A method for stop codon rescue via genetic reassignment using ACE-tRNA.

Abstract

To provide a method for rescuing a termination codon through genetic reassignment using ACE-tRNA.SOLUTION: 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 alpha 1 (EF1 alpha). EF1 alpha recruits aminoacyl-tRNAs to the ribosome and protects the tRNA from being deacylated. Rational nucleotide substitutions result in tuned tRNA: EF1 α interactions that enhance tRNA delivery to the ribosome and protection from de-acylation.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] This invention was made with government support under R01 GM106569, awarded by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0003] DNA molecules carry genetic information in the form of sequences of nucleotide bases that make up the DNA polymer. DNA uses only four types of nucleotide bases: adenine, guanine, cytosine, and thymine. This information is transcribed into messenger RNA (mRNA) in the form of three consecutive bases called codons, and then translated by transfer RNA (tRNA) and ribosomes to form proteins. RNA uses four types of nucleotide bases: adenine, guanine, cytosine, and uracil. The genetic code is the relationship between triplet codons and specific amino acids. 64 possible codon triplets form the genetic code, and the three stop codons signal the translation mechanism (the ribosomes in the cell) to stop protein production at the location of the codon. The other 61 triplets in a codon correspond to one of 20 standard amino acids. See Figure 1.

[0004] DNA is translated by ribosomes, which link together each amino acid one by one to form a polypeptide according to the genetic instructions specifically given by the DNA. When the ribosome reaches a stop codon, protein elongation terminates. The three stop codons are UAG (amber), UAA (ochre), and UGA (opal). Mutations that change an amino acid-coding codon into a stop codon are called "nonsense mutations." These nonsense mutations can lead to significant truncation / shortening of the polypeptide sequence, causing drastic changes in the genetic phenotype. Therefore, when a ribosome reaches a mutant stop signal, it terminates translation, resulting in an incomplete protein, and even if the gene that directs its expression is present, a critically important protein may not be produced.

[0005] Transfer RNA (tRNA) translates mRNA into protein on ribosomes. Each tRNA contains an "anticodon" region that hybridizes with a complementary codon on the mRNA. The tRNA that carries its designated amino acid is called a "charged" tRNA. When a tRNA is one of 61 tRNAs that carry an amino acid (i.e., one without a stop signal), it usually attaches that amino acid to the growing peptide. The structural gene of a tRNA is approximately 72-90 nucleotides long and folds into a cloverleaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenetic split promoters that become part of the mature tRNA coding sequence (Sharp SJ, 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] A "nonsense suppressor" is an allele of a tRNA gene that contains a modified anticodon that inserts an amino acid in response to a termination codon instead of triggering a "stop" signal. For example, the ochre mutation results in the creation of a UAA codon in the mRNA. The ochre suppressor gene produces a tRNA with an AUU anticodon that inserts an amino acid at the UAA site, thereby allowing continuous translation of the mRNA despite the presence of a codon that would normally cause translation to stop.

[0007] Numerous nonsense suppressor tRNA alleles have been identified in prokaryotes and eukaryotes such as yeast and C. elegans. Different suppressor tRNAs exhibit different suppression efficiencies. In E. coli and other systems, amber suppressors are relatively more efficient, ochre suppressors less so, and opal suppressors the least efficient. This suggests that amber codons are not used as frequently to terminate protein synthesis, while ochre and opal codons are more often used as natural termination signals.

[0008] Undesirable errors in the DNA blueprint can cause disease. For example, an unexpected "termination" signal occurring in the middle of a protein rather than at the end of the blueprint can lead to the production of truncated or shortened proteins with altered or no function at all. Many human diseases, such as cystic fibrosis, muscular dystrophy, beta-thalassemia, and Liddle syndrome, are caused by undesirable termination signals in the DNA read frame for proteins critical to proper lung, blood, muscle, or kidney function, respectively.

[0009] Therefore, there is a need to provide novel modified nonsense suppressor tRNAs that are stabilized compared to the corresponding unmodified nonsense suppressor tRNAs, and nonsense suppressor tRNAs that have increased activity in suppressing gene termination associated with cystic fibrosis. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Sharp SJ, Schaack J., Coolen L., Burke DJ and Soll D., “Structure and transcription of eukaryotic tRNA genes”, Crit.Rev.Biochem, 19:107-144 (1985) [Non-Patent Document 2] Geiduschek EO, and Tocchini-Valentini, “Transcription by RNA polymerase III, Annu.Rev.Biochem.57:873-914 (1988) [Overview of the project] [Means for solving the problem]

[0011] In one embodiment, the present 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 1α1 (EF1α). EF1α recruits aminoacyl-tRNA to the ribosome and protects the tRNA from deacylation. Rational nucleotide substitutions result in a tuned tRNA:EF1α interaction that enhances tRNA delivery to the ribosome and protection from deacylation.

[0012] In one embodiment, the present invention provides modified transfer RNA (tRNA) of sequence numbers 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 substituted with uracil.

[0013] In one embodiment, the present invention provides a modified transfer RNA (tRNA) which is one of the modified transfer RNAs (tRNAs) of sequence numbers 1 to 538, wherein thymidine is substituted with uracil.

[0014] In one embodiment, the modified tRNAs are sequence numbers 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 One of the following: 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, where thymidine is substituted with uracil.

[0015] In one embodiment, the present invention provides a modified transfer RNA (tRNA) comprising a T stem, a D stem, an anticodon loop, and an acceptor stem, wherein (a) the anticodon arm comprises a trinucleotide anticodon, the anticodon being 5'-UCA-3', and recognizing a TGA stop codon, and the acceptor arm is operably ligated to arginine, tryptophan, or glycine; (b) the anticodon arm comprises a trinucleotide anticodon, the anticodon being 5'-UUA-3', and recognizing a TAA stop codon, and the acceptor arm is operably ligated to glutamine or glutamic acid; or (c) the anticodon arm comprises a trinucleotide anticodon, the anticodon being 5'-CUA-3', and recognizing a TAG stop codon, and the acceptor arm is operably ligated to tryptophan, glutamic acid, or glutamine. In one embodiment, the T-arm contains a rationally modified nucleotide sequence that modulates its interaction with EF1α, thereby enhancing its inhibitory activity and increasing its therapeutic potential. tRNA having a modulated interaction with EF1α exhibits enhanced nonsense inhibition and provides enhanced therapeutic properties.

[0016] In one embodiment, the present invention provides an oligonucleotide sequence encoding the modified tRNA described above, wherein the oligonucleotide has a total length of less than 150 nucleotides. In one embodiment, the oligonucleotide is DNA.

[0017] In one embodiment, the present invention provides an oligonucleotide comprising a first oligonucleotide sequence and a second oligonucleotide sequence, wherein the first and second oligonucleotide sequences independently encode the modified tRNA described above, the first and second oligonucleotides independently have a total length of less than 150 nucleotides, and the two sequences are in tandem.

[0018] In one embodiment, the present invention provides an expression cassette comprising a promoter and a nucleic acid or oligonucleotide encoding a modified tRNA.

[0019] In one embodiment, the present invention provides a vector comprising the above-described oligonucleotide or expression cassette.

[0020] In one embodiment, the vector is a viral vector or a plasmid vector.

[0021] In one embodiment, the present invention provides a composition comprising the above-mentioned modified tRNA, oligonucleotide, or vector and a pharmaceutically acceptable carrier.

[0022] In one embodiment, the carrier is a liposome.

[0023] In one embodiment, the present invention provides cells containing the above-mentioned vector.

[0024] The present invention provides a method for treating a stop codon-related gene disorder, comprising administering the modified tRNA composition to a patient who requires treatment for the stop codon-related gene disorder.

[0025] In one embodiment, the genetic disorders associated with immature stop codons are cystic fibrosis, muscular dystrophy, β-thalassemia, or Liddle syndrome.

[0026] In one embodiment, the present invention provides a method for restoring translation to a nucleotide sequence containing a nonsense mutation in a cell, the method comprising introducing the composition into the cell.

[0027] In one embodiment, the present invention provides a method for identifying anti-codon edited (ACE) tRNAs by high-throughput cloning and screening using the suppression of nonsense codons in luciferase enzymes such as NanoLuc. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1. Table of the genetic code.

[0029] [Figure 2] Figure 2. tRNA has a typical four-arm structure, including a T arm, a D arm, an anticodon arm, and an acceptor arm. These arms will also be referred to as “loops” throughout this specification.

[0030] [Figure 3] Figure 3. ACE-tRNA for nonsense suppression (Homo sapiens tRNATrp TGA).

[0031] [Figure 4] Figure 4. Anticodon-edited (ACE)-tRNA encoded in a vector used to identify functional ACEtRNA sequences. This vector sequence contains a nanoluciferase reporter system. The vector shown was used to identify ACEtRNAs with TGA repression. TAA and TAG variants were used for appropriate tRNA screening (see Figures 14-17).

[0032] [Figure 5] Figure 5. Schematic diagram of the rescue of proteins and ion channels containing stop codons via suppressor tRNA.

[0033] [Figure 6]Figures 6A and 6B. Nonsense codon rescue using human ACE-tRNA. Figure 6A. Schematic diagram of anticodon-editing (ACE) Trp tRNA and cherry-TGA-eGFP-HA construct. Figure 6B. Rescue of cherry-TGA eGFP-HA construct by ACE tryptophan tRNA #4.

[0034] [Figure 7] Figure 7. Rationale and frequency of nonsense codons observed in human diseases. Twenty native amino acid codons were ranked in relation to their contribution to human diseases, with darkly diagonally marked codons being the most frequent (TGG, TAC, TAT, TCA, and TTA) and dotted codons being the least frequent. All diagonally marked codon sequences require a single nucleotide mutation to convert from the intended amino acid to the stop codon. Right panel, the most common disease-causing nonsense codons within the cystic fibrosis membrane conductance regulator (CFTR). Novel tRNA sequences were discovered here for repairing the indicated mutations.

[0035] [Figure 8] Figure 8. Identification of tRNA sequences for tryptophan-TGA and glycine-TGA repair. The left axis shows multipliers above background for luciferase activity. The majority of tRNAs with mutant anticodon loops lack rescue activity.

[0036] [Figure 9] Figure 9. Rescue of CFTR1282x using Trpchr17.trna39 and Glychr19.trna2 ACE-tRNAs. Biochemical Western blot data of the CFTR W1282X channel co-expressed in HEK cells with the denoted tRNAs. Expression vectors containing 4 copies of the denoted tRNAs show higher CFTR protein rescue. The "C" band indicates the rescue of fully matured glycosylated CFTR proteins. The antibody used was M3A7 from Cystic Fibrosis Therapeutics at a 1:1000 dilution.

[0037] [Figure 10-1] Figures 10A and 10B. Expression of ACE-tRNATrp and ACE-tRNAGly results in the specific incorporation of congener amino acids into nonsense codons. Figure 10A) Co-expression of the model protein histidinol dehydrogenase (HDH)-His-Strep N94-TGA with ACE-tRNATrp (left) and ACE-tRNAGly (right) results in a full-length HDH protein (asterisk) detectable by silver staining after affinity purification. Figure 10B) Spectra of WT HDH (top), HDH-N94+ACE-tRNAGly (center), and HDH-N94+ACE-tRNATrp (bottom). The spectra reveal amino acid mass differences at the N94 position that specifically match glycine (-57Da) and tryptophan (+72Da), indicating insertion of ACE-tRNA congener amino acids. [Figure 10-2] Figures 10A and 10B. Expression of ACE-tRNATrp and ACE-tRNAGly results in the specific incorporation of congener amino acids into nonsense codons. Figure 10A) Co-expression of the model protein histidinol dehydrogenase (HDH)-His-Strep N94-TGA with ACE-tRNATrp (left) and ACE-tRNAGly (right) results in a full-length HDH protein (asterisk) detectable by silver staining after affinity purification. Figure 10B) Spectra of WT HDH (top), HDH-N94+ACE-tRNAGly (center), and HDH-N94+ACE-tRNATrp (bottom). The spectra reveal amino acid mass differences at the N94 position that specifically match glycine (-57Da) and tryptophan (+72Da), indicating insertion of ACE-tRNA congener amino acids.

[0038] [Figure 11] Figure 11. Flowchart of the cloning process for constructing a tRNA library.

[0039] [Figure 12-1]Figures 12A-12B. Targeted mutations in nucleotides within the t-stem region further enhance ACE-tRNA rescue function. Figure 12A. Trpchr17.tRNA39 was systematically mutagenesized within the t-stem region. These efforts led to the identification of ACE tRNA TS-10 52-62 GC (Figure 12B), represented by the diagonal bars in the plot, indicating an approximately 250% increase in biological activity. [Figure 12-2] Figures 12A-12B. Targeted mutations in nucleotides within the t-stem region further enhance ACE-tRNA rescue function. Figure 12A. Trpchr17.tRNA39 was systematically mutagenesized within the t-stem region. These efforts led to the identification of ACE tRNA TS-10 52-62 GC (Figure 12B), represented by the diagonal bars in the plot, indicating an approximately 250% increase in biological activity.

[0040] [Figure 13-1] Figures 13A-13F. ACE-tRNA is selective for nonsense codons and is more efficient than aminoglycoside nonsense repression. Figure 13A) ACE-tRNATrp#5 and Figure 13B) ACE-tRNAGly#16 were cloned into NanoLuc reporter plasmids containing TGA, TAA, or TAG nonsense codons. Nonsense repression was measured only in NanoLuc-TGA constructs after transfusion. Figures 13C and 13D) Repression of NanoLuc-TGA by addition of gentimicin (40 μM) and G418 (150 μM) and co-transfection of ACE-tRNATrp#5 and ACE-tRNAGly#16 was measured in HEK293 cells at Figure 13C) 24 hours and Figure 13D) 48 hours. (Figures 13E and 13F) HEK293 cells stably expressing NanoLuc-TGA were treated with gentimycin (40 μM) and G418 (150 μM), and transfected with ACE-tRNATrp#5 and ACE-tRNAGly#16. Nonsense suppression was measured at 24 hours (Figure 13E) and 48 hours (Figure 13F) after treatment. [Figure 13-2]Figures 13A-13F. ACE-tRNA is selective for nonsense codons and is more efficient than aminoglycoside nonsense repression. Figure 13A) ACE-tRNATrp#5 and Figure 13B) ACE-tRNAGly#16 were cloned into NanoLuc reporter plasmids containing TGA, TAA, or TAG nonsense codons. Nonsense repression was measured only in NanoLuc-TGA constructs after transfusion. Figures 13C and 13D) Repression of NanoLuc-TGA by addition of gentimicin (40 μM) and G418 (150 μM) and co-transfection of ACE-tRNATrp#5 and ACE-tRNAGly#16 was measured in HEK293 cells at Figure 13C) 24 hours and Figure 13D) 48 hours. (Figures 13E and 13F) HEK293 cells stably expressing NanoLuc-TGA were treated with gentimycin (40 μM) and G418 (150 μM), and transfected with ACE-tRNATrp#5 and ACE-tRNAGly#16. Nonsense suppression was measured at 24 hours (Figure 13E) and 48 hours (Figure 13F) after treatment.

[0041] [Figure 14] Figure 14. ACE-tRNA-Arg-TGA. Identification of ACE-tRNA for the repair of arginine-TGA nonsense codons.

[0042] [Figure 15] Figure 15. ACE-tRNA-Gln TAG. Identification of ACE-tRNA for repair of glutamine tag nonsense codons.

[0043] [Figure 16] Figure 16. Identification of ACE-tRNA for the repair of glutamine TAA nonsense codons.

[0044] [Figure 17]Figure 17. Identification of ACE-tRNA for the repair of glutamate-TAG nonsense codons.

[0045] [Figure 18] Figure 18. Identification of ACE-tRNA for the repair of ACE-tRNA-Gln TAA nonsense codons.

[0046] [Figure 19] Figure 19. Identification of ACE-tRNA-Trp TAG nonsense codon repair.

[0047] [Figure 20-1] Figures 20A-20D. Delivery of ACE-tRNA as a small RNA supports robust suppression of G542X and W1282X nonsense mutations. Figure 20A) CFTR cRNAs containing the G542X or W1282X cystic fibrosis-causing nonsense mutations were co-injected into Xenopus oocytes along with serial dilutions of pre-folded ACE-tRNAGly and ACE-tRNATrp, respectively. CFTR Cl-current was recorded after 36 hours using a two-electrode voltage clamp method. The current-voltage relationship shows that increasing the amount of pre-folded RNA (ACE-tRNATrp) and ACE-tRNAGly (Figure 20B) leads to an increase in CFTR function (measured CFTR Cl-current), which is achieved in ACE-tRNAGly experiments, along with WT CFTR. Figure 20D) Dose-response of rescue by G542X ACE-tRNAGly (black circles) and W1282X ACE-tRNATrp (white squares) (CFTR Cl-current induced at +40mV was normalized to WT CFTR Cl-current at +40mV). The dose-dependency of ACE-tRNAGly (EC50 = approx. 20ng; Hill coefficient approx. 1.4) shows clear saturation at WT CFTR levels, while ACE-tRNATrp shifts to the right (EC50 = approx. 94ng; Hill coefficient 1.24). [Figure 20-2]Figures 20A-20D. Delivery of ACE-tRNA as a small RNA supports robust suppression of G542X and W1282X nonsense mutations. Figure 20A) CFTR cRNAs containing the G542X or W1282X cystic fibrosis-causing nonsense mutations were co-injected into Xenopus oocytes along with serial dilutions of pre-folded ACE-tRNAGly and ACE-tRNATrp, respectively. CFTR Cl-current was recorded after 36 hours using a two-electrode voltage clamp method. The current-voltage relationship shows that increasing the amount of pre-folded RNA (ACE-tRNATrp) and ACE-tRNAGly (Figure 20B) leads to an increase in CFTR function (measured CFTR Cl-current), which is achieved in ACE-tRNAGly experiments, along with WT CFTR. Figure 20D) Dose-response of rescue by G542X ACE-tRNAGly (black circles) and W1282X ACE-tRNATrp (white squares) (CFTR Cl-current induced at +40mV was normalized to WT CFTR Cl-current at +40mV). The dose-dependency of ACE-tRNAGly (EC50 = approx. 20ng; Hill coefficient approx. 1.4) shows clear saturation at WT CFTR levels, while ACE-tRNATrp shifts to the right (EC50 = approx. 94ng; Hill coefficient 1.24).

[0048] [Figure 21]Figures 21A-21B. Nonsense mutation suppression screening to identify candidate anticodon-editing tRNAs (ACE-tRNAs). Figure 21A, a schematic diagram, illustrates the essential interaction of ACE-tRNAs with the translation mechanism. After delivery, ACE-tRNAs are recognized by endogenous aminoacyl-tRNA synthetase and filled with their congener amino acids (aminoacylated). The aminoacylated ACE-tRNAs are recognized by endogenous elongation factor 1α, which protects the ACE-tRNAs from deacylation and delivers the aminoacyl ACE-tRNAs to the ribosomes for suppression of immature termination codons (UGA in this case). Figure 21B shows that individual ACE-tRNAs were cloned into a High Throughput Cloning Nonsense Reporter plasmid using Golden Gate combined with CcdB negative selection. This all-in-one plasmid contains the NLuc luciferase reporter, along with either UGA, UAG, or UAA PTC at p.162, between the large portion of the enzyme and the small essential C-terminal portion.

