FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. During reverse transcription, central initiation of the plus-strand DNA at the cPPT and central termination at the CTS lead to the formation of a three- stranded DNA structure: a central DNA flap. The DNA flap acts as a cis-active determinant of lentiviral genome nuclear import and / or increases the titer of the virus. In an embodiment, the retroviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. In an embodiment, a transfer plasmid includes a FLAP element. In an embodiment, a vector comprises a FLAP element isolated from HIV-1.

[0122] In an embodiment, the retroviral vector further comprises an export element. The term "export element" refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. RNA export elements include the human immunodeficiency virus (HIV) RRE and the hepatitis B virus post-transcriptional regulatory element (HPRE). The RNA export element is placed within the 3' UTR of a gene and is inserted as one or multiple copies.

[0123] In an embodiment, the retroviral vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements increase expression of a heterologous nucleic acid. The posttranscriptional regulatory element is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or the posttranscriptional regulatory element present in hepatitis B virus (HPRE). The posttranscriptional regulatory element is positioned at the 3' end of the heterologous nucleic acid sequence. This configuration results in synthesis of an mRNA transcript whose 5' portion comprises the heterologous nucleic acid coding sequences and whose 3' portion comprises the posttranscriptional regulatory element sequence. In an embodiment, vectors lack or do not comprise a posttranscriptional regulatory element such as a WPRE or HPRE because in some instances these elements increase the risk of cellular transformation and / or do not substantially or significantly increase the amount of mRNA transcript or increase mRNA stability.

[0124] In an embodiment, the retroviral vector further comprises a polyadenylation signal. The term "polyadenylation signal" or "polyadenylation sequence" denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyadenylation signal are unstable and are rapidly degraded. The polyadenylation signal includes an ideal polyadenylation sequence (AATAAA, ATT AAA, AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit P-globin polyadenylation sequence (rPgpA), or another suitable heterologous or endogenous polyadenylation sequence.

[0125] In an embodiment, the retroviral vector further comprises an insulator element. Insulator elements contribute to protecting retrovirus-expressed sequences, such as therapeutic genes, from integration site effects, which are mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (position effect). In an embodiment, the retroviral vector comprises an insulator element in one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome. Insulators include the chicken P-globin insulator. In an embodiment, the insulator element is chicken HS4.Adenoviral Vectors

[0126] In an embodiment, the viral vector is an adenoviral vector. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The term "adenovirus" refers to any virus in the genus Adenoviridiae including human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. An adenoviral vector is generated by introducing one or more mutations (a deletion, insertion, or substitution) into the adenoviral genome of the adenovirus so as to accommodate the insertion of a non-native nucleic acid sequence, such as for gene transfer, into the adenovirus.

[0127] In an embodiment, a human adenovirus is used as the source of the adenoviral genome for the adenoviral vector. The adenoviral vector is derived from human adenovirus subgroup A, which includes serotypes 12, 18, and 31. In an embodiment, the adenoviral vector is derived from human adenovirus subgroup B, which includes serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50. In an embodiment, the adenoviral vector is derived from human adenovirus subgroup C, which includes serotypes 1, 2, 5, and 6. In an embodiment, the adenoviral vector is derived from human adenovirus subgroup D, which includes serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48. In an embodiment, the adenoviral vector is derived from human adenovirus subgroup E, which includes serotype 4. In an embodiment, the adenoviral vector is derived from human adenovirus subgroup F, which includes serotypes 40 and 41. In an embodiment, the adenoviral vector is derived from an unclassified serogroup, which includes serotypes 49 and 51. Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, Va.).

[0128] In an embodiment, a non-human adenovirus is used to generate the adenoviral vector. The non-human adenovirus serves as a source of the adenoviral genome for the adenoviral vector. In an embodiment, the adenoviral vector is derived from ape adenovirus. In an embodiment, the adenoviral vector is derived from simian adenovirus, including both new world and old world monkey adenoviruses. In an embodiment, the adenoviral vector is derived from avian adenovirus. In an embodiment, the adenoviral vector is derived from canine adenovirus. In an embodiment, the adenoviral vector is derived from ovine adenovirus. In an embodiment, the adenoviral vector is derived from bovine adenovirus. In an embodiment, the adenoviral vector is derived from gorilla adenovirus. In an embodiment, the adenoviral vector comprises a combination of subtypes and thereby is a chimeric adenoviral vector.

[0129] In an embodiment, the adenoviral vector is replication-competent. A replication-competent adenoviral vector replicates in typical host cells, which are cells typically capable of being infected by an adenovirus. In an embodiment, the adenoviral vector is conditionally replication-competent. A conditionally replication-competent adenoviral vector is an adenoviral vector that has been engineered to replicate under pre-determined conditions. Replication-essential gene functions, such as gene functions encoded by the adenoviral early regions, are operably linked to an inducible, repressible, or tissue-specific transcription control sequence, such as a promoter.

[0130] In an embodiment, the adenoviral vector is replication-deficient. A replicationdeficient adenoviral vector is an adenoviral vector that requires complementation of one or more gene functions or regions of the adenoviral genome that are required for replication, as a result of a deficiency in one or more replication-essential gene function or regions, such that the adenoviral vector does not replicate in typical host cells, especially those in a human to be infected by the adenoviral vector.

[0131] In an embodiment, the replication-deficient adenoviral vector requires complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome for propagation to form adenoviral vector particles. In an embodiment, the replication-deficient adenoviral vector is deficient in one or more replication-essential gene functions of only the early regions (E1-E4 regions) of the adenoviral genome. In an embodiment, the replication-deficient adenoviral vector is deficient in one or more replication-essential gene functions of only the late regions (L1-L5 regions) of the adenoviral genome. In an embodiment, the replication-deficient adenoviral vector is deficient in one or more replication-essential gene functions of both the early and late regions of the adenoviral genome. In an embodiment, the replication-deficient adenoviral vector is deficient in all adenoviral genes, which is referred to as a high capacity adenovector (HC-Ad).

[0132] The replication-deficient adenoviral vector is produced in complementing cell lines that provide gene functions not present in the replication-deficient adenoviral vector but required for viral propagation at appropriate levels in order to generate high titers of viral vector stock. In an embodiment, the complementing cell line is 293 cells. In an embodiment, the complementing cell line is PER.C6 cells. In an embodiment, the complementing cell line is 293-ORF6 cells. In an embodiment, the complementing cells have been generated topropagate adenoviral vectors encoding transgenes whose expression inhibits viral growth in host cells.Viral Vector Production Methods

[0133] Methods for producing viral vectors are known in the art. A virus of interest is produced in a suitable host cell line using conventional techniques including culturing a transfected or infected host cell under suitable conditions so as to permit the production of infectious viral particles. Nucleic acids encoding viral genes and / or tRNAs are incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques.

[0134] In an embodiment, the host cells for viral vector production are human cell lines. In an embodiment, the host cells are HEK293 cells. In an embodiment, the host cells are human embryonic kidney 293 (HEK 293) cells. In an embodiment, the host cells are 911 cells. In an embodiment, the host cells are A549 cells. In an embodiment, the host cells are HER96 cells. In an embodiment, the host cells are PER-C6 cells. In an embodiment, the host cells are HeLa cells. In an embodiment, the host cells are HeLa-S3 cells.

[0135] In an embodiment, the host cells for viral vector production are non-human mammalian cell lines. In an embodiment, the host cells are Chinese hamster ovary (CHO) cells. In an embodiment, the host cells are baby hamster kidney (BHK) cells. In an embodiment, the host cells are monkey kidney cells (COS). In an embodiment, the host cells are COS cells. In an embodiment, the host cells are Vero cells. In an embodiment, the host cells are human hepatocellular carcinoma cells. In an embodiment, the host cells are Hep G2 cells. In an embodiment, the host cells are myeloma cells.

[0136] In an embodiment, the host cells for viral vector production are bacterial cells. In an embodiment, the host cells are E. coli cells. E. coli cells are used for production of plasmid DNA encoding viral components and for amplification of recombinant DNA constructs.

[0137] In an embodiment, the host cells for viral vector production are insect cells. Insect cells are used for production of AAV vectors and other viral vectors using baculovirus expression systems.

[0138] Transformed host cells are grown under conditions that permit the host cells to express the genes that encode the tRNAs and viral components. Specific expression andpurification conditions vary depending upon the expression system employed and the viral vector being produced.

[0139] In an embodiment, producer cells are directly administered to a subject. In an embodiment, following production, infectious viral particles are recovered from the culture and optionally purified. Purification steps include plaque purification, centrifugation, clarification, enzymatic treatment, and chromatographic steps. In an embodiment, centrifugation comprises cesium chloride gradient centrifugation. In an embodiment, enzymatic treatment comprises benzonase treatment. In an embodiment, enzymatic treatment comprises protease treatment. In an embodiment, chromatographic steps comprise ion exchange chromatography. In an embodiment, purification steps comprise filtration steps.Cells Comprising Expression Vectors

[0140] A cell comprises the expression vector described herein. The cell containing the expression vector expresses the suppressor tRNA molecules encoded by the expression insert within the expression vector. The suppressor tRNA molecules expressed by the cell hybridize to nonsense stop codons during translation and permit incorporation of amino acids at positions corresponding to the nonsense stop codons.

[0141] In an embodiment, the cell is a mammalian cell. Mammalian cells provide the cellular machinery for transcription and processing of tRNA molecules encoded by the expression insert. Mammalian cells express endogenous aminoacyl-tRNA synthetases that aminoacylate the suppressor tRNA molecules with appropriate amino acids. The aminoacylated suppressor tRNA molecules participate in translation and permit readthrough of premature stop codons in the mammalian cell.

[0142] In an embodiment, the mammalian cell is a human cell. Human cells are target cells for therapeutic applications of the expression vectors described herein. Human cells containing the expression vector express suppressor tRNA molecules that hybridize to premature stop codons in human genes affected by nonsense mutations. The suppressor tRNA molecules permit production of full-length proteins from genes that would otherwise produce truncated proteins due to premature stop codons.

[0143] In an embodiment, the cell is selected from the group consisting of a fibroblast, an epithelial cell, a neuronal cell, a muscle cell, and a stem cell. Fibroblasts are connective tissue cells that are affected by nonsense mutations in genes associated with connective tissuedisorders. Epithelial cells line body surfaces and cavities and are affected by nonsense mutations in genes associated with epithelial disorders. Neuronal cells are nerve cells that are affected by nonsense mutations in genes associated with neurological disorders. Muscle cells are contractile cells that are affected by nonsense mutations in genes associated with muscular disorders. Stem cells are undifferentiated cells that have the capacity to differentiate into various cell types and are targets for gene therapy applications.

[0144] In an embodiment, the cell comprises a gene having a nonsense mutation. The nonsense mutation introduces a premature stop codon within the coding sequence of the gene. The premature stop codon causes premature termination of translation, resulting in production of a truncated protein from the gene having the nonsense mutation. The suppressor tRNA encoded by the expression insert hybridizes to a premature stop codon resulting from the nonsense mutation. When the suppressor tRNA hybridizes to the premature stop codon, the suppressor tRNA permits incorporation of an amino acid at the position corresponding to the premature stop codon, thereby allowing translation to continue and enabling production of a full-length protein from the gene having the nonsense mutation.

[0145] In an embodiment, the gene having the nonsense mutation encodes a tumor suppressor protein. Nonsense mutations in tumor suppressor genes result in production of truncated tumor suppressor proteins that lack tumor suppressor function. The suppressor tRNA encoded by the expression insert hybridizes to the premature stop codon in the tumor suppressor gene and permits production of full-length tumor suppressor protein. In an embodiment, the gene having the nonsense mutation is the TP53 gene. The TP53 gene encodes the p53 tumor suppressor protein. Nonsense mutations in the TP53 gene result in production of truncated p53 protein. The suppressor tRNA encoded by the expression insert hybridizes to the premature stop codon in the TP53 gene and permits production of full-length p53 protein.

