Recombinant viral vector and nucleic acid for its production

Abstract

Described herein are nucleic acids, AAV transfer cassettes, and plasmids used in the production of recombinant adeno-associated virus (rAAV) vectors. The disclosed nucleic acids, cassettes, and plasmids contain sequences that express one or more transgenes that have therapeutic efficacy in the amelioration, treatment, and / or prevention of one or more diseases or disorders.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 770,202, filed on November 21, 2018, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates to the fields of molecular biology and gene therapy. More specifically, the present disclosure relates to compositions and methods for the production of recombinant viral vectors.

[0003] Description of Electronically Submitted Text Files The contents of the text files electronically submitted with this specification are hereby incorporated by reference in their entirety. A copy of the Sequence Listing in computer - readable format (file name: STRD - 011 - 01WO_Sequence_Listing.txt, date of record November 21, 2019, file size approximately 145 kilobytes).

Background Art

[0004] Recombinant viral vectors (including adeno - associated viral vectors (AAV)) are useful as gene delivery agents and are powerful tools for human gene therapy. Using AAV, high - frequency stable DNA integration and expression can be achieved in various cells in vivo and in vitro. Unlike some other viral vector systems, AAV does not require active cell division for stable integration in target cells.

[0005] Recombinant AAV vectors can be produced in culture using a viral producer cell line. Production of recombinant AAV typically requires the presence in the cell of three elements: 1) a nucleic acid containing the transgene flanked by AAV inverted terminal repeat (ITR) sequences, 2) the AAV rep and cap genes, and 3) helper viral protein sequences. These three elements can be provided on one or more plasmids and transfected or transduced into the cell.

[0006] The production and use of recombinant AAV vectors has been limited by the inability to efficiently package transgene DNA into the viral capsid and to effectively express the transgene in target cells. Accordingly, there is a need in the art for improved compositions and methods for the production of recombinant AAV vectors. SUMMARY OF THE INVENTION

[0007] Nucleic acids comprising an AAV transfer cassette are described herein. The disclosed nucleic acids can be used in the production of recombinant adeno-associated virus (AAV) vectors. The disclosed nucleic acids and transfer cassettes comprise the sequences of one or more transgenes having therapeutic efficacy in the remission, treatment, and / or prevention of one or more diseases or disorders.

[0008] In some embodiments, the present disclosure provides a nucleic acid comprising, from 5' to 3', a 5' inverted terminal repeat (ITR), a promoter, a transgene sequence, a polyadenylation signal, and a 3' ITR. In some embodiments, the transgene sequence encodes a frataxin (FXN) protein. The FXN protein can be, for example, a human FXN protein. In some embodiments, the FXN protein has the sequence of SEQ ID NO: 65, or a sequence that is at least 95% identical thereto. In some embodiments, the nucleic acid comprises any one of the sequences of SEQ ID NOs: 28-64, or a sequence that is at least 95% identical thereto.

[0009] In some embodiments, the 5' ITR is the same length as the 3' ITR. In some embodiments, the 5' ITR and the 3' ITR have different lengths. In some embodiments, at least one of the 5' ITR and the 3' ITR is about 110 to about 160 nucleotides in length. At least one of the 5' ITR and the 3' ITR can be isolated from or derived from the genome of, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO: 1, or a sequence that is at least 95% identical thereto. In some embodiments, the 3' ITR comprises the sequence of SEQ ID NO: 2, or a sequence that is at least 95% identical thereto. In some embodiments, the 3' ITR comprises the sequence of SEQ ID NO: 3, or a sequence that is at least 95% identical thereto.

[0010] The promoter can drive the expression of the transgene. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a modified form of a wild-type promoter. For example, due to packaging restrictions for AAV, the length of the promoter can be reduced. In some embodiments, the promoter is a truncated form of a wild-type promoter.

[0011] The promoter can be, for example, the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus (MMTV) promoter, Rous sarcoma virus (RSV) promoter, polyhedrin promoter, chicken β-actin (CBA) promoter, EF-1α promoter, EF-1α short promoter, EF-1α core promoter, dihydrofolate reductase (DHFR) promoter, GUSB240 promoter, GUSB379 promoter, or phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter comprises a sequence selected from any one of SEQ ID NOs: 6 to 12, or a sequence that is at least 95% identical thereto.

