WO2025036281A1

WO2025036281A1 - Engineered suppressor trna molecules and methods

Info

Publication number: WO2025036281A1
Application number: PCT/CN2024/110998
Authority: WO
Inventors: Jianguo ZHU; Lingjie Kong; Rong Huang; Yu Ding; Jianfeng DONG; Kangyun DONG; Anrui LU
Original assignee: Shanghai Codone Biotechnology Co
Current assignee: Shanghai Codone Biotechnology Co
Priority date: 2023-08-11
Filing date: 2024-08-09
Publication date: 2025-02-20
Anticipated expiration: 2026-02-11
Also published as: TW202521693A

Abstract

Provided are engineered transfer RNA (tRNA) molecules that restore translation of a coding nucleic acid containing a premature UGA/TGA stop codon by inserting an arginine (Arg) amino acid into the polypeptide in decoding the UGA/TGA codon. Also provided are expression cassettes and vectors comprising nucleic acids encoding an engineered tRNA, pharmaceutical compositions comprising the expression cassettes, vectors, and/or the engineered tRNA, and methods of restoring translation and/or disease treatment involving engineered tRNA molecules.

Description

ENGINEERED SUPPRESSOR TRNA MOLECULES AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT International Application No. PCT/CN2023/112612, filed on August 11, 2023, and PCT International Application No. PCT/CN2024/100922, filed on June 24, 2024, which are hereby incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (302442000141SEQLIST. xml; Size: 204, 154 bytes; and Date of Creation: August 8, 2024) is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates in some aspects to engineered transfer RNA (tRNA) molecules that restore translation of a coding nucleic acid containing a premature UGA/TGA stop codon by inserting an arginine (Arg) amino acid into the polypeptide in decoding the UGA/TGA codon. The present disclosure further relates to expression cassettes and vectors comprising nucleic acids encoding an engineered tRNA, pharmaceutical compositions comprising the expression cassettes, vectors, and/or the engineered tRNA, and methods of restoring translation and/or disease treatment involving engineered tRNA molecules.

BACKGROUND

Rare diseases, also known as orphan diseases, are defined strictly according to their prevalence. In the United States, a disease is considered to be “rare” when it affects less than one person out of 1500; in Europe, less than one person out of 2000. To date, 6172 unique rare diseases have been described. 71.9%of these originated from genetic mutations, with the remainder being induced by viral or bacterial infections, allergies, or environmental effects (Nguengang Wakap, S., et al. (2020) Estimating cumulative point prevalence of rare diseases: analysis of the Orphanet database. Eur J Hum Genet. Feb; 28 (2) : 165-173. doi: 10.1038/s41431-019-0508-0. Epub 2019 Sep 16. PMID: 31527858; PMCID: PMC6974615) . About 263-446 million people are estimated to suffer from rare diseases globally at any point in time (Nguengang Wakap, S., et al. (2020) Estimating cumulative point prevalence of rare diseases: analysis of the Orphanet database. Eur J Hum Genet. Feb; 28 (2) : 165-173. doi: 10.1038/s41431-019-0508-0. Epub 2019 Sep 16. PMID: 31527858; PMCID: PMC6974615) . Unfortunately, 75%of rare disease patients are children, 30%of whom have life expectancies of less than 5 years (Benhabiles, H., et al. (2016) . Pathologies Susceptible to be Targeted for Nonsense Mutation Therapies. 10.1016/B978-0-12-804468-1.00002-6) .Due to the low prevalence of each rare disease, the market has not been able to support the resources needed to study pathogenesis of these diseases or discover new drugs or treatments.

Genetically-based rare diseases can be caused by insertion or deletion of one or more nucleotides (such as a frameshift mutation) and/or point mutations (such as nonsense or missense mutations) in a gene, leading to absent or nonfunctional gene products (Benhabiles, H., et al. (2016) . Pathologies Susceptible to be Targeted for Nonsense Mutation Therapies. 10.1016/B978-0-12-804468-1.00002-6) . Nonsense mutations are well-studied point mutations that result in a premature termination codon (PTC) in the messenger RNA (mRNA) , which leads to decreased mRNA stability and truncated protein products (Khajavi, M., et al. (2006) Nonsense-mediated mRNA decay modulates clinical outcome of genetic disease. Eur J Hum Genet. Oct; 14 (10) : 1074-81. doi: 10.1038/sj. ejhg. 5201649. Epub 2006 Jun 7. PMID: 16757948) . So far, 410, 743 gene mutations have been found responsible for various human inherited diseases, of which about 11% (45, 689) are nonsense mutations (data from the Human Gene Mutation Database https: //www. hgmd. cf. ac. uk/ac/index. php, accessed July 4, 2023) . These nonsense mutations are present in the mRNAs of various genes, and occur at different frequencies depending on the stop codon, including 41.8% (19, 101) TAG, 36.5% (16, 680) TGA, and 21.7% (9, 908) TAA PTCs. Remarkably, the codon decoding Arginine (Arg) to TGA nonsense mutation counts for the most frequent transitions (9302) (data from the Human Gene Mutation Database https: //www. hgmd. cf. ac. uk/ac/index. php, accessed July 4, 2023) .

Human inherited diseases caused by nonsense mutations are mainly associated with truncated protein products and rapid degradation of PTC-containing mRNA. Degradation and elimination of PTC-containing mRNAs is triggered by the evolutionarily conserved RNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD) (Chang, Y.F., et al. (2007) The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem. 76: 51-74. doi: 10.1146/annurev. biochem. 76.050106.093909. PMID: 17352659) . The NMD pathway relies on the ribosome for scanning of stop codons, as well as the presence of exon-junction complexes (EJCs) , which are deposited approximately 20-24 nt upstream of exon-exon junctions. When a ribosome pauses at a stop codon and an EJC is present more than 50 nt downstream of it, the SURF complex is recruited to the mRNA. The SURF complex consists of a protein kinase (SMG1) , an RNA helicase (UPF1) , as well as the peptide release factors eRF1 and eRF3. Phosphorylation of UPF1 further leads to the recruitment of SMG5, SMG6 and SMG7, which provide a link to the mRNA decay pathway mediated by exonucleases and endonucleases (Nickless, A, et al. (2017) Control of gene expression through the nonsense-mediated RNA decay pathway. Cell Biosci. May 19; 7: 26. doi: 10.1186/s13578-017-0153-7. PMID: 28533900; PMCID: PMC5437625) .

In the past two decades, clinical studies of PTC therapeutics have focused on enhancing PTC readthrough by incorporation of an amino acid to restore the production and function of faulty protein (Spelier, S., et al. (2023) Readthrough compounds for nonsense mutations: bridging the translational gap. Trends Mol Med. Apr; 29 (4) : 297-314. doi: 10.1016/j. molmed. 2023.01.004. Epub 2023 Feb 22. PMID: 36828712) . The dominant strategy in the field has been to apply small molecules to promote PTC readthrough based on their role in inhibiting ribosome proofreading (e.g., aminoglycosides, Ataluren) , the NMD pathway (e.g., amlexanox) , and inducing mRNA-tRNA mispairing (e.g., pseudouridylation) (Spelier, S., et al. (2023) Readthrough compounds for nonsense mutations: bridging the translational gap. Trends Mol Med. Apr; 29 (4) : 297-314. doi: 10.1016/j. molmed. 2023.01.004. Epub 2023 Feb 22. PMID: 36828712) . Although many small molecules have had positive outcomes in clinical trials to treat diseases caused by PTCs, the uncertainty of which amino acid is incorporated increases the risk of yielding an inappropriate protein product and/or producing off-target effects. Another approach is to implement a gene therapy strategy to ensure the production of the correct protein, for instance, CRISPR-based gene editing of a faulty gene or delivery of transgene (Porter, J.J., et al. (2021) Therapeutic promise of engineered nonsense suppressor tRNAs. Wiley Interdiscip Rev RNA. Jul; 12 (4) : e1641. doi: 10.1002/wrna. 1641. Epub 2021 Feb 10. PMID: 33567469; PMCID: PMC8244042) . However, the application of this approach is limited by the single target specificity and the risk of off-target effects.

Following the development of in vivo delivery systems and genome-wide, transcriptome-wide, and proteome-wide analysis technologies, suppressor tRNAs have drawn attention for their potential to treat diseases caused by PTCs (Wang, J., et al. (2022) AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice. Nature. Apr; 604 (7905) : 343-348. doi: 10.1038/s41586-022-04533-3. Epub 2022 Mar 23. PMID: 35322228; PMCID: PMC9446716) . Suppressor tRNAs are derived from naturally occurring tRNAs, with alterations of the anticodon to target a PTC (e.g., UAA, UAG, UGA) , and they carry a desired amino acid to restore full-length protein products for mRNAs containing PTCs. In comparison with other PTC rescue strategies, suppressor tRNAs provide the potential to target various PTC induced genetic diseases with a single component and minor side effects (Wang, J., et al. (2022) AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice. Nature. Apr; 604 (7905) : 343-348. doi: 10.1038/s41586-022-04533-3. Epub 2022 Mar 23. PMID: 35322228; PMCID: PMC9446716) .

All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference.

SUMMARY

One aspect of the present application provides an engineered transfer RNA (tRNA) for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16; and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA contains no more than about 5 nucleotide substitutions relative to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA comprises a U to C substitution at the 4^th nucleotide 3’ to the anticodon. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA has nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8 and 61-64. In some embodiments of this aspect that may be combined with any of the preceding embodiments, the engineered tRNA exhibits an increased stability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

In some embodiments of this aspect that may be combined with any of the preceding embodiments, the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence.

Another aspect of the present application provides a nucleic acid encoding the engineered tRNA of any one of the preceding embodiments. Another aspect of the present application provides a vector comprising the nucleic acid of the preceding aspect. In some embodiments of this aspect, the vector is a viral vector or a plasmid. In some embodiments of this aspect, the vector is a viral vector, and wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector. In some embodiments of this aspect, the vector is an AAV 2/2 vector, an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector.

Another aspect of the present application provides a pharmaceutical composition comprising the engineered tRNA of any one of the preceding embodiments, the nucleic acid of any one of the preceding embodiments, or the vector of any one of the preceding embodiments, further comprising a pharmaceutically acceptable carrier. In some embodiments of this aspect, the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle.

Another aspect of the present application provides a method of restoring translation of a coding nucleic acid of interest containing a premature UGA/TGA stop codon in a cell, comprising introducing to the cell the engineered tRNA of any one of the preceding embodiments, the nucleic acid of any one of the preceding embodiments, or the vector of any one of the preceding embodiments, wherein the engineered tRNA introduced into the cell or produced from the nucleic acid or vector recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid of interest containing the premature UGA/TGA stop codon. In some embodiments of this aspect, the engineered tRNA restores at least 5%of the translation of the coding nucleic acid of interest containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon.

Another aspect of the present application provides a method of treating a disease associated with a premature UGA/TGA stop codon in an individual, comprising: administering to the individual an effective amount of the pharmaceutical composition of any one of the preceding embodiments. In some embodiments of this aspect, the premature-stop-codon-associated disease is cystic fibrosis, muscular dystrophy, Alport syndrome, Stargardt disease, dilated cardiomyopathy, β-thalassemia or Liddle's syndrome. In some embodiments of this aspect, the individual is human.

Another aspect of the present application provides an engineered tRNA of any one of the preceding embodiments, wherein the engineered tRNA exhibits increased PTC readthrough efficiency in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

FIGS. 1A-1C show NanoLuc luciferase assays used to screen engineered tRNAs for PTC suppression efficiency. FIG. 1A shows a schematic of two versions of a luciferase construct for a nonsense mutation suppression screen involving rescuing full-length luciferase and its activity. The top and bottom panels ( “Luc_V2_R3” and “Luc_V3_R3” ) depict different versions of the construct. The rectangles labeled “NanoLuc” depicts a modified Luciferase protein coding genes (Promega, Madison, Wisconsin, USA) ) containing a UGA/TGA stop codon (labeled PTC) after base pair positions 195 (Luc_V2_R3) or 474 (Luc_V3_R3) . Modified NanoLuc protein coding-genes containing the GGA (glycine) codon after base pair positions 195 or 474 were used as corresponding controls for Luc_V2_R3 and Luc_V3_R3, respectively. The rectangles with hatched shading on the left and right ends of the luciferase protein depict a GS (Glycine and Serine) linker, and the rectangles shaded with circles or plus signs represent 3x Myc and 3x FLAG tags on the N-and C-termini, respectively, of the luciferase, which can be used for protein pull-down assays and further mass spectrometric analyses. The vertical line within each luciferase depicts the position at which translation would be terminated due to a nonsense mutation in the mRNA (not pictured) if the tRNA being screened did not effectively suppress the nonsense mutation. FIGS. 1B-1C show results from two versions (Luc_V2_R3, shown in FIG. 1B, and Luc_V3_R3, shown in FIG. 1C) of a NanoLuc luciferase assay as described in FIG. 1A on engineered suppressor tRNAs R3-1, R3-2, R3-3, R3-4, R3-5, R3-6, R3-7, R3-8, R3-9, R3-10, R3-11, R3-12, R3-13, R3-14, R3-15, R3-147, R3-148, R3-149, R3-150 and R3-151 and positive controls V2_R4, and V3_R4, respectively, as described in Example 1. The engineered tRNA tested is indicated on the x-axis. The measured luciferase activity in relative luminescence unit (RLU) is indicated on the y-axis. Error bars indicate the standard deviation of biological triplicates.

FIGS. 1D-1G illustrate the PTC suppression efficiency of engineered tRNAs at both the protein and mRNA levels in cell lines with PTCs in the p53 gene. FIGS. 1D-1E show Western Blots of rescued full-length p53 expression (top row) compared to truncated p53 expression (middle row) and GAPDH expression (bottom row) in DMS1114 cells (FIG. 1D) or Calu6 cells (FIG. 1E) transfected with R3-7, R3-8, R3-13, R3-147, R3-148, R3-149, Scr1-4 (ascrambled sequence derived from R3-147, used as the negative control for transfection) , or treated with 500 μg/mL or 5 mg/mL of G418 (labeled as G418-0.5 and G418-5, respectively; G418 is an aminoglycoside with known PTC readthrough ability) , compared to the positive control cell line A549. FIGS. 1F-1G show RT-qPCR results of the level of the premature UGA stop codon-containing p53 mRNA in DMS1114 cells (FIG. 1F) or Calu6 cells (FIG. 1G) transfected with plasmids expressing R3-7, R3-8, R3-13, R3-147, R3-148, R3-149, Scr1-4, or blanks, compared to the positive control cell line A549. The sample source is shown on the x-axes. The relative abundance of premature UGA stop codon-containing p53 mRNA normalized to the non-PTC containing p53 mRNA in the A549 control is shown on the y-axes. Error bars indicate the standard deviation of biological triplicates.

FIGS. 1I-1O present results from next-generation tRNA sequencing, detailing the expression levels of engineered tRNAs (FIGS. 1I-1J) , their corresponding endogenous tRNAs (FIG. 1K) , and the homeostasis of the endogenous tRNAome (FIGS. 1L-1O) . FIGS. 1I-1K feature line plots that show the normalized total reads detected for selected engineered and endogenous tRNAs across different samples. The horizontal axes represent the Sprinzl positions from the 5’ to 3’ ends of the reference tRNAs, while the vertical axes display normalized total read counts. Each line in these plots (FIGS. 1I-1K) corresponds to a sample: Luc_V2_R3 cell line transfected with plasmids expressing engineered tRNAs (R3-8, black dashed line; R3-147, black solid line; R3-147-M54, black dotted line) , with Scr1-4 (grey solid line) used as a negative control, and cells treated with H₂O (grey dashed line) used as a blank control. The reference sequence to which the reads are aligned is indicated at the top of each plot, including R3-8 (FIG. 1I, left panel, from naturally occurring tRNA Arg-CCT-4-1) , R3-147 (FIG. 1I, right panel, from naturally occurring tRNA Arg-TCT-1-1) , R3-147-M54 (FIG. 1J, also referred to as M54) , and naturally occurring tRNAs Arg-CCT-4-1 (FIG. 1K, left panel) and Arg-TCT-1-1 (FIG. 1K, right panel) . FIGS. 1L-1O compare endogenous tRNA expression levels between Luc_V2_R3 cell lines transfected with Scr1-4 (plotted on all y-axes) and various engineered tRNAs (plotted on all x-axes) , including R3-8 (FIG. 1L) , R3-147 (FIG. 1M) , R3-147-M54 (FIG. 1N) , and the blank control (FIG. 1O) . Total normalized read counts are shown on each axis in log10 format. The Pearson correlation coefficient for each comparison is displayed in the top left of each panel. Each figure (FIGS. 1L-1O) is divided into three panels: the left panel shows the analysis of all cytoplasmic tRNAs at the single tRNA level, the middle panel presents the isodecoder level analysis, and the right panel focuses on the isoacceptor level analysis.

FIG. 1P displays Western blot results for the modified NanoLuc protein rescued by various engineered tRNAs. "WCE" denotes the whole cell extract, which serves as a loading control to confirm the presence and enrichment of the targeted NanoLuc protein. "FT" refers to the flow-through sample collected during immunoprecipitation, used to assess the success of the immunoprecipitation, while "IP" indicates the immunoprecipitated sample. The engineered tRNAs R3-8, R3-147 and R3-147-M54 (referred to as M54) were transfected with plasmids and expressed in the LUC_V2_R3 cell line. The upper band represents the rescued NanoLuc protein, as shown in FIG. 1A, while the lower band represents the NanoLuc protein without the 3x Myc tag and the front GS linker (resulting from translation starting at the NanoLuc gene's start codon instead of the Myc tag's start codon) . Scr1-4 is used as a negative control. The x-axis shows the sample identity indicated along the top of the figure.

FIGS. 1Q-1S present the results of NanoLuc luciferase assays (left panel) and transduction efficiency (right panel, measured by the proportion of cells exhibiting GFP expression) for the Luc-V2-R3 cell line, which was transduced with lentivirus (denoted as "LV" in the bar plot) carrying engineered tRNA genes, including R3-5 (FIG. 1Q) , R3-8 (FIG. 1R) , and R3-147 (FIG. 1S) . Two different MOIs (multiplicities of infection) of the lentivirus were used, as indicated on the x-axis. The y-axes of the bar plot in the left panel show relative luminescence units, while the y-axes of the bar plot in the right panel display the percentage of GFP-positive (GFP+) cells. Error bars represent the standard deviation from biological triplicates. A blank control was also performed with no lentivirus transduction.

FIGS. 1AA-1BB illustrate the construct schematic of recombinant Adeno-Associated Virus (rAAV) and the results of NanoLuc luciferase assays in the Luc-V2-R3 cell line transduced with rAAV carrying engineered suppressor tRNA. FIG. 1AA presents the schematic of an scAAV-1xU6 vector, which includes, from left to right (5’ to 3’ direction) : a mutated inverted terminal repeat (AAV2 ΔITR) , a U6 cassette (comprising the U6 promoter, an engineered tRNA, and a poly-T termination signal) , the human cytomegalovirus (CMV) enhancer, the chicken β-actin promoter, the reporter gene mCherry, and a normal AAV2 ITR. FIG. 1BB shows the results of NanoLuc luciferase assays for cells transduced with rAAV containing engineered suppressor tRNA genes in the scAAV-1xU6 vector, as depicted in FIG. 1AA. Various gradient MOIs (multiplicity of infection) of the rAAVs were transduced into the Luc-V2-R3 cell line, as indicated on the x-axes, increasing from right to left. The y-axes of the bar plot display the relative luminescence units. The strand type and serotype of the AAV are indicated at the bottom of the bar plot. The specific engineered suppressor tRNA genes used in the assay are indicated at the bottom of the bar plot, including R3-5 (left panel) , R3-8 (middle panel) , and R3-147 (right panel) . Error bars represent the standard deviation of biological triplicates.

FIG. 1H shows the results of NanoLuc luciferase assay as described in FIG. 1A, comparing the engineered tRNAs M2, M3, M4, M5, M6, M7, and M8 with R3-147 (left-most bar) , water (right-most bar) , Scr1-4, and G418 (3rd bar from the right) in the Luc-V2-R3 cell line. The x-axis indicates the sample identity, and the y-axis indicates the measured luciferase activity in relative luminescence unit (RLU) . Error bars indicate the standard deviation from biological triplicates.

FIGS. 1T-1Z present results from next-generation tRNA sequencing, detailing the expression levels of engineered tRNAs (FIGS. 1T-1U) , their corresponding endogenous tRNA (FIG. 1V) , scrambled sequence (FIG. 1U, right panel) , and the homeostasis of the endogenous tRNAome (FIGS. 1W-1Z) . FIGS. 1T-1V illustrate line plots showing the normalized total reads detected for selected engineered and endogenous tRNAs across different samples. The horizontal axes represent the Sprinzl positions from the 5’ to 3’ ends of the reference tRNAs, while the vertical axes display normalized total read counts. Each line in these plots (FIGS. 1T-1V) corresponds to a sample: Luc_V2_R3 cell line transfected with plasmids expressing engineered tRNAs (R3-147, grey solid line; M2, black solid line; M3, black dashed line; M4, black dotted line) , with Scr1-4 (grey dotted line) used as a negative control. The reference sequence to which the reads are aligned is indicated at the top of each plot, including R3-147 (FIG. 1T, left panel) , M2 (FIG. 1T, right panel) , M4 (FIG. 1U, left panel) , scrambled sequence Scr1-4 (FIG. 1U, right panel) , and naturally occurring tRNAs Arg-TCT-1-1 (FIG. 1V) . The horizontal solid line in FIG. 1U (right panel) represents the lower limit of detection (LLOD) . FIGS. 1W-1Z compare endogenous tRNA expression levels between Luc_V2_R3 cell lines transfected with Scr1-4 (plotted on all y-axes) and various engineered tRNAs (plotted on all x-axes) , including R3-147 (FIG. 1W) , M2 (FIG. 1X, also referred to as R3-147-M2) , M3 (FIG. 1Y, also referred to as R3-147-M3) , and M4 (FIG. 1Z, also referred to as R3-147-M4) . Total normalized read counts are shown on each axis in log10 format. The Pearson correlation coefficient for each comparison is displayed in the top left of each panel. Each figure (FIGS. 1W-1Z) is divided into three panels: the left panel shows the analysis of all cytoplasmic tRNAs at the single tRNA level, the middle panel presents the isodecoder level analysis, and the right panel focuses on the isoacceptor level analysis.

