Methods for saving termination codons via genetic redistribution using ace-tRNA

Abstract

The present invention relates to a method for rescuing termination codons via genetic redistribution using ACE-tRNA. The present invention relates to a method for rescuing termination codons via genetic redistribution using ACE-tRNA. Specifically, the present invention provides a modified transfer RNA (tRNA) comprising a T-arm, a D-arm, an anticodon arm, and a receptor arm, where the T-arm comprises a nucleotide that interacts with an elongation factor 1 [alpha] protein, and methods of use thereof.

Description

[0001] Invention priority This application claims priority to U.S. Provisional Application No. 62 / 580,887 filed on November 2, 2017, and U.S. Provisional Application No. 62 / 687,015 filed on June 19, 2018. The entire contents of the above-mentioned applications are hereby incorporated by reference.

[0002] Statement Regarding Federally Funded Research This invention was made with government support under R01 GM106569 awarded by the National Institutes of Health. The government has certain rights in this invention. background The DNA molecule carries genetic information in the form of a sequence of nucleotide bases that make up the DNA polymer. Only four nucleotide bases are used in DNA: adenine, guanine, cytosine, and thymine. This information in the form of a codon of three consecutive bases is transcribed into messenger RNA (mRNA) and then translated by transfer RNA (tRNA) and ribosomes to form proteins. Four nucleotide bases are used in RNA: adenine, guanine, cytosine, and uracil. The genetic code is the relationship between a triplet codon and a specific amino acid. 64 possible codon triplets form the genetic code, of which three stop (also called terminating) codons provide a signal to the translation machinery (the cell's ribosome) to terminate protein production at a specific codon. The other 61 triplets in the code correspond to one of the 20 standard amino acids. See Figure 1 .

[0004] DNA is translated by ribosomes, and according to the genetic instructions specifically provided by DNA, each amino acid is linked together one by one to form a polypeptide. When the ribosome reaches the stop codon, the extension of the protein is terminated. The three stop codons are UAG (amber), UAA (ochre) and UGA (opal). Mutations that change the codons encoding amino acids to stop codons are called "nonsense mutations". These nonsense mutations can lead to significant truncation / shortening of polypeptide sequences and can cause profound changes in genetic phenotypes. Therefore, even if there may be gene-guided expression, key proteins may not be produced because when the ribosome reaches the mutant stop signal, it terminates translation, resulting in unfinished proteins.

[0005] Transfer RNA translates mRNA into protein on the ribosome. Each tRNA contains an "anticodon" region that hybridizes with the complementary codon on the mRNA. The tRNA that carries its designated amino acid is called a "loaded" tRNA. If the tRNA is one of the 61 amino acid-related (i.e., not the termination signal-related) tRNAs, it usually attaches its amino acid to the growing peptide. The structural gene of tRNA is about 72-90 nucleotides long and folds into a cloverleaf structure. The tRNA is transcribed by RNA polymerase III and contains its own intragenic split promoter, which becomes part of the mature tRNA coding sequence (Sharp S. J., Schaack J., Coolen L., Burke DJ and Soll D., "Structure and transcription of eukaryotic tRNA genes", Crit. Rev. Biochem, 19: 107-144 (1985); Geiduschek EO and Tocchini-Valentini, "Transcription by RNA polymerase III, Annu. Rev. Biochem. 57: 873-914 (1988)).

[0006] "Nonsense suppressors" are alleles of tRNA genes that contain altered anticodons so that instead of triggering a "stop" signal, they insert an amino acid in response to a stop codon. For example, the ochre mutation results in the production of a UAA codon in an mRNA. The ochre suppressor gene produces a tRNA with an AUU anticodon that inserts an amino acid at the UAA site, which allows continued translation of the mRNA despite the presence of a codon that normally triggers translation termination.

[0007] Many nonsense suppressor tRNA alleles have been identified in prokaryotes and eukaryotes such as yeast and C. elegans. Different suppressor tRNAs differ in their suppression efficiency. In E. coli and other systems, amber suppressors are relatively more effective, ochre suppressors are less effective, and opal is the least efficient, suggesting that amber codons are rarely used to terminate protein synthesis, while ochre and opal codons are more frequently used as natural termination signals.

[0008] Unwanted errors in the DNA blueprint can cause disease. For example, an unexpected "stop" signal appears in the middle of a protein rather than at the end of the blueprint, resulting in a truncated or shortened protein with altered function or no function at all. Many human diseases, such as cystic fibrosis, muscular dystrophy, beta thalassemia, and Liddle's syndrome, result from unwanted stop signals in the DNA reading frame for proteins that are important for proper lung, blood, muscle, or kidney function, respectively.

[0009] Therefore, there is a need to provide novel modified nonsense suppressor tRNAs that are stable compared to the corresponding unmodified nonsense suppressor tRNA, as well as nonsense suppressor tRNAs that have enhanced activity to inhibit termination of genes associated with cystic fibrosis.

[0010] Overview In certain embodiments, the invention provides a modified transfer RNA (tRNA) comprising a T arm, a D arm, an anticodon arm and an acceptor arm, wherein the T arm comprises a T stem having a nucleotide that interacts with elongation factor 1-α1 (EF1α). EF1α recruits aminoacyl tRNA to ribosomes and protects tRNA from deacylation. Rational nucleotide substitutions result in tuned tRNA:EF1α interactions that enhance tRNA delivery to ribosomes and protection from deacylation.

[0011] In certain embodiments, the invention provides a modified transfer RNA (tRNA) of SEQ ID NO: 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, or 55, wherein thymidine is replaced with uracil.

[0012] In certain embodiments, the invention provides a modified transfer RNA (tRNA) of any one of SEQ ID NOs: 1-538, wherein thymidine is replaced with uracil.

[0013] In certain embodiments, the modified tRNA is SEQ ID NO: 56-60, 62-66, 84-86, 90-111, 113, 128-143, 147-149, 153-156, 161-174, 176, 178, 181, 184-186, 192, 196-197, 199-201, 205, 213-240, 246, 255-256, 258-285, 299, 305-312, 314, 318-332, 335-344, 346, 350-354, 357-360, 361-364, 367-368, 369-370, 371-371, 372-373, 374-375 any of 62, 365-370, 372-383, 388-390, 392, 394-401, 403-407, 414-416, 418, 422, 425, 428-433, 437, 444-445, 452, 455, 459-463, 470, 472-474, 476, 487-492, 525, 530-539, 545-550, 553-555, 561-563, and 567-579, wherein thymidine is replaced by uracil.

[0014] In certain embodiments, the invention provides a modified transfer RNA (tRNA) comprising a T stem, a D stem, an anticodon loop and an acceptor stem, wherein (a) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-UCA-3' and recognizes a TGA stop codon, and wherein the acceptor arm is operably connected to arginine, tryptophan or glycine; (b) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-UUA-3' and recognizes a TAA stop codon, and wherein the acceptor arm is operably connected to glutamine or glutamate; or (c) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-CUA-3' and recognizes a TAG stop codon, and wherein the acceptor arm is operably connected to tryptophan, glutamate or glutamine. In certain embodiments, the T arm comprises a rationally altered nucleotide sequence that tunes the interaction with EF1α, enhances its inhibitory activity, and thereby increases its therapeutic potential. tRNAs with tuned interactions with EF1α have enhanced nonsense suppression and offer enhanced therapeutic properties.

[0015] In certain embodiments, the invention provides an oligonucleotide sequence encoding a modified tRNA as described above, wherein the oligonucleotide has a total length of less than 150 nucleotides. In certain embodiments, the oligonucleotide is DNA.

[0016] In certain embodiments, the present invention provides an oligonucleotide comprising a first oligonucleotide sequence and a second oligonucleotide sequence, wherein the first oligonucleotide sequence and the second oligonucleotide sequence independently encode a modified tRNA as described above, wherein the first oligonucleotide and the second oligonucleotide independently have a total length of less than 150 nucleotides, and wherein the two sequences are tandem.

[0017] In certain embodiments, the invention provides an expression cassette comprising a promoter and a nucleic acid encoding a modified tRNA or an oligonucleotide as described above.

[0018] In certain embodiments, the present invention provides a vector comprising the above-mentioned oligonucleotide or expression cassette.

[0019] In certain embodiments, the vector is a viral vector or a plasmid vector.

[0020] In certain embodiments, the present invention provides a composition comprising the above-mentioned modified tRNA, oligonucleotide or vector, and a pharmaceutically acceptable carrier.

[0021] In certain embodiments, the carrier is a liposome.

[0022] In certain embodiments, the present invention provides cells comprising the above-described vectors.

[0023] The present invention provides a method for treating a stop codon-related genetic disease, comprising administering the above-mentioned modified tRNA composition to a patient in need thereof.

[0024] In certain embodiments, the genetic disease associated with a premature stop codon is cystic fibrosis, muscular dystrophy, beta thalassemia, or Liddle's syndrome.

[0025] In certain embodiments, the present invention provides a method of restoring translation of a nucleotide sequence comprising a nonsense mutation in a cell, comprising introducing the above-described composition into the cell.

[0026] In certain embodiments, the invention provides methods for identifying anticodon editing (ACE) tRNAs by high-throughput cloning and screening using nonsense codon suppression in luciferases, including NanoLuc. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 .Genetic code table.

[0028] Figure 2 .tRNA has a general four-arm structure, which includes a T arm, a D arm, an anticodon arm and an acceptor arm. These arms are also referred to as "loops" throughout the text.

[0029] Figure 3.ACE-tRNA for nonsense suppression (H. sapiens tRNA Trp TGA ).

[0030] Figure 4 .Anticoncodon editing (ACE)-tRNA encoded in a vector for identification of functional ACE tRNA sequences. This vector sequence includes a Nanoluciferase reporter system. The depicted vector was used to identify ACE tRNAs with TGA inhibition. TAA and TAG variants were used for appropriate tRNA screening (see Figures 14 to 17 ).

[0031] Figure 5 Schematic diagram of the rescue of proteins and ion channels by stop codons via suppressor tRNA.

[0032] Fig. 6A and 6B . Nonsense codon rescue using human ACE-tRNA. Fig. 6A . Schematic diagram of the anticodon editing (ACE)Trp tRNA and cherry-TGA-eGFP-HA construct. Figure 6B . Rescue by cherry TGAeGFP-HA construct of ACE tryptophan tRNA#4.

[0033] Figure 7 . Principle and prevalence of nonsense codons observed in human disease. Twenty natural amino acid codons are ranked by their contribution to human disease, with dark cross-hatched codons being the most prevalent (TGG, TAC, TAT, TCA, and TTA) and dotted codons being the least prevalent. All cross-hatched codon sequences require a single nucleotide mutation to convert from the intended amino acid to a stop codon. Right, the most common pathogenic nonsense codon within the cystic fibrosis transmembrane conductance regulator (CFTR). Here, novel tRNA sequences have been discovered for repairing the indicated mutations.

[0034] Figure 8 Identification of tRNA sequences for repair of tryptophan-TGA and glycine-TGA. The left axis indicates the fold over background of luciferase activity. Most tRNAs with mutant anticodon loops lack rescue activity.

[0035] Fig. 9.CFTR 1282X rescue using Trpchr17.trna39 and Glychr19.trna2 ACE-tRNA. Biochemical western blot data of CFTR W1282X channels co-expressed in HEK cells with the indicated tRNAs. Expression vectors containing four copies of the indicated tRNAs showed higher rescue of CFTR protein. The "C" band indicates rescue of fully mature glycosylated CFTR protein. The antibody used was M3A7 from Cystic Fibrosis Therapeutics at a dilution of 1:1000.

[0036] Fig. 10A and 10B .ACE-tRNA Trp and ACE-tRNA Gly Expression of the gene results in specific incorporation of the cognate amino acid into nonsense codons. Fig. 10A ) Model protein histidinol dehydrogenase (HDH)-His-Strep N94-TGA and ACE-tRNA Trp (Left) and ACE-tRNA Gly (right) Co-expression of , resulting in full-length HDH protein (asterisk) detectable by silver staining after affinity purification. Fig. 10B )WT HDH (top), HDH-N94+ACE-tRNA Gly (Middle) and HDH-N94+ACE-tRNA Trp (Bottom) The spectrum highlights the difference in amino acid mass at position N94, which specifically matches glycine (-57 Da) and tryptophan (+72 Da), indicating the insertion of the ACE-tRNA cognate amino acid.

[0037] Fig.11 . Cloning workflow for construction of tRNA libraries.

[0038] Figures 12A-12B Targeted mutagenesis of nucleotides within the t-stem region further enhanced the rescue function of ACE-tRNA. Fig. 12A .Trpchr17.tRNA 39 was systematically mutagenized within the t-stem region. These efforts identified ACE tRNA TS-1052-62G-C ( Fig. 12B ) and the cross-hatched lines in the graph, which show -250% increased bioactivity.

[0039] Figure 13A-13F. ACE-tRNA is selective for nonsense codons and is more effective than aminoglycoside nonsense suppression. Figure 13A) ACE-tRNA Trp #5 and Fig. 13B )ACE-tRNAGly #16 was cloned into a NanoLuc reporter plasmid containing TGA, TAA or TAG nonsense codons. After transfection, nonsense suppression was only measured in the NanoLuc-TGA construct. Figure 13C and Fig.13D ) by adding gentamicin (40uM) and G418 (150uM), as well as with ACE-tRNA Trp #5 and ACE-tRNA Gly Co-transfected NanoLuc-TGA of #16 inhibited the expression of 1:1 protein in HEK293 cells at 24 h and 37 °C (Figure 13C). Fig.13D )Measurement was performed for 48 hours. Figure 13E and Fig.13F HEK293 cells stably expressing NanoLuc-TGA were treated with gentamicin (40uM) and G418 (150uM) and ACE-tRNA Trp #5 and ACE-tRNA Gly #16 was transfected. Figure 13E) 24 hours after treatment and Fig.13F ) Nonsense suppression was measured for 48 hours.

[0040] Fig.14 .ACE-tRNA-Arg-TGA. Identification of ACE-tRNA for repair of the arginine-TGA nonsense codon.

[0041] Fig.15 .ACE-tRNA-Gln TAG. Identification of ACE-tRNA for repair of glutamine TAG nonsense codon.

[0042] Fig.16 .ACE-tRNA-Gln TAA. Identification of ACE-tRNA for repair of glutamine TAA nonsense codon.

[0043] Fig.17 .ACE-tRNA-Glu TAG. Identification of ACE-tRNA for repair of the glutamate-TAG nonsense codon.

[0044] Fig.18 .ACE-tRNA-Gln TAA. Identification of ACE-tRNA for repair of the glutamate TAA nonsense codon.

[0045] Fig.19 .ACE-tRNA-Trp TAG. Identification of ACE tRNA for repair of the tryptophan TAG nonsense codon.

[0046] Figures 20A-20DDelivery of ACE-tRNA as small RNA supports robust suppression of G542X and W1282X nonsense mutations. Fig. 20A ) CFTR cRNA with G542X or W1282X nonsense mutations causing cystic fibrosis was co-injected with serial dilutions of prefolded ACE-tRNAGly and ACE-tRNATrp, respectively, in Xenopus oocytes. Two-electrode voltage clamp recordings of CFTRCl- current were performed 36 hours later. The current-voltage relationship showed that increasing amounts of Fig. 20B )ACE-tRNATrp and Fig. 20C ) ACE-tRNAGly prefolded RNA resulted in increased CFTR function (measured CFTR Cl- current) achieved with WT CFTR in ACE-tRNAGly experiments. Fig.20D ) Dose response of rescue by G542X ACE-tRNAGly (filled circles) and W1282X ACE-tRNATrp (open squares) (CFTR Cl- currents elicited at +40 mV were normalized to WT CFTR Cl- currents at +40 mV). The dose dependence of ACE-tRNAGly (EC50 = ~20 ng; Hill coefficient ~1.4) showed clear saturation at WT CFTR levels, while ACE-tRNATrp was right-shifted (EC50 = ~94 ng; Hill coefficient 1.24).

[0047] Figures 21A-21B Nonsense mutation suppression screening to identify candidate anticodon-editing tRNAs (ACE-tRNAs). Fig.21A , schematic diagram shows the necessary interaction of ACE-tRNA with the translation machinery.After delivery, ACE-tRNA is recognized by endogenous aminoacyl tRNA synthetase and loaded with (aminoacylated) its cognate amino acid.Aminoacylated ACE-tRNA is recognized by endogenous elongation factor 1-α, which protects ACE-tRNA from deacylation and delivers aminoacyl ACE-tRNA to ribosomes for suppression of premature termination codons, in this case UGA. Fig. 21B , using Golden Gate paired with CcdB negative selection, individual ACE-tRNAs were cloned into a high-throughput cloning nonsense reporter plasmid. The all-in-one plasmid contains the NLuc luciferase reporter gene with UGA, UAG or UAAPTC at p.162 between the enzymatic big bit and the essential C-terminal small bit.

[0048] Fig. 22The ACE-tRNA gene family was screened using a high-throughput cloning nonsense mutation reporter platform. For each ACE-tRNA family, the indicated anticodon editing PTC sequence was tested, which was one nucleotide away from the endogenous anticodon sequence. Fig.25 Multiple high-performance suppressor tRNAs were identified for each category. Data are displayed on a Log10 scale based on normalized NLuc luminescence. Each tRNA dataset was obtained in triplicate and is shown with SEM, with corresponding ANOVA statistical analysis in Table 2. The encoded identifiers and corresponding tRNA sequences are shown in Fig.26 and Table 9.

[0049] Figures 23A-23C Cognate-encoded and high-fidelity suppression by engineered tRNA. Fig.23A , Tryptic fragment of histidinol dehydrogenase (HDH), where "X" indicates the suppressed PTC codon. MS / MS spectra of the tryptic fragment for WT (top), N94G (middle), and N94W (bottom) HDH, with the masses of y and b ions indicated. The b9 ion mass is shifted by -57Da and +72Da from the predicted mass of WT asparagine, indicating that the ACE-tRNA Gly and ACE-tRNA Trp Encodes the homologous amino acids glycine and tryptophan. Fig. 23B , ACE-TGA-tRNA Gly (Glychr19.t2) selectively suppresses the UGA stop codon in transiently transfected HEK293 cells. Fig.23C ) After 48 hours of incubation in Hek293 cells stably expressing NLuc-UGA, ACE-tRNA Gly Transfection was superior to both gentamicin (40uM) and G418 (140uM).