[0012] In some embodiments, the transgene sequence is CpG-optimized. In some embodiments, the transgene sequence comprises SEQ ID NO: 19 or 20, or a sequence that is at least 95% identical thereto.

[0013] In some embodiments, the nucleic acid comprises a Kozak sequence immediately 5' of the transgene sequence. The Kozak sequence can comprise, for example, the sequence of SEQ ID NO: 17 or 18, or a sequence that is at least 95% identical thereto.

[0014] In some embodiments, the polyadenylation signal is selected from the polyadenylation signals of simian virus 40 (SV40), human α-globin, rabbit α-globin, human β-globin, rabbit β-globin, human collagen, polyomavirus, human growth hormone (hGH), and bovine growth hormone (bGH). In some embodiments, the polyadenylation signal comprises any one of the sequences of SEQ ID NOs: 21 to 24, or a sequence that is at least 95% identical thereto.

[0015] In some embodiments, the nucleic acid further comprises an enhancer. The enhancer can be, for example, a CMV enhancer. In some embodiments, the enhancer comprises the sequence of SEQ ID NO: 4 or 5, or a sequence that is at least 95% identical thereto.

[0016] In some embodiments, the nucleic acid further comprises an intronic sequence. The intronic sequence can be, for example, a chimeric or hybrid sequence. In some embodiments, the intronic sequence comprises a sequence isolated from or derived from one or more of the following genes: β-globin, triβ-actin, murine minute virus, and human IgG. In some embodiments, the intronic sequence comprises any one of the sequences of SEQ ID NOs: 13-16, or a sequence that is at least 95% identical thereto.

[0017] In some embodiments, the nucleic acid further comprises at least one stuffer sequence (e.g., 1, 2, 3, 4, or 5 stuffer sequences). In some embodiments, the at least one stuffer sequence comprises any one of the sequences of SEQ ID NOs: 25-27, or a sequence that is at least 95% identical thereto.

[0018] Vectors (e.g., AAV vectors or plasmids) comprising the nucleic acids of the present disclosure are also provided herein.

[0019] Cells comprising the nucleic acids of the present disclosure are also provided.

[0020] A method of producing a recombinant AAV vector, comprising contacting AAV-producing cells with the nucleic acids or plasmids / bacmids of the present disclosure, is also provided. Recombinant AAV vectors produced by this method are also provided. The recombinant AAV vector may include capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. In some embodiments, the AAV vector may include a capsid protein having one or more substitutions or mutations compared to the wild-type AAV capsid protein. In some embodiments, the recombinant AAV vector is single-stranded (ssAAV). In some embodiments, the recombinant AAV vector is self-complementary (scAAV).

[0021] Compositions comprising the nucleic acids, plasmids, bacmids, cells, or recombinant AAV vectors of the present disclosure are also provided.

[0022] A method of treating a subject, comprising administering a therapeutically effective amount of the nucleic acids, plasmids, cells, or recombinant AAV vectors of the present disclosure to a subject in need of treatment, is also provided. In some embodiments, the subject is a human subject. In some embodiments, the subject has Friedreich's ataxia (FRDA).

[0023] These and other embodiments are treated in more detail in the detailed description of the invention, which is set forth below.

Brief Description of the Drawings

[0024]

Figure 1

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Mode for Carrying Out the Invention

[0025] Gene therapy has high promise for the treatment and prevention of genetic diseases and disorders, such as Friedreich's ataxia (FRDA). FRDA is an autosomal recessive disorder typically caused by mutations in the frataxin (FXN) gene. Approximately 1 in 50,000 individuals in the United States has FRDA. The typical age of onset is between about 5 and about 18 years. Symptoms vary among subjects and can include (i) loss of coordination of the arms and legs (ataxia), (ii) fatigue / energy depletion and loss of muscle strength, (iii) visual impairment, hearing loss, and slurred speech, (iv) invasive scoliosis (curvature of the spine), (v) diabetes (typically insulin-dependent), and (vi) severe heart conditions, such as hypertrophic cardiomyopathy and arrhythmias. The mental abilities of individuals with FRDA remain unimpaired. There is currently no treatment for FRDA, and subjects are monitored for symptom management. Accordingly, there is a need in the art for compositions and methods for treating and / or preventing FRDA.

[0026] Nucleic acids comprising an AAV transfer cassette for the production of an AAV vector are provided herein. The AAV vector can be used for gene therapy applications to deliver, for example, a therapeutic transgene to a cell or to a subject in need thereof. The AAV transfer cassettes and vectors of the disclosure can be used to treat or prevent various genetic diseases and disorders (FRDA).