[0049] [Figure 22] Figure 22. Screening of ACE-tRNA gene families using a high-throughput cloning nonsense mutation reporter platform. Figure 25 shows that the descriptive anticodon-edited PTC sequence was tested for each ACE-tRNA family located one nucleotide away from the endogenous anticodon sequence. Multiple high-performance suppressor tRNAs were identified for each class. Data are presented on a Log10 scale with respect to normalized NLuc luminescence. Each tRNA dataset was acquired in triplets and is shown SEM-encoded, along with the corresponding ANOVA statistical analysis in Table 2. The coded identity and corresponding tRNA sequence are shown in Figure 26 and Table 9, respectively.

[0050] [Figure 23-1]Figures 23A-23C. Homocoding and high-fidelity repression by manipulated tRNAs. Figure 23A, trypsin digestion fragment of histidinol dehydrogenase (HDH), where "X" indicates the repressed PTC codon. MS / MS spectra of trypsin digestion fragments with expressed y and b ion masses for WT (top), N94G (middle), and N94W (bottom) HDH. The b9 ion masses shift by the predicted masses of -57 Da and +72 Da from WT asparagine, indicating that the homogeneous amino acids glycine and tryptophan are encoded by ACE-tRNAGly and ACE-tRNATrp, respectively. Figure 23B, ACE-TGA-tRNAGly (Glychr19.t2) selectively represses the UGA stop codon in transiently transfected HEK293 cells. Figure 23C) ACE-tRNAGly translocation was superior to both gentamicin (40 μM) and G418 (140 μM) after 48 hours of incubation in Hek293 cells stably expressing NLuc-UGA. [Figure 23-2] Figures 23A-23C. Homocoding and high-fidelity repression by manipulated tRNAs. Figure 23A, trypsin digestion fragment of histidinol dehydrogenase (HDH), where "X" indicates the repressed PTC codon. MS / MS spectra of trypsin digestion fragments with expressed y and b ion masses for WT (top), N94G (middle), and N94W (bottom) HDH. The b9 ion masses shift by the predicted masses of -57 Da and +72 Da from WT asparagine, indicating that the homogeneous amino acids glycine and tryptophan are encoded by ACE-tRNAGly and ACE-tRNATrp, respectively. Figure 23B, ACE-TGA-tRNAGly (Glychr19.t2) selectively represses the UGA stop codon in transiently transfected HEK293 cells. Figure 23C) ACE-tRNAGly translocation was superior to both gentamicin (40 μM) and G418 (140 μM) after 48 hours of incubation in Hek293 cells stably expressing NLuc-UGA.

[0051] [Figure 24-1] Figures 24A-24B. Ribosome profiling of ACE-tRNA at the 3'UTR of the entire transcriptome. Figure 24A, ribosome footprint density on the 3'UTR is plotted as a log2 multiplier change against read information from treated cells compared to a control (puc57GG empty vector), as described in Materials and Methods. Transcripts were grouped by their endogenous TAA, TAG, and TGA stop codons. Each point represents the mean of two replicates for the transcript. Error bars show the mean ± standard deviation of the log2 multiplier change. Figure 24B, the mean log2 multiplier change of normalized ribosome footprint occupancy is plotted for each nucleotide between -50 and +50 nt around the stop codon of the transcriptome (18,101 sequences). The illustration shows an offset of approximately 15 nucleotides from the 5' end of the ribosome footprint to the first base position of the stop codon in the ribosome A region. [Figure 24-2] Figures 24A-24B. Ribosome profiling of ACE-tRNA at the 3'UTR of the entire transcriptome. Figure 24A, ribosome footprint density on the 3'UTR is plotted as a log2 multiplier change against read information from treated cells compared to a control (puc57GG empty vector), as described in Materials and Methods. Transcripts were grouped by their endogenous TAA, TAG, and TGA stop codons. Each point represents the mean of two replicates for the transcript. Error bars show the mean ± standard deviation of the log2 multiplier change. Figure 24B, the mean log2 multiplier change of normalized ribosome footprint occupancy is plotted for each nucleotide between -50 and +50 nt around the stop codon of the transcriptome (18,101 sequences). The illustration shows an offset of approximately 15 nucleotides from the 5' end of the ribosome footprint to the first base position of the stop codon in the ribosome A region.

[0052] [Figure 25]Figure 25. Typical codon usage of PTCs. The diagonal lines indicate the most common codons and corresponding amino acid types that can be converted to stop codons through nucleotide substitution. For each type, an engineered tRNA was developed.

[0053] [Figure 26-1] Figure 26. ACE-tRNA activity plots referenced by numbers. [Figure 26-2] Same as above. [Figure 26-3] Same as above. [Figure 26-4] Same as above. [Figure 26-5] Same as above. [Figure 26-6] Same as above. [Figure 26-7] Same as above. [Figure 26-8] Same as above. [Figure 26-9] Same as above. [Figure 26-10] Same as above. [Figure 26-11] Same as above. [Figure 26-12] Same as above. [Figure 26-13] Same as above. [Figure 26-14] Same as above.

[0054] [Figure 27] Figure 27. Alignment of glycine-tRNA sequences. The 21 tRNAGly human sequences show high sequence homology among tRNA clades. The patterns in the tRNA diagram correspond to the patterned boxes in the sequences.

[0055] [Figure 28] Figure 28. The type of side chain at p.162 in nanoluciferase does not affect activity. Total luminescence activity is shown for each mutation at the site.

[0056] [Figure 29A-1]Figures 29A-29C. Analysis of ACE-tRNATrp sequences obtained from multiple species and suppressor tRNA mutations. Figures 29A-29B. Sequence alignment. Figure 29C. NLuc-UGA + ACE-tRNATrp / NLuc-UGA. [Figure 29A-2] Same as above. [Figure 29B-1] Figures 29A-29C. Analysis of ACE-tRNATrp sequences obtained from multiple species and suppressor tRNA mutations. Figures 29A-29B. Sequence alignment. Figure 29C. NLuc-UGA + ACE-tRNATrp / NLuc-UGA. [Figure 29B-2] Same as above.

[0057] [Figure 30] Figures 30A-30C. The histidinol dehydrogenase (HDH) His(8)-streptactin expression construct enables efficient one-step isolation of the protein from HEK293 cells. Figure 30A) Protein sequence of the HDH expression construct. Underlined sequences indicate regions covered by mass spectrometry. The bold underlined asparagine (amino acid position 94) is the residue mutated to TGA PTC to determine the fidelity of ACE-tRNA. Double affinity tags are shown in bold italics. Figure 30B) Silver staining of HDH protein after PTC suppression using Trpchr17.trna39 and Figure 30C) Glychr19.trna2.

[0058] [Figure 31] Figure 31. Stop codon specificity is maintained for ACE-tRNATrp. Repressive activity against tRNA TrpTGATrpchr17.trna39, Figure 22, which is the highest-performing TrpTGA suppressor tRNA, is shown.36 This tRNA was co-expressed with the pNano-STOP plasmid.

[0059] [Figure 32]Figures 32A-32D. ACE-tRNA is more efficient than aminoglycoside PTC repression. Figure 32A) raw and Figure 32B) normalized luminescence measured 24 hours after addition of gentamicin (40 μM) and G418 (150 μM) and transfusion of Trpchr17.trna39 and Glychr19.trna2 in HEK293 cells stably expressing the PTC reporter Nluc-UGA. Figure 32C) raw and Figure 32D) normalized luminescence measured 24 hours after addition of gentamicin (40 μM) and G418 (150 μM) and cotransfusion of Trpchr17.trna39 and Glychr19.trna2 in HEK293 cells.

[0060] [Figure 33] Figure 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, but only 5 μL of the reaction mixture was added to the cells to reduce the effect of the translocation reagent on cell viability. ACE-tRNA delivered as RNA (white symbols) was more rapid in rescuing PTC reporter expression than the cDNA construct (black circles). However, when expressed from cDNA, ACE-tRNA activity continued to increase over 48 hours and decreased as RNA deliverable. [Modes for carrying out the invention]

[0061] Over the years, researchers have identified hundreds of unique point mutations that result in established nonsense codons in human genes. These types of mutations can lead to, 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, breast cancer, fibrous histiocytoma, SRY sex reversal, triose phosphate isomerase anemia, diabetes, and rickets. The BRACA-1 and BRACA-2 genes, which are associated with breast cancer, also have similar mutations.

[0062] Hundreds of nucleotide sequences encoding human tRNAs are known and are generally available to those skilled in the art through sources such as Genbank. The structure of tRNAs is highly conserved, and tRNAs often function across species. For this reason, tRNA sequences from bacteria or other eukaryotes are also potential sources for the stabilized tRNA oligonucleotides of the present invention. Whether a particular tRNA sequence functions in desired mammalian cells can be determined through standard experimental procedures. Further unknown potential tRNA sequences can be modified as described herein for stabilization through standard experimental procedures.

[0063] tRNA genes possess potent promoters that are active in all cell types. In eukaryotes, the promoter for tRNA genes is contained within the structural sequence that encodes the tRNA molecule itself. While elements regulating transcriptional activity exist in the 5' upstream region, the length of the active transcription unit can be significantly less than 500 base pairs, thus facilitating its inclusion in delivery vectors. Once transcription and processing occur, tRNA exhibits a low degradation rate. Finally, gene therapy using nonsense suppressors maintains endogenous physiological regulation of target genes containing nonsense codons. Nonsense mutation

[0064] Transfer RNA (tRNA) is a type of RNA molecule that functions in the sequencing of messenger RNA (mRNA) sequences into proteins. tRNA functions at specific sites within ribosomes during translation, which synthesizes proteins from mRNA molecules. Nonsense mutations, also known as immature termination codons (PTCs), account for approximately 10–15% of single base pair mutations that cause human diseases, including cystic fibrosis (Peltz et al., Annu Rev Med., 64:407-25, 2013). Due to nearly complete loss of gene expression and activity, and the potential for dominant-negative effects of the truncated product, nonsense mutations generally have more serious consequences than missense mutations. PTCs result in immature translation termination and accelerated mRNA transcript degradation via the nonsense mutation-dependent degradation (NMD) pathway.

[0065] Current research shows that molecular editing of the anticodon sequence within tRNA can alter the specific site in the RNA transcript from which tRNA delivers its amino acid. This approach has made it possible to effectively and therapeutically restore immature termination codons (PTCs) to their original, lost amino acids. Anticodon-edited tRNAs (ACE-tRNAs) form a new class of biotherapeutic agents.

[0066] Manipulated tRNAs enable the "re-editing" of disease-causing nonsense codons into specific amino acids. These manipulated tRNAs target only one type of stop codon, such as TGA rather than TAC or TAA. Since the tRNA+ promoter is only about 300 bp, the small size of these tRNA molecules allows for immediate expression (ready). It is suitable for expression. In short, oligonucleotides containing structural components of tRNA genes that function in human cells are synthesized. The sequences of these oligonucleotides are designed based on known sequences that have substitutions made in the anticodon region of a tRNA that cause that particular tRNA to recognize nonsense or other specific mutations.

[0067] Several small molecules were screened to inhibit nonsense stop codons through interaction with ribosomes, with G418, gentamicin, and PTC124 being the most prominent. PTC124, or ataluren, recently completed a Phase III clinical trial for use as a treatment for cystic fibrosis. Ataluren and aminoglycosides promote the read-through of each of the three nonsense codons by incorporating near-cognate amino acids, thereby changing nonsense mutations into missense mutations. (Roy et al., PNAS 2016 Nov 1;113(44):12508-12513). Anticodon-editing tRNA (ACE-tRNA)

[0068] tRNA has a typical four-arm structure, including a T arm, a D arm, an anticodon arm, and an acceptor arm (Figure 2).

[0069] The T-arm consists of a "T-stem" and a "TψC loop." In one embodiment, the T-stem is modified to increase the stability of the tRNA. In one embodiment, ACE-tRNA has a modified T-stem that increases the biological activity of inhibiting the stop site compared to the endogenous T-stem sequence.

[0070] In one embodiment, the present invention includes a composition containing stabilized tRNA that can be used with greater efficacy to treat a wide variety of nonsense mutation-related diseases. In Tables 1-8, the following sequences are written as DNA, but in RNA (transcribed DNA), "T: thymidine" is "U: uracil". Therefore, all tRNAs transcribed from the following sequences contain uracil instead of thymidine.

[0071] In one embodiment, the tRNA has the following sequence (where thymidine is replaced by uracil): [ka]

[0072] Table 1 Table 1-1 Table 1-2

[0073] Table 2 Table 2

[0074] Table 3 Table 3-1 Table 3-2

[0075] Table 4 Table 4

[0076] Table 5 Table 5

[0077] Table 6 Table 6

[0078] Table 7 Table 7-1 Table 7-2

[0079] Table 8 [Table 8]

[0080] In one embodiment, the ACE-tRNA for nonsense suppression is as shown in Figure 3 (Homo sapiens tRNA). Trp TGA ).

[0081] Human UAA, UAG, and UGA suppressor tRNAs were designed in accordance with the present invention. Screening identified codon-editing tRNAs for the repair of Trp-TGA, Trp-TAG, Arg-TGA, Gln-TAG, Gln-TA, Glu-TAG, and Glu-TAA. The tRNAs are approximately 100 nucleotides long and can be introduced into cells to suppress nonsense codon mutations where wild-type amino acids should be present. Oligonucleotides can be introduced directly into recipient cells or ligated in tandem to increase the potency of the oligonucleotides. Expression cassettes and vectors

[0082] In one embodiment, 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 does not need to be included in a separate transcription unit, but may be provided.

[0083] In one embodiment of the present invention, the nucleotide expression system of the present invention is then incorporated into a suitable gene delivery vehicle used to transduce cells to express a suppressor tRNA. The gene delivery vehicle can be any delivery vehicle known in the art and may include any of the numerous vectors, including naked DNA facilitated by receptor and / or lipid-mediated transduction. Such vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (e.g., bacterial vectors), and viral vectors, including, but not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors (including human and porcine), herpesvirus vectors, Epstein-Barr virus vectors, SV40 virus vectors, poxvirus vectors, and pseudotyped virus vectors.

[0084] In one embodiment, ACT-tRNA (PTC) is encoded in the vector. Figure 4. In one embodiment, the viral vector is a retrovirus or adenovirus vector. Examples of retrovirus vectors that may be used include, but are not limited to, vectors derived from retroviruses such as Moloney's mouse leukemia virus, splenic necrosis virus, and Rous sarcoma virus, Harvey's sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Retrovirus; Retrovirus vector

[0085] The term "retrovirus" is used to describe RNA viruses that use reverse transcriptase during their replication cycle. The retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus can be incorporated into the chromosome of an infected cell, and once incorporated, it is called a "provirus." The provirus acts as a template for RNA polymerase II, inducing the expression of RNA molecules that encode structural proteins and enzymes necessary to produce new viral particles. At each end of a provirus is a structure called a "long terminal repeat" or "LTR." LTRs contain numerous regulatory signals, including transcriptional regulators, polyadenylation signals, and sequences necessary for replication and incorporation of the viral genome. The retroviridae family, which includes Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, lentiviruses, and spumaviruses, includes several genera. Some retroviruses are oncogenic (i.e., tumorigenic), while others are not. Oncoviruses induce sarcomas, leukemias, lymphomas, and breast cancers in susceptible species. Retroviruses can infect a wide variety of species and can be transmitted horizontally and vertically. Retroviruses can be incorporated into host DNA and transmit the host DNA sequence from cell to cell. This has led to the development of retroviruses as vectors for various purposes, including gene therapy.

[0086] Retroviruses, including human foam virus (HFV) and human immunodeficiency virus (HIV), have attracted considerable attention in recent years because their target cells are not limited to dividing cells, and their limited host cell targeting can be easily extended through pseudotyping by the vesicular stomatitis virus G (VSV-G) coat glycoprotein (see, for example, JCBurns et al., Proc. Natl. Acad. Sci. USA 90:8033-8037

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

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

[1996] ).

[0087] A vector system typically comprises a DNA vector containing a small portion of the retroviral sequence (the long terminal repeat sequence or "LTR" of the virus and the packaging or "psi" signal) and a packaging cell line. The gene to be transferred is inserted into the DNA vector. The viral sequence present on the DNA vector provides the signals necessary for the insertion or packaging of the vector RNA into the viral particle and for 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, an FIV system using a 3 plasmid translocation production method in 293T cells was used (Johnston et al.). (al., J. Virol. 1999 73:4991-5000). A virus lacking replication ability was successfully produced.

[0088] Vector DNA is introduced into packaging cells by any of the following techniques (e.g., calcium phosphate coprecipitation, lipofection, electroporation). Viral proteins produced by the packaging cells mediate the insertion of the RNA-based vector sequence into viral particles that are released into the culture supernatant.

[0089] For cells that divide naturally or are stimulated to divide by growth factors, simple retroviruses, such as mouse leukemia virus (MLV) vectors, are suitable delivery systems. However, a major limitation of the use of many retroviral vectors commonly used in gene transfer is that most vectors are limited to dividing cells. When non-dividing cells are the target cells, lentiviruses that can infect non-dividing cells can be used.

[0090] As used herein, the term “lentivirus” refers to a group (or genus) of retroviruses that cause slowly developing diseases. Viruses within this group include HIV (human immunodeficiency virus; including HIV1 and HIV2), the virulence factor of human acquired immunodeficiency syndrome (AIDS); Visna maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; canine 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 possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in subhuman primates. Diseases caused by these viruses are characterized by long incubation periods and prolonged courses. Typically, these viruses latently infect monocytes and macrophages, from where they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

[0091] Lentiviruses, including HIV, SIV, FIV, and Equine Infectious Anemia Virus (EIAV), rely on several viral regulatory genes in addition to simple structural gag-pol-env genes for efficient intracellular replication. Thus, lentiviruses employ more complex strategies for gene regulation and viral replication than classical retroviruses, and packaging signals appear to be spread throughout the entire viral genome. These additional genes exhibit a regulatory network during the lentiviral life cycle. For example, upon HIV-1 infection, transcription is upregulated by the expression of Tat through interaction with RNA targets (TARs) in the LTR. Subsequently, the expression of full-length and spliced ​​mRNA is regulated by the function of Rev, which interacts with RNA elements (RREs) present in the gag and env regions (S. Schwartz et al., J. Virol., 66:150-159

[1992] ). Nuclear export of gag-pol and env mRNA depends on Rev function. In addition to these two essential regulatory genes, a series of accessory genes, including vif, vpr, vpx, vpu, and nef, are also present in the viral genome, and their effects on efficient viral production and infectivity have been demonstrated, although they are not absolutely necessary 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 structural description of the exemplary lentivirus HIV-1 is given in U.S. Patent No. 6,531,123.

[0092] A “source” or “original” retrovirus is a wild-type retrovirus from which a pseudotyped retrovirus is derived, or a wild-type retrovirus used as a starting point during packaging or construction of the transgene vector for the preparation of one or more genetic elements of the vector. A genetic element may be used unchanged, or it may be mutated (but not beyond the point where it lacks statistically significant sequence similarity to the original element). A vector may have more than one source retrovirus, and different source retroviruses may 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.

