Methods of rescuing stop codons via genetic reassignment with ace-trna

Abstract

To provide compositions and methods for treating diseases and disorders associated with premature termination codon (PTC).SOLUTION: The present invention provides compositions and methods for treating diseases and disorders associated with PTC, the methods comprising administering an anticodon edited (ACE) tRNA (ACE-tRNA) or a nucleic acid molecule encoding the same. Specifically, the methods comprise administering to a subject at least one ACE-tRNA specific to UGA, UAA, or UAG to treat the associated disorder. Furthermore, nucleic acid sequences of the ACE-tRNA are disclosed.SELECTED DRAWING: Figure 1

Description

Background Art

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 580,887, filed November 2, 2017, and U.S. Provisional Application No. 62 / 687,015, filed June 20, 2018, the contents of which are hereby incorporated by reference in their entirety.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under R01 GM106569 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] DNA molecules carry genetic information in the form of the sequence of nucleotide bases that make up DNA polymers. Only four nucleotide bases, adenine, guanine, cytosine, and thymine, are used in DNA. This information is transcribed into messenger RNA (mRNA) in the form of codons of three consecutive bases, and then translated by transfer RNA (tRNA) and ribosomes to form proteins. Four nucleotide bases, adenine, guanine, cytosine, and uracil, are used in RNA. The genetic code is the relationship between triplet codons and specific amino acids. The 64 possible codon triplet forms make up the genetic code, and three stop (also called termination) codons provide signals to the translation machinery (cellular ribosomes) to terminate protein production at a particular codon. The other 61 triplets in the code correspond to one of the 20 standard amino acids. See Figure 1.

[0004] DNA is translated by ribosomes to form polypeptides by linking each amino acid one by one according to the genetic instructions specifically provided by the DNA. When the ribosome reaches a stop codon, the elongation of the protein ends. The three stop codons are UAG (amber), UAA (ochre), and UGA (opal). Mutations that change amino acid - coding codons to stop codons are called "nonsense mutations". These nonsense mutations can result in significant truncation / shortening of the polypeptide sequence and can cause profound changes in the gene phenotype. Thus, although there may be a gene that directs expression, when the ribosome reaches a mutant stop signal, translation ends, resulting in an incomplete protein, and important proteins may not be produced.

[0005] Transfer RNA translates mRNA on ribosomes into proteins. Each tRNA contains an "anticodon" region that hybridizes with a complementary codon on the mRNA. A tRNA carrying its designated amino acid is called a "charged" tRNA. When the tRNA is one of the 61 amino - acid - related (i.e., not related to the stop signal) tRNAs, it usually attaches the amino acid to the growing peptide. The structural genes of tRNAs are about 72 - 90 nucleotides long and fold into a clover - leaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenic split promoters that become part of the mature tRNA - coding sequence (Sharp S.J., Schaack J., Coolen L., Burke D.J. and Soll D., "Structure and transcription of eukaryotic tRNA genes", Crit. Rev. Biochem, 19:107 - 144(1985), Geiduschek E.O., 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 containing an altered anticodon such that, instead of triggering a "stop" signal, it inserts an amino acid in response to a stop codon. For example, an 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 continued translation of the mRNA despite the presence of a codon that normally causes termination in translation.

[0007] Many nonsense suppressor tRNA alleles have been identified in prokaryotes and eukaryotes such as yeast and C. elegans. Different suppressor tRNAs differ in their suppression efficiency. In E. coli and other systems, amber suppressors are relatively efficient, ochre suppressors are not efficient, and opal is the least efficient, suggesting that the amber codon is used less frequently to terminate protein synthesis and that the ochre and opal codons are used more frequently as natural termination signals.

[0008] Errors undesirable in DNA blueprints can cause disease. For example, an unexpected "stop" signal occurs not at the end of the blueprint, but in the middle of a protein, resulting in the production of truncated or shortened proteins with altered or no function at all. Many human diseases, including cystic fibrosis (Cheng et al., 1990, Cell, 63, 827-834), Duchenne muscular dystrophy, spinal muscular atrophy (Lefebvre et al., 1995, Cell, 80, 155-165), infantile neuronal ceroid lipofuscinosis (Das et al., 1998, J Clin Invest, 102, 361-370), β o-thalassemia (Chang and Kan, 1979, Proc Natl Acad Sci U S A, 76, 2886-2889), cystinosis (Kalatzis et al., 2002, Hum Mutat, 20, 439-446), X-linked nephrogenic diabetes insipidus (Pan et al., 1992, Nat Genet, 2, 103-106), Hurler syndrome (Ballabio et al., 2009, Biochim Biophys Acta, 1793, 684-696), Usher syndrome type 11, polycystic kidney disease, Riddle syndrome, xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes, and kuru, are caused by unwanted stop signals in the DNA reading frame of proteins. In addition, nonsense mutations occur within the tumor suppressor genes p53 and ATM, further suggesting their role in these diseases. Thus, PTC represents a unique group of diseases that affect over 30 million people worldwide and account for 10-15% of all genetic diseases.

[0009] Accordingly, there is a need in the art for compositions and methods generalizable to multiple tRNA gene families for the treatment of diseases and disorders associated with PTC. The present invention addresses this unmet need in the art

Summary of the Invention

[0010] In certain embodiments, the present invention provides a modified transfer RNA (tRNA) comprising a T arm, a D arm, and an anticodon arm and an acceptor arm, the T arm comprising a T stem having nucleotides that interact with elongation factor 1-alpha 1 (EF1α). EF1α replenishes aminoacyl-tRNA to the ribosome and protects the tRNA from deacylation. Rational nucleotide substitutions result in enhanced EF1α interactions that modulate tRNA delivery to the ribosome and protection from deacylation.

[0011] In certain embodiments, the present invention provides a modified transfer RNA (tRNA) of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 (where thymidine is replaced by uracil).

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

[0013] In certain embodiments, the modified tRNA is any one of SEQ ID NOs: 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, 388-390, 392, 394-401, 403-407, 414-416, 418, 422, 425, 428-433, 437, 444-445, 452, 455, 459-463, 470, 472-474, 476, 487-492, 525, 530-539, 545-550, 553-555, 561-563, and 567-579 (wherein thymidine is replaced by uracil).

[0014] In certain embodiments, the present invention provides a modified transfer RNA (tRNA) comprising a T-stem, a D-stem, and an anticodon loop, and an acceptor stem, wherein (a) the anticodon arm comprises a trinucleotide anticodon which is 5'-UCA-3', recognizes the TGA stop codon, and the acceptor arm is operably linked to arginine, tryptophan or glycine, (b) the anticodon arm comprises a trinucleotide anticodon which is 5'-UUA-3', recognizes the TAA stop codon, and the acceptor arm is operably linked to glutamine or glutamic acid, or (c) the anticodon-arm comprises a trinucleotide anticodon which is 5'-CUA-3', recognizes the TAG stop codon, and the acceptor arm is operably linked to tryptophan, glutamic acid or glutamine. In certain embodiments, the T-arm comprises a rationally varied nucleotide sequence that modulates the interaction with EF 1α, enhancing its inhibitory activity and thereby increasing its therapeutic potential. The tRNA that modulates the interaction with EF 1α has enhanced nonsense suppression and provides improved therapeutic properties.

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

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

[0017] In certain embodiments, the present invention provides a vector comprising the oligonucleotide or expression cassette described above.

[0018] In certain embodiments, the vector is a viral vector or a plasmid vector.

[0019] In certain embodiments, the present invention provides a composition comprising the modified tRNA, oligonucleotide, or vector described above, and a pharmaceutically acceptable carrier.

[0020] In certain embodiments, the carrier is a liposome.

[0021] In certain embodiments, the present invention provides a cell comprising the vector described above.

[0022] The present invention provides a method for treating a genetic disease associated with a stop codon, which includes administering the modified tRNA composition described above to a patient in need of treatment for the disease.

[0023] In certain embodiments, the genetic diseases associated with premature termination codons are cystic fibrosis, muscular dystrophy, β-thalassemia or Riddle syndrome.

[0024] In certain embodiments, the present invention provides a method for restoring translation into a nucleotide sequence containing a nonsense mutation in a cell, the method comprising introducing the above-described composition into the cell.

[0025] In certain embodiments, the present invention provides a method for identifying anti-codon editing (ACE) tRNA by high-throughput cloning and screening using suppression of nonsense codons of luciferase enzymes containing NanoLuc.

[0026] In certain embodiments, the present invention provides a method for treating a disease associated with a PTC in a subject in need of treatment for the disease, the method comprising administering to the subject at least one composition comprising an ACE-tRNA or a nucleic acid molecule encoding the same.

[0027] In certain embodiments, the disease is a disease or disorder associated with a UGA PTC, and the method comprises administering at least one ACE-tRNA specific for UGA.

[0028] In certain embodiments, the disease is a disease or disorder associated with a UAA PTC, and the method comprises administering at least one ACE-tRNA specific for UAA.

[0029] In certain embodiments, the disease is a disease or disorder associated with a UAG PTC, and the method comprises administering at least one ACE-tRNA specific for UAG.

[0030] In certain embodiments, the method comprises administering at least two ACE-tRNAs, each of the at least two ACE-tRNAs being specific for incorporating at least two different amino acid molecules into a polypeptide chain.

[0031] In certain embodiments, the method comprises administering at least two ACE-tRNAs, each of the at least two ACE-tRNAs being specific for incorporating the same amino acid molecule into a polypeptide chain.

[0032] In certain embodiments, at least two ACE-tRNAs are encoded on the same nucleic acid molecule.

[0033] In certain embodiments, at least two ACE-tRNAs are encoded on different nucleic acid molecules.

[0034] In certain embodiments, the method comprises an ACE-tRNA Arg , an ACE-tRNA Gly , and an ACE-tRNA Trp and administering at least one ACE-tRNA specific for UGA selected from the group consisting of.

[0035] In certain embodiments, the disease is selected from the group consisting of Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia telangiectasia, Tay-Sachs disease, cystic fibrosis, Wilms tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich disease, β-thalassemia, type 2A and type 3 von Willebrand disease, Robinow syndrome, brachydactyly type B (shortening of the fingers and middle phalanges), genetic susceptibility to mycobacterial infection, hereditary retinal diseases, hereditary bleeding disorders, hereditary blindness, congenital sensorineural deafness and colonic agangliosis as well as hereditary neurodevelopmental disorders including sensorineural deafness, colonic agangliosis, peripheral neuropathy and central hypomyelinating leukodystrophy, Riddle syndrome, xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes and kuru.

Brief Description of the Drawings

[0036]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0037] Over the years, researchers have identified hundreds of unique point mutations that result in nonsense codons established in human genes. These types of mutations cause, for example, Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia telangiectasia, Tay-Sachs disease, cystic fibrosis, Wilms tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich disease, β-thalassemia, type 2A and type 3 von Willebrand disease, Robinow syndrome, brachydactyly type B (shortening of the fingers and middle phalanges), genetic susceptibility to mycobacterial infection, hereditary retinal diseases, hereditary bleeding tendencies, hereditary blindness, congenital sensorineural deafness and agangliosis of the colon and hereditary neurodevelopmental disorders including sensorineural deafness, agangliosis of the colon, peripheral neuropathy and central dysmyelinating leukodystrophy, Riddle syndrome, xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes and kuru. The BRACA-1 and BRACA-2 genes associated with breast cancer also have similar mutations.

[0038] The nucleotide sequences encoding hundreds of human tRNAs are known and generally available to those of skill in the art through sources such as Genbank. The structure of tRNA is highly conserved and tRNA often functions across species. Thus, bacterial or other eukaryotic tRNA sequences are also potential sources of the oligonucleotides of the stabilized tRNAs of the present invention. Determination of whether a particular tRNA sequence functions in a desired mammalian cell can be confirmed through routine experimentation. Additional potentially additional tRNA sequences that are not yet known can be modified as described herein to be stabilized through routine experimentation.

[0039] tRNA genes have strong promoters that are active in all cell types. The promoters of eukaryotic tRNA genes are contained within the structural sequences that encode the tRNA molecule itself. There are elements in the 5′ upstream region that control transcriptional activity, but the length of the active transcription unit can be quite short, even less than 500 base pairs, and thus it is easy to accommodate within a delivery vector. Once they are transcribed and processed, tRNAs have a low degradation rate. Finally, gene therapy with nonsense suppressors maintains the endogenous physiological control over target genes containing nonsense codons.

[0040] Nonsense mutation Transfer RNA (tRNA) is a type of RNA molecule that functions in the decoding of messenger RNA (mRNA) sequences into proteins. tRNAs function at specific sites within ribosomes during translation to synthesize proteins from mRNA molecules. Nonsense mutations, also called premature termination codons (PTCs), account for approximately 10–15% of single base pair mutations that cause human diseases, such as cystic fibrosis (Peltz et al., Annu Rev Med., 64:407-25, 2013). In general, nonsense mutations have a more profound impact than missense mutations because gene expression and activity are almost completely lost and there is a potential dominant inhibitory effect of truncated products. PTCs result in premature translation termination and accelerated mRNA transcript degradation via the nonsense mutation-dependent decay (NMD) pathway.

[0041] Current research shows that tRNAs can change specific sites within the RNA transcript that delivers their amino acid by molecular editing of the anticodon sequence within the tRNA. This approach has made it possible to effectively and therapeutically revert premature termination codons (PTCs) back to the original lost amino acid. Anticodon-edited tRNAs (ACE-tRNAs) form a new class of biological therapeutics.

[0042] The engineered tRNAs enable the "recoding" of disease-causing nonsense codons into specific amino acids. These engineered tRNAs target only one type of stop codon, such as TGA via TAC or TAA. The small size of these tRNA molecules makes them suitable for rapid expression because the tRNA + promoter is only about 300 bp. Briefly, oligonucleotides containing the structural elements of tRNA genes that function in human cells are synthesized. The sequence of this oligonucleotide is designed based on known sequences with substitutions made in the anticodon region of the tRNA, enabling specific tRNAs to recognize nonsense or other specific mutations.

[0043] Several small molecules have been screened to suppress nonsense stop codons through interaction with ribosomes, and the most promising molecules are G418, gentamicin, and PTC124. PTC124 or ataluren has recently been released from phase III clinical trials for use as a cystic fibrosis therapeutic. Ataluren and aminoglycosides promote readthrough of each of the three nonsense codons by inserting near-cognate amino acids that change the nonsense mutation to a missense mutation. (Roy et al., PNAS 2:016 Nov 1;113(44):12508-12513)

[0044] Anticodon-edited tRNA (ACE-tRNA) tRNA has a general four-arm structure including a T arm, a D arm, an anticodon arm, and an acceptor arm (Figure 2).

[0045] The T arm consists of a "T stem" and a "TΨC loop". In certain embodiments, the T stem is modified to increase the stability of the tRNA. In certain embodiments, ACE-tRNA has a modified T stem that increases the biological activity for suppressing the termination site compared to the endogenous T stem sequence.

[0046] In one embodiment, the present invention includes a composition comprising a stabilized tRNA that can be used with higher efficacy to treat a variety of nonsense mutation-related diseases. The following sequences in Tables 1-8 are described as DNA, but as RNA (transcribed DNA), "T: thymidine" is "U: uracil". Accordingly, all tRNAs transcribed from the following sequences contain uracil instead of thymidine.

[0047] In certain embodiments, the tRNA has the following sequence (where thymidine is replaced by uracil):

Chemical formula

Table 1

Table 2

Table 3

Table 4

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Table 6

Table 7

Table 8

[0048] In one embodiment, the ACE-tRNA for nonsense suppression is as shown in FIG. 3 (H. sapiens tRNA Trp TGA ).

[0049] According to the present invention, human UAA, UAG, and UGA suppressor tRNAs have been designed. The screen identifies codon-editing tRNAs for the repair of Trp-TGA, Trp-TAG, Arg-TGA, Gln-TAG, Gln-TA, Glu-TAG, Glu-TAA. The tRNA is approximately 100 nucleotides in length and can be introduced into cells to suppress nonsense codon mutations where a wild-type amino acid should be present. The oligonucleotide can be introduced directly into recipient cells or can be ligated in tandem to enhance the efficacy of the oligonucleotide.

[0050] Expression cassette and vector In certain embodiments, the ACT-tRNA is encoded by an expression cassette. In yet another embodiment, the suppressor tRNAs 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 sequence encoding the tRNA, the tRNA sequence can be provided but does not need to be included in a separate transcription unit.

[0051] In one embodiment of the present invention, the nucleotide expression system of the present invention is an appropriate gene delivery vehicle and is contained within the gene delivery vehicle that is then used to transduce cells to express the suppressor tRNA. The gene delivery vehicle can be any delivery vehicle known in the art and can include naked DNA facilitated by receptor and / or lipid-mediated transfection, as well as any of a number of vectors. Such vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (e.g., bacterial vectors, etc.), and viral vectors including, but not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors (including others such as human and porcine), herpes viral vectors, Epstein-Barr viral vectors, SV40 viral vectors, poxviral vectors, and pseudotyped viral vectors.

[0052] In certain embodiments, the ACT-tRNA(PTC) is encoded by a vector. Figure 4. In certain embodiments, the viral vector is a retroviral or adenoviral vector. Examples of retroviral vectors that can be employed include vectors derived from retroviruses such as Moloney murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus, but are not limited thereto.

[0053] Retrovirus, retroviral vector The term "retrovirus" is used for RNA viruses that utilize 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 integrated into the chromosome of the infected cell and, once integrated, is called a "provirus." "The provirus functions as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes necessary to produce new virus particles. At each end of the provirus, there is a structure called a "long terminal repeat" or "LTR." The LTR contains numerous control signals, including transcriptional regulatory factors, polyadenylation signals, and sequences necessary for replication and integration of the viral genome. The family Retroviridae includes several genera, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus. Some retroviruses are oncogenic (i.e., tumorigenic), while others are not. Tumor viruses induce sarcomas, leukemias, lymphomas, and breast cancers in susceptible species. Retroviruses infect a wide variety of species and can be transmitted horizontally as well as vertically. They can be integrated into the host DNA and can transfer sequences of the host DNA from cell to cell. As a result, retroviruses have been developed as vectors for various purposes, including gene therapy.

[0054] Retroviruses, including human foamy virus (HFV) and human immunodeficiency virus (HIV), have recently attracted much attention because their target cells are not limited to dividing cells and their restricted host cell tropism can be easily expanded through pseudotyping with vesicular stomatitis virus G (VSV-G) envelope glycoprotein (see, for example, J.C. Burns et al., Proc. Natl. Acad. Sci. USA 90:8033-8037

[1993] ; A.M.L. Lever, Gene Therapy. 3:470-471

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

[1996] ).

[0055] Vector systems generally have a DNA vector containing a small portion of retroviral sequences (viral terminal repeats or "LTR" and packaging or "psi" signals) and a packaging cell line. The gene to be introduced is inserted into the DNA vector. The viral sequences present on the DNA vector provide signals necessary for the insertion or packaging of vector RNA into viral particles and the expression of the inserted gene. The packaging cell line provides the viral proteins necessary for particle assembly (D. Markowitz et al., J. Virol., 62:1120

[1988] ). In one embodiment of the present invention, an FIV system employing a three-plasmid transfection production method in 293T cells was used (Johnston et al., J Virol. 199973:4991-5000). A replication-incompetent virus was successfully produced.