FIGS. 2A-2DD show schematics of the secondary structures of human native tRNA Arg-TCT-1-1 (HGNC: 34695, NCBI Gene: 100189133, corresponding to the sequence set forth in SEQ ID NO: 43) (FIG. 2A) and its derived engineered tRNAs (FIGS. 2B-2DD) . Each panel depicts the secondary structure of the respective tRNA, with A, G, C, and U representing the relative positions of the bases adenine (A) , guanine (G) , cytosine (C) , and uracil (U) , dots representing pairing between bases, and numbers indicating the Sprinzl position of the sequence from 5’ to 3’ . In each panel, the T-arm is shown to the right, the D-arm is shown to the left, the anticodon arm is shown at the bottom (with the tri-nucleotide anticodon comprising the three bases at the bottom of each schematic and underlined) , and the acceptor arm is shown at the top. The arrow in FIG. 2B indicates the change in R3-147 compared to human tRNA Arg-TCT-1-1. The nucleotide “A” on the 3’side of the anticodon (at Sprinzl position 36) in FIGS. 2B-2DD indicates that the nucleotide at that position has been changed from U to A in the respective engineered tRNA compared to human tRNA Arg-TCT-1-1. Boxed nucleotides indicate additional differences in the respective engineered tRNA compared to R3-147. FIG. 2A shows human tRNA Arg-TCT-1-1 (corresponding to the sequence set forth in SEQ ID NO: 43) . FIG. 2B shows engineered tRNA R3-147 (corresponding to the sequence set forth in SEQ ID NO: 1) , which has the same sequence as human tRNA Arg-TCT-1-1 except that R3-147 has a UCA anticodon instead of the UCU anticodon present in human tRNA Arg-TCT-1-1. FIG. 2C shows engineered tRNA M2 (corresponding to the sequence set forth in SEQ ID NO: 2, also referred to as R3-147-M2) , which has the same sequence as R3-147 except that it contains a C at Sprinzl position 40, corresponding to a U to C substitution at the 4th nucleotide 3’ to the anticodon compared to R3-147. FIG. 2D shows engineered tRNA M3 (corresponding to the sequence set forth in SEQ ID NO: 3, also referred to as R3-147-M3) , which has the same sequence as R3-147 except that it contains a CCA sequence attached to the acceptor arm at the 3’ end. FIG. 2E shows engineered tRNA M4 (corresponding to the sequence set forth in SEQ ID NO: 4, also referred to as R3-147-M4) , which has the same sequence as R3-147 except that it contains a G at Sprinzl position 37, corresponding to an A to G substitution at the 1st nucleotide 3’ to the anticodon compared to R3-147. FIG. 2F shows engineered tRNA M5 (corresponding to the sequence set forth in SEQ ID NO: 5, also referred to as R3-147-M5) , which contains both the M2 and M3 substitutions. FIG. 2G shows engineered tRNA M6 (corresponding to the sequence set forth in SEQ ID NO: 6, also referred to as R3-147-M6) , which contains both the M2 and M4 substitutions. FIG. 2H shows engineered tRNA M7 (corresponding to the sequence set forth in SEQ ID NO: 7, also referred to as R3-147-M7) , which contains both the M3 and M4 substitutions. FIG. 2I shows engineered tRNA M8 (corresponding to the sequence set forth in SEQ ID NO: 8, also referred to as R3-147-M8) , which contains all three of the M1, M2, and M3 substitutions. FIG. 2J shows engineered tRNA M22 (corresponding to the sequence set forth in SEQ ID NO: 65, also referred to as R3-147-M22) , which contains the M2 substitutions. FIG. 2K shows engineered tRNA M23 (corresponding to the sequence set forth in SEQ ID NO: 66, also referred to as R3-147-M23) , which contains the M2 substitutions. FIG. 2L shows engineered tRNA M24 (corresponding to the sequence set forth in SEQ ID NO: 67, also referred to as R3-147-M24) , which contains the M2 substitutions. FIG. 2M shows engineered tRNA M25 (corresponding to the sequence set forth in SEQ ID NO: 68, also referred to as R3-147-M25) , which contains the M2 substitutions. FIG. 2N shows engineered tRNA M26 (corresponding to the sequence set forth in SEQ ID NO: 69, also referred to as R3-147-M26) , which contains the M2 substitutions. FIG. 2O shows engineered tRNA M46 (corresponding to the sequence set forth in SEQ ID NO: 70, also referred to as R3-147-M46) , which contains the M2 substitutions. FIG. 2P shows engineered tRNA M50 (corresponding to the sequence set forth in SEQ ID NO: 71, also referred to as R3-147-M50) , which contains the M2 substitutions. FIG. 2Q shows engineered tRNA M54 (corresponding to the sequence set forth in SEQ ID NO: 72, also referred to as R3-147-M54) , which contains the M2 substitutions. FIG. 2R shows engineered tRNA M55 (corresponding to the sequence set forth in SEQ ID NO: 73, also referred to as R3-147-M55) , which contains the M2 substitutions. FIG. 2S shows engineered tRNA M56 (corresponding to the sequence set forth in SEQ ID NO: 74, also referred to as R3-147-M56) , which contains the M4 substitutions. FIG. 2T shows engineered tRNA M57 (corresponding to the sequence set forth in SEQ ID NO: 75, also referred to as R3-147-M57) , which contains the M2 substitutions. FIG. 2U shows engineered tRNA M58 (corresponding to the sequence set forth in SEQ ID NO: 76, also referred to as R3-147-M58) , which contains the M2 substitutions. FIG. 2V shows engineered tRNA M59 (corresponding to the sequence set forth in SEQ ID NO: 77, also referred to as R3-147-M59) , which contains the M2 substitutions. FIG. 2W shows engineered tRNA M62 (corresponding to the sequence set forth in SEQ ID NO: 78, also referred to as R3-147-M62) , which contains the M2 substitutions. FIG. 2X shows engineered tRNA M63 (corresponding to the sequence set forth in SEQ ID NO: 79, also referred to as R3-147-M63) , which contains the M2 substitutions. FIG. 2Y shows engineered tRNA M65 (corresponding to the sequence set forth in SEQ ID NO: 80, also referred to as R3-147-M65) , which contains the M2 substitutions. FIG. 2Z shows engineered tRNA M67 (corresponding to the sequence set forth in SEQ ID NO: 81, also referred to as R3-147-M67) , which contains the M2 substitutions. FIG. 2AA shows engineered tRNA M100 (corresponding to the sequence set forth in SEQ ID NO: 61, also referred to as R3-147-M100) , which contains the M2 substitutions. FIG. 2BB shows engineered tRNA M102 (corresponding to the sequence set forth in SEQ ID NO: 62, also referred to as R3-147-M102) , which contains the M2 substitutions. FIG. 2CC shows engineered tRNA M103 (corresponding to the sequence set forth in SEQ ID NO: 63, also referred to as R3-147-M103) , which contains the M2 substitutions. FIG. 2DD shows engineered tRNA M108 (corresponding to the sequence set forth in SEQ ID NO: 64, also referred to as R3-147-M108) , which contains the M4 substitutions.

[Rectified under Rule 91, 04.09.2024]
FIGS. 3A and 3B show schematics of the secondary structures of engineered tRNAs. Each panel depicts the secondary structure of the respective tRNA, with A, G, C, and U representing the relative positions of the bases adenine (A) , guanine (G) , cytosine (C) , and uracil (U) , dots representing pairing between bases, and numbers indicating the Sprinzl position of the sequence from 5’ to 3’ . In each panel, the T-arm is shown to the right, the D-arm is shown to the left, the anticodon arm is shown at the bottom (with the tri-nucleotide anticodon comprising the three bases at the bottom of each schematic and underlined) , and the acceptor arm is shown at the top. FIG. 3A shows engineered tRNA R3-5 (corresponding to the sequence set forth in SEQ ID NO: 13) , which has the same sequence as human tRNA Arg-CCT-1-1 (corresponding to the sequence set forth in SEQ ID NO: 32) except that R3-5 has a UCA anticodon instead of the CCU anticodon present in human tRNA Arg-CCT-1-1. FIG. 3B shows engineered tRNA R3-8 (corresponding to the sequence set forth in SEQ ID NO: 16) , which has the same sequence as human tRNA Arg-CCT-4-1 (corresponding to the sequence set forth in SEQ ID NO: 35) except that R3-8 has a UCA anticodon instead of the CCU anticodon present in human tRNA Arg-CCT-4-1.

FIG. 4A presents the results of a NanoLuc luciferase assay, as described in FIG. 1A, comparing engineered tRNAs with the positive control R3-147 and the negative control Scr1-4. The vertical axis shows the fold change in relative luminescence unit for cell lines Luc-V2-R3 transfected with plasmids expressing the engineered suppressor tRNAs (shown on the horizontal axis) compared to a cell line transfected with plasmids expressing tRNA R3-147. Error bars indicate the standard deviation of biological triplicates. The horizontal dotted line represents a fold change of 1.

FIGS. 4B-4C illustrate the outcomes of PTC readthrough assessments on X-linked Alport syndrome-associated PTC mutants within the gene COL4A5 (Collagen alpha-5 (IV) ) treated with lentivirus (referred to as "LV" in the bar plot) containing various engineered tRNAs. The PTC mutations were introduced into the A549 cell line, resulting in a conversion from an arginine codon to a stop codon at positions 1563 ( “COL4A5 R1563*” ) (FIG. 4B, left panel) and 373 ( “COL4A5 R373*” ) (FIG. 4B, right panel) within the COL4A5 protein sequence (NCBI Reference Sequence: NP_000486.1) . In FIG. 4B, RT-qPCR results show the relative levels of COL4A5 mRNA in samples treated with engineered suppressor tRNAs R3-147 and R3-147-M54 transduced by lentivirus, as indicated on the x-axes. The mRNA levels of COL4A5 in each sample were normalized to the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plotted as fold changes relative to the WT sample (A549 cell) on the y-axes. “Blank” indicates a negative control where the PTC cells are untreated with the lentivirus. Error bars represent the standard deviation of biological triplicates. FIG. 4C presents the integrated density of the COL4A5 signal normalized to cell counts in immunofluorescence analysis, showing the restoration of full-length COL4A5 protein in the COL4A5 R373*PTC cell line treated with lentivirus containing engineered suppressor tRNA R3-147-M54, as indicated on the x-axes. "Blank" refers to a negative control where PTC cells are untreated with the lentivirus. Cells treated solely with the secondary antibody are included to confirm that the observed signal is due to nonspecific binding.

FIGS. 4D-4F illustrate the construct schematic of recombinant Adeno-Associated Virus (rAAV) and the results of NanoLuc luciferase assays in the Luc-V2-R3 cell line transduced with rAAV carrying engineered suppressor tRNA. FIG. 4D presents the schematic of an ssAAV-2xU6 vector, which contains, from left to right (5’ to 3’ direction) : an Adeno-associated virus inverted terminal repeat (AAV2 ITR) , two identical copies of a U6 cassette (including the U6 promoter, an engineered tRNA, and a poly-T termination signal) , the human cytomegalovirus (CMV) enhancer, the chicken β-actin promoter, the reporter gene mCherry, a poly A signal, and another AAV2 ITR. FIGS. 4E-4F show NanoLuc luciferase assay results (left panel) and transduction efficiency (right panel, via measurement of the proportion of cells showing mCherry expression) for cells transduced with rAAV containing engineered suppressor tRNA genes in the ssAAV-2xU6 vector, as shown in FIG. 4D. Various gradient MOIs (multiplicity of infection) of the rAAVs were transduced into the Luc-V2-R3 cell line, as indicated on the x-axes, increasing from right to left. The relative luminescence units are shown on the y-axes of the bar plot in the left panel, and the percentage of mCherry-positive cells is shown on the y-axes of the bar plot in the right panel. The strand type and serotype of the AAV are indicated at the bottom of the bar plot. The specific engineered suppressor tRNA genes used in the assay are indicated at the bottom of the bar plot, including R3-147 (FIG. 4E) and R3-147-M54 (FIG. 4F) . Error bars represent the standard deviation of biological triplicates.

FIGS. 4G-4H depict the relative levels of urinary proteins in male mice with X-linked Alport syndrome, injected with ssAAV2/9 containing the construct detailed in FIG. 4D. These mice possess an X-linked COL4A5 premature termination codon (PTC) mutation at position 471 (arginine to PTC) ( “COL4A5-R471*” ) within the COL4A5 protein sequence (NCBI Reference Sequence: NP_000486.1) . The ratio of urine albumin (U-ALB) to urine creatinine (U-CRE) , an indicator of disease progression, is plotted on the y-axis, while the age of the mice in weeks is plotted on the x-axis. In FIG. 4G, the mice are categorized by the genotype of the injected rAAV: mice injected with saline ( "Normal Saline" , n=9) are shown by a black solid line, those injected with rAAV (carrying engineered suppressor tRNA R3-147) at a dose of 5E11 v.g. per mouse ( "ssAAV2/9_R3-147 5E11 v.g. " , n=9) by a black dashed line, mice injected with rAAV (carrying engineered suppressor tRNA R3-147) at a dose of 1.5E12 v.g. per mouse ( "ssAAV2/9_R3-147 1.5E12 v.g. " , n=9) by a black dotted line, and control Col4a5 wild-type mice ( "Col4a5 WT/Y mouse" , n=3) by a grey solid line. The intravenous injection occurs at 4 weeks of age, as indicated by the arrow below the line plot. Error bars represent the standard deviation of replicates. In FIG. 4H, the mice are grouped by the genotype of the injected rAAV: mice injected with saline ( "Normal Saline" , n=6) are shown with a grey solid line, those injected with rAAV (carrying engineered suppressor tRNA R3-147-M54) at a dose of 1.5E11 v.g. per mouse ( "ssAAV2/9_R3-147-M54 1.5E11 v.g. " , n=6) by a black solid line, mice injected with rAAV (carrying engineered suppressor tRNA R3-147-M54) at a dose of 5E11 v.g. per mouse ( "ssAAV2/9_R3-147-M54 5E11 v.g. " , n=6) by a black dashed line, and mice injected with rAAV (carrying engineered suppressor tRNA R3-147-M54) at a dose of 1.5E12 v.g. per mouse ( "ssAAV2/9_R3-147-M54 1.5E12 v.g. " , n=6) by a black dotted line. The intravenous injection is performed at 4 weeks of age, as indicated by the arrow below the line plot. Error bars represent the standard deviation of replicates.

DETAILED DESCRIPTION

The present application provides novel engineered arginine-carrying tRNA for suppressing a premature UGA/TGA stop codon (also referred to herein as “engineered suppressor tRNAs” ) . These engineered suppressor tRNAs contain one or more modifications relative to a suppressor tRNA identified through extensive screening as being particularly efficient in suppressing UGA/TGA stop codons. The modifications include, for example, i) an A to G substitution at the 1st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4th nucleotide 3’ to the anticodon; and iii) a CCA sequence attached to the acceptor arm at the 3’ end introduced through a nucleic acid encoding the suppressor tRNA. The one or more modifications introduced to the suppressor tRNA result in a further increase in readthrough efficiency, making the modified tRNAs particularly suitable for suppressing premature stop codons in vivo. In a naturally occurring process, a 3’ CCA sequence is added to the acceptor arm of a tRNA post-transcriptionally. Surprisingly, a CCA introduced through the nucleic acid encoding the suppressor tRNA, as compared to those being added post-transcriptionally, allows for a higher readthrough efficiency. The findings that minor changes to the nucleotide sequence of the suppressor tRNA can significantly improve the properties of the suppressor tRNA is remarkable and unexpected.

Thus, the present application in one aspect provides engineered tRNAs for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16; and wherein the one or more modifications comprises: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In another aspect, there are provided nucleic acids encoding the engineered tRNA, as well as expression cassettes and vectors comprising such nucleic acids. Also provided are methods of using the engineered tRNAs, nucleic acids encoding the engineered tRNAs, expression cassettes, and vectors.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some embodiments, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

The term “transfer ribonucleic acid” , “transfer RNA” , or “tRNA” as used herein refers to a nucleic acid molecule that helps translate mRNA to protein. The terms “anti codon” , “anticodon” and “anti-codon” are synonymous used interchangeably herein. Each tRNA can be “charged with” an amino acid corresponding to the mRNA codon that is recognized by the tRNA’s anticodon. “Charging” can be mediated by aminoacyl tRNA synthetase.

The terms “premature stop codon” , “premature termination codon” , and “PTC” are used synonymously herein to refer to a stop codon that, when present, results in an unintended truncation of a polypeptide relative to a wild-type counterpart.

The term “engineered tRNA” as used herein can refer to transfer RNAs having at least one difference in the sequence of the engineered tRNA relative to a comparable wild type tRNA, such as, for example, relative to human tRNA Arg-TCT-1-1. The terms “suppressor tRNA” “tRNA suppressor” or “engineered tRNA suppressor” as used herein refer to an engineered tRNA capable of suppressing premature stop codon halt in an mRNA.

The term “effective amount” as used herein can refer to a quantity sufficient to achieve a desired effect.

The term “restoring” as used herein in relation to expression of a protein can refer to the ability to establish expression of the full-length version of the protein from an mRNA comprising a PTC, where previous protein expression was truncated due to the PTC.

The term “mutation” as used herein can refer to an alteration to a nucleic acid sequence and/or a polypeptide sequence relative to a reference sequence.

The terms “disease” , “condition” , “disease state” or “disease phenotype” as used herein refer to one or more characteristics of a mammalian cell that results from a stop codon within the coding region of a gene inside the cell (e.g., that results from a nonsense mutation) .

The term “transduction of cells” as used herein refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus rather than by transfection. An RNA virus (i.e., a retrovirus) for transferring a nucleic acid into a cell is referred to herein as a transducing chimeric retrovirus. Exogenous genetic material contained within a retrovirus is incorporated into the genome of the transduced cell. A cell that has been “transduced” with a chimeric DNA virus does not incorporate the exogenous genetic material into its genome but can express the exogenous genetic material extra-chromosomally within the cell. A “transduced gene” is a gene that has been introduced into the cell via retroviral or vector infection and provirus integration.

The term “expression cassette” as used herein refers to a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell.

The term “operably-linked” as used herein refers to the association of nucleic acid sequences on single nucleic acid fragment such that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is considered “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide when the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter) .

The use of ordinal terms such as “first” , “second” , “third” , etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a) , b) , etc., or i) , ii) , etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

As used in this specification and the appended claims, the singular forms “a, ” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a molecule” includes a plurality of such molecules, and the like.

The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se.

As used herein, the term “nucleic acid” generally refers to a polymer comprising one or more nucleic acid subunits or nucleotides. A nucleic acid may include one or more subunits selected from adenosine (A) , cytosine (C) , guanine (G) , thymine (T) and uracil (U) , or variants thereof. A nucleotide can include A, C, G, T or U, or variants thereof. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (e.g., A or G, or variant thereof) or a pyrimidine (e.g., C, T or U, or variant thereof) . A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil-counterparts thereof) to be resolved. In some examples, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) , or derivatives thereof. A nucleic acid may be single-stranded or double-stranded.

The terms “polynucleotide” , “nucleic acid molecule” , “nucleic acid sequence” or “nucleotide sequence” as used herein generally refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, with a given sequence of nucleotides, of which it may be desired to know the presence or amount. Thus, this term comprises, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA) , or modified or substituted sugar or phosphate groups. The nucleotide sequence can comprise RNA or DNA, or a sequence derived from RNA or DNA. Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA including genomic DNA and messenger RNA.

The term “sequencing, ” as used herein, generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as DNA or RNA, including variants or derivatives thereof (e.g., single stranded DNA) . Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Sanger sequencing, Pacific BiosciencesOxfordor Life Technologies (Ion) . Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR) , or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human) , as generated by the systems from a sample provided by the subject.

As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient, including but not limited to one or more therapeutic benefits and/or prophylactic benefits. A “therapeutic benefit” in this sense can refer to eradication or amelioration of symptoms or of an underlying disorder being treated. A “prophylactic effect” in this sense includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof, even though a diagnosis of this disease may not have been made.

II. TRANSFER RNA SEQUENCE AND STRUCTURE

Transfer RNAs (tRNAs) are generally RNA molecules of about 70-90 nucleotides in length. They fold into a clover leaf-like secondary structure that contains three hairpin loop “arms” and an acceptor arm comprising the 3’ and 5’ ends of the tRNA. From 5’ to 3’ , a tRNA includes: a D-stem with a D-loop at the end of the stem, an anticodon stem with an anticodon loop at the end of the stem (comprising a tri-nucleotide anti-codon sequence) , a variable region, and a T-stem with a T-loop at the end of the stem. The anticodon recognizes the corresponding codon on the mRNA. In a naturally occurring process, the 3’ CCA is usually not present in the DNA sequence encoding the tRNA in mammalian cells, but is rather added post-transcriptionally.

tRNAs serve as adaptors to link an mRNA and its protein products by both recognizing the codons on mRNA and carrying the amino acid to append to the peptide chain. Upon recognition of the codon corresponding to its anticodon, a charged tRNA can transfer its amino acid to the growing amino acid chain to form a polypeptide or protein. Endogenous tRNAs can be charged by endogenous aminoacyl tRNA synthetase.

tRNA biogenesis entails multiple steps, including transcription by a polymerase into a precursor tRNA (pre-tRNA) and subsequent intron splicing by a splicing endonuclease (for tRNAs containing introns; not every tRNA has an intron) , 5’ end and 3’ end cleavage by endo-and/or exonucleases, addition of the CCA tail by nucleotidyltransferase, and modifications on multiple nucleotide residues (Sekulovski S, Trowitzsch S. Transfer RNA processing -from a structural and disease perspective. Biol Chem. 2022 Jun 21;403 (8-9) : 749-763. doi: 10.1515/hsz-2021-0406. PMID: 35728022) . Each step requires a series of protein enzymes and energy (Sekulovski S, Trowitzsch S. Transfer RNA processing -from a structural and disease perspective. Biol Chem. 2022 Jun 21; 403 (8-9) : 749-763. doi: 10.1515/hsz-2021-0406. PMID: 35728022) .

The canonical Sprinzl position system assigns numbers to each nucleotide based on the tRNA cloverleaf secondary structure. Nucleotides in positions 1-7 pair with those in positions 72-66 to form the acceptor arm. Positions 8-9 act as spacers between the acceptor arm and the D-arm. Nucleotides in positions 10-13 pair with those in positions 25-22 to form the D-stem, while the D-loop consists of nucleotides from positions 14 to 21 (if a tRNA has more than 8 nucleotides in the D-loop, the positions are listed as 14-17, 17A, 18, 19, 20, 20A, 20B, 21, depending on the tertiary structure) . Position 26 serves as a spacer between the D-arm and the anticodon arm. Nucleotides in positions 27-31 pair with those in positions 43-39 to form the anticodon stem. The anticodon loop includes nucleotides from positions 32 to 38, with the anticodon located at positions 34-36. The variable region consists of nucleotides from positions 44 to 48 (if a tRNA has more than 5 nucleotides in the variable region, the positions are listed as 44, 45, e11-e17, e1-e5, e27-e21, 46, 47, 48) . Nucleotides in positions 49-53 pair with those in positions 65-61 to form the T-stem, while the T-loop consists of nucleotides from positions 54 to 60. Additionally, the nucleotide at position 73 is the discriminator, which is post-transcriptionally attached to the CCA tail.

The present invention provides engineered tRNAs for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16; and wherein the one or more modifications comprises: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon;

ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 12 (such as no more than about any of 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 80% (e.g., at least about any of 85%, 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 4.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 2.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 3.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 6.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 7.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 4^th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 5.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 8.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to A substitution at the 7th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, an A to U substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a U to A substitution at position 27, a U to C substitution at position 40, and an A to U substitution at position 43 relative to a tRNA having the sequence of SEQ ID NO: 1, wherein positions are labeled according to Sprinzl position. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 61.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a G to U substitution at the 5th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, a C to A substitution at the 5th nucleotide 3’ to the anticodon, an A to G substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a G to U substitution at position 29, a U to C substitution at position 40, a C to A substitution at position 41, and an A to G substitution at position 43 relative to a tRNA having the sequence of SEQ ID NO: 1, wherein positions are labeled according to Sprinzl position. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 62.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 7th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, an A to G substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a U to C substitution at position 27, a U to C substitution at position 40, and an A to G substitution at position 43 relative to a tRNA having the sequence of SEQ ID NO: 1, wherein positions are labeled according to Sprinzl position. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 63.

In some embodiments, there is provided an engineered tRNA for carrying an arginine comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a G to C substitution at the 32nd nucleotide 5’ to the anticodon, a U to C substitution at the 30th nucleotide 5’ to the anticodon, a C to A substitution at the 28th nucleotide 5’ to the anticodon, an A to G substitution at the 8th nucleotide 5’ to the anticodon, an A to G substitution at the 1st nucleotide 3’ to the anticodon, a G to U substitution at the 31st nucleotide 3’ to the anticodon, an A to G substitution at the 33rd nucleotide 3’ to the anticodon, a U to G substitution at the 35th nucleotide 3’ to the anticodon, a C to U substitution at the 36th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a G to C substitution at position 2, a U to C substitution at position 4, a C to A substitution at position 6, A to G substitution at position 26, an A to G substitution at position 37, aG to U substitution at position 67, an A to G substitution at position 69, a U to G substitution at position 71, and a C to U substitution at position 72 relative to a tRNA having the sequence of SEQ ID NO: 1, wherein positions are labeled according to Sprinzl position. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 80% (e.g., at least about any of 85%, 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 12 (such as no more than about any of 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiment, the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 64.