[0050] Figures 24A-24B .Ribosome profiling of ACE-tRNA at 3'UTRs across the transcriptome. Fig.24A , as described in Materials and Methods, the ribosome footprint density on the 3'UTR is plotted as the log2 fold change of the treated cell reading relative to the control (puc57GG empty vector). Transcripts are grouped by their endogenous TAA, TAG, and TGA stop codons. Each point represents the average of two replicates for the transcript. Error bars show the mean ± SD of the log2 fold change. Fig. 24B, the average log2 fold change of normalized ribosome footprint occupancy is plotted for each nucleotide from -50 to +50 nt around the stop codon of the transcriptome (18,101 sequences). The sketch shows the ~15 nt shift from the 5' end of the ribosome footprint to the first base position of the stop codon in the ribosomal A site.

[0051] Fig.25 . Codon usage for common PTCs. The cross-hatching indicates the most common codons and corresponding amino acid types, which can be converted to stop codons via nucleotide substitution. Engineered tRNAs have been developed for each type.

[0052] Fig.26 . ACE-tRNA activity diagram with numbered references.

[0053] Fig. 27 Alignment of glycine tRNA sequences. 21 tRNAGly human sequences confirm high sequence homology in the tRNA clade. The patterns in the tRNA images correspond to the pattern boxes in the sequences.

[0054] Fig.28 The side chain identity at p.162 in Nanoluciferase does not affect activity. The total luminescent activity is indicated for each mutation at the site.

[0055] Figures 29A-29B ACE-tRNA from multiple species Trp Sequence and analysis of suppressor tRNA mutations. Fig.29A .Sequence alignment. Fig.29B .NLuc-UGA+ACE-tRNA Trp / NLuc-UGA.

[0056] Figures 30A-30C .Histidinol dehydrogenase (HDH) His(8)-streptactin expression construct allows efficient one-step isolation of the protein from HEK293 cells. Fig. 30A ) Protein sequence of the HDH expression construct. The underlined sequence indicates coverage by mass spectrometry. The bold, underlined asparagine (amino acid position 94) is the residue mutated to TGA PTC for determination of ACE-tRNA fidelity. The dual affinity tag is indicated in bold italics. Fig. 30B )Trpchr17.trna39 and Fig. 30C ) Silver staining of HDH protein after PTC inhibition of Glychr19.trnA2.

[0057] Fig.31 .Termination codon specificity for ACE-tRNATrp is maintained. For tRNA Trp TGA Trpchr17.trna39 (the best performing Trp TGA suppressor tRNA) inhibitory activity 36, Fig. 22 This tRNA was co-expressed with the indicated pNano-STOP plasmids.

[0058] Figure 32A-32D. ACE-tRNA is more effective than aminoglycoside PTC inhibition. Figure 32A) Raw luminescence and fluorescence measured 24 hours after addition of gentamicin (40uM), G418 (150uM), and transfection with Trpchr17.trna39 and Glychr19.trna2 in HEK293 cells stably expressing the PTC reporter Nluc-UGA. Fig.32B Figure 32C) Raw luminescence and measured 24 hours after adding gentamicin (40uM), G418 (150uM) and co-transfecting with Trpchr17.trna39 and Glychr19.trna2 in HEK293 cells Fig.32D ) Standardized luminescence.

[0059] Fig.33 Comparison of the time course of ACE-tRNA activity after delivery as RNA or cDNA. ACE-tRNA was delivered to HEK293 cells stably expressing pNanoLuc-UGA, however, only 5 μl of the reaction mixture was added to the cells to reduce the effect of the transfection reagent on cell viability. ACE-tRNA delivered as RNA (open symbols) was faster than cDNA constructs (filled circles) in rescuing the expression of PTC reporters. However, when expressed by cDNA, ACE-tRNA activity continued to rise within 48 hours and decreased with the deliverable RNA. DETAILED DESCRIPTION OF THE INVENTION Over the years, researchers have identified hundreds of unique point mutations that result in nonsense codons established in human genes. These types of mutations result in, for example, muscular dystrophy, xeroderma pigmentosum, cystic fibrosis, hemophilia, anemia, hypothyroidism, p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer, esophageal cancer, bone cancer, ovarian cancer, esophageal cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes, and rickets. The BRACA-1 and BRACA-2 genes associated with breast cancer also have similar mutations.

[0061] Nucleotide sequences encoding hundreds of human tRNAs are known and generally available to those skilled in the art through sources such as Genbank. The structure of tRNA is highly conserved, and tRNA often spans species and is functional. Therefore, bacteria or other eukaryotic tRNA sequences are also potential sources of oligonucleotides for the stabilized tRNA of the present invention. Whether a specific tRNA sequence is functional in a desired mammalian cell can be determined by routine experiments. Further additional potential tRNA sequences that are still unknown can be modified as described herein to be stabilized by routine experiments.

[0062] The tRNA gene has a strong promoter that is active in all cell types. The promoter for the eukaryotic tRNA gene is contained in the structural sequence encoding the tRNA molecule itself. Although there are elements regulating transcriptional activity in the 5' upstream region, the length of the active transcription unit can be significantly less than 500 base pairs, and therefore the adaptation in the delivery vector is direct. Once they have been transcribed and processed, tRNA has a low degradation rate. Finally, gene therapy with nonsense suppressor genes maintains endogenous physiological control of target genes containing nonsense codons.

[0063] Nonsense mutation Transfer RNA (tRNA) is a class of RNA molecules that play a role in decoding messenger RNA (mRNA) sequences into proteins. tRNA acts at specific sites on the ribosome during the translation process, which synthesizes proteins from mRNA molecules. Nonsense mutations, also known as premature termination codons (PTCs), account for ~10-15% of single base pair mutations that cause human disease, and cystic fibrosis follows. (Peltz et al., Annu Rev Med., 64: 407-25, 2013). In general, nonsense mutations have more serious consequences than missense mutations due to the almost complete loss of gene expression and activity, and the possibility of dominant negative effects with truncated products. PTCs lead to premature translation termination and accelerated degradation of mRNA transcripts through the nonsense-mediated degradation (NMD) pathway.

[0064] Current studies have shown that the specific site within the RNA transcript to which a tRNA delivers its amino acid can be altered by molecular editing of the anticodon sequence within the tRNA. This approach allows the premature termination codon (PTC) to be efficiently and therapeutically restored to the originally missing amino acid. Anticodon editing tRNAs (ACE-tRNAs) constitute a new class of biotherapeutics.

[0065] The modified tRNA allows pathogenic nonsense codons to be "re-edited" into specific amino acids. These modified tRNAs target only one type of stop codon, such as TGA over TAC or TAA. The small size of these tRNA molecules makes them amenable to easy expression because the tRNA+promoter is only ~300bp. In short, oligonucleotides containing structural components of tRNA genes that are functional in human cells were synthesized. The sequence of this oligonucleotide is designed based on a known sequence, accompanied by substitutions made in the anticodon region of the tRNA, causing specific tRNAs to recognize nonsense mutations or other specific mutations.

[0066] Several small molecule screens have been performed to inhibit nonsense stop codons by interacting with the ribosome, with the most prominent molecules being G418, gentamicin, and PTC124. PTC124 or atalulum has recently been withdrawn from Phase 3 clinical trials as a therapeutic agent for cystic fibrosis. Atalulum and aminoglycosides promote read-through of each of the three nonsense codons by importing near-homologous amino acids that convert nonsense mutations into missense mutations. (Roy et al., PNAS 2016 Nov 1; 113(44): 12508-12513).

[0067] Anticodon editing tRNA (ACE-tRNA) tRNA has a general four-arm structure, which includes a T arm, a D arm, an anticodon arm, and an acceptor arm ( Figure 2 ).

[0068] The T arm is composed of a "T stem" and a "TΨC loop". In certain embodiments, the T stem is modified to increase the stability of the tRNA. In certain embodiments, the ACE-tRNA has a modified T stem that increases biological activity relative to the endogenous T stem sequence to inhibit the termination site.

[0069] In one embodiment, the present invention includes a composition comprising stabilized tRNA, which can be used with greater effectiveness to treat a wide variety of nonsense mutation-related diseases. The following sequences in Tables 1-8 are written as DNA, but as RNA (transcribed DNA), "T: thymidine" is "U: uracil". Therefore, the tRNA transcribed from the following sequences all contain uracil instead of thymidine.

[0070] In certain embodiments, the tRNA has the following sequence (wherein thymidine is replaced with uracil): TS-36: GGCCTCGTGGCGCAACGGTAGCGCGTCTGACTtCAGATCAGAAGGtTGCGgGTTCAAATCcCGTCGGGGTCA (SEQ ID NO: 1) TS-37: GGCCTCGTGGCGCAACGGTAGCGCGTCTGACTtCAGATCAGAAGGtTaCGgGTTCAAATCcCGTCGGGGTCA (SEQ ID NO: 2) TS-38: GGCCTCGTGGCGCAACGGTAGCGCGTCTGACTtCAGATCAGAAGGtTcCGgGTTCAAATCcCGgCGGGGTCA (SEQ ID NO: 3) Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 In one embodiment, the ACE-tRNA used for nonsense suppression is Figure 3 As depicted in (Homo sapiens tRNA T rp TGA ).

[0071] According to the present invention, human UAA, UAG and UGA suppressor gene tRNAs have been designed. The screen has identified codon-edited tRNAs for repairing Trp-TGA, Trp-TAG, Arg-TGA, Gln-TAG, Gln-TA, Glu-TAG, Glu-TAA. The tRNA is approximately 100 nucleotides in length and can be introduced into cells to suppress nonsense codon mutations that should be present for wild-type amino acids. The oligonucleotides can be introduced directly into recipient cells, or can be connected in series to increase the efficacy of the oligonucleotides.

[0072] Expression cassettes and vectors In certain embodiments, the ACT-tRNA is encoded by an expression cassette. In yet another embodiment, the suppressor tRNA of the present invention can be introduced into cells using standard conventional genetic engineering techniques through the use of a vector. Due to the internal promoter sequence of the tRNA coding sequence, the tRNA sequence need not be included in a separate transcription unit, although one transcription unit can be provided.

[0073] In one embodiment of the invention, the nucleotide expression system of the present invention is included in a suitable gene transfer vector, which is subsequently used to transduce cells to express suppressor gene tRNA. The gene delivery vehicle can be any delivery vehicle known in the art, and can include naked DNA promoted by receptor and / or lipid-mediated transfection, and any of many vectors. Such vectors include but are not limited to eukaryotic vectors, prokaryotic vectors (such as bacterial vectors) and viral vectors, including but not limited to retroviral vectors, adenoviral vectors, adeno-associated viral vectors, slow virus vectors (human and other, including porcine animals), herpes virus vectors, Epstein-Barr virus vectors, SV40 virus vectors, pox virus vectors and pseudotyped virus vectors.

[0074] In certain embodiments, ACT-tRNA(PTC) is encoded in a vector. Figure 4 In certain embodiments, the viral vector is a retroviral or adenoviral vector. Examples of retroviral vectors that can be used include, but are not limited to, Moloney murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.

[0075] Retrovirus; Retroviral vector The term "retrovirus" is used to refer to RNA viruses that utilize reverse transcriptase during their replication cycle. Retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus can be integrated into the chromosomes of infected cells; once integrated, it is called a "provirus". The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes required to produce new virus particles. At each end of the provirus is a structure called "long terminal repeat" or "LTR". LTR contains numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. There are several genera within the Retroviridae family, including pool virus A, tumor virus A, tumor virus B, tumor virus C, tumor virus D, slow virus, and foamy virus. Some retroviruses are oncogenic (i.e., tumorigenic), while others are not. Tumor viruses induce sarcomas, leukemias, lymphomas, and breast cancers in susceptible species. Retroviruses infect a wide variety of species and can be transmitted horizontally or vertically. They are integrated into the host DNA and are able to transfer sequences of the host DNA between cells. This has led to the development of retroviruses as vectors for a variety of purposes including gene therapy.

[0076] Retroviruses, including human foamy virus (HFV) and human immunodeficiency virus (HIV), have received much attention recently because their target cells are not restricted to dividing cells and their limited host cell tropism can be easily expanded via pseudotyping with the vesicular stomatitis virus G (VSV-G) envelope glycoprotein (see, e.g., JC Burns et al., Proc. Natl. Acad. Sci. USA 90:8033-8037

[1993] ; AM Lever, Gene Therapy. 3:470-471

[1996] ; and D. Russell and AD Miller, J. Virol., 70:217-222

[1996] ).

[0077] Vector systems generally have a DNA vector and a packaging cell line, wherein the DNA vector contains a small portion of the retroviral sequence (the viral long terminal repeat or "LTR" and the packaging or "psi" signal). The gene to be transferred is inserted into the DNA vector. The viral sequences present on the DNA vector provide the signals required for the insertion or packaging of the vector RNA into the viral particles and the expression of the inserted gene. The packaging cell line provides the viral proteins required for particle assembly (D. Markowitz et al., J. Virol., 62: 1120

[1988] ). In one embodiment of the present invention, the FIV system using a three-plasmid transfection production method in 293T cells is used (Johnston et al., J Virol. 1999 73: 4991-5000). Replication-incompetent viruses have been successfully produced.

[0078] By any of a variety of techniques (e.g., calcium phosphate coprecipitation, lipofection, electroporation), vector DNA is introduced into packaging cells. Viral proteins produced by packaging cells mediate the insertion of vector sequences in RNA form into viral particles, which are then shed into the culture supernatant.

[0079] For cells that naturally divide or are stimulated to divide by growth factors, simple retroviruses such as murine leukemia virus (MLV) vectors are suitable delivery systems. However, a major limitation of using many commonly used retroviral vectors in gene transfer is that many vectors are limited to dividing cells. If non-dividing cells are the target cells, a lentivirus that can infect non-dividing cells can be used.

[0080] As used herein, the term "slow virus" refers to a group (or genus) of retroviruses that cause slowly developing diseases. Included in this group are viruses such as HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), the causative agent of human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; caprine arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possible central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in human-like primates. Diseases caused by these viruses are characterized by long incubation periods and prolonged courses. Typically, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV and SIV also readily infect T lymphocytes (ie, T cells).

[0081] Lentiviruses, which include HIV, SIV, FIV, and equine infectious anemia virus (EIAV), rely on several viral regulatory genes plus a simple structural gag-pol-env gene for efficient intracellular replication. Thus, lentiviruses use a more complex strategy for gene regulation and viral replication than classical retroviruses, in which packaging signals are evident throughout the viral genome. These additional genes exhibit a range of regulatory functions during the lentiviral life cycle. For example, following HIV-1 infection, transcription is upregulated by the expression of Tat through interaction with an RNA target (TAR) in the LTR. The expression of full-length mRNA and spliced ​​mRNA is then regulated by the function of Rev, which interacts with RNA elements present in the gag region and the env region (RRE) (S. Schwartz et al., J. Virol., 66: 150-159

[1992] ). Nuclear export of gag-pol and env mRNAs depends on Rev function. In addition to these two essential regulatory genes, a series of auxiliary genes, including vif, vpr, vpx, vpu and nef, are present in the viral genome and their role in efficient virus production and infectivity has been demonstrated, although they are not absolutely essential for viral replication (K. and F. Wong-Staal, Microbiol. Rev., 55: 193-205 (1991]; RA Subbramanian and EA Cohen, J. Virol. 68: 6831-6835

[1994] ; and D. Trono, Cell 82: 189-192

[1995] ). A detailed description of the structure of an exemplary lentivirus, HIV-1, is given in U.S. Pat. No. 6,531,123.

[0082] A "source" or "original" retrovirus is a wild-type retrovirus from which a pseudotyped retrovirus is derived, or is used as a starting point in the construction of a packaging or transgenic vector, one or more genetic elements used to prepare a vector. A genetic element can be used without change, or it can be mutated (but not beyond the point where it lacks statistically significant sequence similarity to the original element). A vector can have more than one source retrovirus, and the different source retroviruses can be, for example, MLV, FIV, HIV-1 and HIV-2, or HIV and SIV. The term "genetic element" includes, but is not limited to, genes.

[0083] Homologous retrovirus is a wild-type retrovirus, and the vector in question has the maximum sequence identity percentage at the nucleic acid level. Usually, this will be the same as the source retrovirus. However, if the source retrovirus is widely mutated, it is conceivable that the vector will more closely resemble some other retroviruses later. Homologous retrovirus is not necessarily the physical starting point for construction; Direct synthetic genetic elements, especially mutant elements, can be selected, rather than first obtaining the original elements, which are then modified. The term "homologous" can be similarly applied to proteins, genes or genetic elements (for example, splicing donor sites or packaging signals). When referring to homologous proteins, the sequence identity percentage is measured at the amino acid level.

[0084] The term "homologous" retrovirus may be difficult to interpret in extreme cases, i.e., if all retroviral genetic elements have been replaced with alternative non-lentiviral genetic elements. In this case, the previously mentioned source retroviral strain is arbitrarily considered a homologous retrovirus.

[0085] As used herein, the term "replication" in reference to a virus or vector does not refer to the normal replication of proviral DNA in chromosomes due to cell reproduction, nor does it refer to the autonomous replication of plasmid DNA due to the presence of a functional replication origin. Instead, "replication" refers to the completion of the complete viral life cycle, in which infectious virus particles containing viral RNA enter cells, the RNA is reverse transcribed into DNA, the DNA is integrated into the host chromosome as a provirus, and the infected cell produces virion proteins and assembles them together with the full-length viral genomic RNA into new equivalent infectious particles.

[0086] The term "replication competent" refers to a wild-type virus or a mutant virus that is capable of replication such that replication of the virus in an infected cell results in the production of infectious virions that, upon infecting another previously uninfected cell, result in the latter cell likewise producing such infectious virions. The present invention contemplates the use of replication-defective viruses.