[0093] A cognate retrovirus is a wild-type retrovirus that has the greatest percentage sequence identity at the nucleic acid level for the vector in question. Typically, this will be identical to the source retrovirus. However, if the source retrovirus is extensively mutated, the vector may more closely resemble some other retrovirus. A cognate retrovirus does not necessarily have to be a physical starting point for construction; rather than obtaining the original element first and then modifying it, one may choose to synthesize the genetic element, particularly the mutant element, directly. The term "cognate" can be applied similarly to proteins, genes, or genetic elements (e.g., splice donor sites or packaging signals). When referring to cognate proteins, percentage sequence identity is determined at the amino acid level.

[0094] The term "congenital" retrovirus can be difficult to interpret in extreme cases, namely when all the retrovirus's genetic elements are replaced by alternative non-lentiviral genetic elements. In such cases, the aforementioned source retrovirus strain can be arbitrarily considered a congenital retrovirus.

[0095] Where used herein in reference to a virus or vector, the term “replication” does not refer to the normal replication of proviral DNA in a chromosome as a result of cell replication or the autonomous replication of plasmid DNA as a result of the presence of a functional origin of replication. Instead, “replication” refers to the completion of the complete viral life cycle, in which an infectious viral particle containing viral RNA enters a cell, the RNA is reverse transcribed into DNA, the DNA is incorporated into the host chromosome as a provirus, the infected cell produces virion proteins, and these are assembled into new, equally infectious particles with full-length viral genomic RNA.

[0096] The term "replication-capable" refers to a wild-type or mutant virus that is capable of replicating so that viral replication in an infected cell results in the production of infectious virions, and these infectious virions then infect another previously uninfected cell, causing that uninfected cell to similarly produce such infectious virions. This invention envisions the use of replication-deficient viruses.

[0097] As used herein, the term “attenuated virus” refers to any virus (e.g., an attenuated lentivirus) that has been modified to substantially reduce its pathogenicity in the intended subject. The virus can be attenuated to the point where it is clinically nonpathogenic; that is, the subject exposed to the virus does not exhibit a statistically significant increase in disease compared to a control subject.

[0098] The present invention envisions the preparation and use of modified retroviruses. In some embodiments, the retroviruses are variants of viruses consisting of parts of one or more retrovirus species, such as mouse leukemia virus, human immunodeficiency virus type 1, human immunodeficiency virus type 2, feline immunodeficiency virus, monkey immunodeficiency virus, Visna-Maedi, canine arthritis encephalitis virus, equine infectious anemia virus, and bovine immunodeficiency virus, or viruses (e.g., hybrids consisting of parts of MLV, FIV, HIV-1 and HIV-2 or HIV-1 and / or SIV).

[0099] A reference virus is a virus whose genome is used when describing the components of a mutant virus. For example, certain genetic elements of a mutant virus may be said to differ from congeneral elements of the reference virus due to various substitutions, deletions, or insertions. The mutant virus does not actually need to originate from the reference virus.

[0100] A preferred reference FIV sequence can be found in Talbott et al., Proc Natl Acad Sci USA. 1989 86:5743-7; Genbank access#NC_001482. In some embodiments, a three-plasmid transient transfection method can be used to produce non-replicating pseudotyped retroviruses (e.g., FIV). A general method is described in Wang et al., J Clin Invest. 1999 104:R55-62 and Johnston et al., J Virol. 1999 73:4991-5000. Retrovirus vectors

[0101] The present invention envisions a retroviral gene amplification and transfer system comprising a transgene vector, one or more compatible packaging vectors, an envelope vector, and a suitable host cell. The vectors used may be derived from retroviruses (e.g., lentiviruses). The retroviral vector enables (1) transtransfer of the packaging vector and envelope vector into a host cell to form a packaging cell line that produces viral particles that do not essentially contain the packaging vector RNA, (2) transtransfer of the transgene vector into the packaging cell line, (3) packaging of the transgene vector RNA into infectious viral particles by the packaging cell line, and (4) delivery of the particles to target cells so that such cells are transduced and subsequently express the transgene.

[0102] The particles are either administered directly to the subject in vivo, or the subject's cells are extracted, infected with the particles in vitro, and then returned to the subject's body.

[0103] The packaging vectors and transgene vectors of the present invention produce non-replicating viruses. Vectors selected for incorporation into a given vector system of the present invention are vectors such that, without further mutation of the packaging vector(s) or transgene vector, cotransferred cells cannot produce replicative viruses by homologous recombination of (one or more) packaging vectors(s) and transgene vectors alone. The coat proteins used in this system may be retroviral coats, synthetic or chimeric coats, or coats from viruses covered with non-retroviral coats (e.g., baculoviruses). Packaging signal

[0104] As used herein, the terms “packaging signal” or “packaging sequence” refer to a sequence located within the retroviral genome or vector that is required for, or at least facilitates, the insertion of viral RNA or vector RNA into the viral capsid or particle. A packaging signal in RNA identifies that RNA as the RNA to be packaged within the virion. For convenience, the term “packaging signal” is also used to refer to a vector DNA sequence that is transcribed into a functional packaging signal. A particular packaging signal may be part of a gene, but is recognized more in RNA form than as a peptide portion of the encoded protein.

[0105] A key difference between packaging vectors and transgene vectors is that in packaging vectors, major packaging signals are inactivated, whereas in transgene vectors, major packaging signals remain functional. Ideally, all packaging signals should be inactivated in packaging vectors, and all packaging signals should remain functional in transgene vectors. However, countervailing considerations, such as maximizing viral titer or inhibiting homologous recombination, can make such constructs less desirable. Packaging systems; packaging vectors; packaging cell lines

[0106] A packaging system is a single vector or a set of vectors that collectively provides, in an expressible form, all the genetic information necessary for the introduction gene incorporated into the RNA to be reverse transcribed within the target cell and integrated into the host genome in an expressible manner. This is achieved by producing virions capable of enclosing appropriate RNA in a capsid, transporting virions from the virion-producing cell, delivering virions to target cells, and then integrating the incorporated RNA into the host genome in an expressible manner. However, the packaging system must not be substantially packaged itself. Rather, the packaging system packages a separate introduction gene vector.

[0107] In this invention, the packaging vector provides functional equivalents of the gag and pol genes ("GP" vectors). Env genes(s) are provided by envelope vectors. Theoretically, if one desires to construct different gag and pol genes on separate vectors and operably link them to different controllable promoters (or one to a controllable promoter and the other to a constitutive promoter) so that their relative expression levels can be appropriately regulated, a three-vector system ("G", "P", and "E" vectors) is possible.

[0108] A packaging cell line is a suitable host cell into which a packaging system producing viral particles is transfused under achievable conditions. As used herein, the term “packaging cell line” typically refers 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 may have a functional psi at one chromosomal site within its genome. + It is genetically engineered to have a 5'-LTR-gag-pol-3'-LTR fragment (denoted as Δ-psi) lacking a sequence, and a 5'-LTR-env-3'-LTR fragment, which is also a Δ-psi located in a different chromosomal region. Both of these segments are constitutively transcribed, but psi +Because the region is lost and the produced viral RNA molecule is smaller than its full size, an empty viral particle is formed.

[0109] If host cells are transfused solely by a packaging vector, they will produce virtually only viral particles without a full-length packaging vector. In one example, less than 10% of the viral particles produced by packaging cells contain full-length packaging vector-derived RNA. However, because packaging vectors lack functional primer-binding sites, even if these particles infect new cells, the packaging vector RNA is not reverse-transcribed into DNA, and therefore the new cells do not produce virions. Thus, on its own, a packaging vector is a non-replicating virus.

[0110] In some embodiments, the packaging cells and / or cell lines contain a transgene vector. The packaging cell line packages the transgene vector into infectious particles. Such cell lines are referred to herein as “transgenic virion-producing cell lines.”

[0111] Packaging can be either inducible or non-inducible. In inducible packaging cells and cell lines, retroviral particles are produced in response to at least one inducible factor. In non-inducible packaging cell lines and cells, no inducible factor is required for retroviral particle production to occur.

[0112] A packaging vector is necessarily different from a reproducible wild-type retroviral genome by inactivating at least one packaging signal of the congeneral wild-type genome. More than one packaging signal may be inactivated. In one example, the only retroviral gene provided by the packaging vector is a retroviral gene encoding a structural protein or an essential regulatory protein. Transgene vector

[0113] The transgene vector is an expression vector containing the desired expressible non-retroviral gene and at least one functional retroviral packaging signal, such that after the transgene vector is transfected into a packaging cell line, the transgene vector is transcribed into RNA, and this RNA is packaged into infectious viral particles. Subsequently, these particles infect target cells, their RNA is reverse transcribed into DNA, and the DNA is incorporated into the host cell genome as a proviral component, thereby delivering the desired gene to the target cells.

[0114] As used herein, the term “transduction” refers to the delivery of a gene using a viral or retroviral vector by infection, rather than by translocation. In some embodiments, the retroviral vector is transduced. Thus, the “transduced gene” is a gene introduced into a cell via retroviral infection or vector infection and proviral incorporation. In some embodiments, a viral vector (e.g., “transgene vector”) transduces a gene into a “target cell” or host cell. The present invention encompasses transgene vectors suitable for use in the present invention that are linked to any target gene (or a “marker gene” or “reporter gene” used to indicate the infection or expression of a gene).

[0115] As used herein, the term “long-term transduction” refers to a vector that can remain transduced in host or target cells for a longer period than observed with other vectors. For example, the present invention provides a retroviral vector that can remain transduced for at least 120 days, at least one year, or for the lifetime of the subject or for the duration of treatment. The duration of expression is a function of the promoter selection and the type of target cell, rather than the vector selection.

[0116] The terms "stable transduction" or "stable transduction" refer to the introduction and integration of foreign DNA into the genome of a transduced cell. The term "stable transduction" refers to a cell that has foreign DNA stably integrated into its genomic DNA.

[0117] The terms "transient transduction" or "transiently transduced" refer to the introduction of foreign DNA into a cell in which the foreign DNA is not integrated into the genome of the transduced cell. The foreign DNA persists in the nucleus of the transduced cell for several days. During this period, the foreign DNA undergoes regulatory control that governs the expression of endogenous genes in the chromosome. The term "transient transdextrin" refers to a cell that has taken up foreign DNA but has not been able to integrate this DNA.

[0118] In some embodiments, the target cells and / or host cells of the present invention are “non-dividing” cells. These cells include cells such as nerve cells that do not normally divide. However, the present invention is not intended to be limited to non-dividing cells (including, but not limited to, muscle cells, leukocytes, spleen cells, hepatocytes, ophthalmic cells, and epithelial cells).

[0119] In some embodiments, the vector and its offspring are at least 10 5 It is possible to transduce multiple target cells to achieve a vector titer of cfu / ml. The multiple of infections (MOI) can be at least 1 (i.e., an average of one infection per cell) or even at least 2. Expression cassettes and vectors

[0120] The present invention also provides an expression cassette containing a sequence encoding ACE-tRNA.

[0121] In one embodiment, the expression cassette further contains a promoter. In one embodiment, the promoter is a controllable promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is a PGK, CMV, RSV, H1, or U6 promoter (PolII and PolIII promoters).

[0122] The present invention provides a vector containing the expression cassette described above. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a viral vector based on adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, HSV, or mouse Maloney.

[0123] As used herein, “expression cassette” means a nucleic acid sequence capable of inducing the expression of a specific nucleotide sequence in a suitable host cell, and which may include a promoter operably ligated to the nucleotide sequence of interest, which can be operably ligated to a termination signal. An expression cassette may also include sequences required for the proper translation of the nucleotide sequence. The coding region typically encodes the protein of interest. An expression cassette containing the nucleotide sequence of interest may be a chimera. An expression cassette may be naturally occurring but obtained in a recombinant form useful for heterologous expression. The expression of a nucleotide sequence in an expression cassette may be regulated by a constitutive promoter or a controllable promoter that 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 particular tissue or organ or developmental stage.

[0124] "Operatively ligated" refers to the association of multiple nucleic acid sequences on a single nucleic acid fragment such that the function of one sequence is influenced by another sequence. For example, if two sequences are positioned such that a regulatory DNA sequence influences the expression of a coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional regulation of a promoter), the regulatory DNA sequence is said to be "operatively ligated to" or "associated with" the DNA sequence encoding the RNA or polypeptide. The coding sequence can be operatively ligated to the regulatory sequence in sense or antisense orientation. Adeno-associated virus (AAV)

[0125] Adeno-associated viruses (AAVs) are small, non-pathogenic viruses belonging to the Parvoviridae family. AAVs differ from other members of this family in that they rely on helper viruses for replication. In the absence of helper viruses, AAVs can be incorporated in a locus-specific manner within the q arm of chromosome 19. The approximately 5kb genome of AAV consists of a single segment of single-stranded DNA with either positive or negative polarity. The ends of the genome are short terminal inversion sequences that fold into hairpin structures and can serve as starting points for viral DNA replication. Physically, parvovirus virions are not enclosed in a sheath, and their icosohedral capsid is approximately 20 nm in diameter.

[0126] To date, numerous serologically distinct AAVs have been identified, with more than 12 being isolated from humans or primates. The AAV2 genome is 4680 nucleotides long and contains two reading frames (ORFs). The left ORF encodes non-structural Rep proteins, Rep40, Rep52, Rep68, and Rep78, which are involved in the regulation of replication and transcription, in addition to the production of single-stranded progeny genomes. Furthermore, two of these Rep proteins have been associated with the preferential integration of the AAV genome into the q-arm region of human chromosome 19. Rep68 / 78 has also been shown to possess NTP-binding activity as well as DNA and RNA helicase activity. The Rep proteins have several potential phosphorylation sites in addition to nuclear localization signals. Mutations in one of these kinase sites have resulted in loss of replication activity.

[0127] The ends of the genome are short terminal inversion sequences (ITRs) with the potential to fold into T-shaped hairpin structures, serving as starting points for viral DNA replication. Within the ITR region, two elements central to ITR function have been described: the GAGC repeat motif and terminal resolution sites (trs). This repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin configuration. This binding plays a role in positioning Rep68 / 78 for cleavage at the trs, and this occurs in a site-specific and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep-binding site with adjacent trs. These elements have been shown to be functional and necessary for locus-specific integration.

[0128] AAV virions are icosahedral particles, approximately 25 nm in diameter, that are not enclosed in a sheath, and consist of three related proteins designated VP1, VP2, and VP3. The ORF on the right encodes the capsid proteins, VP1, VP2, and VP3. These proteins are found in a ratio of 1:1:10, and all originate from the ORF on the right. These capsid proteins differ from each other due to alternative splicing and the use of abnormal start codons. Deletion analysis has shown that the removal or modification of VP1, which is translated from the alternatively spliced ​​message, results in a reduced production of infectious particles. Mutations within the VP3 coding region prevent the production of any single-stranded progeny DNA or infectious particles. AAV particles are viral particles containing the AAV capsid protein. The AAV capsid polypeptide can encode the complete VP1, VP2, and VP3 polypeptides. The particles may contain AAV2 and other AAV capsid proteins (i.e., chimeric proteins such as AAV1 and AAV2). Variations in the amino acid sequence of the AAV2 capsid protein are assumed herein, as long as the resulting viral particles contain the AAV2 capsid and remain antigenically or immunologically distinct from AAV1, as can be systematically determined by standard methods. Specifically, for example, ELISA and Western blotting can be used to determine whether the viral particles are antigenically or immunologically distinct from AAV1. Furthermore, AAV2 viral particles preferably retain tissue-specific properties different from AAV1.

[0129] AAV2 particles are viral particles containing the AAV2 capsid protein. The AAV2 capsid polypeptide, which encodes the complete VP1, VP2, and VP3 polypeptides, can have at least about 63% homology (or identity) overall with a polypeptide having the amino acid sequence encoded by the nucleotides described in NC_001401 (the nucleotide sequence encoding the AAV2 capsid protein). The capsid protein can have about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% homology with the protein encoded by the nucleotide sequence described in NC_001401. The capsid protein can have approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% identity with the protein encoded by the nucleotide sequence described in NC_001401. The particle may be a particle containing another AAV and the AAV2 capsid protein, i.e., a chimeric protein. Variations in the amino acid sequence of the AAV2 capsid protein are assumed herein, as long as the resulting viral particles containing the AAV2 capsid remain antigenically or immunologically different from AAV4, as can be systematically determined by standard methods. Specifically, for example, ELISA and Western blotting can be used to determine whether the viral particles are antigenically or immunologically different from AAV1. Furthermore, while AAV2 virus particles preferably retain tissue-specific properties different from AAV1, such as those exemplified in the examples herein, AAV2 chimeric particles containing at least one AAV2 coat protein may have different tissue-specific properties than AAV2 particles consisting solely of AAV2 coat proteins.

[0130] In one embodiment, the present invention further provides AAV2 particles containing a vector comprising a pair of AAV2 terminal inversion sequences, i.e., AAV2 particles encapsulated in a capsid. The nucleotide sequence of the AAV2 ITR is known in the art. Furthermore, the particles may be particles containing both AAV1 and AAV2 capsid proteins, i.e., chimeric proteins. Furthermore, the particles may be particles encapsulating a vector comprising a pair of AAV terminal inversion sequences from other AAVs (e.g., AAV1-AAV9 and AAVrh10) in a capsid. The capsid-encapsulated vector in the particle may further contain exogenous nucleic acids inserted between the terminal inversion sequences.

[0131] The following characteristics of AAV have made it an attractive vector for gene transfer: AAV vectors have been shown to be stably integrated into the cellular genome in vitro, to have a broad host range, to transduce both dividing and non-dividing cells in vitro and in vivo, and to maintain high levels of expression of the transduced gene. Viral particles are thermally stable, tolerant of solvents, surfactants, pH changes, and temperature, and can be enriched on a CsCl gradient or by other means. This invention provides a method for administering AAV particles, recombinant AAV vectors, and recombinant AAV virions. For example, AAV2 particles are viral particles containing the AAV2 capsid protein, or AAV1 particles are viral particles containing the AAV1 capsid protein. Recombinant AAV2 vectors are nucleic acid constructs containing at least one specific nucleic acid of AAV2. Recombinant AAV2 virions are particles containing the recombinant AAV2 vector. In order to be considered to fall under the term "ITR," the nucleotide sequence must retain one or both of the following features that distinguish AAV2 ITR from AAV1 ITR: (1) three "GAGC" repeats (instead of four in AAV1) and (2) in the AAV2 ITR Rep-binding site, the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0132] The promoter for inducing the expression of the sequence encoding the tRNA to be delivered can be any desired promoter selected by known considerations, such as the level of expression of the nucleic acid functionally linked to the promoter and the type of cell in which the vector should be used. The promoter can be exogenous or endogenous. Promoter examples may include known potent promoters such as the SV40 or inducible metallothionein promoter, or AAV promoters such as the AAVp5 promoter. Further examples of promoters include those derived from actin genes, immunoglobulin genes, cytomegalovirus (CMV), adenoviruses, bovine papillomavirus, adenoviruses such as the major late promoter, inducible heat shock promoters, respiratory multinuclear viruses, Rous sarcoma virus (RSV), and others. Further examples include controlled promoters.