[0146] In an embodiment, the gene having the nonsense mutation encodes a structural protein. Nonsense mutations in genes encoding structural proteins result in production of truncated structural proteins that lack proper function. In an embodiment, the gene having the nonsense mutation encodes an ion channel protein. Nonsense mutations in genes encoding ion channel proteins result in production of truncated ion channel proteins that lack proper function. In an embodiment, the gene having the nonsense mutation encodes an enzyme. Nonsense mutations in genes encoding enzymes result in production of truncated enzymes that lack catalytic activity.Pharmaceutical Compositions

[0147] A composition comprises a suppressor tRNA encoded by the expression insert described herein and a pharmaceutically acceptable carrier. The composition provides a formulation for delivering the suppressor tRNA to cells and tissues for therapeutic applications. The suppressor tRNA in the composition hybridizes to premature stop codons during translation and permits incorporation of amino acids at positions corresponding to the premature stop codons.

[0148] The pharmaceutically acceptable carrier is selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle. Polymeric nanoparticles are colloidal particles composed of biodegradable or non-biodegradable polymers that encapsulate or adsorb the suppressor tRNA for delivery. Liposomes are spherical vesicles composed of lipid bilayers that encapsulate the suppressor tRNA in an aqueous interior or within the lipid bilayer. Micelles are aggregates of amphiphilic molecules that form a hydrophobic core and hydrophilic shell, providing a carrier structure for the suppressor tRNA.

[0149] In an embodiment, the suppressor tRNA in the composition comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine. The suppressor tRNA that recognizes UGA stop codons and is aminoacylated with arginine permits arginine to be incorporated into a growing polypeptide chain at positions corresponding to premature UGA stop codons. The aminoacylation of the suppressor tRNA with arginine occurs through the action of endogenous aminoacyl-tRNA synthetases in target cells or through aminoacylation prior to formulation of the composition.

[0150] The composition is formulated for intravenous, intramuscular, subcutaneous, or intrathecal administration. Formulations for intravenous administration comprise sterile solutions or suspensions suitable for injection into the bloodstream. Formulations for intramuscular administration comprise sterile solutions or suspensions suitable for injection into muscle tissue. Formulations for subcutaneous administration comprise sterile solutions or suspensions suitable for injection beneath the skin. Formulations for intrathecal administration comprise sterile solutions suitable for injection into the cerebrospinal fluid surrounding the spinal cord.

[0151] The composition further comprises a buffer, a stabilizer, or a combination thereof. Buffers maintain the pH of the composition within a range suitable for stability of thesuppressor tRNA and compatibility with administration to a subject. Stabilizers protect the suppressor tRNA from degradation during storage and administration.

[0152] The pharmaceutical composition contains formulation materials for modifying, maintaining, or preserving the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In an embodiment, the formulation materials include amino acids such as glycine, glutamine, asparagine, arginine, and lysine. In an embodiment, the formulation materials include antimicrobials. In an embodiment, the formulation materials include antioxidants such as ascorbic acid, sodium sulfite, and sodium hydrogen-sulfite. In an embodiment, the formulation materials include buffers such as borate, bicarbonate, Tris-HCl, citrates, and phosphates. In an embodiment, the formulation materials include bulking agents such as mannitol and glycine. In an embodiment, the formulation materials include chelating agents such as ethylenediamine tetraacetic acid (EDTA). In an embodiment, the formulation materials include complexing agents such as caffeine, polyvinylpyrrolidone, betacyclodextrin, and hydroxypropyl-beta-cyclodextrin.

[0153] In an embodiment, the formulation materials include fillers. In an embodiment, the formulation materials include monosaccharides. In an embodiment, the formulation materials include disaccharides. In an embodiment, the formulation materials include carbohydrates such as glucose, mannose, and dextrins. In an embodiment, the formulation materials include proteins such as serum albumin, gelatin, and immunoglobulins. In an embodiment, the formulation materials include coloring agents, flavoring agents, and diluting agents. In an embodiment, the formulation materials include emulsifying agents. In an embodiment, the formulation materials include hydrophilic polymers such as polyvinylpyrrolidone. In an embodiment, the formulation materials include low molecular weight polypeptides. In an embodiment, the formulation materials include salt-forming counterions such as sodium.

[0154] In an embodiment, the formulation materials include preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide. In an embodiment, the formulation materials include solvents such as glycerin, propylene glycol, and polyethylene glycol. In an embodiment, the formulation materials include sugar alcohols such as mannitol and sorbitol. In an embodiment, the formulation materials include suspending agents. In an embodiment, the formulation materials include surfactants orwetting agents such as pluronics, PEG, sorbitan esters, polysorbates, triton, tromethamine, lecithin, cholesterol, and tyloxapal. In an embodiment, the formulation materials include stability enhancing agents such as sucrose and sorbitol. In an embodiment, the formulation materials include tonicity enhancing agents such as alkali metal halides, sodium chloride, potassium chloride, mannitol, and sorbitol. In an embodiment, the formulation materials include delivery vehicles, diluents, excipients, and pharmaceutical adjuvants.

[0155] The pharmaceutical composition contains sustained- or controlled-delivery formulations. In an embodiment, the sustained- or controlled-delivery formulation comprises liposome carriers. In an embodiment, the sustained- or controlled-delivery formulation comprises bio-erodible microparticles. In an embodiment, the sustained- or controlled-delivery formulation comprises porous beads. In an embodiment, the sustained- or controlled-delivery formulation comprises depot injections. In an embodiment, the sustained- or controlled-delivery formulation comprises porous polymeric microparticles. In an embodiment, the sustained- or controlled-delivery formulation comprises semipermeable polymer matrices in the form of shaped articles, films, or microcapsules.

[0156] In an embodiment, the sustained release matrices include polyesters. In an embodiment, the sustained release matrices include hydrogels. In an embodiment, the sustained release matrices include polylactides. In an embodiment, the sustained release matrices include copolymers of L-glutamic acid and gamma ethyl-L-glutamate. In an embodiment, the sustained release matrices include poly(2-hydroxyethyl-methacrylate). In an embodiment, the sustained release matrices include ethylene vinyl acetate. In an embodiment, the sustained release matrices include poly-D(-)-3 -hydroxybutyric acid. The sustained release matrices provide controlled release of the suppressor tRNA over an extended period following administration.tRNA Production and Delivery Methods

[0157] tRNA molecules described herein are produced by various methods. In an embodiment, tRNA molecules are produced by extracellular production using synthetic chemical methods. Synthetic chemical methods for tRNA production include solid-phase synthesis and enzymatic ligation of chemically synthesized oligonucleotides. In an embodiment, tRNA molecules are produced by intracellular production using recombinant DNA methods. Recombinant DNA methods for tRNA production include expression oftRNA-encoding sequences from expression vectors in host cells, followed by isolation and purification of the expressed tRNA molecules. In an embodiment, tRNA molecules are produced by purification from natural sources. Purification from natural sources includes isolation of tRNA molecules from cells or tissues that naturally express the tRNA of interest.

[0158] Aminoacylation of tRNA molecules is performed by chemical aminoacylation methods or by enzymatic aminoacylation methods. Chemical aminoacylation involves covalent attachment of an amino acid to the 3' end of a tRNA molecule using chemical reagents and reaction conditions that promote formation of an ester bond between the amino acid and the terminal adenosine of the tRNA. Enzymatic aminoacylation involves attachment of an amino acid to a tRNA molecule by an aminoacyl-tRNA synthetase enzyme. Aminoacyl-tRNA synthetases recognize specific tRNA molecules and catalyze the attachment of cognate amino acids to the tRNA molecules. In an embodiment, the tRNA is aminoacylated with a desired amino acid prior to introduction into a cell or administration to a subject.

[0159] The tRNA is modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. Conjugation of cholesterol to tRNA molecules increases the lipophilicity of the tRNA, facilitating passage across cell membranes and uptake into target cells. The cholesterol conjugation also protects the tRNA from degradation by endo- and exo-nucleases in vivo. In an embodiment, the lipophilic group is attached to the tRNA through a linker moiety that connects the lipophilic group to a nucleotide within the tRNA molecule.

[0160] The tRNA molecules are conjugated to or otherwise associated with an aptamer. Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules with high affinity and specificity. Conjugation of tRNA molecules to aptamers provides targeting capability, directing the tRNA to specific cell types or tissues that express receptors or surface molecules recognized by the aptamer. The aptamer-tRNA conjugate binds to target cells through aptamer-receptor interactions, facilitating uptake of the tRNA into the target cells.

[0161] In an embodiment, the tRNA is delivered using lipid nanoparticles. Lipid nanoparticles are composed of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-conjugated lipids that self-assemble into nanostructures capable of encapsulating nucleic acids. The ionizable lipids facilitate endosomal escape following cellular uptake, permitting release of the tRNA into the cytoplasm. In an embodiment, the lipid nanoparticlecomprises an ionizable cationic lipid selected from DLin-MC3-DMA, ALC-0315, SM-102, or derivatives thereof. In an embodiment, the lipid nanoparticle comprises a helper lipid such as distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE). In an embodiment, the lipid nanoparticle comprises a polyethylene gly col-lipid conjugate such as PEG-DMG or PEG-DSPE. The lipid nanoparticle formulation may be optimized for delivery to specific tissues by adjusting the lipid composition, particle size, and surface charge.

[0162] In an embodiment, the tRNA is delivered using polymer-based nanoparticles. Polymer nanoparticles include particles formed from biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), and chitosan. In an embodiment, the polymer nanoparticle comprises PLGA with a molecular weight ranging from 10 kDa to 100 kDa. In an embodiment, the polymer nanoparticle comprises chitosan or a chitosan derivative that provides cationic surface charge for nucleic acid complexation. Polymer nanoparticles may be surface-modified with targeting ligands, cell-penetrating peptides, or polyethylene glycol to enhance cellular uptake, tissue targeting, or circulation time.

[0163] In an embodiment, the tRNA or expression vector encoding the tRNA is delivered by electroporation. Electroporation involves application of electrical pulses to cells to transiently permeabilize the cell membrane, permitting entry of nucleic acids into the cytoplasm. Electroporation parameters including pulse voltage, pulse duration, pulse number, and pulse interval may be optimized for different cell types to achieve efficient delivery while maintaining cell viability. In an embodiment, electroporation is performed using a square wave pulse generator. In an embodiment, electroporation is performed using an exponential decay pulse generator.

[0164] In an embodiment, the tRNA or expression vector encoding the tRNA is delivered by ex vivo cell modification followed by administration of the modified cells to a subject. Ex vivo delivery involves isolation of cells from a subject, introduction of the tRNA or expression vector into the isolated cells outside the body, and subsequent administration of the modified cells back to the subject. In an embodiment, the cells modified ex vivo are autologous cells derived from the subject to be treated. In an embodiment, the cells modified ex vivo are allogeneic cells derived from a donor. In an embodiment, the cells modified ex vivo are hematopoietic stem cells, T cells, natural killer cells, mesenchymal stem cells, orinduced pluripotent stem cells. The ex vivo modification may be performed using electroporation, nucleofection, lipofection, or viral transduction.

[0165] In an embodiment, the tRNA is delivered using nucleofection. Nucleofection is a specialized electroporation technique that delivers nucleic acids directly into the cell nucleus using cell type-specific electrical parameters and proprietary nucleofection solutions.Nucleofection may provide higher transfection efficiency in difficult-to-transfect cell types such as primary cells and stem cells compared to conventional electroporation methods.

[0166] In an embodiment, the tRNA is delivered using microinjection. Microinjection involves direct injection of nucleic acids into individual cells using a fine glass micropipette under microscopic guidance. Microinjection permits precise delivery of defined quantities of tRNA into the cytoplasm or nucleus of target cells.

[0167] In an embodiment, the tRNA is delivered using cell-penetrating peptides. Cellpenetrating peptides are short peptide sequences that facilitate translocation of associated cargo molecules across cell membranes. In an embodiment, the cell-penetrating peptide is TAT peptide derived from HIV-1 transactivator of transcription protein. In an embodiment, the cell-penetrating peptide is penetratin derived from Antennapedia homeodomain. In an embodiment, the cell-penetrating peptide is a polyarginine peptide. The tRNA may be covalently conjugated to the cell-penetrating peptide or non-covalently complexed with the cell-penetrating peptide for delivery.

[0168] In an embodiment, the tRNA is delivered using exosomes or extracellular vesicles. Exosomes are naturally occurring membrane-bound vesicles secreted by cells that may be loaded with therapeutic nucleic acids for delivery. In an embodiment, exosomes are isolated from producer cells and loaded with tRNA by electroporation, sonication, or incubation methods. Exosomes may provide advantages for delivery including low immunogenicity, ability to cross biological barriers, and natural targeting properties based on the cell type from which they are derived.