[0087] As used herein, the term "attenuated virus" refers to any virus (e.g., an attenuated lentivirus) that has been modified so that its pathogenicity in the intended subject is substantially reduced. A virus can be attenuated to the point where it is non-pathogenic from a clinical standpoint, i.e., subjects exposed to the virus do not show a statistically significantly increased level of pathology relative to control subjects.

[0088] The present invention contemplates the preparation and use of modified retroviruses. In some embodiments, the retrovirus is a mutant of murine leukemia virus, human immunodeficiency virus type 1, human immunodeficiency virus type 2, feline immunodeficiency virus, simian immunodeficiency virus, Vesna-Medi, caprine arthritis-encephalitis virus, equine infectious anemia virus, and bovine immunodeficiency virus, or a virus composed of portions of more than one retroviral species (e.g., a hybrid composed of portions of MLV, FIV, HIV-1 and HIV-2, or HIV-1 and / or SIV).

[0089] A reference virus is a virus whose genome is used to describe the components of a mutant virus. For example, a specific genetic element of a mutant virus can be said to differ from a homologous element of a reference virus by various substitutions, deletions, or insertions. A mutant virus is not necessarily actually derived from a reference virus.

[0090] Preferred reference FIV sequences are found in Talbott et al., Proc Natl Acad Sci US A. 198986:5743-7; Genbank accession number NC_001482. In certain embodiments, a three-plasmid transient transfection method can be used to produce a pseudotyped retrovirus (e.g., FIV) without replication competence. General methods are described in Wang et al., J Clin Invest. 1999104:R55-62 and Johnston et al., J Virol. 199973:4991-5000.

[0091] Retroviral vector system The present invention contemplates a retroviral gene amplification and transfer system comprising a transgenic vector, one or more compatible packaging vectors, an envelope vector, and a suitable host cell. The vector used can be derived from a retrovirus (e.g., a lentivirus). Retroviral vectors allow (1) transfection of the packaging vector and the envelope vector into a host cell to form a packaging cell line that produces viral particles that are substantially free of packaging vector-RNA, (2) transfection of the transgenic vector into the packaging cell line, (3) packaging of the transgenic vector RNA into infectious viral particles by the packaging cell line, and (4) administration of the particles to target cells so that such cells are transduced and subsequently express the transgene.

[0092] The particles are administered directly to the subject in vivo, or cells from the subject are removed, infected with the particles ex vivo, and returned to the subject.

[0093] The packaging vectors and transgenic vectors of the present invention will generate replication-incompetent viruses. The vectors selected for incorporation into a given vector system of the present invention are such that, without further mutation of the packaging vector or transgenic vector, the co-transfected cells are unable to generate replication-competent viruses by homologous recombination of the packaging vector and transgenic vector alone. The envelope protein used in the present system can be a retroviral envelope, a synthetic or chimeric envelope, or an envelope from a non-retroviral enveloped virus (e.g., a baculovirus).

[0094] Packaging Signals As used herein, the term "packaging signal" or "packaging sequence" refers to a sequence located within a retroviral genome or vector that is required for inserting the viral or vector RNA into a viral capsid or particle, or at least facilitates the insertion of the viral or vector RNA into a viral capsid or particle. The packaging signal in the RNA identifies the RNA as the RNA to be packaged into the viral particle. For convenience, the term "packaging signal" is also used to refer to a vector DNA sequence that is transcribed into a functional packaging signal. Some packaging signals may be part of a gene, but are recognized in the form of RNA rather than as a peptide portion of a coded protein.

[0095] The key difference between packaging vectors and transgenic vectors is that in packaging vectors, the major packaging signal is inactivated, while in transgenic vectors, the major packaging signal is functional. Ideally, in packaging vectors, all packaging signals are inactivated, while in transgenic vectors, all packaging signals are functional. However, opposing considerations, such as maximizing viral titers or inhibiting homologous recombination, may make such constructs less desirable.

[0096] packaging system; packaging vector; packaging cell line The packaging system is one or more vectors that together provide all the genetic information required to produce a virion in an expressible form that can encapsidate the appropriate RNA, transport it from the cell producing the virion, deliver it to the target cell, and in the target cell, cause the RNA to be reverse transcribed and integrated into the host genome in such a way that the transgene incorporated into the RNA can be expressed. However, the packaging system must not be substantially able to package itself. Instead, it packages a separate transgene vector.

[0097] In the present invention, the packaging vector will provide functional equivalents of the gag and pol genes ("GP" vector). The env gene will be provided by the envelope vector. In theory, if one wishes to construct the different gag and pol genes on separate vectors and operably link them to different regulatable promoters (or one operably linked to a regulatable promoter and the other to a constitutive promoter) so that their relative expression levels can be appropriately adjusted, a three-vector system ("G", "P" and "E" vectors) is possible.

[0098] A packaging cell line is a suitable host cell transfected with a packaging system that produces viral particles under conditions that allow the production of viral particles. As used herein, the term "packaging cell line" is generally used to refer to a cell line that expresses viral structural proteins (e.g., gag, pol, and env) but does not contain a packaging signal. For example, a cell line has been genetically engineered to carry a lack of a functional psi gene at a chromosomal site within its genome. + The 5'-LTR-gag-pol-3'-LTR fragment with a sequence (designated as Δ-psi) and a 5'-LTR-env-3'-LTR fragment located at another chromosomal locus also with Δ-psi. Although both fragments are constitutively transcribed, the lack of psi + region and the resulting viral RNA molecules are smaller than full size, thus forming empty viral particles.

[0099] If a host cell is transfected with a packaging vector alone, it essentially produces only viral particles, without the full-length packaging vector. In one example, less than 10% of the viral particles produced by the packaging cells contained full-length packaging vector-derived RNA. However, because the packaging vector lacks a functional primer binding site, even if these particles infect new cells, the packaging vector RNA is not reverse transcribed back into DNA, and thus the new cells do not produce viral particles. Therefore, the packaging vector itself is a replication-incompetent virus.

[0100] In some embodiments, the packaging cell and / or cell line contains the transgenic vector. The packaging cell line packages the transgenic vector into infectious particles. Such cell lines are referred to herein as "transgenic virion production cell lines".

[0101] It is contemplated that packaging can be inducible, as well as non-inducible. In inducible packaging cells and packaging cell lines, retroviral particles are produced in response to at least one inducing agent. In non-inducible packaging cell lines and packaging cells, no inducing agent is required in order for retroviral particle production to occur.

[0102] The packaging vector must be different from the wild-type, replication-competent retroviral genome due to the inactivation of at least one packaging signal of the homologous wild-type genome. More than one packaging signal may be inactivated. In one example, the only retroviral genes provided by the packaging vector are those encoding structural proteins or essential regulatory proteins.

[0103] Transgenic vector The transgenic vector is an expression vector that carries an expressible non-retroviral target gene and includes at least one functional retroviral packaging signal, so that after the transgenic vector is transfected into the packaging cell line, the transgenic vector is transcribed into RNA, and this RNA is packaged into infectious viral particles. These particles in turn infect target cells, their RNA is reverse transcribed into DNA, and the DNA is incorporated into the host cell genome as a proviral element, thereby delivering the target gene to the target cell.

[0104] As used herein, the term "transduction" refers to gene delivery using a viral vector or retroviral vector by means of infection rather than by transfection. In certain embodiments, a retroviral vector is transduced. Therefore, a "transduced gene" is a gene that has been introduced into a cell by retrovirus or vector infection and proviral integration. In certain embodiments, a viral vector (such as a "transgenic vector") transduces genes into a "target cell" or host cell. The present invention encompasses transgenic vectors suitable for use in the present invention, which are connected to any target gene (or a "marker gene" or "reporter gene" for indicating infection or expression of a gene).

[0105] As used herein, the term "long-term transduction" refers to a vector that is able to maintain transduction in a host cell or target cell for a period of time longer than those observed with other vectors. For example, the present invention provides retroviral vectors that are able to maintain transduction for at least 120 days, at least one year, or the life span of the subject or the necessary course of treatment. The duration of expression is a function of the promoter and target cell type selection, not the selection of the vector.

[0106] The term "stable transduction" or "stably transduced" refers to the introduction and integration of foreign DNA into the genome of the transduced cell. The term "stable transductant" refers to a cell that has stably integrated foreign DNA into its genomic DNA.

[0107] The term "transient transduction" or "transiently transduced" refers to the introduction of exogenous DNA into a cell, wherein the exogenous DNA fails to integrate into the genome of the transduced cell. The exogenous DNA persists in the nucleus of the transduced cell for several days. During this period, the exogenous DNA is under regulatory control that controls the expression of endogenous genes in the chromosomes. The term "transient transducent" refers to a cell that has taken up exogenous DNA but has failed to integrate the DNA.

[0108] In some embodiments, the target and / or host cells of the present invention are "non-dividing" cells. These cells include cells that do not normally divide, such as neuronal cells. However, it is not intended to limit the present invention to non-dividing cells (including but not limited to muscle cells, white blood cells, spleen cells, liver cells, eye cells, epithelial cells).

[0109] In some embodiments, the vector and vector progeny are capable of transducing multiple target cells so as to achieve at least 10 5 The vector titer is in cfu / ml. The multiplicity of infection (MOI) may be at least one (ie, an average of one hit per cell), or even at least two.

[0110] Expression cassettes and vectors The present invention also provides an expression cassette comprising a sequence encoding ACE-tRNA.

[0111] In certain embodiments, the expression cassette further comprises a promoter. In certain embodiments, the promoter is a regulatable promoter. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a PGK, CMV, RSV, H1 or U6 promoter (Pol II and Pol III promoter).

[0112] The present invention provides a vector containing the above expression cassette. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a viral vector based on adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, HSV or mouse Moloney.

[0113] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to a target nucleotide sequence, which may be operably linked to a termination signal. It may also include sequences required for the correct translation of the nucleotide sequence. The coding region usually encodes a target protein. The expression cassette including the target nucleotide sequence may be chimeric. The expression cassette may also be naturally occurring, but that which has been obtained in a recombinant form that can be used for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter, which initiates transcription only when the host cell is exposed to certain specific stimuli. In the case of multicellular organisms, the promoter may also be specific to a specific tissue or organ or developmental stage.

[0114] "Operably linked" refers to the combination of nucleic acid sequences on a single nucleic acid fragment such that the function of one of the sequences is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked" or "bound" to a DNA sequence encoding an RNA or polypeptide if the two sequences are located such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0115] Adeno-associated virus (AAV) Adeno-associated virus (AAV) is a small non-pathogenic virus of the Parvoviridae family. The difference between AAV and other members of the family is that it relies on a helper virus for replication. In the absence of a helper virus, AAV may be integrated into the q arm of chromosome 19 in a locus-specific manner. The approximately 5kb genome of AAV consists of a segment of single-stranded DNA of positive or negative polarity. The end of the genome is a short reverse terminal repeat, which can be folded into a hairpin structure and serves as the starting point for viral DNA replication. Physically, the parvovirus particle is non-enveloped, and its icosahedral capsid is about 20nm in diameter.

[0116] So far, numerous serologically different AAVs have been identified, and more than a dozen have been isolated from humans or primates. The genome of AAV2 is 4680 nucleotides in length and contains two open reading frames (ORFs). The left ORF encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which, in addition to the production of single-stranded progeny genomes, are also involved in the regulation of replication and transcription. In addition, two Rep proteins have been preferentially integrated into the region of the q arm of human chromosome 19 with the AAV genome. Rep68 / 78 has also been shown to have NTP binding activity and DNA and RNA helicase activity. Rep protein has a nuclear localization signal and several potential phosphorylation sites. A mutation in one of these kinase sites results in the loss of replication activity.

[0117] The end of the genome is a short reverse terminal repeat (ITR), which has the potential to be folded into a T-shaped hairpin structure, which serves as the starting point for viral DNA replication. In the ITR region, two elements essential to the function of ITR have been described: the GAGC repeat motif and the terminal melting site (trs). When ITR is in linear or hairpin configuration, the repeat motif has been shown to bind Rep. This combination acts to locate Rep68 / 78 for the cutting at the trs, and the cutting occurs in a site and strand-specific manner. Except for its role in replication, these two elements seem to be essential for viral integration. What is included in No. 19 chromosome integration loci is the Rep binding site with adjacent trs. These elements have been shown to be functional, and are essential for locus-specific integration.

[0118] AAV virions are non-enveloped icosahedral particles with a diameter of approximately 25 nm, composed of three related proteins called VP1, VP2 and VP3. The right ORF encodes the capsid proteins VP1, VP2 and VP3. These proteins are found in a ratio of 1:1:10, respectively, and are all derived from the right ORF. The capsid proteins differ from each other by using alternative splicing and rare start codons. Deletion analysis has shown that the removal or alteration of VP1 translated from alternative splicing information leads to a reduced yield of infectious particles. Mutations in the VP3 coding region result in the inability to produce any single-stranded progeny DNA or infectious particles. AAV particles are viral particles containing AAV capsid proteins. AAV capsid polypeptides can encode the entire VP1, VP2 and VP3 polypeptides. Particles can be particles (i.e., chimeric proteins, such as AAV1 and AAV2) containing AAV2 and other AAV capsid proteins. Changes in the amino acid sequence of the AAV2 capsid protein are contemplated herein, as long as the resulting viral particles comprising the AAV2 capsid remain antigenically or immunologically distinct from AAV1, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blot can be used to determine whether the viral particles are antigenically or immunologically distinct from AAV1. In addition, the AAV2 viral particles preferably retain a tissue tropism that is distinct from that of AAV1.

[0119] AAV2 particles are virus particles comprising AAV2 capsid proteins. AAV2 capsid polypeptides encoding the entire VP1, VP2 and VP3 polypeptides may generally have at least about 63% homology (or identity) with a polypeptide having an amino acid sequence encoded by the nucleotides shown in NC_001401 (nucleotide sequence encoding AAV2 capsid protein). The capsid protein may have about 70% homology, about 75% homology, 80% homology, 85% homology, 90% homology, 95% homology, 98% homology, 99% homology or even 100% homology with the protein encoded by the nucleotide sequence shown in NC_001401. The capsid protein may be about 70% identical, about 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 98% identical, 99% identical or even 100% identical to the protein encoded by the nucleotide sequence shown in NC_001401. The particle may be a particle comprising another AAV and AAV2 capsid protein, i.e., a chimeric protein. Changes in the amino acid sequence of the AAV2 capsid protein are contemplated herein, as long as the resulting viral particles comprising the AAV2 capsid remain antigenically or immunologically different from AAV4, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blot can be used to determine whether the viral particles are antigenically or immunologically different from AAV1. In addition, the AAV2 viral particles preferably retain a tissue tropism different from AAV1, such as that exemplified in the examples herein, although AAV2 chimeric particles comprising at least one AAV2 coat protein may have a different tissue tropism than AAV2 particles consisting only of AAV2 coat proteins.

[0120] In certain embodiments, the present invention further provides AAV2 particles, which contain, i.e., encapsidate a vector containing a pair of AAV2 inverted terminal repeats. The nucleotide sequence of AAV2 ITR is known in the art. In addition, the particle can be a particle containing both AAV1 and AAV2 capsid proteins, i.e., a chimeric protein. In addition, the particle can be an encapsidate particle containing a pair of AAV inverted terminal repeats from other AAVs (e.g., AAV1-AAV9 and AAVrh10). The encapsidated vector in the particle can further contain an exogenous nucleic acid inserted between the inverted terminal repeats.

[0121] The following characteristics of AAV have made it an attractive vector for gene transfer. AAV vectors have been shown to stably integrate into the cell genome in vitro; have a wide host range; transduce both dividing and non-dividing cells in vitro and in vivo, and maintain high expression levels of the transduced gene. The virus particles are thermostable, resistant to solvents, detergents, changes in pH, temperature, and can be concentrated on a CsCl gradient or by other means. The present invention provides methods for administering AAV particles, recombinant AAV vectors, and recombinant AAV virions. For example, an AAV2 particle is a virus particle comprising an AAV2 capsid protein, or an AAV1 particle is a virus particle comprising an AAV1 capsid protein. A recombinant AAV2 vector is a nucleic acid construct comprising at least one unique nucleic acid of AAV2. Recombinant AAV2 virions are particles containing a recombinant AAV2 vector. To be considered within the term "AAV2 ITR", a nucleotide sequence must retain one or both of the features described herein that distinguish AAV2 ITRs from AAV1 ITRs: (1) three (rather than four as in AAV1) "GAGC" repeats, and (2) in the AAV2 ITR Rep binding site, the fourth nucleotide in the first two "GAGC" repeats is C rather than T.

[0122] The promoter driving the expression of the sequence encoding the tRNA to be delivered can be any desired promoter, which is selected by known considerations, such as the expression level of the nucleic acid functionally connected to the promoter and the cell type in which the vector is to be used. The promoter can be an exogenous promoter or an endogenous promoter. The promoter can include, for example, a known strong promoter such as SV40, or an inducible metallothionein promoter, or an AAV promoter such as an AAV p5 promoter. Other examples of promoters include promoters derived from: actin genes, immunoglobulin genes, cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenovirus promoters such as adenovirus major late promoters, inducible heat shock promoters, respiratory syncytial virus, Rous sarcoma virus (RSV), etc. Other examples include regulated promoters.

[0123] The AAV vector may further include an exogenous (heterologous) nucleic acid functionally connected to a promoter. "Heterologous nucleic acid" means that any heterologous or exogenous nucleic acid can be inserted into a vector for transfer into a cell, tissue or organism. For example, a nucleic acid can encode tRNA. As known in the art, "functional connection" means that a promoter can promote the expression of a heterologous nucleic acid, such as the appropriate orientation of a promoter relative to a heterologous nucleic acid. In addition, as known in the art, a heterologous nucleic acid preferably has all appropriate sequences for nucleic acid expression, to functionally encode, i.e., to allow the nucleic acid to be expressed. Nucleic acid may include, for example, an expression control sequence, such as an enhancer. Nucleic acid can encode more than one gene product, limited only by the size of the nucleic acid that can be packaged.