[0133] AAV vectors may further contain exogenous (heterogeneous) nucleic acids functionally linked to a promoter. “Heterogeneous nucleic acids” means that any heterogeneous or exogenous nucleic acid can be inserted into the vector for transfer into cells, tissues, or organisms. The nucleic acid may, for example, encode tRNA. “Functionally linked” means that the promoter can promote the expression of the heterogeneous nucleic acid, as is known in the art, including the appropriate orientation of the promoter toward the heterogeneous nucleic acid. Furthermore, the heterogeneous nucleic acid preferably has all the appropriate sequences for nucleic acid expression to functionally encode, i.e., to enable the nucleic acid to be expressed, as is known in the art. The nucleic acid may include expression regulatory sequences, such as enhancers. The nucleic acid may encode more than one gene product, limited only by the size of the nucleic acid that can be packaged.

[0134] AAV1 particles are viral particles containing the AAV1 capsid protein. Variations in the amino acid sequence of the AAV1 capsid protein are assumed herein, as long as the resulting viral particles containing the AAV1 capsid remain antigenically or immunologically distinct from other AAV capsids, as can be determined systematically by standard methods. Specifically, for example, ELISA and Western blotting can be used to determine whether viral particles are antigenically or immunologically distinct from other AAV serotypes.

[0135] As used herein, the term "polypeptide" refers to a polymer of amino acids, including full-length proteins and their fragments. For this reason, "protein" and "polypeptide" are often used interchangeably herein.

[0136] This method provides a method for delivering nucleic acids to cells, comprising administering AAV particles containing a vector with nucleic acids inserted between pairs of AAV terminal inverted sequences to cells, thereby delivering the nucleic acids to the cells. Administration to cells can be achieved by any means, including simply bringing particles, optionally contained in a desired liquid such as tissue culture medium or buffered saline solution, into contact with the cells. The particles can remain in contact with the cells for any desired length of time, typically administered and then retained indefinitely. For such in vitro methods, the virus can be administered to cells by standard viral transduction methods, such as those known in the art and illustrated herein. The titer of the virus to be administered may vary depending particularly on the type of cell, but the titer generally used for AAV transduction is typical. Furthermore, the titer used to transduce specific cells in this embodiment can be used. Cells may include any desired cells from humans and other large (non-rodent) mammals such as primates, horses, sheep, goats, pigs, and dogs.

[0137] The present invention further provides a method for delivering nucleic acids to cells in a target, comprising administering AAV particles containing nucleic acids inserted between pairs of AAV terminal inversion sequences to a target, thereby delivering nucleic acids to cells in the target.

[0138] One embodiment of this disclosure provides cells containing a viral vector as described herein. AAV Vector

[0139] In one embodiment, the viral vector of this disclosure is an AAV vector. The term “AAV” vector represents an adeno-associated virus and may be used to represent the naturally occurring wild-type virus itself or its derivatives. Unless otherwise required, the term encompasses all subtypes, serotypes, and pseudotypes, and includes both naturally occurring and recombinant forms. As used herein, the term “serotype” represents an AAV that is identified and distinguished from other AAVs based on the reactivity of its 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 represent an AAV that contains the capsid protein encoded from the cap gene of AAV2 as well as a genome containing the 5' and 3' ITR sequences from the same AAV2 serotype. As used herein, for example, rAAV1 may be used to represent an AAV having both a capsid protein and a 5'-3'ITR derived from the same serotype, or rAAV1 may represent an AAV having a capsid protein derived from one serotype and a 5'-3'ITR derived from a different AAV serotype, for example, an AAV having a capsid derived from AAV serotype 2 and an ITR derived from AAV serotype 5. For each example illustrated herein, the description of vector design and preparation will specify the serotypes of the capsid and 5'-3'ITR sequences. The abbreviation "rAAV" represents recombinant adeno-associated virus and is also referred to as a recombinant AAV vector (or "rAAV vector").

[0140] The term "AAV virus" or "AAV virus particle" refers to a viral particle composed of at least one AAV capsid protein (preferably all of the capsid proteins of wild-type AAV) and polynucleotides enclosed within the capsid. If the particle contains heterogeneous polynucleotides (i.e., polynucleotides other than those in the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as "rAAV".

[0141] In one embodiment, the AAV expression vector is constructed using known techniques to provide at least the transcription, regulatory elements including a transcription start region, the DNA of interest, and components operably ligated toward the transcription termination region. The regulatory elements are selected to be functional in mammalian cells. The resulting construct containing the operably ligated components is flanked (5' and 3') by a functional AAV ITR sequence.

[0142] "Adeno-associated virus terminal inversion sequences" or "AAV ITRs" refer to regions recognized in this field that are found at each end of the AAV genome and function together in cis as origins for DNA replication and as packaging signals for the virus. AAV ITRs, along with AAV rep coding regions, provide efficient excision and rescue from mammalian cell genomes, as well as integration into the mammalian cell genome of nucleotide sequences interposed between two adjacent ITRs.

[0143] The nucleotide sequences of the AAV ITR region are known. As used herein, “AAV ITR” does not need to have the indicated wild-type nucleotide sequence and can be modified, for example, by nucleotide insertion, deletion, or substitution. Furthermore, AAV ITR can originate from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, and AAV7. Moreover, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in the AAV vector do not necessarily need to be identical, nor do they need to originate from the same AAV serotype or isolate, insofar as they function as intended—that is, to enable the excision and rescue of the desired sequence from the host cell genome or vector, and to enable the incorporation of a heterologous sequence into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0144] In one embodiment, the AAV ITR can be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, and AAV7. Furthermore, the 5' and 3' ITRs adjacent to the selected nucleotide sequence in the AAV expression vector do not necessarily have to be identical, nor do they have to be derived from the same AAV serotype or isolate, as long as they function as intended, i.e., to enable the excision and rescue of the desired sequence from the host cell genome or vector, and to enable the integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present in the cell.

[0145] In one embodiment, the AAV capsid may be derived from AAV2. Suitable DNA molecules for use in AAV vectors are known in the art to be of sizes 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, and less than about 2.5 kb.

[0146] In one embodiment, a selected nucleotide sequence is operably ligated to a regulatory element that induces its transcription or expression in vivo in the subject. Such regulatory elements may include regulatory sequences typically associated with the selected gene, or heterologous regulatory sequences may be utilized. Useful heterologous regulatory sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoters such as the CMV very early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the pol II promoter, the pol III promoter, synthetic promoters, and hybrid promoters. Furthermore, sequences derived from non-viral genes, such as the mouse metallothionein gene, can also be used in this invention. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, Calif.).

[0147] In one embodiment, both heterogeneous promoters and other regulatory elements, such as tissue-specific and inducible promoters and enhancers, are particularly useful. Examples of heterogeneous promoters include the CMV promoter. Examples of inducible promoters include DNA responsive elements to ecdysone, tetracycline, hypoxia, and aufin.

[0148] In one embodiment, an AAV expression vector containing a target DNA molecule bordered by an AAV ITR can be constructed by directly inserting a selected sequence into the AAV genome from which a major AAV reading frame ("ORF") has been excised. Other parts of the AAV genome can be removed, as long as a sufficient portion of the ITR still allows for replication and packaging functions. Such constructs can be designed using techniques well known in the art.

[0149] Alternatively, AAV ITR can be excised from the viral genome or from an AAV vector containing AAV ITR, and the 5' and 3' ends of a selected nucleic acid construct present in another vector can be fused using standard ligation techniques. For example, ligation may be performed using 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, and 10 mM DTT, 33 μg / mL BSA, 10 mM–50 mM NaCl and 40 μM ATP, 0.01–0.02 (Weiss) units of T4 DNA ligase, 0°C (for "sticky end" ligation) or 1 mM ATP, 0.3–0.6 (Weiss) units of T4 DNA ligase, 14°C (for "blunt end" ligation). Intermolecular "sticky end" ligation is typically performed at a total DNA concentration of 30–100 μg / mL (total final concentration of 5–100 nM). AAV vector containing ITR.

[0150] Furthermore, chimeric genes can be synthetically produced to contain AAV ITR sequences positioned at the 5' and 3' positions of one or more selected nucleic acid sequences. Complete chimeric sequences are assembled from duplicate oligonucleotides prepared by standard methods.

[0151] To produce rAAV virions, AAV expression vectors are introduced into suitable host cells using known techniques, such as transduction. Numerous transduction techniques are commonly known in this field. See, for example, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transduction methods include calcium phosphate coprecipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.

[0152] In one embodiment, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of heterologous DNA molecules. The term includes the offspring of the translocated original cell. Thus, “host cell” as used herein generally refers to a cell translocated with an exogenous DNA sequence. Cells derived from a stable human cell line, 293 (e.g., readily available from the U.S. Cell Culture Line Preservation Service under accession number ATCC CRL1573) may be used in the practice of this disclosure. In particular, human cell line 293 is a human fetal kidney cell line transformed with adenovirus type 5 DNA fragments and expresses adenovirus E1a and E1b genes. The 293 cell line is readily translocated and provides a particularly convenient platform for producing rAAV virions.

[0153] The term "AAV rep coding region" refers to the region of the AAV genome recognized in this art that encodes the replication proteins Rep78, Rep68, Rep52, and Rep40. These Rep expression products have been shown to have many functions, including recognition, binding, and nick formation of AAV origins in DNA replication, DNA helicase activity, and modulation of transcription from AAV (or other heterologous) promoters. Rep expression products are collectively required for AAV genome replication. Suitable homologs of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene, which is also known to mediate AAV-2 DNA replication.

[0154] The term "AAV cap coding region" refers to the region of the AAV genome recognized in this art that encodes the capsid proteins VP1, VP2, and VP3, or their functional homologs. These cap expression products provide the packaging functions collectively required to package the viral genome.

[0155] In one embodiment, AAV helper function is introduced into host cells by transposing the host cells with an AAV helper construct, either before or in conjunction with the transposition of an AAV expression vector. Thus, AAV helper constructs are used to provide at least transient expression of AAV rep and / or cap genes to complement lost AAV function necessary for productive AAV infection. AAV helper constructs lack AAV ITR and cannot replicate or package on their own. These constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Numerous AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode expression products of both Rep and Cap. Numerous other vectors encoding Rep and / or Cap expression products have also been described.

[0156] Methods of viral vector delivery include injecting AAV into the subject. Generally, rAAV virions can be introduced into cells using either in vivo or in vitro transduction techniques. When transduced in vitro, the desired recipient cells are removed from the subject, transduced with rAAV virions, and then reintroduced into the subject. Alternatively, syngeneic or heterogeneic cells may be used, and these cells will not produce an inappropriate immune response in the subject.

[0157] Appropriate methods for the delivery and introduction of transduced cells into a subject are described. For example, cells can be transduced in vitro by combining recombinant AAV virion with cells in a suitable medium, and screening for cells possessing the DNA of interest can be done using conventional techniques, e.g., Southern blotting and / or PCR, or by using selectable markers. The transduced cells can then be formulated into pharmaceutical compositions, which are described in more detail below, and which can be introduced into a subject by various techniques, e.g., transplantation, intramuscular, intravenous, subcutaneous and intraperitoneal injection.

[0158] In one embodiment, the pharmaceutical composition contains sufficient genetic material to produce a therapeutically effective amount of the nucleic acid of interest, i.e., an amount sufficient to reduce or alleviate the symptoms of the disease condition in question, or an amount sufficient to provide a desired benefit. The pharmaceutical composition also contains pharmaceutically acceptable excipients. 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, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Among these, pharmaceutically acceptable salts may include, for example, mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc., and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Furthermore, auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., may be present in such vehicle. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0159] It should be understood that more than one transgene may be expressed by the delivered viral vector. Alternatively, separate vectors expressing one or more different transgenes may also be delivered to the subject as described herein. Furthermore, viral vectors delivered by the methods of this disclosure are also intended to be combined with other suitable compositions and therapies.

[0160] As will be apparent to those skilled in the art in light of the teachings of this specification, the effective amount of viral vector that must be added can be determined experimentally. Administration can be carried out in one dose, continuously or intermittently throughout the course of treatment. The most effective means of administration and methods of determining dosage amounts are well known to those skilled in the art and vary depending on the viral vector, the composition of the treatment, the target cells, and the subject being treated. Single and multiple administrations can be carried out at dosage levels and patterns selected by the physician performing the treatment.

[0161] In certain embodiments, rAAV is administered at a dose of about 0.3 - 2 mL of 1×10 5 ~1×10 16 vg / mL. In certain embodiments, rAAV is administered at a dose of about 1 - 3 mL of 1×10 7 ~1×10 14 vg / mL. In certain embodiments, rAAV is administered at a dose of about 1 - 2 mL of 1×10 8 ~1×10 13 vg / mL.

[0162] Formulations containing rAAV particles contain an effective amount of rAAV particles in a vehicle, and the effective amount can be readily determined by those skilled in the art. rAAV particles typically range from about 1% - about 95% (w / w) of the composition, or, as appropriate, higher or lower ranges. The amount to be administered depends on factors such as the age, weight, and physical condition of the animal or human subject for which the treatment is contemplated. Effective dosages can be established by those skilled in the art through standard tests to establish a dose-response curve. The subject is treated by administration of rAAV particles at one or more doses. Multiple doses can be administered if it is necessary to maintain sufficient enzyme activity.

[0163] Vehicles containing water, aqueous saline, artificial CSF, or other known substances can be used in 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 in combination with an anti-inflammatory agent, and packaged for use.

[0164] The present invention provides a method for increasing the level of a target protein in cells by introducing a protein, or a nucleic acid molecule encoding the protein, into cells in an amount sufficient to increase the level of the target protein in the cells. In one embodiment, 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%. 39 Nucleic acids that code for therapeutic agents

[0165] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single-stranded or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate, and a base that is either a purine or a pyrimidine. Unless otherwise specified, the term encompasses nucleic acids that contain known analogs of natural nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0166] A "nucleic acid fragment" is a part of a given nucleic acid molecule. The term "substantial identity" of a polynucleotide sequence means that, when compared to a reference sequence using one of the described alignment programs with standard parameters, a given polynucleotide contains a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% sequence identity, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or at least 95%, 96%, 97%, 98%, or 99% sequence identity. Methods for introducing genetic material into cells

[0167] Exogenous genetic material (e.g., DNA encoding one or more therapeutic ACE-tRNAs) is introduced into cells in vivo by gene transfer methods such as transduction or transfection to provide genetically modified cells. Various expression vectors (i.e., vehicles for facilitating the delivery of exogenous genetic material into target cells) are known to those skilled in the art.

[0168] As used herein, “cellular translocation” refers to the acquisition of new genetic material by a cell through the uptake of added DNA. Therefore, translocation refers to the insertion of nucleic acids into a cell by physical or chemical means. Several translocation techniques are known to those skilled in the art, including calcium phosphate DNA coprecipitation; DEAE-dextran; electroporation; cationic liposome-mediated translocation; and tungsten particle-assisted microparticle guns. Strontium phosphate DNA coprecipitation is another possible translocation method.

[0169] In contrast, "transduction of cells" refers to a method of introducing nucleic acids into cells using DNA or RNA viruses. RNA viruses (i.e., retroviruses) used to introduce nucleic acids into cells are referred to herein as transduction chimeric retroviruses. Exogenous genetic material contained within the retrovirus is incorporated into the genome of the transduced cell. Cells transduced with a chimeric DNA virus (e.g., an adenovirus having cDNA encoding a therapeutic agent) do not have exogenous genetic material incorporated into their genome, but can express exogenous genetic material that is retained outside the chromosome within the cell.

[0170] Typically, exogenous gene material contains a heterologous gene (usually in the form of a cDNA containing exons encoding a therapeutic protein) along with a promoter to regulate the transcription of the new gene. The promoter characteristically has a specific nucleotide sequence required to initiate transcription. If necessary, the exogenous gene material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcriptional activity. In this discussion, “enhancer” simply means any untranslated DNA sequence that works adjacent to the coding sequence (located in cis) to alter the basal transcription level determined by the promoter. Exogenous gene material can be introduced into the cellular genome immediately downstream of the promoter so that the promoter and coding sequence are operationally linked to allow transcription of the coding sequence. Retroviral expression vectors may contain exogenous promoter elements to regulate the transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inductive promoters.

[0171] Naturally occurring constitutive promoters regulate the expression of essential cellular functions. As a result, genes regulated by constitutive promoters are expressed under all conditions of cell growth. Exemplary constitutive promoters include those for the following genes encoding certain constitutive or "housekeeping" functions: the promoters for 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. Furthermore, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40; the long terminal repeat (LTR) and other retroviruses of Moloney's leukemia virus; and the thymidine kinase promoter of herpes simplex virus. Therefore, any of the constitutive promoters cited above can be used to regulate the transcription of heterologous gene insertions.

[0172] Genes regulated by inductive promoters are expressed only in the presence of an inducer, or more in the presence of an inducer (for example, transcription under the regulation of a metallothionein promoter is significantly increased in the presence of a certain metal ion). Inductive promoters contain a response element (RE) that stimulates transcription when bound to its inducer. Examples of REs exist for serum factors, steroid hormones, retinoic acid, and cyclic AMP. To obtain an inductive response, a promoter containing a specific RE can be selected, and in some cases, the RE itself can be bound to a different promoter, thereby conferring inductivity to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive vs. inductive: strong vs. weak), it is possible to regulate both the presence and level of therapeutic agent expression in genetically modified cells. If the gene encoding a therapeutic agent is regulated by an inductive promoter, in-situ delivery of the therapeutic agent can be induced by exposing the genetically modified cells to conditions that allow for the transcription of the therapeutic agent, for example, by intraperitoneal injection of a specific inducer of the inductive promoter that regulates the transcription of the therapeutic agent. For example, the in-situ expression of therapeutic agents encoded by genes regulated by the metallothionein promoter in genetically modified cells can be enhanced by bringing the genetically modified cells into situ contact with a solution containing the appropriate (i.e., inducing) metal ions.

[0173] Therefore, the amount of therapeutic agent delivered in situ is controlled by regulating the following factors: (1) the nature of the promoter used to induce transcription of the inserted gene (i.e., whether the promoter is constitutive or inductive, potent or weak); (2) the number of copies of the exogenous gene inserted into the cell; (3) the number of transduced / transplanted cells administered to the patient (e.g., transplanted); (4) the size of the transplanted tissue (e.g., graft or embedded expression system); (5) the number of transplanted tissues; (6) the length of time the transduced / transplanted cells or transplanted tissues are implanted; and (7) the rate of therapeutic agent production by the genetically modified cells. The selection and optimization of these factors for delivering a therapeutically effective dose of a particular therapeutic agent is considered to be within the realm of the art of the art, without excessive experimentation, taking into account the factors disclosed above and the patient's clinical profile.

[0174] In addition to at least one promoter and at least one heterogeneous nucleic acid encoding a therapeutic agent, the expression vector may include a selection gene, such as a neomycin resistance gene, to facilitate the selection of cells into which the expression vector has transfected or transduced. Alternatively, cells may be transfected with two or more expression vectors, where at least one vector contains the gene encoding the therapeutic agent and the other vector contains the selection gene. The selection of appropriate promoters, enhancers, selection genes, and / or signal sequences (described below) is considered to be within the realm of the art without excessive experimentation. Disease condition and treatment methods

[0175] In one embodiment, the present invention includes compositions and methods for treating cystic fibrosis by reversing the effects of existing mutations associated with nonsense mutations through the introduction of the synthetic oligonucleotide suppressor tRNA of the present invention.