[0169] Alternative delivery systems such as lipid nanoparticles, polymeric nanoparticles, or ex vivo cell modification may be employed using standard techniques known in the art for small RNA or expression cassette delivery.

[0170] The tRNA is delivered using drug delivery systems including a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Nanoparticles encapsulate or adsorb tRNA molecules and protect the tRNA from degradation during circulation anddelivery to target cells. Dendrimers are branched polymeric molecules that form complexes with tRNA molecules through electrostatic interactions between the positively charged dendrimer surface and the negatively charged tRNA. Polymers form complexes with tRNA molecules and provide protection from degradation and facilitate cellular uptake. Liposomes are lipid vesicles that encapsulate tRNA molecules within an aqueous interior or within the lipid bilayer, providing protection and facilitating delivery to target cells.

[0171] Cationic delivery systems facilitate binding of tRNA molecules and enhance interactions at cell membranes to permit efficient uptake of tRNA by cells. Positively charged cationic lipids, dendrimers, or polymers bind to the negatively charged tRNA molecules through electrostatic interactions. The cationic delivery systems also interact with the negatively charged cell membrane, promoting fusion or endocytosis and facilitating entry of the tRNA into the cell. In an embodiment, cationic lipids, dendrimers, or polymers are bound to the tRNA. In an embodiment, cationic lipids, dendrimers, or polymers are induced to form a vesicle or micelle that encases the tRNA.

[0172] Drug delivery systems for systemic delivery of tRNAs include DOTAP, Oligofectamine, solid nucleic acid lipid particles, cardiolipin, polyethyleneimine, Arg-Gly-Asp (RGD) peptides, and polyamidoamines. DOTAP is a cationic lipid that forms complexes with tRNA molecules for systemic delivery. Oligofectamine is a lipid-based transfection reagent that facilitates delivery of tRNA molecules to cells. Solid nucleic acid lipid particles are lipid nanoparticles that encapsulate tRNA molecules for systemic administration.Cardiolipin is a phospholipid that forms complexes with tRNA molecules. Polyethyleneimine is a cationic polymer that forms complexes with tRNA molecules through electrostatic interactions. Arg-Gly-Asp (RGD) peptides are cell-targeting peptides that bind to integrin receptors on cell surfaces and facilitate targeted delivery of tRNA molecules.Polyamidoamines are dendritic polymers that form complexes with tRNA molecules for delivery.

[0173] The tRNA forms a complex with cyclodextrin for systemic administration.Cyclodextrins are cyclic oligosaccharides that form inclusion complexes with various molecules, including nucleic acids. The cyclodextrin-tRNA complex provides protection of the tRNA from degradation and facilitates systemic delivery. In an embodiment, the cyclodextrin is a beta-cyclodextrin or a derivative thereof. The cyclodextrin-tRNA complex is administered by intravenous injection or other systemic routes of administration.

[0174] Routes of administration for tRNA molecules and compositions containing tRNA molecules include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. Intravenous administration involves injection of the tRNA composition directly into the bloodstream for systemic distribution. Intradermal administration involves injection of the tRNA composition into the dermis layer of the skin. Inhalation administration involves delivery of the tRNA composition to the respiratory tract through aerosol or nebulized formulations. Transdermal administration involves delivery of the tRNA composition across the skin using patches or topical formulations. Topical administration involves application of the tRNA composition directly to a body surface. Transmucosal administration involves delivery of the tRNA composition across mucosal membranes. Intrathecal administration involves injection of the tRNA composition into the cerebrospinal fluid surrounding the spinal cord for delivery to the central nervous system. Rectal administration involves delivery of the tRNA composition through the rectum using suppositories or enema formulations.Manufacturing Methods

[0175] The expression inserts, expression vectors, and suppressor tRNA molecules described herein are manufactured using recombinant DNA techniques, molecular cloning methods, and viral vector production systems. The manufacturing methods encompass construction of nucleic acid sequences encoding suppressor tRNAs, assembly of expression inserts containing multiple copies of suppressor tRNA-encoding sequences with spacer elements, incorporation of expression inserts into expression vectors, and production of viral particles for delivery of the expression vectors to target cells.

[0176] Pol III transcription drives expression of suppressor tRNA molecules from the expression inserts described herein. RNA polymerase III (Pol III) is the eukaryotic RNA polymerase responsible for transcription of small non-coding RNAs, including transfer RNAs, 5S ribosomal RNA, and U6 small nuclear RNA. The suppressor tRNA genes within the expression inserts contain intragenic split promoters that are recognized by Pol III transcription machinery. The intragenic promoters comprise A box and B box elements located within the tRNA coding sequence. The A box element is positioned in the D-loop region of the tRNA gene, and the B box element is positioned in the T-loop region of the tRNA gene. Transcription factor IIIC (TFIIIC) binds to the A box and B box elements andrecruits transcription factor IIIB (TFIIIB) to a position upstream of the transcription start site. TFIIIB positions Pol III at the transcription start site and directs initiation of transcription.

[0177] In an embodiment, the expression insert comprises a U6 promoter operably linked to each nucleic acid encoding the suppressor tRNA. The U6 promoter is an external Pol III promoter that provides transcriptional control elements upstream of the tRNA coding sequence. The U6 promoter comprises a TATA box, a proximal sequence element (PSE), and a distal sequence element (DSE). The TATA box is recognized by TATA-binding protein (TBP), which is a component of TFIIIB. The PSE is recognized by the small nuclear RNA activating protein complex (SNAPc). The DSE contains binding sites for transcription factors that enhance transcription from the U6 promoter. The U6 promoter drives high levels of Pol Ill-mediated transcription of the suppressor tRNA in a variety of cell types.

[0178] Transcription termination of suppressor tRNA genes occurs at a termination sequence located downstream of the tRNA coding sequence. In an embodiment, the termination sequence is a hexa-thymine sequence (TTTTTT). The hexa-thymine sequence signals Pol III to terminate transcription and release the nascent RNA transcript. The termination sequence is positioned at the 3' end of each suppressor tRNA coding sequence within the expression insert. The spacer sequences are positioned between the termination sequence of one suppressor tRNA gene and the promoter of the subsequent suppressor tRNA gene.

[0179] Vector assembly involves construction of expression vectors containing the expression inserts described herein. DNA molecules encoding the suppressor tRNA sequences, promoter sequences, termination sequences, and spacer sequences are synthesized chemically or amplified from template DNA using polymerase chain reaction (PCR) techniques. The synthesized or amplified DNA fragments are ligated together using DNA ligase enzymes to form the expression insert. The expression insert is then ligated into an expression vector backbone to generate the complete expression vector.

[0180] In an embodiment, the expression vector backbone is a plasmid vector. The plasmid vector comprises an origin of replication for propagation in bacterial host cells, a selectable marker gene for selection of transformed bacterial cells, and restriction enzyme recognition sites for insertion of the expression insert. The expression insert is ligated into the plasmid vector at the restriction enzyme recognition sites using T4 DNA ligase or other DNA ligase enzymes. The ligated plasmid is transformed into competent bacterial cells, andtransformed cells are selected based on expression of the selectable marker gene. Plasmid DNA is isolated from selected bacterial colonies and verified by restriction enzyme digestion analysis and DNA sequencing.

[0181] In an embodiment, the expression vector backbone is a viral vector backbone. The viral vector backbone comprises cis-acting elements for viral replication, packaging, and integration, as well as restriction enzyme recognition sites or recombination sites for insertion of the expression insert. The expression insert is inserted into the viral vector backbone by restriction enzyme digestion and ligation or by site-specific recombination using recombinase enzymes. The assembled viral vector is verified by restriction enzyme digestion analysis and DNA sequencing.

[0182] AAV packaging involves production of recombinant AAV particles containing the expression vector genome. AAV packaging is performed in producer cells that express the AAV rep and cap genes in trans and provide helper virus functions. The producer cells are transfected with the AAV vector plasmid containing the expression insert flanked by AAV inverted terminal repeats (ITRs), a plasmid encoding the AAV rep and cap genes, and a plasmid encoding adenoviral helper functions. In an embodiment, the producer cells are HEK293 cells. In an embodiment, the producer cells are HEK293T cells.

[0183] The AAV rep genes encode Rep proteins (Rep78, Rep68, Rep52, and Rep40) that are responsible for replication of the AAV genome. The AAV cap genes encode capsid proteins (VP1, VP2, and VP3) that form the AAV capsid shell. The adenoviral helper functions include Ela, Elb55K, E2a, E4orf6, and VA RNA genes. The adenoviral helper functions are provided by the HEK293 cell line, which stably expresses the adenoviral El genes, and by a helper plasmid encoding the remaining adenoviral helper genes.

[0184] Following transfection, the producer cells are cultured under conditions that permit expression of the AAV rep and cap genes and replication and packaging of the AAV vector genome. The AAV vector genome is replicated by the Rep proteins and packaged into AAV capsids formed by the VP1, VP2, and VP3 capsid proteins. The recombinant AAV particles accumulate within the producer cells and are released into the culture medium upon cell lysis.

[0185] In an embodiment, the AAV particles are harvested from the producer cells by cell lysis. Cell lysis is performed by freeze-thaw cycles, detergent treatment, or mechanical disruption. The cell lysate containing the AAV particles is clarified by centrifugation toremove cell debris. The clarified lysate is treated with benzonase nuclease to digest residual cellular DNA and plasmid DNA that are not protected within AAV capsids.

[0186] The AAV particles are purified from the clarified lysate using density gradient centrifugation, chromatography, or a combination thereof. In an embodiment, the AAV particles are purified by cesium chloride (CsCl) density gradient centrifugation. The clarified lysate is layered onto a CsCl gradient and centrifuged at high speed. The AAV particles band at a characteristic density within the CsCl gradient and are collected by fractionation. In an embodiment, the AAV particles are purified by iodixanol density gradient centrifugation, lodixanol gradients provide separation of AAV particles from cellular contaminants based on density differences.

[0187] In an embodiment, the AAV particles are purified by affinity chromatography. Affinity chromatography utilizes ligands that bind specifically to AAV capsid proteins. The clarified lysate is applied to an affinity chromatography column, and AAV particles are retained on the column through binding to the affinity ligand. Contaminants are washed from the column, and the AAV particles are eluted using conditions that disrupt the affinity interaction. In an embodiment, the AAV particles are purified by ion exchange chromatography. Ion exchange chromatography separates AAV particles from contaminants based on differences in surface charge.

[0188] The purified AAV particles are formulated in a pharmaceutically acceptable buffer for storage and administration. The formulation buffer comprises salts, pH buffering agents, and stabilizers that maintain the integrity and infectivity of the AAV particles. In an embodiment, the formulation buffer comprises phosphate buffered saline (PBS). In an embodiment, the formulation buffer comprises Tris-buffered saline. In an embodiment, the formulation buffer comprises a cryoprotectant such as glycerol or sucrose for storage at low temperatures.

[0189] The titer of the purified AAV particles is determined by quantitative PCR (qPCR), which measures the number of vector genome copies per unit volume. The qPCR assay utilizes primers and probes that hybridize to sequences within the AAV vector genome, such as the ITR sequences or sequences within the expression insert. The titer is expressed as vector genomes per milliliter (vg / mL) or genome copies per milliliter (gc / mL).

[0190] In an embodiment, the infectivity of the purified AAV particles is determined by transduction assays. Transduction assays measure the ability of AAV particles to deliver thevector genome to target cells and express the encoded transgene. Target cells are transduced with serial dilutions of the AAV preparation, and expression of the suppressor tRNA or a reporter gene is measured. The infectious titer is expressed as transducing units per milliliter (TU / mL) or infectious units per milliliter (lU / mL).

[0191] Lentiviral vector production involves transfection of producer cells with plasmids encoding the lentiviral vector genome, packaging genes, and envelope protein. In an embodiment, the producer cells are HEK293T cells. The lentiviral vector plasmid contains the expression insert flanked by lentiviral long terminal repeats (LTRs) and packaging signal sequences. The packaging plasmid encodes the Gag, Pol, and Rev genes. The envelope plasmid encodes the VSV-G envelope protein or another envelope protein that confers broad tropism.

[0192] Following transfection, the producer cells are cultured under conditions that permit expression of the lentiviral genes and assembly of lentiviral particles. The lentiviral particles are released into the culture medium by budding from the producer cell membrane. The culture medium containing the lentiviral particles is harvested at intervals following transfection.