In some embodiments, the engineered tRNAs contain modifications at locations in the sequence of the engineered tRNA are described relative to the 5’ to 3’s equence of a tRNA reference sequence, wherein the 5’ end of the reference sequence is position 1. In some embodiments, the tRNA reference sequence is human tRNA-Arg-TCT-1-1. In some embodiments, the tRNA reference sequence is R3-147 (SEQ ID NO: 1) . For example, in some embodiments, there is provided an engineered transfer RNA (tRNA) for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, and wherein the one or more modifications comprise: i) a G at nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1; ii) a C at nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA comprises a G at nucleic acid position 37. In some embodiments the engineered tRNA comprises a C at nucleic acid position 40. In some embodiments, the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end.

In some embodiments, the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 5.

In some embodiments, the engineered tRNA comprises an A at the 7th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 27, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a U at the 7th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 43, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 61.

In some embodiments, the engineered tRNA comprises a U at the 5th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 29, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , an A at the 5th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 41, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 7th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 43, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 62.

In some embodiments, the engineered tRNA comprises a C at the 7th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 27, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 7th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 43, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 63.

In some embodiments, the engineered tRNA comprises a C at the 32nd nucleotide 5’ to the anticodon (corresponding to nucleic acid position 2, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a C at the 30th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 4, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , an A at the 28th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 6, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 8th nucleotide 5’ to the anticodon (corresponding to nucleic acid position 26, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a U at the 31st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 67, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 33rd nucleotide 3’ to the anticodon (corresponding to nucleic acid position 69, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a G at the 35th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 71, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a U at the 36th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 72, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 64.

In some embodiments, the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) . In some embodiments, the engineered tRNA comprises a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) . In some embodiments, the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) and a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) . In some embodiments, the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) and the engineered tRNA has the RNA sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6, 8, 65-69, 70, 71, 72-73, 75-77, 78-79, 80, 81, 61, 62, and 63. In some embodiments, the engineered tRNA comprises a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) and the engineered tRNA has the RNA sequence selected from the group consisting of SEQ ID NOs: 4, 6-8, 74, and 64.

In some embodiments, the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) and a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) . In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 6.

In some embodiments, the engineered tRNA comprises a G at the 1^st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 7.

In some embodiments, the engineered tRNA comprises a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1) , and a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has the RNA sequence set forth in SEQ ID NO: 8.

In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, or 8 can be from about 70%to about 80%, from 80%to about 90%, from about 85%to about 95%, from about 90%to about 95%, from about 95%to about 99%. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8 can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, or 8 can be at most about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, or less.

In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 13. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 16. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 1, 13, 16, 61, 62, 63, or 64 can be from about 70%to about 80%, from 80%to about 90%, from about 85%to about 95%, from about 90%to about 95%, from about 95%to about 99%. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 1, 13, 16, 61, 62, 63, or 64 can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the sequence identity between an engineered tRNA and any of the sequences of SEQ ID NOs: 1, 13, 16, 61, 62, 63, or 64 can be at most about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, or less.

In some embodiments, the %identity can be measured over a range of 90%of the length of nucleotides of an engineered tRNA. In some embodiments, the %identity can be measured over a range of 95%of the length of nucleotides of an engineered tRNA. In some embodiments, the %identity can be measured over 100%of the length of nucleotides of an engineered tRNA. The engineered tRNA can be a sequence of any of SEQ ID NOs: 2-8, 61, 62, 63, and 64. In some embodiments, the engineered tRNA sequence can comprise any of SEQ ID NOs: 2-8, 61, 62, 63, and 64.

In some embodiments, the engineered tRNA contains no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide substitutions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64. In some embodiments, the engineered tRNA contains no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide substitutions relative to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than about 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

The engineered tRNAs described herein can contain further modifications relative to SEQ ID NO: 1 (e.g., further nucleotide substitutions other than those described above) . In some embodiments, the modification can be a mutation, such as an insertion, a deletion (such as, for example, deletion of a single nucleotide from the variable loop) , or a substitution of one or more nucleotides. In some embodiments, the insertion is an intron of any length in the tRNA. In some embodiments, the engineered suppressor tRNA contains an intron that is spliced during tRNA maturation. In some embodiments, the intron is of variable length.

In some embodiments, the modification can be a chemical modification of one or more nucleotides in the sequence of the engineered tRNA. The chemical modification can comprise pseudouridine, inosine, wyosine, wybutosine, an acetyl group, an isopentenyl group, an hydroxy group, a peroxy group, an ribosyl group, a carbamoyl group, a carboxyl group, a methoxy group, a carbonyl group, a methyl group, a dimethyl group, a trimethyl group, a formyl group, a cyano group, a galactosyl group, a glutamyl group, a fluoro group, a methoxyethyl group, an ethyl group, a phosphate group, an amide group, an ester group, an amino group, or one or more additional modifications as described in “Modified residues” (Genesilico, https: //genesilico. pl/modomics/modifications, accessed on July 25, 2023) , or any combination thereof. In some embodiments, the engineered tRNA can comprise a chemical modification comprising a methyl group, a fluoro group, a methoxyethyl group, an ethyl group, a phosphate group, an amide group, an ester group, or any combination thereof.

Also provided are engineered pre-tRNAs that are processed into any of the engineered tRNAs described herein.

In some embodiments, the engineered tRNA is synthesized exogenously. In some embodiments, the engineered tRNA comprises both DNA and RNA. In some embodiments in which the engineered tRNA comprises both DNA and RNA, the engineered tRNA comprises a single RNA nucleotide among DNA nucleotides, or a single DNA nucleotide among RNA nucleotides. In some embodiments in which the engineered tRNA comprises both DNA and RNA, the engineered tRNA comprises a stretch of DNA and RNA sequence. In some embodiments, the engineered tRNA is transcribed from DNA, or its reverse, complement, or reverse-complement counterpart.

In some embodiments, the arginine with which the engineered tRNA is charged is detectably labeled to enable detection in vivo. In some embodiments, the labeling comprise, for example, click chemistry wherein an azide/alkyne containing unnatural amino acid can be added by the orthogonal tRNA/synthetase pair and, thus, can be detected using alkyne/azide comprising fluorophore or other such molecule.

III. NUCLEIC ACIDS ENCODING ENGINEERED TRNAS

The engineered tRNAs described herein may be encoded by nucleic acids. Accordingly, in some aspects, there is provided a nucleic acid encoding an engineered tRNA described herein. In some embodiments, the nucleic acid is an RNA. In some embodiments, the nucleic acid is a tRNA.

In another aspect, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In another aspect, there is provided a nucleic acid encoding an engineered pre-tRNAs that is processed into an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, and wherein the one or more modifications comprise: i) an A to G substitution at the 1st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the matured tRNA derived from engineered pre-tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the matured tRNA derived from engineered pre-tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1.

In another aspect, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 13, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In another aspect, there is provided a nucleic acid encoding an engineered pre-tRNAs that is processed into an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 13, and wherein the one or more modifications comprise: i) an A to G substitution at the 1st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4th nucleotide 3’ to the anticodon; and iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the matured tRNA derived from engineered pre-tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 13. In some embodiments, the matured tRNA derived from engineered pre-tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13.

In another aspect, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 16, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In another aspect, there is provided a nucleic acid encoding an engineered pre-tRNAs that is processed into an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 16, and wherein the one or more modifications comprise: i) an A to G substitution at the 1st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the matured tRNA derived from engineered pre-tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 16. In some embodiments, the matured tRNA derived from engineered pre-tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 4.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 3.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 6.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 7.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 4^th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1 and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 5. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 5.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 13 or SEQ ID NO: 16. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 16.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises an A to G substitution at the 1^st nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a U to C substitution at the 4th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1, and a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in in isolated tRNA) . In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, or 2 nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 8.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to A substitution at the 7th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, and an A to U substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 61. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 61.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a G to U substitution at the 5th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, a C to A substitution at the 5th nucleotide 3’ to the anticodon, and an A to G substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 62. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 62.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a U to C substitution at the 7th nucleotide 5’ to the anticodon, a U to C substitution at the 4th nucleotide 3’ to the anticodon, and an A to G substitution at the 7th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 63. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 63.

In some embodiments, there is provided a nucleic acid encoding an engineered tRNA for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anti-codon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises a G to C substitution at the 32nd nucleotide 5’ to the anticodon, a U to C substitution at the 30th nucleotide 5’ to the anticodon, a C to A substitution at the 28th nucleotide 5’ to the anticodon, an A to G substitution at the 8th nucleotide 5’ to the anticodon, an A to G substitution at the 1st nucleotide 3’ to the anticodon, a G to U substitution at the 31st nucleotide 3’ to the anticodon, an A to G substitution at the 33rd nucleotide 3’ to the anticodon, a U to G substitution at the 35th nucleotide 3’ to the anticodon, and a C to U substitution at the 36th nucleotide 3’ to the anticodon relative to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA comprises a nucleotide sequence that is at least about 85%(e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 12 (such as no more than about any of 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a nucleotide sequence of SEQ ID NO: 64. In some embodiments, the nucleic acid encoding the engineered tRNA comprises a DNA sequence encoding the nucleotide sequence of SEQ ID NO: 64.

In some embodiments, there is provided a nucleic acid encoding an engineered transfer RNA (tRNA) for carrying an arginine (or an expression cassette comprising such nucleic acid, or a vector (e.g., a viral vector) comprising such expression cassette) , the engineered tRNA comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16, and wherein the one or more modifications comprise: i) a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16) ; ii) a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40, wherein the nucleic acid position corresponds to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16) ; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA comprises a G at the 1st nucleotide 3’ to the anticodon (corresponding to nucleic acid position 37) . In some embodiments the engineered tRNA comprises a C at the 4th nucleotide 3’ to the anticodon (corresponding to nucleic acid position 40) . In some embodiments, the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end. Provided in some embodiments are nucleic acid molecules comprising RNA encoding any of the engineered tRNAs described herein. In some embodiments, the RNA comprises a sequence encoding a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 61, 62, 63, and 64. Provided in some embodiments are nucleic acid molecules comprising DNA encoding any of the engineered tRNAs described herein. In some embodiments, the DNA comprises a sequence encoding a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 61, 62, 63, and 64.

IV. NONSENSE MUTATION SUPPRESSION

Provided herein are engineered arginine transfer RNA (Arg tRNA) molecules that suppress the UGA premature stop codon to enable readthrough of the premature UGA stop codon in a template mRNA. These are also referred to as “engineered tRNA suppressor molecules” , “engineered suppressor tRNAs” , “suppressor tRNAs” , “sup-tRNAs” , “engineered tRNA suppressors” or “Arg-UGA sup-tRNAs” . As used herein, “suppression” and “readthrough” can be used interchangeably to refer to the activity of the engineered tRNAs disclosed herein.

The engineered tRNAs described herein have an anticodon sequence that base pairs with the UGA premature stop codon (also referred to herein as a PTC) , thus enabling the engineered tRNA, when charged with the arginine amino acid, to add the arginine to a growing polypeptide molecule, thus effecting readthrough of the premature stop codon.

PTCs can be introduced into a coding sequence through, for example, a mutation. A mutation can occur in a DNA molecule, an RNA molecule (e.g., tRNA, mRNA) , in a polypeptide or protein, or any combination thereof. In some embodiments, the reference sequence is obtained from a database, such as the NCBI Reference Sequence (RefSeq) Database. In some embodiments, the mutation comprises a substitution, a deletion, an insertion, an inversion, and/or a conversion in one or more nucleotides or one or more amino acids. A mutation can include two or more sequence changes in different alleles, or two or more sequence changes in a single allele. A mutation can include two different nucleotides at one position in one allele, such as a mosaic or a chimeric. A mutation can be present in a malignant tissue. A mutation can comprise a sequence variant, a sequence variation, a sequence alteration, and/or an allelic variant. A mutation can result in the presence of a PTC. In some embodiments, a mutation changes an Arg-encoding codon to a UGA/TGA PTC. Presence or an absence of a mutation can indicate an increased risk to develop a disease or condition. A presence or an absence of a mutation can indicate a presence of a disease or condition. A mutation can be present in a benign tissue. Absence of a mutation can but does not necessarily indicate that a tissue or sample can be benign. The methods described herein can comprise identifying the presence of a mutation in a sample.

An engineered tRNA as described herein can recognize a premature UGA stop codon in an mRNA encoding a polypeptide and at least partially transform interpretation of a premature stop codon as a sense arginine codon, such as, for example by adding the correct (e.g., non-disease-causing) arginine amino acid to the growing peptide. Such transformation, also known as stop codon readthrough or PTC readthrough (also referred to synonymously herein as “read-through” ) , can produce a substantially full-length polypeptide at an efficiency of from about 1%to about 100%relative to a comparable polypeptide produced using a comparable mRNA that lacks the premature stop codon. In some embodiments, an efficiency can be at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%90%, 95%, or 99%. In some embodiments, an efficiency can be at least about: 1%to about 10%, 5%to about 20%, 10%to about 35%, 25%to about 50%, 40%to about 70%, 60%to about 80%, 75%to about 90%or about 85%to about 100%.

In some embodiments, an efficiency of PTC readthrough can comprise an in vivo efficiency of PTC readthrough. In some embodiments, in vivo efficiency of PTC readthrough can be determined by at least partially treating a disease or condition. For example, in vivo efficiency of PTC readthrough can be measured by at least partially improving, for instance, the ability to produce a specific full-length protein, the absence and/or truncation of which is associated with a disease or disorder. Additional, non-limiting examples of possible in vivo measurements of efficiency of PTC readthrough include improving the ability to hear, improving the ability to see, improving motor ability, cognitive ability or any combination thereof. In some embodiments, an efficiency of PTC readthrough can comprise an in vitro efficiency of PTC readthrough, such as an in vitro efficiency of PTC readthrough as determined by, for example: (a) transfecting a first vector encoding an engineered tRNA and a second vector encoding a screening mRNA encoding a first marker protein, such as a luciferase protein or a green fluorescent protein into a first cell, such as a first human cell, where the screening mRNA encoding the first marker fluorescent protein can comprise a premature stop codon (this can be referred to herein as a (e.g., “broken” marker) ; (b) transfecting a third vector encoding a comparable screening mRNA encoding a second marker protein into a second cell, such as a second human cell, wherein the comparable screening mRNA does not comprise a premature stop codon; and (c) comparing an amount of a measurable output of the marker protein (e.g., fluorescence) from the first and second cells. In some embodiments, a gene or mRNA encoding a marker protein can comprise at least two premature stop codons. In some instances, the premature stop codons can be the same stop codons, or different stop codons.

In some embodiments, the engineered tRNA exhibits an increased PTC readthrough ability as compared to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, increased PTC readthrough ability is caused by increased tRNA stability. In some embodiments, increased PTC readthrough ability is caused by increased tRNA capability. Increased capability of the engineered tRNA may be determined by, for instance, NanoLuc luciferase assay, RT-qPCR, Western Blot, protein pull down, or the mass spectrometry. In some embodiments, increased PTC readthrough ability is caused by increased tRNA abundance. In some embodiments, increased PTC readthrough ability is caused by switched tRNA chemical modifications. In some embodiments, increased PTC readthrough ability is caused by increased affinity for aminoacyl tRNA synthetase. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the docking partner elongation factor EF-Tu/EF1A1. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the ribosome. In some embodiments, increased PTC readthrough ability is caused by changes in other properties of the engineered tRNA as compared to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, increased PTC readthrough ability is caused by one or more (such as any of 2, 3, or 4) mechanisms discussed above.

In some embodiments, the engineered tRNA exhibits an increased PTC readthrough ability as compared to a tRNA having the sequence of SEQ ID NO: 13. In some embodiments, increased PTC readthrough ability is caused by increased tRNA stability. In some embodiments, increased PTC readthrough ability is caused by increased tRNA capability. Increased capability of the engineered tRNA may be determined by, for instance, NanoLuc luciferase assay, RT-qPCR, Western Blot, protein pull down, or the mass spectrometry. In some embodiments, increased PTC readthrough ability is caused by increased tRNA abundance. In some embodiments, increased PTC readthrough ability is caused by switched tRNA chemical modifications. In some embodiments, increased PTC readthrough ability is caused by increased affinity for aminoacyl tRNA synthetase. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the docking partner elongation factor EF-Tu/EF1A1. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the ribosome. In some embodiments, increased PTC readthrough ability is caused by changes in other properties of the engineered tRNA as compared to a tRNA having the sequence of SEQ ID NO: 13. In some embodiments, increased PTC readthrough ability is caused by one or more (such as any of 2, 3, or 4) mechanisms discussed above.

In some embodiments, the engineered tRNA exhibits an increased PTC readthrough ability as compared to a tRNA having the sequence of SEQ ID NO: 16. In some embodiments, increased PTC readthrough ability is caused by increased tRNA stability. In some embodiments, increased PTC readthrough ability is caused by increased tRNA capability. Increased capability of the engineered tRNA may be determined by, for instance, NanoLuc luciferase assay, RT-qPCR, Western Blot, protein pull down, or the mass spectrometry. In some embodiments, increased PTC readthrough ability is caused by increased tRNA abundance. In some embodiments, increased PTC readthrough ability is caused by switched tRNA chemical modifications. In some embodiments, increased PTC readthrough ability is caused by increased affinity for aminoacyl tRNA synthetase. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the docking partner elongation factor EF-Tu/EF1A1. In some embodiments, increased PTC readthrough ability is caused by increased tRNA affinity to the ribosome. In some embodiments, increased PTC readthrough ability is caused by changes in other properties of the engineered tRNA as compared to a tRNA having the sequence of SEQ ID NO: 16. In some embodiments, increased PTC readthrough ability is caused by one or more (such as any of 2, 3, or 4) mechanisms discussed above.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, in vivo efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vivo from an mRNA containing a premature UGA stop codon relative to the amount of a comparable polypeptide produced in vivo from a comparable mRNA that lacks a premature stop codon.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 13. In some embodiments, in vivo efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vivo from an mRNA containing a premature UGA stop codon relative to the amount of a comparable polypeptide produced in vivo from a comparable mRNA that lacks a premature stop codon.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 16. In some embodiments, in vivo efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vivo from an mRNA containing a premature UGA stop codon relative to the amount of a comparable polypeptide produced in vivo from a comparable mRNA that lacks a premature stop codon.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vitro as compared to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, in vitro PTC read-through ability is measured by in vitro efficiency of PTC readthrough. In some embodiments, in vitro efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vitro from an mRNA containing a premature UGA stop codon relative to a comparable polypeptide produced in vitro from a comparable mRNA that lacks a premature stop codon.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vitro as compared to a tRNA having the sequence of SEQ ID NO: 13. In some embodiments, in vitro PTC read-through ability is measured by in vitro efficiency of PTC readthrough. In some embodiments, in vitro efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vitro from an mRNA containing a premature UGA stop codon relative to a comparable polypeptide produced in vitro from a comparable mRNA that lacks a premature stop codon.

In some embodiments, the engineered tRNA exhibits an increased PTC read-through ability in vitro as compared to a tRNA having the sequence of SEQ ID NO: 16. In some embodiments, in vitro PTC read-through ability is measured by in vitro efficiency of PTC readthrough. In some embodiments, in vitro efficiency of PTC readthrough is measured as a percentage of substantially full-length polypeptides produced in vitro from an mRNA containing a premature UGA stop codon relative to a comparable polypeptide produced in vitro from a comparable mRNA that lacks a premature stop codon.

An in vitro efficiency of PTC readthrough can be from about 1%to 100%, from about 10%to 100%, from about 20%to 100%, from about 30%to 100%, from about 40%to 100%, from about 50%to 100%, from about 60%to 100%, from about 70%to 100%, from about 75%to 100%. from about 80%to 100%, from about 85%to 100%, from about 90%to 100%, from about 95%to 100%, or from about 98%to 100%of the amount of a comparable polypeptide produced in vitro from a comparable mRNA that lacks a premature stop codon. In some embodiments, an in vitro efficiency of PTC readthrough can be from about 30%to 50%of the amount of a comparable polypeptide produced in vitro from a comparable mRNA that lacks a premature stop codon.

The engineered tRNAs disclosed herein are capable of premature stop codon readthrough, as disclosed herein. For example, the engineered tRNAs are capable of premature stop codon readthrough of a UGA/TGA stop codon mutation. In some embodiments, the premature stop codon is an Arg-to-UGA/TGA stop codon mutation. In some embodiments, the premature stop codon is of a UGA/TGA stop codon mutation from a codon encoding an amino acid other than arginine. In some embodiments, the protein resulting from the PTC readthrough has an identical sequence to a protein produced from a version of the coding sequence not containing a PTC. In some embodiments, the protein resulting from the PTC readthrough has a different sequence from a protein produced from a version of the coding sequence not containing a PTC.

In some embodiments, determining the amount of full-length protein can be used to measure premature stop codon readthrough. In some embodiments, the Arg-to-UGA/TGA stop codon mutation is in a mammalian cell. In some embodiments, the Arg-to-UGA/TGA stop codon mutation is in a human cell. In some embodiments, the presence of the Arg-to-UGA/TGA stop codon mutation is associated with a disease or condition.

In some embodiments, the engineered tRNA does not read through natural stop codons (that is, not premature; the termination signal that ends translation to result in a full-length functional protein/polypeptide, such that it is the last codon in a protein coding sequence (CDS) ) other than the premature UGA /TGA stop codon. In some embodiments, the engineered tRNA does read through stop codons other than the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA reads through stop codons other than the premature UGA/TGA stop codon with a reduced efficiency compared to the efficiency with which the engineered tRNA reads through the UGA/TGA stop codon. In some embodiments, the efficiency of read through of other stop codons is about 1%or less, about 10%or less, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or between about 90%and 100%compared to the efficiency with which the engineered tRNA reads through the UGA/TGA stop codon. In some embodiments, the efficiency of read through of other stop codons is less than about 10%. In some embodiments, the efficiency of read through of other stop codons is less than about 1%. In some embodiments, read through of other stop codons alters the function of the protein into which the engineered tRNA incorporates the amino acid. In some embodiments, read through of other stop codons does not alter the function of the protein into which the engineered tRNA incorporates the amino acid.

In some embodiments, an engineered tRNA can be aminoacylated with a canonical amino acid. In some embodiments, the engineered tRNA provided herein is aminoacylated with arginine. In some embodiments, the engineered tRNA provided herein is aminoacylated with a canonical amino acid other than arginine. In some embodiments, an engineered tRNA can be aminoacylated with a non-canonical amino acid. A non-canonical amino acid can comprise p-Acetylphenylalanine, p-Propargyloxyphenylalanine, p-Azidophenylalanine, O-methyltyrosine, p-Iodophenylalanine, 3-Iodotyrosine, Biphenylalanine, 2-Aminocaprylic acid, p-Benzoylphenylalanine, o-Nitrobenzylcysteine, o-Nitrobenzylserine, 4, 5-Dimethoxy-2-nitrobenzyl serine, o-Nitrobenzyllysine, Dansylalanine, Acetyllysine, Methylhistidine, 2-Aminononanoic acid, 2-Aminodecanoic acid, Cbz-lysine, Boc-lysine, or Allyloxycarbonyllysine. In some embodiments in which the engineered tRNA is aminoacylated with an amino acid other than arginine, the function of the protein into which the engineered tRNA incorporates the amino acid is not altered compared to a protein produced from a version of the mRNA not comprising a PTC. In some embodiments in which the engineered tRNA is aminoacylated with an amino acid other than arginine, the function of the protein into which the engineered tRNA incorporates the amino acid is altered compared to a protein produced from a version of the mRNA not comprising a PTC.

The engineered tRNAs described herein, in some embodiments, can exhibit improved suppression efficiency as compared with a corresponding parental tRNA, such as, for example, as compared with SEQ ID NO: 1. SEQ ID NO: 1 corresponds to the RNA sequence of an Arg-TGA sup-tRNA known as R3-147, which is a derivative of the human tRNA Arg-TCT-1-1.

In some embodiments, suppression efficiency can be more than or equal to about 30%, 50%, 70%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400%higher than the suppression efficiency of SEQ ID NO: 1. In some embodiments, suppression efficiency can be more than about 300%higher than the suppression efficiency of SEQ ID NO: 1.