[0027] All papers, publications, and patents cited herein are hereby incorporated by reference as if each individual paper, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials related to the cited publications.

[0028] Unless the context otherwise indicates, it is specifically intended that the various features described herein can be used in any combination. The section headings are used herein for purposes of organization and are not intended to be limiting.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the detailed description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0030] Definitions The following terms are used in the description herein and in the appended claims.

[0031] The singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0032] Furthermore, as used herein, the term "about", when referring to a measurable value (such as the amount or length of polynucleotides or polypeptides, sequences, dosages, times, temperatures, and the like), means a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or in some cases ±0.1% of the specified amount.

[0033] Furthermore, as used herein, "and / or" encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0034] "Nucleic acid" or "polynucleotide" refers to a sequence of nucleotide bases, such as RNA, DNA, or a hybrid sequence of DNA-RNA (including both naturally occurring nucleotides and non-naturally occurring nucleotides). In some embodiments, the nucleic acids of the present disclosure are single-stranded DNA sequences or double-stranded DNA sequences. The nucleic acid can be 1 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1 million, or more than 1 million nucleotides in length. The nucleic acid will generally contain phosphodiester bonds, but in some cases, alternative backbones containing, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite, or P-ethoxy linkages, or nucleic acid analogs having a peptide nucleic acid backbone and linkages are included. Other analogous nucleic acids include those with a positive backbone, a non-ionic backbone, and a non-ribose backbone. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids. These modifications of the ribose-phosphate backbone can facilitate the addition of labels or increase the stability and half-life of such molecules in a physiological environment. The nucleic acids of the present disclosure can be linear or circular (e.g., plasmids).

[0035] The terms "protein", "peptide", and "polypeptide" are used interchangeably herein and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that can make up the sequence of a protein or peptide.

[0036] As used herein, the terms "viral vector," "virulent vector," or "gene delivery vector" refer to viral particles that function as nucleic acid delivery vehicles and contain a vector genome packaged within the virion. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0037] Adeno-associated virus or AAV belongs to the genus Dependovirus of the family Parvoviridae. The wild-type AAV genome of 4.7 kb encodes two major open reading frames. The rep gene expresses viral replication proteins, and the cap gene expresses viral capsid proteins. At the ends of the AAV genome, there are inverted terminal repeats (ITRs) that form a T-shaped hairpin structure. Mature AAV virions are infectious in mammalian cells, but in the replicative AAV life cycle, helper functions from, for example, adenovirus or herpesvirus are required. Recombinant AAV vectors can be generated by replacing the wild-type AAV open reading frames with transgene expression cassettes.

[0038] As described herein, AAV can be AAV serotype 1, AAV serotype 2, AAV serotype 3 (including 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV rh32.33, AAV rh8, AAV rh10, AAV rh74, AAV hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and other AAVs that are currently known or later discovered. See, for example, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of AAV serotypes and clades have been identified (see, for example, Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383; and Table 1).

Table 1-1

Table 1-2

Table 1-3

[0039] The term "self-complementary AAV" or "scAAV" refers to a recombinant AAV vector that forms a dimeric inverted repeat DNA molecule that spontaneously anneals and results in faster and more robust transgene expression compared to a conventional single-stranded (ss) AAV genome. Notably, scAAV can only carry a genome of only about 2.4 kb, which is half the size of a conventional AAV vector. In some embodiments, a dual-vector strategy can be used to overcome the small packaging capacity of AAV. For example, cis-activation, trans-splicing, overlapping, and hybrid systems can be used.

[0040] The term "AAV transfer cassette" refers to a nucleic acid that contains a transgene with the first and second ITR sequences in proximity. The AAV transfer cassette is packaged into an AAV vector during AAV vector production.

[0041] The term "viral production cell", "viral production cell line", or "viral producer cell" refers to a cell that is used to produce a viral vector. HEK293 cells and 293T cells are common viral production cell lines. Table 2 below lists exemplary viral production cell lines for various viral vectors.