[0124] AAV1 particles are viral particles comprising AAV1 capsid proteins. Changes in the amino acid sequence of AAV1 capsid proteins are contemplated herein, as long as the resulting viral particles comprising AAV1 capsids remain antigenically or immunologically distinct from other AAV capsids, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blots can be used to determine whether viral particles are antigenically or immunologically distinct from other AAV serotypes.

[0125] As used herein, the term "polypeptide" refers to a polymer of amino acids, and includes full-length proteins and fragments thereof. Therefore, "protein" and "polypeptide" are often used interchangeably herein.

[0126] The present method provides a method for delivering nucleic acid to cells, which includes administering a vector containing AAV particles to cells, the vector comprising a nucleic acid inserted between a pair of AAV reverse terminal repeats, thereby delivering nucleic acid to cells. Administration to cells can be completed by any means, including simply contacting particles optionally contained in a desired liquid such as tissue culture medium or a buffered saline solution with cells. Particles can be allowed to remain in contact with cells for any desired length of time, and generally, particles are administered and allowed to be maintained indefinitely. For such in vitro methods, as known in the art and as illustrated herein, viruses can be administered to cells by standard viral transduction methods. The viral titer administered can vary, particularly depending on the cell type, but is generally a typical titer for AAV transduction. In addition, the titer for transducing specific cells in the present embodiment can be utilized. These cells can include any desired cells in humans and other large (non-rodent) mammals, such as primates, horses, sheep, goats, pigs and dogs.

[0127] The present invention further provides a method of delivering a nucleic acid to a cell in a subject, comprising administering to the subject an AAV particle comprising a nucleic acid inserted between a pair of AAV inverted terminal repeats, thereby delivering the nucleic acid to the cell in the subject.

[0128] Certain embodiments of the present disclosure provide cells comprising a viral vector as described herein.

[0129] AAV vectors In one embodiment, the viral vector of the present disclosure is an AAV vector. An "AAV" vector refers to an adeno-associated virus, and can be used to refer to the naturally occurring wild-type virus itself or a derivative thereof. The term encompasses all subtypes, serotypes, and pseudotypes, as well as both naturally occurring and recombinant forms, unless otherwise required. As used herein, the term "serotype" refers to an AAV that is identified and distinguished from other AAVs based on the reactivity of the capsid protein with a defined antiserum, for example, there are eight known serotypes of primate AAVs, AAV-1 to AAV-9 and AAVrh10. For example, serotype AAV2 is used to refer to an AAV that contains a capsid protein encoded by the cap gene of AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype. As used herein, for example, rAAV1 can be used to refer to an AAV having both capsid proteins and 5'-3' ITRs from the same serotype, or it can refer to an AAV having capsid proteins from one serotype, and 5'-3' ITRs from a different AAV serotype, such as a capsid from AAV serotype 2 and an ITR from AAV serotype 5. For each example presented herein, the description of vector design and production describes the serotype of the capsid and 5'-3' ITR sequences. The abbreviation "rAAV" refers to a recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").

[0130] "AAV virus" or "AAV virus particle" refers to a virus particle composed of at least one AAV capsid protein (preferably all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is usually referred to as "rAAV".

[0131] In one embodiment, an AAV expression vector is constructed using known techniques to provide at least control elements, including a transcription initiation region, a target DNA, and a transcription termination region, as operably linked components in the transcription direction. The control elements are selected to be functional in mammalian cells. The resulting construct containing the operably linked components is flanked (5' and 3') by functional AAV ITR sequences.

[0132] "Adeno-associated virus inverted terminal repeats" or "AAV ITRs" refers to the art-recognized regions found at each end of the AAV genome that function together in cis as an origin of DNA replication and a packaging signal for the virus. The AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue of nucleotide sequences inserted between the two flanking ITRs, as well as integration into the mammalian cell genome.

[0133] The nucleotide sequence of the AAV ITR region is known. As used herein, "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be changed, for example, by insertion, deletion or substitution of nucleotides. In addition, AAV ITR may be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, etc. In addition, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector are not necessarily identical or derived from the same AAV serotype or isolate, as long as they function as expected, i.e., allowing the excision and rescue of the target sequence from the host cell genome or the vector, and when the AAV Rep gene product is present in the cell, allowing the heterologous sequence to be integrated into the recipient cell genome.

[0134] In one embodiment, the AAV ITRs may be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, etc. Furthermore, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV expression vector do not have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, i.e., allowing excision and rescue of the sequence of interest from the host cell genome or vector, and allowing integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0135] In one embodiment, the AAV capsid can be derived from AAV2. Suitable DNA molecules for use in AAV vectors are less than about 5 kilobases (kb), less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, less than about 2.5 kb in size, and are known in the art.

[0136] In one embodiment, the selected nucleotide sequence is operably linked to a control element that directs its transcription or expression in a subject. Such control elements may include control sequences that are generally associated with the selected gene. Alternatively, heterologous control sequences may be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, pol II promoter, pol III promoter, synthetic promoter, hybrid promoter, etc. In addition, sequences derived from non-viral genes, such as mouse metallothionein genes, may also be used herein. Such promoter sequences are commercially available from, for example, Stratagene (San Diego, Calif.).

[0137] In one embodiment, heterologous promoters and other control elements, such as tissue-specific and inducible promoters, enhancers, etc., are of particular use. Examples of heterologous promoters include the CMV promoter. Examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and aufin.

[0138] In one embodiment, an AAV expression vector having a DNA molecule of interest bound by an AAV ITR can be constructed by inserting the selected sequence directly into the AAV genome, which already has the major AAV open reading frame ("ORF") excised therefrom. Other portions of the AAV genome may also be deleted, as long as sufficient ITR portions are retained to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art.

[0139] Alternatively, the AAV ITRs can be excised from the viral genome or an AAV vector containing the same 5' and 3' fused nucleotide constructs as those present in another vector using standard ligation techniques. For example, ligation can be performed in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 μg / ml BSA, 10 mM-50 mM NaCl and 40 μM ATP at 0°C, 0.01-0.02 (Weiss) units of T4 DNA ligase (for "sticky end" ligation), or 1 mM ATP at 14°C, 0.3-0.6 (Weiss) units of T4 DNA ligase (for "blunt end" ligation). Intermolecular "sticky end" ligation is typically performed at a total DNA concentration of 30-100 μg / ml (total end concentration of 5-100 nM). The AAV vector contains ITRs.

[0140] Alternatively, chimeric genes can be generated synthetically to include AAV ITR sequences arranged 5' and 3' to one or more selected nucleic acid sequences. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods.

[0141] To produce rAAV virions, the AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. Many transfection techniques are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate coprecipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-speed microprojectiles.

[0142] In one embodiment, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells and mammalian cells, which can be or have been used as recipients of heterologous DNA molecules. The term includes the offspring of transfected original cells. Therefore, as used herein, "host cells" generally refer to cells transfected with exogenous DNA sequences. Cells from a stable human cell line 293 (easily available through, for example, the American Type Culture Collection with accession number ATCC CRL1573) can be used in the practice of the present disclosure. In particular, the human cell line 293 is a human embryonic kidney cell line that has been transformed with type 5 adenovirus DNA fragments and expresses adenovirus E1a and E1b genes. The 293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV virions therein.

[0143] "AAV rep coding region" means the region of the AAV genome recognized in the art that encodes the replication proteins Rep 78, Rep 68, Rep 52, and Rep 40. These Rep expression products have been shown to have many functions, including recognition, binding, and nicking of the DNA replication origin of AAV, DNA helicase activity, and modulation of transcription from AAV (or other heterologous) promoters. Rep expression products are commonly required for replication of the AAV genome. Suitable homologs of the AAV rep coding region include the human herpes virus 6 (HHV-6) rep gene, which is also known to mediate AAV-2 DNA replication.

[0144] "AAV cap coding region" refers to the art-recognized AAV genome region that encodes capsid proteins VP1, VP2 and VP3 or their functional homologs. These Cap expression products provide packaging functions, which are commonly required for packaging viral genomes.

[0145] In one embodiment, by transfecting the host cell with an AAV helper construct before or simultaneously with the AAV expression vector transfection, the AAV helper function is introduced into the host cell. The AAV helper construct is therefore used to provide at least transient expression of the AAV rep and / or cap genes to supplement the AAV functions necessary for the productive AAV infection of the deletion. The AAV helper construct lacks the AAV ITR and can neither replicate nor package itself. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products have been described.

[0146] The delivery method of the viral vector includes injecting AAV into the subject. Generally, the rAAV virions can be introduced into the cells using in vivo or in vitro transduction techniques. If transduced in vitro, the desired recipient cells are removed from the subject, transduced with rAAV virions and reintroduced into the subject. Alternatively, isogenic cells or xenogeneic cells can be used, wherein those cells do not generate an inappropriate immune response in the subject.

[0147] Suitable methods for delivering and introducing transduced cells into a subject have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with cells, for example in an appropriate culture medium, and those cells with the target DNA can be screened using conventional techniques (e.g., Southern blots and / or PCR), or by using selectable markers. The transduced cells can then be formulated into a pharmaceutical composition described more fully below, and the composition can be introduced into a subject by various techniques, such as by transplantation, intramuscular, intravenous, subcutaneous, and intraperitoneal injection.

[0148] In one embodiment, the pharmaceutical composition comprises enough genetic material to produce a therapeutically effective amount of the target nucleic acid, that is, an amount sufficient to reduce or improve the symptoms of the disease state in question, or an amount sufficient to confer the desired benefit. The pharmaceutical composition also contains a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80 and liquids, such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc., may be included. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may be present in such vehicles. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0149] It should be understood that more than one transgene can be expressed by the delivered viral vector. Alternatively, separate vectors, each expressing one or more different transgenes, can also be delivered to a subject as described herein. In addition, it is also contemplated that the viral vectors delivered by the methods of the present disclosure can be combined with other suitable compositions and therapies.

[0150] As will be apparent to those skilled in the art, in view of the teachings of this specification, the effective amount of the viral vector that must be added can be determined empirically. Administration can be achieved in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means of administration and dosage are well known to those skilled in the art and will vary with the viral vector, the composition of the therapy, the target cell, and the subject to be treated. Single and multiple administrations can be performed with dosage levels and patterns selected by the treating physician.

[0151] In certain embodiments, the rAAV is administered at a dose of about 0.3-2 ml 1x105-1x1016 vg / ml. In certain embodiments, the rAAV is administered at a dose of about 1-3 ml 1x107-1x1014 vg / ml. In certain embodiments, the rAAV is administered at a dose of about 1-2 ml 1x108-1x1013 vg / ml.

[0152] The formulation containing rAAV particles will contain an effective amount of rAAV particles in a vehicle, which is easily determined by a person skilled in the art. The rAAV particles can generally range from about 1% to about 95% (w / w) of the composition, or even higher or lower when appropriate. The amount to be administered depends on factors such as the age, weight and physical condition of the animal or human subject being considered for treatment. One of ordinary skill in the art can determine the effective dose by routine experiments to establish a dose response curve. The subject is treated by administering rAAV particles in one or more doses. Multiple doses can be administered as needed to maintain sufficient enzyme activity.

[0153] Vehicles, including water, aqueous saline, artificial CSF or other known substances, can be used with the present invention. To prepare the formulation, the purified composition can be isolated, lyophilized and stabilized. The composition can then be adjusted to an appropriate concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0154] The present invention provides methods for increasing the level of a target protein in a cell by introducing a protein or a nucleic acid molecule encoding the protein into a cell in an amount sufficient to increase the level of the target protein in the cell. In certain embodiments, the accumulation of the target protein is increased by at least 10%. The accumulation of the target protein is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[0155] Nucleic acids encoding therapeutic agents The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, which are composed of monomers (nucleotides) containing sugars, phosphates, and bases that are purines or pyrimidines. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0156] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence using one of the alignment programs described with standard parameters.

[0157] Methods for introducing genetic material into cells By genetic transfer methods, such as transfection or transduction, exogenous genetic material (e.g., DNA encoding one or more therapeutic ACE-tRNAs) is introduced into cells in vivo to provide genetically modified cells. Various expression vectors (i.e., vehicles for facilitating delivery of exogenous genetic material into target cells) are known to those of ordinary skill in the art.

[0158] As used herein, "transfection of cells" refers to the acquisition of new genetic material by cells by incorporation of added DNA. Thus, transfection refers to the insertion of nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art, including: calcium phosphate DNA coprecipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; and tungsten particle-facilitated microprojectile bombardment. Strontium phosphate DNA coprecipitation is another possible transfection method.

[0159] In contrast, "transduction of cells" refers to the process of transferring nucleic acids into cells using DNA or RNA viruses. RNA viruses (i.e., retroviruses) used to transfer nucleic acids into cells are referred to herein as transducing chimeric retroviruses. The exogenous genetic material contained in the retrovirus is incorporated into the genome of the transduced cells. Cells that have been transduced with chimeric DNA viruses (e.g., adenoviruses carrying cDNA encoding therapeutic agents) do not have exogenous genetic material incorporated into their genomes, but are able to express exogenous genetic material retained outside the chromosomes in the cells.

[0160] Typically, exogenous genetic material includes a heterologous gene (usually in the form of a cDNA containing exons encoding therapeutic proteins), together with a promoter that controls the transcription of the new gene. The promoter uniquely has a specific nucleotide sequence necessary for initiating transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required for obtaining the desired gene transcription activity. For the purposes of this discussion, an "enhancer" is simply any non-translated DNA sequence that works adjacent to the coding sequence (cis) to change the basal transcription level determined by the promoter. Exogenous genetic material can be introduced into the cell genome in the immediate downstream of the promoter so that the promoter and the coding sequence are operably connected to allow transcription of the coding sequence. Retroviral expression vectors can include exogenous promoter elements to control the transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive promoters and inducible promoters.

[0161] Naturally occurring constitutive promoters control the expression of basic cellular functions. As a result, genes under the control of constitutive promoters are expressed under all cell growth conditions. Exemplary constitutive promoters include promoters for the following genes encoding certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include, in particular, 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. Therefore, any of the constitutive promoters mentioned above can be used to control the transcription of heterologous gene inserts.

[0162] Genes under the control of inducible promoters are expressed only in the presence of an inducing agent or expressed to a higher degree (for example, transcription under the control of metallothionein promoters greatly increases in the presence of certain metal ions). Inducible promoters include response elements (REs) that stimulate transcription when their inducing factors bind. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain inducible responses, and in some cases, REs themselves can be attached to different promoters, thereby giving induction to recombinant genes. Therefore, by selecting appropriate promoters (constitutive versus inducible; strong versus weak), both the presence and expression level of the therapeutic agent in genetically modified cells can be controlled. If the gene encoding the therapeutic agent is under the control of an inducible promoter, then by exposing the genetically modified cells in situ to conditions that allow the therapeutic agent to transcribe, for example, by intraperitoneal injection of the specific inducer of the inducible promoter (which controls the transcription of the therapeutic agent), the in situ delivery of the therapeutic agent is triggered. For example, in situ expression of a therapeutic agent encoded by a gene under the control of a metallothionein promoter by a genetically modified cell is enhanced by contacting the genetically modified cell in situ with a solution containing the appropriate (ie, inducing) metal ion.

[0163] Thus, the amount of therapeutic agent delivered in situ is regulated by controlling such factors as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak); (2) the number of copies of the exogenous gene inserted into the cell; (3) the number of transduced / transfected cells administered to (e.g., implanted into) the patient; (4) the size of the implant (e.g., a graft or encapsulated expression system); (5) the number of implants; (6) the length of time the transduced / transfected cells or implants remain in situ; and (7) the productivity of the therapeutic agent by the genetically modified cells. The selection and optimization of these factors for delivering a therapeutically effective dose of a particular therapeutic agent, taking into account the factors disclosed above and the clinical profile of the patient, is deemed to be within the ordinary skill of the art without undue experimentation.

[0164] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may also include a selection gene, such as a neomycin resistance gene, for facilitating selection of cells transfected or transduced with the expression vector. Alternatively, cells are transfected with two or more expression vectors, at least one vector containing a gene encoding a therapeutic agent and another vector containing a selection gene. The selection of suitable promoters, enhancers, selection genes and / or signal sequences (described below) is considered to be within the scope of ordinary skill in the art without undue experimentation.

[0165] Disease conditions and treatments In one embodiment, the invention includes compositions and methods for treating cystic fibrosis by reversing the effects of existing nonsense mutation-related mutations by introducing the synthetic oligonucleotide suppressor tRNA of the invention.

[0166] Certain embodiments of the present disclosure provide methods of treating a disease in a mammal, comprising administering to the mammal a protein as described herein or a vector encoding a therapeutic agent (e.g., a modified and / or stabilized ACE-tRNA). In certain embodiments, the mammal is a human.

[0167] Certain embodiments of the present disclosure provide the use of a therapeutic agent as described herein or a vector encoding a therapeutic agent for the preparation of a medicament useful for treating a disease in a mammal. In certain embodiments, the disease is cystic fibrosis.

[0168] The disclosure also provides mammalian cells containing the vectors described herein. The cells can be human.

[0169] Certain aspects of the present disclosure relate to polynucleotides, polypeptides, vectors and genetically engineered cells (modified in vivo), and uses thereof. In particular, the present disclosure relates to methods for gene therapy that are capable of systemically delivering therapeutically effective doses of therapeutic agents.

[0170] According to one aspect, a cell expression system for expressing a therapeutic agent in a mammalian recipient is provided. The expression system (also referred to herein as "genetically modified cells") comprises a cell and an expression vector for expressing a therapeutic agent. Expression vectors include, but are not limited to, viruses, plasmids, and other vectors for delivering heterologous genetic material to cells. Therefore, as used herein, the term "expression vector" refers to a vector for delivering heterologous genetic material to cells. In particular, the expression vector is a recombinant adenovirus, an adeno-associated virus, or a lentiviral or retroviral vector.