[0056] Vector DNA is introduced into packaging cells by any of various techniques (e.g., calcium phosphate co-precipitation, lipofection, electroporation). The viral proteins produced by the packaging cells mediate the insertion of the vector sequences in the form of RNA into viral particles, which are secreted into the culture supernatant.

[0057] For cells that naturally divide or are stimulated to divide by growth factors, simple retroviruses such as murine leukemia virus (MLV) vectors are suitable delivery systems. However, a major limitation in the use of many commonly used retroviral vectors in gene transfer is that many of the vectors are limited to dividing cells. Therefore, when non-dividing cells are the target cells, lentiviruses that can infect non-dividing cells can be used.

[0058] As used herein, the term "lentivirus" refers to a group (or genus) of retroviruses that cause disease gradually. Viruses included in this group are HIV, the pathogen of human acquired immunodeficiency syndrome (AIDS) (including human immunodeficiency virus, HIV type 1 and HIV type 2), visna-maedi that causes encephalitis (visna) or pneumonia (maedi) in sheep, caprine arthritis encephalitis virus that causes immunodeficiency, arthritis, and encephalopathy in goats, equine infectious anemia virus that causes autoimmune hemolytic anemia and encephalopathy in horses, feline immunodeficiency virus (FIV) that causes feline immunodeficiency, bovine immunodeficiency virus (BIV) that causes lymphadenopathy, lymphocytosis, and possibly central nervous system infections in cattle, and simian immunodeficiency virus (SIV) that causes immunodeficiency and encephalopathy in primates close to humans. The diseases caused by these viruses are characterized by a long incubation period and a protracted course. Usually, the virus first infects monocytes or macrophages and then spreads to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

[0059] Lentiviruses, including HIV, SIV, FIV, and equine infectious anemia virus (EIAV), depend on several viral regulatory genes in addition to the simple structural gag-pol-env genes for efficient intracellular replication. Thus, lentiviruses use more complex strategies for gene regulation and viral replication than classical retroviruses, and the packaging signal clearly spreads throughout the viral genome. These additional genes represent a web of regulatory functions during the lentiviral life cycle. For example, upon HIV-1 infection, transcription is upregulated by the expression of Tat through its interaction with the RNA target (TAR) in the LTR. Subsequently, the expression of full-length and spliced mRNAs is regulated by the function of Rev, which interacts with RNA sequences present in the gag region and the env region (RRE) (S. Schwartz et al., J. Virol., 66:150-159

[1992] ). The nuclear export of gag-pol and env mRNAs depends on the Rev function. In addition to these two essential regulatory genes, a list of accessory genes, including vif, vpr, vpx, vpu, and nef, is also present in the viral genome, and although they are not absolutely required for viral replication, their effects on efficient virus production and infectivity have been demonstrated ((K. and F. Wong-Staal, Microbiol. Rev., 55:193-205 (1991), R. A. Subbramanian and E. A. Cohen, J. Virol. 68:6831-6835

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

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

[0060] A "source" or "original" retrovirus is the wild-type retrovirus from which a pseudotyped retrovirus is derived, or is used as a starting point for preparing one or more of the genetic elements of a vector during the construction of a packaging or transgene vector. The genetic elements may be used without change or may be mutated (provided that they do not exceed a statistically significant lack of sequence similarity with the original element). A vector may have two or more source retroviruses, and the 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.

[0061] A cognate retrovirus is the wild-type retrovirus with which the vector in question has the greatest percentage of sequence identity at the nucleic acid level. Usually, this will be the same as the source retrovirus. However, if the source retrovirus mutates extensively, the vector may then be more closely similar to some other retrovirus. The cognate retrovirus need not be the physical starting point of the construction, and instead of first obtaining and then modifying the original element, one may choose to directly synthesize the genetic element, particularly the variant element. The term "cognate" may similarly be applied to proteins, genes, or genetic elements (e.g., splice donor sites or packaging signals). When referring to cognate proteins, the percentage of sequence identity is determined at the amino acid level.

[0062] The term "cognate" retrovirus can be difficult to interpret in extreme cases, i.e., when all retroviral genetic elements have been replaced by alternative non-lentiviral genetic elements. In this case, the aforementioned source retrovirus strain is optionally considered to be the cognate retrovirus.

[0063] As used herein with respect 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 propagation, 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 full viral life cycle in which infectious virus particles containing viral RNA enter a cell, the RNA is reverse transcribed into DNA, the DNA is integrated into the host chromosome as a provirus, the infected cell produces viral proteins, and these are assembled together with full-length viral genomic RNA into new, similar infectious particles.

[0064] The term "replication competent" refers to a wild-type or mutant virus that can replicate such that the replication of the virus in an infected cell results in the production of infectious virus particles that, after infecting another, previously uninfected cell, also produce such infectious virus particles in the latter cell. The present invention contemplates the use of replication-deficient viruses.

[0065] As used herein, the term "attenuated virus" refers to any virus (e.g., an attenuated lentivirus) that has been modified such that its pathogenicity in the intended subject is substantially reduced. The virus can be attenuated until it is non-pathogenic from a clinical perspective, i.e., until the subject exposed to the virus does not show a statistically significant increased level of pathology compared to a control subject.

[0066] The present invention contemplates the preparation and use of modified retroviruses. In some embodiments, the retrovirus is a mutant of murine leukemia virus, human immunodeficiency virus type 1, human immunodeficiency virus type 2, feline immunodeficiency virus, simian immunodeficiency virus, visna-maedi, caprine arthritis encephalitis virus, equine infectious anemia virus, and bovine immunodeficiency virus, or a virus consisting of parts of one or more retroviral species (e.g., a hybrid consisting of parts of MLV, FIV, HIV-1 and HIV-2, or HIV-1 and / or SIV).

[0067] A standard virus is a virus whose genome is used when describing the components of a mutant virus. For example, a particular genetic element of a mutant virus can be said to differ from its cognate element in the standard virus by various substitutions, deletions, or insertions. The mutant virus need not actually be derived from the standard virus.

[0068] Preferred standard FIV sequences are found in Talbott et al., Proc Natl Acad Sci U S A. 1989 86:5743-7; Genbank access #NC_001482. In certain embodiments, a three-plasmid transient transfection method can be used to produce replication-incompetent pseudotyped retroviruses (e.g., FIV). General methods are described in Wang et al., J Clin Invest 1999 104:R55-62 and Johnston et al., J Virol. 1999 73:4991-5000.

[0069] Retroviral vector system The present invention contemplates a retroviral gene amplification and delivery system comprising a transgene vector, one or more compatibility packaging vectors, an envelope vector, and a suitable host cell. The vectors used can be derived from retroviruses (e.g., lentiviruses). The retroviral vector enables (1) transfection of the packaging vector and the envelope vector into a host cell to form a packaging cell line that produces essentially packaging vector-RNA-free virus particles, (2) transfection of the transgene vector into the packaging cell line, (3) packaging of the transgene vector RNA by the packaging cell line into infectious virus particles, and (4) administration of the particles to target cells such that the cells are transduced and subsequently express the transgene.

[0070] Either the particles are directly administered in vivo to the subject, or the subject's cells are removed, the particles are infected in vitro, and then returned to the subject's body.

[0071] The packaging vector and the transgene vector of the present invention generate non-replicable viruses. The vector selected for integration into a given vector system of the present invention is such that co-transfected cells cannot generate replication-competent viruses by homologous recombination of only the packaging vector(s) and the transgene vector without further mutation of the packaging vector(s) or the transgene vector. The envelope protein used in this system can be a retroviral envelope, a synthetic or chimeric envelope, or an envelope derived from a non-retroviral envelope virus (e.g., baculovirus).

[0072] Packaging signal As used herein, the terms "packaging signal" or "packaging sequence" refer to a sequence located within a retroviral genome or vector that is required for, or at least facilitates, the insertion of viral or vector RNA into the viral capsid or particle. The packaging signal in RNA is identified as that which packages the RNA into the virion. The term "packaging signal" is also used for convenience to refer to the vector DNA sequence that is transcribed into a functional packaging signal. A particular packaging signal may be part of a gene, but is recognized in the form of RNA, not as a peptide portion of the encoded protein.

[0073] The main difference between the packaging vector and the transgene vector is that in the packaging vector, the main packaging signal is inactivated, while in the transgene vector, the main packaging signal is functional. Ideally, in the packaging vector, all packaging signals are inactivated, and in the transgene vector, all packaging signals function. However, offsetting considerations such as maximizing virus titer or inhibiting homologous recombination can make such constructs less desirable.

[0074] Packaging system, packaging vector, packaging cell line A packaging system is a vector or a plurality of vectors that collectively provide all the genetic information necessary to produce a virion that can encapsidate suitable RNA, transport it from virion-producing cells, transfer it to target cells, and in the target cells, integrate the RNA into the host genome so that the RNA can be reverse transcribed and the transgene incorporated into the aforementioned RNA can be expressed. However, the packaging system must be substantially non-packagable by itself. Rather, it packages a separate transgene vector.

[0075] In the present invention, the packaging vector provides functional equivalents of the gag and pol genes (the "GP" vector). The env gene(s) are provided by an envelope vector. Theoretically, it is possible to construct different gag and pol genes on separate vectors, operably link them to different regulatable promoters (or one to a regulatable promoter and the other to a constitutive promoter), and adjust their relative expression levels appropriately, in which case three vector systems (the "G", "P", and "E" vectors) are possible.

[0076] A packaging cell line is a suitable host cell transfected by a packaging system that produces virus particles under achievable conditions. As used herein, the term "packaging cell line" is typically used with respect to cell lines that express viral structural proteins (e.g., gag, pol, and env) but do not contain a packaging signal. For example, the cell line lacks a psi + sequence (designated as Δ-psi) at one chromosomal locus within its genome and is genetically engineered to be carried by a 5'-LTR-gag-pol-3'-LTR fragment and a 5'-LTR-env-3'-LTR fragment where Δ-psi is also located at another chromosomal locus. Both of these segments are constitutively transcribed, but the psi + region is defective, and the resulting viral RNA molecules are less than full size, so empty virus particles are formed.

[0077] When the host cell is transfected with only the packaging vector(s), it produces substantially only virus particles without the full-length packaging vector. In one example, less than 10% of the virus particles produced by the packaging cells contain RNA derived from the full-length packaging vector. However, since the packaging vector lacks a functional primer binding site, even if these particles infect new cells, the packaging vector RNA is not reverse transcribed into DNA, and thus the new cells do not produce virions. Thus, by itself, the packaging vector is a replication-incompetent virus.

[0078] In some embodiments, the packaging cell and / or cell line contains 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."

[0079] Packaging is contemplated to be either inducible or non - inducible. In inducible packaging cells and packaging cell lines, retroviral particles are produced in response to at least one inducer. In non - inducible packaging cell lines and packaging cells, an inducer is not required to cause retroviral particle production.

[0080] A packaging vector is necessarily different from a wild - type replication - competent retroviral genome by inactivation of at least one packaging signal of the cognate wild - type genome. Two or more packaging signals can be inactivated. In one embodiment, only the retroviral genes provided by the packaging vector encode structural or essential regulatory proteins.

[0081] Transgene vector A transgene vector is an expression vector that carries a non - retroviral gene of interest that is expressible and contains at least one functional retroviral packaging signal. Thus, after the transgene vector is transfected into a packaging cell line, the transgene vector is transcribed into RNA, and this RNA is packaged into infectious virus particles. These particles then infect target cells, and their RNA is reverse - transcribed into DNA, which is integrated into the host cell genome as a proviral sequence, thereby delivering the gene of interest to the target cells.

[0082] As used herein, the term "transduction" refers to the delivery of gene(s) using a viral or retroviral vector by infection, rather than by transfection. In certain embodiments, a retroviral vector is transduced. Thus, a "transduced gene" is a gene that has been introduced into a cell via retroviral or vector infection and proviral integration. In certain embodiments, a viral vector (e.g., a "transgene vector") transduces a gene into a "target cell" or host cell. The present invention encompasses a transgene vector suitable for use in the present invention, linked to any gene of interest (or a "marker gene" or "reporter gene" used to indicate infection or gene expression).

[0083] As used herein, the term "long-term transduction" refers to a vector that can remain transduced in a host or target cell for a longer period of time than is observed with other vectors. For example, the present invention provides a retroviral vector that can maintain transduction for at least 120 days, at least one year, or for the duration of the lifespan of the subject or the treatment. The duration of expression is a function of the promoter selection and the target cell type, rather than the vector selection.

[0084] The terms "stable transduction" or "stably transduced" refer to the introduction and integration of foreign DNA into the genome of a transduced cell. The term "stable transductant" refers to a cell that has stably integrated foreign DNA into its genomic DNA.

[0085] The terms "transient transduction" or "transiently transduced" refer to the introduction of foreign DNA into a cell where 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 is under the control of regulatory elements that govern the expression of endogenous genes on the chromosome. The term "transient transduction agent" refers to a cell that has taken up foreign DNA but has been unable to integrate this DNA.

[0086] 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, white blood cells, spleen cells, liver cells, eye cells, epithelial cells).

[0087] In some embodiments, vectors and vector progeny can transduce multiple target cells to achieve a vector titer of at least 10 5 cfu / ml. The multiplicity of infection (MOI) can be at least one (i.e., an average of 1 hit per cell), or at least two.

[0088] Expression Cassette and Vector The present invention also provides an expression cassette comprising a sequence encoding ACE-tRNA.

[0089] In certain embodiments, the expression cassette further contains a promoter. In certain embodiments, the promoter is a regulatable promoter. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a PGK, CMV, RSV, H1 or U6 promoter (Pol II and Pol III promoters).

[0090] The present invention provides a vector containing the above-described expression cassette. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, HSV, or murine Moloney-based viral vector

[0091] As used herein, an "expression cassette" means a nucleic acid sequence capable of inducing the expression of a particular nucleotide sequence in a suitable host cell, which may include a promoter operably linked to a nucleotide sequence of interest that can be operably linked to a termination signal. It may also include sequences necessary for proper translation of the nucleotide sequence. The coding region usually encodes the protein of interest. An expression cassette containing a nucleotide sequence of interest may be chimeric. An expression cassette may be naturally occurring but obtained in a recombinant form useful for heterologous expression. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter that initiates transcription only when the host cell is exposed to some specific stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0092] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence encoding an RNA or polypeptide when the two sequences are arranged such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence may be operably linked to regulatory sequences in the sense or antisense orientation.

[0093] Adeno-associated virus (AAV) Adeno-associated virus (AAV) is a small non-pathogenic virus of the Parvoviridae family. AAV differs from other members of this family in that its replication is dependent on a helper virus. In the absence of a helper virus, AAV can integrate into the q arm of chromosome 19 in a locus-specific manner. The approximately 5 kb genome of AAV consists of a segment of single-stranded DNA of either plus or minus polarity. The ends of the genome are folded into hairpin structures and are short inverted terminal repeats that can function as origins of viral DNA replication. Physically, the parvovirus virion has no envelope, and its icosahedral capsid is approximately 20 nm in diameter.

[0094] To date, a number of serologically distinct AAVs have been identified, and more than 12 have been isolated from humans or primates. The genome of AAV2 is 4,680 nucleotides in length and contains two open reading frames (ORFs). The left ORF encodes the 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 the Rep proteins have been associated with the preferential integration of the AAV genome into regions of the q arm of human chromosome 19. Rep68 / 78 has also been shown to have NTP-binding activity as well as DNA and RNA helicase activity. The Rep proteins have a nuclear localization signal as well as several potential phosphorylation sites. Mutation of one of these kinase sites resulted in a loss of replication activity.

[0095] The ends of the genome are short inverted terminal repeats (ITRs) that have the potential to fold into a T-shaped hairpin structure that functions as the origin of viral DNA replication. Within the ITR region, two elements central to the function of the ITR, the GAGC repeat motif, and the terminal resolution site (trs) are described. The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin structure. This binding serves to position Rep68 / 78 for cleavage at the trs, which occurs in a site-specific and strand-specific manner. These two elements appear to be central to viral integration in addition to their role in replication. The integration locus contained within chromosome 19 is a Rep binding site with adjacent trs. These elements have been shown to be functional and necessary for locus-specific integration.

[0096] An AAV virion is an icosahedral particle without an envelope, approximately 25 nm in diameter, consisting of three related proteins called VP1, VP2, and VP3. The right ORF encodes the capsid proteins VP1, VP2, and VP3. These proteins are found in a ratio of 1:1:10, respectively, and all are derived from the right ORF. The capsid proteins differ from each other by alternative splicing and the use of unusual start codons. Deletion analysis has shown that the removal or modification of VP1 translated from the message by alternative splicing results in a decrease in the yield of infectious particles. Mutations within the VP3 coding region render it unable to produce any single-stranded progeny DNA or infectious particles. An AAV particle is a viral particle containing an AAV capsid protein. The AAV capsid polypeptide can encode the entire VP1, VP2, and VP3 polypeptides. The particle can be a particle containing AAV2 and other AAV capsid proteins (i.e., chimeric proteins such as AAV1 and AAV2). Changes in the amino acid sequence of the AAV2 capsid protein are contemplated herein, as long as the resulting viral particle containing the AAV2 capsid remains antigenically or immunologically different from AAV1, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blot can be used to determine whether the viral particle is antigenically or immunologically different from AAV1. Furthermore, the AAV2 viral particle preferably retains a tissue tropism different from that of AAV1.

[0097] An AAV2 particle is a viral particle containing an AAV2 capsid protein. The AAV2 capsid polypeptide encoding the entire VP1, VP2, and VP3 polypeptides can have an overall homology (or identity) of at least about 63% to the 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% homology, about 75% homology, 80% homology, 85% homology, 90% homology, 95% homology, 98% homology, 99% homology, or even 100% homology to the protein encoded by the nucleotide sequence described in NC_001401. The capsid protein can have about 70% identity, about 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, 99% identity, or even 100% identity to the protein encoded by the nucleotide sequence described in NC_001401. The particle can be a particle containing another AAV and AAV2 capsid protein, i.e., a chimeric protein. Changes in the amino acid sequence of the AAV2 capsid protein are contemplated herein as long as the resulting viral particle containing the AAV2 capsid remains antigenically or immunologically different from AAV4, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blot can be used to determine whether the viral particle is antigenically or immunologically different from AAV1. Furthermore, the AAV2 viral particle preferably retains a tissue tropism different from that of AAV1 as exemplified in the examples herein, but an AAV2 chimeric particle containing at least one AAV2 coat protein can have a tissue tropism different from that of an AAV2 particle consisting of only the AAV2 coat protein.

[0098] In certain embodiments, the invention further provides a vector containing a pair of AAV2 terminal inverted repeats, i.e., an AAV2 particle that forms a capsid. The nucleotide sequence of the AAV2 ITR is known in the art. Further, the particle can be a particle containing both AAV1 and AAV2 capsid proteins, i.e., a chimeric protein. Further, the particle can be a particle that forms a capsid around a vector containing a pair of AAV terminal inverted repeats from other AAVs (e.g., AAV1 - AAV9 and AAVrh10). The vector formed into a capsid within the particle can further contain a foreign nucleic acid inserted between the terminal inverted repeats.