In some embodiments, the engineered tRNA described herein can exhibit increased stability as compared with the stability of a corresponding parental tRNA, such as compared with the stability of SEQ ID NO: 1. In some embodiments, the stability is measured in vivo. In some embodiments, the stability is measured in vitro. Stability can be measured in different ways, for example, in terms of thermodynamic stability or shielding an engineered tRNA from degradation. Changing a base pair to G-C bond can at least provide a thermodynamic as well as a degradation shield, therefore stabilizing an engineered tRNA from degradation. In some embodiments, the engineered tRNA exhibits an increased stability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1. In some embodiments, the engineered tRNA exhibits an increased PTC readthrough capability in vivo or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 1.

In some embodiments, the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence. In some embodiments, the engineered tRNA stability increases by less than about 10%, by about 10%-20%, by about 20%-30%, by about 30%-40%, by about 40%-50%, by about 50%-60%, by about 60%-70%, by about 70%-80%, by about 80%-90%, by about 90%-100%, by about 100%-200%, by about 200%-300%, or by more than about 300%compared to a tRNA having the sequence of SEQ ID NO: 1.

The engineered tRNAs described herein, in some embodiments, can exhibit improved suppression efficiency as compared with a corresponding parental tRNA, such as, for example, as compared with SEQ ID NO: 13. SEQ ID NO: 13 corresponds to the RNA sequence of an Arg-TGA sup-tRNA known as R3-5, which is a derivative of the human tRNA Arg-CCT-1-1.

In some embodiments, suppression efficiency can be more than or equal to about 30%, 50%, 70%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400%higher than the suppression efficiency of SEQ ID NO: 13. In some embodiments, suppression efficiency can be more than about 300%higher than the suppression efficiency of SEQ ID NO: 13.

In some embodiments, the engineered tRNA described herein can exhibit increased stability as compared with the stability of a corresponding parental tRNA, such as compared with the stability of SEQ ID NO: 13. In some embodiments, the stability is measured in vivo. In some embodiments, the stability is measured in vitro. Stability can be measured in different ways, for example, in terms of thermodynamic stability or shielding an engineered tRNA from degradation. Changing a base pair to G-C bond can at least provide a thermodynamic as well as a degradation shield, therefore stabilizing an engineered tRNA from degradation. In some embodiments, the engineered tRNA exhibits an increased stability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 13 In some embodiments, the engineered tRNA exhibits an increased PTC readthrough capability in vivo or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 13.

In some embodiments, the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence. In some embodiments, the engineered tRNA stability increases by less than about 10%, by about 10%-20%, by about 20%-30%, by about 30%-40%, by about 40%-50%, by about 50%-60%, by about 60%-70%, by about 70%-80%, by about 80%-90%, by about 90%-100%, by about 100%-200%, by about 200%-300%, or by more than about 300%compared to a tRNA having the sequence of SEQ ID NO: 13.

The engineered tRNAs described herein, in some embodiments, can exhibit improved suppression efficiency as compared with a corresponding parental tRNA, such as, for example, as compared with SEQ ID NO: 16. SEQ ID NO: 16 corresponds to the RNA sequence of an Arg-TGA sup-tRNA known as R3-8, which is a derivative of the human tRNA Arg-CCT-4-1.

In some embodiments, suppression efficiency can be more than or equal to about 30%, 50%, 70%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400%higher than the suppression efficiency of SEQ ID NO: 16. In some embodiments, suppression efficiency can be more than about 300%higher than the suppression efficiency of SEQ ID NO: 16.

In some embodiments, the engineered tRNA described herein can exhibit increased stability as compared with the stability of a corresponding parental tRNA, such as compared with the stability of SEQ ID NO: 16. In some embodiments, the stability is measured in vivo. In some embodiments, the stability is measured in vitro. Stability can be measured in different ways, for example, in terms of thermodynamic stability or shielding an engineered tRNA from degradation. Changing a base pair to G-C bond can at least provide a thermodynamic as well as a degradation shield, therefore stabilizing an engineered tRNA from degradation. In some embodiments, the engineered tRNA exhibits an increased stability in vivo as compared to a tRNA having the sequence of SEQ ID NO: 16. In some embodiments, the engineered tRNA exhibits an increased PTC readthrough capability in vivo or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 16.

In some embodiments, the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence. In some embodiments, the engineered tRNA stability increases by less than about 10%, by about 10%-20%, by about 20%-30%, by about 30%-40%, by about 40%-50%, by about 50%-60%, by about 60%-70%, by about 70%-80%, by about 80%-90%, by about 90%-100%, by about 100%-200%, by about 200%-300%, or by more than about 300%compared to a tRNA having the sequence of SEQ ID NO: 16.

In some embodiments, the engineered tRNA restores less than 10%of the translation of the coding nucleic acid containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA restores at least 5%of the translation of the coding nucleic acid containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA restores at least 10%of the translation of the coding nucleic acid containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA restores between about 10-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%of the translation of the coding nucleic acid containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon.

In some embodiments, the engineered tRNA exhibits a different chemical modification pattern compared to a native tRNA. In some embodiments, presence of the engineered tRNA results in restoration of the function of a protein comprising a PTC. In some embodiments, an mRNA targeted by the engineered tRNA comprises one premature UGA stop codon. In some embodiments, an mRNA targeted by the engineered tRNA can comprise one, two, three, four, five, or more than five premature UGA stop codons. In some embodiments, different alleles of a given gene comprise one or more different TGA stop codons in different positions. In some embodiments, an individual has multiple different TGA -stop-codon-containing alleles of a gene, such as, for example, in the case of compound heterozygosity. In some embodiments, every copy of a gene present in an individual contains a TGA stop codon. In some embodiments, the presence of one or more UGA/TGA stop codons in an individual results in complete loss of function of the protein encoded by the gene. In some embodiments, the presence of one or more UGA/TGA stop codons in an individual results in partial loss of function of the protein encoded by the gene. Accordingly, an engineered tRNA as described herein can produce readthrough of any or all of the one or more premature stop codons, thereby at least partially restoring a substantially full-length polypeptide. In some embodiments, at least partially restoring a substantially full-length polypeptide can comprise at least partially treating a disease or condition.

In some embodiments, the engineered tRNA can reduce or prevent nonsense-mediated decay (NMD) of an mRNA containing one or more UGA PTCs. NMD can be a quality control pathway that degrades PTC-containing mRNAs. Some embodiments can include a method of inhibiting NMD comprising contacting a cell with an engineered tRNA described herein. In some embodiments, the contact can result in an increase in abundance of mRNAs containing UGA codons in the cell relative to a baseline measurement. Some embodiments can include a method of inhibiting NMD of an mRNA containing one or more UGA PTCs in a subject, comprising administering to a subject an engineered tRNA. In some embodiments, the administration can result in a prevention or decrease of NMD-induced degradation of an mRNA containing one or more UGA PTCs in the subject. In some embodiments, the administration can result in an increase in abundance of an mRNA containing one or more UGA PTCs relative to a baseline measurement. The increase in the target mRNA abundance can be measured by comparing abundance in a second sample taken from the subject to a baseline target mRNA measurement in a first sample taken from the subject. The first and/or second samples can comprise a tissue or fluid sample described herein.

In some embodiments, the engineered tRNA can decrease NMD-induced degradation of an mRNA containing one or more UGA PTCs by: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%90%, 95%, 99%or 100%, or a range of any two of the aforementioned percentages. In some embodiments, the engineered tRNA can decrease NMD-induced degradation of an mRNA containing one or more UGA PTCs by at least about: 1%to about 10%, 5%to about 20%, 10%to about 35%, 25%to about 50%, 40%to about 70%, 60%to about 80%, 75%to about 90%or about 85%to about 100%.

V. EXPRESSION CASSETTES, VECTORS, PHARMACEUTICAL COMPOSITIONS, AND DELIVERY

A. Expression Cassettes

In some embodiments, the engineered tRNA is encoded by an expression cassette comprising a nucleic acid encoding the engineered tRNA. In some embodiments, the engineered tRNA is be introduced to cells using standard conventional genetic engineering techniques through use of vectors. In some embodiments, the engineered tRNA sequence is provided without a separate promoter, because of the internal promoter sequences of tRNA encoding sequences. In some embodiments, the engineered tRNA sequence is provided with a separate promoter.

Expression cassettes may include, for example, one or more promoter sequences operably linked to a nucleotide sequence of interest, which may further be operably linked to one or more termination sequences. Expression cassettes may also include, for example, sequences required for proper transcription of the nucleotide sequence. The coding region may code, for example, for an engineered tRNA. An expression cassette including a nucleotide sequence of interest may be chimeric. An expression cassette may contain naturally occurring sequences in a recombinant form useful for heterologous expression. Expression of a nucleotide sequence in the expression cassette may be under the control of, for example, a constitutive promoter, a cell-type specific promoter, a temporal promoter, and/or a regulatable promoter that, for example, initiates transcription only when the host cell is exposed to a particular stimulus. In embodiments relating to multicellular organisms, the promoter can also be specific to, for example, a particular tissue, organ, and/or or stage of development. In some embodiments, expression of the exogenous genetic material is driven by RNA polymerase III promoter, such as, for example, U6 promoter, H1 promoter and 7SK promoter. In some embodiments, expression of the exogenous genetic material is driven by a constitutive promoter, such as, for example, a promoter from any of the following “housekeeping” genes: hypoxanthine phosphoribosyl transferase (HPRT) , dihydrofolate reductase (DHFR) , adenosine deaminase, phosphoglycerate kinase (PGK) , pyruvate kinase, phosphoglycerate mutase, the actin promoter; or, for example, a viral promoters that functions constitutively in eukaryotic cells, such as, for example, the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among others. Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

B. Vectors

In some embodiments, the nucleotide expression cassette or nucleic acid of the invention is included within an appropriate gene transfer vehicle which is then used to transduce cells to express the suppressor tRNA. The gene delivery vehicle can be any delivery vehicle known in the art, and can include, for example, naked DNA that is facilitated by a receptor, and/or lipid mediated transfection, and/or vectors. Such vectors include but are not limited to eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors) , and viral vectors. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentivirus vectors (human and other including porcine) , Herpes virus vectors, Epstein-Barr viral vectors, SV40 virus vectors, pox virus vectors, and pseudotyped viral vectors. In some embodiments, retroviral vectors are transduced. In some embodiments, viral vectors (e.g., “transgene vectors” ) transduce genes into “target cells” or host cells. The present invention encompasses transgene vectors that are suitable for use in the present invention that are linked to any gene of interest (or a “marker gene” or “reporter gene, ” used to indicate infection or expression of a gene) .

In some embodiments, the engineered tRNA is encoded in a vector. In some embodiments, the engineered tRNA is encoded in a viral vector. In some embodiments, the viral vector is a retroviral, adeno-associated vector, or adenoviral vector. Exemplary retroviral vectors include, but are not limited to, spleen necrosis virus, Moloney Murine Leukemia Virus, and vectors derived from retroviruses such as avian leukosis virus, human immunodeficiency virus, Rous Sarcoma Virus, Harvey Sarcoma Virus, myeloproliferative sarcoma virus, and mammary tumor virus.

Vectors encoding the engineered tRNA molecules of the present disclosure are also provided herein. The vectors are viral vectors in some cases. Also disclosed herein are various constructs for packaging engineered tRNA suppressor molecules in viral vectors, including packaging of: one or multiple engineered tRNA suppressor payloads, markers such as GFP or mCherry, stuffer sequences, or any combination thereof. Methods of delivering the engineered tRNA or vector encoding the engineered tRNA optionally packaged into a virus, are also provided herein.

In some embodiments, a vector can encode for an engineered tRNA or an engineered pre-tRNA. In some embodiments, a composition can comprise a vector. In some embodiments, a vector can comprise a plasmid or a viral vector. In some embodiments, a vector encoding the engineered tRNA or engineered pre-tRNA is administered to a subject. Exemplary viral vectors can include an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a portion of any of these, or any combination thereof. In some embodiments, the vector comprises DNA, such as double-stranded DNA or single-stranded DNA. In some embodiments, the vector comprises RNA. In some embodiments, the vector comprises a recombinant vector. In some embodiments, the vector is modified from a naturally occurring vector. In some embodiments, the RNA comprises a base modification. In some embodiments, the vector comprises at least a portion of a non-naturally occurring vector. Any vector can be utilized.

In some embodiments, the vector comprises an AAV vector. A vector can be modified to include a modified VP1 protein (such as an AAV vector modified to include a VP1 protein) . In some embodiments, the AAV is a recombinant AAV (rAAV) vector. rAAVs can be composed of substantially similar capsid sequence and structure as found in wild-type AAVs (wtAAVs) , except that rAAVs encapsidate genomes that are substantially devoid of AAV protein-coding sequences and have therapeutic gene expression cassettes, such as engineered tRNA expression cassettes, designed in their place. In some embodiments, sequences of viral origin can be inverted terminal repeats (ITRs) , which can guide genome replication and packaging during vector production. Suitable AAV vectors can be selected from any AAV serotype or combination of serotypes. For example, an AAV vector can be any one of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, or any combination thereof. In some embodiments, a vector is selected based on its natural tropism. In some embodiments, a vector serotype is selected based on its ability to cross the blood brain barrier, such as, for example AAV9 and/or AAV10. In some embodiments, an AAV vector is a chimera of at least two serotypes. In some embodiments, an AAV vector can be a chimera of serotypes AAV2 and AAV5. In some embodiments, a chimeric AAV vector can comprise rep and ITR sequences from AAV2 and a cap sequence from AAV5. In some embodiments, an AAV vector does not comprise a rep sequence. In some embodiments, a rep gene (such as, for example, from AAV2) is used in trans for AAV production. In some embodiments, an AAV vector can be self-complementary. In some embodiments, an AAV vector can be single-stranded AAV vector. In some embodiments, an AAV vector can be a self-complementary AAV vector. In some embodiments, an AAV vector can comprise an inverted terminal repeat (ITR) . In some embodiments, an AAV vector can comprise a self-complementary inverted terminal repeat (scITR) sequence. In some embodiments, rep, cap, and ITR sequences can be combined from any or all the of the AAV serotypes provided herein. In some embodiments, a suitable AAV vector can be further modified to encompass modifications such as in a capsid or rep protein. Such modifications can include deletions, insertions, mutations, and combinations thereof. In some embodiments, a modification to a vector can be made to reduce immunogenicity to allow for repeated dosing. In some embodiments, a serotype of a vector that can be utilized can be changed when repeated dosing can be performed to reduce and/or eliminate immunogenicity.

In some embodiments, an AAV vector is from an AAV having a serotype comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or a pseudotype comprising AAV-DJ, AAV-DJ/8, AAV-Rhl0, AAV-Rh74, AAV-retro, AAV-PHP. B, AAV8-PHP. eB, AAV-PHP. Sor AAV-2i8. In some embodiments, the AAV vector comprises a genome comprising a replication gene and inverted terminal repeats from a first AAV serotype and a capsid protein from a second AAV serotype. In some embodiments, the AAV vector comprises an AAV 2/2 vector, an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector.

In some embodiments, the vector comprises from 2 to 6 copies of nucleic acids encoding an engineered tRNA per viral genome. In some embodiments, the vector comprises different engineered suppressor tRNAs with multiple copies per viral genome. In some embodiments, the vector comprises from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 1 to 10, from 2 to 3, from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 7, from 2 to 8, from 2 to 9, or from 2 to 10 copies per viral genome. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies per viral genome. In some embodiments, the vector comprises from 1 to 5, from 1 to 10, from 1 to 15, from 1 to 20, from 1 to 25, from 1 to 30, from 1 to 35, from 1 to 40, from 1 to 45, or from 1 to 50 copies per viral genome.

A retroviral transgene vector is an expression vector bearing an expressible non-retroviral gene of interest and further includes at least one functional retroviral packaging signal. Thus, after the transgene vector is transfected into a packaging cell line, the transgene vector is transcribed into RNA, and this RNA is packaged into an infectious viral particle, which then infects target cells. Upon infection, the RNA in the viral particle is reverse transcribed into DNA, and the DNA is incorporated into the cell genome as a proviral element, thereby transmitting the gene of interest to the target cells.

C. Pharmaceutical compositions

In some embodiments, an engineered tRNA or an expression cassette, vector, and/or nucleic acid encoding an engineered tRNA is present in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. an excipient, and/or a diluent. In some embodiments, the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle.

In some embodiments, liposomes are used to mediate delivery of the engineered tRNA. In some embodiments, lipid nanoparticles (LNPs) are used to mediate delivery of the engineered tRNA. In some embodiments, LNPs are used to deliver synthetic RNA.

Provided herein are engineered tRNA molecules or vectors encoding the engineered tRNA molecules that can be packaged into a virus for virus particle-mediated delivery of the engineered tRNA molecules to a target cell or tissue in vivo. Compositions described herein comprising the engineered tRNA molecule (s) or the vector (s) encoding the engineered tRNA molecule (s) can employ an AAV vector for delivery to a subject. AAV vector delivery can achieve long-term benefit with single dose and can provide opportunity for multiplexed targeting.

In some embodiments, an engineered tRNA, a vector encoding the engineered tRNA, or both, can be present in a delivery system. In some embodiments, the delivery system comprises a viral particle, a liposome, a nanoparticle, an exosome, an extracellular vesicle, a nanomesh, a charged polymer, an uncharged polymer, a surfactant, a penetrating enhancer, a gene transfer agent, a phospholipid, a micelle, a synthetic vector, a macromolecule, a dendrimer, a biopolymer, or any combination thereof. In some embodiments, an engineered tRNA or a vector encoding the engineered tRNA can be present in a viral particle, a liposome, a nanoparticle, an exosome, an extracellular vesicle, a nanomesh, or any combination thereof. In some embodiments, the vector is inside a polypeptide coat.

In some embodiments, a composition comprises an excipient. An excipient can be, for example, added to a stem cell or co-isolated with the stem cell from a source. An excipient can comprise a cryo-preservative, such as DMSO, glycerol, polyvinylpyrrolidone (PVP) , or any combination thereof. An excipient can comprise a cryo-preservative, such as a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof. An excipient can comprise a pH agent (for example, to minimize oxidation or reduction of a component of the composition) , a stabilizing agent (for example, to prevent modification or degradation of a component of the composition) , a buffering agent (for example, to enhance temperature stability) , a solubilizing agent (for example, to increase protein solubility) , or any combination thereof. An excipient can comprise a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof. An excipient can comprise sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG) , human serum albumin (HSA) , sorbitol, sucrose, trehalose, sodium phosphate, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, arginine, sodium acetate, HC1, disodium edetate, lecithin, glycerine, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. An excipient can be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986) .

A composition described herein can comprise a naturally-occurring or non-naturally-occurring carrier. In some embodiments, the carrier is inert (for example, a detectable agent or label) . In some embodiments, the carrier is active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. In some embodiments, a carrier comprises a pharmaceutically acceptable carrier. In some embodiments, a carrier includes pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers) , which can be present singly or in combination, comprising alone or in combination 1-99.99%by weight or volume. Exemplary protein excipients include serum albumins such as human serum albumin (HSA) , recombinant human albumin (rHA) , gelatin, casein, and the like. Representative amino acid components, antibody components, or both, which can also function in a buffering capacity, include alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Some embodiments comprise carbohydrate excipients, such as, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) , and myoinositol.

Compositions provided herein can comprise a diluent, such as, for example, water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the diluent is an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or similar. In some embodiments, the diluent is an alkaline metal carbonate such as calcium carbonate; an alkaline metal phosphate such as calcium phosphate; an alkaline metal sulphate such as calcium sulphate; a cellulose derivative such as cellulose, microcrystalline cellulose, or cellulose acetate; magnesium oxide, dextrin, fructose, dextrose, glyceryl palmitostearate, lactitol, choline, lactose, maltose, mannitol, simethicone, sorbitol, starch, pregelatinized starch, talc, xylitol and/or anhydrates, hydrates and/or pharmaceutically acceptable derivatives thereof or combinations thereof.

D. Delivery

In some embodiments, the nucleic acid encoding the engineered tRNA is encoded by a nucleic acid that can be delivered to a host cell via viral or non-viral based methods. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or gene-gun, naked DNA and artificial virions. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) .

The use of RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.

In certain embodiments according to any one of the methods described herein, the method comprises introducing a viral vector (such as an AAV or a lentiviral vector) encoding the nucleic acid encoding the engineered tRNA to the host cell. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the construct is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the construct is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector further comprises a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid is located upstream or downstream of the nucleic acid encoding the tRNA. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector comprises first nucleic acid sequence encoding the nucleic acid encoding the engineered tRNA and a second nucleic acid sequence encoding a complement of the nucleic acid encoding the engineered tRNA, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence. In some embodiments, the vector is encapsidated in a rAAV particle. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAV-PHP. B, AAV-PHP. eB, AAV-BR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or rAAV2/HboV1 serotype capsid.

In some embodiments, the method comprises introducing a plasmid encoding the nucleic acid encoding the engineered tRNA to the host cell. In some embodiments, the method comprises electroporation of the nucleic acid encoding the engineered tRNA (e.g., synthetic nucleic acid encoding the engineered tRNA) into the host cell. In some embodiments, the method comprises transfection of the nucleic acid encoding the engineered tRNA into the host cell.

In some embodiments, the engineered tRNA is delivered to a subject. In some embodiments, the engineered tRNA is delivered to a cell. In some embodiments, the cell is in a subject. In some embodiments, the subject and/or cell is eukaryotic. In some embodiments, the subject and/or cell is mammalian. In some embodiments, the subject and/or cell is human.

VI. METHODS OF SUPPRESSING PTC AND DISEASE TREATMENT

The present application further provides methods of suppressing PTC by using the engineered tRNAs provided herein. In some embodiments, the engineered tRNA (or nucleic acid encoding the engineered tRNA) is used to treat a disease associated with a premature UGA/TGA codon. In some embodiments, a method of treating a disease associated with a premature UGA/TGA stop codon in an individual comprises administering to the individual an effective amount of a pharmaceutical composition comprising an engineered tRNA, an expression cassette comprising a nucleic acid encoding an engineered tRNA, and/or a vector comprising an expression cassette comprising a nucleic acid encoding an engineered tRNA, and further comprising a pharmaceutically acceptable carrier. In the context of therapeutic or prophylactic applications, the effective amount can depend on, for example, the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and/or tolerance to pharmaceutical compositions. In some embodiments, an effective amount can be an amount that can be required to at least partially treat a patient with a disease associated with the presence of a PTC. In some embodiments related to in vitro applications, the effective amount depends on the size and nature of the particular application and/or on the nature and/or sensitivity of the in vitro target and the methods in use. In some embodiments, the effective amount comprises one or more administrations of a composition.

In some embodiments, there is provided a method of restoring translation of a coding nucleic acid containing a premature UGA/TGA stop codon in (or a method of reading through a nucleic acid containing a premature UGA/TGA stop codon) a host cell, comprising introducing to the host cell an engineered tRNA for carrying an arginine (or an expression cassette comprising a nucleic acid encoding the engineered tRNA, or a vector comprising an expression cassette comprising a nucleic acid encoding the engineered tRNA) , wherein the engineered tRNA comprises a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) , and wherein the engineered tRNA introduced into the cell (or produced by the expression cassette or vector) recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1. In some embodiments, the method is carried out ex vivo. In some embodiments, the method of carried out in vivo.

In some embodiments, there is provided a method of restoring translation of a coding nucleic acid containing a premature UGA/TGA stop codon in (or a method of reading through a nucleic acid containing a premature UGA/TGA stop codon) a host cell, comprising introducing to the host cell an engineered tRNA for carrying an arginine (or an expression cassette comprising a nucleic acid encoding the engineered tRNA, or a vector comprising an expression cassette comprising a nucleic acid encoding the engineered tRNA) , wherein the engineered tRNA comprises a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 13, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) , and wherein the engineered tRNA introduced into the cell (or produced by the expression cassette or vector) recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 13. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 13. In some embodiments, the method is carried out ex vivo. In some embodiments, the method of carried out in vivo.