[0171] The expression vector further comprises a promoter for controlling transcription of the heterologous gene. The promoter may be an inducible promoter (described herein). The expression system is suitable for administration to a mammalian recipient. The expression system may comprise a plurality of non-immortal genetically modified cells, each containing at least one recombinant gene encoding at least one therapeutic agent.

[0172] The cell expression system is formed in vivo. According to another aspect, a method for treating a mammalian recipient in vivo is provided. The method includes, for example, introducing an expression vector for expressing a heterologous gene product into a patient's cells in situ via intravenous administration. To form an expression system in vivo, an expression vector for expressing a therapeutic agent is introduced iv into a mammalian recipient in vivo.

[0173] According to yet another aspect, a method for treating a mammalian recipient in vivo is provided. The method comprises introducing a target therapeutic agent into the patient in vivo.

[0174] The expression vector used to express the heterologous gene may include an inducible promoter for controlling the transcription of the heterologous gene product. Thus, the in situ delivery of the therapeutic agent is controlled by exposing the cell in situ to conditions that induce transcription of the heterologous gene.

[0175] The present disclosure provides methods for treating diseases in mammals by administering expression vectors to cells or patients. For gene therapy methods, those of ordinary skill in the art of molecular biology and gene therapy can determine the appropriate dosage and administration route of the expression vectors used in the novel methods of the present disclosure without undue experimentation.

[0176] According to one embodiment, cells are transformed or otherwise genetically modified in vivo. Cells from a mammalian recipient are transformed (i.e., transduced or transfected) in vivo with a vector containing exogenous genetic material for expressing a heterologous (e.g., recombinant) gene encoding a therapeutic agent and delivering the therapeutic agent in situ.

[0177] As used herein, "exogenous genetic material" refers to a natural or synthetic nucleic acid or oligonucleotide that is not naturally found in a cell; or if it is naturally found in a cell, it is not transcribed or expressed at a biologically significant level by the cell. Thus, "exogenous genetic material" includes, for example, non-naturally occurring nucleic acids that can be transcribed into tRNA.

[0178] The therapeutic agents and conditions amenable to gene therapy disclosed above are exemplary only and are not intended to limit the scope of the present disclosure. The selection of suitable therapeutic agents for treating known conditions is deemed to be within the ordinary skill in the art without undue experimentation.

[0179] In certain embodiments, the therapy has a potential use for treating / managing diseases caused by premature termination codons (PTC), including but not limited to cystic fibrosis, muscular dystrophy, beta thalassemia and Liddle syndrome. The advantage of this therapy is that it provides improved termination codon suppression specificity. The therapeutic ACE-tRNA of the present invention targets specific termination codons, such as TGA, thereby reducing the off-target effect at termination codons unrelated to the disease. The advantage of this therapy is also that it provides amino acid specificity. The expressed tRNA is modified to specifically replace the amino acids lost via the insertion of disease termination codons, thus eliminating any spurious effects on protein stability, folding and transport.

[0180] In certain embodiments, the present system is modular, and therefore each possible disease PTC can be "individualized". For example, there are nine individual tryptophan tRNAs recognized by Trp synthetase in the human genome, all of which suppress mRNA UGG codons. Therefore, each of these nine Trp tRNAs provides an opportunity for codon re-editing tolerance (UGG → UGA). In addition, in view of its proximity to the stop codon in the genetic code, it is common for arginine codons to mutate into PTC nonsense codons in diseases. There are more than thirty Arg tRNAs that can be tested for codon editing tolerance and suppression efficacy.

[0181] A further advantage of the present invention is that it provides for easy expression and cell-specific delivery, since the entire system (tRNA+promoter sequence) is compact.

[0182] Dosages, formulations and routes of administration of the agents of the present invention The agents of the invention are administered so as to result in a reduction in at least one symptom associated with a genetic disease (e.g., cystic fibrosis). The amount administered will vary depending on various factors, including, but not limited to, the composition selected, the specific disease, the weight, physical condition, and age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by a clinician using animal models or other test systems well known in the art.

[0183] The present invention contemplates treating genetic diseases (e.g., cystic fibrosis) by administering an agent of the present invention, such as ACE-tRNA, an expression vector, or a viral particle. Administration of the therapeutic agent according to the present invention may be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is treatment or prevention, and other factors known to skilled practitioners. Administration of the agent of the present invention may be substantially continuous over a preselected time period, or may be a series of spaced doses. Both local administration and systemic administration are contemplated.

[0184] One or more suitable unit dosage forms having a therapeutic agent of the invention, which, as discussed below, may optionally be formulated for sustained release (e.g., using microencapsulation), may be administered by various routes, including parenteral, including by intravenous and intramuscular routes, and by direct injection into diseased tissue. Where appropriate, the formulation may be conveniently presented in discrete unit dosage forms and may be prepared by any method well known in pharmacy. Such methods may include the steps of combining the therapeutic agent with a liquid carrier, a solid matrix, a semisolid carrier, a subdivided solid carrier, or a combination thereof, and then, when desired, introducing the product into or forming the desired delivery system.

[0185] When the therapeutic agents of the present invention are prepared for administration, they can be combined with pharmaceutically acceptable carriers, diluents or excipients to form pharmaceutical preparations or unit dosage forms. The total active ingredients in such preparations account for 0.1 to 99.9% of the preparation by weight. "Pharmaceutically acceptable" is a carrier, diluent, excipient and / or salt that is compatible with the other ingredients of the preparation and is harmless to its recipient. The active ingredient for administration can be presented as a powder or granules; as a solution, suspension or emulsion.

[0186] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. The therapeutic agents of the invention can also be formulated into solutions suitable for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes.

[0187] Pharmaceutical formulations of the therapeutic agents of the invention may also take the form of aqueous or nonaqueous solutions or dispersions, or alternatively emulsions or suspensions.

[0188] Therefore, the therapeutic agent can be formulated for parenteral administration (e.g., by injection, such as push injection or continuous infusion), and can be present in a unit dosage form in an ampoule, a prefilled syringe, a small volume infusion container, or a multi-dose container with an added preservative. The active ingredient can take such forms as a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the active ingredient can be in the form of a powder obtained by aseptic separation of a sterile solid or by lyophilization from a solution, for preparation with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0189] It should be understood that the unit content of one or more active ingredients contained in each aerosol dose of each dosage form does not itself constitute an effective amount for treating a specific indication or disease, because the necessary effective amount can be achieved by administering multiple dosage units. In addition, an effective amount can be obtained using a dose smaller than that in the dosage form, either individually or in a series of administrations.

[0190] The pharmaceutical preparation of the present invention may include a pharmaceutically acceptable carrier, diluent, solubilizer or emulsifier, and salts of a type well known in the art as optional ingredients. Specific non-limiting examples of carriers and / or diluents that can be used in the pharmaceutical preparation of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions and water at pH 7.0-8.0.

[0191] Defining Disease State: For purposes of the present invention, a "disease state" or "disease phenotype" is a characteristic of a mammalian cell that results from a stop codon within the coding region of a gene within the cell (e.g., resulting from a nonsense mutation). For example, an increasing number of human genetic diseases are thought to be caused by nonsense mutations (see, e.g., Atkinson et al., Nuc. Acids Res. 22:1327, 1994). To name a few, beta thalassemia, Duchenne muscular dystrophy, xeroderma pigmentosum, Fanconi's anemia, and cystic fibrosis can all be caused by nonsense mutations in identified genes.

[0192] Endogenous tRNA synthetase: A tRNA synthetase is considered "endogenous" to a cell if it is present in the cell into which the tRNA is introduced according to the invention. As will be apparent to one of ordinary skill in the art, a tRNA synthetase may be considered endogenous for these purposes whether it is naturally found in the relevant type of cell or the particular cell in question has been engineered or otherwise artificially manipulated to contain or express it.

[0193] Suppressor tRNA: A "suppressor tRNA" is one whose anticodon is complementary to a codon that would otherwise terminate translation, allowing detectable readthrough to occur under the conditions of the experiment. Standard stop codons are the amber (UAG), ochre (UAA), and opal (UGA) codons. However, non-standard stop codons (e.g., 4-nucleotide codons) have also been employed in the literature (see, e.g., Moore et al., J. Mol. Biol. 298:195, 2000; Hohsaka et al., J. Am. Chem. Soc. 121:12194, 1999).

[0194] The invention will now be illustrated by the following non-limiting examples.

[0195] Example 1 The genetic code uses four nucleotides, which in turn form triplet codons that form the basis for DNA to protein translation. There are 64 codons in total, 61 of which are used to encode amino acids, and three of which (TAG, TGA and TAA) encode protein terminal "stop" or "nonsense" codons.

[0196] 5 to 10% of cystic fibrosis cases are caused by "nonsense" mutations that result in premature truncation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. An example of this "class 1" mutation is p.Trp1282X, which causes a premature stop codon (PTC) that results in loss of CFTR function and a severe cystic fibrosis phenotype. Certain compounds such as ataluren promote stop readthrough of disease-producing nonsense mutations but are only moderately successful as therapeutic agents due to a number of caveats, including poor stop codon specificity and unexpectedly inefficient codon skipping in vivo. However, the widespread use of these compounds and the finding that endogenous stop codon readthrough is prevalent in metazoans suggest that auxiliary inhibition may be feasible if delivered to a subset of cell types, namely the airway epithelium. However, when therapeutically assisted stop codon readthrough is successful, non-selective incorporation of amino acids at the position of the nonsense codon has the potential to affect protein folding, trafficking, and function (as in the case of CFTR 1282X); and therefore, additional therapeutic intervention is required. Therefore, there is a pressing unmet need to understand the nature of the disease PTC as well as potential therapeutic inhibitors, and more effective treatments of PTC disease in general.

[0197] This example characterizes anticodon editing (ACE) Trp-tRNA for CFTR p.Trp1282X channel rescue. Such tRNAs are modified to "suppress" the disease-causing TGA stop codon, and the original amino acid Trp is incorporated at p.Trp1282XCFTR, in fact, genetically reconstructing the wild-type CFTR protein. The data confirm that this general method (nonsense suppression) produces a stable rescue of transcripts carrying in-frame stop codons by transient transfection of tRNA and its corresponding synthetase in adherent cells, or its delivery to more natural airway cell types, such as A549 airway cells based on viruses. This method provides many significant benefits over existing strategies: 1) Improved codon specificity-expressed tRNA can be directed to specific stop codons, thus reducing off-target effects at stop codons that are not related to the disease. 2) Amino acid specificity-expressed tRNA and / or synthetase can be modified to replace amino acids lost via the insertion of disease stop codons, thus eliminating any spurious effects on CFTR stability, folding and transport. 3) Tunability - In theory, the system can be personalized for each type of tRNA and PTC mutation. 4) Ease of expression - The entire system is compact (<1 kb) and can be easily packaged and expressed transiently or via nanoparticle delivery of tRNA. 5) Proof of principle for a general strategy - In-frame stop codons are a major cause of human disease and few therapeutic options exist; the experiments performed here on p.Trp1282X are expected to lead to insights into the mechanisms of other CFTR nonsense codons.

[0198] The data show that the ACE-tRNA stop codon suppressor tRNA is effective in "rescuing" transcripts containing an introduced stop site ( Fig. 6A and 6B ), suggesting that this type of tRNA has the potential to interfere with nonsense-mediated degradation (NMD), which is a major biological obstacle in the therapeutic rescue of disease termination sites. Therefore, it opens the possibility of using suppressor tRNAs to gain more molecular insights into NMD in disease.

[0199] result We wonder if it is possible to express eukaryotic tRNAs that have been anticodon edited to suppress termination sites such as TGA, rather than their designated codons. This was tested in five human tryptophan tRNAs with a test construct consisting of a fluorescent protein (cherry) in frame with an eGFP sequence separated by a linker containing a TGA site. To indicate the production of full-length protein, the HA epitope was added to the C-terminus of the eGFP reading frame. This test system is useful because the visual appearance of the cherry signal indicates the delivery and expression of the plasmid, and displays TGA inhibition in combination with the eGFP rescue. Fig. 6A and 6B The data in show Western blot data for the ability of five anticodon-edited Trp tRNAs to inhibit the TGA termination site in the short linker between the cherry and eGFP reading frames using this test construct. Of these constructs, candidates 1, 2, 3, and 5 showed modest activity in this regard. This may be due to structural intolerance to mutations, or the possibility that even changing the anticodon by only a single base disrupts the ability of the Trp synthetase to recognize and / or acylate the tRNA with tryptophan. However, number 4 of these test tRNAs (tRNA#4) showed significant inhibitory activity at the TGA site, producing a full-length cherry-eGFP-HA protein ( Figure 6B ). Further, in the absence of co-expressed tRNA, no read-through was observed in the last lane. Figure 6B .

[0200] method Trp tRNAs were examined for codon editing tolerance (TGG→TGA) and their ability to inhibit targeting to the TGA test site in transiently transfected tandem fluorophores (mCherry-TGA-GFP) and CFTR Trp1282X. A preliminary screen of 5 / 9 Trp tRNAs identified anti-codon editing Trp-tRNAs that were transiently transfected in HEK cells and had “stand-alone” functionality to rescue the cherry-TGA-eGFP-HA test construct, Figure 6B The selective presence of the HA epitope indicated successful rescue, as did confocal examination of cherry and eGFP fluorescence at the single cell level (not shown). This result provides proof-of-principle data that a) some ACE-tRNAs can tolerate anticodon editing, b) these tRNAs retain the ability to be acylated by Trp by endogenous tryptophan synthetases, and c) these tRNAs can inhibit TGA sites embedded in open protein reading frames.

[0201] The remaining four Trp-tRNAs functionally checked the tolerance of anticodon editing from TAA to TGA suppressor. The ability of these anticodon-edited tRNAs to rescue cherry-TGA-eGFPHA clones was tested. Biochemical (western blot) data on cherry and eGFP signals and HA epitopes were obtained. Here, cherry expression served as a positive transfection control. Confocal imaging verified cherry and eGFP fluorescence at the single cell level.

[0202] By mass spectrometry analysis of the tryptic fragment of the purified rescued cherry-Trp-eGFPHA protein, the fidelity of the endogenous Trp synthetase to load ACE-Trp tRNA with tryptophan amino acids was determined. The predicted mass of the tryptic fragment generated by the linker between the cherry and eGFP reading frames is: KPINQWPANTHER, with a predicted mass of 1590.8135; bold W indicates the incorporation site, Figure 10. Therefore, this represents the first example of nonsense codon repair and replacement with wild-type amino acids, and is therefore a significant improvement over existing methods such as therapeutic ataluren. In the latter example, the compound promotes the reading of nonsense codons with incorrect amino acids, and therefore, it is important to provide the discovery and identification of new tRNA sequences that provide strict repair.

[0203] Rescue of transiently transfected CFTR 1282X channels by the ACE-tRNA identified above was assessed by standard biochemical methods for full maturation of the B- and C-glycosylated CFTR bands 20. Thus, the channel has been repaired with wild-type amino acids, is fully functional and successfully trafficked to the plasma membrane.

[0204] The next step is to functionally characterize the CFTR Trp1282X channel rescued by the ACE-tRNA system identified above using electrophysiological methods (single cell patch clamp and Ussing chamber) and biochemical methods. The efficacy of the expressed tRNA to reduce nonsense-mediated degradation (NMD) of 1282X mRNA is evaluated using quantitative rtPCR. Reprogrammed human airway cells are used to test the Trp-tRNA rescue of codon editing of the expression of the natural 1282X CFTR channel.

[0205] It was demonstrated that anticodon editing is tolerated in the identified human Trp tRNA and that this 75-bp transfer RNA is able to suppress in-frame TGA codons within the tested constructs. These experiments extrapolated this finding to characterize the ability of this ACE–tRNA to interact with CFTR 1282TGA mRNA and generate functional CFTR channels in model cells (FRT and A549) as well as p.1282X human reprogrammed airway cells.

[0206] Biochemical determination of rescue levels in transiently expressed CFTR 1282X channels and reprogrammed airway cells. Antibody M3A7 is used to identify rescued CFTR (epitope is aa 1370-1380), and detects all CFTR rescued and unrescued, antibodies bound to the N-terminus, such as MM13-4 (epitope aa 25-36) available from EMD Millipore. Alternatively, L12B4 (epitope aa 386-412, EMD Millipore) or 660 (epitope aa 576-585) can be obtained from CysticFibrosis Foundation Therapeutics.

[0207] Surface functionality was examined by electrophysiological methods, patch clamp and Ussing chamber recordings. The stability and abundance of 1282X mRNA was determined by quantitative rtPCR of RNA extracts from transiently expressing cells and reprogrammed airway cells.

[0208] Bioinformatic analysis of RNA transcriptome data from human airway cells identified the abundance, background, and identity of transcripts containing the TGA codon. The top 10 expressed transcripts using TGA for their normal termination site were followed at the level of individual transcripts using protein biochemistry before and after ACE–tRNA expression. Biochemical and immunohistological probes for apoptosis were also used to examine the impact of ACE-tRNA in cell death.

[0209] In summary, the data show that ion channel genes with in-frame stop sites are amenable to this type of “rescue” ( Fig. 9 ), and the components of the system can be virally expressed in airway cells. Further, a highly simplified version of this idea, ACE-tRNA of human origin, demonstrated the “independent” ability to rescue the in-frame CFTR TGA codon in mammalian cell lines ( Fig. 9This approach has many advantages over existing stop codon strategies and warrants further examination with respect to the ability of ACE-tRNA to 1) abrogate nonsense-mediated degradation, 2) function in lung cell preparations, and 3) specifically rescue CFTR 1282X.