[0099] Due to the following characteristics of AAV, it has become an attractive vector for gene transfer. AAV vectors have been shown in vitro to stably integrate into the cellular genome, have a broad host range, transduce both dividing and non - dividing cells in vitro and in vivo, and maintain high expression levels of the transduced gene. The viral particles are heat - stable, resistant to solvents, detergents, pH changes, and temperature changes, and can be concentrated by CsCl gradient or other means. The present invention provides methods of administering AAV particles, recombinant AAV vectors, and recombinant AAV virions. For example, an AAV2 particle is a viral particle containing the AAV2 capsid protein, or an AAV1 particle is a viral particle containing the AAV1 capsid protein. A recombinant AAV2 vector is a nucleic acid construct containing at least one unique nucleic acid of AAV2. A recombinant AAV2 virion is a particle containing the recombinant AAV2 vector. For purposes of being considered within the term "AAV2 ITR", the nucleotide sequence must retain one or both of the features described herein that distinguish the AAV2 ITR from the AAV1 ITR: (1) three (not four as in AAV1) "GAGC" repeats, and (2) at the AAV2 ITR Rep binding site, the fourth nucleotide in the first two "GAGC" repeats is C rather than T.

[0100] The promoter driving the expression of the sequence encoding the tRNA to be delivered can be any desired promoter selected based on known considerations such as the expression level of the nucleic acid functionally linked to the promoter and the cell type in which the vector is used. The promoter can be a foreign or endogenous promoter. Examples of promoters include known strong promoters such as the SV40 or inducible metallothionein promoter, or AAV promoters such as the AAV p5 promoter. Further examples of promoters include promoters derived from the actin gene, immunoglobulin gene, cytomegalovirus (CMV), adenovirus, bovine papillomavirus, adenovirus promoter such as the adenovirus major late promoter, inducible heat shock promoter, respiratory syncytial virus, Rous sarcoma virus (RSV), etc. Additional examples include regulated promoters.

[0101] The AAV vector can further contain a foreign (heterologous) nucleic acid functionally linked to the promoter. "Heterologous nucleic acid" means that any heterologous or foreign nucleic acid can be inserted into the vector for introduction into a cell, tissue or organism. The nucleic acid can, for example, encode a tRNA. "Functionally linked" means that the promoter can promote the expression of the heterologous nucleic acid, as is known in the art, such as the appropriate orientation of the promoter with respect to the heterologous nucleic acid. Further, the heterologous nucleic acid preferably has all the sequences appropriate for the expression of the nucleic acid, as is known in the art, to functionally encode, i.e., to express the nucleic acid. The nucleic acid can include expression control sequences such as enhancers. The nucleic acid can encode two or more gene products, limited only by the size of the nucleic acid that can be packaged.

[0102] An AAV1 particle is a viral particle containing an AAV1 capsid protein. Changes in the amino acid sequence of the AAV1 capsid protein are contemplated herein, so long as the resulting viral particles containing the AAV1 capsid remain antigenically or immunologically distinct from other AAV capsids, as can be routinely determined by standard methods. Specifically, for example, ELISA and Western blot can be used to determine whether the viral particles are antigenically or immunologically distinct from other AAV serotypes.

[0103] As used herein, the term "polypeptide" refers to a polymer of amino acids and includes full-length proteins and fragments thereof. Thus, the terms "protein" and "polypeptide" are often used interchangeably herein.

[0104] The method provides a method of delivering a nucleic acid to a cell, comprising administering to the cell an AAV particle containing a vector comprising a nucleic acid inserted between a pair of AAV terminal inverted repeats, thereby delivering the nucleic acid to the cell. Administration to the cell can be accomplished by any means, including optionally contacting the cell with the particles contained in a desired liquid such as tissue culture medium or buffered saline solution. The particles can remain in contact with the cell for any desired length of time and typically, the particles can be administered and retained indefinitely. Such in vitro methods are known in the art and, as exemplified herein, the virus can be administered to the cell by standard viral transduction methods. The titer of the virus administered can vary, particularly depending on the cell type, but is generally representative of those used for AAV transduction. Additionally, the titer used to transduce the specific cells in this example can be utilized. The cells can include any desired cells in humans as well as other large (non-rodent) mammals such as primates, horses, sheep, goats, pigs, and dogs.

[0105] The present invention further provides a method for delivering a nucleic acid to a target cell, the method comprising administering to the target an AAV particle comprising a nucleic acid inserted between a pair of AAV terminal inverted repeats, thereby delivering the nucleic acid to the target cell.

[0106] Certain embodiments of the present disclosure provide cells comprising the viral vectors described herein.

[0107] AAV vector In one embodiment, the viral vector of the present disclosure is an AAV vector. The term "AAV" vector refers to an adeno-associated virus and can be used to refer to the naturally occurring wild-type virus itself or a derivative thereof. The term encompasses all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, unless otherwise required. As used herein, the term "serotype" refers to an AAV that is identified based on its capsid protein reactivity with a defined antiserum and is distinguishable from other AAVs. For example, there are eight known serotypes of primate AAV, AAV-1 to AAV-9, and AAVrh10. For example, serotype AAV2 is used to refer to an AAV comprising a capsid protein encoded by the cap gene of AAV2 and a genome comprising 5' and 3' ITR sequences from the same AAV2 serotype. As used herein, for example, rAAV1 may be used to refer to an AAV having both a capsid protein and 5'-3' ITR from the same serotype, or may refer to a capsid protein from one AAV serotype and 5'-3' ITR from a different AAV serotype, for example, a capsid from AAV serotype 2 and an ITR from AAV serotype 5. For each example illustrated herein, the description of vector design and production describes the serotype of the capsid and the 5'-3' ITR sequence. The abbreviation "rAAV" refers to a recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").

[0108] "AAV virus" or "AAV virus particle" refers to a virus particle consisting of at least one AAV capsid protein (preferably, by all of the capsid proteins of wild-type AAV) and a polynucleotide encapsulated. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is typically referred to as "rAAV".

[0109] In one embodiment, the AAV expression vector is constructed to provide control elements, including a transcription start region, DNA of interest, and a transcription termination region, as components that are at least operably linked in the transcriptional direction using known techniques. The control elements are selected to be functional within mammalian cells. The resulting construct containing the operably linked components is adjacent to (5' and 3') the functional AAV ITR sequence.

[0110] "Adeno-associated virus inverted terminal repeat" or "AAV ITR" means the regions recognized in the art found at each end of the AAV genome, which function together cis as an origin of DNA replication and as a viral packaging signal. The AAV ITR, together with the AAV rep coding region, provides for efficient excision and rescue, as well as integration, of the nucleotide sequence inserted between two adjacent ITRs into the mammalian cell genome.

[0111] The nucleotide sequences of the AAV ITR regions are known. As used herein, "AAV ITR" need not have the indicated wild-type nucleotide sequence and can be altered, for example, by nucleotide insertions, deletions, or substitutions. In addition, the AAV ITR can be derived from any of several AAV serotypes including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, etc. Further, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector and enable integration of the heterologous sequence into the recipient cell genome when the AAV Rep gene product is present intracellularly.

[0112] 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, AAV7, etc. Further, the 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., enable excision and rescue of the sequence of interest from the host cell genome or vector and enable integration of the DNA molecule into the recipient cell genome when the AAV Rep gene product is present intracellularly.

[0113] In one embodiment, the AAV capsid can be derived from AAV2. Suitable DNA molecules for use in an AAV vector are less than about 5 kilobases (kb), less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, less than about 2.5 kb in size and are known in the art.

[0114] In one embodiment, the selected nucleotide sequence is operably linked to a control element that induces transcription or its expression in vivo in a subject. Such control elements can include control sequences normally associated with the selected gene. Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the pol II promoter, the pol III promoter, synthetic promoters, hybrid promoters, and the like, but are not limited thereto. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene are also found to be useful herein. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, Calif.).

[0115] In one embodiment, heterologous promoters, together with other control elements such as tissue-specific and inducible promoters, enhancers, etc., are particularly useful. Examples of heterologous promoters include the CMV promoter. Examples of inducible promoters include the DNA response sequences of ecdysone, tetracycline, hypoxia, and aufin.

[0116] In one embodiment, an AAV expression vector containing a DNA molecule of interest bound by an AAV ITR can be constructed by directly inserting the selected sequence(s) into an AAV genome having the major AAV open reading frame ("ORF") excised therefrom. Other portions of the AAV genome can also be deleted as long as sufficient portions of the ITR remain to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art.

[0117] Alternatively, the AAV ITR can be excised from the viral genome or an AAV vector containing the same and fused to the 5' and 3' of a selected nucleic acid construct present in another vector using standard ligation techniques. For example, ligation can be achieved in 20 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 10 mM DTT, 33 μg / ml BSA, 10 mM to 50 mM NaCl, and at 0°C with 40 μM ATP, 0.01 to 0.02 (Weiss) units of T4 DNA ligase (for "sticky end" ligation), or at 14°C with 1 mM ATP, 0.3 to 0.6 (Weiss) units of T4 DNA ligase (for "blunt end" ligation). Intermolecular "sticky end" ligation is usually performed at a total DNA concentration of 30 to 100 μg / ml (total end concentration of 5 to 100 nM). The AAV vector containing the ITR

[0118] In addition, chimeric genes can be synthetically produced to include AAV ITR sequences located 5' and 3' of one or more selected nucleic acid sequences. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods.

[0119] To produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques such as transfection. Many transfection techniques are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using a high-speed microprojectile.

[0120] 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. This term includes the progeny of the original transfected cells. Thus, as used herein, "host cell" generally refers to a cell transfected with an exogenous DNA sequence. Cells derived from the stable human cell line 293 (e.g., available under accession number ATCC CRL1573 through the American Type Culture Collection) can be used in the practice of the present disclosure. In particular, the human cell line 293 is a human embryonic kidney cell line transformed with adenovirus type 5 DNA fragments and expresses the adenovirus E1a and E1b genes. The 293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV virions.

[0121] The "AAV rep coding region" means the region of the AAV genome recognized in the 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 nicking of the AAV origin of DNA replication, DNA helicase activity, and regulation of transcription from AAV (or other heterologous) promoters. The 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.

[0122] The "AAV cap coding region" means the region of the AAV genome recognized in the art that encodes the capsid proteins VP1, VP2, and VP3, or functional homologs thereof. These Cap expression products provide the packaging functions that are collectively required to package the viral genome.

[0123] In one embodiment, the AAV helper function is introduced into the host cell by transfecting the host cell with an AAV helper construct before or at the same time as transfection of the AAV expression vector. Thus, the AAV helper construct is used to provide at least transient expression of the AAV rep and / or cap genes to complement the defective AAV functions required for productive AAV infection. The AAV helper construct lacks AAV ITRs and cannot replicate or package itself. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Several other vectors encoding Rep and / or Cap expression products have been described.

[0124] The method of delivering the viral vector includes 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 reintroduced into the subject. Alternatively, syngeneic or allogeneic cells can be used if these cells do not generate an inappropriate immune response in the subject.

[0125] Suitable methods for delivering and introducing the transduced cells into the subject have been described. For example, the cells can be transduced in vitro, for example, by combining the recombinant AAV virions with the cells in a suitable medium, and screening for cells having the DNA of interest can be performed using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which is described in more detail below, and compositions introduced into the subject by various techniques such as transplantation, intramuscular, intravenous, subcutaneous, and intraperitoneal injection are also included.

[0126] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the nucleic acid of interest, i.e., an amount sufficient to produce a reduction or alleviation of the symptoms of the disease state in question, or an amount sufficient to provide the desired benefit, of the genetic material. 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 undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts can include therein mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate, and salts of organic acids such as acetate, propionate, malonate, benzoate. Further, such vehicles can contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0127] It should be understood that two or more transgenes can be expressed by the delivered viral vector. Alternatively, separate vectors, each expressing one or more different transgenes, can also be delivered to the subject as described herein. Further, the viral vectors delivered by the methods of the present disclosure are also intended to be combined with other suitable compositions and therapies.

[0128] In view of the teachings of this specification, as will be apparent to those of ordinary skill in the art, the effective amount of viral vector to be added can be determined empirically. Administration can be performed in a single dose, either continuously or intermittently throughout the course of treatment. Methods for determining the most effective means and amounts of administration are well known to those of ordinary skill in the art and will vary depending on the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out at dosage levels and patterns selected by the treating physician.

[0129] In certain embodiments, rAAV is administered at a dose of 1×105 - 1×1016 vg / ml in about 0.3 - 2 ml. In certain embodiments, rAAV is administered at a dose of 1×107 - 1×1014 vg / ml in about 1 - 3 ml. In certain embodiments, rAAV is administered at a dose of 1×108 - 1×1013 vg / ml in about 1 - 2 ml.

[0130] Formulations containing rAAV particles will contain an effective amount of rAAV particles in a vehicle, and the effective amount can be readily determined by those of ordinary skill in the art. The rAAV particles will typically be in the range of about 1% - about 95% (w / w) of the composition, or higher or lower ranges as appropriate. The amount administered depends on factors such as the age, weight, and health status of the animal or human subject being considered for treatment. The effective dose can be established by those of ordinary skill in the art through routine tests to establish a dose-response curve. The subject is treated by administering rAAV particles at one or more doses. Multiple doses can be administered as needed to maintain sufficient enzyme activity.

[0131] Vehicles containing water, saline, artificial CSF, or other known substances can be used with the present invention. To prepare the formulation, the purified composition can be isolated, lyophilized, and stabilized. The composition can then be adjusted to the appropriate concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0132] The present invention provides a method for increasing the level of a target protein in a cell by introducing into the cell an amount of the above-described protein or a nucleic acid molecule encoding the protein sufficient to increase the level of the target protein in the cell. In certain embodiments, the accumulation of the target protein is increased by at least 10%. The accumulation of the target protein is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. 39

[0133] a nucleic acid encoding a therapeutic agent The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and polymers thereof, composed of monomers (nucleotides) containing a sugar, a phosphate, and a base which is either a purine or a pyrimidine. Unless otherwise specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0134] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. The term "substantial identity" of polynucleotide sequences means that a sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity when compared to a reference sequence using one of the alignment programs described using standard parameters.

[0135] In one embodiment, the nucleic acid molecule is an RNA molecule comprising ACE-tRNA.

[0136] In one embodiment, the nucleic acid molecule is a DNA or cDNA molecule encoding one or more ACE-tRNAs. In one embodiment, the nucleic acid molecule is a nucleic acid molecule encoding two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more ACE-tRNA molecules under the control of a single promoter.

[0137] In one embodiment, the present invention provides a nucleic acid molecule encoding two or more ACE-tRNAs separated by a cleavage site. In one embodiment, the cleavage site is a P2A site. In one embodiment, the cleavage site is a protease cleavage site. In various embodiments, protease cleavage sites include, but are not limited to, sites targeted by caspases (e.g., caspase 1 to caspase 10), enterokinase, factor Xa, granzyme B, HRV3C protease, hydroxylamine, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, TEV protease, thermolysin, or thrombin. In one embodiment, the protease for cleavage of the protease cleavage site is encoded downstream of two or more genes encoding reporter molecules as part of the same polypeptide. In one embodiment, the nucleotide sequence encoding the protease is ligated to the nucleotide sequences of two or more genes encoding ACE-tRNAs by the protease cleavage site.

[0138] In one embodiment, the nucleic acid molecule comprises one or more reporter molecules. A reporter is a molecule whose expression confers upon a cell a detectable trait. In various embodiments, reporters include, but are not limited to, chloramphenicol acetyltransferase (CAT), β-galactosyltransferase, horseradish peroxidase, luciferase, NanoLuc®, alkaline phosphatase, and fluorescent proteins such as, but not limited to, green fluorescent protein (e.g., GFP, TagGFP, T-Sapphire, Azami Green, Emerald, mWasabi, mClover3), red fluorescent protein (e.g., tdTomato, mRFP1, JRed, HcRed1, AsRed2, AQ143, mCherry, mRuby3, mPlum), yellow fluorescent protein (e.g., EYFP, mBanana, mCitrine, PhiYFP, TagYFP, Topaz, Venus), orange fluorescent protein (e.g., DsRed, Tomato, Kusabria Orange, mOrange, mTangerine, TagRFP), cyan fluorescent protein (e.g., CFP, mTFP1, Cerulean, CyPet, AmCyan1), blue fluorescent protein (e.g., Azurite, mtagBFP2, EBFP, EBFP2, Y66H) and near-infrared fluorescent protein (e.g., iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720), infrared fluorescent protein (e.g., IFP1.4) and photoactivatable fluorescent protein (e.g., Kaede, Eos, IrisFP, PS-CFP).

[0139] Method for introducing genetic material into cells Exogenous genetic material (e.g., DNA encoding one or more therapeutic ACE-tRNAs) is introduced into cells in vivo by gene delivery methods such as transfection or transduction to provide genetically modified cells. A variety of expression vectors (i.e., vehicles for facilitating delivery of exogenous genetic material to target cells) are known to those of skill in the art.

[0140] As used herein, "cell transfection" refers to the acquisition of new genetic material by a cell by incorporating additional DNA. Thus, transfection refers to inserting nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those skilled in the art and include calcium phosphate DNA coprecipitation, DEAE-dextran, electroporation, cationic lipid-mediated transfection, and particle bombardment with tungsten particles. Strontium phosphate DNA coprecipitation is another possible transfection method.

[0141] In contrast, "cell transduction" refers to the process of introducing nucleic acids into cells using DNA or RNA viruses. RNA viruses (i.e., retroviruses) for introducing nucleic acids into cells are referred to herein as transduction chimeric retroviruses. The foreign genetic material contained within the retrovirus is integrated into the genome of the transduced cell. Cells transduced with chimeric DNA viruses (e.g., adenoviruses carrying cDNA encoding a therapeutic agent) do not integrate foreign genetic material into their genome but can express foreign genetic material retained episomally within the cell.

[0142] Typically, exogenous genetic material includes a heterologous gene (usually cDNA in the form containing exons encoding a therapeutic protein) along with a promoter, which controls the transcription of the new gene. The promoter characteristically has the specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) necessary to obtain the desired gene transcription activity. For the purposes of this discussion, an “enhancer” is any untranslated DNA sequence that acts adjacent to the coding sequence (within cis) to alter the basal transcription level directed by the promoter. The exogenous genetic material can be introduced into the cellular genome immediately downstream of the promoter such that the promoter and the coding sequence are operably linked to enable transcription of the coding sequence. A retroviral expression vector may include an exogenous promoter element that controls the transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive promoters and inducible promoters.

[0143] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes under the control of a constitutive promoter are expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters of the following genes that encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus, among others. Thus, any of the above-described constitutive promoters can be used to control the transcription of a heterologous gene insert.

[0144] Genes under the control of inducible promoters are expressed only or more highly in the presence of an inducer (e.g., transcription under the control of the metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters contain responsive elements (REs) that stimulate transcription when their inducer factors bind. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. To obtain an inducible response, a promoter containing a specific RE can be selected, and in some cases, the RE itself can be ligated to a different promoter, thereby conferring inducibility on a recombinant gene. Thus, by selecting an appropriate promoter (constitutive vs. inducible; strong vs. weak), it is possible to control both the presence and the expression level of a therapeutic agent in a genetically modified cell. When a gene encoding a therapeutic agent is under the control of an inducible promoter, in situ delivery of the therapeutic agent is caused, for example, by exposing the genetically modified cells to conditions that allow for in situ transcription of the therapeutic agent by intraperitoneal injection of a specific inducer of the inducible promoter that controls transcription of the agent. For example, in situ expression by genetically modified cells of a therapeutic agent encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducible) metal ion in situ.