In some embodiments, there is provided a method of restoring translation of a coding nucleic acid containing a premature UGA/TGA stop codon in (or a method of reading through a nucleic acid containing a premature UGA/TGA stop codon) a host cell, comprising introducing to the host cell an engineered tRNA for carrying an arginine (or an expression cassette comprising a nucleic acid encoding the engineered tRNA, or a vector comprising an expression cassette comprising a nucleic acid encoding the engineered tRNA) , wherein the engineered tRNA comprises a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 16, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end (e.g., introduced through a nucleic acid encoding the suppressor tRNA or in an isolated tRNA) , and wherein the engineered tRNA introduced into the cell (or produced by the expression cassette or vector) recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid containing the premature UGA/TGA stop codon. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 16. In some embodiments, the method is carried out ex vivo. In some embodiments, the method of carried out in vivo.

In some embodiments, the method of suppressing PTC by using the engineered tRNAs provided herein can comprise delivery of a mixture of multiple types of engineered tRNAs and/or nucleic acids encoding multiple types of engineered tRNAs, such as simultaneous or non-simultaneous delivery of 2 or more, 3 or more, 4 or more, or 5 or more different engineered tRNAs, and/or simultaneous or non-simultaneous delivery of 2 or more, 3 or more, 4 or more, or 5 or more different nucleic acids, each encoding a different engineered tRNA, and/or delivery of only 1 nucleic acid that encodes multiple different engineered tRNAs. In some embodiments, the different engineered tRNAs suppress the same PTC (e.g., UGA) . In some embodiments each of the different engineered tRNAs suppresses a different PTC (e.g., one or more engineered tRNAs suppress the UGA PTC, and one or more different engineered tRNAs suppress a different PTC) . In some embodiments, at least one of the different engineered tRNAs is an engineered Arg-UGA engineered tRNA described herein. In some embodiments, all of the different engineered tRNAs are different engineered Arg-UGA engineered tRNA described herein.

By “read through, ” it is meant that the engineered tRNA affects translation of a PTC-containing mRNA, resulting in the incorporation of an arginine at the PTC in the nascent growing polypeptide chain, rather than termination of translation and generation of a truncated protein, which would otherwise occur.

Methods for determining whether an engineered tRNA result in read through are known in the art, and include, for example, western blot analysis, immunohistochemistry, flow cytometry, mass spectrometry, as well as cell-based reporter assays such as those described in the Examples section. In some aspects, improvement in one or more clinical parameters allows for determining whether a PTC correction agent is effectively increasing levels of full-length protein. In some aspects, even modest or slight increases in the amount of full-length protein are beneficial in alleviating some disease states. For example, the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H, caused by nonsense mutation resulting in decreased levels of iduronidase encoded by the IDUA gene) , has a low threshold for correction, since <1%of wild-type iduronidase function can significantly moderate the clinical phenotype (Ashton et al., Am. J. Hum. Genet. 1992, 50: 787-794, Bunge et al., Biochim. Biophys. Acta. 1998, 1407: 249-256) . Thus, increasing the amount of full-length protein, in some aspects, to reach 1%of wild-type levels, is beneficial in treating some diseases caused by nonsense mutations. In some examples, the method results in an increase of at least 5%, such as 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or a 100%increase, in the amount of full length protein produced, for example as compared to wild type levels (e.g., levels of expression of the wild-type protein wherein the gene/mRNA does not contain a nonsense mutation) . In other examples, such as the disease β-thalassemia, increased levels of the full-length beta chains of hemoglobin leads to improved or ameliorated disease states, such as decreased or no anemia, decreased tiredness, decreased breathlessness, and increased exercise tolerance. Methods for monitoring improvement in the β-thalassemia disease state are known, and include, for example, pulse oximetry, hemoglobin electrophoresis; serum transferrin, ferritin, Fe binding capacity analysis; urine urobilin &urobilinogen assays; peripheral blood smear test; hematocrit analysis; and serum bilirubin analysis.

In some embodiments, the host cell is a eukaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell. In some embodiments, the host cell is a murine cell. In some embodiments, the host cell is a plant cell or a fungal cell. In some embodiments, the host cell is a diseased cell. In some embodiments, the host cell comprises one or more mutations, such as a nonsense mutation.

In some embodiments, the host cell is a cell line, such as Neuro-2a, HEK293T, HT29, NCI-60, MCF-7, HL-60, A549, HepG2, RD, SF268, SW13, LHCN differentiated, LHCN undifferentiated, Saos-2, CHO, or HeLa cells. In some embodiments, the host cell is a primary cell, such as fibroblast, epithelial, or immune cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a post-mitosis cell. In some embodiments, the host cell is a cell of the central nervous system (CNS) , such as a brain cell, e.g., a cerebellum cell. In some embodiments, the host cell is an immortalized PTC containing cell. In some embodiments, the host cell is a PTC-containing stem cell or IPS cell. In some embodiments, the host cell is from a PTC-containing stem cell-or IPS cell-differentiated cells or organoids.

In some embodiments, the host cell is a neuron, a photoreceptor cell (e.g. a S cone cell, a L cone cell, a M cone cell, a rod cell) , a retinal pigment epithelium cell, a glia cell (e.g. an astrocyte, an oligodendrocyte, a microglia) , a muscle cell (e.g. a myoblast, a myotube) , a hepatocyte, or a lung epithelial cell. In some embodiments, a cell can be a horizontal cell, a ganglion cell, or a bipolar cell.

In some embodiments, the host cell is in an individual, such as a human individual. In some embodiments, the host cell is an ex vivo cell population.

In some embodiments, the coding nucleic acid containing a premature UGA/TGA stop codon is an mRNA. In some embodiments, the premature UGA/TGA stop codon (also referred to as “PTC” ) results from a nonsense mutation, such as a disease-causing mutation (such as a disease-causing mutation described herein) . The disease can be caused by the rapid turnover (e.g., by NMD) of the mRNA, a lack or reduced production of functional protein due to a truncated protein product, insufficient levels of a truncated protein product having normal or partial function, or combinations thereof. In some embodiments, the PTC is a PTC resulting from abnormal or inefficient biogenesis of mRNAs. In some embodiments, the PTC is not the result of a mutation, such as, for example as the result of one or more errors introduced during transcription of non-mutated DNA.

In some aspects, mutations resulting in a PTC have important consequences on gene expression, such as in the context of disease. For example, a PTC will terminate mRNA translation prior to completion of a full-length polypeptide, leading to production of truncated proteins that are often partially functional, nonfunctional, unstable, and/or have detrimental function. In addition, PTC-containing mRNAs are also frequently unstable because the mRNAs are degraded by NMD, resulting in a severe reduction in steady-state mRNA levels. In some examples, the combination of these PTC-induced events reduce the level of functional protein produced to such an extent that a severe disease state results.

The PTC-containing mRNA described herein can be transcribed from any gene of interest. In some embodiments, the PTC-containing mRNA is transcribed from a gene selected from the group consisting of IDUA, CFTR, DMD, HBB, and MECP2. In some embodiments, the PTC-containing mRNA is transcribed from IDUA. Additional nonsense mutations can be found in, for example, the Human Gene Mutation Database (HGMD) and the ClinVar (Landrum, M.J., et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res . 2018 Jan 4. PubMed PMID: 29165669) .

In some embodiments, a composition described herein (e.g., a composition comprising an engineered tRNA or a nucleic acid encoding the engineered tRNA) can be administered to prevent a disease or condition as described herein. For example, in some embodiments, a composition as described herein can be administered prophylactically to prevent an incidence of a disease or condition. Prevention can at least partially reduce an appearance, onset, or incidence of one or more symptoms of a disease or condition. In some embodiments, there is provided a method of treating a disease associated with a premature UGA/TGA stop codon in an individual, comprising: administering to the individual an effective amount of a pharmaceutical composition comprising an engineered transfer RNA (tRNA) for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm (or an expression cassette comprising a nucleic acid encoding the engineered tRNA, or a vector comprising an expression cassette comprising a nucleic acid encoding the engineered tRNA) , wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16, and wherein the one or more modifications comprise: i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon; ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or iii) a CCA sequence attached to the acceptor arm at the 3’ end. In some embodiments, the engineered tRNA has a nucleotide sequence that is at least about 85% (e.g., at least about any of 87%, 90%, 92%, or 95%) identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16. In some embodiments, the engineered tRNA contains no more than 5 (such as no more than about any of 4, 3, 2, or 1) nucleotide substitutions relative to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16) .

Also provided are compositions (such as pharmaceutical compositions) described herein for use in treating a disease associated with a premature UGA/TGA stop codon in an individual. In some embodiments, there are provided uses of the compositions (such as pharmaceutical compositions) described herein for the manufacture of medicament for treating a disease associated with a premature UGA/TGA stop codon in an individual.

In some embodiments, a composition described herein can be administered to treat a disease or condition as described herein. For example, in some embodiments, a composition as described herein can be administered after onset or diagnosis of a disease to prevent continuing or worsening of the disease or condition, and/or to reduce the severity of symptoms, such as by at least partially reduce an appearance, onset, or incidence of one or more symptoms of a disease or condition. Any disease associated with a nonsense allele may be treated using the compositions and methods provided herein. Other diseases or disorders that are also treatable using the compositions and methods provided herein include, but are not limited to, Shwachman-Diamond syndrome, Alport syndrome, Stargardt disease, Usher syndrome, ataxia telangiectasia, hemophilia A and B, Hailey-Hailey disease, Ullrich disease, methylmalonic acidemia, carnitine palmitoyltransferase 1A deficiency, peroxisome biogenesis disorders, limb girdle muscular dystrophy, Schmid metaphyseal chondrodysplasia, Sandhoff disease, Marfan syndrome, anemia, epidermolysis bullosa simplex, Tay-Sachs disease, triose phosphate isomerase deficiency, Alzheimer's disease, long-QT syndrome, insulin resistance, maple syrup urine disease, hereditary fructose intolerance, X-linked severe combined immunodeficiency, infantile neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, polycystic kidney disease, Liddle's syndrome, xeroderma pigmentosum, Fanconi's anemia, p53-associated cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian carcinoma) , esophageal carcinoma, osteosarcoma, ovarian carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian carcinoma, SRY sex reversal, triosephosphate isomerase-anemia, inherited cancers such as those due to BRCA1 nonsense mutations, carbohydrate metabolism disorders, amino acid metabolism disorders, lipoprotein metabolism disorders, lipid metabolism disorders, lysosomal enzymes metabolism disorders, steroid metabolism disorders, purine metabolism disorders, pyrimidine metabolism disorders, metal metabolism disorders, porphyrin metabolism disorders, and heme metabolism disorders.

Any suitable subject can be administered a composition as described herein or treated by a method as described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like) , domestic animals (e.g., dogs and cats) , farm animals (e.g., horses, cows, goats, sheep, pigs) , and experimental animals (e.g., mouse, rat, rabbit, guinea pig) . In some embodiments, the subject is a human. The subject can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero) . In some embodiments, a human can be an embryo, a fetus, a child, or an adult. In some embodiments, a human can be from about: 1 day to about 7 days old, 1 week to about 5 weeks old, 1 month to about 12 months old, 1 year to about 10 years old, 6 months to about 15 years old, 5 years to about 25 years old, 20 years to about 50 years old, 40 years to about 80 years old, 75 years to about 100 years old, or about 90 years to about 130 years old. A mammal can be male or female. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, a subject can be a pregnant subject, such as a pregnant human at an age appropriate for reproduction. In some embodiments, the patient can about 20 years of age. In some embodiments, the patient can 10-30 years of age. In some embodiments, a subject has or is suspected of having a disease or condition. In some embodiments, a subject has or is suspected of having one or more PTCs associated with a disease or condition. In some embodiments, the subject has a disease or condition caused by one or more PTCs. In some embodiments, the subject has one or more Arg-to-stop PTCs.

In some embodiments, a subject has received a diagnosis of a disease or a condition. In some embodiments, a subject has not received a diagnosis of a disease or condition. A diagnosis can include a blood test, a clinical diagnosis based on one or more symptoms, or any combination thereof. A diagnostic (such as a blood test) can confirm a presence or an absence of a mutation (e.g., a UGA PTC) in an mRNA encoding a polypeptide. In some embodiments, a diagnostic test can comprise sequencing (e.g. Sanger sequencing, Illumina sequencing, or sequencing by synthesis) a biological sample from a subject. A presence or an absence of a mutation in a portion of an mRNA can include a plurality of mutations (such as from about 1 to about 200 mutations) . A clinical diagnosis can be based on one or more symptoms, such as, for example, loss of speech, loss of purposeful use of hands, involuntary hand movements, loss of mobility, gait disturbances, loss of muscle tone, seizures, scoliosis, sleep disturbances, slowed growth rate, difficulty breathing, loss of hearing, muffing of speech and sounds, difficulty understanding words, trouble hearing consonants, a loss of vision, a restricted vision field, a cloudiness of vision, a blurred vision, eye discomfort, a cough, a cough with phlegm, fatty stools, infertility, weight loss, salty skin, or any combination thereof.

In some embodiments, a pregnant female subject is administered a composition described herein at one or more stages of pregnancy. In some embodiments, a female subject is administered the composition during a prenatal period. In some embodiments, an embryo or a fetus can be administered a composition as described herein in the womb. In some embodiments, an embryo can be administered a composition as described herein in an in vitro setting.

In some embodiments, a disease or condition can comprise, for example, Rett syndrome, autism, West syndrome, Lennox-Gastaut syndrome, epileptic encephalopathy (EEP) , Pitt-Hopkins syndrome, comprise cystic fibrosis, deafness (e.g. autosomal dominant 17 deafness, autosomal dominant 13 deafness, autosomal dominant 11 deafness) retinitis pigmentosa, Tay-Sachs, Parkinson’s, Cystic Fibrosis, Usher syndrome, Wolman disease, a liver disease (Alpha-1 antitrypsin (AAT) deficiency) , a neurodegenerative disease, a muscular disorder, a metabolic disorder, an ocular disorder (e.g. an ocular disease) , a cancer, albinism, Alzheimer’s disease, Amyotrophic lateral sclerosis, Asthma, β-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, Chronic Obstructive Pulmonary Disease (COPD) , dementia, Distal Spinal Muscular Atrophy (DSMA) , Dystrophic Epidermolysis bullosa, Epidermylosis bullosa, Fabry disease, Factor V Leiden associated disorders, Familial Adenomatous, Polyposis, Galactosemia, Gaucher's Disease, Glucose-6-phosphate dehydrogenase, Hemophilia, Hereditary Hematochromatosis, Hunter Syndrome, Huntington's disease, Hurler Syndrome, Inflammatory Bowel Disease (IBD) , Inherited polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, Mucopolysaccharidosis, Myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-esol related cancer, Parkinson's disease, Peutz-Jeghers Syndrome, Phenylketonuria, Pompe's disease, Primary Ciliary Disease, Prothrombin mutation related disorders, such as the Prothrombin G20210A mutation, Pulmonary Hypertension, Retinitis Pigmentosa, Sandhoff Disease, Severe Combined Immune Deficiency Syndrome (SCID) , Sickle Cell Anemia, Spinal Muscular Atrophy, Stargardt Disease, Dilated cardiomyopathy, X-linked immunodeficiency, various forms of cancer (e.g., BRCA1 and 2 linked breast cancer and ovarian cancer) , muscular dystrophy, an ornithine transcarbamylase deficiency, prostate cancer, a lung cancer, a skin cancer, Stargardt macular dystrophy, Charcot-Marie-Tooth disease, or any combination thereof. A disease or condition can comprise a muscular dystrophy, such as, for example, myotonic, Duchenne, Becker, Limb-girdle, facioscapulohumeral, congenital, oculopharyngeal, distal, Emery-Dreifuss, or any combination thereof. A disease or condition can comprise pain, such as chronic pain. Pain can include neuropathic pain, nociceptive pain, or a combination thereof. Nociceptive pain can include visceral pain, somatic pain, or a combination thereof.

In some embodiments, treatment comprises administration to a subject one or more compositions as described herein. In some embodiments, treatment comprises administration of a co-therapy to a subject, such as, for example, co-therapy comprising the engineered tRNA alongside, for example, a cancer treatment (e.g. radiotherapy, chemotherapy, CAR-T therapy, immunotherapy, hormone therapy, cryoablation) , surgery, antibiotics, antivirals, or any combination thereof. In some embodiments, co-therapy comprises a mucus thinner, cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies, a lung transplant, bronchodilator, airway clearance, an anti inflammatory medication, nebulizer treatment, an oral pancreatic enzyme, a stool softener, elexacaftor, ivacaftor and tezacaftor, lumacaftor, or any combination thereof. In some embodiments, a co-therapy comprises physical therapy, hydrotherapy, occupational therapy, speech-language therapy, feeding assistance, an antiepileptic drug, an antireflux drug, levocarnitine, steroid therapy, nonsteroidal anti-inflammatory drug (NSAID) , vision aids (e.g. glasses, and/or corrective eye surgery) , hearing treatment (e.g., hearing aids) , or any combination thereof. In some embodiments, co-therapy comprises an RNA or DNA editing technology. In some embodiments, treatment includes curing a disease or condition. In some embodiments, treatment includes substantially reducing one or more symptoms of a disease or condition.

In some embodiments, administration of the engineered tRNA results in a reduction in at least one symptom associated with a genetic disease. The amount and form of tRNA or nucleic acid encoding tRNA administered will vary depending on various factors such as, for example, the composition chosen, the particular disease, weight, physical condition, and age of the subject, and whether prevention or treatment is the goal. In some embodiments, the reduction in at least one symptom occurs with less than about 1%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%of restoration of translation of a mRNA comprising a PTC. In some embodiments, the reduction in at least one symptom occurs with less than about 10%of restoration of translation of a mRNA comprising a PTC. In some embodiments, restoration of translation is measured as the amount of full-length protein produced in a sample comprising an mRNA comprising the PTC relative to a comparative sample comprising a comparative mRNA encoding the same protein but not comprising the PTC.

In some embodiments, such as embodiments relating to mammalian subjects, the endogenous DNA sequences encoding the endogenous tRNAs of the subject do not comprise a CCA sequence at the 3’ end of the sequence encoding the acceptor arm. In some embodiments, such as embodiments relating to mammalian subjects, a CCA sequence is attached post-transcriptionally to the acceptor arm at the 3’ end of endogenous tRNAs.

In some embodiments, compositions disclosed herein are in unit dose forms. In some embodiments, compositions disclosed herein are in multiple-dose forms. Unit dose forms, as used herein, can refer to physically discrete units suitable for administration to human or non-human subjects (e.g., animals) . In some embodiments, unit dose forms are packaged individually. In some embodiments, unit dose forms can comprise a mixture of multiple types of engineered tRNAs. For example, a single unit dose can comprise a mixture of 2 or more, 3 or more, 4 or more, or 5 or more different engineered tRNAs. In some embodiments, the different engineered tRNAs suppress the same PTC (e.g., UGA) . In some embodiments each of the different engineered tRNAs suppresses a different PTC (e.g., one or more engineered tRNAs suppress the UGA PTC, and one or more different engineered tRNAs suppress a different PTC) . In some embodiments, at least one of the different engineered tRNAs is an engineered Arg-UGA engineered tRNA described herein. In some embodiments, all of the different engineered tRNAs are different engineered Arg-UGA engineered tRNA described herein.

Each unit dose can contain a predetermined quantity of an active ingredient (s) that can be sufficient to produce the desired therapeutic effect in association with pharmaceutical carriers, diluents, excipients, or any combination thereof. Examples of unit dose forms include ampoules, syringes, and individually packaged tablets and capsules.

In some embodiments, a unit dose form is packaged in a disposable syringe. In some embodiments, a unit dose form is administered in fractions or multiples. A multiple-dose form can be a plurality of identical unit dose forms packaged in a single container, which can be administered in segregated a unit dose form. Examples of a multiple-dose form can include vials, bottles of tablets or capsules, or bottles of pints or gallons. In some embodiments, a multiple-dose form comprises multiple doses of the same pharmaceutically active agents. In some embodiments, a multiple-dose form comprises doses of different pharmaceutically active agents.

In some embodiments, a composition described herein is administered to enable the delivery of an engineered tRNA or a vector encoding an engineered tRNA to a desired site of biological action. In some embodiments, administration includes, for example, oral administration, topical administration, intravenous administration, inhalation administration, or any combination thereof. In some embodiments, delivery includes injection, catheterization, gastrostomy tube administration, intraosseous administration, ocular administration, intracerebroventricular administration, otic administration, transdermal administration, oral administration, rectal administration, nasal administration, intravaginal administration, intracavernous administration, transurethral administration, sublingual administration, intracranial injection, intracranial injection into the parenchyma, intracisternal magna (ICM) , intra-cerebroventricular (ICV) or a combination thereof.

In some embodiments, delivery includes direct application to the affected tissue or region of the body. In some embodiments, topical administration comprises administering a lotion, a solution, an emulsion, a cream, a balm, an oil, a paste, a stick, an aerosol, a foam, a jelly, a foam, a mask, a pad, a powder, a solid, a tincture, a butter, a patch, a gel, a spray, a drip, a liquid formulation, an ointment to an external surface of a surface, such as a skin. In some embodiments, delivery comprises injection, such as, for example, parenchymal injection, intracistemal injection, intraarterial injection, intracistemal injection, intramuscular injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, epidural, or any combination thereof. In some embodiments, delivery is by parenteral administration, such as, for example, an intravenous administering, an intra-arterial administering, an intrathecal administering, an intraocular administering, an otic administering, an intracerebroventricular administering, or an intraperitoneal administering.

In some embodiments, delivery is from a device, such as a pump, an infusion pump, or a combination thereof. In some embodiments, delivery is by an enema, an eye drop, a nasal spray, or any combination thereof. In some embodiments, a subject can administer the composition in the absence of supervision. In some embodiments, a subject can administer the composition under the supervision of a medical professional. In some embodiments, a medical professional administers the composition. In some embodiments, administering can be oral ingestion, such as, for example, comprising ingestion of a tea, an elixir, a food, a drink, a beverage, a syrup, a liquid, a gel, a capsule, a tablet, an oil, a tincture, or any combination thereof. In some embodiments, delivery can be by a capsule or a tablet. In some instances, the capsule comprises hydroxymethylcellulose, gelatin, hydroxypropylmethyl cellulose, pullulan, or any combination thereof. In some embodiments, capsules can comprise a coating, for example, an enteric coating. In some embodiments, capsules are vegetarian vegan, such as a hypromellose capsule. In some embodiments, delivery comprises inhalation by an inhaler, a diffuser, a nebulizer, a vaporizer, or a combination thereof.

In some embodiments, administration is intermittent. In some embodiments, the compositions described herein is administered at a first time point and a second time point. In some embodiments, there is 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more between administrations.

Administration or application of a composition disclosed herein can be performed for a treatment duration of about at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 consecutive or nonconsecutive days.

For example, in some embodiments comprising AAV delivery, the treatment is administered or applied once, twice, or no more than a few times during the life of the subject. Alternatively, for example, in some embodiments comprising delivering synthetic tRNA through carriers such as LNP, the treatment is administered or applied periodically. In embodiments involving, for example, treatment of cystic fibrosis, the treatment may be administered or applied approximately once a week for the lifetime of the subject. In other embodiments, such as embodiments involving, for example, cancer, the treatment may be administered or applied at different intervals, such as one injection approximately every few days for about one, two, three, or four months, or one injection about every one, two, three, or four months.

In some embodiments, a treatment duration can be from about 1 to about 30 days, from about 2 to about 30 days, from about 3 to about 30 days, from about 4 to about 30 days, from about 5 to about 30 days, from about 6 to about 30 days, from about 7 to about 30 days, from about 8 to about 30 days, from about 9 to about 30 days, from about 10 to about 30 days, from about 11 to about 30 days, from about 12 to about 30 days, from about 13 to about 30 days, from about 14 to about 30 days, from about 15 to about 30 days, from about 16 to about 30 days, from about 17 to about 30 days, from about 18 to about 30 days, from about 19 to about 30 days, from about 20 to about 30 days, from about 21 to about 30 days, from about 22 to about 30 days, from about 23 to about 30 days, from about 24 to about 30 days, from about 25 to about 30 days, from about 26 to about 30 days, from about 27 to about 30 days, from about 28 to about 30 days, or from about 29 to about 30 days. Administration or application of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. Administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject. Administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more.