[0210] Example 2 Several different nonsense mutations cause CF, thus accounting for approximately 10% of all CF disease. Figure 7 These cases clustered within ten specific genetic lesions: E60X, R75X, G542X, R553X, Q890X, Y1092X, R1158X, R1162X, and W1282X. We propose that with the right approach, it should be feasible to screen existing human tRNA sequences for modification and tolerance to anticodon editing. To this end, approximately 144 ACE-tRNAs were candidates for testing those that could be used to promote repair of pathogenic nonsense codons and full-length protein expression. Specifically, using Fig.11 A tRNA library was generated to identify novel tRNA sequences encoding ACE-tRNAs that have the ability to repair the apical CF-causing nonsense mutation according to the protocol described in Specifically, 10 ng of annealed oligonucleotides encoding ACE-tRNA were combined with 50 ng of NanoLuc reporter plasmid, 1 ul 10xCutSmart Buffer (NEB), 1 ul T4 ligase (NEB), 10 mM ATP, and 1 ul BbsI (NEB) and cycled in a thermal cycler as described in . Fig.11 1 ul of the reaction was transformed into competent E. coli and the transformants were plated on ampicillin agar plates. One transformant was picked per plate, grown in 1 ml LB under ampicillin selection, microprepped and sequence verified.

[0211] Screening studies were first performed to identify the best ACE-tRNA candidates from tryptophan and glycine. 125 ng of sequence-verified miniprep cDNA of the NanoLuc reporter plasmid with ACE-tRNA was transfected into HEK cells using calcium phosphate. HEK cells were cultured the day before at 4x10 4 Plates were plated in 96-well plates. 24 hours after transfection, the medium was replaced with 20ul PBS and 15ul NanoGlo reagent (Promega) was added. Plates were read on SpectraMax i3 (Molecular Devices). Data are replicates of 3 or more times. Figure 8The data show that most tRNAs demonstrate weak codon editing tolerance. However, clear high-performing tRNAs emerged from the screen, with the identification of ACE-Trp and ACE-Gly tRNAs, which demonstrated rescue of nonsense-codon-containing proteins at 20- to 130-fold above background.

[0212] In order to evaluate whether these novel tRNAs can rescue CFTR channels with nonsense codons, they are co-expressed in mammalian HEK cells together with CFTRW1282X cDNA plasmids. By standard biochemical methods, cell preparations are analyzed via the Western blot evaluation of CFTR protein. This method is very advantageous for this purpose, because the CFTR protein displays the multi-band pattern that is fully established. Specifically, "B" and "C" bands represent the full length and fully mature, post-translation processed CFTR protein at the cell surface, respectively. In this case, two kinds of rescues with Trpchr17.trna39 and Glychr19.trna2 ACT-tRNA all produce the solid colony of "B" and "C" CFTR immunopositive (antibody MA37) bands, showing that the ion channel protein of full length, successful transportation is promoted by the tRNA. Fig. 9 .

[0213] Example 3 T stem modification significantly improves nonsense suppression. Figure 10. In this article, we propose additional modifications of tRNA to further achieve its function for suppressing nonsense codons and promoting protein expression. This hypothesis is based on the possibility that mutations rationally introduced within the tRNA "t stem" loop shown in Figure 10 will produce tRNA molecules that are more stable and functionally more effective for nonsense codon suppression. To this end, single and double mutations were directly engineered into the t stem loop of tRNATrpchr17.trna39 (an ACE-tRNA identified to have activity in rescuing tryptophan TGA nonsense codons). 38 tRNA t stem variants were thus generated and used in Figure 4In the HEK cells of the nonsense rescue reporter construct transient transfection shown in , screened.24 hours after transfection, the luciferase activity of the cells was determined, as shown in Figure 10.Data show strong variation and identify novel tRNA sequences with different t stem-loop sequences with enhanced inhibitory activity.It is worth noting that a kind of such mutant TS-3852-62G-C enhances the inhibitory ability of Trpchr17.trna39 to approximately 250% (Figure 12).We therefore propose that this is a generalizable modification, that is, the generalizable modification of the new tRNA sequence identified by embodiment 1 and 2 can be better performed (about its ability to rescue nonsense codons) by further principle modification.Such methods contribute to the therapeutic utility of ACE-tRNA for tissue types with low abundance target RNA or wherein tRNA delivery may be limited.

[0214] Example 4 To allow identification of nucleotide composition and functional ability to suppress nonsense codons by novel tRNAs, an all-in-one plasmid with a one-pot cloning reaction for high-throughput cloning was invented. Fig.11 This method allows for the easy study of ACE-tRNA activity via luciferase activity in a standard 96-well format. Briefly, a synthetic nucleotide sequence encoding tRNA was ligated into the NanoLuc Reporter plasmid, where Fig.11 An example of a TGA nonsense reporter plasmid variant is shown. TAA (opal) and TAG (amber) stop codon rescue vectors have been Figure 16-19 The benefit of this approach is that, in the presence of restriction enzymes and ligases, DNA oligonucleotides encoding the tRNA library can be ligated into the NanoLuc reporter plasmid, where the reaction proceeds to almost 100% incorporation of the tRNA insert ( Fig.11 ) - hence the "one pot" name. The reaction is transformed into E. coli, where the resulting cDNA is purified by standard methods. Another benefit of the method of the present invention is that the tRNA and reporter gene are located in a single expression cassette, thereby reducing biological variability and improving the quality of data obtained in the resulting tRNA inhibitory activity screen. The purified cDNA plasmids are then screened for their ability to repair nonsense codons in a high-throughput 96-well format by inferred luciferase activity. The method is suitable for high-throughput screening of hundreds to thousands of tRNAs for novel therapeutic activity.

[0215] Fig.11 The "one-pot" cloning and expression system described in has been successfully used to identify unique tRNA sequences for repairing tryptophan and glycine ACE-tRNA (Figure 13), ACE-tRNA-Arg ( Fig.14 )、ACE-tRNA-Gln TAG( Fig.15 )、ACE-tRNA-Gln TAA( Fig.16 )、ACE-tRNA-Glu TAG( Fig.17 )、ACE-tRNA-Gln TAA( Fig.18 ) and ACE-tRNA-Trp TAG( Fig.19 ). Figures 20A-20D It was shown that delivery of ACE-tRNA as small RNA supported robust suppression of G542X and W1282X nonsense mutations.

[0216] Example 5 Engineered transfer RNA for suppression of premature stop codons summary Early termination codon (PTC) is responsible for 10-15% of all genetic diseases. PTC inhibition during translation provides a promising method for treating various genetic disorders, however, small molecules that promote PTC read-through have produced mixed performance in clinical trials. A high-throughput assay based on cells is presented to identify the transfer RNA (ACE-tRNA) of the anticodon transformation that can effectively inhibit PTC in the reading frame and faithfully encode its homologous amino acids. In short, ACE-tRNA is identified as having a high inhibitory activity of the most common human pathogenic nonsense codons targeted. The whole genome transcriptome ribosome profile analysis of cells expressing ACE-tRNA at the level of repairing PTC indicates that there is limited interaction with the translation termination codon. These ACE-tRNAs show high inhibitory efficacy in mammalian cells, Xenopus (Xenopus) oocytes and mice, and PTC repair is produced in a variety of genes, and the gene is included in the pathogenic mutation in the cystic fibrosis transmembrane conductance regulator (CFTR).

[0217] introduction Premature termination codons (PTCs) arise from single nucleotide mutations that convert a canonical triplet nucleotide codon to one of three termination codons, such as TAG, TGA, or TAA. PTCs are often more harmful than missense mutations because they result in loss of protein expression. In addition, mRNA abundance is reduced by nonsense-mediated degradation (NMD), and in some cases, truncated proteins may have dominant negative functions. 1-3 It is not surprising, therefore, that PTC is associated with a number of severe disease phenotypes, including cystic fibrosis. 4 , Duchenne muscular dystrophy, spinal muscular atrophy 5 Infantile neuronal ceroid lipofuscinosis 6 , beta thalassemia 7 , Cystinosis 8 , X-linked nephrogenic diabetes insipidus 9Hurler syndrome 10 Usher syndrome 11 and polycystic kidney disease. In addition, nonsense mutations occur in the tumor suppressor genes p53 and ATM 12 , further suggesting its role in disease. The amino acid codons most susceptible to PTC conversion are those with single nucleotide substitutions from stop codons: tryptophan, tyrosine, cysteine, glutamate, lysine, glutamine, serine, leucine, arginine, and glycine ( Fig.25 ). PTCs therefore represent a unique group of diseases that affect more than 30 million people worldwide and account for 10-15% of all genetic diseases. 13 .

[0218] Small molecules such as aminoglycosides 14 , dipeptide 15 and oxadiazole 16 , promoting the “readthrough” or “suppression” of nonsense mutations. These compounds have been shown to 17、18 , mammalian cell lines 19 and some animal disease models 16、20 However, this approach results in the encoding of nearly homologous amino acids 21 , effectively generating a missense mutation at the PTC, which itself may have deleterious effects on protein folding, trafficking, and function. In addition, aminoglycosides are ototoxic and nephrotoxic. 22 , while the pioneering oxadiazole atalulum exhibited unexpectedly low efficacy in patient populations (ACT DMD Phase 3 clinical trial, NCT01826487; ACT CF, NCT02139306), thus limiting its utility as a PTC therapeutic. Recent and ongoing advances in CRISPR / Cas9-mediated genome editing potentially offer a permanent solution for diseases arising from nonsense mutations. However, various aspects of this technology pose obstacles to its rapid use as a therapeutic agent. 23、24 This is not limited to the requirement for a “precise” or “individualized” diagnosis for each mutation based on the context of each patient’s genetic variability.

[0219] A PTC repair approach was identified that demonstrates the versatility of small molecules and the precision of gene editing. tRNAs that meet these criteria were investigated, in which their anticodons have been modified by mutagenesis to recognize and suppress UGA, UAA, or UAGPTC codons. To be effective, the anticodon-edited tRNA, also known as ACE-tRNA, should still be recognized by the endogenous translational cellular machinery, including aminoacyl-tRNA synthetases for charging ACE-tRNA with its cognate amino acid and eukaryotic elongation factor 1a (eEF-1α) for delivering the charged tRNA to the ribosome, Fig.21A Such suppressor tRNAs have been shown to rescue, in a limited manner, β-thalassemia-related 25 , Xeroderma pigmentosum 26 and transgenic PTC reporter gene 27 Related in-frame stop codons.

[0220] It is shown here that the anticodon editing method can be generalized to a variety of tRNA gene families, indicating that many annotated tRNAs are biologically feasible. Further, it is confirmed that the suppressor gene tRNA of anticodon editing encodes its cognate amino acid, lacks significant interaction with the terminal stop codon, and effectively inhibits PTC in vivo. In summary, the data support the possibility that such modified tRNAs meet the broad requirements of covering pathogenic PTCs and therefore represent a promising new class of RNA therapeutics.

[0221] result The rationale for this study stems from the observation that for a given cognate amino acid (isoacceptor), there are multiple tRNA genes with unique sequences (isodecoders), resulting in >400 tRNAs annotated in the human genome (http: / / lowelab.ucsc.edu / GtRNAdb / ). 28、29 First, tRNA genes were examined to identify individual ACE-tRNAs that retained the inhibitory efficacy of PTC in mammalian cells. To maximize sequence coverage, an all-in-one cDNA plasmid was generated that supported both high-throughput cloning (HTC) of ACE-tRNAs and quantitative detection of PTC inhibition using luminescence after delivery to mammalian cells. Fig. 21B Use with ccdB negative selection 31 Paired Golden Gate clones 30 , the ACE-tRNA sequence was cloned as a DNA oligonucleotide into the HTC plasmid. This strategy yielded ~100% cloning efficiency. ACE-tRNA inhibition efficiency was read out from the split NanoLuc luciferase (NLuc) NanoBiT platform using a 96-well format, where the PTC of interest (UGA, UAA, or UAG) was introduced in frame at the junction between the Bigbit and Littlebit domains, Fig. 21B 32 , and normalized to the background obtained in NLuc-PTC expressing cells. Twenty-one glycine ACE-tRNAs were first evaluated for inhibition of UGA PTC. Fig. 22 , top left, column 1 (purple). Most ACE-tRNA GlyThe sequences failed to inhibit UGA NLuc PTC, however, three Gly-tRNAs with high inhibition yields (~100-fold over background) were identified UGA Given the high sequence conservation in Gly-tRNAs screened for anticodon tolerance ( Fig. 27 ), it is difficult to predict a priori which tRNA is most amenable to anticodon editing.

[0222] Next, for each of the possible single nucleotide mutations that could produce pathogenic PTC, a screen was performed for codon-editing tRNAs: Arg-tRNA UGA , Gln-tRNA UAA , Gln-tRNA UAG Trp-tRNA UGA , Trp-tRNA UAG Glu-tRNA UAA Glu-tRNA UAG 、Cys-tRNA UGA 、Tyr-tRNA UAG 、Tyr-tRNA UAA 、Ser-tRNAUAG、Leu-tRNA UAG , Leu-tRNA UAA , Lys-tRNA UAG , Lys-tRNA UGA and Ser-tRNA UAG The enzymatic activity of NLuc was not significantly affected by the introduced amino acids ( Fig.28 ), thus attributing the differences in NLuc luminescence to ACE-tRNA inhibitory ability. This screen identified multiple ACE-tRNAs for each amino acid and stop codon type, with inhibitory coverage for all three stop codons, Fig. 22 Many of these ACE-tRNAs showed strong activity, with >100-fold PTC inhibition over background, which was significantly higher than the aminoglycosides used in this study. Interestingly, some ACE-tRNAs displayed clear preferences for specific anticodon editing, likely reflecting altered aminoacyl-tRNA synthetase binding to tRNA anticodon isoacceptor sequences. 33 For example, the rescue of tryptophan conversion to UAG inhibition is the same as ACE-tRNA Trp However, the opposite was true for glutamine, where a clear preference for UAA over UAG was shown. Notably, in each case, multiple high-performing inhibitors were identified, and this was not the case for Arg UGA Twenty effective ACE-ArgUGA Inhibitors. In other cases, such as those showing functional ACE-tRNA Glu , the inhibition efficiency is roughly equal for UAA and UAG. Lys A similar pattern was found in , where the coding for suppression via UAG or UGA was strongly mirrored. UAA , resulting in an inhibitory signal >2,000-fold above background. Of the ACE-tRNAs identified in the screen, the tryptophan tRNA gene family exhibited the weakest inhibitory activity against UGA PTC. There are only 6 unique human ACE-tRNAs Trp Sequences available for screening, expanding UGA inhibitory ACE-tRNA using tRNAs from a range of species Trp Library. Except for the miscoded A9C tRNA Trp and bacterial Hirsh Trp repressors 34-36 , tryptophan tRNA genes with unique sequences from yeast, fly, mouse, rat, rabbit, and frog were tested for UGA anticodon editing tolerance, Fig.29A This effort was unsuccessful in identifying ACE-tRNAs beyond the human ACE Trp tRNA. Trp UGA PTC inhibitory activity, Fig.29B In summary, the tRNA screen identified multiple engineered tRNAs (for each amino acid and stop codon type) that displayed potent suppression and were therefore generally tolerant to anticodon editing.

[0223] Next, we determined whether the ACE-tRNAs identified in the screen could be functionalized by their cognate aminoacyl-tRNA synthetases at the expense of aminoacylation stringency. To this end, mass spectrometry was used to examine PTC inhibition in a model soluble protein, histidinol dehydrogenase (HDH), Fig.23A . The TGA codon was introduced at asparagine 94 (N94) ( Fig. 30A -C), and with the ACE-tRNAs encoding Glychr19.trna2 or Trpchr17.trna39 (the highest performing glycine and tryptophan ACE-tRNAs, respectively). UGA ) were tandemly co-expressed in HEK293 cells. The resulting full-length, inhibited HDH protein was purified by C-terminal affinity tag and analyzed by mass spectrometry. Fig.23A ( Fig.28Subsequent data searches identified Asn to Trp modifications (+72 Da) for Trp chr17.trna39 and modifications (-57 Da) for Glychr19.trna2, thus confirming the true encoding of the cognate amino acids for each ACE-tRNA type. Importantly, >98% of the peptides identified at the HDHp.N94X site had the encoded cognate tryptophan and glycine in each case. Further, both ACE-tRNAs retained selectivity for the UGA stop codon over UAA and UAG, Fig. 23B (ACE-tRNA Gly )and Fig.31 (ACE-tRNA Trp ). Finally, when transiently expressed, ACE-tRNA Gly In terms of its ability to inhibit NLuc-UGA stably expressed in HEK293 cells, it outperformed conventional small molecule inhibitors gentamicin (40 μM) and G418 (140 μM). Fig.23C Even for ACE-tRNA Trp This is also true for , which has lower inhibition efficiency than G418, but exceeds PTC rescue, Fig.33 .

[0224] The question was raised whether ACE-tRNAs that display efficient suppression of premature stop codons could also induce global readthrough of natural stop codons. To address this potential “off-target” suppression, a transcriptome-wide quantitative profile of ribosomes actively engaged on all cellular transcripts was obtained by generating ribosome footprint libraries from HEK293 cells expressing exogenous ACE-tRNA or a control mock plasmid (puc57GG). Streptomycin was removed from the growth medium to prevent readthrough artifacts. For comparison, ribosome footprint libraries were also generated from cells in the presence or absence of G418 (150 μM, 48 h). Fig.24AThe ribosome footprint density of G418 and five ACE-tRNAs compared with the control (log2 fold change) on the 3'UTR region is shown. Only transcripts with a minimum threshold of 5RPKM for the coding sequence and a minimum threshold of 0.5RPKM in the 3'UTR in two repeated libraries are included for quantitative comparison (254 transcripts in G418 and 495-748 transcripts in ACE-tRNA). In this system, for any of the three endogenous stop codon groups, G418 has no observable effect on the 3'UTR ribosome density of the whole transcriptome. The ACE-tRNA examined here has no detected changes in the 3'UTR ribosome density, except for the cognate stop codon complementary to the ACE-tRNA anticodon, inducing ACE-tRNA Gln-UAA and Arg-UGA, which increase the 3'UTR ribosome density by about 2 times. Understanding the biological significance of the 2-fold readthrough of protein termination will require further study, but this effect is significantly lower than the 100- to 1000-fold inhibition of PTC by the same ACE-tRNA.