[0176] One embodiment of this disclosure provides a method for treating a disease in a mammal, comprising administering a protein or vector encoding a therapeutic agent described herein (e.g., modified and / or stabilized ACE-tRNA) to the mammal. In one embodiment, the mammal is a human.

[0177] One embodiment of this disclosure provides the use of a therapeutic agent or a vector encoding a therapeutic agent described herein for preparing a pharmaceutical useful for treating a disease in a mammal. In one embodiment, the disease is cystic fibrosis.

[0178] This disclosure also provides mammalian cells containing the vectors described herein. The cells may be human cells.

[0179] Certain aspects of this disclosure relate to polynucleotides, polypeptides, vectors, and genetically modified cells (modified in vivo) and their uses. In particular, this disclosure relates to a gene therapy method that enables bisystemic delivery of a therapeutically effective dose of a therapeutic agent.

[0180] According to one embodiment, 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 cell”) comprises cells and an expression vector for expressing the therapeutic agent. The expression vector includes, but is not limited to, viruses, plasmids, and other vehicles for delivering heterologous genetic material to cells. Thus, as used herein, the term “expression vector” refers to a vehicle for delivering heterologous genetic material to cells. In particular, the expression vector is a recombinant adenovirus, adeno-associated virus, or lentivirus or retroviral vector.

[0181] The expression vector further comprises a promoter for regulating the transcription of heterologous genes. The promoter may be an inducible promoter (as described herein). The expression system is suitable for administration to mammalian recipients. The expression system may comprise a plurality of non-immortalized genetically modified cells, each containing at least one recombinant gene encoding at least one therapeutic agent.

[0182] The cell expression system is formed in vivo. In yet another embodiment, a method is provided for treating a mammalian recipient in vivo. This method includes, for example, introducing an expression vector for expressing a heterologous gene product into the patient's cells in situ, via intravenous administration. To form an expression system in vivo, the expression vector for expressing the therapeutic agent is introduced in vivo into the mammalian recipient by intravenous administration.

[0183] In yet another embodiment, a method is provided for treating a mammalian recipient in vivo. This method includes introducing a targeted therapeutic agent into the patient in vivo.

[0184] Expression vectors for expressing heterologous genes may contain inducible promoters to regulate the transcription of heterologous gene products. Therefore, in-situ delivery of therapeutic agents is regulated by exposing cells in-situ to conditions that induce the transcription of heterologous genes.

[0185] This disclosure provides methods for treating diseases in mammals by administering expression vectors to cells or patients. Regarding gene therapies, those skilled in molecular biology and gene therapy should be able to determine appropriate doses and routes of administration of the expression vectors used in the novel methods of this disclosure without excessive experimentation.

[0186] According to one embodiment, cells are transformed or otherwise genetically modified in vivo. Cells obtained from a mammalian recipient are transformed in vivo (i.e., transduced or transmigrated) with a vector containing exogenous genetic material for expressing a heterologous (e.g., recombinant) gene encoding a therapeutic agent, and the therapeutic agent is delivered in situ.

[0187] As used herein, “exogenous genetic material” refers to a nucleic acid or oligonucleotide, whether natural or synthetic, that is not found naturally in cells, or, if found naturally in cells, is not transcribed or expressed by cells at a biologically significant level. Therefore, “exogenous genetic material” includes, for example, a nucleic acid that is not naturally occurring and can be transcribed into tRNA.

[0188] The therapeutic agents and conditions disclosed above that are suitable for gene therapy are merely illustrative and are not intended to limit the scope of this disclosure. The selection of an appropriate therapeutic agent for treating known conditions is considered to be within the realm of the art, without excessive experimentation.

[0189] In one embodiment, this therapy has potential applications for the treatment / management of diseases caused by immature termination codons (PTCs), including but not limited to cystic fibrosis, muscular dystrophy, β-thalassemia, and Liddle syndrome. This therapy is advantageous in that it provides improved stop codon suppression specificity. The therapeutic ACE-tRNA of the present invention targets specific stop codons, e.g., TGA, thus reducing off-target effects at disease-unrelated stop codons. This therapy is also advantageous in that it provides amino acid specificity. The expressed tRNA is engineered to specifically replace amino acids lost through the insertion of disease stop codons, thus eliminating any spurious effects on protein stability, folding, and transport.

[0190] In one embodiment, the system is modular and therefore can be "individualized" for any possible disease PTC. For example, each of the nine tryptophan tRNAs recognized by Trp synthase exists in the human genome, and all of them repress the mRNA UGG codon. Thus, each of these nine Trp tRNAs offers an opportunity for codon re-editing tolerance (UGG→UGA). Furthermore, considering the proximity to the stop codon in the gene code, mutations of the arginine codon to a PTC nonsense codon are common in the disease. There are more than 30 Arg tRNAs that can be tested for codon editing tolerance and repressive effects.

[0191] A further advantage of the present invention is that the entire system (tRNA + promoter sequence) is compact, which allows for easy expression and cell-specific delivery. Dosage, formulation, and route of administration of the drug of the present invention

[0192] The agents of the present invention are administered to reduce at least one symptom associated with a genetic disorder (e.g., cystic fibrosis). The dose administered varies depending on a variety of factors, including, but not limited to, the selected composition, the specific disease, the mammal's body weight, physical condition and age, 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.

[0193] The present invention envisions treating genetic disorders (e.g., cystic fibrosis) by administering the agents of the present invention, such as ACE-tRNA, expression vectors, or viral particles. Administration of therapeutic agents according to the present invention may be continuous or intermittent, depending, for example, the recipient's physiological condition, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the agents of the present invention may be essentially continuous over a pre-selected period, or it may be a series of intermittently spaced doses. Both topical and systemic administration are envisioned.

[0194] As described below, one or more suitable unit dosage forms having the therapeutic agent(s) of the present invention, which may be formulated for controlled release as needed (e.g., using microencapsulation), can be administered by various routes including parenteral routes such as intravenous and intramuscular routes, as well as by direct injection into diseased tissue. The formulations may conveniently be provided in separate unit dosage forms and may be prepared by any of the methods well known in pharmacy. Such methods may include the step of bringing the therapeutic agent into association with a liquid carrier, a solid matrix, a semi-solid carrier, a micronized solid carrier or a combination thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0195] When the therapeutic agent of the present invention is prepared for administration, the therapeutic agent of the present invention may be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical preparation or a unit dosage form. The total active ingredient in such a preparation comprises 0.1 to 99.9% by weight of the preparation. "Pharmaceutically acceptable" means a carrier, diluent, excipient and / or salt that is compatible with the other ingredients of the preparation and not harmful to its recipient. The active ingredient for administration may be present as a powder or as granules; as a solution, suspension or emulsion.

[0196] The pharmaceutical preparation containing the therapeutic agent of the present invention can be prepared by known and readily available ingredients by techniques known in the art. The therapeutic agent of the present invention can also be formulated as a solution suitable for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes. [[ID=!0]]

[0197] The pharmaceutical preparation of the therapeutic agent of the present invention can also take the form of an aqueous or anhydrous liquid or dispersion or the form of an emulsion or suspension.

[0198] Therefore, therapeutic agents may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be administered in unit dose form in ampoules, pre-filled syringes, small-volume infusion containers, or in multi-dose containers with added preservatives. The active ingredient may take such form as a suspension, liquid, or emulsion in an oily or aqueous vehicle and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form obtained by sterile isolation of a sterile solid or by lyophilization from a solution, for preparation using a suitable vehicle, e.g., sterile water free of pyrogens, before use.

[0199] Since the required effective dose can be achieved through the administration of multiple dosing units, it is understood that the unit content of one or more active ingredients contained in the individual aerosol doses of each dosage form does not, in itself, need to constitute an effective amount to treat a specific indication or disease. Furthermore, an effective dose may be achieved using less than the dose in the dosage form, either individually or in a series of administrations.

[0200] The pharmaceutical formulations of the present invention may, as necessary, include pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers and salts of a type well known in the art. Non-limiting specific examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered salt solutions such as phosphate-buffered salt solution pH 7.0-8.0 and water. definition

[0201] Disease Condition: In this invention, “disease condition” or “disease phenotype” refers to a characteristic of a mammalian cell resulting from a stop codon in the coding region of a gene within the cell (e.g., a nonsense mutation). For example, an increasing number of human genetic disorders are thought to be caused by nonsense mutations (see, e.g., Atkinson et al., Nuc. Acids Res. 22:1327, 1994). To name just a few, β-thalassemia, Duchenne muscular dystrophy, xeroderma pigmentosum, Fanconi anemia, and cystic fibrosis can all be caused by nonsense mutations in identified genes.

[0202] Endogenous tRNA synthetase: If tRNA is present in a cell into which tRNA is introduced according to the present invention, then tRNA synthetase can be considered "endogenous" to the cell. As will be obvious to those skilled in the art, tRNA synthetase can be considered endogenous for these purposes, whether it is found naturally in the relevant type of cell, or whether the cell is engineered to contain or express tRNA synthetase, or otherwise skillfully handled by human hands.

[0203] Suppressor tRNA: A "suppressor tRNA" is one whose anticodon is complementary to a codon that terminates translation, allowing for detectable read-through under experimental conditions. Standard termination codons are amber (UAG), ochre (UAA), and opal (UGA) codons. However, non-standard termination codons (e.g., 4-nucleotide codons) have also been used 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).

[0204] Herein, the present invention is illustrated by the following non-limiting embodiments. [Examples]

[0205] [Example 1] The genetic code uses four nucleotides to form sequential triplet codons, which form the basis of DNA translation into proteins. There are a total of 64 codons, of which 61 are used to code for amino acids, and three of these (TAG, TGA, and TAA) code for protein termination "stop" or "nonsense" codons.

[0206] 5–10% of cystic fibrosis cases are caused by “nonsense” mutations that result in immature truncation of the cystic fibrosis membrane conductance regulator (CFTR) protein. An example of this “class 1” mutation is p.Trp1282X, an immature termination codon (PTC) that causes loss of CFTR function and a severe cystic fibrosis phenotype. Some compounds, such as ataleren, facilitate the readthrough of termination of disease-causing nonsense mutations, but have had little success as therapeutic agents due to several caveats, including insufficient stop codon specificity and unexpectedly low codon skipping efficiency in vivo. However, the widespread use of these compounds and the finding that endogenous stop codon readthrough is common in metazoans suggest that assisted suppression may be viable if delivered to a subset of cell types, namely airway epithelium. However, even when therapeutically assisted stop codon readthrough is successful, non-selective uptake of amino acids at nonsense codon sites can affect protein folding, transport, and function (as in the case of CFTR1282X), thus requiring further therapeutic intervention. Therefore, there is an urgent and unmet need to understand the nature of disease-related PTCs, potentially therapeutic suppressors, and, in general, more effective treatments for PTC diseases.

[0207] This embodiment determines the properties of anticodon-editing (ACE) Trp-tRNA for the rescue of the CFTR p.Trp1282X channel. Such tRNAs are engineered to "repress" the disease-causing TGA stop codon, incorporating the original amino acid Trp in the p.Trp1282X CFTR and effectively genetically reconstructing the wild-type CFTR protein. The data demonstrate that this general approach (nonsense repression) results in robust rescue of transcripts containing stop codons in frame through transient translocation of tRNA and its homologous synthase in adherent cells or through virus-based delivery of these to more native airway cell types such as A549 airway cells. This approach offers numerous significant benefits beyond existing strategies: 1) Improved codon specificity – Since expressed tRNAs can be induced toward specific stop codons, off-target effects on disease-independent stop codons are reduced. 2) Amino acid specificity - The expressed tRNA and / or synthase can be manipulated to replace the lost amino acids through the insertion of disease stop codons, thus counteracting any spurious effects on CFTR stability, folding, and transport. 3) Tunability - This system can theoretically be individualized for each type of tRNA and PTC mutation. 4) Easy expression - The entire system is compact (less than 1kb), easily packaged, and can be expressed transiently or via nanoparticle delivery of tRNA. 5) Proof of principle for general strategies - In-frame stop codons are a major cause of human disease, and there are few treatment options; here, experiments performed on p.Trp1282X are expected to provide insights into the mechanisms of other CFTR nonsense codons.

[0208] The data demonstrate that ACE-tRNA stop codon suppressor tRNAs are efficient at “saving” transcripts containing introduced stop sites (Figures 6A and 6B), suggesting that such tRNAs have the ability to inhibit nonsense mutation-dependent degradation (NMD), a major biological impediment in the therapeutic rescue of disease stop sites. This opens up the possibility of using suppressor tRNAs to gain more molecular insights into NMD in disease. result

[0209] The inventors questioned whether it is possible to express anticodon-edited eukaryotic tRNAs that suppress the termination site, e.g., TGA, and not suppress the specified codon. This was tested with five human tryptophan tRNAs on a test construct consisting of an eGFP sequence and an in-frame fluorescent protein (cherry), separated by a linker containing the TGA site. To indicate the production of full-length protein, an HA epitope was added to the C-terminus of the eGFP reading frame. This test system is useful because the appearance of the cherry signal indicates plasmid delivery and expression, and, combined with eGFP rescue, indicates TGA suppression. The data in Figures 6A and 6B show Western blot data from assays using this test construct to determine the ability of five anticodon-edited human Trp tRNAs to suppress the TGA termination site in the short linker between the cherry and the eGFP reading frame. Of these constructs, candidates 1, 2, 3, and 5 show moderate activity in this regard. This could be due to structural intolerance to the mutation, or possibly because altering the anticodon by just one base disrupted the ability of Trp synthase to recognize and / or acylate tryptophan-containing tRNAs. However, tRNA number 4 of these test tRNAs (tRNA#4) showed significant repressive activity at the TGA site and produced full-length cherry-eGFP-HA protein (Figure 6B). Furthermore, no readthrough was observed in the absence of co-expressed tRNA, last lane, Figure 6B. method

[0210] Trp tRNAs were investigated for codon editing tolerance (TGG→TGA) and their ability to suppress the targeted TGA test site in transiently transfected tandem fluorescent labeling reagent (m-cherry-TGA-GFP) and CFTR Trp1282X. Initial screening of 5 out of 9 Trp tRNAs revealed an anticodon-editing Trp-tRNA that transiently transfected into HEK cells and possessed "unparalleled" functionality for rescuing the cherry-TGA-eGFP-HA test construct, Figure 6B. The selective presence of the HA epitope demonstrates successful rescue and confocal examination of both cherry and eGFP fluorescence at the single-cell level (not shown). These results provide proof of principle data that a) some ACE-tRNAs can tolerate anticodon editing, b) these tRNAs retain the ability to be acylated with Trp by endogenous tryptophan synthase, and c) these tRNAs can suppress the TGA site embedded within the protein reading frame.

[0211] The remaining four Trp-tRNAs are functionally investigated for their tolerance to anticodon editing from TAA to TGA suppressor. These anticodon-edited tRNAs are tested for their ability to rescue the cherry-TGA-eGFPHA clone. Biochemical (Western blot) data are obtained for cherry and eGFP signals as well as the HA epitope. Here, cherry expression serves as a positive translocation control. Confocal images demonstrate cherry and eGFP fluorescence at the single-cell level.

[0212] The fidelity of endogenous Trp synthase, which introduces tryptophan amino acids into ACE-Trp tRNA, is determined by mass spectrometry of trypsin-digested fragments of purified, rescued cherry-Trp-eGFPHA protein. The expected mass for the trypsin-digested fragment produced from the linker between the cherry- and eGFP reading frames is as follows: It has an expected mass of 1590.8135; [ka] ; The "W" in bold indicates the site of incorporation, Figure 10. Thus, this represents the first example of nonsense codon repair and replacement with wild-type amino acids, and is therefore a significant advance compared to existing approaches such as Ataluran, which are useful for treatment. In the latter example, the compound promotes read-through of the nonsense codon by incorrect amino acids, and thus the discovery and identification of new tRNA sequences that provide precise repair is important.

[0213] The rescue of the transiently transfected CFTR1282X channel by the ACE-tRNA identified above is evaluated by standard biochemical methods for the complete maturation of the B and C glycosylated CFTR bands 20. Thus, this channel is repaired with wild-type amino acids, is fully functional, and is successfully transported to the cell membrane.

[0214] The next step is to functionally characterize the CFTR Trp1282X channel rescued by the ACE-tRNA system identified above using electrophysiological (single-cell patch clamp and Ussing chamber) and biochemical approaches. The efficacy of the expressed tRNA to attenuate nonsense mutation-dependent decay (NMD) of 1282X mRNA can be evaluated using quantitative rtPCR. Reprogrammed human airway cells are used to test the rescue of the native 1282X CFTR channel by the expressed codon-edited Trp-tRNA.

[0215] In the identified human Trp tRNA, it has been demonstrated that anticodon editing is tolerated and that this 75-base pair transfer RNA can suppress the in-frame TGA codon within the test construct. These experiments extrapolate this discovery to characterize the ability of this ACE-tRNA to interact with CFTR 1282TGA mRNA and produce functional CFTR channels in model cells (FRT and A549) and p.1282X human reprogrammed airway cells.

[0216] Biochemical determination of rescue levels in transiently expressed CFTR1282X channels and in reprogrammed airway cells. To recognize rescued CFTR (epitope amino acids 1370-1380) and detect all rescued and unrescueable CFTR, antibody M3A7, which binds to the N-terminal-like MM13-4 (epitope amino acids 25-36), available from EMD Millipore, is used. Alternatively, L12B4 (epitope amino acids 386-412, EMD Millipore) or 660 (epitope amino acids 576-585) are available through Cystic Fibrosis Foundation Therapeutics.

[0217] Surface functionality is investigated through electrophysiological approaches such as patch-clamp and Ussing chamber recordings. The stability and abundance of 1282X mRNA are assayed by quantitative rtPCR of RNA extracts from transiently expressing cells and reprogrammed airway cells.

[0218] Bioinformatics analysis of RNA transcriptome data obtained from human airway cells identifies the abundance, context, and identity of transcripts containing TGA codons. The top 10 expressed transcripts using TGA for their normal termination sites are tracked at the individual transcript level using protein biochemistry before and after ACE-tRNA expression. Biochemical and immunohistochemical probes of cellular apoptosis are also used to investigate the effects of ACE-tRNA on cell death.

[0219] In conclusion, these data indicate that ion channel genes with in-frame termination sites are well-suited for this type of “rescue” (Figure 9), and that components of the system can be expressed by viruses in airway cells. Furthermore, in a highly simplified form of this idea, human-derived ACE-tRNA demonstrates a “unique” ability to rescue in-frame CFTR TGA codons in mammalian cell lines (Figure 9). This approach has many advantages over existing stop-codon strategies and warrants further investigation regarding the ability of ACE-tRNA to 1) suppress nonsense mutation-dependent degradation, 2) function in lung cell preparations, and 3) specifically rescue CFTR1282X. [Example 2]

[0220] Several different nonsense mutations cause CF, thus underlie approximately 10% of all CF disorders. Figure 7. These cases can be aggregated into the following 10 specific genetic lesions: E60X, R75X, G542X, R553X, Q890X, Y1092X, R1158X, R1162X, and W1282X. We propose that, with the appropriate approach, it should be feasible to screen existing human tRNA sequences for modification and tolerance to anticodon editing. For this purpose, approximately 144 ACE-tRNAs were candidates to be tested for ACE-tRNAs that can be used to promote the repair of disease-causing nonsense codons and the expression of full-length proteins. Specifically, using the scheme described in Figure 11, we constructed tRNA libraries to identify novel tRNA sequences encoding ACE-tRNAs capable of repairing the higher-level CF-causing nonsense mutations. Specifically, as shown in Figure 11, 10 ng of annealed oligonucleotides encoding ACE-tRNA were cycled in a thermocycler with 50 ng of NanoLuc reporter plasmid, 1 μL of 10xCutSmart Buffer (NEB), 1 μL of T4 ligase (NEB), 10 mM ATP, and 1 μL of BbsI (NEB). 1 μL of the reaction mixture was used to transform transformation-receptive E. coli, and the transformants were seeded onto ampicillin agar plates. One transformant per plate was isolated and grown in 1 mL of LB under ampicillin selectivity, followed by miniprep and sequence confirmation.