Administration or application of composition disclosed herein can be performed at least 1, 2, 3, 4, 5, or more times a day. In some embodiments, administration or application of composition disclosed herein is performed at least 1, 2, 3, 4, 5, 6, 7, or more times in a week. In some embodiments, administration or application of composition disclosed herein is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more times a month. In some embodiments, administration is continuous or essentially continuous over a preselected period of time. In some embodiments, administration is local. In some embodiments, administration is systemic.

In some embodiments, exogenous genetic material (e.g., encoding one or more engineered tRNAs) is introduced into a cell in vivo by genetic transfer methods, such as transfection or transduction, to provide a genetically modified cell. Exemplary transfection techniques include, but are not limited to, calcium phosphate DNA co-precipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment; and/or strontium phosphate DNA co-precipitation.

VII. KITS

In some embodiments, the engineered tRNA of the present invention is packaged in a kit. In some embodiments, a kit comprises an engineered tRNA. In some embodiments, a kit can comprise a composition described herein and a container. For example, in some embodiments, a kit comprises a pharmaceutical composition, which can comprise an engineered tRNA, a polynucleotide (e.g., vector) encoding the engineered tRNA, or both. In some embodiments, a kit comprises a pharmaceutical composition described herein (e.g. an engineered tRNA and a pharmaceutically acceptable excipient, carrier or diluent, optionally in a dose unit form) . In some embodiments, a kit comprises a packaging or a container. In some embodiments, a kit comprises packaging. In some embodiments, the kit can comprise a container. In some embodiments, the container can be made of plastic, glass, metal, or any combination thereof. In some embodiments, a kit can comprise instructions for use, such as instructions for administration to a subject.

In some embodiments, a packaged product comprising a composition described herein comprises labels. In some embodiments, the pharmaceutical composition described herein is manufactured according to current good manufacturing practice (cGMP) and applicable labeling regulations. In some embodiments, a pharmaceutical composition and/or kit disclosed herein can be aseptic.

Some embodiments relate to one or more methods of making a kit. In some embodiments, the method includes contacting the composition with a packaging or container. In some embodiments, the method includes contacting the composition with a packaging. In some embodiments, the method can includes contacting the composition with a container.

The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

EXAMPLES

Example 1: GENERATION OF ENGINEERED TRNAS AND APPLICATION OF PLASMID-DELIVERED ENGINEERED SUPPRESSOR tRNAS FOR RECOVERY OF PTC-CONTAINING PROTEINS

The following Example describes screening of engineered tRNAs for carrying an arginine that recognize a UGA/TGA stop codon. It also illustrates the use of plasmids to deliver various engineered suppressor tRNAs, enabling the restoration of full-length NanoLuc and p53 proteins containing PTCs.

Materials and Methods

Cloning of suppressor tRNA plasmids

The cassette encoding engineered suppressor tRNAs consisted of a U6 promoter, an engineered suppressor tRNA, and a poly T tail. The engineered suppressor tRNAs are derived from various human non-intron and intron-spliced Arginine tRNAs including tRNA-Arg-ACG-1-1 (TRR-ACG1-1, HGNC: 34964) , tRNA-Arg-ACG-2-1 (TRR-ACG2-1, HGNC: 34664) , tRNA-Arg-CCG-1-1 (TRR-CCG1-1, HGNC: 34933) , tRNA-Arg-CCG-2-1 (TRR-CCG2-1, HGNC: 12346) , tRNA-Arg-TCG-1-1 (TRR-TCG1-1, HGNC: 34827) , tRNA-Arg-TCG-2-1 (TRR-TCG2-1, HGNC: 34979) , tRNA-Arg-TCG-3-1 (TRR-TCG3-1, HGNC: 34939) , tRNA-Arg-TCG-4-1 (TRR-TCG4-1, HGNC: 12345) , tRNA-Arg-TCG-5-1 (TRR-TCG5-1, HGNC: 34593) , tRNA-Arg-TCG-6-1 (TRR-TCG6-1, HGNC: 34584) , tRNA-Arg-CCT-1-1 (TRR-CCT1-1, HGNC: 34638) , tRNA-Arg-CCT-2-1 (TRR-CCT2-1, HGNC: 34744) , tRNA-Arg-CCT-3-1 (TRR-CCT3-1, HGNC: 34730) , tRNA-Arg-CCT-4-1 (TRR-CCT4-1, HGNC: 34975) , tRNA-Arg-CCT-5-1 (TRR-CCT5-1, HGNC: 34833) , tRNA-Arg-TCT-1-1 (TRR-TCT1-1, HGNC: 34695) , tRNA-Arg-TCT-2-1 (TRR-TCT2-1, HGNC: 12343) , tRNA-Arg-TCT-3-1 (TRR-TCT3-1, HGNC: 34670) , tRNA-Arg-TCT-4-1 (TRR-TCT4-1, HGNC: 34728) , tRNA-Arg-TCT-5-1 (TRR-TCT5-1, HGNC: 34571) with substitution of the anticodon with tri-nucleotides UCA, which can base pair with stop codon UGA. The engineered suppressor tRNA sequences are provided as SEQ ID NO: 1 to SEQ ID NO: 27. The original human non-intron and intron-spliced Arginine tRNA sequences are provided as SEQ ID NO: 28 to SEQ ID NO: 47. The name of engineered suppressor tRNAs and their corresponding original human Arginine tRNAs are listed in Table 1.

Table 1: Information on engineered suppressor tRNA

After confirmation of the sequences by Sanger sequencing (GenScript Biotech Corporation) , the cassettes were assembled into the parental plasmid pSpCas9 (BB) -2A-GFP (PX458) by replacing the gRNA cassettes. The resulting plasmids were further constructed by removal of Cas9 cassette and the AAV2 ITR sequence. The DNA sequence of parental plasmid pSpCas9 (BB) -2A-GFP (PX458) is provided as SEQ ID NO: 48. The DNA sequence of constructed suppressor tRNA plasmid ptRNA-GFP is provided as SEQ ID NO: 49. The engineered suppressor tRNA expression plasmids were further generated by replacing the region indicated by the dashed line shown in the portion of the plasmid ptRNA-GFP highlighted in grey in SEQ ID NO: 49) with individual engineered suppressor tRNA sequences as listed in SEQ ID NO: 1-27 and 61-81. As an example, the constructed suppressor tRNA plasmid ptRNA-GFP-R3-147 sequence with engineered suppressor tRNA R3-147 (which originated from human tRNA-Arg-TCT-1-1) is provided as SEQ ID NO: 50. The remaining EGFP expression cassette was used as a loading control to monitor the plasmid expression status after cell transfection.

As a negative control, a scrambled sequence Scr1-4 (also referred to as “Scr” ) derived from the suppressor tRNA R3-147 was constructed into the plasmid ptRNA-GFP-R3-147 by replacing the engineered suppressor tRNA R3-147. The RNA sequence of Scr1-4 is provided as SEQ ID NO: 51.

Suppressor tRNA transfection

The engineered suppressor tRNA screening was carried out in two HEK293 cell lines: Luc_V2_R3 and Luc_V3_R3, each of which stably expressed a different modified NanoLuc luciferase. These modifications include the insertion of stop codon TGA after 195 bp or 474bp in the NanoLuc luciferase gene in the Luc_V2_R3 and Luc_V3_R3 cells, respectively, resulting the premature termination codon and truncated non-functional NanoLuc luciferase, as shown in FIG. 1A. In addition, the modified UGA-containing NanoLuc luciferases were fused with 3x Myc and 3x FLAG at their N-and C-termini, respectively. The DNA sequence encoding the modified NanoLuc Luc_V2_R3 and Luc_V3_R3 are provided as SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

HEK293 cell lines stably expressing a different modified NanoLuc luciferase in which the premature TGA stop codon was replaced with the glycine-coding codon GGA were used as the corresponding positive control, named Luc_V2_R4 (also referred to as V2_R4) , and Luc_V3_R4 (also referred to as V3_R4) , respectively. The DNA sequence encoding the modified NanoLuc Luc_V2_R4 and Luc_V3_R4 are provided as SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

For further evaluation of the suppressor tRNA read through efficiency, the Western Blot and RT-qPCR were carried out in DMS114 cells and Calu6 cells that comprised the premature UGA stop codon in the p53 mRNA. The DMS114 cells contained a homozygous nonsense mutation resulting in the transition of arginine codon into a UGA stop codon, and a truncated p53 protein 212 amino acid in length. Similarly, the Calu6 cells contained an arginine codon mutated into a UGA stop codon, resulting a truncated p53 protein 195 amino acids in length. In addition, A549 cells were used as the positive control, as they expressed the wild-type p53 protein.

The cells were seeded with 1 × 10⁴ cells/well in a 96-well plate in 100 μL high glucose DMEM supplemented with 10%FBS and 2 mM L-Glutamine. Once the confluency reached 80%, the cells were transfected with suppressor tRNA plasmids using HD Transfection Reagent (Promega) according to the manufacturer’s instructions. The mixture of the HD Transfection Reagent and DNA in the ratio of 3: 1 was incubated at room temperature for 10 min, and 5 μL of the mixture was added into cells in a 96-well plate containing 100μl of medium per well. The plasmid with scrambled sequence Scr1-4 was used as a negative control. Cells cultured under medium supplemented with 500 μg/mL or 5 mg/mL G418 was used as a positive control. For each sample, triplicate replications were carried out on the same plate and adjacent to each other. After 48 hours of culturing, PTC suppression by suppressor tRNA was quantified by NanoLuc luciferase assay, qPCR, and Western blot as described below. The cells used for the suppressor tRNA screening are listed in Table 2.

Table 2: Information on cells used

NanoLuc luciferase assay

The NanoLuc luciferase assay was carried out withLuciferase Assay System (Promega) according to the manufacturer’s instructions. Luciferase Assay Reagent was prepared freshly by mixing 1 volume ofLuciferase Assay Substrate with 50 volumes ofLuciferase Assay Buffer. The cell culture plate was equilibrated at room temperature for 10 min, and 100 μL ofLuciferase Assay Reagent was added into each well. After consistent shaking at room temperature for 5 min in the dark, the mixture was further transferred into white solid plate (Greiner) . The luminescence was detected by SpectaMax i3x plate reader (Molecular Devices) with endpoint mode and 200 ms integration time.

Western blot

After washing twice with PBS, the cells were scraped in the presence of ice-cold RIPA lysis buffer (Sangon Biotech) and lysed on ice for 10 min. The supernatant was collected after 10 min centrifugation with 21,000 g at 4℃, and the protein concentration was determined with Modified BCA Protein Assay Kit (Sangon Biotech) on SpectaMax i3x plate reader (Molecular Devices) . After adjusting the protein concentration, 5x SDS loading buffer was added to the sample, which was further denatured at 98℃ for 10 min. The sample was then flash frozen in liquid nitrogen and stored at -80℃. To separate the protein sample, 20 μg of proteins were loaded on 4-20%SurePAGE^TM Bis-Tris (Genscript) and run in MES buffer (Genscript) at 150 V for 1 hour via a BIO-RAD system. The protein bands were transferred to 0.22 μm PVDF membrane at 20 V for 7 min via iBlot^TM 2 Gel Transfer Device (Invitrogen) . The membrane was blocked with 3%BSA (Beyotime) in 1x TBST for 1 hour at room temperature, and incubated with primary antibody (Table 3) for 1 hour at 4℃. After the three 10 min washes with 1x TBST, the membrane was incubated with the secondary antibody (Table 3) hr at room temperature. Following another three 10 min washes with 1x TBST, the targeted protein was detected with High sensitive Plus ECL luminescence reagent (Sangon Biotech) , and imaged using ChemiDoc MP Imaging System (BioRad) .

Table 3: Antibody information used in Western Blot

RNA isolation

After washing twice with PBS, the cells were scraped in the presence of 1 mL of TRIzol^TM Reagent (Thermofisher Scientific: 15596026) and incubated for 5 min at room temperature. Cell debris were removed by centrifugation for 5 min at 12,000 g and 4 ℃, the supernatant was transferred into a new tube containing 0.2 mL of chloroform and mixed thoroughly by shaking. After 5 min incubation at room temperature and 15 min centrifugation at 12,000 g and 4 ℃, the mixture was separated into three phases. The upper aqueous phase was then transferred into a new tube containing 0.5 mL of isopropanol and mixed by vortexing. After a 10 min incubation at 4 ℃, the RNA was precipitated by centrifugation for 10 min at 12,000 g and 4 ℃. The RNA pellet was further washed by 1 mL of 75 %ethanol, air dried, and resuspended in 50 μL of RNase-free water. The RNA was further treated with DNase I (New England Biolabs, M0303S) to remove potential DNA contamination and stored in -80 ℃.

RT-qPCR

The concentration of RNA was determined by nanodrop, and equal amounts of RNAs were reverse-transcribed with PrimeScript RT Reagent Kit (TAKARA, RR037A) according to the manufacturer’s instructions. The obtained cDNAs were then used for gene expression quantification with TB Green Premix Ex Taq II (TAKARA, RR82WR) according to the manufacturer’s instructions. The qPCR was performed on the 480 System (Roche) , and the program was set up as: pre-denaturing step at 95 ℃ for 30 seconds, 40 times of amplification cycle including denaturing at 95 ℃ for 5 seconds and annealing plus extending at 60 ℃ for 30 seconds. The primers used for the qPCR are listed in Table 4.

Table 4: Primer information used in qPCR

Results

Native Arginine tRNA-derived suppressor tRNAs exhibit variable PTC suppression efficacy

Native non-intron or intron-spliced Arginine tRNA tRNA-Arg-ACG-1-1, tRNA-Arg-ACG-2-1, tRNA-Arg-CCG-1-1, tRNA-Arg-CCG-2-1, tRNA-Arg-CCT-1-1, tRNA-Arg-CCT-2-1, tRNA-Arg-CCT-3-1, tRNA-Arg-CCT-4-1, tRNA-Arg-CCT-5-1, tRNA-Arg-TCG-1-1, tRNA-Arg-TCG-2-1, tRNA-Arg-TCG-3-1, tRNA-Arg-TCG-4-1, tRNA-Arg-TCG-5-1, tRNA-Arg-TCG-6-1, tRNA-Arg-TCT-1-1 (FIG. 2A) , tRNA-Arg-TCT-2-1, tRNA-Arg-TCT-3-1, tRNA-Arg-TCT-4-1 and tRNA-Arg-TCT-5-1 were engineered and synthesized with substitution of the native anticodon with the tri-nucleotide UCA, which can base pair with the stop codon UGA. These engineered suppressor tRNAs R3-1, R3-2, R3-3, R3-4, R3-5, R3-6, R3-7, R3-8, R3-9, R3-10, R3-11, R3-12, R3-13, R3-14, R3-15, R3-147, R3-148, R3-149, R3-150 and R3-151 were further cloned into suppressor tRNA plasmid ptRNA-GFP (SEQ ID NO: 49) by replacing the dashed line shown in SEQ ID NO: 49 with individual suppressor tRNA, which were then transfected into NanoLuc reporter cell lines Luc_V2_R3 and Luc_V3_R3 for a NanoLuc luciferase assay as depicted in FIG. 1A. Engineered suppressor tRNAs that read through the PTC in the NanoLuc gene of the NanoLuc reporter cell line result in the production of full-length, functional NanoLuc protein. This protein reacts with the substrate in the reagent, emitting luminescence. The differing readthrough capabilities of various engineered suppressor tRNAs led to variations in the yield of the full-length, functional NanoLuc protein, which is reflected in the changes in luminescence intensity. The data from NanoLuc luciferase assay showed unequal PTC suppression efficacy (FIGS. 1B-1C) , suggesting that portions of the tRNA sequence other than the anticodon affect the capability of suppressor tRNA. Among the screened suppressor tRNAs, R3-8 and R3-147, derived from human tRNA-Arg-CCT-4-1 and tRNA-Arg-TCT-1-1, respectively, showed the highest luciferase activities in both NanoLuc reporter cell lines (FIGS. 1B-1C) , whereas the negative control transfected with Scr1-4 exhibited minor luciferase activity.

Six candidates, R3-7, R3-8, R3-13, R3-147, R3-148, and R3-149, were chosen for further testing by Western blot and RT-qPCR. The Western blots and RT-qPCR were carried out using two distinct p53-UGA cell lines, Calu6 and DMS114, which were transfected with the selected suppressor tRNA. As an aminoglycoside, G418 exhibited the ability in PTC read through, and was used as another positive control. In both cell lines, R3-147 rescued the most full-length p53 protein, to a comparable level as in the positive control cell line A549 (FIGS. 1D-1E) . No full-length p53 protein was detected in the negative controls, which included the DMS114 cell only and cell lines transfected with Scr1-4 (FIGS. 1D-1E) . Cell lines treated with different concentrations of G418 showed no or very low amounts of full-length p53 protein production (FIGS. 1D-1E) . The RT-qPCR results showed a high level of the premature UGA stop codon containing p53 mRNA in both cell lines transfected with R3-147, at more than 4.5 times higher (FIG. 1F) or 1.5 times higher (FIG. 1G) than the p53 mRNA level in the positive control cell line A549, demonstrating the escape of the PTC containing p53 mRNA from the NMD pathway. These results demonstrate that engineered suppressor tRNAs can effectively read through PTCs at various positions in different genes to produce full-length, functional proteins, and that they can partially prevent the degradation of mRNA containing PTCs.

Modifications of the suppressor tRNAs boost PTC suppression efficiency

Based on R3-147, multiple modifications were introduced into the anticodon arm and the tail to produce engineered tRNAs as described in FIGS. 2C-2I, and the PTC suppression efficiency of these were measured using the NanoLuc luciferase assay described in FIG. 1A. Engineered tRNA “M2” (FIG. 2C, also referred to as R3-147-M2) has a C at the 4th nucleotide 3’ to the anticodon (labeled as nucleic acid position 40) , while R3-147 has a U at position 40 (FIG. 2B) . Engineered tRNA “M3” (FIG. 2D, also referred to as R3-147-M3) has a 3’ CCA tail encoded at the DNA level. Engineered tRNA “M4” (FIG. 2E, also referred to as R3-147-M4) has a G at the 1st nucleotide 3’ to the anticodon (labeled as position 37) , while R3-147 has an A at position 37 (FIG. 2B) . Engineered tRNA “M5” (FIG. 2F, also referred to as R3-147-M5) has the modifications of both M2 and M3. Engineered tRNA “M6” (FIG. 2G, also referred to as R3-147-M6) has the modifications of both M2 and M4. Engineered tRNA “M7” (FIG. 2H, also referred to as R3-147-M7) has the modifications of both M3 and M4. Engineered tRNA “M8” (FIG. 2I, also referred to as R3-147-M8) has the modifications of M2, M3, and M4.

As shown in FIG. 1H, each modified engineered suppressor tRNA-M2, M3, M4, M5, M6, M7, and M8-exhibited varying degrees of PTC read-through efficiency. Specifically, M2, M3, M4, M5, and M7 demonstrated nearly or more than a 2-fold increase in PTC read-through efficiency, indicating that modifications at these specific sites significantly enhance the suppressor tRNAs'capability to overcome PTCs. In contrast, M6 and M8 showed relatively weaker improvements in PTC read-through efficiency, likely due to the simultaneous alteration of the 1st and 4th nucleotides 3’ to the anticodon, which may diminish the enhancement achieved by individual modifications.

Based on M2 and M4, additional modifications were introduced to the tRNA molecules, successfully creating the engineered suppressor tRNA M22 (FIG. 2J, also referred to as R3-147-M22) , M23 (FIG. 2K, also referred to as R3-147-M23) , M24 (FIG. 2L, also referred to as R3-147-M24) , M25 (FIG. 2M, also referred to as R3-147-M25) , M26 (FIG. 2N, also referred to as R3-147-M26) , M46 (FIG. 2O, also referred to as R3-147-M46) , M50 (FIG. 2P, also referred to as R3-147-M50) , M54 (FIG. 2Q, also referred to as R3-147-M54) , M55 (FIG. 2R, also referred to as R3-147-M55) , M56 (FIG. 2S, also referred to as R3-147-M56) , M57 (FIG. 2T, also referred to as R3-147-M57) , M58 (FIG. 2U, also referred to as R3-147-M58) , M59 (FIG. 2V, also referred to as R3-147-M59) , M62 (FIG. 2W, also referred to as R3-147-M62) , M63 (FIG. 2X, also referred to as R3-147-M63) , M65 (FIG. 2Y, also referred to as R3-147-M65) , M67 (FIG. 2Z, also referred to as R3-147-M67) , M100 (FIG. 2AA, also referred to as R3-147-M100) , M102 (FIG. 2BB, also referred to as R3-147-M102) , M103 (FIG. 2CC, also referred to as R3-147-M103) , and M108 (FIG. 2DD, also referred to as R3-147-M108) . The PTC suppression efficiency of these engineered suppressor tRNAs were measured using the NanoLuc luciferase assay described in FIG. 1A.

NanoLuc luciferase assays revealed that M54 exhibited approximately 11 times the read-through efficiency of R3-147 (Figure 4A) . In contrast, M2 showed only about twice the read-through efficiency of R3-147 in the same assay (FIG. 1H) . This indicates that these additional modifications significantly enhance the suppressor tRNA's ability to overcome PTCs.

As shown in FIG. 4A, many of the engineered tRNAs demonstrated increased PTC suppression efficiency compared to R3-147 in the NanoLuc luciferase assay. Specifically, engineered tRNAs M23, M26, and M108 exhibited more than twice the luciferase activity of R3-147. Engineered tRNA M55 displayed about 7-times greater activity. Engineered tRNAs M54, M59, M63, and M67 showed approximately 12-times greater activity. Engineered tRNA M62 exhibited about 18-times greater activity, and engineered tRNA M65 showed about 30-times greater activity (FIG. 4A) . These results indicate that the additional modifications significantly enhance the suppressor tRNAs’a bility to overcome PTCs.

Example 2: DETERMINATION OF THE AMINO ACID CARRIED BY ENGINEERED SUPPRESSOR TRNA

The following example describes the determination of the amino acid carried by the engineered suppressor tRNA recognizing a UGA/TGA stop codon.

Materials and Methods

Suppressor tRNA transfection

Suppressor tRNA transfection into NanoLuc reporter cell line Luc -V2-R3 was conducted following the methodology outlined in Example 1.

Immunoprecipitation

After two washes with PBS, the cells were scraped in the presence of ice-cold RIPA lysis buffer (Sangon Biotech) supplemented with protease inhibitor cocktail (Roche) and benzonase (Sigma) , and then lysed on ice for 10 minutes. The supernatant (whole cell extract, WCE) was collected after centrifugation at 12,000 g for 10 minutes at 4℃. The anti-FLAG Magnetic Beads (Thermo Scientific) were equilibrated at room temperature and then washed three times with the lysis buffer. The whole cell extract was incubated with the anti-FLAG Magnetic Beads at room temperature with mixing for 30 minutes. The supernatant (flow-through, FT) was collected, and the beads were washed twice with PBS and once with water. Subsequently, the protein bound to the beads was eluted with 50 μL of 2x SDS-PAGE Sample Buffer and denatured at 95℃ for 10 minutes with rotation at 1000 rpm. The supernatant (immunoprecipitated, IP) was collected, flash-frozen in liquid nitrogen, and stored at -80℃.

Western Blot

Western blot was conducted following the methodology outlined in Example 1.