[0145] Thus, the amount of therapeutic agent delivered in situ is regulated by controlling factors such as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive, inducible, strong, or weak), (2) the copy number of the foreign gene inserted into the cell, (3) the number of transduced / transfected cells administered (e.g., implanted) to the patient, (4) the size of the implant (e.g., graft or encapsulated expression system), (5) the number of implants, (6) the length of time the transduced / transfected cells or implant are retained at a given location, and (7) the rate of production of the therapeutic agent by the genetically engineered cells. The selection and optimization of these factors for the delivery of a therapeutically effective dose of a particular therapeutic agent are considered to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the factors and clinical profile of the patient as disclosed above.

[0146] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may include a selectable gene, such as a neomycin resistance gene, to facilitate the selection of cells transfected or transduced with the expression vector. Alternatively, the cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), and another vector containing the selectable gene. The selection of suitable promoters, enhancers, selectable genes, and / or signal sequences is considered to be within the scope of one of ordinary skill in the art without undue experimentation.

[0147] The ACE-tRNA construct of the present invention can be inserted into any type of target or host cell. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means.

[0148] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0149] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from, for example, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0150] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicles).

[0151] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes via a linking molecule associated with both liposomes and oligonucleotides, trapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained within micelles, or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure as micelles or have a "collapsed" structure. They can also be simply dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include fat droplets that occur naturally within the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0152] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO, dicetyl phosphate (“DCP”) can be obtained from K&K Laboratories (Plainview, NY), cholesterol (“Choi”) can be obtained from Calbiochem-Behring, dimyristoyl phosphatidylglycerol (“DMPG”) and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent since it evaporates more readily than methanol. “Liposome” is a general term encompassing various single and multi-layer lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in an excess of aqueous medium. The lipid components undergo self-arrangement before a closed structure is formed, confining water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures in solution that differ from normal vesicular structures are also included. For example, the lipids can be presumed to be in a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0153] The nucleic acid molecules of the present invention can be administered via electroporation, for example, by the method described in U.S. Patent No. 7,664,545, the content of which is incorporated herein by reference. Electroporation can be performed by the methods and / or devices described in U.S. Patent Nos. 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the entire content of which is incorporated herein by reference. Electroporation can be performed via a minimally invasive device.

[0154] A minimally invasive electroporation device ("MID") can be a device for injecting the above-described compositions and associated fluids into body tissue. The device can comprise a hollow needle, a DNA cassette, and fluid delivery means, and the device is adapted to operate the fluid delivery means in use so as to simultaneously (e.g., automatically) inject DNA into the body tissue while inserting the needle into the body tissue. This has the advantage that the ability to gradually inject DNA and associated fluids while the needle is inserted leads to a more even distribution of the fluid through the body tissue. The pain experienced during injection can be reduced due to the distribution of the DNA being injected over a larger area.

[0155] The MID may inject the composition into the tissue without using a needle. The MID can inject the composition as a small stream or jet with a force such that the composition penetrates the tissue surface and enters the underlying tissue and / or muscle. The force behind the small stream or jet can be provided by expanding a compressed gas, such as carbon dioxide, through a micro-orifice within a fraction of a second. Examples of minimally invasive electroporation devices and methods of using them are described in published U.S. Patent Application No. 2008 / 023465, U.S. Patent No. 6,520,950, U.S. Patent No. 7,171,264, U.S. Patent No. 6,208,893, U.S. Patent No. 6,009,347, U.S. Patent No. 6,120,493, U.S. Patent No. 7,245,963, U.S. Patent No. 7,328,064, and U.S. Patent No. 6,763,264, the contents of each of which are incorporated herein by reference.

[0156] The MID may include an injector that creates a high-speed jet of liquid that painlessly penetrates the tissue. Such needleless injectors are commercially available. Examples of needleless injectors that can be utilized herein include those described in U.S. Patent Nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the contents of each of which are incorporated herein by reference.

[0157] A desired composition in a form suitable for direct or indirect electrotransport is typically introduced (e.g., injected) into the tissue being treated by using a needleless injector in contact with the tissue surface to activate the delivery of a jet of the agent with a force sufficient to cause penetration of the composition into the tissue. For example, if the tissue being treated is mucosa, skin, or muscle, the agent is projected toward the mucosa or skin surface with a force sufficient to penetrate the stratum corneum and into the dermal layer or to penetrate the underlying tissue and muscle, respectively.

[0158] Needleless injectors are suitable for delivering compositions to all types of tissue, particularly the skin and mucosa. In some embodiments, a needleless injector can be used to propel a liquid containing a composition onto the surface and into the skin or mucosa of a subject. Representative examples of various types of tissue that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, sublingual, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessel, or any combination thereof.

[0159] The MID can have needle electrodes for electroporating tissue. For example, by generating pulses between multiple electrode pairs in a plurality of electrode arrays arranged in a rectangular or square pattern, it provides improved results compared to generating pulses between a pair of electrodes. For example, U.S. Patent No. 5,702,359 entitled "Needle Electrodes for Mediated Delivery of Drug and Genes" discloses an array of needles where multiple pairs of needles can be pulsed during treatment. In this application, which is incorporated herein by reference in its entirety, the needles were arranged in a circular array, but had connectors and switching devices that enabled pulsing between opposing pairs of needle electrodes. A pair of needle electrodes can be used to deliver a recombinant expression vector to cells. Such devices and systems are described in U.S. Patent No. 6,763,264, the content of which is incorporated herein by reference. Alternatively, a single-needle device that allows for DNA injection and electroporation with a single needle similar to a conventional injection needle and applies a lower voltage pulse than that delivered by currently used devices can be used, thus reducing the electrical sensation experienced by the patient.

[0160] The MID may include one or more electrode arrays. The arrays may include two or more needles of the same diameter or different diameters. The needles may be spaced evenly or unevenly. The needles may be from 0.005 inches to 0.03 inches, from 0.01 inches to 0.025 inches, or from 0.015 inches to 0.020 inches. The needles may be 0.0175 inches in diameter. The needles may be at intervals of 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more.

[0161] The MID may be composed of a pulse generator and two or more needle composition injectors that deliver the composition and the electroporation pulse in a single step. The pulse generator may enable flexible programming of the pulses and injection parameters, as well as comprehensive recording and storage of electroporation and patient data, via a personal computer operated by a flash card. The pulse generator can deliver various voltage pulses in a short period of time. For example, the pulse generator can deliver three 15-volt pulses with a duration of 100 milliseconds. An example of such an MID is the Elgen 1000 system by Inovio Biomedical Corporation, the content of which is described in U.S. Patent No. 7,328,064, which is incorporated herein by reference.

[0162] The MID can be the CELLECTRA (Inovio Pharmaceuticals, Blue Bell PA) device and system, a modular electrode system that facilitates the introduction of macromolecules such as DNA into cells of selected tissues within the body or in plants. The modular electrode system may include a plurality of needle electrodes, a subcutaneous needle, an electrical connector that provides an electrically conductive link from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. The operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into selected tissues within the body or in plants. The macromolecule is then delivered to the selected tissue via the subcutaneous needle. The programmable constant current pulse controller is activated, and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the macromolecule into the cells between the plurality of electrodes. Cell death due to overheating of the cells is minimized by limiting the power consumption in the tissue by the constant current pulse. The Cellectra device and system are described in U.S. Patent No. 7,245,963, which is incorporated herein by reference.

[0163] The MID can be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device that provides a hollow needle and a fluid delivery means, where the device is adapted to operate the fluid delivery means during use to simultaneously (e.g., automatically) inject a fluid, which is a composition described herein, into the body tissue while inserting the needle into the body tissue. The advantage is that the fluid can be gradually injected while the needle is being inserted, leading to a more even distribution of the fluid throughout the body tissue. Also, the pain experienced during injection is thought to be reduced by the distribution of the volume of the fluid being injected over a larger area.

[0164] In addition, the automatic injection of the fluid facilitates the automatic monitoring and registration of the actual dosage of the injected fluid. This data can be stored by the control unit for documentation purposes, if needed.

[0165] The injection rate can be either linear or non-linear, and it is understood that the injection can be performed after the needle is inserted through the skin of the subject being treated and while they are further inserted into the body tissue.

[0166] Suitable tissues into which the fluid can be injected by the device of the present invention include tumor tissue, skin or liver tissue, but can also be muscle tissue.

[0167] The device further includes needle insertion means for guiding the insertion of the needle into the body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that both the needle insertion and injection of the injection fluid can be controlled to match the insertion rate to the injection rate as required. It also makes it easier for the user to operate. Means can be provided to automatically insert the needle into the body tissue if required.

[0168] The user can select when to start the injection of the liquid. However, ideally, the injection is started when the tip of the needle reaches the muscle tissue, and the device can include means for sensing when the needle has been inserted to a depth sufficient for the injection to start. This means that it can prompt the automatic start of the injection of the liquid when the needle reaches the desired depth (usually the depth at which the muscle tissue begins). The depth at which the muscle tissue begins can be a preset needle insertion depth such as a value of 4 mm which is considered sufficient for the needle to pass through the skin layer.

[0169] The sensing means may include an ultrasonic probe. The sensing means may include means for detecting a change in impedance or resistance. In this case, the means may be adapted to sense a change in impedance or resistance as the needle moves from different types of body tissue into muscle, rather than record the depth of the needle in the body tissue as such. Either of these alternatives provides a relatively accurate and simple operating means for the sensing means at which injection can be initiated. The depth of needle insertion can be further recorded as required and used to control the injection of fluid such that the volume of fluid injected is determined when the depth of needle insertion is recorded.

[0170] The apparatus may further comprise a base for supporting the needle and a housing for receiving the base therein, the base being movable relative to the housing such that the needle is retracted within the housing when the base is in a first rearward position relative to the housing and the needle extends outside the housing when the base is in a second forward position within the housing. This is advantageous for the user as the housing can be placed on the patient's skin and then the needle can be inserted into the patient's skin by moving the housing relative to the base.

[0171] As described above, it is desirable to achieve a controlled rate of fluid injection such that the fluid is uniformly distributed over the entire length of the needle as the needle is inserted into the skin. The fluid delivery means may include piston drive means adapted to inject the fluid at a controlled rate. The piston drive means can be actuated, for example, by a servo motor. However, the piston drive means can be actuated by a base that is axially moved relative to the housing. It will be understood that alternative means of fluid delivery may be provided. Thus, for example, a closed container that can be compressed for fluid delivery at a controlled or uncontrolled rate can be provided in place of a syringe and piston system.

[0172] The above-described device can be used for any type of injection. However, it is considered to be particularly useful in the field of electroporation, and thus, it may further include means for applying a voltage to the needle. Thereby, the needle can be used not only for injection but also as an electrode during electroporation. This is particularly advantageous because it means that the electric field is applied to the same region as the injected fluid. Conventionally, in electroporation, it has been very difficult to accurately align the electrodes with the previously injected fluid. Therefore, users tend to inject a larger volume of fluid than necessary in a larger area and apply the electric field in a higher area to ensure overlap between the injected substance and the electric field. Using the present invention, a good match between the electric field and the fluid can be achieved while reducing both the volume of the injected fluid and the magnitude of the applied electric field.

[0173] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of a recombinant nucleic acid sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot) or other assays well known to those skilled in the art.

[0174] Disease conditions and treatment methods In one embodiment, the present invention includes compositions and methods for treating cystic fibrosis by reversing the effect of existing mutations associated with nonsense mutations through the introduction of the synthetic oligonucleotide suppressor tRNA of the present invention.

[0175] Certain embodiments of the present disclosure provide a method of treating a mammalian disease, comprising administering to a mammal a protein or vector encoding a therapeutic agent (e.g., a modified and / or stabilized ACE-tRNA) described herein. In certain embodiments, the mammal is a human.

[0176] Certain embodiments of the present disclosure provide for the use of a therapeutic agent described herein or a vector encoding a therapeutic agent for preparing a medicament useful for treating mammalian diseases. Diseases or disorders associated with PTC include, but are not limited to, variations of Duchenne and Becker muscular dystrophy due to PTC in dystrophin, retinoblastoma due to PTC in RB1, neurofibromatosis due to PTC in NF1 or NF2, ataxia telangiectasia due to PTC in ATM, Tay-Sachs disease due to PTC in HEXA, cystic fibrosis due to PTC in CFTR, Wilms tumor due to PTC in WT1, hemophilia A due to PTC in factor VIII, hemophilia B due to PTC in factor IX, p53-related cancers due to PTC in p53, Menkes disease, Ullrich disease, β-thalassemia due to PTC in β-globin, type 2A and type 3 von Willebrand disease due to PTC in von Willebrand factor, Robinow syndrome, brachydactyly type B (shortening of fingers and metacarpal bones), genetic susceptibility to mycobacterial infection due to PTC in IFNGR1, hereditary retinal diseases due to PTC in CRX, hereditary bleeding tendency due to PTC in coagulation factor X, hereditary blindness due to PTC in rhodopsin, congenital sensorineural deafness and agangliosis coli due to PTC in SOX10, and hereditary neurodevelopmental disorders including sensorineural deafness, agangliosis coli, peripheral neuropathy and central dysmyelinating leukodystrophy due to PTC in SOX10, Riddle syndrome, pigmentary mosaicism, Fanconi anemia, anemia, hypothyroidism, p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes and kuru, and many other diseases or disorders.

[0177] The present disclosure also provides mammalian cells containing the vectors described herein. The cells can be human.

[0178] Certain aspects of the present disclosure relate to polynucleotides, polypeptides, vectors, and genetically engineered cells (modified in vivo), and their use. In particular, the present disclosure relates to methods for gene therapy that enable both systemic delivery of a therapeutically effective dose of a therapeutic agent.

[0179] According to one aspect, a cell expression system for expressing a therapeutic agent in a mammalian recipient is provided. The expression system (also referred to herein as a "genetically recombinant cell") includes a cell and an expression vector for expressing the therapeutic agent. Expression vectors include, but are 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 a lentivirus or retroviral vector.

[0180] The expression vector further includes a promoter for controlling transcription of the heterologous gene. The promoter can be an inducible promoter (described herein). The expression system is suitable for administration to a mammalian recipient. The expression system can include a plurality of non-immortalized genetically recombinant cells, each cell containing at least one recombinant gene encoding at least one therapeutic agent.

[0181] The cell expression system is formed in vivo. According to another aspect, a method for treating a mammalian recipient in vivo is provided. The method includes introducing an expression vector for expressing a heterologous gene product into the patient's cells in situ via intravenous administration or the like. To form the expression system in vivo, an expression vector for expressing the therapeutic agent is introduced intravenously in vivo into the mammalian recipient.

[0182] According to another aspect, a method for treating a mammalian recipient in vivo is provided. The method includes introducing a target therapeutic agent into the patient in vivo.

[0183] An expression vector for expressing a heterologous gene may include an inducible promoter for controlling the transcription of the heterologous gene product. Thus, in situ delivery of a therapeutic agent is controlled by exposing the cells in situ to conditions that induce transcription of the heterologous gene.

[0184] The present disclosure provides a method of treating a disease in a mammal by administering an expression vector to a cell or a patient. With respect to gene therapy methods, those of ordinary skill in the art of molecular biology and gene therapy will be able to determine the appropriate dosage and route of administration of the expression vectors used in the novel methods of the present disclosure without undue experimentation.

[0185] The present disclosure provides a method of treating a disease in a mammal by administering at least one ACE-tRNA to a cell or a patient. With respect to gene therapy methods, those of ordinary skill in the art of molecular biology and gene therapy will be able to determine the appropriate dosage and route of administration of the ACE-tRNA used in the novel methods of the present disclosure without undue experimentation.

[0186] In one embodiment, the present disclosure provides a method of treating a disease in a mammal by administering to a cell or a patient at least two, at least three, at least four, or more than four ACE-tRNAs or nucleic acid molecules encoding ACE-tRNAs. In one embodiment, the method comprises administering a plurality of ACE-tRNAs or nucleic acid molecules encoding a plurality of ACE-tRNAs, each of the plurality of ACE-tRNAs being specific for the incorporation of a distinct amino acid from another ACE-tRNA in the composition. For example, in one embodiment, the method comprises administering a combination of a first ACE-tRNA for incorporating Arg into a polypeptide and a second ACE-tRNA for incorporating Gly into a polypeptide. In one embodiment, the plurality of ACE-tRNAs are specific for a PTC (e.g., UGA). In one embodiment, the plurality of ACE-tRNAs are specific for different PTCs.

[0187] According to one embodiment, cells are transformed or otherwise genetically engineered in vivo. Cells derived from a mammalian recipient are transformed in vivo (i.e., transduced or transfected) 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.

[0188] As used herein, "exogenous genetic material" refers to any natural or synthetic nucleic acid or oligonucleotide that is not naturally found within a cell or, if naturally found within the cell, is not transcribed or expressed at a biologically significant level by the cell. Thus, "exogenous genetic material" includes, for example, nucleic acids of non-natural origin that can be transcribed into tRNA.

[0189] The above-disclosed therapeutic agents and states suitable for gene therapy are merely exemplary and are not intended to limit the scope of the present disclosure. The selection of a suitable therapeutic agent for treating a known condition is considered to be within the scope of one of ordinary skill in the art without undue experimentation.

[0190] In certain embodiments, the therapy has potential use in the treatment / management of diseases caused by premature termination codons (PTCs), including, but not limited to, Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia telangiectasia, Tay - Sachs disease, cystic fibrosis, Wilms tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich disease, β - thalassemia, von Willebrand disease types 2A and 3, Robinow syndrome, brachydactyly type B (shortening of the fingers and metacarpals), genetic susceptibility to mycobacterial infection, hereditary retinal diseases, hereditary bleeding tendencies, hereditary blindness, congenital sensorineural deafness and Hirschsprung disease, and hereditary neurodevelopmental disorders including sensorineural deafness, Hirschsprung disease, peripheral neuropathy and central dysmyelinating leukodystrophy, Riddle syndrome, xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53 - related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes and kuru. This therapy is advantageous in that it provides improved termination codon suppression specificity. The therapeutic ACE - tRNA of the present invention targets, for example, the specific termination codon TGA, thus reducing off - target effects at termination codons unrelated to the disease. This therapy is also advantageous in providing amino acid specificity. The expressed tRNA is designed to specifically substitute the amino acid lost through the insertion of the disease stop codon, thus negating any spurious effects on protein stability, folding and transport.

[0191] In certain embodiments, the system is modular and thus can be "customized" for any possible disease PTC. For example, there are nine individual tryptophan tRNAs in the human genome recognized by tryptophan synthase, all of which suppress the mRNA UGG codon. Thus, each of these nine Trp tRNAs provides an opportunity for codon recoding resistance (UGG→UGA). Further, considering the proximity to the stop codon in the genetic code, mutations of arginine codons to PTC nonsense codons are common in diseases. There are more than 30 Arg tRNAs that can be tested for codon editing resistance and suppression effects.

[0192] A further advantage of the present invention is that the entire system (tRNA + promoter sequence) is compact, thus providing easy expression and cell-specific delivery.

[0193] Dosage, formulation and route of administration of the medicament of the present invention The medicament of the present invention is administered to bring about a reduction in at least one symptom associated with a genetic disease (e.g., cystic fibrosis). The amount administered will vary depending on a variety of factors including the composition selected, the particular disease, body weight, health status, and age of the mammal, as well as whether prevention or treatment is to be achieved, among others. Such factors can be readily determined by a clinician using animal models or other test systems well known in the art.