[0221] To identify the best ACE-tRNA candidates from tryptophan and glycine, a screening study was first conducted. Along with ACE-tRNA, sequenced miniprep cDNA of 125 ng of NanoLuc reporter plasmid was transfused into HEK cells using calcium phosphate. The day before, 4 × 10⁶ cells were used. 4Nine HEK cells were plated in a 96-well plate. 24 hours after translocation, the medium was replaced with 20 μL of PBS and 15 μL of NanoGlo reagent (Promega) was added. The plate was read on a SpectraMax i3 (Molecular Devices). Data are 3 or more replicates. Figure 8. The data show that many tRNAs exhibit poor codon editing tolerance. However, clearly high-performance tRNAs emerged from the above screening study, and ACE-Trp and ACE-Gly tRNAs were identified that demonstrate 20- to 130-fold rescue of nonsense codon-containing proteins compared to the background.

[0222] To evaluate whether these novel tRNAs can rescue CFTR channels containing nonsense codons, they were co-expressed in mammalian HEK cells along with the CFTR W1282X cDNA plasmid. Cell preparations were analyzed by a standard biochemical approach via Western blotting of the CFTR protein. This method is highly advantageous for this purpose because the CFTR protein exhibits a well-established multiband pattern. Specifically, the "B" and "C" bands correspond to full-length and fully mature, post-translationally processed CFTR proteins on the cell surface, respectively. In this case, both rescues by Trpchr17.trna39 ACT-tRNA and Glychr19.trna2 ACT-tRNA produced robust populations of "B" and "C" CFTR immunopositive (antibody MA37) bands, indicating that these tRNAs facilitate the successful transport of full-length ion channel proteins. (Figure 9) [Example 3]

[0223] Modification of the t-stem significantly improves nonsense repression. Figure 10. Here, we propose further modification of tRNA to further confer function to tRNA for the purpose of repressing nonsense codons and promoting protein expression. This hypothesis is based on the possibility that mutations rationally introduced within the tRNA "t-stem" loop, as shown in Figure 10, result in a more stable and functionally more potent tRNA molecule with respect to nonsense codon repression. For this purpose, single and double mutations were directly manipulated in the t-stem of tRNA Trpchr17.trna39, an ACE-tRNA whose activity for rescue of tryptophan TGA nonsense codons has been identified. Thus, 38 tRNA t-stem variants were generated and screened in HEK cells transiently transfected with a nonsense rescue reporter construct shown in Figure 4. 24 hours after transfection, the cells were assayed for luciferase activity, as shown in Figure 10. The data identify novel tRNA sequences with altered t-stem-loop sequences exhibiting strong variability and enhanced inhibitory activity. In particular, one such variant, TS-38 52-62 GC, enhances the inhibitory capacity of Trpchr17.trna39 by approximately 250% (Figure 12). Therefore, we propose that this is a generalizable modification, i.e., the modifications of the novel tRNA sequences identified by Examples 1 and 2 can be further improved through rational modifications (in terms of their ability to rescue nonsense codons). Such an approach would support the therapeutic utility of ACE-tRNAs directed to tissue types with low abundance of target RNA or where tRNA delivery may be limited. [Example 4]

[0224] To enable the identification of nucleotide composition and the functional ability of novel types of tRNA to suppress nonsense codons, we invented an All-In-One Plasmid With A One-Pot Cloning Reaction for High Throughput Cloning, Figure 11. This approach allows for easy investigation of ACE-tRNA activity via luciferase activity in a standard 96-well format. Briefly, a synthetic nucleotide sequence encoding tRNA is ligated into a NanoLuc Reporter plasmid, and an example of a variant of the TGA nonsense reporter plasmid is shown in Figure 11. In Figures 16–19, TAA (Opal) and TAG (Amber) stop codon rescue vectors were successfully designed and implemented. The advantage of this approach is that the DNA oligo encoding the tRNA library can be ligated into the NanoLuc reporter plasmid in the presence of restriction enzymes and ligases, and the reaction proceeds to nearly 100% tRNA insert incorporation (Figure 11), hence the term "one-pot." This reaction product is transformed into E. coli, and the resulting cDNA is purified by standard methods. Another advantage of the invented method is that the tRNA and reporter gene (gen) reside in a single expression cassette, thus reducing biological variability and improving the quality of data obtained in screening for tRNA repressive activity. The purified cDNA plasmid is then screened in a high-throughput 96-well format for its ability to repair nonsense codons by presumed luciferase activity. This approach is suitable for high-throughput screening of hundreds to thousands of tRNAs for novel therapeutic activity.

[0225] The "one-pot" cloning and expression system described in Figure 11 was successfully used to identify specific tRNA sequences for the repair of tryptophan and glycine ACE-tRNA (Figure 13), ACE-tRNA-Arg (Figure 14), ACE-tRNA-Gln TAG (Figure 15), ACE-tRNA-Gln TAA (Figure 16), ACE-tRNA-Glu TAG (Figure 17), ACE-tRNA-Gln TAA (Figure 18), and ACE-tRNA-Trp TAG (Figure 19). Figures 20A–20D show that delivery of ACE-tRNA as small RNA supports robust suppression of G542X and W1282X nonsense mutations. [Example 5] Manipulated transfer RNA for suppression of immature termination codons summary

[0226] Immature termination codons (PTCs) are responsible for 10–15% of all genetic disorders. Intertranslational PTC suppression offers a promising approach to treat various genetic disorders, but small molecules that promote PTC readthrough have given inconsistent results in clinical trials. Anticodon-engineered molecules that can effectively suppress in-frame PTCs and faithfully encode their homologous amino acids ( a nti c odon e A cell-based, high-throughput assay is provided for identifying ngineered (transfer RNA) (ACE-tRNA). Collectively, ACE-tRNAs with high repressive activity targeting nonsense codons that cause the most common human diseases were identified. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNAs at PTC repair levels indicates limited interaction with translation termination codons. These ACE-tRNAs exhibit high repressive activity in mammalian cells, Xenopus oocytes, and mice in vivo, resulting in PTC repair in multiple genes, including disease-causing mutations within the cystic fibrosis membrane conductance regulator (CFTR). Introduction

[0227] Immature termination codons (PTCs) arise from mononucleotide mutations that convert a regular triplet nucleotide codon into one of three stop codons, such as TAG, TGA, or TAA. PTCs are often more detrimental than missense mutations because they result in loss of protein expression. Furthermore, mRNA abundance is reduced through nonsense mutation-dependent degradation (NMD), and in some cases, the truncated protein may have dominant-negative function. 1-3 Therefore, PTC is a type of cystic fibrosis. 4 Duchenne muscular dystrophy, spinal muscular atrophy 5 Infantile neuronal ceroid lipofuscinosis 6 β-thalassemia 7 cystinosis 8 , X-linked nephrogenic diabetes insipidus 9 Hurler syndrome 10 Usher syndrome 11 It is not surprising that this is associated with many severe disease phenotypes, including polycystic kidney disease. Furthermore, nonsense mutations are associated with the tumor suppressor genes p53 and ATM 12 This occurs within the body and further suggests the role of nonsense mutations in the disease. The amino acid codons most susceptible to PTC conversion are those with a single nucleotide substitution from a stop codon: tryptophan, tyrosine, cysteine, glutamic acid, lysine, glutamine, serine, leucine, arginine, and glycine (Figure 25). Therefore, PTCs are a distinct group of diseases affecting more than 30 million people worldwide and account for 10–15% of all genetic disorders. 13 .

[0228] aminoglycosides 14 , dipeptide 15 and oxadiazole 16 These small molecules promote the "readthrough" or "suppression" of nonsense mutations. These compounds are used in model organisms. 17、18 mammalian cell lines 19and several animal disease models 16、20 This is effective in that regard. However, this approach results in encoding of closely related amino acids. 21 Furthermore, it can effectively induce missense mutations in PTCs, which themselves can have detrimental effects on protein folding, transport, and function. In addition, aminoglycosides have ototoxic and nephrotoxic effects. 22 The groundbreaking oxadiazole drug ataluren showed unexpectedly low efficacy in the patient population (ACT DMD Phase 3 clinical trial, NCT01826487; ACT CF, NCT02139306), thus limiting its usefulness as a treatment for PTC. Recent ongoing advances in CRISPR / Cas9-mediated genome editing potentially offer a permanent solution to diseases caused by nonsense mutations. However, aspects of this technology hinder its early use as a therapeutic agent. 23、24 This is not limited to the requirement of “precision” or “personalized” diagnostics for each variant based on the circumstances of each patient’s genetic variability.

[0229] PTC repair approaches demonstrating the versatility of small molecules and the precision of gene editing were identified. To satisfy these criteria, tRNAs in which the anticodon was manipulated via mutagenesis to recognize and repress UGA, UAA, or UAG PTC codons were examined. For effectiveness, anticodon-edited tRNA, also known as ACE-tRNA, must be an endogenous translational cellular mechanism containing aminoacyl-tRNA synthetase to introduce its homologous amino acids into the ACE-tRNA and eukaryotic elongation factor 1a (eEF-1α) to deliver the introduced tRNA to the ribosome. Figure 21A, which should still be recognized by cellular machinery. Such suppressor tRNAs are limited in their mode of action against β-thalassemia. 25 , xeroderma pigmentosum 26 and transgenic PTC reporter gene 27It has been shown to rescue in-frame stop codons associated with this.

[0230] This example demonstrates that the anticodon editing approach is generalizable to multiple tRNA gene families, suggesting that many annotated tRNAs are biologically viable. Furthermore, it is demonstrated that anticodon-edited suppressor tRNAs encode their homologous amino acids, lack significant interaction with termination codons, and are effective in suppressing PTCs in vivo. Overall, these data support the possibility that such manipulated tRNAs meet a broad range of requirements for covering disease-causing PTCs, thus representing a promising new class of RNA therapeutics. result

[0231] The theoretical basis for this study is the observation that there are multiple tRNA genes (isodecoders) that have specific sequences for certain homologous amine acids (isoacceptors), and more than 400 tRNAs have been annotated in the human genome (http:lowelab.ucsc.edu / GtRNAdb / ). 28,29 First, to identify individual ACE-tRNAs that retain PTC repressive efficacy in mammalian cells, we examined tRNA genes. To maximize the covered sequences, we constructed an all-in-one cDNA plasmid that supports both high-throughput cloning (HTC) of ACE-tRNAs and quantitative measurement of PTC repression using luminescence after delivery to mammalian cells, Figure 21B. ccdB negative selection 31 Golden Gate cloning combined with 30Using this method, ACE-tRNA sequences were cloned as DNA oligos into HTC plasmids. This strategy yielded approximately 100% cloning efficiency. ACE-tRNA suppression efficiency was read from another NanoLuc luciferase (NLuc) NanoBiT platform, which uses a 96-well format to introduce the target PTC (UGA, UAA, or UAG) in-frame at the junction between the large bit domain and the small bit domain, as shown in Figure 21B. 32 The results were normalized to a background obtained in cells expressing NLuc-PTC. First, 21 glycine ACE-tRNAs were evaluated for their role in suppressing UGA PTC, Figure 22, upper left, column 1 (violet). ACE-tRNA Gly Most of the sequences failed to suppress UGA NLuc PTC, but three Gly-tRNAs showed high suppression yields (approximately 100 times compared to the background). UGA Given the high sequence conservation among the Gly-tRNAs screened for anticodon tolerance (Figure 27), it will be difficult to novelly predict which tRNA is best suited for anticodon editing.

[0232] Next, the screening was performed on codon-edited tRNAs for each of the following single nucleotide variants that could potentially produce disease-causing PTCs: 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 UAGLys-tRNA UGA and Ser-tRNA UAG The enzymatic activity of NLuc was not significantly affected by the introduced amino acid (Figure 28), and therefore, the difference in NLuc luminescence is due to ACE-tRNA repressive ability. Screening identified multiple ACE-tRNAs for each amino acid and stop codon species, and repression was covered for all three stop codons (Figure 22). Many of these ACE-tRNAs showed strong activity, with PTC repression exceeding 100-fold compared to the background, which was significantly higher than that of the aminoglycosides used in this study. Interestingly, some ACE-tRNAs showed a clear preference for specific anticodon editing, likely reflecting the binding of altered aminoacyl-tRNA synthetase to the tRNA anticodon isoacceptor sequence. 33 For example, the conversion of tryptophan to UAG repression is the same ACE-tRNA Trp This resulted in a tenfold increase in rescue compared to UGA editing. However, the opposite was true for glutamine, which showed a clear preference for UAA over UAG. In particular, multiple high-performance suppressors were identified in each case, which is a PTC that plays a very large role in human diseases, Arg UGA This is particularly evident in the following, and here we present 20 efficient ACE-Arg UGA A suppressor was identified. In other cases, ACE-tRNA was identified. Glu Among those that demonstrated function, the repression efficiency was roughly the same for UAA and UAG. ACE-tRNAs that encoded very similarly via UAG or UGA repression. Lys A similar pattern was found in Gln-tRNA. UAA Regarding this, the inhibitory activity resulted in an inhibitory signal 2,000 times stronger than the background. Among the ACE-tRNAs identified in the screening, the tryptophan-tRNA gene family showed the weakest inhibitory activity against UGA PTC. Unique human ACE-tRNAs TrpSince only six sequences were available for screening, we used tRNAs obtained from diverse species to create UGA-repressive ACE-tRNAs. Trp The library has been expanded. Miscoded A9C tRNA. Trp In addition to the bacterial Hirsh Trp suppressor, UGA anticodon editing tolerance was tested for tryptophan tRNA genes with unique sequences obtained from yeast, flies, mice, rats, rabbits, and frogs. 34,36 Figures 29A-29B. This attempt aims to surpass the repressive activity of human ACE Trp tRNA in ACE-tRNA Trp Figure 29C failed to identify UGA PTC inhibitory activity. Overall, tRNA screening identified multiple manipulated tRNAs (for each amino acid and stop codon type) that showed potent inhibitory activity, thus demonstrating general tolerance to anticodon editing.

[0233] Next, we determined whether the ACE-tRNAs identified by screening were functionalized at the expense of the rigor of aminoacylation by congener aminoacyl-tRNA synthetases. For this purpose, we used mass spectrometry to examine PTC repression in the model soluble protein, histidinol dehydrogenase (HDH), Figure 23A. The TGA codon was introduced into asparagine 94 (N94) (Figures 30A-C), and the most effective glycine and tryptophan ACE-tRNAs were identified, respectively. UGAThe plasmids encoding Glychr19.trna2 or Trpchr17.trna39 ACE-tRNAs were co-expressed in tandem in HEK293 cells. The resulting full-length repressed HDH protein was purified via a Strep-Tactin® C-terminal affinity tag and analyzed by mass spectrometry, Figure 23A (Figure 28). Subsequent data analysis identified modifications of Asn to Trp (+72Da) for Trpchr17.trna39 and (-57Da) for Glychr19.trna2, confirming faithful encoding of congeneral amino acids for each ACE-tRNA type. Importantly, in each case, over 98% of the peptides identified at the HDH p.N94X site encoded congeneral tryptophan and glycine. Furthermore, both ACE-tRNAs exhibited superior selectivity for the UGA stop codon compared to UAA and UAG, as shown in Figure 23B (ACE-tRNA). Gly ) and Figure 31 (ACE-tRNA Trp ). Finally, when transiently expressed, ACE-tRNA Gly It was superior to the conventional small molecule suppressors gentamicin (40 μM) and G418 (140 μM) in its ability to suppress NLuc-UGA stably expressed in HEK293 cells, Figure 23C. ACE-tRNA had lower suppression efficiency. Trp The same was true for the G418, where PTC rescue was superior, as shown in Figures 33A-D.

[0234] The question arose as to whether ACE-tRNAs that effectively suppress immature stop codons could also broadly induce readthrough of native stop codons. To address this potential "off-target" suppression, quantitative profiles of ribosomes actively involved in all cellular transcripts were obtained across the entire transcriptome by constructing ribosome footprint libraries from HEK293 cells expressing exogenous ACE-tRNA or a control pseudoplasmid (puc57GG). Streptomycin was removed from the growth medium to prevent artificial influence of readthrough. For comparison, ribosome footprint libraries were also constructed from cells (150 μM, 48 hours) in and out of the presence of G418. Figure 24A shows the ribosome footprint density of G418 and five ACE-tRNAs compared to the control on the 3'UTR region (log-2 multiplier change). In the two replication libraries, only transcripts with a minimum threshold of 5 RPKM for the coding sequence and 0.5 RPKM for the 3'UTR were included for quantitative comparison (254 transcripts for G418 and 495-748 transcripts for ACE-tRNA). In this system, G418 had no observable effect on the 3'UTR ribosome density across the entire transcriptome for any of the three endogenous stop codon groups. With the exception of ACE-tRNA Gln-UAA and Arg-UGA, which induced approximately a twofold increase in 3'UTR ribosome density for complementary congeneral stop codons to the ACE-tRNA anticodon, the ACE-tRNAs examined in this example did not show any detectable changes in 3'UTR ribosome density. Further research is needed to understand the biological significance of twofold readthrough of protein termination, but this effect is substantially lower compared to the 100- to 1000-fold repression of PTC for the same ACE-tRNA.