Mass spectrometry and data analysis

The remaining IP samples were subsequently forwarded to the service company (Applied Protein Technology) for mass spectrometry analysis. Mass spectrometry (MS) analysis was performed using an HPLC system (Easy-nLC 1000, Thermo Fisher Scientific) coupled with a mass spectrometer (Q Exactive, Thermo Fisher Scientific) . For data processing, the raw data were analyzed using MaxQuant 1.6.14 and a library containing the modified NanoLuc proteins with 20 different amino acid insertions after position 66 (nucleotide position 195) . The amino acid sequence of the full-length modified NanoLuc protein is provided as SEQ ID NO: 56. The potentially generated proteins after PTC readthrough were created by replacing the region indicated by the dashed line in the modified NanoLuc protein (highlighted in grey in SEQ ID NO: 56) with one of the 20 different amino acids. For example, an exemplary amino acid sequence of the full-length modified NanoLuc protein, which incorporates the amino acid arginine, is provided as SEQ ID NO: 57. Parameters included a maximum of 2 missed cleavages, 20 PPM peptide mass tolerance, 0.1 Da fragment mass tolerance, and trypsin digestion, with Carbamidomethyl (C) specified as the fixed modification and Oxidation (M) as the variable modification. The resulting dataset was further analyzed at the peptide level, considering only peptides with Andromeda scores greater than 40. The fraction of amino acids carried by the engineered suppressor tRNA was determined by comparing the intensity of peptides with specific amino acid insertions to the total intensity of all peptides containing an inserted amino acid.

Results

The resulting full-length NanoLuc protein after PTC readthrough incorporates the amino acid arginine at the desired position

Luc-V2-R3 cells were transfected with plasmids containing various engineered suppressor tRNAs. These cells carry the NanoLuc gene with a PTC, which, when read through by the engineered suppressor tRNAs, produces full-length NanoLuc protein fused with a C-terminal FLAG tag (FIG. 1A, top panel) . After immunoprecipitation, the resulting immunoprecipitated protein samples were analyzed via Western blot. As shown in FIG. 1P, the full-length modified NanoLuc protein was significantly more abundant in the immunoprecipitation (IP) samples compared to the whole cell extract (WCE) samples. No signal was observed in the flow-through (FT) samples or in the negative control Scr1-4, confirming the read-through capability of the engineered suppressor tRNAs and the successful enrichment of full-length NanoLuc protein through immunoprecipitation. Additionally, mass spectrometry and subsequent data analysis of the protein bands identified full-length proteins with the insertion of only the amino acid arginine, as shown in Table 5. This result clearly demonstrates that these engineered suppressor tRNAs exclusively carry the amino acid arginine.

Table 5: The fraction of amino acid carried by the engineered suppressor tRNA

Example 3: EXPRESSION OF ENGINEERED SUPPRESSOR TRNAS AND THEIR IMPACT ON TRNAOME HOMEOSTASIS DYNAMICS

The following example describes the expression levels and resulting products of some exemplary engineered suppressor tRNAs and their impact on the dynamics of tRNAome homeostasis.

Materials and Methods

Suppressor tRNA transfection

Small RNA isolation

Small RNA isolation from cells was performed following the protocol provided with the RNAiso for Small RNA Kit (Takara, 9753A) , adhering to the manufacturer’s guidelines. The concentration of small RNA was measured using a Nanodrop spectrophotometer and stored at -80℃.

Library preparation and data processing

The preparation of libraries for small RNA sequencing and RNA sequencing using an Illumina sequencer were conducted according to industry standards. The obtained sequencing data underwent rigorous quality assessment using FastQC software and cleaning with Trimmomatic software. The cleaned data were then compared to a tRNA reference sequence (https: //gtrnadb. ucsc. edu/index. html, Homo sapiens (GRCh38/hg38) ) , which includes all human tRNA sequences with CCA attached at the 3' end, using Bowtie2 software. This enabled a comprehensive analysis of all mature tRNAs within the cells. Total counts for each endogenous mature tRNA were used as normalization parameters for processing the different samples. Additionally, tRNAs were categorized based on their characteristics into isodecoders (tRNAs with the same anticodon) and isoacceptors (tRNAs carrying the same amino acid) for statistical analysis.

Results

The suppressor tRNAs are expressed and present in full-length form

The plasmids containing the suppressor tRNAs were transfected into the NanoLuc reporter cell line Luc_V2_R3, and all the tRNAs were isolated and analysed following the method outlined by Hu et al. (2021) (Hu, J. et al. (2021) . “Quantitative mapping of the cellular small RNA landscape with AQRNA-seq. ” Nature biotechnology. 39: 8: 978-988. doi: 10.1038/s41587-021-00874-y. PMID: 33859402) .

As illustrated in the left panel of FIG. 1I, the engineered suppressor tRNA R3-8 was expressed in cells transfected with the R3-8 plasmid, whereas no R3-8 signal was detected in cells transfected with other plasmids. Similarly, the engineered suppressor tRNA R3-147 was only expressed in cells transfected with the R3-147 plasmid, as shown in the right panel of FIG. 1I.

Importantly, all these engineered suppressor tRNAs were present in their full-length forms, with no fragments observed. Additionally, endogenous tRNAs corresponding to R3-8 and R3-147, specifically Arg-CCT-4-1 (FIG. 1K, left panel) and Arg-TCT-1-1 (FIG. 1K, right panel) , were detected in the transfected cells, with expression levels similar to those in cells transfected with Scr1-4 or treated with water ( "Blank" ) as negative controls. The observed decreases in specific positions in the line graph were attributed to nucleotide jumping during reverse transcription at nucleotides with specific chemical modifications in the tRNAs.

The modified suppressor tRNA M2-M8 were also subjected to tRNA sequencing. As shown in the left panel of FIG. 1T, the engineered suppressor tRNA R3-147 was detected in cells transfected with both the R3-147 and M3 plasmids. This finding is due to R3-147 and M3 having identical sequences after transcription and maturation in the cells. The tRNA R3-147 signal in cells transfected with the M3 plasmid was notably higher than in cells transfected with the R3-147 plasmid, suggesting that the addition of the CCA sequence at the DNA level enhances the abundance of exogenous tRNA in cells. This increase is likely due to improved tRNA stability or prevention of degradation from errors in CCA tail addition, rather than increased transcription levels (as the same U6 promoter was used) . Similarly, as shown in the right panel of FIG. 1T, the engineered suppressor tRNA M2 was observed only in cells transfected with the M2 plasmid, with expression levels comparable to those of mature tRNA R3-147 in cells transfected with the R3-147 plasmid. The left panel of FIG. 1U shows that the engineered suppressor tRNA M4 was present only in cells transfected with the M4 plasmid, with levels slightly lower than those of mature tRNA R3-147. Importantly, all engineered suppressor tRNAs were present in their full-length forms. Sequencing of cells transfected with Scr1-4 revealed very low expression levels of Scr1-4 (FIG. 1U, right panel) , with reads only slightly above the lower limit of detection (LLOD) . While some Scr1-4 sequence signals were detected in cells transfected with engineered suppressor tRNAs, these were very low and below the LLOD, thus considered background noise. Furthermore, as shown in FIG. 1V, endogenous tRNA Arg-TCT-1-1 corresponding to the engineered suppressor tRNAs was detected in all transfected cells, with expression levels similar to those in cells transfected with the Scr1-4 negative control. This confirmed that modifications to tRNA did not affect the levels of endogenous related tRNAs.

The remarkably successful suppressor tRNA M54, which demonstrated approximately a 12-fold increase in luminescence signal compared to R3-147, was also analyzed through tRNA sequencing. As shown in FIG. 1J, expression of the engineered suppressor tRNA M54 was observed only in cells transfected with the M54 plasmid, and it was present in its full-length form with no fragments. Additionally, the corresponding endogenous tRNA Arg-TCT-1-1 was detected in the transfected cells (FIG. 1K, right panel) , with expression levels comparable to those in cells transfected with Scr1-4 or treated with water ("Blank") as negative controls. Notably, the expression level of M54 was approximately 6-times higher than that of R3-147 (FIG. 1I, right panel) and about 10-times higher than the corresponding endogenous tRNA Arg-TCT-1-1 (FIG. 1K, right panel) .

The expression of suppressor tRNAs maintains the homeostasis of mature cytoplasmic tRNAs

Compared to cells transfected with the negative control Scr1-4, cells transfected with plasmids carrying engineered suppressor tRNAs showed no significant alterations in the levels of mature endogenous tRNAs, whether measured at the single tRNA level (FIGS. 1L-1N and 1W-1Z, left panel) , the isodecoder level (FIGS. 1L-1N and 1W-1Z, middle panel) , or the isoacceptor level (FIGS. 1L-1N and 1W-1Z, right panel) . Even with high levels of M54 expression, no notable differences in mature endogenous tRNA abundance were observed compared to the negative control Scr1-4 (FIG. 1N) . These findings suggest that the expression of engineered suppressor tRNAs does not disturb tRNA homeostasis. Additionally, no significant differences were found between the water-treated blank control (FIG. 1O) and cells transfected with the negative control Scr1-4.

Example 4: APPLICATION OF ENGINEERED SUPPRESSOR tRNAs DELIVERED BY LENTIVIRUS IN THE RECOVERY OF NANOLUC PROTEIN

The following example illustrates the use of lentiviruses to deliver various engineered suppressor tRNAs, thereby restoring the function of the NanoLuc protein containing a PTC.

Materials and Methods

Lentiviral transduction

The cell line Luc_V2_R3 was digested with 0.25%trypsin and plated into 24-well plates at a density of 10,000 cells per well for lentivirus transduction after 24 hours. Based on the provided virus infection titer, the required amounts of lentiviruses LV_R3-5, LV_R3-8 and LV_R3-147 were calculated to achieve MOI (multiplicity of infection) of 2 and 20. The viruses were diluted in 0.5 mL DMEM medium with 2%FBS, and polybrene was added to a final concentration of 8 μg/mL. The original medium was discarded, the virus-containing medium was added individually, and the cells were incubated overnight at 37℃ in a 5%CO2 cell culture incubator. After 24 hours, replace the medium with fresh normal medium and perform NanoLuc luciferase assays 48 hours later.

NanoLuc luciferase assay

NanoLuc luciferase assay was conducted following the methodology outlined in Example 1.

Flow Cytometry (FCM) Analysis

The GFP expression levels were analyzed using a flow cytometer. For each sample, 10,000 cells were gated based on forward light scatter. The GFP fluorescence signal was collected through a 488 nm band-pass filter, and the GFP fluorescence intensity was defined as the GFP-A(green fluorescence signal) mean of 10,000 cells. P1 and P2 regions were determined to separate the lower fluorescence cluster (P1) from the higher fluorescence cluster (P2) . P1 cell clusters were defined as GFP-non-expressing cells, while P2 cell clusters were defined as GFP-expressing cells. The percentage of P2 cell clusters represented the transduction efficiency of the lentivirus.

Results

The lentivirus-delivered engineered tRNAs restores function of NanoLuc protein containing a PTC

The lentiviral vector RNA sequence (SEQ ID NO: 58) was modified by replacing the region marked with dashed lines and highlighted in gray with the reverse complementary RNA sequences of the engineered suppressor tRNAs R3-5 (FIG. 3A, corresponding to SEQ ID NO: 13) , R3-8 (FIG. 3B, corresponding to SEQ ID NO: 16) , and R3-147 (FIG. 2B, corresponding to SEQ ID NO: 1) . This created lentiviral (LV) vectors carrying the engineered suppressor tRNAs. Additionally, the vector included a green fluorescent protein (GFP) expression cassette as a marker for monitoring transduction efficiency. The lentivirus encapsulating this vector was then used in experiments.

When the lentivirus carrying the engineered suppressor tRNAs was used to infect Luc-V2-R3 cell lines, a high proportion of GFP-positive cells was observed (FIGS. 1Q-1S) . This indicates that the vector not only successfully transduced the cells but also integrated into the cell genome. Additionally, these samples displayed NanoLuc luciferase activity (FIGS. 1Q-1S) , confirming the proper expression of the engineered suppressor tRNAs within the cells and demonstrating that these tRNAs can effectively perform their PTC readthrough function within the lentiviral vector. Furthermore, as the multiplicity of infection (MOI) decreased, both the proportion of GFP-positive cells and luminescence signal showed a gradual decline (FIGS. 1Q-1S) . This indicates that the expression level of the engineered suppressor tRNAs in the cells is affected by the MOI, which indirectly confirms that the observed luminescence signals are due to the production of full-length NanoLuc protein through readthrough, rather than background noise. It also reinforces stable expression of the engineered suppressor tRNAs by the lentiviral vector.

Example 5: APPLICATION OF ENGINEERED SUPPRESSOR TRNAS ON READING THROUGH OF PTC IN THE COL4A5 GENE ASSOCIATED WITH ALPORT SYNDROME

The following Example describes the surprising ability of engineered suppressor tRNA to restore mRNA and protein levels of the COL4A5 (COLLAGEN IV ALPHA V) gene containing a PTC in in-vitro lentivirus assays. The PTC in the COL4A5 gene can induce Alport syndrome.

Materials and Methods

Cell culture

The A549 cell line was selected for generating PTC cell lines as COL4A5 R373*, where the codon coding arginine at position 373 was mutated to a stop codon. Additionally, another PTC cell line, COL4A5 R1563*, was created by changing the codon coding arginine at position 1563 to a stop codon. These cell lines-A549, A549 COL4A5 R373*, and R1563*-were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10%FBS (Thermo Fisher Scientific) .

Lentiviral transduction

The A549 and COL4A5 PTC cell lines were digested with 0.25%trypsin and plated into 24-well plates at a density of 30,000 cells per well for lentivirus transduction after 24 hours. Based on the provided virus infection titer, the required amounts of lentiviruses LV_R3-147 and LV_R3-147-M54 were calculated to achieve an MOI (multiplicity of infection) of 20. The viruses were diluted in 0.5 mL DMEM medium with 2%FBS, and polybrene was added to a final concentration of 8 μg/mL. The original medium was discarded, the virus-containing medium was added individually, and the cells were incubated overnight at 37℃ in a 5%CO2 cell culture incubator.

Cell screening

Twenty-four hours after lentiviral transduction, the virus-containing medium was replaced, and the cells were transferred to a 6-well plate. Puromycin was added to the medium at a final concentration of 1 μg/mL to select for cells with integrated lentivirus. Once the cells were growing stably, they were cultured in normal medium for further experiments.

RNA isolation

Total RNA extraction from cells was performed following the protocol provided with the Total RNA Miniprep Kit (New England Biolabs, T20120S) , adhering to the manufacturer’s guidelines. Subsequently, the RNA underwent DNase I treatment (New England Biolabs, M0303S) to eliminate potential DNA contamination. The concentration of total RNA was measured using a Nanodrop spectrophotometer and stored at -80℃.

RT-qPCR

Equivalent amounts of RNA were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814) following the manufacturer’s protocol. The resulting cDNAs were then utilized for gene expression quantification employing the TaqMan^TM Fast Advanced Master Mix (Thermo Fisher Scientific, 4444556) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted on the 480 System (Roche) , with the following program setup: a pre-denaturation step at 95 ℃ for 30 seconds, followed by 40 cycles of amplification comprising denaturation at 95 ℃ for 5 seconds and annealing plus extension at 60 ℃ for 30 seconds. The primers utilized for the qPCR are detailed in Table 6.

Table 6: Primer information used in qPCR

Immunofluorescence analysis

Cells were plated at a density of 50,000 cells per well in a 24-well plate and incubated in a 5%CO2 cell incubator at 37℃ for 24 hours. Subsequently, the cells were fixed with 500 μL of 4%paraformaldehyde (in PBS, pH 7.4) for 10 minutes and washed three times for 10 minutes each with 500 μL of ice-cold PBS. Afterward, the cells were permeabilized with 500 μL of PBS containing 0.1%Triton-X 100 for 10 minutes and washed three times for 10 minutes each with 500 μL of PBS. To block nonspecific antibody binding, the cells were treated with 500 μL of PBS containing 10%goat serum for one hour. Following three washes of 5 minutes each with 500 μL of PBS, the cells were incubated overnight at 4℃ with the primary antibody Anti-COL4A5 H53 (Table 3) at a 1: 500 dilution in PBS containing 10%goat serum. After washing three times for 10 minutes each with PBST (PBS + 0.1%Tween 20) , the cells were incubated with the secondary antibody Goat anti-Rat AF647 (Table 3) at a dilution of 1: 1000 in PBS for 1 hour in the dark at room temperature. Subsequently, the cells were washed three times for 10 minutes each with PBST, and the nuclei were stained with Hoechst 33342 at a dilution of 1: 200 in PBST for 5 minutes. After three additional washes for 10 minutes each with PBST, the cells were stored at 4℃ in 500 μL of PBS solution. Immunofluorescence signals were visualized using a confocal microscope (Nikon) . ImageJ software was utilized to analyze the integrated density of the COL4A5 signal, and to count the cells based on the Hoechst 33342 signal.

Results

The lentivirus-delivered engineered suppressor tRNA restores mRNA levels of COL4A5 gene containing PTCs

The lentiviral vector RNA sequence (SEQ ID NO: 58) was modified by replacing the region marked with dashed lines and highlighted in gray with the reverse complementary RNA sequences of the engineered suppressor tRNAs R3-147 (FIG. 2B, corresponding to SEQ ID NO: 1) and R3-147-M54 (FIG. 2Q, corresponding to SEQ ID NO: 72) . This created lentiviral (LV) vectors carrying the engineered suppressor tRNAs. Additionally, the vector included a puromycin resistance gene (PuroR) as a selection marker. The lentivirus encapsulating this vector was then used in experiments.

As shown in FIG. 4B (left panel) , compared to untreated PTC cells, the mRNA levels in the COL4A5 R1563*cell line transduced with the lentivirus carrying the engineered suppressor tRNA increased. Specifically, cells transduced with LV_R3-147 showed an approximate 1-fold increase in COL4A5 mRNA levels, reaching about one-quarter of the normal mRNA levels found in wild-type A549 cells. Cells transduced with LV_R3-147-M54 exhibited an approximate 3-fold increase in COL4A5 mRNA levels, achieving about 60%of the normal mRNA levels in wild-type A549 cells. Consistent with the NanoLuc luciferase assay results, M54 exhibited a much higher readthrough capability for the PTC compared to R3-147.

Similarly, in another PTC containing cell line COL4A5 R373* (FIG. 4B, right panel) , cells transduced with the engineered suppressor tRNA R3-147-M54 also showed a significant improvement. The mRNA levels increased approximately 3-fold compared to untreated PTC cells, reaching about 45%of the normal mRNA levels in wild-type A549 cells. These experimental results demonstrate that the engineered suppressor tRNA can be effectively transduced into different cells using a lentiviral vector to achieve expression and function. Additionally, the engineered suppressor tRNA R3-147-M54 can effectively restore mRNA levels for PTCs located at various positions within a gene.

The lentivirus-delivered engineered suppressor tRNA restores full-length protein level of COL4A5 gene containing PTC

Since the engineered suppressor tRNA restores COL4A5 mRNA levels, its effect on the restoration of full-length COL4A5 protein levels in PTC cell lines was further evaluated. Quantitative results from immunofluorescence analysis of full-length COL4A5 protein are shown in FIG. 4C. Compared to untreated PTC cell lines, the expression of full-length COL4A5 protein in the COL4A5 R373*cell line increased 3-fold after transduction with LV_R3-147-M54, correlating with the mRNA restoration levels. These results demonstrate that the engineered suppressor tRNA not only restores the mRNA levels of PTC-containing genes but also restores full-length protein expression proportionally, validating their efficiency and reliability.

Example 6: APPLICATION OF ENGINEERED SUPPRESSOR tRNAs DELIVERED BY AAV IN THE RECOVERY OF NANOLUC PROTEIN

The following example illustrates the use of AAVs with different strand types and serotypes to deliver engineered suppressor tRNA, thereby restoring the function of the NanoLuc protein containing a PTC.

Materials and Methods

AAV transduction

The NanoLuc reporter cell line Luc-V2-R3 was digested with 0.25%trypsin and plated into 24-well plates at a density of 10, 000 cells per well for AAV transduction after 24 hours. Based on the provided virus physical titer, the required amounts of AAV were calculated to achieve a series of MOI (multiplicity of infection) gradients, ranging from 2.00E+05 to 2.5E+04 vg/cell for ssAAV2/9 and 5.00E+04 to 1.25E+04 vg/cell for scAAV2/9. The viruses were diluted in 0.2 mL of DMEM medium with 2%FBS. The original medium was discarded, and the virus-containing medium was individually added to each well. The cells were incubated at 37℃ in a 5%CO2 cell culture incubator for 1 hour, after which another 0.3 mL of DMEM medium with 20%FBS was added to each well. The plate was then incubated at 37℃ in a 5%CO2 cell culture incubator for another 72 hours before detection.

NanoLuc luciferase assay

Flow Cytometry (FCM) Analysis

The mCherry expression levels were analyzed using a flow cytometer. For each sample, 10,000 cells were gated based on forward light scatter. The mCherry fluorescence signal was collected through a 587 nm band-pass filter, and the mCherry fluorescence intensity was defined as the mCherry-A (red fluorescence signal) mean of 10,000 cells. P1 and P2 regions were determined to separate the lower fluorescence cluster (P1) from the higher fluorescence cluster (P2) . P1 cell clusters were defined as mCherry-non-expressing cells, while P2 cell clusters were defined as mCherry-expressing cells. The percentage of P2 cell clusters represents the transduction efficiency of AAV.

Results

The scAAV2/9-delivered engineered tRNAs restores function of NanoLuc protein containing a PTC

The self-complementary AAV (scAAV) vector DNA sequence (SEQ ID NO: 60) was modified by replacing the regions marked with dashed lines and highlighted in gray with the DNA sequences of the engineered suppressor tRNAs R3-5 (FIG. 3A, corresponding to SEQ ID NO: 13) , R3-8 (FIG. 3B, corresponding to SEQ ID NO: 16) , and R3-147 (FIG. 2B, corresponding to SEQ ID NO: 1) . This created scAAV vectors carrying the engineered suppressor tRNAs, as shown in FIG. 1AA, which were packaged into AAV2/9 capsids by a vendor.

Infecting the Luc-V2-R3 cell line with these packaged scAAV2/9 particles resulted in NanoLuc luciferase activity (FIG. 1BB) . This indicates that the scAAV vector not only successfully transduced the cells but also ensured proper expression of the engineered suppressor tRNA, demonstrating its PTC readthrough function. Additionally, as the multiplicity of infection (MOI) decreased, luminescence signal gradually declined (FIG. 1BB) . This indicates that the expression level of the engineered suppressor tRNA is affected by the MOI, indirectly confirming that the observed luminescent signal is produced by the full-length NanoLuc protein generated through readthrough, rather than by background noise.

The ssAAV2/9-delivered engineered suppressor tRNA restores function of NanoLuc protein containing a PTC

The single-stranded AAV (ssAAV) vector DNA sequence (SEQ ID NO: 59) was modified by replacing the regions marked with dashed lines and highlighted in gray with the DNA sequences of the engineered suppressor tRNAs R3-147 (FIG. 2B, corresponding to SEQ ID NO: 1) and R3-147-M54 (FIG. 2Q, corresponding to SEQ ID NO: 72) . This created ssAAV vectors carrying the engineered suppressor tRNAs, as shown in FIG. 4D, which were packaged into AAV2/9 capsids by a vendor. Additionally, the vector included a red fluorescent protein (mCherry) expression cassette as a marker to monitor transduction efficiency.

As depicted in FIGS. 4E-4F, Luc-V2-R3 cell lines infected with ssAAV2/9 carrying various engineered suppressor tRNAs all exhibited a portion of mCherry positive cells and displayed luciferase activities, indicating successful transduction and suppressor tRNA expression with the ssAAV vector. With decreasing MOI, both the proportion of mCherry positive cells and the luciferase activities gradually decreased, implying a correlation between suppression efficiency and the quantity of engineered suppressor tRNAs expressed by ssAAV2/9 transduced into the cells. Moreover, the transduction efficiency remained consistent when transducing ssAAV2/9 carrying different engineered suppressor tRNAs at the same MOI, suggesting that the presence of engineered suppressor tRNAs does not affect transduction efficiency. Additionally, cells transduced with the same MOI of ssAAV2/9 and exhibiting similar transduction efficiency showed varying luciferase activities: R3-147-M54 displayed an activity approximately 50 times higher than R3-147, indicating the enhanced readthrough efficiency of the modified suppressor tRNA compared to the template R3-147.