Table 2

[0042] "HEK293" refers to a cell line derived from human fetal kidney cells originally grown in tissue culture. The HEK293 cell line grows readily in culture and is commonly used for virus production. As used herein, "HEK293" can also refer to one or more variant HEK293 cell lines (i.e., cell lines derived from the original HEK293 cell line that contain one or more additional genetic changes). Many variant HEK293 strains have been developed and optimized for one or more specific applications. For example, the 293T cell line contains the SV40 large T antigen that enables episomal replication of transfected plasmids containing the SV40 origin of replication, leading to increased expression of the desired gene product.

[0043] "Sf9" refers to an insect cell line that is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE. Sf9 cells can be grown in the absence of serum and can be cultured either adherently or in suspension.

[0044] "Transfection reagent" means a composition that facilitates the transfer of nucleic acid into cells. Some transfection reagents commonly used in the art include one or more lipids (e.g., Lipofectamine™) that bind to nucleic acids and to the cell surface.

[0045] Inverted terminal repeat The sequence of the inverted terminal repeat or ITR is the minimal sequence required for AAV proviral integration and for packaging of AAV DNA into virions. The ITR is involved in various activities in the AAV life cycle. For example, the ITR sequence plays a role in excision from plasmids after transfection, replication of the vector genome, and integration and rescue from the host cell genome.

[0046] The nucleic acids of the present disclosure may include 5’ ITR and / or 3’ ITR. The ITR sequences may be about 110 to about 160 nucleotides (e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides) in length. In some embodiments, the 5’ ITR is the same length as the 3’ ITR. In some embodiments, the 5’ ITR and the 3’ ITR have different lengths. In some embodiments, the 5’ ITR is longer than the 3’ ITR, and in other embodiments, the 3’ ITR is longer than the 5’ ITR.

[0047] The ITR may be isolated from or derived from the genome of any AAV (e.g., AAVs listed in Table 1). In some embodiments, at least one of the 5’ ITR and the 3’ ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, at least one of the 5’ ITR and the 3’ ITR may be a wild-type ITR or a mutant ITR isolated from or derived from a member of another parvovirus species other than AAV. For example, in some embodiments, the ITR may be a wild-type ITR or a mutant ITR isolated from or derived from bocavirus or parvovirus B19.

[0048] In some embodiments, the ITRs include modifications that enhance the production of self-complementary AAV (scAAV). In some embodiments, the modification that enhances the production of scAAV is a deletion of the terminal resolution sequence (TRS) from the ITR. In some embodiments, the 5' ITR is a wild-type ITR and the 3' ITR is a mutant ITR lacking the terminal resolution sequence. In some embodiments, the 3' ITR is a wild-type ITR and the 5' ITR is a mutant ITR lacking the terminal resolution sequence. In some embodiments, the terminal resolution sequence is absent in both the 5' ITR and the 3' ITR. In other embodiments, the modification that enhances the production of scAAV is a substitution with a different hairpin-forming sequence (such as an shRNA-forming sequence) of the ITR.

[0049] In some embodiments, the 5' ITR or the 3' ITR may include the sequence of SEQ ID NO: 1, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the 5' ITR or the 3' ITR may include the sequence of SEQ ID NO: 2, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the 5' ITR or the 3' ITR may include the sequence of SEQ ID NO: 3, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the 5' ITR includes the sequence of SEQ ID NO: 1 and the 3' ITR includes the sequence of SEQ ID NO: 2. In some embodiments, the 5' ITR includes the sequence of SEQ ID NO: 1 and the 3' ITR includes the sequence of SEQ ID NO: 3.

[0050] In some embodiments, the nucleic acid may include one or more “surrogate” ITRs (i.e., non-ITR sequences that serve the same function as an ITR). See, e.g., Xie, J. et al., Mol. Ther., 25(6):1363-1374 (2017). In some embodiments, the ITR is replaced by a surrogate ITR. In some embodiments, the surrogate ITR includes a hairpin-forming sequence. In some embodiments, the surrogate ITR is a short hairpin (sh) RNA-forming sequence.

[0051] Promoters, enhancers, repressors, and other regulatory sequences Gene expression can be controlled by nucleotide sequences such as promoters, enhancers, and / or repressors operably linked to the gene. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0052] In some embodiments, the nucleic acids or AAV transfer cassettes described herein include a promoter. Those promoters can be, for example, constitutive promoters or inducible promoters. In some embodiments, the promoter is a tissue-specific promoter. As used herein, the term “promoter” refers to one or more nucleic acid control sequences that direct transcription of an operably linked nucleic acid. A promoter can include nucleic acid sequences near the start site of transcription (such as a TATA element). A promoter can also include cis-acting polynucleotide sequences that can be bound by transcription factors. A “constitutive” promoter is a promoter that is active under most environmental and most developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.