[0235] Multiple in-frame stop codons are often found at the ends of genes. 37-39ACE-tRNA and G418 treatment may induce slight differences in overall 3'UTR ribosome density. Ribosome occupation was examined at each nucleotide in the 3'UTR within the 60-nucleotide region downstream of the stop codon. Figure 24B demonstrates ribosome occupation around the native stop codon for each nucleotide within the region from -35 to +65 nt relative to the first nucleotide of the stop codon. Reads were normalized to a total of 1 million mapped reads and compared to control cells, and reported as log2 multiplier changes as Panel A. Over 5,200 transcripts were mapped to at least one footprint in the region of interest. ACE-tRNA Gln-UAA and Arg-UGA not only showed a significant increase in ribosome occupation in the initial region but also exhibited characteristic 3-nucleotide periodicity, indicating that ribosomes followed codon-by-codon movement rather than being randomly distributed. ACE-tRNA or G418 against UGA-Trp, UGA-Gly, and UAG-Glu consistently showed no observable changes in ribosome occupancy, even in the early region of the 3'UTR. Taken together, ribosome profiling data indicate that the efficiency of innate stop codon repression by ACE-tRNA is generally low, significantly lower than the level of PTC repression. Consideration

[0236] PTCs cause numerous human diseases, and there are no established therapeutic options for managing them. High-throughput cloning, identification, characterization, and functional analysis of anticodon-edited tRNAs exhibiting effective PTC reversal in eukaryotic cells and mouse skeletal muscle are reported in this embodiment. In particular, the screening as a whole identifies ACE-tRNAs capable of repairing the majority of known human disease-causing PTCs. The manipulated tRNAs faithfully encode their homologous amino acids, thus eliminating any spurious effects on downstream protein stability, folding, and transport, and consequently negating the need for tandem therapies involving protein folding or transport factors. Transmuted as cDNA, ACE-tRNAs rescued multiple full-length proteins, an NLuc luciferase reporter, and two disease nonsense mutations in the model proteins HDH and CFTR via PTC repression. Arg cDNA demonstrated potent and stable in vivo suppression of PTC in mouse skeletal muscle, suggesting particularly high levels of cellular tolerance to ACE-tRNA activity. Identifying ACE-tRNA active against arginine in muscle is promising for treating dystrophin disorders caused by nonsense mutations. As with many genetic disorders, over 10% of dystrophin disorders are caused by nonsense mutations. 43 The CGA→TGA mutation is the most common. 43 Efficient repression can also be achieved by ACE-tRNA delivered as synthetic RNA transcripts, enabling the development of nanoparticle formulations. Further research is needed to evaluate the ideal tRNA delivery strategy for each tissue and disease type, and the rapidly expanding technologies for nucleic acid delivery will likely be helpful in this endeavor.

[0237] Factors that suppress PTC also have the potential to produce read-through of native stop codons. RNA profiling data presented herein suggest that this is generally not true in cells and for the tested codon-editing tRNAs. Arg-tRNA UGA and Gln-tRNA UAA However, a detectable readthrough was found, but Glu-tRNA UAG UGA-Gly-tRNA UGA and Trp-tRNA UGA However, no significant effect was measured on broad-spectrum translation termination. This behavior was clearly not separated by the type of stop codon or the intrinsic PTC repressive activity of the tRNA. One potential reason why ACE-tRNA does not effectively facilitate true stop codon read-through may be due to the sequence landscape of the contextual area around the translation termination. 44 This possibility is supported by the finding that the composition of the termination complex in PTC differs from that in natural terminations. 45、46 However, when lower levels of readthrough occur, multiple cellular mechanisms are appropriately in place to constrain both normal termination readthrough and its harmful effects. Multiple in-frame termination codons are often found at the ends of genes. 37-39 Specialized ubiquitin ligauze 47 and ribosome-associated pathways 48 However, it is known to identify and degrade proteins with incorrect translation terminations. Nevertheless, despite the limited effects observed in this example in mammalian cells, similar ribosome profiling experiments should be performed in desired cell or tissue types regarding ACE-tRNA delivery and expression.

[0238] Previous studies have shown that the surrounding mRNA sequence affects the intrinsic stop codon repressive efficacy of aminoglycosides and atalurene PTCs. 49-52, ACE-tRNA can be similarly affected. Furthermore, gene addition strategies for replacing PTC-containing genes via viral delivery or non-viral delivery have achieved short-term benefits in some situations, but it can be difficult to control the expression levels of transgenes. In contrast, the amount of protein rescue via ACE-tRNA suppression is related to the endogenous cellular RNA levels, and thus, higher levels of expression are essentially controlled. The biological purpose remains unknown for most of the variable isoacceptor tRNA sequences in the human genome, and nearly half of these genes have been speculated to be transcriptionally silent pseudogenes 53 , and the individually presented data 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 in this example further demonstrate that most tRNA gene sequences support viable activity when removed from the genomic context, deepening the mystery regarding the biological necessity for multiple tRNAs and codon usage. Thus, the high-throughput suppression strategy described in this example is useful for identifying new types of tRNA sequences with unique suppression characteristics, and such research has the ability to advance the production of new RNA reagents as well as the molecular understanding of tRNA expression and suppression. Materials and Methods Nonsense Reporter HTC Plasmid

[0239] The parent plasmid used was pcDNA3.1(+). Gibson Assembled (New England Biolabs, USA) was performed on the cDNA encoding pNLuc within the HindIII and XhoI restriction sites. Glycine (codon gga), tryptophan (tgc), amber (tag), opal (tga), and ochre (taa) were added at amino acid position 160 between the cDNA pcr. Using a pcr-based Gibson Assembly, the pcDNA3.1(+) polyA sequence was replaced with one without the BbsI restriction site. First, two BbsI restriction sites (bold italicized) were then added. [ka] The following is the T7 promoter sequence (italicized): [ka] Human tRNA with (Ye et al., 2008) upstream Tyr The 5' leader sequence of the gene (bold) followed by the reverse T3 primer sequence (italicized) and the 3' termination sequence (bold). [ka] By inserting this, a high-throughput ACE-tRNA Golden Gate cloning site was created. HTC in the ACE-tRNA library

[0240] tRNA gene sequences were obtained from the tRNA database, tRNAscan-SE (http: / / gtrnadb.ucsc.edu / index.html; PMID:26673694). All tRNA gene sequences used in this study are numbered in Figure 26 and Table 9. The tRNA sequences were synthesized as complementary ultramers obtained from Integrated DNA Technologies (IDT, USA) in a 200 pmol scale, 96-well format, with appropriate mutations in their corresponding anticodons (UAG, UGA, or UAA). All tRNA sequences were synthesized to have CGAC and GGAC overhangs (denoted as 5'→3') on forward and reverse oligos, respectively. Ultramers were annealed by resuspending them in annealing buffer (100 mM potassium acetate; 30 mM HEPES, pH 7.5) to a concentration of 100 ng / μL, heated to 96°C for 2 minutes, and cooled to 4°C at 1°C / min in a thermocycler. In a 96-well PCR plate, each well contained 10 ng of HTC plasmid with appropriate PTC codons, 2 ng of ACE-tRNA double-stranded plasmid, and 1 mM ATP, 10 mM DTT, 400 units of T4 DNA ligase, and 10 units of BbsI-HF were arranged in ddH2O in 10 μL (queued) portions. The 96-well plates were cycled in a thermocycler as follows: ([5 min at 37°C, 5 min at 20°C] × 30 cycles, 10 min at 37°C, 10 min at 80°C, and cooled to 4°C. In a deep-well 96-well plate, 10 μL of chemically transformable DH5α cells (ThermoFisher, USA) were added to 1 μL of Golden Gate reaction mixture, and a heat shock was applied at 42°C for 30 seconds, followed by 100 μL of Super Optimal The cells were resuspended in Broth (SOC; Thermofisher, USA). Transformants were grown at 37°C for 1 hour at 250 rpm, then added to 2 mL of Luria-Bertani liquid medium (LB) supplemented with 100 μg / mL carbenicillin, and grown in a covered deep 48-well plate at 37°C for 20 hours at 300 rpm. E. coli growth was performed in deep-well plates and in Enzyscreen (http: / / www.enzyscreen.com) clamps. The E. coli suspension cultures were allowed to settle (10 minutes at room temperature, 4,000 g), plasmid DNA was prepared, and diluted to 125 ng / μL (IBI Scientific, USA). All clones were sequenced. 100% cloning efficiency was achieved using this method. HTS of ACE-tRNA libraries

[0241] On the day before translocation, HEK293 cells (less than 40 passages) were placed in 96-well cell culture plates treated with Dulbecco's Modified Basic Medium (DMEM) (Thermofisher, USA) supplemented with 10% FBS, 1% Pen / Step, and 2 mM L-glutamine, in a 1.4 × 10⁶ well. 4Cells were seeded per well. Three all-in-one nonsense reporters containing the ACE-tRNA gene were transfused per plate using Calfectin (Signagen, USA). Sixteen hours after transfusion, the culture medium was aspirated and 20 μL of PBS was added to each well. 15 μL of soluble Nano-Glo® Luciferase Assay Reagent was added to each well (1:50). Reagent versus buffer (Promega, USA). After rotational shaking, plates were incubated for 2 minutes and read using a SpectraMax i3 plate reader (Molecular Devices, USA; integration time, 200 msec; all wavelengths were collected in endpoint mode). For each experiment, luminescence was averaged across three wells, and all ACE-tRNAs were repeated more than three times in this manner. Each plate also contained three wells transfected with an all-in-one nonsense reporter without ACE-tRNA to serve as a control for translocation efficiency and baseline PTC readthrough. All values ​​are reported as the ratio of ACE-tRNA luminescence above baseline PTC readthrough luminescence ± SEM. One-way ANOVA was performed on all ACE-tRNAs in a given amino acid family using Tukey's post-hoc analysis. CFTR, HDH-his-strep, and 4×ACE-tRNA expression plasmids

[0242] For expression in mammalian cells, cDNA for the coding region and 200 base pairs of the 3' untranslated region (UTR) of human CFTR was ligated into pcDNA3.1(+) (Promega, USA) using KpnI and XbaI restriction enzymes. The G542tga and W1282tga mutations were introduced using QuickChange XL II (Stratagene, USA). For expression in African clawed frog (Xenopus laevis) oocytes, cDNA for the coding region and the 140 base pairs of the 5' and 244 base pairs of the 3'UTR of human CFTR was ligated into pGEM-HE (Promega, USA). Both the G542tga and W1282tga mutations were introduced using QuickChange XL II. cDNA encoding *E. coli* histidinol dehydrogenase was codon-optimized for *Mus musculus* and synthesized with a c-terminal 8×His-Strep-tag for protein purification from mammalian cells (BioBasic Inc, Canada). The synthesized cDNA was ligated into pcDNA3.1(+) using EcoRI and XhoI restriction sites. Nonsense mutant tags, taa and tga, were introduced using QuickChange XL II. To construct a multiplexed ACE-tRNA expression plasmid, a BbsI "multiplication site" was added between the EcoRI and HindIII restriction sites. [ka] A novel parent Golden Gate pUC57(amp) plasmid was constructed by inserting the directional BbsI recognition sequence (in italics) and the unique 4-base pair overhang for ligation (in bold). pUC57(amp) was selected as the parent plasmid due to its relatively small size and lack of the skeletal BbsI restriction site and T7 and T3 promoter sequences. Features present in the HTS plasmid include the T7 and T3 promoter sequences adjacent to the ACE-tRNA cassette, and an ideal equivalent melting temperature (T) for PCR amplification. mThis provides a universal primer-binding sequence having ). Using the NEB Golden Gate Assembly Tool (https: / / goldengate.neb.com / editor), PCR primers annealing to T7 and T3 flanking sequences were constructed, and after cleaving the distal BbsI recognition sequence, a unique 4-base pair overhang was created. The final result was the production of four ACE-tRNA PCR products using universal PCR primers that could be "daisy-chained" through complementary 4-base pair overhangs and ligated into the puc57 Golden Gate plasmid using a one-pot Golden Gate reaction. All clones were sequenced. Cell culture, protein expression, and Western blotting

[0243] HEK293T cells (ATCC, USA) were grown at 37°C and 5% CO2 in a standard growth medium (v / v) containing 10% FBS (HiClone, USA), 1% Pen Strep, and 1% L-Glut in high-glucose DMEM (Gibco, USA). cDNA was transfused at a concentration of 75% using Calfectin according to a standard protocol (SignaGen Laboratories, USA). After 36 hours, the cells were scraped and pelleted 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, and 0.75 mM benzamidine. For CFTR-expressing cells, the cell pellet was vigorously down-homogenized (dounced) 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, and 50 mM Tris-HCl pH 7.4, and centrifuged at 100,000 g to separate the entire membrane from soluble cytoplasmic proteins. The pellet was solubilized 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, and 0.75 mM benzamidine. Equal cell lysates were loaded onto 3-15% separation gradient SDS-pages with 4% stacking gels in the presence of 1% 2-mercaptoethanol, separated at 55V O / N, and 0.45 μM LF. Cells were transferred to PVDF (Bio-Rad, USA). PVDF was immunoblotted using the anti-CFTR antibody M3A7 (1:1000; Millipore, USA) in 2% skim milk and imaged on the LI-COR Odyssey Imaging System (LI-COR, USA). For HDH-His-Strep-expressing cells, the cell pellet was vigorously downhomogenized 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. The lysates were centrifuged at 100,000 g for 30 minutes at 4°C. The supernatant (soluble cellular proteins) was separated on a 4-12% Bis-Tris SDS-page acrylamide gel (ThermoFisher, USA) in the presence of 1% 2-mercaptoethanol, transferred to 0.22 μM LF PVDF (Bio-Rad, USA), immunoblotting was performed using anti-Strep antibody (1:5000; iba, Germany) in 2% skim milk, and imaging was performed on the LI-COR Odyssey Imaging System (LI-COR, USA). mass spectrometry

[0244] Fragmentation data for purified HDH-His-Strep protein were obtained at the University of Iowa Proteomics Facility. Briefly, HDH-His-Strep protein obtained from the fast-spin soluble fraction was passed through a Strep Trap 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. The protein was eluted with a wash buffer supplemented with 10 mM d-desthobiotin and concentrated in a 30 kDA cutoff Amicon-Ultrafiltration column (Millipore, USA). The concentrated protein was loaded onto a NuPage 4-12% Bis-Tris precast gel (Invitrogen, USA) and separated at 150V for 1.5 hours. The gel was stained using a Pierce mass spectrometry-compatible silver staining kit (ThermoFisher Scientific, USA).

[0245] Intragel trypsin digestion. Briefly, the targeted protein band is manually cut out from the SDS-PAGE gel, and then 1 mm 3The gels were cut into 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 destaining. Further treatment of the gel pieces with ACN was performed, and they were dried using a speed vac. After drying, the gel pieces were reduced in 50 μL of 10 mM DTT at 56°C for 60 minutes, and then alkylated with 55 mM IAM at room temperature for 30 minutes. To remove excess DTT and IAM, the gel pieces were washed twice with 25 mM ammonium bicarbonate:acetonitrile (1:1, v / v). After drying, the gel pieces were placed on ice in 50 μL of 10 ng / μL trypsin solution in 25 mM ammonium bicarbonate and incubated on ice for 60 minutes. Digestion was then performed at 37°C for 16 hours. Peptide extraction was performed twice using 100 μL of 50% acetonitrile / 0.2% formic acid for 0.5 hours each. The combined extract was concentrated to approximately 15 μL in a Speed ​​Vac.

[0246] LC-MS / MS. Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) connected to an Eksigent Ekspert® nanoLC 425 System (Sciex). A Trap-Elute Jumper Chip (product number: 800-00389) and a connected 1 / 16'' 10-port Valco directed loading (by gradient 1 pump) and final elution (by gradient 2 pump). The column assembly was designed as two tandem 75 μm × 15 cm columns (ChromXP C18-CL, 3 μm 120A, Eksigent portion of AB SCIEX) mounted in an Ekspert® cHiPLC system. Approximately 0.5 μg of total digestate was loaded for each injection. The peptides were separated in-line by mass spectrometry using a 120-minute gradient consisting of linear and steady-state segments, with buffer A being 0.1% formic acid and buffer B being 95% ACN and 0.1% formic acid. The gradient was first maintained at 4% for 3 minutes, followed by the following transitions (%B, min): (26, 48), (35, 58), (35, 64), (50, 72), (50, 78), (94, 84), (94, 96), (4, 100), (4, 120).

[0247] The tandem mass spectrometry scan sequence on the LUMOS Orbitrap began with a full survey (m / z 350-1500) acquired on an Orbitrap Fusion Lumos mass spectrometer (Thermo) at a resolution of 60,000 in the off-axis Orbitrap segment (MS1). Gradient MS1 scans were acquired every 3 seconds during the 120-minute gradient. The most abundant precursor was selected from 2-8 charged state ions at a threshold of 2.0E5. If an ion was targeted twice in the previous 30 seconds, it was dynamically excluded for 30 seconds. The selected ions were isolated by a multi-segment quadrupole with a mass window at m / z 2 and then sequentially subjected to both CID and HCD activation conditions in IT and ion routing multipoles, respectively. The AGC target for CID was 4.0E04, with a collision energy of 35%, an activation Q of 0.25, and a maximum filling time of 100 ms. The targeted precursor was also fragmented by high-energy collision-induced dissociation (HCD) at a collision energy of 40% and a Q of 0.25. HCD fragment ions were analyzed using Orbitrap (AGC 1.2E05, maximum injection time 110 ms, and resolution set to 30,000 at 400 Th). Both MS2 channels were recorded as centroids, and the MS1 survey scan was recorded in profile mode.

[0248] Proteomics search. The initial spectral search was performed by Proteome Discoverer version 2.1.1.21 (ThermoFisher Scientific, USA) using Sequest HT. The spectra were also searched by the Byonic search engine (Protein Metrics) version 2.8.2. The search databases consisted of Uniprot KB for species 9606 (human) downloaded on October 24, 2016 containing 92645 sequences and UniprotKB for taxonomy 562 (Escherichia coli) downloaded on November 8, 2016 containing 10079 sequences. For the Byonic search, these two databases were concatenated directly. In both searches, an equal number of decoy inputs were created and searched simultaneously by reversing the original inputs in the Target database.

[0249] In vitro cRNA transcription. G542X UGA , W1282X UGA and WT CFTR pGEMHE (Mense et al., 2006; PMID: 1703051) plasmids were linearized with a 10-fold excess of NheI-HF restriction enzyme (site located 3’ of the coding region) (New England Biolabs, USA) for 3 hours at 37°C and purified using standard cDNA precipitation methods. All cRNA was transcribed using the mMessage mMachine T7 kit (ThermoFisher Scientific, USA). Purification of cRNA from the transcription reaction was performed on columns obtained from the RNeasy Mini Kit (Qiagen, Germany). The concentration was determined by absorbance measurement at 260 nm and the quality was confirmed on a 1% agarose gel (RNase-free). All cRNA was queued at 1 μg / mL prior to use and all results were obtained from two or more cRNA preparations.

[0250] In vitro tRNA transcription. Using the CellScript T7-Scribe Standard RNA IVT Kit (CELLSCRIPT, USA), the best-performing Trp and Gly ACE-tRNAs, Trpchr17.trna39 and Glychr19.trna2, were transcribed in vitro. Equimolar concentrations of T7 oligonucleotides (5'-taatacgactcactata-3') were annealed to ACE-tRNA PAGE-purified Ultramer (20 μg; Integrated DNA Technologies, Coralville, IA) encoding the ACE-tRNA and preceded by a T7 promoter (italicized). Importantly, three terminal nucleotides containing CCA were included (bold). [ka]

[0251] The total reaction volume was adjusted to 100 μL, and the kit reagents were added in the following amounts: 10 μL of 10X T7-Scribe transcription buffer, 7.5 μL of each nucleotide (100 mM stock solution), 10 μL of 100 mM dithiothreitol, 2.5 μL of ScriptGuard RNase inhibitor, and 10 μL of T7-Scribe enzyme solution. After incubating the reaction mixture at 37°C for 4-5 hours, the DNA template was digested with 5 μL of DNase (1 U / μL) provided with the kit for 30-60 minutes. ACE-tRNA was extracted from the reaction mixture using acidic phenol-chloroform (5:1, pH 4.5) and precipitated with ethanol. The precipitated ACE-tRNA was pelleted, washed, dried, resuspended in 100 μL of DEPC-treated water, and further purified using a Chroma Spin-30 column (Clontech, USA). This procedure yielded approximately 100 μL of about 5 μg / μL ACE-tRNA. The ACE-tRNA was again pelletized in 20 μg aliquots, washed, lyophilized, and stored at -80°C until use. All results were obtained from two or more ACE-tRNA preparations.