[0193] The lentiviral particles are concentrated and purified from the culture medium. In an embodiment, the lentiviral particles are concentrated by ultracentrifugation. The culture medium is centrifuged at high speed, and the lentiviral particles pellet at the bottom of the centrifuge tube. The pellet is resuspended in a smaller volume of buffer to achieve concentration of the lentiviral particles. In an embodiment, the lentiviral particles are concentrated by ultrafiltration using tangential flow filtration or centrifugal filter devices.

[0194] The titer of the lentiviral particles is determined by transduction assays or by qPCR. Transduction assays measure the ability of lentiviral particles to integrate the vector genome into target cell chromosomes and express the encoded transgene. The titer is expressed as transducing units per milliliter (TU / mL). qPCR assays measure the number of integrated vector copies in transduced cells or the number of vector RNA copies in the lentiviral preparation.

[0195] Adenoviral vector production involves transfection of producer cells with the adenoviral vector genome. In an embodiment, the producer cells are HEK293 cells or PER.C6 cells. The adenoviral vector genome is introduced into producer cells by transfection of a plasmid containing the adenoviral vector sequences or by infection with a seed stock ofadenoviral vector. The producer cells provide the El gene functions that are deleted from replication-deficient adenoviral vectors.

[0196] Following transfection or infection, the producer cells are cultured under conditions that permit replication and packaging of the adenoviral vector genome. The adenoviral particles accumulate within the producer cells and are released upon cell lysis. The adenoviral particles are harvested by cell lysis and purified by cesium chloride density gradient centrifugation or chromatography methods.

[0197] Quality control testing of the manufactured viral vectors includes assays for identity, purity, potency, and safety. Identity testing confirms that the viral vector contains the correct expression insert sequence. Purity testing measures the levels of residual host cell proteins, host cell DNA, and process-related impurities. Potency testing measures the biological activity of the viral vector, such as the ability to transduce target cells and express the suppressor tRNA. Safety testing includes assays for replication-competent virus, sterility, endotoxin levels, and mycoplasma contamination.Methods of Treating Stop-Codon- Associated Genetic Diseases

[0198] A method of treating a stop-codon-associated genetic disease in a subject in need thereof comprises administering to the subject a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA. The expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA. The spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. A promoter is operably linked to each nucleic acid encoding the suppressor tRNA.

[0199] The term "stop-codon-associated genetic disease" refers to a disorder that is mediated, enhanced, or otherwise facilitated by or associated with a premature stop codon in a gene. Premature stop codons arise from nonsense mutations that introduce a stop codon within the coding sequence of a gene, resulting in premature termination of translation and production of truncated, nonfunctional proteins. The suppressor tRNA administered to the subject hybridizes to the premature stop codon and permits incorporation of an amino acid atthe position corresponding to the premature stop codon, thereby restoring production of full-length protein.

[0200] In an embodiment, the stop-codon-associated genetic disease is selected from the group consisting of P-thalassemia, Choroideremia (CHM), Cystic Fibrosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Hurler Syndrome, KIF1 A, Liddle's Syndrome, a Lysosomal Storage Disease, Marfan Syndrome, Smith-Lemli-Opitz Syndrome, Spinal Muscular Atrophy, an epilepsy disorder, an epileptic encephalopathy, Lennox-Gastaut Syndrome, Kleefstra Syndrome, KCNQ2 Encephalopathy, SYNGAP1 Encephalopathy, Parkinson's with GBA, CDKL5, SLC6A1, BRMUTD, Sotos Syndrome, GLUT1 Deficiency Syndrome, 5q-syndrome, Adams-Oliver syndrome 1, Alagille syndrome 1, Autoimmune lymphoproliferative syndrome type 1 A, Carney complex type I, CHARGE syndrome, Coffin-Siris Syndrome, Duane Syndrome, Ehlers-Danlos Syndrome, Feingold Syndrome 1, Denys-Drash syndrome / Frasier Syndrome, DiGeorge Syndrome (TBX1 -associated), and Cleidocranial dysplasia.

[0201] In an embodiment, the Lysosomal Storage Disease is selected from the group consisting of Maroteaux-Lamy Syndrome, Niemann Pick Disease, and Sanfilippo Syndrome. Lysosomal Storage Diseases result from deficiencies in lysosomal enzymes caused by nonsense mutations that produce truncated, nonfunctional enzyme proteins. Administration of the suppressor tRNA restores production of full-length lysosomal enzymes in cells of the subject.

[0202] In an embodiment, the stop-codon-associated genetic disease is a central nervous system (CNS)-related disorder. CNS-related disorders caused by nonsense mutations include neurological conditions affecting brain and spinal cord function. In an embodiment, the stopcodon-associated genetic disease is a non-CNS-related disorder. Non-CNS-related disorders caused by nonsense mutations include conditions affecting tissues and organs outside the central nervous system.

[0203] In an embodiment, the stop-codon-associated genetic disease is cancer. Premature stop codon mutations in tumor suppressor genes result in production of truncated tumor suppressor proteins that lack tumor suppressor function, contributing to cancer development and progression. Administration of the suppressor tRNA restores production of full-length tumor suppressor proteins in cancer cells.

[0204] In an embodiment, the cancer is selected from the group consisting of p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian carcinoma, breast cancer, and colorectal cancer. In an embodiment, the cancer is esophageal carcinoma. In an embodiment, the cancer is osteocarcinoma. In an embodiment, the cancer is fibrous histiocytoma. The cancers listed result from nonsense mutations in tumor suppressor genes, including the TP53 gene, that produce premature stop codons and truncated tumor suppressor proteins.

[0205] In an embodiment, the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine. The suppressor tRNA that recognizes UGA stop codons and is aminoacylated with arginine permits arginine to be incorporated into a growing polypeptide chain at positions corresponding to premature UGA stop codons. UGA stop codons arise frequently from CGA to UGA mutations caused by deamination of 5-methylcytosine at CpG sites in tumor suppressor genes.

[0206] In an embodiment, the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In an embodiment, the suppressor tRNA comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6. The suppressor tRNA of SEQ ID NO: 6 is a tRNAArg / Opsuppressor that hybridizes to UGA opal stop codons and is aminoacylated with arginine.

[0207] In an embodiment, the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle. The pharmaceutically acceptable carrier facilitates delivery of the suppressor tRNA or expression vector to target cells and tissues in the subject. The polymeric nanoparticle, liposome, or micelle protects the suppressor tRNA or expression vector from degradation and enhances cellular uptake.

[0208] A therapeutically effective amount of active component is administered to the subject. In an embodiment, the therapeutically effective amount of active component is in the range of 0.1 mg / kg to 100 mg / kg. In an embodiment, the therapeutically effective amount of active component is in the range of 1 mg / kg to 100 mg / kg. In an embodiment, the therapeutically effective amount of active component is in the range of 1 mg / kg to 10 mg / kg. The amount administered depends on variables including the type and extent of disease orindication to be treated, the overall health of the subject, the in vivo potency of the suppressor tRNA, the pharmaceutical formulation, and the route of administration.

[0209] In an embodiment, a therapeutically effective amount of a viral expression vector is administered to the subject. In an embodiment, the therapeutically effective amount of the 2 15viral expression vector is in the range of 10 to 10 plaque forming units (pfus). In an embodiment, the therapeutically effective amount of the viral expression vector is in the range of 10 to 10 plaque forming units. In an embodiment, the therapeutically effective amount of the viral expression vector is in the range of 10 to 10 plaque forming units. In an embodiment, the therapeutically effective amount of the viral expression vector is in the range of 105to 1015plaque forming units. In an embodiment, the therapeutically effective amount of the viral expression vector is in the range of 105to 1010plaque forming units. In an embodiment, the therapeutically effective amount of the viral expression vector is in the range of 1010to 1015plaque forming units.

[0210] The dosing frequency for administration of the composition varies depending on factors including the route of administration, dosage amount, serum half-life, and the disease being treated. In an embodiment, the dosing frequency is once per day. In an embodiment, the dosing frequency is once per week. In an embodiment, the dosing frequency is once every two weeks.Methods of Restoring Translation

[0211] A method of restoring translation to a nucleotide sequence comprising a nonsense mutation in a cell comprises introducing into the cell a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA. The expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA. The spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. A promoter is operably linked to each nucleic acid encoding the suppressor tRNA. The suppressor tRNA hybridizes to a premature stop codon in the nucleotide sequence and permits incorporation of an amino acid at a position corresponding to the premature stop codon.

[0212] The method of restoring translation addresses the effects of nonsense mutations that introduce premature stop codons within coding sequences of genes. When a premature stop codon is present in a nucleotide sequence, translation terminates at the position of the premature stop codon rather than at the natural termination codon, resulting in production of a truncated protein. The suppressor tRNA introduced into the cell recognizes and hybridizes to the premature stop codon during translation. Upon hybridization of the suppressor tRNA to the premature stop codon, the suppressor tRNA permits incorporation of an amino acid carried by the suppressor tRNA into the growing polypeptide chain at the position corresponding to the premature stop codon. Translation continues beyond the position of the premature stop codon, and a full-length protein is produced from the nucleotide sequence comprising the nonsense mutation.

[0213] In an embodiment, the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine. The UGA stop codon is an opal stop codon that arises from nonsense mutations in genes. The suppressor tRNA with an anticodon complementary to the UGA stop codon recognizes and hybridizes to UGA stop codons during translation. The suppressor tRNA is aminoacylated with arginine by endogenous aminoacyl-tRNA synthetases in the cell or is aminoacylated with arginine prior to introduction into the cell. When the suppressor tRNA aminoacylated with arginine hybridizes to a premature UGA stop codon, arginine is incorporated into the growing polypeptide chain at the position corresponding to the premature stop codon.

[0214] In an embodiment, the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In an embodiment, the suppressor tRNA comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6. The suppressor tRNA of SEQ ID NO: 6 is a tRNAArg / Opsuppressor that comprises an anticodon that hybridizes to UGA opal stop codons. The tRNAArg / Opsuppressor is recognized by endogenous arginyl-tRNA synthetases and is aminoacylated with arginine. The tRNAArg / Opsuppressor permits arginine to be incorporated at positions corresponding to premature UGA stop codons, restoring translation of nucleotide sequences comprising UGA nonsense mutations.

[0215] In an embodiment, the expression vector is an adeno-associated viral vector. The adeno-associated viral vector delivers the expression insert encoding the suppressor tRNA to target cells. The adeno-associated viral vector transduces the cell and delivers the expression insert to the cell nucleus, where the expression insert directs transcription of the suppressor tRNA. The transcribed suppressor tRNA is processed and aminoacylated in the cell, and the aminoacylated suppressor tRNA participates in translation by hybridizing to premature stop codons and permitting incorporation of amino acids at positions corresponding to the premature stop codons.

[0216] In an embodiment, the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAV10. The serotype of the adeno-associated viral vector is selected based on the target cell type and tissue tropism for the intended application. AAV9 transduces various tissues and organs including the central nervous system, heart, liver, and skeletal muscle. AAV2 transduces the central nervous system, kidney, and photoreceptor cells. AAV8 transduces the central nervous system, heart, liver, pancreas, photoreceptor cells, retinal pigment epithelium, and skeletal muscle.

[0217] The composition comprising the suppressor tRNA or expression vector encoding the suppressor tRNA is introduced into the cell by transfection, transduction, or other delivery methods. In an embodiment, the composition is introduced into the cell by transduction with a viral vector. In an embodiment, the composition is introduced into the cell by transfection with a non-viral delivery system. In an embodiment, the composition is introduced into the cell by lipofection using liposomes or lipid nanoparticles. In an embodiment, the composition is introduced into the cell by electroporation.

[0218] The cell into which the composition is introduced comprises a gene having a nonsense mutation that produces a premature stop codon. The premature stop codon in the gene causes premature termination of translation and production of a truncated protein from the gene. Upon introduction of the composition into the cell, the suppressor tRNA expressed from the expression vector or the suppressor tRNA directly introduced into the cell hybridizes to the premature stop codon during translation of the gene. The suppressor tRNA permits incorporation of an amino acid at the position corresponding to the premature stop codon, and translation continues to produce a full-length protein from the gene having the nonsense mutation.