Example 7: APPLICATION OF ENGINEERED SUPPRESSOR tRNAs DELIVERED BY AAV IN THE TREATMENT OF A MOUSE MODEL WITH ALPORT SYNDROME

On the cellular level, the PTC readthrough capability of the engineered suppressor tRNA described herein has been validated across different genes, cell types, and vector systems. The following example demonstrates the utilization of engineered suppressor tRNAs delivered by ssAAV2/9 to mitigate symptoms in mice afflicted with Alport syndrome.

Detection of urinary Albumin and Creatinine

Since heightened urinary albumin levels and an elevated urinary albumin-to-creatinine ratio are indicative symptoms of Alport syndrome, these levels were evaluated in mice harboring an X-linked COL4A5 PTC R471*mutation (associated with Alport syndrome) . The measurement of albumin and creatinine was performed by a vendor.

Results

The ssAAV2/9-delivered engineered suppressor tRNA (in U6 cassette) decreases the relative ratio of urinary albumin to creatinine

Male PTC mice COL4A5-R471*/Y, aged 4 weeks, received intravenous injections via the tail vein of the ssAAV2/9-coated vector ssAAV-2xU6 (FIG. 4D) , containing nucleotide sequences encoding the engineered suppressor tRNAs R3-147 or R3-147-M54 (corresponding to SEQ ID NO: 1 or SEQ ID NO: 72, respectively) . Urinary albumin and creatinine levels were regularly monitored to assess symptom relief. Mice with a wild-type genetic background served as the healthy control, as depicted in FIG. 4G.

In PTC mice COL4A5-R471*/Y, the relative ratio of urinary albumin to creatinine showed no significant recovery between mice injected with saline and those injected with ssAAV2/9-R3-147, at either 5E11 v.g. or 1.5E12 v.g. doses (FIG. 4G) , indicating the limited effectiveness of engineered suppressor tRNA R3-147 at these doses in treating Alport syndrome. However, the relative ratio of urinary albumin to creatinine significantly decreased in mice injected with middle (5E11 v.g. ) and high (1.5E12 v.g. ) doses of ssAAV2/9-R3-147-M54, compared to those injected with a low (1.5E11 v.g. ) dose of ssAAV2/9-R3-147-M54 or saline (FIG. 4H) , suggesting the robust effectiveness of modified suppressor tRNA R3-147-M54 in treating Alport syndrome, with higher doses being more effective than lower doses. This result confirms that the engineered suppressor tRNA described herein can also perform the PTC readthrough in vivo.

ENUMERATED EMBODIMENTS

Embodiment 1. An engineered transfer RNA (tRNA) for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm, wherein the anticodon-arm comprises a tri-nucleotide anti-codon, wherein the anticodon is 5’ -UCA-3’ and recognizes a UGA stop codon, wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16; and

wherein the one or more modifications comprise:

i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon;

ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or

iii) a CCA sequence attached to the acceptor arm at the 3’ end.

Embodiment 2. The engineered tRNA of embodiment 1, wherein the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

Embodiment 3. The engineered tRNA of embodiment 1 or 2, wherein the engineered tRNA contains no more than about 9 nucleotide substitutions relative to SEQ ID NO: 1, no more than about 5 nucleotide substitutions relative to SEQ ID NO: 1, no more than about 5 nucleotide substitutions relative to SEQ ID NO: 13, or no more than about 5 nucleotide substitutions relative to SEQ ID NO: 16.

Embodiment 4. The engineered tRNA of any one of embodiments 1-3, wherein the engineered tRNA comprises an A to G substitution at the 1st nucleotide 3’ to the anticodon.

Embodiment 5. The engineered tRNA of any one of embodiments 1-4, wherein the engineered tRNA comprises a U to C substitution at the 4^th nucleotide 3’ to the anticodon.

Embodiment 6. The engineered tRNA of any one of embodiments 1-5, wherein the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end.

Embodiment 7. The engineered tRNA of any one of embodiments 1-6, wherein the engineered tRNA has nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8 and 61-64.

Embodiment 8. The engineered tRNA of any one of embodiments 1-7, wherein the engineered tRNA exhibits an increased PTC readthrough capability in vivo and/or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

Embodiment 9. The engineered tRNA of embodiment 8, wherein the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence.

Embodiment 10. A nucleic acid encoding the engineered tRNA of any one of embodiments 1-9.

Embodiment 11. A vector comprising the nucleic acid of embodiment 10.

Embodiment 12. The vector of embodiment 11, wherein the vector is a viral vector or a plasmid.

Embodiment 13. The vector of embodiment 12, wherein the vector is a viral vector, and wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.

Embodiment 14. The vector of embodiment 13, wherein the vector is an AAV 2/2 vector, an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector.

Embodiment 15. A pharmaceutical composition comprising the engineered tRNA of any one of embodiments 1-9, the nucleic acid of embodiment 10, or the vector of any one of embodiments 11-14, further comprising a pharmaceutically acceptable carrier.

Embodiment 16. The pharmaceutical composition of embodiment 15, wherein the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle.

Embodiment 17. A method of restoring translation of a coding nucleic acid of interest containing a premature UGA/TGA stop codon in a cell, comprising introducing to the cell the engineered tRNA of any one of embodiments 1-9, the nucleic acid of embodiment 10, or the vector of any one of embodiments 11-14, wherein the engineered tRNA introduced into the cell or produced from the nucleic acid or vector recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid of interest containing the premature UGA/TGA stop codon.

Embodiment 18. The method of embodiment 17, wherein the engineered tRNA restores at least 5%of the translation of the coding nucleic acid of interest containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon.

Embodiment 19. A method of treating a disease associated with a premature UGA/TGA stop codon in an individual, comprising: administering to the individual an effective amount of the pharmaceutical composition of embodiment 15 or 16.

Embodiment 20. The method of embodiment 19, wherein the premature stop-codon-associated disease is cystic fibrosis, muscular dystrophy, Alport syndrome, Stargardt disease, dilated cardiomyopathy, β-thalassemia or Liddle's syndrome.

Embodiment 21. The method of embodiment 19 or 20, wherein the individual is human.

Embodiment 22. The engineered tRNA of any one of embodiments 1-7, wherein the engineered tRNA exhibits increased PTC readthrough efficiency in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

Embodiment 23. The engineered tRNA of any one of embodiments 1-7, wherein the engineered tRNA exhibits an increased stability in vivo and/or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.

Embodiment 24. The engineered tRNA of embodiment 23, wherein the stability is determined by a proxy measurement, a half-life measurement, an amino acid charging efficiency measurement, or a measurement of binding to a docking partner elongation factor EF-Tu/EF1A1 or ribosomal machinery.

SEQUENCE DESCRIPTIONS

SEQ ID NOs 1-27 and 61-81: engineered suppressor tRNAs derived from human non-intron and intron-spliced arginine tRNA by changing the anticodon. SEQ ID NO: 1 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-147 originated from intron-spliced human tRNA-Arg-TCT-1-1, from 5’ to 3’ . SEQ ID NO: 2 = Nucleotide (RNA) sequence of engineered suppressor tRNA M2 (also referred to as R3-147-M2) modified base on the suppressor tRNA R3-147 with substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 3 = Nucleotide (RNA) sequence of engineered suppressor tRNA M3 (also referred to as R3-147-M3) modified base on the suppressor tRNA R3-147 with addition of CCA tail at the 3’ end, from 5’ to 3’ . SEQ ID NO: 4 = Nucleotide (RNA) sequence of engineered suppressor tRNA M4 (also referred to as R3-147-M4) modified base on the suppressor tRNA R3-147 with substitution of A with G at position 37, from 5’ to 3’ . SEQ ID NO: 5 = Nucleotide (RNA) sequence of engineered suppressor tRNA M5 (also referred to as R3-147-M5) modified base on the suppressor tRNA R3-147 with substitution of U with C at position 40 and addition of CCA tail at the 3’ end, from 5’ to 3’ . SEQ ID NO: 6 = Nucleotide (RNA) sequence of engineered suppressor tRNA M6 (also referred to as R3-147-M6) modified base on the suppressor tRNA R3-147 with substitution of U with C at position 40 and substitution of A with G at position 37, from 5’ to 3’ . SEQ ID NO: 7 = Nucleotide (RNA) sequence of engineered suppressor tRNA M7 (also referred to as R3-147-M7) modified base on the suppressor tRNA R3-147 with substitution of A with G at position 37 and addition of CCA tail at the 3’ end, from 5’ to 3’ . SEQ ID NO: 8 = Nucleotide (RNA) sequence of engineered suppressor tRNA M8 (also referred to as R3-147-M8) modified base on the suppressor tRNA R3-147 with substitution of U with C at position 40 and substitution of A with G at position 37 and addition of CCA tail at the 3’ end, from 5’ to 3’ . SEQ ID NO: 9 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-1 originated from human tRNA-Arg-ACG-1-1, from 5’ to 3’ . SEQ ID NO: 10 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-2 originated from human tRNA-Arg-ACG-2-1, from 5’ to 3’ . SEQ ID NO: 11 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-3 originated from human tRNA-Arg-CCG-1-1, from 5’ to 3’ . SEQ ID NO: 12 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-4 originated from human tRNA-Arg-CCG-2-1, from 5’ to 3’ . SEQ ID NO: 13 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-5 originated from human tRNA-Arg-CCT-1-1, from 5’ to 3’ . SEQ ID NO: 14 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-6 originated from human tRNA-Arg-CCT-2-1, from 5’ to 3’ . SEQ ID NO: 15 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-7 originated from human tRNA-Arg-CCT-3-1, from 5’ to 3’ . SEQ ID NO: 16 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-8 originated from human tRNA-Arg-CCT-4-1, from 5’ to 3’ . SEQ ID NO: 17 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-9 originated from human tRNA-Arg-CCT-5-1, from 5’ to 3’ . SEQ ID NO: 18 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-10 originated from human tRNA-Arg-TCG-1-1, from 5’ to 3’ . SEQ ID NO: 19 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-11 originated from human tRNA-Arg-TCG-2-1, from 5’ to 3’ . SEQ ID NO: 20 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-12 originated from human tRNA-Arg-TCG-3-1, from 5’ to 3’ . SEQ ID NO: 21 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-13 originated from human tRNA-Arg-TCG-4-1, from 5’ to 3’ . SEQ ID NO: 22 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-14 originated from human tRNA-Arg-TCG-5-1, from 5’ to 3’ . SEQ ID NO: 23 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-15 originated from human tRNA-Arg-TCG-6-1, from 5’ to 3’ . SEQ ID NO: 24 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-148 originated from human intron-spliced tRNA-Arg-TCT-2-1, from 5’ to 3’ . SEQ ID NO: 25 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-149 originated from human intron-spliced tRNA-Arg-TCT-3-1, from 5’ to 3’ . SEQ ID NO: 26 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-150 originated from human tRNA-Arg-TCT-4-1, from 5’ to 3’ . SEQ ID NO: 27 = Nucleotide (RNA) sequence of engineered suppressor tRNA R3-151 originated from human intron-spliced tRNA-Arg-TCT-5-1, from 5’ to 3’ . SEQ ID NO: 65 = Nucleotide (RNA) sequence of engineered suppressor tRNA M22 (also referred to as R3-147-M22) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 66 = Nucleotide (RNA) sequence of engineered suppressor tRNA M23 (also referred to as R3-147-M23) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 67 = Nucleotide (RNA) sequence of engineered suppressor tRNA M24 (also referred to as R3-147-M24) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 68 = Nucleotide (RNA) sequence of engineered suppressor tRNA M25 (also referred to as R3-147-M25) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 69 = Nucleotide (RNA) sequence of engineered suppressor tRNA M26 (also referred to as R3-147-M26) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 70 = Nucleotide (RNA) sequence of engineered suppressor tRNA M46 (also referred to as R3-147-M46) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 71 = Nucleotide (RNA) sequence of engineered suppressor tRNA M50 (also referred to as R3-147-M50) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 72 = Nucleotide (RNA) sequence of engineered suppressor tRNA M54 (also referred to as R3-147-M54) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 73 = Nucleotide (RNA) sequence of engineered suppressor tRNA M55 (also referred to as R3-147-M55) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 74 = Nucleotide (RNA) sequence of engineered suppressor tRNA M56 (also referred to as R3-147-M56) , which contains a substitution of A with G at position 37, from 5’ to 3’ . SEQ ID NO: 75 = Nucleotide (RNA) sequence of engineered suppressor tRNA M57 (also referred to as R3-147-M57) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 76 = Nucleotide (RNA) sequence of engineered suppressor tRNA M58 (also referred to as R3-147-M58) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 77 = Nucleotide (RNA) sequence of engineered suppressor tRNA M59 (also referred to as R3-147-M59) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 78 = Nucleotide (RNA) sequence of engineered suppressor tRNA M62 (also referred to as R3-147-M62) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 79 = Nucleotide (RNA) sequence of engineered suppressor tRNA M63 (also referred to as R3-147-M63) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 80 = Nucleotide (RNA) sequence of engineered suppressor tRNA M65 (also referred to as R3-147-M65) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 81 = Nucleotide (RNA) sequence of engineered suppressor tRNA M67 (also referred to as R3-147-M67) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 61 = Nucleotide (RNA) sequence of engineered suppressor tRNA M100 (also referred to as R3-147-M100) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 62 = Nucleotide (RNA) sequence of engineered suppressor tRNA M102 (also referred to as R3-147-M102) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 63 = Nucleotide (RNA) sequence of engineered suppressor tRNA M103 (also referred to as R3-147-M103) , which contains a substitution of U with C at position 40, from 5’ to 3’ . SEQ ID NO: 64 = Nucleotide (RNA) sequence of engineered suppressor tRNA M108 (also referred to as R3-147-M108) , which contains a substitution of A with G at position 37, from 5’ to 3’ .

SEQ ID NOs: 28-47 are the native human endogenous tRNAs with the removal of introns, presented in the same order as their derived engineered suppressor tRNA from SEQ ID NOs: 9-27 with the exception of R3-147. SEQ ID NO: 28 = Nucleotide (RNA) sequence of human tRNA-Arg-ACG-1-1, from 5’ to 3’ . SEQ ID NO: 29 = Nucleotide (RNA) sequence of human tRNA-Arg-ACG-2-1, from 5’ to 3’ . SEQ ID NO: 30 = Nucleotide (RNA) sequence of human tRNA-Arg-CCG-1-1, from 5’ to 3’ . SEQ ID NO: 31 = Nucleotide (RNA) sequence of human tRNA-Arg-CCG-2-1, from 5’ to 3’ . SEQ ID NO: 32 = Nucleotide (RNA) sequence of human tRNA-Arg-CCT-1-1, from 5’ to 3’ . SEQ ID NO: 33 = Nucleotide (RNA) sequence of human tRNA-Arg-CCT-2-1, from 5’ to 3’ . SEQ ID NO: 34 = Nucleotide (RNA) sequence of human tRNA-Arg-CCT-3-1, from 5’to 3’ . SEQ ID NO: 35 = Nucleotide (RNA) sequence of human tRNA-Arg-CCT-4-1, from 5’ to 3’ . SEQ ID NO: 36 = Nucleotide (RNA) sequence of human tRNA-Arg-CCT-5-1, from 5’ to 3’ . SEQ ID NO: 37 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-1-1, from 5’ to 3’ . SEQ ID NO: 38 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-2-1, from 5’ to 3’ . SEQ ID NO: 39 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-3-1, from 5’ to 3’ . SEQ ID NO: 40 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-4-1, from 5’ to 3’ . SEQ ID NO: 41 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-5-1, from 5’ to 3’ . SEQ ID NO: 42 = Nucleotide (RNA) sequence of human tRNA-Arg-TCG-6-1, from 5’ to 3’ . SEQ ID NO: 43 = Nucleotide (RNA) sequence of human tRNA-Arg-TCT-1-1, from 5’ to 3’ . SEQ ID NO: 44 = Nucleotide (RNA) sequence of human tRNA-Arg-TCT-2-1, from 5’ to 3’ . SEQ ID NO: 45 = Nucleotide (RNA) sequence of human tRNA-Arg-TCT-3-1, from 5’ to 3’ . SEQ ID NO: 46 = Nucleotide (RNA) sequence of human tRNA-Arg-TCT-4-1, from 5’ to 3’ . SEQ ID NO: 47 = Nucleotide (RNA) sequence of human tRNA-Arg-TCT-5-1, from 5’ to 3’ .

SEQ ID NOs: 48-50 are relevant parental and constructed plasmids used herein. SEQ ID NO: 48 = Nucleotide (DNA) sequence of the parental plasmid pSpCas9 (BB) -2A-GFP (PX458) used for construction of engineered suppressor tRNAs expression. SEQ ID NO: 49 = Nucleotide (DNA) sequence of the constructed plasmid ptRNA-GFP used for engineered suppressor tRNA expression. SEQ ID NO: 50 = Nucleotide (DNA) sequence of the constructed plasmid ptRNA-GFP-R3-147 used for engineered suppressor tRNAs R3-147 expression.

SEQ ID NO: 51 = Nucleotide (RNA) sequence of scrambled sequence Scr1-4 derived from the suppressor tRNA R3-147, from 5’ to 3’ .

SEQ ID NOs: 52-57 are modified NanoLuc nucleic acids and proteins used herein. SEQ ID NO: 52 = Nucleotide (DNA) sequence of the modified NanoLuc Luc_V2_R3 with premature TGA stop codon after 195bp of NanoLuc gene. SEQ ID NO: 53 = Nucleotide (DNA) sequence of the modified NanoLuc Luc_V3_R3 with premature TGA stop codon after 474bp of NanoLuc gene. SEQ ID NO: 54 = Nucleotide (DNA) sequence of the modified NanoLuc Luc_V2_R4 with glycine coding codon GGA after 195bp of NanoLuc gene. SEQ ID NO: 55 = Nucleotide (DNA) sequence of the modified NanoLuc Luc_V3_R4 with glycine coding codon GGA after 474bp of NanoLuc gene. SEQ ID NO: 56 = amino acid (protein) sequence of the full-length modified NanoLuc protein produced in the NanoLuc Luc_V2_R3 cell line after engineered suppressor tRNA readthrough, with the insertion of a specific amino acid positioned after the 65th amino acid. SEQ ID NO: 57 = the exemplary amino acid (protein) sequence of the full-length modified NanoLuc protein produced in the NanoLuc Luc_V2_R3 cell line after engineered suppressor tRNA readthrough, with the insertion of an amino acid arginine positioned after the 65th amino acid.

SEQ ID NOs: 58-60 are DNA/RNA sequences relevant to viral vectors utilized herein. SEQ ID NO: 58 = Nucleotide (RNA) sequence of the lentivirus LV, employed for delivering engineered tRNA expressed under the U6 promoter. SEQ ID NO: 59 = Nucleotide (DNA) sequence of the single-stranded AAV vector (ssAAV) carrying two copies of tRNAs, employed for delivering engineered tRNA expressed under the U6 promoter. SEQ ID NO: 60 = Nucleotide (DNA) sequence of the self-complementary AAV vector (scAAV) carrying one copy of tRNA, used for delivering engineered tRNA expressed under the U6 promoter

SEQ ID NOs: 83-88 are nucleotide (DNA) sequences related to the “fuse” tags used in the NanoLuc assays. SEQ ID NO: 83 = 5’ fused tag (3 x Myc and GS linker) of SEQ ID NOs: 52-55. SEQ ID NO: 84 = 3’ fused tag (3 x Flag and GS linker) of SEQ ID NOs: 52-55. SEQ ID NO: 85 = SEQ ID NO: 52 without fused tags. SEQ ID NO: 86 = SEQ ID NO: 53 without fused tags. SEQ ID NO: 87 = SEQ ID NO: 55 without fused tags. SEQ ID NO: 88 = SEQ ID NO: 54 without fused tags.

SEQ ID NOs: 89-92 are primers used in qPCR herein, from 5’ to 3’ . SEQ ID NO:89 = primer p53_forward. SEQ ID NO: 90 = primer p53_reverse. SEQ ID NO: 91 = GAPDH_forward.SEQ ID NO:92 = GAPDH_reverse.

Claims

An engineered transfer RNA (tRNA) for carrying an arginine comprising a T-arm, a D-arm, an anticodon arm, a variable region, and an acceptor arm,

wherein the anticodon-arm comprises a tri-nucleotide anti-codon,

wherein the anticodon is 5’-UCA-3’ and recognizes a UGA stop codon,

wherein the engineered tRNA comprises one or more modifications relative to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16; and

wherein the one or more modifications comprise:

i) an A to G substitution at the 1^st nucleotide 3’ to the anticodon;

ii) a U to C substitution at the 4^th nucleotide 3’ to the anticodon; and/or

iii) a CCA sequence attached to the acceptor arm at the 3’ end.
The engineered tRNA of claim 1, wherein the engineered tRNA has a nucleotide sequence that is at least about 85%identical to SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.
The engineered tRNA of claim 1 or 2, wherein the engineered tRNA contains no more than about 9 nucleotide substitutions relative to SEQ ID NO: 1, no more than about 5 nucleotide substitutions relative to SEQ ID NO: 1, no more than about 5 nucleotide substitutions relative to SEQ ID NO: 13, or no more than about 5 nucleotide substitutions relative to SEQ ID NO: 16.
The engineered tRNA of any one of claims 1-3, wherein the engineered tRNA comprises an A to G substitution at the 1st nucleotide 3’ to the anticodon.
The engineered tRNA of any one of claims 1-4, wherein the engineered tRNA comprises a U to C substitution at the 4^th nucleotide 3’ to the anticodon.
The engineered tRNA of any one of claims 1-5, wherein the engineered tRNA comprises a CCA sequence attached to the acceptor arm at the 3’ end.
The engineered tRNA of any one of claims 1-6, wherein the engineered tRNA has nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8 and 61-64.
The engineered tRNA of any one of claims 1-7, wherein the engineered tRNA exhibits an increased PTC readthrough capability in vivo or in vitro as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.
The engineered tRNA of claim 8, wherein the PTC readthrough capability is determined by a NanoLuc luciferase assay, RT-qPCR, Western Blot, Immunoprecipitation, Mass Spectrometry, and/or Immunofluorescence.
A nucleic acid encoding the engineered tRNA of any one of claims 1-9.
A vector comprising the nucleic acid of claim 10.
The vector of claim 11, wherein the vector is a viral vector or a plasmid.
The vector of claim 12, wherein the vector is a viral vector, and wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.
The vector of claim 13, wherein the vector is an AAV 2/2 vector, an AAV 2/5 vector, an AAV 2/6 vector, an AAV 2/7 vector, an AAV2/8 vector, or an AAV 2/9 vector.
A pharmaceutical composition comprising the engineered tRNA of any one of claims 1-9, the nucleic acid of claim 10, or the vector of any one of claims 11-14, further comprising a pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 15, wherein the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle.
A method of restoring translation of a coding nucleic acid of interest containing a premature UGA/TGA stop codon in a cell, comprising introducing to the cell the engineered tRNA of any one of claims 1-9, the nucleic acid of claim 10, or the vector of any one of claims 11-14, wherein the engineered tRNA introduced into the cell or produced from the nucleic acid or vector recognizes and reads through the premature UGA/TGA stop codon, thereby restoring translation of the coding nucleic acid of interest containing the premature UGA/TGA stop codon.
The method of claim 17, wherein the engineered tRNA restores at least 5%of the translation of the coding nucleic acid of interest containing a premature UGA/TGA stop codon relative to a corresponding coding nucleic acid not containing the premature UGA/TGA stop codon.
A method of treating a disease associated with a premature UGA/TGA stop codon in an individual, comprising: administering to the individual an effective amount of the pharmaceutical composition of claim 15 or 16.
The method of claim 19, wherein the premature stop-codon-associated disease is cystic fibrosis, muscular dystrophy, Alport syndrome, Stargardt disease, dilated cardiomyopathy, β-thalassemia or Liddle's syndrome.
The method of claim 19 or 20, wherein the individual is human.
The engineered tRNA of any one of claims 1-7, wherein the engineered tRNA exhibits increased PTC readthrough efficiency in vivo as compared to a tRNA having the sequence of SEQ ID NO: 1, SEQ ID NO: 13, or SEQ ID NO: 16.