[0194] The present invention contemplates treating a disease or disorder associated with PTC by administration of a medicament of the present invention, such as ACE-tRNA, an expression vector, or viral particles. Administration of the therapeutic agent according to the present invention can be continuous or intermittent, depending on, for example, whether the purpose of administration is therapeutic or prophylactic and other factors known to those skilled in the art, depending on the physiological state of the recipient. Administration of the medicament of the present invention may be essentially continuous over a preselected period of time or may be a series of spaced doses. Both local and systemic administration are contemplated.

[0195] One or more suitable unit dosage forms having the therapeutic agent(s) of the present invention may be formulated for controlled release, optionally (e.g., using microencapsulation), as discussed below, and may be administered by various routes including parenteral, including intravenous and intramuscular routes, as well as by direct injection into diseased tissue. The formulations may, where appropriate, be conveniently presented in individual unit dosage forms and may be prepared by any of the pharmaceutically well-known methods. Such methods may include associating the therapeutic agent with a liquid carrier, solid matrix, semi-solid carrier, finely divided solid carrier or combinations thereof, and then, optionally, introducing or shaping the product into the desired delivery system.

[0196] When the therapeutic agents of the present invention are prepared for administration, they can be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation or unit dosage form. The total active ingredient in such formulations comprises from 0.1 to 99.9% by weight of the formulation. "Pharmaceutically acceptable" carriers, diluents, excipients, and / or salts are those which are compatible with the other ingredients of the formulation and not harmful to its recipient. The active ingredient for administration may be present as a powder or granule, as a solution, suspension or emulsion.

[0197] The pharmaceutical formulations containing the therapeutic agents of the present invention can be prepared by procedures well known in the art using well-known and readily available ingredients. The therapeutic agents of the present invention may be formulated, for example, as a solution suitable for parenteral administration by intramuscular, subcutaneous or intravenous routes.

[0198] The pharmaceutical formulations of the therapeutic agents of the present invention may take the form of aqueous or non-aqueous solutions or dispersions, or alternatively, emulsions or suspensions.

[0199] Accordingly, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, e.g., by bolus injection or continuous infusion) and may be presented in unit dosage form in an ampoule, prefilled syringe, small volume infusion container, or multi-dose container having added preservatives. The active ingredient may take the form of a suspension, solution, or emulsion, etc. in an oily vehicle or an aqueous vehicle, and may contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in powder form obtained by lyophilization from a sterile solid isolation or solution for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0200] It will be understood that the unit content of the active ingredient(s) contained in each aerosol dosage of each dosage form need not itself constitute an effective amount for treating a particular indication or disease, since the effective amount can be reached by administration of a plurality of dosage form units. Further, the effective amount can be achieved using less than the dosage in the dosage form, either individually or in any series of administrations.

[0201] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the types well known in the art. Specific non-limiting examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered aqueous saline solutions, such as phosphate buffered saline solutions pH 7.0 - 8.0 and water.

[0202] Route of Administration Provided herein is a method of treating, protecting, and / or preventing a PTC-related disease in a subject in need of treatment, protection, and / or prevention by administering to the subject one or more of the compositions described herein.

[0203] The dosage of the composition can be 1 μg to 10 mg of the active ingredient / kg body weight / time, and can be 20 μg to 10 mg of the ingredient / kg body weight / time. The composition can be administered once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, once every 30 days, or once every 31 days. The number of administrations of the composition for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0204] The composition can be formulated according to standard techniques well known to those skilled in the pharmaceutical art. Such a composition can be administered at dosages and by techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight, and condition of the particular subject, as well as the route of administration.

[0205] The composition can be administered prophylactically or therapeutically. In therapeutic use, the composition is administered to a subject in need of treatment in an amount sufficient to elicit a therapeutic effect. The amount sufficient to achieve this is defined as a "therapeutically effective dose". The amount effective for this use will depend, for example, on the particular composition of the administered composition regimen, the method of administration, the stage and severity of the disease, the general health of the subject, and the judgment of the prescribing physician.

[0206] The composition can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)), Felgner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996), Felgner (U.S. Patent No. 5,703,055, issued December 30, 1997), and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), the entire contents of all of which are hereby incorporated by reference in their entirety. The DNA of the composition can be complexed, for example, into particles or beads for administration to an individual using a vaccine gun. One of ordinary skill in the art will know that the choice of a pharmaceutically acceptable carrier, which includes physiologically acceptable compounds, depends, for example, on the route of administration of the expression vector.

[0207] The composition can be delivered via various routes. Typical routes of delivery include parenteral administration, for example, intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. In particular, for the DNA of the composition, the composition can be delivered into the interstitial space of an individual's tissue (Felgner et al., U.S. Patents Nos. 5,580,859 and 5,703,055, the entire contents of which are hereby incorporated by reference in their entirety). The composition can also be administered to muscle or via intradermal or subcutaneous injection or transdermally, for example, by iontophoresis. Epidermal administration of the composition can also be used. Epidermal administration can involve mechanically or chemically irritating the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are hereby incorporated by reference in its entirety).

[0208] The composition can also be formulated for administration via the nasal cavity. Formulations suitable for nasal administration, where the carrier is solid, can include, for example, coarse powders having a particle size in the range of about 10 to about 500 microns, which are administered in the manner of taking sniff tobacco, i.e., by rapid inhalation through the nasal cavity from a container of powder held near the nose. The formulation may be by nasal spray, nasal drops, or aerosol administration by nebulizer. The formulation can include an aqueous or oily solution of the composition.

[0209] The composition can be a liquid preparation such as a suspension, syrup, or elixir. The composition can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.

[0210] The composition can be incorporated into liposomes, microspheres, or other polymeric matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd Edition 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids and can be non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0211] ACE-tRNA or a nucleic acid molecule encoding ACE-tRNA can be administered by various routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof. For veterinary use, the composition can be administered as a suitably acceptable formulation following normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The composition may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gone gun", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

[0212] ACE-tRNA or a nucleic acid molecule encoding ACE-tRNA can be delivered to mammals by several well-known techniques including, with and without recombinant vectors such as in vivo electroporation, liposome-mediated, nanoparticle-facilitated, recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia, and DNA injection (also referred to as DNA vaccination). ACE-tRNA or a nucleic acid molecule encoding ACE-tRNA can be delivered together with in vivo electroporation via DNA injection.

[0213] Electroporation Administration of a composition via electroporation can be achieved using an electroporation device configured to deliver an energy pulse effective to form reversible pores within a cell membrane to a desired tissue of a mammal, preferably, the energy pulse is a constant current similar to a preset current input by a user. The electroporation device can include an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include one or more of various elements of the electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch, and can be incorporated. Electroporation can be achieved using an in vivo electroporation device, such as the CELLECTRA EP system (Inovio Pharmaceuticals, Plymouth Meeting, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA), which can facilitate transfection of cells by a plasmid.

[0214] An electroporation component can function as one element of an electroporation device, and other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component can function as two or more elements of the electroporation device and can communicate with still other elements of an electroporation device separate from the electroporation component. Elements of an electroporation device that exist as part of one electromechanical device or mechanical device need not be limited because the elements can function as one device or as separate elements that communicate with each other. The electroporation component can be capable of delivering energy pulses that generate a constant current within a desired tissue and includes a feedback mechanism. The electrode assembly can include an electrode array having a plurality of spatially arranged electrodes, and the electrode assembly receives an energy pulse from the electroporation component and delivers it through the electrodes to the desired tissue. At least one of the plurality of electrodes is neutral during delivery of the energy pulse, measures the impedance within the desired tissue, and communicates the impedance to the electroporation component. The feedback mechanism can receive the measured impedance and adjust the pulse of energy delivered by the electroporation component to maintain a constant current.

[0215] The plurality of electrodes can deliver pulses of energy in a dispersed pattern. The plurality of electrodes can deliver pulses of energy in a dispersed pattern through control of the electrodes under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed sequence can include a plurality of pulses delivered during the sequence, and each pulse of the plurality of pulses is delivered by at least two active electrodes having one neutral electrode that measures impedance, and subsequent pulses of the plurality of pulses are delivered by a different one of the at least two active electrodes having one neutral electrode that measures impedance.

[0216] The feedback mechanism can be implemented by either hardware or software. The feedback mechanism can be implemented by an analog closed-loop circuit. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or substantially instantaneous (i.e., as determined by the available technology for determining the response time). The neutral electrode can measure the impedance within the desired tissue and transmit the impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulses to maintain the constant current at a value similar to the preset current. The feedback mechanism can maintain the constant current continuously and instantaneously during the delivery of the energy pulses.

[0217] Examples of electroporation devices and electroporation methods that can facilitate the delivery of the compositions of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al. and U.S. Patent Publication 2005 / 0052630 filed by Smith, et al. (the contents of which are hereby incorporated by reference in their entirety). Other electroporation devices and electroporation methods that can be used to facilitate the delivery of the composition include those provided in U.S. Patent Application No. 11 / 874,072 filed on October 17, 2007 (claiming the benefit of U.S. Provisional Application No. 60 / 852,149 filed on October 17, 2006 and U.S. Provisional Application No. 60 / 978,982 filed on October 10, 2007 under 35 U.S.C. § 119(e), all of which are hereby incorporated by reference).

[0218] U.S. Patent No. 7,245,963 by Draghia - Akli, et al. describes a modular electrode system and their use for facilitating the introduction of biomolecules into cells of selected tissues in vivo or in plants. The modular electrode system can comprise a plurality of needle electrodes, a subcutaneous needle, an electrical connector providing an electrical link from a programmable constant - current pulse controller to the plurality of needle electrodes, and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and securely insert them into selected tissues in vivo or in plants. Then, biomolecules are delivered to the selected tissues via the subcutaneous needle. The programmable constant - current pulse controller is activated and a constant - current electrical pulse is applied to the plurality of needle electrodes. The applied constant - current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is incorporated herein by reference.

[0219] U.S. Patent Publication No. 2005 / 0052630, filed by Smith, et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues in vivo or in plants. The electroporation device comprises a conductive device (an "EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant - current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and enables the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Publication No. 2005 / 0052630 is incorporated herein by reference.

[0220] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 0052630 can be adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Due to the configuration of the electrode array, an injection needle (for delivering selected biomolecules) is also fully inserted into the target organ, and the injection is administered perpendicular to the target subject in a region predefined by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 005263 are preferably 20 mm in length and 21 gauge.

[0221] In addition, some embodiments that contemplate an electroporation device and its use include electroporation devices as described in the following patents: U.S. Patent No. 5,273,525, issued December 28, 1993; U.S. Patent No. 6,110,161, issued August 29, 2000; U.S. Patent No. 6,261,281, issued July 17, 2001; U.S. Patent No. 6,958,060, issued October 25, 2005; U.S. Patent No. 6,939,862, issued September 6, 2005. Further, patents that encompass the subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, regarding DNA delivery using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, regarding a method of DNA injection, are contemplated herein. The above patents are hereby incorporated by reference in their entirety.

[0222] Method for preparing ACE-tRNA Provided herein is a method for preparing a DNA plasmid containing ACE-tRNA as discussed herein. The DNA plasmid can be used to inoculate cell cultures in a large-scale fermentation tank using methods known in the art after the final subcloning step into a mammalian expression plasmid.

[0223] The DNA plasmids for use in the EP devices of the present invention can be formulated or manufactured using combinations of known devices and techniques, but are preferably manufactured using the optimized plasmid manufacturing techniques described in U.S. Published Application No. 2009 / 0004716, filed May 23, 2007. In some embodiments, the DNA plasmids used in these studies can be formulated at a concentration of 10 mg / mL or greater. The manufacturing techniques also include, in addition to those described in U.S. Patent Application No. 60 / 939,792, which is the subject of a patent issued on July 3, 2007 as U.S. Patent No. 7,238,522, various devices and protocols generally known to those of skill in the art, or incorporate them. The above applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, respectively, are hereby incorporated by reference in their entirety.

[0224] Definitions Disease state: For the purposes of the present invention, a "disease state" or "disease phenotype" is a characteristic of mammalian cells resulting from a stop codon within the coding region of a gene within the cell (e.g., resulting from a nonsense mutation). For example, an increase in human genetic diseases is thought to be caused by nonsense mutations (see, e.g., Atkinson et al., Nuc. Acids Res. 22:1327, 1994). By way of example, β-thalassemia, Duchenne muscular dystrophy, albinism, Fanconi anemia, and cystic fibrosis can all be caused by nonsense mutations in the identified genes.

[0225] Endogenous tRNA synthetase: A tRNA synthetase is considered to be "endogenous" to a cell if it is present within the cell into which the tRNA is introduced according to the present invention. As will be apparent to those of skill in the art, a tRNA synthetase can be considered endogenous for these purposes regardless of whether it is naturally found in the relevant type of cell, or whether the particular cell in question has been engineered, or has been engineered by human hand to contain or express it.

[0226] Suppressor tRNA: A "suppressor tRNA" is one in which the codon and anticodon are complementary to a codon that would otherwise terminate translation and in which detectable readthrough occurs under experimental conditions. The standard termination codons are the amber (UAG), ochre (UAA), and opal (UGA) codons. However, non-standard termination codons (e.g., four-nucleotide codons) have also been used in the literature (see, for example, Moore et al., J. Mol. Biol. 298:195, 2000; Hohsaka et al., J. Am. Chem. Soc. 121:12194, 1999).

[0227] Here, the present invention is illustrated by the following non-limiting examples.

Example

[0228] Example 1 The genetic code uses four nucleotides to form triplet codons that underlie the translation from DNA to protein. There are 64 codons in total, 61 of which are used to code for amino acids, and 3 of which (TAG, TGA, and TAA) code for "stop" or "nonsense" codons at the protein terminus.

[0229] Five to ten percent of cases of cystic fibrosis are caused by "nonsense" mutations that result in premature truncation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. An example of this "class 1" mutation is p.Trp1282X, a premature termination codon (PTC), which causes loss of CFTR function and a severe cystic fibrosis phenotype. Although several compounds, such as ataluren, promote stop codon readthrough of disease-causing nonsense mutations, they have not been very successful as therapies because of many caveats, including poor stop codon specificity and unexpectedly low efficiency of codon skipping in vivo. However, the widespread use of these compounds and the discovery that endogenous stop codon readthrough is common in metazoans suggest that it may be possible to supplement suppression in a subset of cell types, namely airway epithelia, when delivered to them. However, if therapeutically assisted stop codon readthrough is successful, non-selective incorporation of amino acids at the position of the nonsense codon can affect protein folding, trafficking, and function (as in the case of CFTR1282X), necessitating additional therapeutic intervention. Thus, an acute unmet need exists to understand the nature of the disease PTCs and potential therapeutic suppressors, generally, more effective therapies for PTC diseases.

[0230] This example features an anticodon-edited (ACE) Trp-tRNA for the rescue of the CFTRp.Trp 1282X channel. Such tRNAs are engineered to "suppress" the disease-causing TGA stop codon and incorporate the original amino acid Trp in p.Trp1282X CFTR, effectively genetically reconstructing the wild-type CFTR protein. Data show that this general approach (nonsense suppression) results in a robust rescue of transcripts bearing in-frame stop codons through either transient transfection of tRNA and its cognate synthetase in adherent cells or virus-based delivery to their more native airway cell types, such as A549 airway cells. This approach offers several major advantages over existing strategies. 1) Improved codon specificity - the expressed tRNA can be targeted to specific stop codons, reducing off-target effects at stop codons unrelated to the disease. 2) Amino acid specificity - the expressed tRNA and / or synthetase can be engineered to replace the amino acid lost through the insertion of the disease stop codon, thus negating any spurious effects on CFTR stability, folding, and trafficking. 3) Tunability - the system can be theoretically individualized for each type of tRNA and PTC mutation. 4) Easy expression - the entire system is compact (<1 kb) and can be easily packaged and expressed transiently or via nanoparticle delivery of the tRNA. 5) Proof of principle for a general strategy - in-frame stop codons are a major cause of human disease and there are few treatment options. Experiments performed with p.Trp1282X are expected to lead to insights into the mechanisms of other CFTR nonsense codons.

[0231] The data show that an ACE-tRNA stop codon suppressor tRNA is effective in "rescuing" transcripts containing the introduced stop site (Figures 6A and 6B), suggesting that such tRNAs have the potential to interfere with the nonsense mutation-dependent decay mechanism (NMD) as a major biological hurdle in the therapeutic rescue of disease stop sites. Thus, the use of suppressor tRNAs opens up the possibility of obtaining more molecular insights into NMD in disease.

[0232] Results Eukaryotic tRNAs with anticodon editing that suppress stop sites, such as TGA, were developed that are not their designated codons. These were tested on a test construct consisting of a fluorescent protein (cherry) in frame with an eGFP sequence separated by a linker containing the TGA site on five human tryptophan tRNAs. To show production of the full-length protein, an HA epitope was added to the C-terminus of the eGFP reading frame. This test system is useful for showing visual appearance of the cherry signal for plasmid delivery and expression and for showing TGA suppression in combination with eGFP rescue. The data in Figures 6A and 6B show Western blot data using this test construct to assay the ability of five anticodon-edited Trp tRNAs human to suppress the TGA stop site in the short linker between 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 resistance to the mutation or the ability of the Trp synthase to recognize and / or acylate the tRNA with tryptophan, even by changing only a single base in the anticodon. However, the fourth of these test tRNAs (tRNA#4) shows significant suppression activity of the TGA site and produces the full-length cherry-eGFP-HA protein (Figure 6B). Furthermore, in the last lane of Figure 6B, no readthrough was seen in the absence of co-expressed tRNA.

[0233] Methods The Trp tRNA was examined for its ability to suppress the codon editing-resistant (TGG→TGA) and transiently transfected tandem fluorophore (mCherry-TGA-GFP) and the target TGA test site of CFTRTrp1282X. In the initial screening of 5 / 9 Trp tRNA, an anticodon-edited Trp-tRNA was discovered, which has a "stand-alone" function to rescue the cherry-TGA-eGFP-HA test construct when transiently transfected in HEK cells (Figure 6B). The selective presence of the HA epitope indicates successful rescue and confocal examination of both cherry and eGFP fluorescence at the single-cell level (not shown). This result provides proof-of-principle data that a) some ACE-tRNAs can tolerate anticodon editing, b) these tRNAs retain the ability to be acylated with Trp by endogenous tryptophan synthase, and c) these tRNAs can suppress TGA sites embedded within the open protein reading frame.

[0234] The remaining four Trp-tRNAs are functionally tested for their resistance to anticodon editing from TAA to TGA suppressors. These anticodon-edited tRNAs are tested for their ability to rescue the cherry-TGA-eGFP-HA clone. Biochemical (western blot) data are obtained for the cherry and eGFP signals as well as the HA epitope. Here, the expression of cherry serves as a positive transfection control. Confocal imaging verifies cherry and eGFP fluorescence at the single-cell level.