[0219] In an embodiment, the gene having the nonsense mutation encodes a tumor suppressor protein. In an embodiment, the gene having the nonsense mutation is the TP53 gene encoding the p53 tumor suppressor protein. Nonsense mutations in the TP53 gene produce premature UGA stop codons that result in truncated p53 protein lacking tumor suppressor function. The tRNAArg / Opsuppressor hybridizes to the premature UGA stop codon in the TP53 gene and permits arginine to be incorporated at the position corresponding to the premature stop codon, restoring production of full-length p53 protein.

[0220] In an embodiment, the gene having the nonsense mutation encodes a structural protein, an ion channel protein, or an enzyme. Nonsense mutations in genes encoding structural proteins, ion channel proteins, or enzymes produce premature stop codons that result in truncated proteins lacking proper function. The suppressor tRNA hybridizes to the premature stop codon and permits incorporation of an amino acid at the position corresponding to the premature stop codon, restoring production of full-length functional protein.Combination Therapies and Treatment Outcomes

[0221] The methods and compositions described herein are used alone or in combination with other therapeutic agents and / or modalities. The term administered "in combination" means that two or more different treatments are delivered to the subject during the course of the subject's affliction with the disorder, such that the effects of the treatments on the subject overlap at a point in time. In an embodiment, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is referred to as "simultaneous" or "concurrent delivery." In an embodiment, the delivery of one treatment ends before the delivery of the other treatment begins.

[0222] In an embodiment of either case, the treatment is more effective because of combined administration. In an embodiment, the second treatment is more effective, such that an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment. In an embodiment, the analogous situation is seen with the first treatment. In an embodiment, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments is partiallyadditive, wholly additive, or greater than additive. The delivery is such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0223] In an embodiment, the suppressor tRNA constructs are administered in combination with nonsense-mediated mRNA decay (NMD) inhibitors. NMD is a cellular surveillance pathway that recognizes and degrades mRNA transcripts containing premature stop codons. By inhibiting NMD, the stability of mRNA transcripts harboring nonsense mutations may be increased, thereby providing more substrate for suppressor tRNA-mediated readthrough. In an embodiment, the NMD inhibitor is a small molecule that targets components of the NMD pathway. In an embodiment, the NMD inhibitor targets UPF1, UPF2, or UPF3 proteins involved in NMD. In an embodiment, the NMD inhibitor is NMDI-1 or NMDI-14. In an embodiment, the NMD inhibitor is amlexanox. The combination of suppressor tRNA constructs with NMD inhibitors may result in enhanced production of full-length protein compared to administration of either agent alone.

[0224] In an embodiment, the suppressor tRNA constructs are administered in combination with gene editing systems. Gene editing systems may be used to correct nonsense mutations at the genomic level, while suppressor tRNA constructs provide readthrough of premature stop codons at the translational level. In an embodiment, the gene editing system is a CRISPR-Cas system. In an embodiment, the gene editing system comprises a Cas9 nuclease and a guide RNA targeting a sequence proximal to the nonsense mutation. In an embodiment, the gene editing system is a base editing system that converts the mutated nucleotide to restore the wild-type codon. In an embodiment, the gene editing system is a prime editing system. The combination of suppressor tRNA constructs with gene editing systems may provide both immediate translational rescue through suppressor tRNA activity and long-term correction through genomic editing.

[0225] In an embodiment, the suppressor tRNA constructs are administered in combination with protein replacement therapy. Protein replacement therapy involves administration of exogenous functional protein to compensate for deficient or absent endogenous protein production. The combination of suppressor tRNA constructs with protein replacement therapy may provide complementary mechanisms for restoring protein function, wherein the suppressor tRNA constructs promote endogenous production of full-length protein while the exogenous protein provides immediate functional supplementation. In an embodiment, the protein replacement therapy comprises enzyme replacement therapy for lysosomal storage diseases. For example, in the treatment of Hurler Syndrome caused bynonsense mutations in the IDUA gene encoding alpha-L-iduronidase, suppressor tRNA constructs may be administered in combination with laronidase, a recombinant form of alpha-L-iduronidase. The laronidase provides immediate enzymatic activity while the suppressor tRNA constructs promote endogenous production of full-length alpha-L-iduronidase from the mutated IDUA gene. In some cases, the combination therapy may permit reduced dosing of the protein replacement therapy or may provide enhanced therapeutic benefit compared to either treatment alone.

[0226] In an embodiment, the suppressor tRNA constructs are administered in combination with conventional therapies for the stop-codon-associated genetic disease being treated. Conventional therapies may address symptoms or downstream effects of the disease while suppressor tRNA constructs address the underlying molecular defect. In an embodiment, the conventional therapy is enzyme replacement therapy for lysosomal storage diseases. In an embodiment, the conventional therapy is an anti-seizure medication for epilepsy disorders such as Dravet Syndrome. In an embodiment, the conventional therapy is a corticosteroid for Duchenne Muscular Dystrophy. In an embodiment, the conventional therapy is a CFTR modulator for Cystic Fibrosis. In an embodiment, the conventional therapy is chemotherapy or radiation therapy for cancers associated with nonsense mutations in tumor suppressor genes.

[0227] In an embodiment, the method is administered in combination with one or more additional therapies. In an embodiment, the additional therapy is DIACOMIT (stiripentol). In an embodiment, the additional therapy is EPIDIOLEX (cannabidiol). In an embodiment, the additional therapy is a ketogenic diet. In an embodiment, the additional therapy is ONFI (clobazam). In an embodiment, the additional therapy is TOPAMAX (topiramate). In an embodiment, the additional therapy is valproic acid.

[0228] In an embodiment, the stop-codon-associated genetic disease is Dravet Syndrome. Dravet Syndrome is a severe form of epilepsy that begins in infancy and is characterized by frequent, prolonged seizures and developmental delays. Dravet Syndrome is associated with nonsense mutations in the SCN1A gene that produce premature stop codons and truncated sodium channel proteins. The suppressor tRNA administered to the subject hybridizes to the premature stop codon in the SCN1A gene and permits incorporation of an amino acid at the position corresponding to the premature stop codon, restoring production of full-length sodium channel protein.

[0229] In an embodiment, during the treatment of Dravet Syndrome, the method is administered in combination with one or more additional therapies. In an embodiment, the additional therapy is DIACOMIT (stiripentol). In an embodiment, the additional therapy is EPIDIOLEX (cannabidiol). In an embodiment, the additional therapy is a ketogenic diet. In an embodiment, the additional therapy is ONFI (clobazam). In an embodiment, the additional therapy is TOPAMAX (topiramate). In an embodiment, the additional therapy is valproic acid.

[0230] In an embodiment, wherein the stop-codon-associated genetic disease is Dravet Syndrome, the method reduces seizure frequency, seizure severity, and / or cognitive impairment in the subject. The reduction in seizure frequency, seizure severity, and / or cognitive impairment results from restoration of full-length sodium channel protein expression through suppressor tRNA-mediated readthrough of premature stop codons.

[0231] In an embodiment, the method reduces seizure frequency in the subject by at least 10% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 20% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 30% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 40% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 50% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 60% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 70% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 80% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by at least 90% over the period of a day. In an embodiment, the method reduces seizure frequency in the subject by 100% over the period of a day.

[0232] In an embodiment, the method reduces seizure frequency in the subject by at least 10% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 20% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 30% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 40% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 50% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 60% over the period of a week. In an embodiment, the method reducesseizure frequency in the subject by at least 70% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 80% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by at least 90% over the period of a week. In an embodiment, the method reduces seizure frequency in the subject by 100% over the period of a week.

[0233] In an embodiment, the method reduces seizure frequency in the subject by at least 10% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 20% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 30% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 40% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 50% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 60% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 70% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 80% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by at least 90% over the period of a month. In an embodiment, the method reduces seizure frequency in the subject by 100% over the period of a month.

[0234] In an embodiment, the method reduces seizure frequency by 50% over the period of a day, a week, or a month. The 50% reduction in seizure frequency represents a clinically meaningful improvement in seizure control for subjects with Dravet Syndrome and other epilepsy disorders associated with nonsense mutations.Ex Vivo Therapies

[0235] In an embodiment, the suppressor tRNA or expression vector encoding the suppressor tRNA is delivered to cells ex vivo for subsequent administration to a subject. Ex vivo therapy involves isolation of cells from a subject or a donor, introduction of the suppressor tRNA or expression vector into the isolated cells outside the body, and administration of the modified cells to the subject. The ex vivo approach permits modification of cells under controlled conditions and selection or expansion of modified cells prior to administration.

[0236] In an embodiment, the cells modified ex vivo are hematopoietic cells.Hematopoietic cells are blood-forming cells that give rise to all blood cell lineages, including red blood cells, white blood cells, and platelets. Hematopoietic cells include hematopoietic stem cells (HSCs), hematopoietic progenitor cells, and mature blood cells. Hematopoietic stem cells reside in the bone marrow and possess the capacity for self-renewal and differentiation into all blood cell types. Introduction of the suppressor tRNA or expression vector into hematopoietic stem cells permits long-term expression of the suppressor tRNA in the hematopoietic system following engraftment of the modified cells in the subject.

[0237] In an embodiment, the hematopoietic cells are isolated from bone marrow of the subject or a donor. Bone marrow aspiration is performed to obtain a sample containing hematopoietic stem cells and progenitor cells. The hematopoietic stem cells are enriched from the bone marrow sample by immunomagnetic selection or fluorescence-activated cell sorting based on expression of cell surface markers such as CD34. In an embodiment, the hematopoietic cells are isolated from peripheral blood following mobilization with granulocyte colony-stimulating factor (G-CSF) or plerixafor. Mobilization increases the number of hematopoietic stem cells circulating in peripheral blood, permitting collection by apheresis. In an embodiment, the hematopoietic cells are isolated from umbilical cord blood collected at birth.

[0238] In an embodiment, the suppressor tRNA or expression vector is introduced into the hematopoietic cells by transduction with a viral vector. Lentiviral vectors integrate into the genome of transduced cells, providing stable, long-term expression of the suppressor tRNA in the hematopoietic cells and their progeny. In an embodiment, the suppressor tRNA or expression vector is introduced into the hematopoietic cells by electroporation.Electroporation permits delivery of the suppressor tRNA or expression vector without viral integration, providing transient or episomal expression depending on the nature of the delivered nucleic acid.

[0239] Following introduction of the suppressor tRNA or expression vector, the modified hematopoietic cells are administered to the subject by intravenous infusion. The modified hematopoietic stem cells home to the bone marrow and engraft, establishing a population of cells that express the suppressor tRNA. The engrafted modified hematopoietic stem cells undergo self-renewal and differentiation, producing progeny cells that inherit the expression vector and express the suppressor tRNA. In an embodiment, the subject undergoesmyeloablative or non-myeloablative conditioning prior to administration of the modified hematopoietic cells to facilitate engraftment.

[0240] In an embodiment, the ex vivo therapy is autologous cell therapy. Autologous cell therapy involves isolation of cells from the subject to be treated, modification of the isolated cells with the suppressor tRNA or expression vector, and administration of the modified cells back to the same subject. Autologous cell therapy reduces the risk of immune rejection because the modified cells are derived from the subject's own tissues. The subject's immune system recognizes the autologous cells as self and does not mount an immune response against the modified cells.

[0241] In an embodiment, the autologous cells are hematopoietic stem cells isolated from the subject's bone marrow or mobilized peripheral blood. The autologous hematopoietic stem cells are transduced with a lentiviral vector encoding the suppressor tRNA or electroporated with the suppressor tRNA or expression vector. The modified autologous hematopoietic stem cells are administered to the subject by intravenous infusion following conditioning. The modified autologous hematopoietic stem cells engraft in the bone marrow and provide longterm expression of the suppressor tRNA in the hematopoietic system.

[0242] In an embodiment, the autologous cells are T cells isolated from the subject's peripheral blood. T cells are lymphocytes that play a role in cell-mediated immunity.Autologous T cells are isolated from peripheral blood by leukapheresis and enriched by immunomagnetic selection or fluorescence-activated cell sorting. The autologous T cells are activated and expanded in culture, and the suppressor tRNA or expression vector is introduced into the activated T cells by transduction or electroporation. The modified autologous T cells are administered to the subject by intravenous infusion.