[0252] Preparation of Ribosome Footprint Profiling Library: HEK293 cells transiently transfected with ACE-tRNA and control plasmid (puc57GG) were grown in standard growth medium for 48 hours in the absence of Pen-Strep. With minor modifications, as described... 55 The library was prepared. Briefly, the cells were rapidly cooled by adding ice-cold PBS and lysed in 25 ml of lysis buffer (20 mM Tris-HCl / pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% (v / v) Triton X-100). -1 Dissolve in Turbo DNase I) on ice for 10 minutes, then tritulate by passing a 26G needle 10 times. Remove by centrifugation at 16,000g at 4°C for 10 minutes, followed by 200U of RiboLock. Before adding the RNase inhibitor (Thermo Scientific), gently stir at room temperature for 45 minutes. 260 The lysate was digested using 100 U of RNase I (Ambion, USA) per lysate. Then, it was digested in 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). -1Ribosome-protected mRNA fragments were isolated by placing the lysate on a 1M sucrose cushion prepared in RiboLock 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 denatured 12% polyacrylamide gel containing 8M urea. RNA fragments with sizes ranging from 26 to 34 nt were manually excised from the gel stained with SYBR Gold (Invitrogen) and isolated to prepare a library of ribosome-protected fragments. Contaminating rRNA fragments were depleted using the Ribo-Zero kit (Illumina). Secondary rRNA depletion using 3' oligonucleotide adapter ligation, reverse transcription, cyclization, and biotinylated rRNA depletion oligos (Table 9) was performed as described. 55 During PCR amplification, barcodes were affixed to the library using index primers for each sample. The barcoded libraries were then pooled with 3% PhiX (Illumina) and sequenced in an Illumina NextSeq500 according to the manufacturer's protocol to typically generate 18 to 27 million reads per sample.

[0253] Ribosome footprint data analysis. HISAT2.0.3 is used to remove rRNA contaminants from the read data. 56First, data files for each barcoded sample (with the 3' adapter sequence removed) were mapped to four rRNA sequences (RNA5S1;NR_023363, RNA5-8SN5;NR_003285, RNA18SN5;NR_003286, and RNA28SN5;NR_003287). The remaining read information was aligned to the sense strand of the longest transcript variant of each human gene (UCSC RefSeq GRCh38). Transcripts with a 3'UTR length of at least 75 nt (18,101 sequences) were used for sequence analysis. A maximum of two mismatches at the 5' end of the read information was tolerated. All multiple-mapped read information was discarded. Fragment read information with a length of 26–34 nt was defined as a ribosome footprint and used for analysis. The 5' terminal nucleotides from each footprint were annotated and mapped onto each transcript. The location of the ribosome A region occupying the 16th to 18th nucleotides in each footprint 57,58This was used to infer the position of ribosomes on each transcript. RPKM (footprint reads per kilobase of transcript per total million-mapped reads) was calculated for each individual transcript (18,101 sequences). In the two replicated libraries, only transcripts with a minimum threshold of 5 RPKM for coding sequences and 0.5 RPKM for the 3'UTR region (254 transcripts for G418 and 495-748 transcripts for ACE-tRNA) were included for the analysis in Figure 24A. For the metagene plot of the entire transcriptome in Figure 2B, the footprint count for each nucleotide within the region of -35 to +65 nt relative to the first nucleotide of a stop codon was normalized to a total of 1 million mapped reads. All transcripts (18,101 sequences) were used for mapping, and over 5,200 transcripts were mapped to at least one footprint in the region of interest. Next, we investigated the in vivo bioactivity of the ACE-tRNAs Glychr19.trna2 and Trpchr17.trna39 in saving PTCs. Sequence determination data were obtained from the Galaxy platform. 59 The analysis was performed using [tool name]. The graphs were created using Prism 7 (GraphPad Software).

[0254] Construction of a stable NLuc reporter cell line. Using Gibson Assembly (New England Biolabs, USA), cDNA encoding pNLuc, which has tag, taa, and tga stop codons at amino acid position 160, was inserted into the AgeI and NotI restriction sites within the multiple cloning region of the retroviral vector pQCXIP (Clontech, USA). Calfectin (SignaGen PhoenixGP cells (PMID: 7690960) were co-transferred with pNLuc-STOP-pQCXIP and cmv-VSV-G (VSV-G envelope pseudotyping) plasmids using Laboratories (USA), and incubated in a CO2-controlled (5%) cell incubator at 33°C for 48 hours. A culture medium (20 mls) containing retrovirus particles was cooled to 4°C, centrifuged at 10,000 g to remove cell fragments, and filtered through a 0.45 μm MCE membrane syringe filter (Millipore, USA) onto two 10 cm dishes seeded with low-passage HEK293 cells at a concentration of 30%. The cell culture dishes were sealed with Parafilm, centrifuged at 3,500 g at 24°C for 90 minutes, and placed in a CO2-controlled (5%) cell incubator at 37°C. Cells were selected after 24 hours with puromycin (1 μg / mL) until the control dish (uninfected) showed complete cell death. Cells were monodispersed in 96-well plates using FACS, followed by monodispersion of the clonal population. To maintain the selected clones throughout the experimental procedure, puromycin was not used, and standard DMEM medium (DMEM-Dulbecco's modified Eagle medium - 10% FBS, 1% Pen / Step, and 2 mM L-glutamine supplemented; L-glutamine-supplemented high glucose; Thermofisher, USA) was used in all studies.

[0255] RNA translocation. HEK293 cells stably expressing pNLuc-UGA were transferred to 1.4 × 10⁶ wells of Dulbecco's Modified Basic Medium (DMEM) (Thermofisher, USA) in 96-well cell culture plates supplemented with 10% FBS, 1% Pen / Step, and 2 mM L-glutamine. 4Cells were seeded in wells. After 16–24 hours, ACE-tRNA was translocated into the cells using Lipofectamine 2000 (ThermoFisher Scientific, USA). Briefly, 3 μg of ACE-tRNA was suspended in 150 μL of OptiMEM, and 12 μL of Lipofectamine 2000 was mixed with 150 μL of OptiMEM. These volumes were combined, thoroughly mixed, and incubated at room temperature for 10 minutes. 75 μL of the translocation complex was added to each well. PTC repression by ACE-tRNA transcripts was quantified as described above.

[0256] Expression in African clawed frog (Xenopus laevis) oocytes. African clawed frog oocytes (stages V and VI) were purchased from Ecocyte(Austin, TX). Before injection, each ACE-tRNA pellet was resuspended in 2 μL of ddH2O, and the fragments were pelletized at 21,000 × g, 4°C for 25 minutes. To measure the dose-response of ACE-tRNA to CFTR channel rescue, serial dilutions of volume-balanced ACE-tRNA fragments were prepared with ddH2O (200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.562 ng / oocyte). In all experiments, 25 ng of CFTR cRNA was injected per oocyte, with an injection volume of 50 nL. ddH2O was used for background control experiments without ACE-tRNA. After injection, oocytes were preserved in OR-3 (50% Leibowitz medium, 250 mg / L gentamicin, 1 mM L-glutamine, 10 mM HEPES (pH 7.6)) at 18°C ​​for 36 hours.

[0257] Recording using the two-electrode voltage clamp (TEVC) method. CFTR Cl -Currents were recorded in ND96 tank solutions containing the following (in mM): 96NaCl, 2KCl, 1MgCl2, and 5HEPES (pH 7.5) in the presence of the maximum CFTR activation cocktail, forskolin (10 μM; adenylyl cyclase activator), and 3-isobutyl-1-methylxanthine (1 mM; phosphodiesterase inhibitor). Glass microelectrodes refilled with 3M KCl had resistances of 0.5–2 MΩ. Data were filtered at 1 kHz and digitized at 10 kHz using a Digidata 1322A adjusted with pClamp 9.2 software (Molecular Devices, USA). CFTR currents were induced using an OC-725C fixed-voltage amplifier (Warner Instruments, USA) with voltage steps of 5 mV from -60 to +35 mV. - Oocytes with a positive current inversion of -20mV were discarded. Clampfit9.2 software was used for current analysis. All values ​​are expressed as mean ± SEM.

[0258] Animal and in vivo imaging. Nu / J mice were purchased from Jackson Labs. Animal experiments were approved by the Wistar Institute's Animal Experimentation Committee (protocol number: 112762). Mice were treated by electroporation after being injected with 10-20 μg of DNA resuspended in 30 μL of water into the tibialis anterior muscle. Three mice were injected with either 10 μg of pNano-TGA + 10 μg of ArgACE-tRNA (right tibialis anterior muscle) or 10 μg of pNano-TGA + 10 μg of empty pUC57 (left tibialis anterior muscle). As controls, three other mice were injected with either 10 μg of pNano-WT (right tibialis anterior muscle; positive control) or water (left tibialis anterior muscle; negative control). DNA was prepared with 333 IU / mL of hyaluronidase (Sigma). One minute after DNA injection, electroporation was performed using a CELLECTRA 3P instrument (Inovio Pharmaceuticals). Nanoluciferase activity was imaged in mice by injecting 100 μL of flimazine (40-fold dilution of Nano-Glo substrate) into the peritoneal cavity. Five minutes after injection, the mice were imaged on an IVIS Spectrum (Perkin Elmer). Imaging was performed using an open filter, and images were acquired in 40 seconds. The images are Living Images. The analysis was performed using the Perkin Elmer software.

[0259] Table 9. Library of annotated tRNA sequences screened for PTC repressive activity. Italicized text next to each sequence indicates the anticodon editing site. Bold text indicates tRNAs with 5-fold higher repressive activity than the background. Note that in tRNAs, thymidine is replaced with uracil. [Table 9-1] [Table 9-2] [Table 9-3] Table 9-4 Table 9-5 Table 9-6 Table 9-7 Table 9-8 Table 9-9 Table 9-10 Table 9-11 Table 9-12 Table 9-13 Table 9-14 Table 9-15 Table 9-16 Table 9-17 Table 9-18 Table 9-19 Table 9-20 [Table 9-21] [Table 9-22] [Table 9-23] [Table 9-24] [Table 9-25] [Table 9-26] [Table 9-27]

[0260] [Example 5] References [ka] [ka] [ka] [ka] [ka]

[0261] While the above specification and examples fully disclose and enable the implementation of the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended herein.

[0262] All publications, patents, and patent applications are incorporated herein by reference. In the above specification, the present invention is described in relation to certain embodiments thereof, and many details are given for illustrative purposes, but it will be obvious to those skilled in the art that there is room for further embodiments of the present invention, and that some of the details given herein can be modified substantially without departing from the basic principles of the present invention.

[0263] The use of the terms “a,” “an,” “the,” and similar demonstrative pronouns in describing the present invention should be interpreted as encompassing both singular and plural forms, unless otherwise stated herein or unless clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-restrictive terms (i.e., “including, but not limited to”), unless otherwise stated herein. Unless otherwise stated herein, descriptions of value ranges herein are merely intended to serve as a simplified way of representing each individual value belonging to that range individually, and each individual value is incorporated herein as individually described herein. Unless otherwise stated herein or unless clearly inconsistent with the context, all methods described herein may be carried out in any suitable order. All examples or illustrative phrases provided herein (for example, the use of "such as") are intended merely to better illustrate the invention and, unless otherwise claimed, do not limit the scope of the invention. No phrase herein should be construed as indicating that any element not claimed is essential to the invention.

[0264] Embodiments of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. By reading the foregoing, variations of these embodiments may be obvious to those skilled in the art. The inventors anticipate that those skilled in the art will take advantage of such modifications as appropriate, and the inventors intend that the invention will be carried out in ways different from those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise stated herein, or unless it is clearly inconsistent with the context, any combination of the above elements in all possible variations thereof is encompassed by the invention. The present invention provides, for example, the following items. (Item 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 a nucleotide that interacts with elongation factor 1-α1 (EF1α). (Item 2) A modified transfer RNA (tRNA) consisting of one of sequence numbers 1 to 538, wherein thymidine is substituted with uracil. (Item 3) The modified transfer RNA (tRNA) described in item 2, consisting of sequence number 4, in which thymidine is replaced with uracil. (Item 4) The modified transfer RNA (tRNA) described in item 2, consisting of sequence number 16, in which thymidine is replaced with uracil. (Item 5) The modified transfer RNA (tRNA) described in item 2 consists of sequence number 24, in which thymidine is replaced with uracil. (Item 6) The modified transfer RNA (tRNA) described in item 2 consists of sequence number 33, in which thymidine is replaced with uracil. (Item 7) The modified transfer RNA (tRNA) described in item 2, consisting of sequence number 38, in which thymidine is replaced with uracil. (Item 8) The modified transfer RNA (tRNA) described in item 2 consists of sequence number 44, in which thymidine is replaced with uracil. (Item 9) The modified transfer RNA (tRNA) described in item 2 consists of sequence number 48, in which thymidine is replaced with uracil. (Item 10) The modified transfer RNA (tRNA) described in item 2 consists of sequence number 53, in which thymidine is replaced with uracil. (Item 11) Sequence numbers 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, 362, 365-370, 372-383, 388-3 A modified transfer RNA (tRNA) consisting of one of the following: 90, 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 substituted with uracil. (Item 12) A modified transfer RNA (tRNA) comprising a T arm, a D arm, an anticodon arm, and an acceptor arm, (a) The anticodon arm comprises a trinucleotide anticodon, the anticodon is 5'-UCA-3', and recognizes a TGA stop codon, and the acceptor arm is operably linked to arginine, tryptophan, or glycine; (b) The anticodon arm comprises a trinucleotide anticodon, the anticodon is 5'-UUA-3', and recognizes a TAA stop codon, and the acceptor arm is operably linked to glutamine or glutamic acid; or (c) The anticodon arm comprises a trinucleotide anticodon, the anticodon is 5'-CUA-3', and recognizes a TAG stop codon, and the acceptor arm is operably linked to tryptophan, glutamic acid, or glutamine; Modified transfer RNA (tRNA). (Item 13) The modified tRNA described in item 12, comprising reasonable nucleotide substitutions that enhance or modulate the interaction of the T-arm with elongation factor 1-α1 (EF1α). (Item 14) An oligonucleotide sequence encoding a modified tRNA as described in any one of items 1 to 13, wherein the oligonucleotide has a total length of less than 150 nucleotides. (Item 15) An oligonucleotide comprising a first oligonucleotide sequence and a second oligonucleotide sequence, wherein the first and second oligonucleotide sequences independently encode a modified tRNA as described in any one of items 1 to 13, the first and second oligonucleotides independently have a total length of less than 150 nucleotides, and the two sequences are in tandem. (Item 16) The oligonucleotide described in item 15, wherein the oligonucleotide is DNA. (Item 17) An expression cassette comprising a promoter and a nucleic acid encoding a modified tRNA as described in any one of items 1 to 13 or an oligonucleotide sequence as described in any one of items 14 to 16. (Item 18) A vector comprising an oligonucleotide as described in any one of items 14-16 or an expression cassette as described in item 17. (Item 19) The vector described in item 18, wherein the vector is a viral vector or a plasmid vector. (Item 20) A modified tRNA as described in any one of items 1 to 13, an oligonucleotide as described in any one of items 14 to 16, or a vector as described in item 18 or 19, A pharmaceutically acceptable carrier, A composition containing the following: (Item 21) The composition according to item 20, wherein the carrier is a liposome. (Item 22) Cells containing the vector described in item 18 or 19. (Item 23) A method for treating a stop codon-related gene disorder, comprising administering a composition described in item 20 or 21 to a patient who is in need of treatment for a stop codon-related gene disorder. (Item 24) The method according to item 23, wherein the genetic disorder associated with the immature stop codon is cystic fibrosis, muscular dystrophy, β-thalassemia, or Liddle syndrome. (Item 25) A method for restoring translation of a nucleotide sequence containing a nonsense mutation in a cell, comprising introducing a composition described in item 20 or 21 into the cell, wherein the modified tRNA restores translation of the nucleotide sequence containing the nonsense mutation.

Claims

1. A modified transfer RNA (tRNA) comprising a T-arm, a D-arm, an anticodon arm, and an acceptor arm, The anticodon arm comprises a trinucleotide anticodon, wherein the anticodon is 5'-CUA-3' and recognizes a TAG stop codon; The acceptor arm is operably connected to glutamine; and The modified tRNA is a nucleic acid sequence selected from SEQ ID NOs. 139, 128-138, and 140-143, and includes a nucleic acid sequence in which thymine is substituted with uracil. Modified tRNA.

2. The modified tRNA according to claim 1, wherein the modified tRNA comprises the nucleic acid sequence of sequence number 139, wherein thymine is replaced with uracil.

3. The modified tRNA according to claim 1, wherein the modified tRNA comprises the nucleic acid sequence of sequence number 141, wherein thymine is replaced with uracil.

4. The modified tRNA according to claim 1, wherein the modified tRNA comprises the nucleic acid sequence of sequence number 130, wherein thymine is replaced with uracil.

5. The modified tRNA according to claim 1, wherein the modified tRNA comprises the nucleic acid sequence of sequence number 134, wherein thymine is replaced with uracil.

6. The modified tRNA according to claim 1, wherein the modified tRNA comprises the nucleic acid sequence of sequence number 140, wherein thymine is replaced with uracil.

7. An oligonucleotide encoding the modified tRNA according to any one of claims 1 to 6.

8. The oligonucleotide according to claim 7, wherein the oligonucleotide has a total length of less than 150 nucleotides.

9. An oligonucleotide comprising a first oligonucleotide sequence and a second oligonucleotide sequence, wherein the first and second oligonucleotide sequences independently encode the modified tRNA described in Claim 1, the first and second oligonucleotides independently have a total length of less than 150 nucleotides, and the two sequences are in tandem.

10. The oligonucleotide according to claim 9, wherein the oligonucleotide is DNA.

11. An expression cassette comprising a promoter and a nucleic acid encoding a modified tRNA according to any one of claims 1 to 6 or an oligonucleotide according to any one of claims 7 to 10.

12. A vector comprising an oligonucleotide according to any one of claims 7 to 10 or an expression cassette according to claim 11.

13. The vector according to claim 12, wherein the vector is a viral vector or a plasmid vector.

14. A modified tRNA according to any one of claims 1 to 6, an oligonucleotide according to any one of claims 7 to 10, or a vector according to claim 12 or 13, A pharmaceutically acceptable carrier, A composition containing the following:

15. The composition according to claim 14, wherein the carrier is a liposome.

16. A cell comprising the vector according to claim 12 or 13.

17. The composition according to claim 14 or 15 for treating a genetic disorder associated with an immature stop codon.

18. The composition according to claim 17, wherein the genetic disorder associated with the immature stop codon is cystic fibrosis, muscular dystrophy, β-thalassemia, or Liddle syndrome.

19. The composition according to claim 18, wherein the gene disorder associated with the immature stop codon is Duchenne muscular dystrophy.

20. A composition according to claim 14 or 15 for restoring translation of a nucleotide sequence containing a nonsense mutation in a cell, characterized in that the composition is introduced into the cell, and the modified tRNA restores translation of the nucleotide sequence containing the nonsense mutation.

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