[0225] Multiple in-frame stop codons are frequently found at the ends of genes 37-39 , and may cause small differences in overall 3'UTR ribosome density for ACE-tRNA and G418 treatments. The ribosome occupancy at each nucleotide in the 3'UTR within the 60 nt region downstream of the stop codon was examined. Fig. 24B Ribosome occupancy around the natural stop codon was confirmed for each nucleotide in the region from −35 to +65 nt relative to the first nucleotide of the stop codon. Reads were normalized per million mapped reads, compared to control cells, and as Fig.24A Recorded as log2 fold change. More than 5,200 transcripts are mapped to at least 1 footprint in the target region. ACE-tRNA Gln-UAA and Arg-UGA not only show a significantly increased ribosome occupancy in the early region, but also show a characteristic 3-nt periodicity, which indicates that ribosomes are not randomly distributed, but follow the movement of codons one by one. Even in the early region of 3'UTR, ACE-tRNA or G418 about UGA-Trp, UGA-Gly and UAG-Glu do not always show observable changes in ribosome occupancy. In short, the ribosome profile data believe that the efficiency of natural stop codon inhibition by ACE-tRNA is generally low, and is significantly lower than the level of PTC inhibition.

[0226] discuss PTC causes many human diseases, and there are no established treatment options for its therapeutic management. This article reports the high-throughput cloning and identification, characterization and functional analysis of tRNAs that display effective PTC reversal in eukaryotic cells and mouse skeletal muscle. It is worth noting that the screening overall identified ACE-tRNA, which has the ability to repair the vast majority of known human pathogenic PTCs. The modified tRNA faithfully encodes its cognate amino acids, thus canceling the spurious effects on downstream protein stability, folding and transport, and thus eliminating the need for tandem therapies involving protein folding or transport agents. When transfected as cDNA, ACE-tRNA rescued a variety of full-length proteins via PTC inhibition; two disease nonsense mutations in NLuc luciferase reporters, model proteins HDH and CFTR. Effective and stable in vivo PTC inhibition in mouse skeletal muscle is achieved by ACE-tRNA Arg The cDNA revealed an exceptionally high level of cellular tolerance to ACE-tRNA activity. The identification of active ACE-tRNA for arginine in muscle is relevant to the treatment of dystrophinopathies caused by nonsense mutations. As with most genetic diseases, more than 10% of dystrophinopathies are caused by nonsense mutations. 43 , of which CGA->TGA mutation is the most common 43 Effective inhibition was also achieved with ACE-tRNA delivered as a synthetic RNA transcript, thus allowing the development of nanoparticle formulations. Further studies are needed to evaluate the ideal tRNA delivery strategy for each tissue and disease type, efforts that are likely to benefit from the rapidly expanding nucleic acid delivery technology.

[0227] Agents that inhibit PTC have the potential to also produce readthrough of the natural stop codon. The RNA profiling data presented herein suggest that this is generally not the case in cells and for the codon-edited tRNAs tested. UGA and Gln-tRNA UAA Detectable read-through was found, but with Glu-tRNA UAG , UGA-Gly-tRNA UGA and Trp-tRNA UGA No significant effect on overall translation termination was measured. This behavior was apparently not separated from the stop codon type or the intrinsic PTC inhibition activity of the tRNA. One potential reason why ACE-tRNA is ineffective in promoting readthrough at true stop codons may be due to the contextual sequence landscape near the end of translation. 44 This possibility is supported by the finding that the composition of the termination complex at the PTC differs from that at the native termination 45、46However, in cases where lower levels of readthrough occur, multiple cellular mechanisms are in place to limit normal termination readthrough and its damaging effects. Multiple in-frame stop codons are frequently found at the ends of genes. 37-39 , and specialized ubiquitin ligases are known 47 and ribosome-related pathways 48 , to identify and degrade proteins with faulty translation termination. However, despite the limited effects seen here in mammalian cells, similar ribosome profiling experiments should be performed in cell or tissue types desired for delivery and expression of ACE-tRNA.

[0228] Previous studies have shown that surrounding mRNA sequences influence the intrinsic stop codon suppression efficacy of aminoglycosides and ataluren PTC 49-52 , and ACE-tRNA may be similarly affected. Further, although gene addition strategies to replace genes containing PTCs via viral or non-viral delivery have achieved short-term benefits in some settings, it may be difficult to regulate transgene expression levels. In contrast, a large amount of protein rescue via ACE-tRNA inhibition is coupled to native cellular RNA levels, and therefore higher levels of expression are inherently regulated. The biological purpose remains unknown for most of the variable isoacceptor tRNA sequences in the human genome, and it is speculated that almost half of these genes are transcriptionally silent pseudogenes. 53 , however, the data presented here suggest that many annotated tRNAs are viable. Consistent with this possibility, suppression approaches have been used to identify functional isodecoder tRNAs within the Ser and Leu isoacceptor families. 54 The data presented here further demonstrate that most tRNA gene sequences support viable activity when taken out of their genomic context, further deepening the mystery of the biological requirements and codon usage for multiple tRNAs. Thus, the high-throughput inhibition strategy described here can be used to identify novel tRNA sequences with unique inhibitory properties, and such studies have the potential to generate new RNA reagents as well as advance the molecular understanding of tRNA expression and inhibition.

[0229] Materials and methods Nonsense reporter HTC plasmid The parent plasmid used was pcDNA3.1(+). The cDNA encoding pNLuc was Gibson assembled (New England Biolabs, USA) into the restriction sites HindIII and XhoI. During the cDNA PCR process, glycine (codon gga), tryptophan (tgc), amber (tag), opal (tga) and ochre (taa) were added to amino acid position 160. Using PCR-based Gibson Assembly, the pcDNA3.1(+) poly A sequence was replaced for sequences without the BbsI restriction site. The high-throughput ACE-tRNA Golden Gate cloning site was generated by first inserting the human tRNA Tyr The 5' leader sequence of the gene (bold) and the upstream T7 promoter sequence (italics) (Ye et al., 2008), followed by two BbsI restriction sites (bold italics) ( (ccdB box) ) and 3' terminator sequence (bold), followed by the reverse T3 primer sequence (italics)

[0230] The HTCtRNA gene sequences of the ACE-tRNA library were obtained from the tRNA database tRNAscan-SE (http: / / gtrnadb.ucsc.edu / index.html; PMID: 26673694). The sequences of all tRNA genes used in this study are in Fig.26and Table 9. The tRNA sequences were synthesized as complementary Ultramers by Integrated DNA Technologies (IDT, USA) in a 96-well format at a scale of 200 pmol, and their corresponding anticodons were appropriately mutated (UAG, UGA or UAA). All tRNA sequences were synthesized with CGAC and GGAC overhangs (annotated as 5'->3') on the forward and reverse oligonucleotides, respectively. Ultramers were annealed by resuspending to 100 ng / μl in annealing buffer (100 mM potassium acetate; 30 mM HEPES, pH 7.5), heated to 96°C for 2 minutes, and cooled to 4°C at 1°C / min in a thermal cycler. In a 96-well PCR plate, each well contained 10 ng of HTC plasmid with appropriate PTC codons, 2 ng of ACE-tRNA duplex, 1 mM ATP, 10 mM DTT, 400 units of T4 DNA ligase and 10 units of BbsI-HF, which were supplemented to 10 ul with ddH2O. 96-well plates were cycled as follows ([5 minutes at 37°C, 5 minutes at 20°C] x 30 cycles, 10 minutes at 37°C, 10 minutes at 80°C, and cooled to 4°C in a thermal cycler). In a deep-well 96-well plate, 1ul Golden Gate reactants were added to 10ul DH5α chemically competent cells (ThermoFisher, USA), heat-shocked at 42°C for 30 seconds, and then resuspended in 100ul SuperOptimal Broth (SOC; Thermofisher, USA). Transformants were grown at 37°C for 1 hour, 250rpm, then added to 2ml Luria-Bertani liquid medium (LB) supplemented with 100ug / ml carbenicillin, and grown at 37°C in covered deep 48-well plates for 20 hours, 300rpm. E. coli growth was performed in deep-well plates and fixtures from Enzyscreen (http: / / www.enzyscreen.com). The E. coli suspension culture was spun down (10 min, 4,000 g at RT) and plasmid DNA was prepared and diluted to 125 ng / μl (IBI scientific, USA). All clones were sequence verified. Using this method, 100% cloning efficiency was achieved.

[0231] HTS of ACE-tRNA library One day before transfection, HEK293 cells (<40 passages) were plated at 1.4 x 10 4Cells / well were plated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1% Pen / Step, and 2mM L-glutamine (Thermofisher, USA) in 96-well cell culture treated plates. All-in-one nonsense reporter with ACE-tRNA gene was transfected in triplicate / plate using Calfectin (Signagen, USA). 16 hours after transfection, the medium was aspirated and 20ul PBS was added to each well. 15ul lysate was added. Luciferase Assay Reagent was added to each well (1:50 reagent to buffer; Promega, USA). After rotational oscillation, the plate was incubated for 2 minutes and read using a SpectraMax i3 plate reader (Molecular Devices, USA; integration time, 200ms; all wavelengths were collected in endpoint mode). For each experiment, the luminescence across three wells was averaged, and all ACE-tRNAs were repeated >3 times in this way. Each plate also contained triplicate wells transfected with an all-in-one nonsense reporter without ACE-tRNA to serve as a control for transfection efficiency and a baseline PTC read-through. All values ​​are reported as the ratio ± SEM of ACE-tRNA luminescence to baseline PTC read-through luminescence. Across all ACE-tRNAs in a given amino acid family, a one-way ANOVA was performed with Tukey's post hoc analysis.

[0232] CFTR, HDH-his-strep and 4xACE-tRNA expression plasmids For expression in mammalian cells, the cDNA of 200 base pairs of the coding region and 3' untranslated region (UTR) of human CFTR was connected to pcDNA3.1 (+) (Promega, USA) using KpnI and XbaI restriction enzymes. G542tga and W1282tga mutations were introduced using QuickChange XL II (Stratagene, USA). For expression in African clawed frog oocytes, the cDNA of 244 base pairs of the coding region and 140 base pairs of 5'UTR and 3'UTR of human CFTR was connected to pGEM-HE (Promega, USA). G542tga and W1282tga mutations were introduced using QuickChange XL II. The cDNA encoding E. coli histidinol dehydrogenase was codon-optimized for mus musculus and synthesized with a c-terminal 8xHis-Strep-tag (BioBasic Inc, Canada) for protein purification from mammalian cells. The synthesized cDNA was ligated into pcDNA3.1(+) using EcoRI and XhoI restriction sites. Nonsense mutations tag, taa and tga were introduced using QuickChange XL II. To generate multiplexed ACE-tRNA expression plasmids, a BbsI "multiple cloning site" (5'- 3'; directional BbsI recognition sequence is italicized, and the unique four base pair overhang for ligation is bold), generating a new parent Golden Gate pUC57 (amp) plasmid. pUC57 (amp) was selected as the parent plasmid because it is relatively small in size and lacks the backbone BbsI restriction site and T7 and T3 promoter sequences. The HTS plasmid features T7 and T3 promoter sequences flanked by ACE-tRNA cassettes, resulting in an ideal, comparable melting temperature (T m) universal primer binding sequence. Using the NEB Golden Gate Assembly Tool (https: / / goldengate.neb.com / editor), PCR primers were generated that annealed to the T7 and T3 flanking sequences and, following cleavage of the distal BbsI recognition sequence, produced unique four-base pair overhangs. The end result was the generation of four ACE-tRNA pcr products using universal PCR primers that could be "daisy-chained" by complementary four-base pair overhangs and ligated into the puc57 Golden Gate plasmid using a one-pot Golden Gate reaction. All clones were sequence verified.

[0233] Cell culture, protein expression, and western blotting HEK293T cells (ATCC, USA) were grown in standard growth medium containing (% by v / v) 10% FBS (HiClone, USA), 1% PenStrep, 1% L-Glut in high glucose DMEM (Gibco, USA) at 37°C, 5% CO2. cDNA was transfected at 75% confluence using Calfectin according to standard protocols (SignaGen Laboratories, USA). After 36 hours, cells were scraped and centrifuged at 7,000 g for 8 minutes at 4°C in PBS supplemented with 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine. For cells expressing CFTR, cell pellets were vigorously ground in 100 mM sucrose, 150 mM NaCl, 1 mM DTT, 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine, 50 mM Tris-HCl pH 7.4, and centrifuged at 100,000 g to separate total membrane from soluble cytoplasmic proteins. The pellets were dissolved in a buffer containing 1% triton, 250 mM NaCl, 50 mM tris-HCl pH 7.4 and 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine. In the presence of 1% 2-mercaptoethanol, equal amounts of cell lysates were loaded onto 3-15% separation gradient SDS-page with 4% stacking gel, separated at 55V O / N, and transferred to 0.45 μM LF PVDF (Bio-Rad, USA). PVDF was immunoblotted using anti-CFTR antibody M3A7 (1:1000; Millipore, USA) in 2% skim milk and imaged on a LI-COR Odyssey Imaging System (LI-COR, USA). For cells expressing HDH-His-Strep, cell pellets were homogenized by vigorous grinding in 100 mM sucrose, 1 mM DTT, 1 mM EDTA, 20 mM tris-HCl pH 8.0, 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, and 0.75 mM benzamidine. Lysates were centrifuged at 100,000 g for 30 minutes at 4°C.Supernatants (soluble cellular proteins) were separated on 4-12% Bis-Tris SDS-page acrylamide gels (ThermoFisher, USA) in the presence of 1% 2-mercaptoethanol, transferred to 0.22 μM LF PVDF (Bio-Rad, USA), and immunoblotted using anti-Strep antibody (1:5000; iba, Germany) in 2% skim milk and imaged on a LI-COROdyssey Imaging System (LI-COR, USA).

[0234] Mass spectrometry Fragmentation data on purified HDH-His-Strep protein were obtained at the University of Iowa Proteomics Facility. Briefly, HDH-His-Strep protein from the soluble fraction of high-speed spin was passed through a StrepTrap HP column (GE Healthcare, Sweden) and washed with 5 column volumes of 100 mM sucrose, 1 mM DTT, 1 mM EDTA, 20 mM tris-HCl pH 8.0, 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, and 0.75 mM benzamidine. Protein was eluted in a wash buffer supplemented with 10 mM d-desthbiotin and concentrated in an Amicon-Ultra filter column (Millipore, USA) cut off at 30 kDA. The concentrated proteins were loaded onto NuPage 4-12% Bis-Tris precast gels (Invitrogen, USA) and separated for 1.5 hours at 150 V. The gels were stained using the Pierce mass spectrometry compatible silver staining kit (ThermoFisher Scientific, USA).

[0235] Trypsin digestion in gel. Briefly, the target protein band from the SDS-PAGE gel was manually excised, cut into 1 mm3 pieces, and washed in 100 mM ammonium bicarbonate: acetonitrile (1: 1, v / v) and 25 mM ammonium bicarbonate / acetonitrile (1: 1, v / v), respectively, to achieve complete decolorization. The gel pieces were further treated with ACN and dried via speed vac. After drying, the gel pieces were reduced in 50 μl 10 mM DTT at 56 ° C for 60 minutes, and then alkylated by 55 mM IAM at room temperature for 30 minutes. The gel pieces were washed twice with 25 mM ammonium bicarbonate: acetonitrile (1: 1, v / v) to remove excess DTT and IAM. After drying, the gel pieces were placed in 50 μL trypsin solution at 10 ng / μL in 25 mM ammonium bicarbonate on ice and incubated on ice for 60 minutes. Digestion was then performed at 37°C for 16 hours. Peptide extraction was performed twice with 100 μl 50% acetonitrile / 0.2% formic acid for 0.5 hours. The combined extracts were concentrated to ~15 μl in a SpeedVac.

[0236] LC-MS / MS. Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to an Eksigent Ekspert TM nanoLC 425System (Sciex) connector. Trap-Elute Jumper Chip (P / N: 800-00389) with 1 / 16" 10-port Valco connector directs loading via the gradient 1 pump and final elution (via the gradient 2 pump). The column assembly is designed to be mounted on an ekspert TM Two 75 μm x 15 cm columns in series in the cHiPLC system (ChromXP C18-CL, 3 μm 120A, Eksigent Division of AB SCIEX). For each injection, an estimated 0.5 μg of total digest was loaded. Peptides were separated on-line with a mass spectrometer using a 120-minute gradient consisting of linear and static segments, where buffer A was 0.1% formic acid and B was 95% ACN, 0.1% formic acid. The gradient was first held at 4% for 3 minutes, followed by the following transitions (% B, minutes): (26, 48), (35, 58), (35, 64), (50, 72), (50, 78), (94, 84), (94, 96), (4, 100), (4, 120).

[0237] Tandem mass spectrometry on LUMOS Orbitrap. The scanning sequence begins with a comprehensive survey (m / z350-1500) obtained with a resolution of 60,000 in the off-axis Orbitrap segment (MS1) on the Orbitrap Fusion Lumos mass spectrometer (Thermo). During the above-mentioned 120-minute gradient process, a gradient MS1 scan every 3 seconds was obtained. Under the 2.0E5 threshold, the most abundant precursor was selected in 2-8 charge state ions. If the ion was targeted twice in the previous 30 seconds, the ion was dynamically excluded for 30 seconds. The selected ions were separated by a multi-segment quadrupole with a mass window on m / z 2, and then sequentially subjected to CID and HCD activation conditions in IT and ion alignment multipoles, respectively. The AGC target for CID is 4.0E04, 35% collision energy, 0.25 activation Q and a maximum fill time of 100 milliseconds. The targeted precursor was also fragmented by high energy collision induced dissociation (HCD) at 40% collision energy and 0.25 activation Q. Orbitrap (AGC 1.2E05, maximum injection time 110ms and resolution set to 30,000 at 400Th) was used to analyze HCD fragment ions. Both MS2 channels were recorded as centroids, while MS1 survey scans were recorded in profile mode.