[0243] In an embodiment, the autologous cells are fibroblasts isolated from the subject's skin. Fibroblasts are connective tissue cells that are obtained by skin biopsy and expanded in culture. The suppressor tRNA or expression vector is introduced into the autologous fibroblasts by transduction or transfection. The modified autologous fibroblasts are administered to the subject by local injection or implantation at a site affected by the stopcodon-associated genetic disease.

[0244] In an embodiment, the autologous cells are induced pluripotent stem cells (iPSCs) derived from the subject's somatic cells. Somatic cells such as fibroblasts or peripheral blood mononuclear cells are reprogrammed to a pluripotent state by introduction of reprogrammingfactors. The resulting iPSCs possess the capacity to differentiate into any cell type. The suppressor tRNA or expression vector is introduced into the iPSCs, and the modified iPSCs are differentiated into the desired cell type for therapeutic application. The differentiated cells expressing the suppressor tRNA are administered to the subject.

[0245] In an embodiment, the ex vivo therapy is allogeneic cell therapy. Allogeneic cell therapy involves isolation of cells from a donor, modification of the donor cells with the suppressor tRNA or expression vector, and administration of the modified donor cells to a recipient subject. Allogeneic cell therapy permits use of cells from healthy donors who do not carry the nonsense mutation present in the subject. In an embodiment, the allogeneic cells are hematopoietic stem cells from a human leukocyte antigen (HLA)-matched donor. HLA matching reduces the risk of graft-versus-host disease and immune rejection following administration of the allogeneic cells.

[0246] In an embodiment, the ex vivo modified cells are used in the treatment of a stopcodon-associated genetic disease. The modified cells express the suppressor tRNA, which hybridizes to premature stop codons in genes affected by nonsense mutations and permits incorporation of amino acids at positions corresponding to the premature stop codons.Administration of the ex vivo modified cells to the subject provides cells capable of producing full-length proteins from genes that would otherwise produce truncated proteins due to premature stop codons.

[0247] In an embodiment, the ex vivo modified hematopoietic cells are used in the treatment of P -thalassemia caused by nonsense mutations in the P-globin gene. The modified hematopoietic stem cells engraft in the bone marrow and differentiate into red blood cell precursors that express the suppressor tRNA. The suppressor tRNA permits readthrough of the premature stop codon in the P-globin gene, restoring production of full-length P-globin protein and functional hemoglobin in the red blood cells derived from the modified hematopoietic stem cells.

[0248] In an embodiment, the ex vivo modified cells are used in the method of restoring translation to a nucleotide sequence comprising a nonsense mutation. The ex vivo modified cells are introduced into the subject, and the suppressor tRNA expressed by the modified cells hybridizes to the premature stop codon in the nucleotide sequence and permits incorporation of an amino acid at the position corresponding to the premature stop codon.Translation continues beyond the premature stop codon, and full-length protein is produced from the nucleotide sequence comprising the nonsense mutation.

[0249] In an embodiment, the ex vivo therapy is administered in combination with other therapeutic agents or modalities. The combination of ex vivo therapy with other treatments provides complementary mechanisms for addressing the stop-codon-associated genetic disease. In an embodiment, the ex vivo modified cells are administered in combination with direct administration of the suppressor tRNA or expression vector to the subject. The direct administration provides suppressor tRNA expression in tissues not reconstituted by the ex vivo modified cells, while the ex vivo modified cells provide long-term suppressor tRNA expression in the hematopoietic system or other cell populations.

[0250] In an embodiment, the ex vivo modified cells are administered in combination with enzyme replacement therapy, gene editing therapy, or conventional therapies for the stop-codon-associated genetic disease being treated. The combination of ex vivo cell therapy with other treatments addresses the disease through multiple mechanisms, providing enhanced therapeutic benefit compared to administration of either treatment alone.Discussion of Representative Experimental Results

[0251] Experimental data demonstrate that suppressor tRNA constructs containing multiple copies of tRNAArg / Opwith U6 promoters and spacer sequences effectively rescue p53 protein expression in cells harboring opal nonsense mutations. Referring to FIG. 4A, western blot analysis of calu-6 cells, which are homozygous for the TP53 R196X opal nonsense mutation, shows that treatment with RNAiMax-tRNAArg / Oprescues p53 protein expression. As shown in FIG. 4A, the p53 band appears in the RNAiMax-tRNAArg / Optreated sample, indicating restoration of full-length p53 protein production. The truncated p53 band is minimal in the RNAiMax-tRNAArg / Optreated sample compared to the G418 treated sample, indicating that the Arg / Op suppressor tRNA achieves rescue with greater specificity than the aminoglycoside drug.

[0252] With reference to FIG. 4B, genome-wide ribosomal profiling data from calu-6 cells demonstrate that the Arg / Op suppressor tRNA does not cause readthrough at normal termination codons. As shown in FIG. 4B, the degree of right shifting in the ribosomal profiling data indicates readthrough beyond normal stop codons into the 3' UTR. The G418 aminoglycoside drug shows substantial right shifting, indicating readthrough at normal stopcodons. The lysine / amber suppressor tRNA also shows some degree of right shifting. In contrast, the Arg / Op suppressor tRNA does not show right shifting, demonstrating that the Arg / Op suppressor tRNA does not read through normal stop codons. PTC124 does not show right shifting but failed to rescue p53 as shown in FIG. 4A. The data demonstrate that the Arg / Op suppressor tRNA is capable of rescuing p53 in a specific manner with minimal readthrough of normal stop codons.

[0253] Referring to FIG. 5, western blot analysis demonstrates that expression constructs Vec2 and Vec3 induce expression of p53 protein in calu-6 cells. Vec2 contains five copies of a repeating unit containing a U6 promoter operably linked to the tRNAArg / Opsuppressor with spacer sequences positioned between successive copies. Vec3 contains three copies of the repeating unit with spacer sequences positioned between successive copies. As shown in FIG.5, lanes corresponding to Vec2 and Vec3 show p53 protein bands detected with anti-p53 antibody, indicating rescue of p53 expression. The anti-GAPDH bands serve as loading controls and show consistent intensity across the lanes. Vecl, which contains five copies of the suppressor tRNA without U6 promoters, does not show comparable p53 rescue. Vec4 and Vec5, which omit the flanking region around the tRNAArg / Opsuppressor, do not show comparable p53 rescue. The data demonstrate that the combination of U6 promoters, flanking sequences, and spacer sequences in Vec2 and Vec3 provides effective expression of the suppressor tRNA for p53 rescue.

[0254] With reference to FIG. 6A and FIG. 6B, the expression constructs are successfully encapsulated in AAV9 vectors while maintaining cellular activity. FIG. 6A shows microscopy images of calu-6 cells transduced with AAV9 vectors containing a reporter gene encoding enhanced green fluorescent protein (EGFP). The wild-type control column shows no green fluorescence. The MOI=104column shows low-level green fluorescence. The MOI=106column shows substantially higher green fluorescence intensity. As shown in FIG. 6B, the fluorescence intensity increases with increasing multiplicity of infection, with the AAV9-CAG-Calu6-lE+6 condition showing fluorescence intensity of 364 arbitrary units compared to 62 arbitrary units for the AAV9-CAG-Calu6-lE+4 condition and 0 for the negative control. The data confirm that the fluorescence imaging is not an artifact and is above background cellular fluorescence levels, demonstrating that the expression constructs are active in cells following AAV9 encapsulation and transduction.

[0255] Referring to FIG. 7, western blot analysis of calu-6 cells transduced with AAV9-encapsulated expression constructs demonstrates rescue of p53 protein expression. As shownin FIG. 7, Vec3 at MOI=106shows p53 protein expression detected with anti-p53 antibody. Vec2 at MOI=106also shows p53 protein expression. The data are consistent with the results from direct transfection experiments and demonstrate that Vec3 is more effective at rescuing p53 expression than Vec2 when delivered via AAV9 vectors. G418 at 3000 pg / ml also rescues p53 expression as shown in FIG. 7, but G418 is not specific as demonstrated by the ribosomal profiling data and is less effective at rescuing p53 expression than Vec3. The anti-GAPDH bands demonstrate equal protein loading across the samples.

[0256] With reference to FIG. 8, FIG. 9, and FIG. 10, a preliminary safety and pharmacokinetic study in mice demonstrates that Vec3 is well-tolerated and distributes to multiple tissues following systemic administration. The study was a single-dose study using tail-vein injection at a dose of 1.8x 1012GC / mouse with a stock concentration of 2.74* 1013GC / ml. Two test groups were evaluated: a vehicle control group receiving PBS and a Vec3 group receiving the Vec3 construct in an AAV9 vector. The mouse strain used was C57BL / 6 with 4 mice per group (2 female / 2 male). The study duration was 14 days with body weight measurements and clinical observations performed every other day.

[0257] As shown in FIG. 8, no significant changes in body weight were observed between the Control group and the Vec3 group over the 14-day study period. Both groups displayed similar weight trajectories, with weights starting at approximately 22 grams on day 0 and gradually increasing to approximately 23 grams by day 14. The Control and Vec3 lines remain closely aligned throughout the experiment, with the Vec3 group tracking slightly below the Control group at most time points. Error bars at each measurement time point indicate variability in the weight measurements. The data demonstrate that Vec3 administration does not adversely affect body weight in mice.

[0258] Referring to FIG. 9, no significant changes in hematological parameters were observed between the PBS vehicle control and Vec3 treatment groups. FIG. 9 depicts six line graphs showing changes in white blood cell parameters including white blood cell counts, neutrophils, monocytes, eosinophils, lymphocytes, and basophils from the start to the end of the study period. White blood cell counts, neutrophils, monocytes, eosinophils, lymphocytes, and basophils remained within comparable ranges between the PBS vehicle control and Vec3 treatment groups throughout the study. The data demonstrate that Vec3 administration does not cause significant alterations in white blood cell populations, supporting the safety profile of the expression construct.

[0259] With reference to FIG. 10, biodistribution analysis demonstrates that Vec3 is detected in several tissues at 14 days post-dosing. FIG. 10 depicts a series of bar graphs showing mean relative expression levels of Vec3 in various tissues including brain, heart, lung, pancreas, liver, and kidney compared to vehicle control. The liver showed the highest mean relative expression levels among the tissues examined at approximately 350 units, followed by heart at approximately 32 units, lung at approximately 20 units, pancreas at approximately 13 units, kidney at approximately 10 units, and brain at approximately 4.3 units. The Vec3 condition demonstrated substantially higher mean relative expression compared to the Vehicle control condition across all six tissues examined. The data demonstrate that Vec3 delivered via AAV9 vector distributes to multiple tissues following systemic administration via tail-vein injection, with particularly high expression in the liver.

[0260] The collective experimental data demonstrate that suppressor tRNA constructs containing multiple copies of tRNAArg / Opwith U6 promoters and spacer sequences achieve rescue of p53 protein expression in cells harboring opal nonsense mutations with high specificity and minimal readthrough at normal termination codons. The expression constructs are successfully encapsulated in AAV9 vectors while maintaining cellular activity following transduction. Furthermore, the preliminary safety and pharmacokinetic study demonstrates that Vec3 is well-tolerated in mice with no significant changes in body weight or hematological parameters, and the construct distributes to multiple tissues including brain, heart, lung, pancreas, liver, and kidney following systemic administration. These data demonstrate utility for therapeutic applications targeting stop-codon-associated genetic diseases caused by UGA nonsense mutations.

[0261] The present invention may be further understood by reference to the following representative embodiments.

[0262] Provided herein as Embodiment 1 is an expression insert for expressing a suppressor tRNA, comprising:

[0263] two or more copies of a nucleic acid encoding a suppressor tRNA;

[0264] a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and

[0265] a promoter operably linked to each nucleic acid encoding the suppressor tRNA.

[0266] Provided herein as Embodiment 2 is the expression insert of Embodiment 1, wherein the spacer is located between a termination sequence of a first copy of the nucleic acid encoding the suppressor tRNA positioned 5' to the spacer and the promoter positioned 3' to the spacer, wherein the promoter positioned 3' to the spacer is operably linked to a second copy of the nucleic acid encoding the suppressor tRNA.

[0267] Provided herein as Embodiment 3 is the expression insert of Embodiment 1 or 2, wherein the termination sequence comprises a hexa-thymine sequence.

[0268] Provided herein as Embodiment 4 is the expression insert of any one of Embodiments 1 to 3, wherein the expression insert comprises from 2 to 10 copies of the nucleic acid encoding the suppressor tRNA.