[0235] The fidelity of the endogenous Trp synthase that charges ACE-Trp tRNA with tryptophan amino acid is determined by mass spectrometry of tryptic digestion fragments of the purified rescued cherry-Trp-eGFP HA protein. The predicted masses of the tryptic digestion fragments generated from the linker between the reading frames of cherry and eGFP are as follows. KPINQWPANTHER with a predicted mass of 1590.8135, bold W indicates the incorporation site, Figure 10. Thus, this represents the first example of nonsense codon repair and substitution with a wild-type amino acid and is thus a significant advancement over existing approaches such as therapeutic ataluren. In the examples described below, the discovery and identification of new tRNA sequences that provide stringent repair is important because the compound promotes readthrough of nonsense codons with incorrect amino acids.

[0236] Rescue of the CFTR 1282X channel transiently transfected with the ACE-tRNA identified above is evaluated by standard biochemical methods for the full maturation of the B and C glycosylated CFTR bands 20. Thus, the channel is repaired with wild-type amino acids, fully functional, and successfully transported to the cell membrane.

[0237] The next step is to functionally characterize the CFTR Trp1282X channel rescued in the ACE-tRNA system identified above using electrophysiological approaches (single cell patch clamp and Ussing chamber) and biochemical approaches. Evaluate the efficacy of the expressed tRNA by quantitative rtPCR to reduce the nonsense mutation-dependent decay mechanism (NMD) of 1282X mRNA. Use reprogrammed human airway cells to test codon editing Trp-tRNA rescue of the native 1282X CFTR channel.

[0238] Anticodon editing is permitted in the identified human Trp tRNA, and it is shown that this 75 base pair transfer RNA can suppress the in-frame TGA codon within the test construct. These experiments hypothesized this discovery and characterized the ability of this ACE-tRNA to interact with CFTR 1282TGA mRNA and generate functional CFTR channels in model cells (FRT and A549) as well as in asp.1282X human reprogrammed airway cells.

[0239] The transiently expressed CFTR1282X channel, and the rescue level biochemical determination antibody M3A7 in reprogrammed airway cells, recognizes the rescued (epitope is aa 1370 - 1380) and binds to the MM13 - 4 like N-terminus available through EMD Millipore (epitope aa25 - 36) and is used to detect all CFTR, rescued and non-rescued. Alternatively, L12B4 (epitope aa 386 - 412, EMD Millipore) or 660 (epitope aa576 - 585,) are available through Cystic Fibrosis Foundation Therapeutics.

[0240] Surface functionality is examined through electrophysiological approaches, patch clamp, and recordings in Ussing chambers. The stability and abundance of 1282X mRNA are assayed by quantitative rtPCR of RNA extracts from transiently expressing cells and reprogrammed airway cells.

[0241] Bioinformatics analysis of RNA transcriptome data from human airway cells identifies the abundance, context, and identity of TGA codons contained within transcripts. The top 10 expressed transcripts that use TGA as a normal termination site are followed up at the level of individual transcripts by protein biochemistry before and after ACE-tRNA expression. Biochemical and immunohistological probes for cell apoptosis are also used to examine the effect of ACE-tRNA on cell death.

[0242] In conclusion, the data indicate that ion channel genes with in-frame termination sites are suitable for this type of "rescue" (Figure 9), and the components of the system can be expressed by the virus in airway cells. Furthermore, the highly simplified form of this idea, human-derived ACE-tRNA, exhibits "stand-alone" ability to rescue the in-frame CFTR TGA codon in mammalian cell lines (Figure 9). This approach has many advantages compared to existing stop codon strategies and merits further examination with respect to the ability of ACE-tRNA to 1) suppress nonsense mutation-dependent degradation mechanisms, 2) function in lung cell preparations, and 3) specifically rescue CFTR 1282X.

[0243] Example 2 Several different nonsense mutations cause CF and thus account for approximately 10% of all CF diseases. Figure 7. These cases are concentrated in 10 specific genetic lesions: E60X, R75X, G542X, R553X, Q890X, Y1092X, R1158X, R1162X, and W1282X. A screening was developed to screen existing human tRNA sequences for modifications and resistance to anticodon editing. For this purpose, about 144 ACE-tRNAs were candidates to be tested for their ability to promote repair of disease-causing nonsense codons and expression of full-length proteins. Specifically, using the scheme described in Figure 11, a tRNA library was generated to identify novel tRNA sequences encoding ACE tRNAs with the ability to repair the top CF-causing nonsense mutations. Specifically, 10 ng of an annealing oligo encoding ACE-tRNA was combined with 50 ng of NanoLuc reporter plasmid, 1 μl of 10× CutSmart Buffer (NEB), 1 μl of T4 ligase (NEB), 10 mM ATP, and 1 μl of BbsI (NEB) and cycled in a thermocycler as described in Figure 11. 1 μl of the reaction was transformed into competent E. coli and the transformants were plated on ampicillin agar plates. One transformant per plate was picked, grown in 1 ml of LB under ampicillin selection, miniprepped, and the sequence verified.

[0244] First, screening tests were performed to identify the best ACE-tRNA candidates from tryptophan and glycine. 125 ng of sequence-verified miniprep cDNA of the NanoLuc reporter plasmid with ACE-tRNA was transfected into HEK cells using calcium phosphate. HEK cells were seeded at 4×10 4It was plated in a 96-well plate. 24 hours after transfection, the medium was replaced with 20 μl of PBS, and 15 μl of NanoGlo reagent (Promega) was added. The plate was read with a SpectraMax i3 (Molecular Devices). The data are from 3 or more replicates. Figure 8. The data show that most tRNAs exhibit insufficient codon editing resistance. However, through the discrimination of ACE-Trp and ACE-Gly tRNAs, distinct high-performance tRNAs emerge from the screen, which show rescue of nonsense codon-containing proteins at 20- to 130-fold above background.

[0245] To evaluate that these novel tRNAs can rescue the CFTR channel with a nonsense codon, they were co-expressed with the CFTR W1282X cDNA plasmid in mammalian HEK cells. The cell preparations were analyzed by a standard biochemical approach via Western blot evaluation of the CFTR protein. This method is very advantageous for this purpose as the CFTR protein shows an established multi-band pattern. Specifically, the "B" and "C" bands represent the full-length and fully mature, post-translationally progressed CFTR protein at the cell surface, respectively. In this case, rescue by both the ACT-tRNAs of Trpchr17.tRNA39 and Glychr19.trna2 generated a strong population of "B" and "C" CFTR immunoreactive (antibody MA37) bands, indicating promotion of the normally transported ion channel protein by the full-length tRNAs. Figure 9.

[0246] Example 3 Nonsense suppression is significantly improved by T-stem modification. Figure 10. Additional modifications of tRNA were made to further enhance their functions for the purpose of suppressing nonsense codons and promoting protein expression. Without being bound by theory, it was hypothesized that mutations rationally introduced within the tRNA 't-stem' loop shown in Figure 10 would result in a more stable and functionally more potent tRNA molecule for nonsense codon suppression. For this purpose, single and double mutations were directly engineered into the t-stem loop of tRNATrpchr17.trna39, an ACE-tRNA identified for activity in the rescue of the tryptophan TGA nonsense codon. Thus, 38 tRNA t-stem variants were generated and screened in HEK cells transiently transfected with the nonsense rescue reporter construct shown in Figure 4. Twenty-four hours after transfection, the cells were assayed for luciferase activity as shown in Figure 10. The data show strong variability and identify novel tRNA sequences with various t-stem loop sequences that enhance suppression activity. In particular, such a mutant, TS-3852-62G-C, enhances the suppression ability of Trpchr17.trna39 by approximately 250% (Figure 12). This is a generalizable modification, i.e., the new tRNA sequences identified by Examples 1 and 2 can be made better through further theoretical modifications (for the ability to rescue nonsense codons). Such an approach aids in the therapeutic utility of ACE-tRNA for tissue types with low levels of target RNA or where tRNA delivery may be limited.

[0247] Example 4 To enable the identification of the nucleotide composition and functional ability of a new type of tRNA to suppress nonsense codons, an all-in-one plasmid with a one-pot cloning reaction was invented for high-throughput cloning, Figure 11. This approach enables the easy investigation of ACE-tRNA activity via luciferase activity in a standard 96-well format. Briefly, the synthetic nucleotide sequence encoding the tRNA is ligated into the NanoLuc reporter plasmid, along with an example of the TGA nonsense reporter plasmid variant shown in Figure 11. TAA (opal) and TAG (amber) stop codon rescue vectors are well-designed and implemented in Figures 16-19. The advantage of this is that this approach can ligate DNA oligos encoding the tRNA library into the NanoLuc reporter plasmid in a reaction that drives the almost 100% incorporation of the tRNA insert (Figure 11) in the presence of restriction enzymes and ligase, i.e., the reaction called "one-pot". The reactants are transformed into E. coli, and the resulting cDNA is purified by standard methods. Another advantage of the method of the present invention is that since the tRNA and the reporter gene are within a single expression cassette, it reduces biological variability and improves the quality of the data obtained in the screening resulting from the tRNA suppression activity. The purified cDNA plasmid is then screened in a high-throughput 96-well format for its ability to repair nonsense codons by putative luciferase activity. This approach is suitable for high-throughput screening of hundreds to thousands of tRNAs for novel therapeutic activities.

[0248] The "one-pot" cloning and expression system described in FIG. 11 has been used successfully to identify unique tRNA sequences for the repair of tryptophan and glycine ACE-tRNA (FIG. 13), ACE-tRNA-Arg (FIG. 14), ACE-tRNA-GlnTAG (FIG. 15), ACE-tRNA-Gln TAA (FIG. 16), ACE-tRNA-Glu TAG (FIG. 17), ACE-tRNA-Gln TAA (FIG. 18), and ACE-tRNA-Trp TAG (FIG. 19). FIGS. 20A-20D show that delivery of ACE-tRNA as a small molecule RNA supports strong suppression of the G542X and W1282X nonsense mutations.

[0249] Example 5 Engineered Transfer RNAs for Suppression of Premature Termination Codons Abstract Premature termination codons (PTCs) are responsible for 10 - 15% of all genetic diseases. PTC suppression during translation offers a promising approach for treating various genetic disorders, but small molecules that promote PTC readthrough have yielded variable performances in clinical trials. High-throughput, cell-based assays are presented to identify anticodon engineered transfer RNAs (ACE-tRNAs) that can effectively suppress in-frame PTCs and faithfully encode their cognate amino acids. Overall, ACE-tRNAs were identified with high suppression activity targeting nonsense codons that cause the most common human diseases. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNAs at levels that repair PTCs indicates limited interaction with translation termination codons. These ACE-tRNAs show high suppression in vivo in mammalian cells, Xenopus oocytes, and mice, causing PTC repair in multiple genes, including mutations causing disease within the cystic fibrosis conductance regulator (CFTR).

[0250] First Premature termination codons (PTCs) arise from single nucleotide mutations that convert a standard triplet nucleotide codon into one of the three stop codons, e.g., TAG, TGA, or TAA. PTCs are often more harmful than missense mutations because they result in loss of protein expression. In addition, mRNA abundance is reduced via the nonsense-mediated decay mechanism (NMD), and in some cases, the truncated proteins can have a dominant-negative function 1~3 and can be harmful. Therefore, it is natural that PTCs are associated with many severe disease phenotypes, including cystic fibrosis 4 , Duchenne muscular dystrophy, spinal muscular atrophy 5 , neuronal ceroid lipofuscinosis, 6 , β-thalassemia 7 , cystinosis 8 , X-linked nephrogenic diabetes insipidus 9 , Hurler syndrome 10 , Asherman syndrome 11 , and polycystic kidney disease. In addition, nonsense mutations occur within the tumor suppressor genes p53 and ATM 12 and suggest their roles in those diseases. The most vulnerable amino acid codons 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). Thus, PTCs represent a distinct group of diseases that account for 10–15% of all genetic diseases and affect more than 30 million people worldwide. 13

[0251] Aminoglycosides 14 dipeptides 15 , and oxadiazoles 16 and other small molecules promote "readthrough" or "suppression" of nonsense mutations. These compounds are effective in model organisms 17、18 , mammalian cell lines 19 and some animal disease models 16、20 . However, this approach is limited to amino acids that are nearly homologous21 results in encoding, effectively generating missense mutations in PTC, which in itself can have a detrimental impact on protein folding, transport, and function. Furthermore, aminoglycosides are 22 ototoxic and nephrotoxic, and ataluren, the first oxadiazole within its class, has shown unexpectedly low efficacy in patient populations (ACT DMD Phase 3 clinical trial, NCT 01826487; ACT CF, NCT 02139306), thus limiting their usefulness as PTC therapeutics. Recent and ongoing advancements in CRISPR / Cas9-mediated genome editing have the potential to provide a permanent solution for diseases caused by nonsense mutations. However, aspects of this technology pose 23、24 hurdles for rapid use as therapeutics. This is not limited to the requirement for "precision" or "personalized" diagnosis for each mutation, based on the genetic diversity situation of each patient.

[0252] PTC repair approaches have been identified that exhibit the versatility of small molecules and the precision of gene editing. tRNAs were investigated to meet these criteria, and their anticodons were thereby engineered via mutagenesis to recognize and suppress UGA, UAA, or UAG PTC codons. To exert an effect, the anticodon-edited tRNAs, also known as ACE-tRNAs, should still be recognized by the endogenous translation cellular machinery, which includes the aminoacyl-tRNA synthetase for charging their cognate amino acids and eukaryotic elongation factor 1a (eEF-1α) for delivering the charged tRNAs to the ribosome (Figure 21A). Such suppressor tRNAs have been shown to rescue in-frame stop codons associated with 25 β-thalassemia 26 xeroderma pigmentosum 27 and transgenic PTC reporter genes.

[0253] Here, it is shown that the anticodon editing approach can be generalized to multiple tRNA gene families, and that many annotated tRNAs are biologically viable. Furthermore, it has been demonstrated that anticodon-edited suppressor tRNAs encode their cognate amino acids, lack significant interactions with the terminal stop codons, and are effective in vivo for suppressing PTC. Overall, this data supports the possibility that such engineered tRNAs meet the broad requirements for covering disease-causing PTCs and thus represent a promising new class of RNA therapeutics.

[0254] Results The rationale for this study is based on the observation that there are multiple tRNA genes with unique sequences (isodecoders) for a given cognate amino acid (isoacceptor), resulting in over 400 tRNAs annotated in the human genome (http: / / lowelab.ucsc.edu / GtRNAdb / ). First, tRNA genes were examined to identify individual ACE-tRNAs that retain the ability to suppress PTC in mammalian cells. To maximize sequence coverage, an all-in-one cDNA plasmid was generated that supports both high-throughput cloning (HTC) of ACE-tRNAs and quantitative measurement of PTC suppression using luminescence after delivery to mammalian cells (Figure 21B). The ACE-tRNA sequences were cloned into the HTC plasmid as DNA oligos using a combination of ccdB negative selection 28、29 and Golden Gate cloning 31 paired with 30 This strategy yielded a cloning efficiency of approximately 100%. The ACE-tRNA suppression efficiency was read from the split NanoLuc luciferase (NLuc) NanoBiT platform, whereby the PTC of interest (UGA, UAA, or UAG) was introduced in-frame at the junction between the large bit domain and the small bit domain using a 96-well format (Figure 21B). 32, normalized to the background obtained from NLuc-PTC-expressing cells. Twenty-one glycine ACE-tRNAs were first evaluated for suppression of the UGA PTC (Figure 22, upper left, column 1 (violet)). The ACE-tRNA Gly Most of the sequences were unable to suppress the UGA NLuc PTC, but three Gly-tRNAs UGA were identified with high suppression yields (about 100-fold of the background). Considering the high sequence conservation among the Gly-tRNAs screened for anticodon tolerance (Figure 27), it is difficult to newly predict which tRNA is most suitable for anticodon editing.

[0255] Next, for each of the single nucleotide mutations that could potentially produce disease-causing PTCs, screening was performed on codon-edited tRNAs: Arg-tRNA UGA , Gln-tRNA UAA , Gln-tRNA UAG Trp-tRNA UGA , Trp-tRNA UAG , Glu-tRNA UAA , Glu-tRNA UAG , Cys-tRNA UGA , Tyr-tRNA UAG , Tyr-tRNA UAA , Ser-tRNA UAG, Leu-tRNA UAG , Leu-tRNA UAA , Lys-tRNA UAG , Lys-tRNA UGA , and Ser-tRNA UAG。Since the enzyme activity of NLuc was not significantly affected by the introduced amino acids (Figure 28), the difference in NLuc luminescence was due to the ACE-tRNA suppression ability. In the screening, multiple ACE-tRNAs were identified for each of the amino acids and stop codon types, and had suppression coverage for all three stop codons (Figure 22). Many of these ACE-tRNAs showed a strong activity with more than 100-fold PTC suppression over background, which was significantly higher than the aminoglycosides used in this study. Interestingly, some ACE-tRNAs showed a clear preference for specific anticodon editing, potentially reflecting altered aminoacyl-tRNA synthetases that bind to the tRNA anticodon isoacceptor sequence 33 For example, in the conversion of tryptophan to UAG suppression, a 10-fold higher rescue was obtained than for the UGA editing of the same ACE-tRNA Trp . However, for glutamine, the opposite was true, with a clear preference shown for UAA over UAG. In particular, in each case, multiple high-performance suppressors were identified, which was especially evident for the PTC of Arg UGA which plays a particularly large role in human diseases, and 20 efficient ACE-Arg UGA suppressors were identified. In other cases such as ACE-tRNA Glu showing function, the suppression efficiency was approximately the same for UAA and UAG. And a similar pattern was found in ACE-tRNA Lys strongly reflecting coding via UAG or UGA suppression. For Gln-tRNA UAA , the suppression activity resulted in a suppression signal more than 2,000-fold over background. Among the ACE-tRNAs identified during the screening, the tryptophan tRNA gene family showed the weakest suppression activity against the UGA PTC. Since there were only 6 unique human ACE-tRNA Trp sequences that could be screened, the UGA suppression ACE-tRNA Trp library was expanded using tRNAs from various species. UGA anticodon editing resistance was achieved with miscoding A9C tRNATrp and the bacterial Hirsh Trp suppressor 34~36 In addition, tryptophan tRNA genes with unique sequences from yeast, fly, mouse, rat, rabbit, and frog were tested. (Figs. 29A - 29B) This effort did not succeed in identifying ACE - tRNA with UGA PTC suppression activity greater than that of human ACE Trp tRNA (Fig. 29C). Overall, tRNA screening identified multiple engineered tRNAs (for each amino acid and stop codon type) that show strong suppression and thus have general resistance to anticodon editing. Trp Next, it was established whether the ACE - tRNAs identified during screening were functionalized at the expense of aminoacylation stringency by cognate aminoacyl - tRNA synthetases. For this, mass spectrometry was used to examine PTC suppression in the model soluble protein, histidinol dehydrogenase (HDH). (Fig. 23A) A TGA codon was introduced at asparagine 94 (N94) (Figs. 30A - C), and plasmids encoding the top - performing glycine and tryptophan ACE - tRNAs

[0256] which are Glychr19.tRNA2 or Trpchr17.tRNA39 ACE - tRNA, respectively, were co - expressed in HEK293 cells in parallel. The resulting full - length, suppressed HDH protein was purified via a Strep - Tactin® C - terminal affinity tag and analyzed by mass spectrometry (Fig. 23A (Fig. 28)). Subsequent searching of the data identified the modification of Asn to Trp, (+72Da) for Trpchr17.tRNA and (-57Da) for Glychr19.tRNA2, thus confirming the faithful coding of cognate amino acids for each ACE - tRNA type. Importantly, in each case, more than 98% of the peptides identified at the HDHp.N94X site had the encoded cognate tryptophan and glycine. Furthermore, both ACE - tRNAs recognize UAA and UAG, Fig. 23B (ACE - tRNA UGA ) and Fig. 31 (ACE - tRNA Gly ) Trp) also retained selectivity for the UGA stop codon. Finally, when transiently expressed, ACE-tRNA Gly was superior to the conventional small molecule inhibitors gentamicin (40 μM) and G418 (140 μM) in its ability to suppress stably expressed NLuc-UGA in HEK293 cells (Figure 23C). ACE-tRNA Trp was similar, and this ACE-tRNA Trp had a lower suppression efficiency compared to G418 but exceeded PTC rescue (Figures 33A - D).