[0238] Proteomics search. Using Sequest HT, an initial spectral search was performed with Proteome Discoverer version 2.1.1.21 (ThermoFisher Scientific, USA). The spectra were also searched with the Byonic search engine (ProteinMetrics) version 2.8.2. The search database consisted of the Uniprot KB for species 9606 (human) containing 92645 sequences downloaded on October 24, 2016, and the Uniprot KB for taxonomy 562 (Escherichia coli) containing 10079 sequences downloaded on November 8, 2016. For Byonic searches, the two databases were directly connected together. In either search, an equal number of bait entries were created and searched simultaneously by reversing the original entries in the target database.

[0239] In vitro cRNA transcription. G542X UGA 、W1282X UGAThe WT CFTR pGEMHE (Mense et al., 2006; PMID: 1703051) plasmids were linearized by 10x excess NheI-HF restriction enzyme (site located 3' of the coding region) (New England BioLabs, USA) at 37°C for 3 hours and purified using standard cDNA precipitation methods. All cRNAs were transcribed using the mMessagemMachine T7 Kit (ThermoFisher Scientific, USA). Purification of cRNA from transcription reactions was performed on a column from the RNeasy Mini Kit (Qiagen, Germany). Concentrations were determined by absorbance measurement at 260 nm and quality was confirmed on a 1% agarose gel (RNase-free). All cRNAs were made up to 1 μg / ml before use, and all results were generated from ≥2 cRNA preparations.

[0240] In vitro tRNA transcription. Using CellScript T7-Scribe Standard RNA IVT Kit (CELLSCRIPT, USA), Trpchr17.trna39 and Glychr19.trna2, the highest performing Trp and GlyACE-tRNA, were transcribed in vitro. An ACE-tRNA PAGE-purified Ultramer (20ug; Integrated DNA Technologies, Coralville, IA) was annealed with an equimolar concentration of T7 oligonucleotides (5'-taatacgactcactata-3'), which encoded ACE-tRNA and was preceded by a T7 promoter (italics). Importantly, the three terminal nucleotides containing CCA were included (bold).

[0241] Trpchr17.trna39(3'->5'): (3'->5'): The total reaction volume was adjusted to 100 μl, and the kit reagents were added in the following amounts: 10 μl 10X T7-Scribe transcription buffer, 7.5 μl of each nucleotide (100 mM stock solution), 10 μl 100 mM dithiothreitol, 2.5 μl ScriptGuard RNase Inhibitor, 10 μl T7-Scribe enzyme solution. After the reaction was incubated at 37°C for 4–5 hours, the DNA template was digested with 5 μl DNase (1 U / μl) provided by the kit for 30–60 minutes. ACE-tRNA was extracted from the reaction with acid phenol chloroform (5:1, pH 4.5) and precipitated with ethanol. The precipitated ACE-tRNA was centrifuged, washed, dried and resuspended in 100 μl DEPC-treated water and further purified with a Chroma Spin-30 column (Clontech, USA). This procedure produced approximately 100 μl ~5 μg / μl ACE-tRNA. ACE-tRNA was re-centrifuged in 20 ug aliquots, washed, lyophilized and stored at -80°C until use. All results were generated from ≥2 ACE-tRNA preparations.

[0242] ribosome footprinting profiling library preparation. HEK293 cells transiently transfected with ACE-tRNA and a control plasmid (puc57GG) were grown in standard growth medium in the absence of Pen-Strep for 48 h. 55 The library was prepared as described above with minor modifications. Briefly, cells were rapidly cooled by adding ice-cold PBS and lysed in lysis buffer (20 mM Tris-HCl / pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% (v / v) Triton X-100, and 25 U ml - 1 Turbo DNase I) on ice for 10 min, followed by 10 passes through a 26-G needle. After clearing by centrifugation at 16,000 g for 10 min at 4°C, the lysate was treated with 100 U RNase I (Ambion, USA) / A at room temperature. 260 The lysate was digested for 45 min with gentle agitation, and then 200 U RiboLock RNase Inhibitor (Thermo Scientific) was added. The lysate was then loaded into a modified polysome buffer (20 mM Tris-HCl / pH 7.4, 150 mM NaCl, 8.5 mM MgCl2, 0.5 mM DTT, 20 U ml -1RiboLock RNase Inhibitor) and centrifuged at 70,000 rpm for 2 hours at 4°C using a Beckmen TLA-110 rotor. Ribosome pellets containing mRNA footprints were extracted using TRIzol and separated on a denaturing 12% polyacrylamide gel containing 8 M urea. RNA fragments ranging in size from 26 to 34 nt were manually excised from the gel stained with SYBR Gold (Invitrogen) and isolated to generate a ribosome-protected fragment library. Contaminating rRNA fragments were depleted using the Ribo-Zero kit (Illumina). As described 55 , 3' oligonucleotide adapter ligation, reverse transcription, circularization, and secondary rRNA depletion were performed using biotinylated rRNA depletion oligonucleotides (Table 9). During PCR amplification, the library was barcoded using an index primer for each sample. The barcoded library was then combined with 3% PhiX (Illumina) and sequenced in an Illumina NextSeq 500 according to the manufacturer's protocol to typically generate 18-27 million reads / sample.

[0243] Ribosome footprinting data were first analyzed using HISAT 2.0.3 56 , the data file of each barcoded sample (minus the adapter sequence at the 3' end) was mapped to four rRNA sequences (RNA5S1; NR_023363, RNA5-8SN5; NR_003285, RNA18SN5; NR_003286 and RNA28SN5; NR_003287) to eliminate rRNA contaminant reads. The remaining reads were aligned to the sense strand of the longest transcript variant of each human gene (UCSC RefSeq GRCh38). Transcripts (18,101 sequences) with a 3'UTR length of at least 75nt were used for subsequence analysis. A maximum of two mismatches at the 5' end of the read were allowed. All multi-mapped reads were discarded. Fragment reads with a length between 26 and 34nt were defined as ribosomal footprints and used for analysis. The 5' end nucleotides from each footprint were annotated and mapped to each transcript. The position of the ribosomal A site occupying the 16th-18th nucleotides of each footprint 57、58The RPKM (footprint reads / kilobase of transcript / million total mapped reads) was calculated for each individual transcript (18,101 sequences). Only transcripts with a minimum threshold of 5 RPKM for coding sequences and 0.5 RPKM for 3'UTR regions in both replicate libraries (254 transcripts in G418 and 495-748 transcripts in ACE-tRNA) were included for Fig.24A For the analysis in Fig. 24B The whole transcriptome metagenomic map in the , the footprint counts for each nucleotide in the region from -35 to +65 nt relative to the first nucleotide of the stop codon were normalized to the total reads per million mapped. All transcripts (18,101 sequences) were used for mapping, and more than 5,200 transcripts were mapped to at least 1 footprint in the region of interest. Next, we examined the in vivo biological activity of ACE-tRNAs Glychr19.trna2 and Trpchr17.trna39 in rescuing PTC. Sequencing data were analyzed using the Galaxy platform 59 The curves were generated using Prism 7 (GraphPad Software).

[0244] Generation of a stable NLuc reporter cell line. The cDNA encoding pNLuc with tag, taa and tga stop codons at amino acid position 160 was inserted into the AgeI and NotI restriction sites within the multiple cloning site of the retroviral vector pQCXIP (Clontech, USA) using Gibson Assembly (New England Biolabs, USA). PhoenixGP cells (PMID: 7690960) were co-transfected with pNLuc-STOP-pQCXIP and cmv-VSV-G (VSV-G envelope pseudotyped) plasmids using Calfectin (SignaGen Laboratories, USA) and placed in a 33°C CO2-controlled (5%) cell culture incubator for 48 hours. The culture medium (20ml) containing retroviral particles is cooled to 4°C, and spun at 10,000g to remove cell debris, and filtered through a 0.45um MCE membrane syringe filter (Millipore, USA) onto two 10cm dishes inoculated with 30% confluent low-passage HEK293 cells. The cell culture dishes are sealed with Parafilm, and spun at 24°C for 90 minutes at 3,500g, and placed in a cell culture incubator controlled at 37°C CO2 (5%). After 24 hours, cells are selected with puromycin (1ug / ml) until the control dish (no infection) shows complete cell death. Cells are monodispersed into 96-well plates using FACS, and colonies are subsequently cloned. Puromycin was not used to maintain selected clones during the experiments, and standard DMEM medium (DMEM - Dulbecco's Modified Eagle's Medium - High Glucose with L-glutamine, supplemented with 10% FBS, 1% Pen / Step, and 2 mM L-glutamine; ThermoFisher, USA) was used in all studies.

[0245] RNA transfection. HEK293 cells stably expressing pNLuc-UGA were transfected at 1.4 x 10 4Cells / well were plated in Dulbecco's modified Eagle's medium (DMEM) in a 96-well cell culture treated plate, and the DMEM was supplemented with 10% FBS, 1% Pen / Step and 2mM L-glutamine (Thermofisher, USA). After 16-24 hours, cells were transfected with ACE-tRNA using lipofectamine2000 (ThermoFisher Scientific, USA). In brief, 3 μg ACE-tRNA was suspended in 150 μl OptiMEM, and 12 μl of Lipofectamine 2000 was mixed with 150 μl OptiMEM. The volumes were combined, mixed thoroughly and incubated at RT for 10 minutes. 75ul transfection complex was added to each well. As described above, PTC inhibition by ACE-tRNA transcripts was quantified.

[0246] Expression in African clawed frog oocytes. African clawed frog oocytes (V and VI phases) were purchased from Ecocyte (Austin, TX). Before injection, each ACE-tRNA mass was resuspended in 2 μl ddH2O, and the fragments were centrifuged for 25 minutes at 21,000xg, 4°C. In order to determine the dose response of ACE-tRNA to CFTR channel rescue, a serial dilution of ACE-tRNA aliquots (200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.562ng / oocyte) balanced by ddH2O volume was generated. In all experiments, each oocyte was injected with 25ng CFTR cRNA, and the injection volume was 50nl. ddH2O was used in no ACE-tRNA background control experiments. After injection, oocytes were maintained at 18°C ​​for 36 hours in OR-3 (50% Leibovitz medium, 250 mg / l gentamicin, 1 mM L-glutamine, 10 mM HEPES (pH 7.6)).

[0247] Two-electrode voltage clamp (TEVC) recordings. CFTR was recorded in the presence of maximal CFTR activation cocktail, forskolin (10 μM; adenylate cyclase activator) and 3-isobutyl-1-methylxanthine (1 mM; phosphodiesterase inhibitor) in ND96 bath solution containing (in mM) -Current: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5). Glass microelectrodes backfilled with 3 M KCl had a resistance of 0.5–2 MΩ. Data were filtered at 1 kHz and digitized at 10 kHz using a Digidata 1322A controlled by pClamp 9.2 software (Molecular Devices, USA). CFTR currents were elicited using 5 mV voltage steps from -60 to +35 mV using an OC-725C voltage clamp amplifier (Warner Instruments, USA). CFTR Cl was discarded. - Current reversal -20 mV was positive for oocytes. Clampfit 9.2 software was used for current analysis. All values ​​are presented as mean ± SEM.

[0248] Animals and in vivo imaging. Committee) approved (Protocol No. 112762). Mice were treated by injecting 10-20ug DNA resuspended in 30ul water into the tibialis anterior muscle, followed by electroporation. 10ug pNano-TGA+10ugArg ACE-tRNA (right tibialis anterior muscle) or 10ug pNano-TGA+10ug empty pUC57 (left tibialis anterior muscle) were injected into 3 mice. As a control, another 3 mice were injected with 10ug pNano-WT (right tibialis anterior muscle; positive control) or water (left tibialis anterior muscle; negative control). DNA was prepared with 333IU / ml of hyaluronidase (Sigma). One minute after DNA injection, electroporation was performed using a CELLECTRA3P device (Inovio Use and Care Animal Nu / J mice were purchased from Jackson Laboratory. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Wistar Institute (Institutional Pharmaceuticals). Nanoluciferase activity was imaged in mice by intraperitoneal injection of 100 ul furimazine (40x dilution of Nano-Glo substrate), and mice were imaged on an IVIS Spectrum (Perkin Elmer) 5 minutes after injection. Imaging was performed using an open filter and images were acquired at 40 seconds. Images were analyzed using Living Image Software (Perkin Elmer).

[0249] Table 9. tRNA annotated sequence library screened for PTC inhibitory activity. The italic text for each sequence shows the location of the anticodon edit. Bold text indicates tRNAs with inhibitory activity 5 times background. Note that in the tRNA, thymidine is replaced with uracil.

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Kim, D., Langmead, B. & Salzberg, SLHISAT: a fast spliced ​​aligner with low memory requirements. Nat Methods 12, 357-360 (2015). 57. Ingolia, NT., Ghaemmaghami, S., Newman, JR & Weissman, JS Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218-223 (2009). 58. Guydosh, NR & Green, R. Dom34 rescues ribosomes in 3′ untranslated regions. Cell 156, 950-962 (2014). 59. Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyzes: 2016 update. Nucleic, Acids Res 44, W3-W10 (2016). While the foregoing description and examples fully disclose and enable the invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0251] All publications, patents and patent applications are incorporated herein by reference. Although in the foregoing specification the invention has been described with respect to certain embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain details described herein may vary considerably without departing from the basic principles of the invention.

[0252] In the context of describing the present invention, unless otherwise specified herein or clearly contradictory to the context, the use of the terms "a" and "an" and "the" and similar indicators should be interpreted as covering both the singular and the plural. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the description of the range of values ​​in this article is only intended to serve as a shorthand method for individually referring to each individual value falling within the range, and each individual value is introduced into the specification as it is individually narrated in this article. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (such as, "for example") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention. Any language in the specification should not be interpreted as indicating that any unclaimed element is necessary for the practice of the present invention.

[0253] Embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the foregoing description, the changes of those embodiments may become apparent to those of ordinary skill in the art. The inventor expects that a skilled person will appropriately adopt such changes, and the inventor expects that the present invention will be practiced in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter described in the claims attached thereto as permitted by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, any combination of the above elements with all possible variations thereof is encompassed by the present invention.

Claims

1. A modified transfer RNA (tRNA) comprising a T arm, a D arm, an anticodon arm and an acceptor arm, wherein the T arm comprises a T stem having nucleotides that interact with elongation factor 1-α1 (EF1α).

2. A modified transfer RNA (tRNA) consisting of any one of SEQ ID NOs: 1-538, wherein thymidine is replaced by uracil.

3. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 4, wherein thymidine is replaced with uracil.

4. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 16, wherein thymidine is replaced with uracil.

5. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 24, wherein thymidine is replaced with uracil.

6. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 33, wherein thymidine is replaced with uracil.

7. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 38, wherein thymidine is replaced with uracil.

8. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 44, wherein thymidine is replaced with uracil.

9. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 48, wherein thymidine is replaced with uracil.

10. The modified transfer RNA (tRNA) of claim 2, wherein the tRNA consists of SEQ ID NO: 53, wherein thymidine is replaced with uracil.

11. A modified transfer RNA (tRNA) consisting of any one of the following: SEQ ID NO: 56-60, 62-66, 84-86, 90-111, 113, 128-143, 147-149, 153-156, 161-174, 176, 178, 181, 184-186, 192, 196-197, 199-201, 205, 213-240, 246, 255-256, 258-285, 299, 305-312, 314, 318-332, 335-344, 346, 350-354, 357-36 0, 362, 365-370, 372-383, 388-390, 392, 394-401, 403-407, 414-416, 418, 422, 425, 428-433, 437, 444-445, 452, 455, 459-463, 470, 472-474, 476, 487-492, 525, 530-539, 545-550, 553-555, 561-563, and 567-579, in which thymidine is replaced by uracil.

12. A modified transfer RNA (tRNA) comprising a T arm, a D arm, an anticodon arm and an acceptor arm, (a) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-UCA-3' and recognizes a TGA stop codon, and wherein the acceptor arm is operably linked to arginine, tryptophan or glycine; (b) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-UUA-3' and recognizes a TAA stop codon, and wherein the acceptor arm is operably linked to glutamine or glutamate; or (c) wherein the anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-CUA-3' and recognizes a TAG stop codon, and wherein the acceptor arm is operably linked to tryptophan, glutamate, or glutamine.

13. The modified tRNA of claim 12, wherein the T arm comprises rational nucleotide substitutions that enhance or tune the interaction with elongation factor 1-alpha 1 (EF1α).

14. An oligonucleotide sequence encoding the modified tRNA of any one of claims 1 to 13, wherein the oligonucleotide has a total length of less than 150 nucleotides.

15. An oligonucleotide comprising a first oligonucleotide sequence and a second oligonucleotide sequence, wherein the first oligonucleotide sequence and the second oligonucleotide sequence independently encode the modified tRNA of any one of claims 1 to 13, wherein the first oligonucleotide and the second oligonucleotide independently have a total length of less than 150 nucleotides, and wherein the two sequences are tandem.

16. The oligonucleotide of claim 15, wherein the oligonucleotide is DNA.

17. An expression cassette comprising a promoter and a nucleic acid encoding the modified tRNA of any one of claims 1 to 13 or the oligonucleotide sequence of any one of claims 14 to 16.

18. A vector comprising the oligonucleotide of any one of claims 14 to 16 or the expression cassette of claim 17.

19. The vector of claim 18, wherein the vector is a viral vector or a plasmid vector.

20. A composition comprising: The modified tRNA of any one of claims 1 to 13, the oligonucleotide of any one of claims 14 to 16, or the vector of claim 18 or 19, and A pharmaceutically acceptable carrier.

21. The composition of claim 20, wherein the carrier is a liposome.

22. A cell comprising the vector of claim 18 or 19.

23. A method for treating a stop codon-associated genetic disease, comprising administering the composition of claim 20 or 21 to a patient in need thereof.

24. The method of claim 23, wherein the genetic disease associated with a premature stop codon is cystic fibrosis, muscular dystrophy, beta thalassemia, or Liddle's syndrome.

25. A method for restoring translation of a nucleotide sequence comprising a nonsense mutation in a cell, comprising introducing the composition of claim 20 or 21 into the cell, wherein the modified tRNA restores translation of the nucleotide sequence comprising a nonsense mutation.

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