[0269] Provided herein as Embodiment 5 is the expression insert of any one of Embodiments 1 to 4, wherein each copy of the nucleic acid encoding the suppressor tRNA comprises an identical nucleotide sequence.

[0270] Provided herein as Embodiment 6 is the expression insert of any one of Embodiments 1 to 5, wherein at least two copies of the nucleic acid encoding the suppressor tRNA comprise different nucleotide sequences.

[0271] Provided herein as Embodiment 7 is the expression insert of any one of Embodiments 1 to 6, wherein at least one copy of the nucleic acid encoding the suppressor tRNA encodes a modified or engineered suppressor tRNA comprising an anticodon that hybridizes to a nonsense stop codon and is capable of being aminoacylated with an amino acid, such that the suppressor tRNA, when expressed in a cell and aminoacylated with the amino acid, hybridizes to the nonsense stop codon and permits the amino acid to be incorporated into a gene product at a position that would otherwise result in a truncated gene product caused by the nonsense stop codon.

[0272] Provided herein as Embodiment 8 is the expression insert of any one of Embodiments 1 to 7, wherein the nonsense stop codon is a UGA stop codon and wherein the suppressor tRNA is aminoacylated or is capable of being aminoacylated with arginine.

[0273] Provided herein as Embodiment 9 is the expression insert of any one of Embodiments 1 to 8, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

[0274] Provided herein as Embodiment 10 is the expression insert of any one of Embodiments 1 to 9, wherein the promoter is selected from the group consisting of a retroviral LTR, a SV40 promoter, a human cytomegalovirus promoter, a U6 promoter, an adenovirus promoter, a TK promoter, and a B19 parvovirus promoter.

[0275] Provided herein as Embodiment 11 is the expression insert of any one of Embodiments 1 to 10, wherein the promoter is a U6 promoter.

[0276] Provided herein as Embodiment 12 is the expression insert of any one of Embodiments 1 to 11, wherein the expression insert comprises a repeating unit, wherein the repeating unit comprises the promoter, the nucleic acid encoding the suppressor tRNA, and a termination sequence.

[0277] Provided herein as Embodiment 13 is the expression insert of any one of Embodiments 1 to 12, wherein the repeating unit comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9.

[0278] Provided herein as Embodiment 14 is the expression insert of any one of Embodiments 1 to 13, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and wherein the spacer is positioned after a final repeating unit of the expression insert.

[0279] Provided herein as Embodiment 15 is a method of treating a stop-codon-associated genetic disease in a subject in need thereof, comprising:

[0280] administering to the subject a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA,

[0281] wherein the expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA,

[0282] wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and

[0283] wherein a promoter is operably linked to each nucleic acid encoding the suppressor tRNA.

[0284] Provided herein as Embodiment 16 is the method of Embodiment 15, wherein the stop-codon-associated genetic disease is selected from the group consisting of P-thalassemia, Choroideremia (CHM), Cystic Fibrosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Hurler Syndrome, KIF1A, Liddle's Syndrome, a Lysosomal Storage Disease, Marfan Syndrome, Smith-Lemli-Opitz Syndrome, Spinal Muscular Atrophy, an epilepsy disorder, an epileptic encephalopathy, Lennox-Gastaut Syndrome, Kleefstra Syndrome, KCNQ2 Encephalopathy, SYNGAP1 Encephalopathy, Parkinson's with GBA, CDKL5, SLC6A1, BRMUTD, Sotos Syndrome, or GLUT1 Deficiency Syndrome, 5q-syndrome, Adams-Oliver syndrome 1, Alagille syndrome 1, Autoimmune lymphoproliferative syndrome type 1A, Carney complex type I, CHARGE syndrome, Coffin-Siris Syndrome, Duane Syndrome, Ehlers-Danlos Syndrome, Feingold Syndrome 1, Denys-Drash syndrom e / Frasier Syndrome, DiGeorge Syndrome (TBX1 -associated), or Cleidocranial dysplasia.

[0285] Provided herein as Embodiment 17 is the method of any one of Embodiments 15 to 16, wherein the stop-codon-associated genetic disease is cancer.

[0286] Provided herein as Embodiment 18 is the method of any one of Embodiments 15 to 17, wherein the cancer is selected from the group consisting of p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian carcinoma, breast cancer, and colorectal cancer.

[0287] Provided herein as Embodiment 19 is the method of any one of Embodiments 15 to 18, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

[0288] Provided herein as Embodiment 20 is the method of any one of Embodiments 15 to 19, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

[0289] Provided herein as Embodiment 21 is the method of any one of Embodiments 15 to 20, wherein the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle.

[0290] Provided herein as Embodiment 22 is a method of restoring translation to a nucleotide sequence comprising a nonsense mutation in a cell, comprising:

[0291] introducing into the cell a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA,

[0292] wherein the expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA,

[0293] wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and

[0294] wherein a promoter is operably linked to each nucleic acid encoding the suppressor tRNA;

[0295] wherein the suppressor tRNA hybridizes to a premature stop codon in the nucleotide sequence and permits incorporation of an amino acid at a position corresponding to the premature stop codon.

[0296] Provided herein as Embodiment 23 is the method of Embodiment 22, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

[0297] Provided herein as Embodiment 24 is the method of Embodiment 22 or 23, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

[0298] Provided herein as Embodiment 25 is the method of any one of Embodiments 22 to 24, wherein the expression vector is an adeno-associated viral vector.

[0299] Provided herein as Embodiment 26 is an expression vector comprising the expression insert of any one of Embodiments 1 to 14.

[0300] Provided herein as Embodiment 27 is the expression vector of Embodiment 26, wherein the expression vector is a viral vector.

[0301] Provided herein as Embodiment 28 is the expression vector of Embodiment 26 or 27, wherein the viral vector is an adeno-associated viral vector.

[0302] Provided herein as Embodiment 29 is the expression vector of any one of Embodiments 26 to 28, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAV10.

[0303] Provided herein as Embodiment 30 is the expression vector of any one of Embodiments 26 to 29, wherein the expression vector further comprises a reporter gene.

[0304] Provided herein as Embodiment 31 is a cell comprising the expression vector of any one of Embodiments 26 to 30.

[0305] Provided herein as Embodiment 32 is the cell of Embodiment 31, wherein the cell is a mammalian cell.

[0306] Provided herein as Embodiment 33 is the cell of Embodiment 31 or 32, wherein the mammalian cell is a human cell.

[0307] Provided herein as Embodiment 34 is the cell of any one of Embodiments 31 to 33, wherein the cell is selected from the group consisting of a fibroblast, an epithelial cell, a neuronal cell, a muscle cell, and a stem cell.

[0308] Provided herein as Embodiment 35 is the cell of any one of Embodiments 31 to 34, wherein the cell comprises a gene having a nonsense mutation, and wherein the suppressor tRNA encoded by the expression insert hybridizes to a premature stop codon resulting from the nonsense mutation.

[0309] Provided herein as Embodiment 36 is a composition comprising a suppressor tRNA encoded by the expression insert of any one of Embodiments 1 to 14, and a pharmaceutically acceptable carrier.

[0310] Provided herein as Embodiment 37 is the composition of Embodiment 36, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle.

[0311] Provided herein as Embodiment 38 is the composition of Embodiment 36 or 37, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

[0312] Provided herein as Embodiment 39 is the composition of any one of Embodiments 36 to 38, wherein the composition is formulated for intravenous, intramuscular, subcutaneous, or intrathecal administration.

[0313] Provided herein as Embodiment 40 is the composition of any one of Embodiments 36 to 39, wherein the composition further comprises a buffer, a stabilizer, or a combination thereof.

[0314]

[0315] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

[0316] In the context of the present description, all publications, patent applications, patents and other references mentioned herein, if not otherwise indicated, are explicitly incorporated by reference herein in their entirety for all purposes as if fully set forth, and shall be considered part of the present disclosure in their entirety.

[0317] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control.

[0318] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others or ordinary skill in the art to understand the embodiments disclosed herein.

[0319] Except where expressly noted, trademarks are shown in upper case.

[0320] Unless stated otherwise, all percentages, parts, ratios, etc., are by weight.

[0321] When an amount, concentration, or other value or parameter is given as a range, or a list of upper and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper and lower range limits, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range. Further, where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

[0322] Further, unless otherwise explicitly stated to the contrary, when one or multiple ranges or lists of items are provided, this is to be understood as explicitly disclosing any single stated value or item in such range or list, and any combination thereof with any other individual value or item in the same or any other list.

[0323] When the term “about” or "approximately" is used, it is used to mean a certain effect or result can be obtained within a certain tolerance (e.g., ±10%), and the skilled person knows how to obtain the tolerance. When the term "about" is used in describing a value or an end-point of a range, the disclosure should be understood to include the specific value or endpoint referred to.

[0324] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0325] The transitional phrase "consisting of' excludes any element, step, or ingredient not specified in the claim, closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consists of' appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0326] The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. A “consisting essentially of’ claim occupies a middle ground between closed claims that are written in a “consisting of’ format and fully open claims that are drafted in a “comprising” format. Optional additives as defined herein, at a level that is appropriate for such additives, and minor impurities are not excluded from a composition by the term “consisting essentially of’.

[0327] As used herein, an "embodiment" means that a particular feature, structure or characteristic is included in at least one or more manifestations, examples, or implementations of this invention. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art. Combinations of features of different embodiments are all meant to bewithin the scope of the invention, without the need for explicitly describing every possible permutation by example. Thus, any of the claimed embodiments can be used in any combination.

[0328] Further, unless expressly stated to the contrary, “and / or” refers to an inclusive and not to an exclusive. Thus, “and / or” should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. For example, a condition A and / or B, is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0329] The use of "a" or "an" to describe the various elements and components herein is merely for convenience and to give a general sense of the disclosure. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. The terms “including” and “having” are intended to be inclusive such that there may be additional elements other than the listed elements.

[0330] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

[0331] As used herein, the phrases “selected from the group consisting of,” “chosen from,” and the like include mixtures of the specified materials.

[0332] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

[0333] Although this invention has been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.

[0334] The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and itsrequirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

[0335] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.EXAMPLES

[0336] The examples herein are intended to illustrate certain aspects of the present disclosure to one of ordinary skill in the art. However, the examples are not intended to limit the scope of the present disclosure.

[0337] Example 1

[0338] Rescue of TP53 levels in calu6, a cell line with an opal nonsense mutation in the gene (homozygous TP53 R196X), was evaluated for an Arg / opal (Arg / Op) suppressor gene of the present invention compared to Geneticin (or G418; an aminoglycoside drug), Ataluren (or PTC 124; an aminoglycoside drug from PTC Therapeutics), and a lysine amber suppressor.

[0339] Cells were treated with 100 pg of G418, 10 pM of PTC124, or 20 mg RNAiMax-tRNAArg / Op. As shown in the western blot in Fig. 4A, G418 and Arg / Op rescued p53, but G418 had more truncated p53 than Arg / Op suggesting it is not specific. PTC124 failed to rescue p53 in this experiment.

[0340] As shown in Fig. 4, ribosome profiling shows rescue of TP53 (truncated p53) levels in calu6 by the Arg / Op suppressor gene of the present invention without significant NTC readthrough, performing better than known alternative interventions. Fig. 4B shows a graph of a genome-wide ribosomal profiling study using calu6 cells with the indicated treatments. The degree of right shifting indicates read-through beyond normal stops (into the 3' UTR). The data demonstrate that G418 reads through normal stops, as does lysine / amber,to a certain extent, while the Arg / Op tRNA does not. While PTC124 does not read through normal stops, it failed to rescue p53 as shown in Fig. 4A.

[0341] Thus, only the Arg / Op tRNA is capable of rescuing p53 in a specific manner with minimal read through of normal stop codons.