[0257] The question was raised as to whether an ACE-tRNA that effectively suppresses premature stop codons could also induce global readthrough of natural stop codons. To address this potential "off-target" suppression, a quantitative profile of the entire transcriptome of ribosomes actively involved in all cellular transcripts was obtained by generating a library of ribosome footprints from HEK293 cells expressing exogenous ACE-tRNA or a control mock plasmid (puc57GG). Streptomycin was removed from the growth medium to prevent readthrough artifacts. For comparison, ribosome footprint libraries were also generated from cells in the presence or absence of G418 (150 μM, 48 hours). Figure 24A shows the ribosome footprint density (log2-fold change) of G418 and five ACE-tRNAs compared to the control in the 3'UTR region. For quantitative comparison, only transcripts with a minimum threshold of 5 RPKM in the coding sequence and 0.5 RPKM in the 3'UTR in two replicate libraries were included (254 transcripts in G418, 495 - 748 transcripts in ACE-tRNA). In this system, G418 had no observable effect on the 3'UTR ribosome density of the entire transcriptome for any of the three endogenous stop codon groups. The ACE-tRNAs examined here showed no detectable change in 3'UTR ribosome density for cognate stop codons complementary to the ACE-tRNA anticodon that induced an approximately 2-fold increase in 3'UTR ribosome density, except for ACE-tRNA Gln-UAA and Arg-UGA. Further studies will be needed to understand the biological significance of a 2-fold readthrough of protein termination, but this effect is substantially lower compared to 100- to 1000-fold suppression of the PTC by the same ACE-tRNA.

[0258] Multiple in-frame stop codons are genes 37~39Frequently found at the end of [[ID=]], it can cause a slight difference in the overall 3'UTR ribosome density for ACE-tRNA and G418 treatment. The ribosome occupancy was examined at each nucleotide within the 3'UTR in the 60nt region downstream of the stop codon. Figure 24B shows the ribosome occupancy surrounding the natural stop codon of each nucleotide in the region of -35 to +65nt compared to the first nucleotide of the stop codon. Compared with control cells, the read data per 1 million total mapped reads were normalized and reported as log2 fold change as in panel A. More than 5,200 transcripts were mapped to at least one footprint within the region of interest. ACE-tRNA Gln-UAA and Arg-UGA showed not only a significant increased ribosome occupancy in the initial region but also a characteristic 3nt periodicity, indicating that the ribosomes followed a codon-by-codon movement rather than being randomly distributed. ACE-tRNAs of UGA-Trp, UGA-Gly and UAG-Glu, or G418 did not consistently show observable changes in ribosome occupancy in the initial region of the 3'UTR. Overall, ribosome profiling data suggest that the efficiency of natural stop codon suppression by ACE-tRNA is generally low and significantly lower than the level of PTC suppression.

[0259] Discussion PTC causes many human diseases for which there are no established treatment options for their therapeutic management. Here, we report the high-throughput cloning, identification, characterization, and functional analysis of anticodon-edited tRNAs that show efficient PTC reversal in eukaryotic cells and mouse skeletal muscle. In particular, the screening identifies a total of ACE-tRNAs that have the ability to repair most of the PTCs that cause known human diseases. The engineered tRNAs faithfully encode their cognate amino acids and thus suppress spurious effects on downstream protein stability, folding, and transport, thus obviating the need for tandem therapies involving protein folding or transport agents. When transfected as cDNA, ACE-tRNAs rescued multiple full-length proteins via PTC suppression, the NLuc luciferase reporter, the model protein HDH, and two disease nonsense mutations in CFTR. The strength and stability of in vivo PTC suppression in mouse skeletal muscle were demonstrated by ACE-tRNA Arg cDNA, suggesting a particularly high level of cellular resistance to ACE-tRNA activity. The identification of active ACE-tRNA for arginine in muscle is relevant to the treatment of dystrophinopathy caused by nonsense mutations. Following adaptation to most genetic diseases, more than 10 percent of dystrophinopathies are most frequent with CGA->TGA mutations 43 nonsense mutations 43 caused by. Efficient suppression was also achieved using ACE-tRNA delivered as a synthetic RNA transcript, thus enabling the development of nanoparticle formulations. Future studies will be needed to evaluate the ideal tRNA delivery strategies for each tissue and disease type that are likely to benefit from the rapidly expanding technologies for nucleic acid delivery.

[0260] Agents that suppress PTC may also generate readthrough of natural stop codons. The RNA profiling data presented herein generally suggest that this is not the case in the cells and codon-edited tRNAs tested. Arg-tRNA UGAand Gln-tRNA UAA detectable readthrough was found, but for Glu-tRNA UAG , UGA-Gly-tRNA UGA and Trp-tRNA UGA no significant effect on global translation termination was measured. This behavior was not clearly separated by stop codon type or the intrinsic PTC suppression activity of the tRNA. One potential reason that ACE-tRNA does not effectively promote readthrough at actual stop codons may be due to the context sequence landscape near translation termination 44 . This possibility is supported by the finding that the composition of the termination complex at the PTC is different from that at natural termination 45、46 . However, when lower levels of readthrough occur, multiple cellular mechanisms exist to limit both the normal termination readthrough effect and its damaging effects.Multiple in-frame stop codons are frequently found at the end of genes 37~39 , and special ubiquitin ligases 47 and ribosome-associated pathways 48 are known to identify and degrade proteins with incorrect translation termination. However, despite the limited effects seen here in mammalian cells, similar ribosome profiling experiments should be performed in the desired cell or tissue type for ACE-tRNA delivery and expression

[0261] Previous studies have shown that the surrounding mRNA sequence affects the efficiency of aminoglycoside and ataluren PTC 49-52has been shown to affect the intrinsic stop codon suppression efficacy, and ACE-tRNA can similarly have an impact. Furthermore, gene addition strategies for replacing genes containing PTCs via viral or non-viral delivery have achieved short-term benefits in some scenarios, but it can be difficult to regulate the transgene expression level. In contrast, the abundance of protein rescue via ACE-tRNA suppression is tied to natural cellular RNA levels, and thus, the upper expression level is essentially regulated. For most of the variable acceptor tRNA sequences within the human genome, the biological purpose remains unclear, and nearly half of these genes are transcriptionally silent pseudogenes 53 are presumed to be, but the data here suggest that many annotated tRNAs are viable. Consistent with this possibility, suppression approaches have been used to identify functional isodecoder tRNAs within the Ser and Leu acceptor families 54 . The data presented here show that when removed from the genomic context, most of the tRNA gene sequences support viability, further deepening the mystery regarding the biological necessity for multiple tRNAs and further demonstrating codon usage. Thus, the high-throughput suppression strategy described herein is useful for identifying new types of tRNA sequences with intrinsic suppression properties, and such studies have the potential to produce new RNA reagents and facilitate the molecular understanding of tRNA expression and suppression.

[0262] Materials and Methods Nonsense reporter HTC plasmid The parental plasmid used was pcDNA3.1(+). The cDNA encoding pNLuc was inserted into the restriction sites HindIII and XhoI by Gibson Assembly (New England Biolabs, USA). During cDNA pcr, glycine (codon gga), tryptophan (tgc), amber (tag), opal (tga), and ochre (taa) were added at amino acid position 160. The pcDNA3.1(+) polyA sequence was replaced for those without the BbsI restriction site using pcr-based Gibson assembly. The high-throughput ACE-tRNA Golden Gate cloning site was first inserted upstream with the 5’ leader sequence (bold) of the human tRNA Tyr gene as the T7 promoter sequence (italic),

Chemical formula

[0263] HTC of the ACE-tRNA library The tRNA gene sequences are obtained from the tRNA database tRNAscan-SE (http: / / gtrnadb.ucsc.edu / index.html; PMID: 26673694). The sequences of all tRNA genes used in this study are numbered in Figure 26 and Table 9. The tRNA sequences were synthesized as complementary Ultramers from Integrated DNA Technologies (IDT, USA) in a 96-well format with their corresponding anticodons appropriately mutated (UAG, UGA, or UAA) at a scale of 200 pmol. All tRNA sequences were synthesized with CGAC and GGAC overhangs (annotated 5’->3’) on the forward oligo and reverse oligo, respectively. The Ultramers were resuspended and annealed at 100 ng / μl in annealing buffer (100 mM potassium acetate; 30 mM HEPES, pH 7.5), heated at 96 °C for 2 minutes, and cooled to 4 °C at 1 °C / min in a thermoshaker. In a 96-well PCR plate, each well contained 10 ng of the HTC plasmid along with the appropriate PTC codon, 2 ng of the ACE-tRNA duplex, 1 mM ATP, 10 mM DTT, 400 units of T4 DNA ligase, and 10 units of BbsI-HF, and was made up to 10 μl with ddH2O. The 96-well plate was cycled in a thermocycler as follows ([5 minutes at 37 °C, 5 minutes at 20 °C] × 30 cycles, 10 minutes at 37 °C, 10 minutes at 80 °C, and cooled to 4 °C). 1 μl of the Golden Gate reaction product in a deep-well 96-well plate was added to 10 μl of DH5α chemically competent cells (ThermoFisher, USA), heat-shocked at 42 °C for 30 seconds, and resuspended in 100 μl of Super Optimal Broth (SOC; ThermoFisher, USA). The transformants were outgrown 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 at 37 °C for 20 hours at 300 rpm in a covered deep 48-well plate.Growth of E. coli was carried out in deep well plates and clamps from Enzyscreen (http: / / www.enzyscreen.com). The E. coli suspension culture was spin - down (10 min, 4,000 g at room temperature), plasmid DNA was prepared and diluted to 125 ng / μl (IBI scientfic, USA). All clones were sequence - verified. Using this method, 100% cloning efficiency was achieved.

[0264] HTS of the ACE - tRNA library One day before transfection, HEK293 cells (less than 40 passages) were seeded at 1.4×10 in 96 - well cell culture - treated plates in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% FBS, 1% Pen / Step, and 2 mM L - glutamine (Thermofisher, USA). 4Cells were plated in wells. An all-in-one nonsense reporter with the ACE-tRNA gene was transfected in triplicate / plate using Calfectin (Signagen, USA). Sixteen hours after transfection, the medium was aspirated and 20 μl of PBS was added to each well. 15 μl of the Soluble Nano-Glo® Luciferase Assay Reagent was added to each well (1:50 reagent to buffer, Promega, USA). The plate was incubated for 2 minutes after rotary shaking and read using a SpectraMax i3 plate reader (Molecular Devices, USA, integration time 200 ms, all wavelengths collected in endpoint mode). Luminescence was averaged across three wells for each experiment, and all ACE-tRNAs were repeated in this manner more than three times. Each plate also contained triplicate wells transfected with an all-in-one nonsense reporter lacking the ACE-tRNA as a control for transfection efficiency and baseline PTC readthrough. All values were reported as the ratio of ACE-tRNA luminescence to baseline PTC readthrough luminescence ± SEM. One-way ANOVA was performed using Tukey's post hoc analysis across all ACE-tRNAs in a given amino acid family.

[0265] CFTR, HDH-his-strep, and 4xACE-tRNA expression plasmids For expression in mammalian cells, the cDNA of the coding region and 200 base pairs of the 3' untranslated region (UTR) of human CFTR were 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 Xenopus laevis oocytes, the coding region of the cDNA and 140 base pairs of the 5' and 244 base pairs of the 3' UTR of human CFTR were ligated into pGEM-HE (Promega, USA). The G542tga and W1282tga mutations were introduced using QuickChange XL II. The cDNA encoding E. coli histidinol dehydrogenase was codon-optimized for Mus musculus and synthesized with an 8xHis-Strep-tag at the C-terminus for protein purification from mammalian cells (BioBasic Inc, Canada). The synthesized cDNA was ligated into pcDNA 3.1(+) using EcoRI and XhoI restriction sites. The nonsense mutations tag, taa, and tga were introduced using QuickChange XLII. To generate the multiplexed ACE-tRNA expression plasmid, the BbsI "multiple cloning site"

Chemical formula

[0266] Cell culture, protein expression and Western blot HEK293T cells (ATCC, USA) were grown in a standard growth medium containing 10% FBS (HiClone, USA), 1% Pen Strep, 1% L-Glut in high glucose DMEM (Gibco, USA) (v / v%) at 37 °C and 5% CO2. cDNA was transfected at 75% confluence using Calfectin according to a standard protocol (SignaGen Laboratories, USA). After 36 h, cells were scraped and pelleted at 7,000 g for 8 min 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, cell pellets were vigorously 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, 50 mM Tris-HCL ph7.4 and centrifuged at 100,000 g to separate total membranes from soluble cytoplasmic proteins. Pellets were solubilized in a buffer containing 1% Triton, 250 mM NaCl, 50 mM Tris-HCl pH7.4, and 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, 0.75 mM benzamidine. Equal cell lysates were loaded onto a 3–15% separation gradient SDS-page with a 4% stacking gel in the presence of 1% 2-mercaptoethanol and separated at 55 V O / N and transferred to 0.45 μM LF PVDF (Bio-Rad, USA). PVDF was immunoblotted using the anti-CFTR antibody M3A7 (1:1000; Millipore, USA) in 2% non-fat milk and imaged on a LI-COR Odyssey Imaging System (LI-COR, USA).For HDH-His-Strep expressing cells, the cell pellet was vigorously homogenized 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 lysate was centrifuged at 100,000 g for 30 min at 4 °C. The supernatant (soluble cell 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), immunoblotted using anti-Strep antibody (1:5000; Iba, Germany) in 2% non-fat milk, and imaged on a LI-COR Odyssey Imaging System (LI-COR, USA).

[0267] Mass spectrometry Fragmentation data on the purified HDH-His-Strep protein was obtained at the University of Iowa Proteomics Facility. Briefly, the HDH-His-Strep protein from the soluble fraction of high-speed spin was passed through a StrepTrap HP column (GE Healthcare, Sweden) and washed with 5 column volumes of 100 mM sucrose, 1 mM DTT, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.5 μg / ml pepstatin, 2.5 μg / ml aprotinin, 2.5 μg / ml leupeptin, 0.1 mM PMSF, and 0.75 mM benzamidine. The protein was eluted in a wash buffer supplemented with 10 mM d-desthbiotin and concentrated in a 30 kDa cut-off Amicon-Ultra filtration column (Millipore, USA). The concentrated protein was loaded onto a NuPage 4-12% Bis-Tris precast gel (Invitrogen, USA) and separated at 150 V for 1.5 hours. The gel was stained using a Pierce mass spectrometry-compatible silver staining kit (ThermoFisher Scientific, USA).

[0268] In-gel trypsin digestion. Briefly, target protein bands from SDS-PAGE gels were manually excised, cut into 1 mm pieces, washed in 100 mM ammonium bicarbonate:acetonitrile (1:1, v / v) and 25 mM ammonium bicarbonate / acetonitrile (1:1, v / v) to achieve complete de-staining. The gel pieces were further treated with ACN and dried via 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. The gel pieces were washed twice with 25 mM ammonium bicarbonate:acetonitrile (1:1, v / v) to remove excess DTT and IAM. After drying, the gel pieces were placed on ice in 50 μl of 10 ng / μL trypsin solution in 25 mM ammonium bicarbonate and incubated on ice for 60 minutes. Then, digestion was carried out at 37 °C for 16 hours. Peptide extraction was performed twice with 100 μl of 50% acetonitrile / 0.2% formic acid for 0.5 hours. The combined extracts were concentrated to approximately 15 μl in a Speed Vac.

[0269] LC-MS / MS mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to an Eksigent Ekspert (registered trademark) nanoLC 425 System (Sciex). A Trap-Elute Jumper Chip (P / N: 800-00389) and a 1 / 16-inch 10-port Valco directed loading coupled to it were designed as a column assembly that performs gradient 1 pump and final elution (by gradient 2 pump) with two tandem 75 μm × 15 cm columns (ChromXPC18-CL, 3 μm 120A, Eksigent part of AB SCIEX) attached to an ekspert (trademark) cHiPLC system. For each injection, an estimated 0.5 μg of total digest was loaded. Peptides were separated inline with the mass spectrometer using a 120-minute gradient consisting of linear and static segments where buffer A was 0.1% formic acid and buffer B was 95% ACN, 0.1% formic acid. The gradient started with a hold at 4% for 3 minutes and was then carried out with the following transitions (%B, minutes): (26, 48), (35, 58), (35, 64), (50, 72), (50, 78), (94, 84), (94, 96), (4, 100), (4, 120).

[0270] 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). During the 120-minute gradient described above, gradient MS1 scans were acquired every 3 seconds. The most abundant precursors were selected from 2 - 8 charge state ions at a 2.0E5 threshold. Ions were dynamically excluded for 30 seconds if targeted twice in the previous 30 seconds. The selected ions were separated by a multi-segment quadrupole with a mass window of m / z 2 and then sequentially subjected to activation conditions for both CID and HCD by an IT and an ion routing multipole, respectively. The AGC target for CID was 4.0E04, 35% collision energy, 0.25 activation Q and a maximum fill time of 100 milliseconds. The targeted precursors were also fragmented by high energy collision induced dissociation (HCD) at 40% collision energy and an activation Q of 0.25. The HCD fragment ions were analyzed using the Orbitrap (AGC 1.2E05, maximum injection time 110 ms, and a resolution set to 30,000 at 400 Th). Both MS2 channels were recorded as centroid and the MS1 survey scan was recorded in profile mode.

[0271] Proteomics analysis. Initial spectral searches were performed using Sequest HT in Proteome Discoverer version 2.1.1.21 (ThermoFisher Scientific, USA). Spectra were also searched using the Byonic search engine (Protein Metrics) ver. 2.8.2. The search databases consisted of Uniprot KB for species 9606 (human) containing 92,645 sequences downloaded on October 24, 2016, and Uniprot KB for taxonomy 562 (Escherichia coli) containing 10,079 sequences downloaded on November 8, 2016. In the Byonic search, these two databases were directly concatenated. In either search, an equal number of decoy entries were created and searched simultaneously by reversing the original entries in the target database.

[0272] In vitro cRNA transcription of G542X UGA , W1282X UGA , and WT CFTR pGEMHE (Mense et al., 2006; PMID: 1703051) plasmids were linearized at 37 °C for 3 h with a 10-fold excess of NheI-HF restriction enzyme (site located 3’ of the coding region) (New England BioLabs, USA) and purified using the standard cDNA precipitation method. All cRNAs were transcribed using the mMessage mMachine T7 Kit (ThermoFisher Scientific, USA). Purification of cRNA from the transcription reaction was performed on a column from the RNeasy Mini Kit (Qiagen, Germany). The concentration was determined by absorbance measurement at 26o nm, and the quality was confirmed on a 1% agarose gel (RNAase-free). All cRNAs were quantified to 1 μg / ml before use, and all results were generated from ≥2 cRNA preparations.

[0273] In vitro tRNA transcripts Trpchr17.tRNA39 and Glychr19.tRNA2, which execute above Trp and Gly ACE-tRNAs, were transcribed in vitro using the CellScript T7-Scribe Standard RNA IVT Kit (CELLSCRIPT, USA). Equimolar concentrations of T7 oligo (5’-taatacgactcactata-3’) were annealed to the ACE-tRNA PAGE purified ultramer (20 ug; Integrated DNA Technologies, Coralville, IA) code for the ACE-tRNA, preceded by the T7 promoter (italicized). Importantly, the three terminal nucleotides containing CCA were included (bolded).

Chemical formula

[0274] 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), 10 μl of 100 mM dithiothreitol, 2.5 μl of ScriptGuard RNase inhibitor, 10 μl of T7-Scribe enzyme solution. After incubating the reaction at 37 °C for 4 - 5 hours, the DNA template was digested for 30 - 60 minutes with 5 μl of DNase (1 U / μl) provided with the kit. The ACE-tRNA was extracted from the reaction with 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 with a Chroma Spin-30 column (Clontech, USA). This procedure yielded approximately 100 μl of ACE-tRNA at about 5 μg / μl. The ACE-tRNA was repelleted in 20 μg aliquots, washed, lyophilized, and stored at -80 °C until use. All results were generated from ACE-tRNA preparations of ≧2.

[0275] Preparation of ribosome-footprint profiling library. HEK 293 cells transiently transfected with ACE-tRNA and control plasmid (puc57GG) were grown in standard growth medium for 48 h in the absence of Pen-Strep. The library was prepared with some modifications as described in 55 . Briefly, cells were rapidly cooled by adding ice-cold PBS and lysed on ice for 10 min in lysis buffer (20 mM Tris-HCl / pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% (v / v) Triton X-100, and 25 U / ml -1 Turbo DNase I), and then disrupted 10 times by passing through a 26-G needle. After clearing by centrifugation at 16,000 g for 10 min at 4 °C, the lysate was incubated at room temperature with 260 100 U RNase I (Ambion, USA) per lysate for 45 min with gentle stirring, followed by addition of 200 U RiboLock RNase inhibitor (Thermo Scientific). The ribosome-protected mRNA fragments were then purified with modified polysome buffer (20 mM Tris-HCl / pH 7.4, 150 mM NaCl, 8.5 mM MgCl2, 0.5 mM DTT, 20 U / ml -1The lysate was loaded onto a 1 M sucrose cushion prepared with RiboLock RNase inhibitor and isolated by centrifugation at 70,000 rpm for 2 hours at 4 °C using a Beckmen TLA-110 rotor. The ribosome pellet containing the mRNA footprint was extracted using TRIzol and separated on a denaturing 12% polyacrylamide gel containing 8 M urea. RNA fragments in the size range of 26 - 34 nt were manually excised from the gel stained with SYBR Gold (Invitrogen), isolated, and a ribosome-protected fragment library was generated. Ligation of 3' oligonucleotide adapters, reverse transcription, circularization, and secondary rRNA depletion using rRNA-depleted oligos (Table 9) of contaminating rRNA fragments depleted using the Ribo-Zero kit (Illumina) were performed as described55. The library was barcoded using index primers for each sample during PCR amplification. The barcoded libraries were then pooled with 3% PhiX (Illumina) and sequenced on an Illumina NextSeq 500 according to the manufacturer protocol, typically generating 18 million - 27 million read data per sample.

[0276] Ribosome footprint data analysis. The data files of each barcoded sample (minus adapter sequence at the 3' end) were first aligned to the reference genome using HISAT2.0.3 5Using 6, mapping was performed to four rRNA sequences (RNA5S1, NR_023363, RNA5-8SN5, NR_003285, RNA18SN5, NR_003286, and RNA28SN5, NR_003287) to exclude rRNA contaminating read data. The remaining read data was aligned to the sense strand of the longest transcript variant (UCSC Ref Seq GRCh 38) of each human gene. Transcripts with a 3’UTR length of at least 75 nt (18,101 sequences) were used for subsequence analysis. Up to two mismatches were permitted at the 5’ end of the read data. All multi-mapped read data was discarded. Read data of fragments with a length of 26 - 34 nt was defined as ribosome footprints and used for analysis. The 5’-terminal nucleotides from each footprint were annotated and mapped onto each transcript. Each footprint 57、58 Using the position of the ribosome A-site occupying nucleotides 16 - 18 of each footprint, the position of ribosomes on each transcript was inferred. RPKM (reads per kilobase of transcript per million mapped reads) on each individual transcript (18,101 sequences) was calculated. For analysis in Figure 24A, only transcripts with a minimum threshold of 5 RPKM in the coding sequence and 0.5 RPKM in the 3’UTR region in two replicate libraries (254 transcripts in G418 and 495 - 748 transcripts in ACE-tRNA) were included. For the metagene plot of the entire transcriptome in Figure 2B, the footprint count of each nucleotide in the region from -35 nt to +65 nt relative to the first nucleotide of the stop codon was normalized for each million mapped read data. All transcripts (18,101 sequences) were used for mapping, and more than 5,200 transcripts were mapped to at least one footprint in the region of interest. Next, the in vivo biological activity of rescuing the PTCs of ACE-tRNA Glychr19.trna2 and Trpchr17.trna39 was examined. Sequencing data was on the Galaxy platform 59It was analyzed using [specific tool]. Graphs were generated using Prism 7 (GraphPad Software).

[0277] Generation of stable NLuc reporter cell lines. A cDNA encoding pNLuc with a tag at amino acid position 160 and taa and tga stop codons was inserted into the AgeI and NotI restriction sites within the multiple cloning sites of the retroviral vector pQCXIP (Clontech, USA) using Gibson Assembly (New England Biolabs, USA). Phoenix GP cells (PMID: 7690960) were co-transfected with pNLuc-STOP-pQCXIP and cmv-VSV-G (VSV-G envelope pseudotyping) plasmids using Calfectin (SignaGen Laboratories, USA) and placed in a 33 °C, CO2-regulated (5%) cell incubator for 48 hours. The culture medium (20 ml) containing retroviral particles was cooled to 4 °C, centrifuged at 10,000 g to remove cell debris, and filtered through a 0.45 μm MCE membrane syringe filter (Millipore, USA) into two 10 cm dishes seeded with low-passage HEK293 cells at a 30% culture density. The cell culture dishes were sealed with parafilm, rotated at 3,500 g at 24 °C for 90 minutes, and placed in a 37 °C, CO2-regulated (5%) cell culture incubator. Cells were selected with puromycin (1 μg / ml) 24 hours later until the control dish (no infection) showed complete cell death. Cells were dispersed into 96-well plates using FACS, and then the clonal populations were also dispersed. Puromycin was not used to maintain the selected clones during the experiment, and standard DMEM medium (DMEM - Dulbecco's modified Eagle medium - high glucose, 10% FBS, 1% Pen / Step, and 2 mM L-glutamine, ThermoFisher, USA) was used in all studies.

[0278] HEK293 cells stably expressing RNA transfection pNLuc-UGA were plated at 1.4×10 4 cells / well in a 96-well cell culture treated plate of Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% FBS, 1% Pen / Step, and 2 mM L-glutamine (Thermofisher, USA). After 16 - 24 hours, the cells were transfected with ACE-tRNA 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. The volumes were combined, mixed thoroughly, and incubated at room temperature for 10 minutes. 75 μl of the transfection complex was added to each well. PTC suppression by the ACE-tRNA transcript was quantified as described above.

[0279] Expression in Xenopus laevis oocytes. Xenopus laevis 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 debris was pelleted at 21,000×g for 25 minutes at 4 °C. To determine the dose response of ACE-tRNA in CFTR channel rescue, serial dilutions of ACE-tRNA aliquots volume-balanced with ddH2O (200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.562 ng / oocyte) were generated. In all experiments, 25 ng of CFTR cRNA was injected per oocyte, and the injection volume was 50 nl. ddH2O was not used in background control experiments without ACE-tRNA. After injection, the oocytes were held at 18 °C for 36 hours in OR-3 (50% Leibovitz's medium, 250 mg / l gentamicin, 1 mM L-glutamine, 10 mM HEPES (pH 7.6)).

[0280] Two-electrode voltage clamp (TEVC) recording. CFTR Cl- Currents were recorded in ND 96 bath solution containing the following (in mM). (96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5) were contained in the presence of the maximal CFTR activation cocktail, forskolin (10 μM; adenylate cyclase activator) and 3-isobutyl-1-methylxanthine (1 mM; phosphodiesterase inhibitor).) Glass microelectrodes backfilled with 3 M KCl had resistances of 0.5 - 2 MΩ. Data were filtered at 1 kHz and digitized at 10 kHz using Digidata 1322A controlled by pClamp 9.2 software (Molecular Devices, USA). CFTR currents were elicited using an OC-725C voltage clamp amplifier (Warner Instruments, USA) with 5 mV voltage steps from -60 to +35 mV. CFTR Cl - Oocytes in which the current reversed positively at -20 mV were discarded. Clampfit 9.2 software was used for the current analysis. All values are presented as mean ± SEM.

[0281] Animals and in vivo imaging. Nu / J mice were purchased from Jackson labs. Animal experiments were approved by the Institutional Animal Care and Use Committee (protocol number: 112762) of the Wistar Institute. Mice were treated by injecting 10 - 20 μg of DNA resuspended in 30 μl of water into the tibialis anterior muscle, followed by electroporation. 10 μg of pNano-TGA + 10 μg of ACE-tRNA (right tibialis anterior) or 10 μg of pNano-TGA + 10 μg of empty pUC57 (left tibialis anterior) were injected into 3 mice. As a control, 10 μg of pNano-WT (right tibialis anterior; positive control) or water (left tibialis anterior; negative control) were injected into another 3 mice. DNA was formulated with 333 IU / ml of hyaluronidase (Sigma). One minute after DNA injection, electroporation was performed with a CELLECTRA 3P device (Inovio Pharmaceuticals). Nano-luciferase activity was imaged in mice injected intraperitoneally with 100 μl of furimazine (40-fold dilution of the Nano-Glo substrate) and imaged on an IVIS Spectrum (Perkin Elmer) 5 minutes after injection. Imaging was performed with an open filter and images were acquired in 40 seconds. Images were analyzed using Living Image Software (Perkin Elmer).

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

Table 9-28

Table 9-29

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L. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12, 357 - 360 (2015). 57. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome - wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218 - 223 (2009). 58. Guydosh, N. R. & Green, R. Dom34 rescues ribosomes in 3’untranslated regions. Cell 156, 950 - 962 (2014). 59. Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res 44, W3 - W10 (2016).

[0283] Example 6: ACE - tRNA is active in vivo Here, it is demonstrated that the anticodon - edited tRNA shows effective PTC reversion in eukaryotic cells and mouse skeletal muscle.

[0284] The in - vivo activity and stability of ACE - tRNA were examined. The NLuc - UGA PTC reporter cDNA was used with ACE - tRNA ArgFour copies of UGA or a plasmid encoding an “empty vector control” were delivered to mouse skeletal muscle (tibialis anterior) using electroporation (Wheeler et al., 2009, Science, 325, 336 - 339; Wheeler et al., 2007, J Clin Invest, 117, 3952 - 3957; Muthumani et al., 2017, Cancer Immunol Immunother, 66, 1577 - 1588). These data were compared to the expression of WT NLuc. The results showed that ACE - tRNA Arg UGA is a potent in vivo PTC suppressor, generating an expression profile equal to or greater than full - length WT NLuc at several time points (Figure 34A and Figure 35). Signals from the NLuc - UGA plasmid and non - electroporation were not detectable. Furthermore, ACE - tRNA Arg suppression activity was stable, as demonstrated by a similar duration of NLuc activity between the rescue protein and the WT protein (Figure 34B). Furthermore, this duration and intensity of luciferase expression support the claim of negligible effects of high resistance in vivo and increased read - through observed with ACE - tRNA Arg Next, the ability of functional ACE - tRNA delivered as RNA was demonstrated. For this, ACE - tRNA Trp and ACE - tRNA Gly RNA transcripts were transfected into HEK293 cells stably expressing the NLuc - UGA reporter. Here, the results showed that both ACE - tRNAs functioned similarly to when expressed as cDNA plasmids and were accompanied by comparable fold - rescues when delivered as small RNAs (Figure 34C). When the NLuc - UGA PTC reporter cDNA was delivered to mouse skin tissue using electroporation, ACE - tRNA Arg suppression activity was also observed (Figure 36).

[0285] Next, experiments were designed to rescue two diseases caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). This large membrane protein controls anion transport across epithelia in multiple organs, and missense and nonsense mutations within its reading frame cause cystic fibrosis. For this purpose, CFTR p.G542X (c.16524G>T; UGA stop codon) and p.W1282X (c.3846G>A; UGA stop codon) cDNAs were transiently co-expressed with their respective ACE-tRNA expression plasmids in HEK293 cells and analyzed by Western blot using a C-terminal antibody to identify production of the full-length protein (Figure 34D). Both rescue conditions, as well as WTCFTR expression, evidenced normal trafficking of the CFTR protein by the presence of both the fully glycosylated band C form and the core-glycosylated band B CFTR proteins. No signal was seen for either p.G542X or p.W1282X transfected alone, indicating low rates of spontaneous readthrough of the PTCs shown under these conditions. In the absence of delivery or expression considerations, Xenopus leavis oocytes, a non-dividing model cell in which the ACE-tRNA concentration (as RNA) can be controlled and functional expression quantified to better quantify the PTC suppression properties of each ACE-tRNA, were used. Specifically, this expression system is suitable for microinjection and two-electrode voltage clamp (TEVC) analysis, an easy electrophysiological method for assessing ion channel function in the cell membrane. CFTR cRNA (complementary RNA produced in vitro from a cDNA template) was injected alone or together with the indicated ACE-tRNA at increasing concentrations (Figure 34E and Figure 34F). No functional CFTR channels were seen for any of the mutants lacking co-injected ACE-tRNA, even in the presence of the maximal CFTR activation cocktail, forskolin (10 μM; adenylate cyclase activator), and 3-isobutyl-1-methylxanthine (1 mM; phosphodiesterase inhibitor) (Figure 34E, left).However, under the same conditions, when 200 ng of ACE-tRNA Gly chr19.tRNA2 (Figure 34E, upper right) or Trp chr17.tRNA39 (Figure 34E, lower right) was co-injected, CFTR chloride conductance was measured in response to a transient change in membrane potential, indicating that both ACE-tRNAs were highly effective in suppressing the UGA PTCs that cause both diseases. To better quantify the relative expression of the rescued channels, this rescue was compared to WT CFTR cRNA alone (25 ng), and suppression of the PTC in CFTR was evaluated over a range of ACE-tRNA concentrations. The resulting ACE-tRNA dose-response "current-voltage" relationships are shown in Figure 34F. These data were generated by plotting the steady-state ionic current at each voltage against the voltage used to elicit the measured current and are direct measures of channel function and abundance. Expression at WT-like current levels was achieved by Gly chr 19.tRNA2 and approximately 50% achieved for Trp chr17.tRNA 39 ACE-tRNA, consistent with the given suppression activity and cognate amino acid coding for these tRNAs.

[0286] The foregoing specification and examples disclose and enable the present invention, but they are not intended to limit the scope of the invention as defined by the claims appended hereto.

[0287] All publications, patents, and patent applications are hereby incorporated by reference. In the foregoing specification, the invention has been described with reference to specific embodiments and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that the specific details described herein may be significantly modified without departing from the basic principles of the invention.

[0288] In the context of describing the present invention, the use of the terms "a" and "an" and "the" and similar referents should be construed to cover both the singular and plural forms unless otherwise indicated herein or clearly contradicted by the context. The terms "comprising," "having," "including," and "containing" should be construed as open - ended terms (i.e., meaning "including but not limited to") unless otherwise described. The recitation of a range of values herein is merely intended to serve as a shorthand way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or illustrative language (e.g., "for example") provided herein is merely intended to better clarify the invention and is not intended to limit the scope of the invention, particularly unless otherwise claimed. No language in this specification should be construed as indicating any non - claimed element as essential to the practice of the invention.

[0289] Embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately use such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise indicated herein or clearly contradicted by the context, any combination of the above - described elements in all possible variations thereof is included by the present invention.

Claims

**Claim 1** A composition for generating one or more anticodon-edited tRNAs (ACE-tRNAs) or fragments thereof in a subject, comprising one or more nucleic acid molecules or fragments thereof. **Claim 2** The composition according to claim 1, comprising a cDNA molecule encoding an ACE-tRNA. **Claim 3** The composition according to claim 1, comprising an RNA molecule comprising an ACE-tRNA. **Claim 4** The composition according to any one of claims 1 to 3, wherein the one or more nucleic acid molecules are engineered to be within an expression vector. **Claim 5** The composition according to any one of claims 1 to 4, further comprising a pharmaceutically acceptable excipient. **Claim 6** A method of treating a disease associated with PTC in a subject in need thereof, comprising administering to the subject at least one composition according to any one of claims 1 to 5. **Claim 7** The method according to claim 6, wherein the disease is a disease or disorder associated with UGA PTC, and the method further comprises administering at least one ACE-tRNA specific for UGA. **Claim 8** The method according to claim 6, wherein the disease is a disease or disorder associated with UAA PTC, and the method further comprises administering at least one ACE-tRNA specific for UAA. **Claim 9** The method according to claim 6, wherein the disease is a disease or disorder associated with UAG PTC, and the method further comprises administering at least one ACE-tRNA specific for UAG. **Claim 10** The method according to any one of claims 6 to 9, wherein the method comprises administering at least two ACE-tRNAs, each of the at least two ACE-tRNAs being specific for at least two different amino acid molecules on a polypeptide chain. **Claim 11** The method according to any one of claims 6 to 9, wherein the method comprises administering at least two ACE-tRNAs, each of the at least two ACE-tRNAs being specific for incorporating the same amino acid molecule on a polypeptide chain. **Claim 12** The method according to either claim 10 or 11, wherein the at least two ACE-tRNAs are encoded on the same nucleic acid molecule. **Claim 13** The method according to either claim 10 or 11, wherein the at least two ACE-tRNAs are encoded on different nucleic acid molecules.

14. The method comprises administering at least one ACE-tRNA specific for UGA selected from the group consisting of ACE-tRNA Arg , ACE-tRNA Gly , and ACE-tRNA Trp The method according to claim 7.

15. The method according to claim 6, wherein the disease is selected from the group consisting of Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia telangiectasia, Tay-Sachs disease, cystic fibrosis, Wilms tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich disease, β-thalassemia, type 2A and type 3 von Willebrand disease, Robinow syndrome, brachydactyly type B (shortening of fingers and metacarpal bones), genetic susceptibility to mycobacterial infection, hereditary retinal diseases, hereditary bleeding tendency, hereditary blindness, congenital sensorineural deafness and colonic agangliosis, and hereditary neurodevelopmental disorders including sensorineural deafness, colonic agangliosis, peripheral neuropathy and central myelin hypoplasia leukodystrophy, Riddle syndrome, xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer), esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY conversion, triosephosphate isomerase anemia, diabetes and kuru.

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