[0342] Example 2

[0343] The following expression vectors were prepared:Vector DescriptionVecl pAAV[Exp]-5xsup-tRNA(Arg)Vec2 pAAV[Exp]-5x(U6>sup-tRNA(Arg)Vec3 pAAV[Exp]-3x(U6>sup-tRNA(Arg)Vec4 pAAV[Exp]-3x(U6>sup-tRNA(Arg)(noflankingseq)Vec5 pAAV[Exp]-5x(U6>sup-tRNA(Arg)(noflankingseq)

[0344] With respect to the vectors:

[0345] Vecl contained 5 copies of the suppressor tRNA (e.g., tRNAArg / Op) (SEQ ID NO: 6);

[0346] Vec2 contains 5 total copies of a repeating unit containing a U6 promoter, the tRNAArg / Opsuppressor, and a termination sequence (SEQ ID NO: 9), each copy is separated by a spacer (SEQ ID NOs: 1-4, 5' to 3', respectively) and the final copy is followed by a spacer (SEQ ID NO: 5);

[0347] Vec3 contains 3 total copies of a repeating unit containing a U6 promoter, the tRNAArg / Opsuppressor, and a termination sequence (SEQ ID NO: 9), each copy is separated by a spacer (SEQ ID NOs: 1 and 3, 5' to 3', respectively) and the final copy is followed by a spacer (SEQ ID NO: 5);

[0348] Vec4 is similar to Vec3, but omits the flanker region around the tRNAArg / Opsuppressor;

[0349] Vec5 is similar to Vec2, but omits the flanker region around the tRNAArg / Opsuppressor.

[0350] Rescue of p53 levels in calu6, a cell line with an opal nonsense mutation in the gene (homozygous TP53 R196X), was evaluated using the constructs of the present invention incorporating a tRNAArg / Opsuppressor gene. For this study, calu-6 cells were transfected with 1 mg / ml of vector and the samples were harvested 48 hours post-transfection. Western blot analysis was performed using anti-p53 antibodies and anti-GAPDH antibodies (control).

[0351] As shown in Fig. 5, Vec2 and Vec3 significantly induced the expression (i.e., rescue) of p53 protein.

[0352] Example 3

[0353] Encapsulation and cellular activity of Vec2 was studied by using a plasmid construct containing enhanced green fluorescent protein (EGFP). Figs. 6A and 6B show that Vec2 can be encapsulated in AAV9 and are active in calu-6 cells 72 hours post-transfection. Further, Fig. 6B shows that the fluorescence intensity increases with the multiplicity of infection (MOI) confirming that the fluorescence imaging is not an artefact and is above background cellular fluorescence.

[0354] Western blot analysis using anti-p53 antibodies and anti-GAPDH antibodies (control) for calu-6 cells 72 hours post-transfection was performed. Column 4 in Fig. 7 corresponds to Vec2 at MOI=106and Column 6 in Fig. 7 corresponds to Vec3 at MOI=106. This example is consistent with the results in Example 2 and shows that Vec3 is more effective at rescuing p53 expression than Vec2. Further, column 11 of Fig. 7 corresponds to 3000 pg / ml Geneticin (or G418; an aminoglycoside drug). As shown in the western blot in Fig. 4A, G418 also rescued p53, but G418 is not specific (see Example 1) and is less effective at rescuing p53 expression than Vec3.

[0355] Example 4

[0356] A preliminary safety and pharmacokinetic (PK) study was conducted in mice to evaluate Vec3 described in Example 2. The study was a single-dose study using tail-vein injection at a dose of 1.8* 1012GC / mouse with a stock concentration of 2.74* 1013GC / ml. Two test groups were evaluated: a vehicle control group receiving PBS and a Vec3 group receiving the Vec3 construct in an AAV9 vector. The mouse strain used was C57BL / 6 with 4 mice per group (2 female / 2 male). The study duration was 14 days with body weight measurements and clinical observations performed every other day. Clinical chemistry was evaluated pre- and post-treatment. Tissues collected at the end of the study for RNAextraction included liver, lung, pancreas, kidney, brain, and heart, which were stored in RNAlater.

[0357] As shown in Fig. 8, no significant changes in body weight were observed between the Control group and the Vec3 group over the 14-day study period. Both groups displayed similar weight trajectories, with weights starting at approximately 22 grams on day 0 and gradually increasing to approximately 23 grams by day 14.

[0358] As shown in Fig. 9, no significant changes in hematological parameters were observed. White blood cell counts, neutrophils, monocytes, eosinophils, lymphocytes, and basophils remained within comparable ranges between the PBS vehicle control and Vec3 treatment groups from the start to the end of the study period.

[0359] Biodistribution analysis was performed to evaluate the presence of Vec3 in various tissues. As shown in Fig. 10, Vec3 was detected in several tissues including brain, heart, lung, pancreas, liver, and kidney at 14 days post-dosing. The liver showed the highest mean relative expression levels among the tissues examined, followed by heart, lung, pancreas, kidney, and brain. The Vec3 condition demonstrated substantially higher mean relative expression compared to the Vehicle control condition across all six tissues examined.

[0360] The data from this preliminary safety and PK study indicate that Vec3 was well-tolerated in mice with no significant changes in body weight or hematological parameters, and the construct was detected in multiple tissues following systemic administration via tailvein injection.

[0361] The data collected across Examples 1-4 demonstrate that the suppressor tRNA constructs of the present invention, particularly those containing multiple copies of the tRNAArg / Opsuppressor with U6 promoters and novel spacer sequences (Vec2 and Vec3), effectively rescue p53 protein expression in cells harboring opal nonsense mutations. The Arg / Op suppressor tRNA achieves this rescue with high specificity and minimal readthrough at normal termination codons, unlike aminoglycoside drugs such as G418 which exhibit nonspecific readthrough activity. Furthermore, the expression constructs can be successfully encapsulated in AAV9 vectors and maintain cellular activity, demonstrating their utility for therapeutic applications. The preliminary safety and pharmacokinetic study in Example 4 further supports the therapeutic potential of the constructs, as Vec3 was well-tolerated in mice with no significant changes in body weight or hematological parameters, and theconstruct was detected in multiple tissues including brain, heart, lung, pancreas, liver, and kidney following systemic administration.

[0362] Numerous modifications and variations on the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

Claims

CLAIMS1. An expression insert for expressing a suppressor tRNA, comprising:two or more copies of a nucleic acid encoding a suppressor tRNA;a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; anda promoter operably linked to each nucleic acid encoding the suppressor tRNA.

2. The expression insert of claim 1, wherein the spacer is located between a termination sequence of a first copy of the nucleic acid encoding the suppressor tRNA positioned 5' to the spacer and the promoter positioned 3' to the spacer, wherein the promoter positioned 3' to the spacer is operably linked to a second copy of the nucleic acid encoding the suppressor tRNA.

3. The expression insert of claim 2, wherein the termination sequence comprises a hexa-thymine sequence.

4. The expression insert of claim 1, wherein the expression insert comprises from 2 to 10 copies of the nucleic acid encoding the suppressor tRNA.

5. The expression insert of claim 1, wherein each copy of the nucleic acid encoding the suppressor tRNA comprises an identical nucleotide sequence.

6. The expression insert of claim 1, wherein at least two copies of the nucleic acid encoding the suppressor tRNA comprise different nucleotide sequences.

7. The expression insert of claim 1, wherein at least one copy of the nucleic acid encoding the suppressor tRNA encodes a modified or engineered suppressor tRNA comprising an anticodon that hybridizes to a nonsense stop codon and is capable of being aminoacylated with an amino acid, such that the suppressor tRNA, when expressed in a cell and aminoacylated with the amino acid, hybridizes to the nonsense stop codon and permits the amino acid to be incorporated into a gene product at a position that would otherwise result in a truncated gene product caused by the nonsense stop codon.

8. The expression insert of claim 7, wherein the nonsense stop codon is a UGA stop codon and wherein the suppressor tRNA is aminoacylated or is capable of being aminoacylated with arginine.

9. The expression insert of claim 8, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

10. The expression insert of claim 1, wherein the promoter is selected from the group consisting of a retroviral LTR, a SV40 promoter, a human cytomegalovirus promoter, a U6 promoter, an adenovirus promoter, a TK promoter, and a B19 parvovirus promoter.

11. The expression insert of claim 10, wherein the promoter is a U6 promoter.

12. The expression insert of claim 1, wherein the expression insert comprises a repeating unit, wherein the repeating unit comprises the promoter, the nucleic acid encoding the suppressor tRNA, and a termination sequence.

13. The expression insert of claim 12, wherein the repeating unit comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9.

14. The expression insert of claim 12, wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and wherein the spacer is positioned after a final repeating unit of the expression insert.

15. A method of treating a stop-codon-associated genetic disease in a subject in need thereof, comprising:administering to the subject a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA,wherein the expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA,wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, andwherein a promoter is operably linked to each nucleic acid encoding the suppressor tRNA.

16. The method of claim 15, wherein the stop-codon-associated genetic disease is selected from the group consisting of P-thalassemia, Choroideremia (CHM), Cystic Fibrosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Hurler Syndrome, KIF1A, Liddle’s Syndrome, a Lysosomal Storage Disease, Marfan Syndrome, Smith-Lemli-Opitz Syndrome, Spinal Muscular Atrophy, an epilepsy disorder, an epileptic encephalopathy, Lennox-Gastaut Syndrome, Kleefstra Syndrome, KCNQ2 Encephalopathy, SYNGAP1 Encephalopathy, Parkinson's with GBA, CDKL5, SLC6A1, BRMUTD, Sotos Syndrome, or GLUT1 Deficiency Syndrome, 5q-syndrome, Adams-Oliver syndrome 1, Alagille syndrome 1, Autoimmune lymphoproliferative syndrome type 1 A, Carney complex type I, CHARGE syndrome, Coffin-Siris Syndrome, Duane Syndrome, Ehlers-Danlos Syndrome, Feingold Syndrome 1, Denys-Drash syndrome / Frasier Syndrome, DiGeorge Syndrome (TBX1-associated), or Cleidocranial dysplasia.

17. The method of claim 15, wherein the stop-codon-associated genetic disease is cancer.

18. The method of claim 17, wherein the cancer is selected from the group consisting of p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian carcinoma, breast cancer, and colorectal cancer.

19. The method of claim 15, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

20. The method of claim 19, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

21. The method of claim 15, wherein the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle.

22. A method of restoring translation to a nucleotide sequence comprising a nonsense mutation in a cell, comprising:introducing into the cell a composition comprising a suppressor tRNA or an expression vector encoding the suppressor tRNA,wherein the expression vector comprises an expression insert comprising two or more copies of a nucleic acid encoding the suppressor tRNA and a spacer positioned between respective copies of the nucleic acid encoding the suppressor tRNA,wherein the spacer comprises a nucleotide sequence having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, andwherein a promoter is operably linked to each nucleic acid encoding the suppressor tRNA;wherein the suppressor tRNA hybridizes to a premature stop codon in the nucleotide sequence and permits incorporation of an amino acid at a position corresponding to the premature stop codon.

23. The method of claim 22, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

24. The method of claim 23, wherein the suppressor tRNA comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6.

25. The method of claim 22, wherein the expression vector is an adeno-associated viral vector.

26. An expression vector comprising the expression insert of claim 1.

27. The expression vector of claim 26, wherein the expression vector is a viral vector.

28. The expression vector of claim 27, wherein the viral vector is an adeno-associated viral vector.

29. The expression vector of claim 28, wherein the adeno-associated viral vector is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAV10.

30. The expression vector of claim 26, wherein the expression vector further comprises a reporter gene.

31. A cell comprising the expression vector of claim 26.

32. The cell of claim 31, wherein the cell is a mammalian cell.

33. The cell of claim 32, wherein the mammalian cell is a human cell.

34. The cell of claim 31, wherein the cell is selected from the group consisting of a fibroblast, an epithelial cell, a neuronal cell, a muscle cell, and a stem cell.

35. The cell of claim 31, wherein the cell comprises a gene having a nonsense mutation, and wherein the suppressor tRNA encoded by the expression insert hybridizes to a premature stop codon resulting from the nonsense mutation.

36. A composition comprising a suppressor tRNA encoded by the expression insert of claim 1, and a pharmaceutically acceptable carrier.

37. The composition of claim 36, wherein the pharmaceutically acceptable carrier is selected from the group consisting of a polymeric nanoparticle, a liposome, and a micelle.

38. The composition of claim 36, wherein the suppressor tRNA comprises an anticodon that hybridizes to a UGA stop codon and is aminoacylated or is capable of being aminoacylated with arginine.

39. The composition of claim 36, wherein the composition is formulated for intravenous, intramuscular, subcutaneous, or intrathecal administration.

40. The composition of claim 36, wherein the composition further comprises a buffer, a stabilizer, or a combination thereof.

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