[0053] Exemplary promoters that can be used in the nucleic acids and cassettes described herein include the CMV promoter, SV40 promoter (e.g., SV40 early or late promoter), metallothionein promoter, mouse mammary tumor virus (MMTV) promoter, Rous sarcoma virus (RSV) promoter, polyhedrin promoter, chicken β-actin (CBA) promoter, EF-1α promoter, dihydrofolate reductase (DHFR) promoter, GUSB240 promoter (e.g., human GUSB240 (hGUSB240) promoter), GUSB379 promoter (e.g., human GUSB379 (hGUSB379) promoter), and phosphoglycerate kinase (PGK) promoter (e.g., human PGK (hPGK) promoter). In some embodiments, EF-1α is selected from the EF-1α wild-type promoter, EF-1α short promoter, and EF-1α core promoter. In some embodiments, the promoter is selected from the group consisting of the chicken β-actin (CBA) promoter, EF-1α short promoter, EF-1α wild-type promoter, EF-1α core promoter, hPGK promoter, hGUSB240 promoter, and hGUSB379 promoter. In some embodiments, the promoter comprises any one of the sequences of SEQ ID NOs: 6-12, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0054] A non-limiting list of exemplary tissue-specific promoters and enhancers that can be used in the nucleic acids and cassettes described herein includes the HMG-COA reductase promoter; sterol regulatory element 1 (SRE-1); phosphoenolpyruvate carboxykinase (PEPCK) promoter; human C-reactive protein (CRP) promoter; human glucokinase promoter; cholesterol 7-α hydroxylase (CYP-7) promoter; β-galactosidase α-2,6 sialyltransferase promoter; insulin-like growth factor binding protein (IGFBP-1) promoter; aldolase B promoter; human transferrin promoter; type I collagen promoter; prostate acid phosphatase (PAP) promoter; prostate secretory protein 94 (PSP 94) promoter; prostate-specific antigen complex promoter; human glandular kallikrein gene promoter (hgt-1); muscle cell-specific enhancer binding factor MEF-2; mucle creatine kinase promoter; pancreatitis-related protein promoter (PAP); elastase 1 transcriptional enhancer; pancreas-specific amylase and elastase enhancer promoter; pancreatic cholesterol esterase gene promoter; uteroglobin promoter; cholesterol side-chain cleavage (SCC) promoter; γ-γ enolase (neuron-specific enolase, NSE) promoter; neurofilament heavy chain (NF-H) promoter; human CGL-1 / granzyme B promoter; terminal deoxynucleotidyl transferase (TdT), λ5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoter; human CD2 promoter and its 3’ transcriptional enhancer; human NK and T cell-specific activation (NKG5) promoter; pp60c-src tyrosine kinase promoter; organ-specific neoantigen (OSN), molecular weight 40 kDa (p40) promoter; colon-specific antigen-P promoter; human α-lactalbumin promoter;Phosphoenolpyruvate carboxykinase (PEPCK) promoter, HER2 / neu promoter, casein promoter, IgG promoter, carcinoembryonic antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, tyrosine hydroxylase promoter, albumin promoter, alpha-fetoprotein promoter, acetylcholine receptor promoter, alcohol dehydrogenase promoter, alpha or beta globin promoter, T cell receptor promoter, osteocalcin promoter, IL-2 promoter, IL-2 receptor promoter, whey acidic protein (WAP) promoter, and MHC class II promoter are included.;

[0055] Gene expression can be controlled by one or more distal "enhancer" elements or "repressor" elements that can be located several thousand base pairs from the transcription start site. Enhancer elements or repressor elements regulate transcription in a manner similar to cis-acting elements near the transcription start site, except for the exception that enhancer elements can act at a distance from the transcription start site.

[0056] In some embodiments, the nucleic acids or AAV transfer cassettes described herein include an enhancer. The enhancer can be operably linked to a promoter. The enhancer can be, for example, a CMV enhancer. In some embodiments, the enhancer comprises the sequence of SEQ ID NO: 4 or 5, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0057] Transgene The nucleic acids and AAV transfer cassettes described herein can include a transgene sequence for expression in a target cell.

[0058] The transgene can be any heterologous nucleic acid sequence(s) of interest. The nucleic acid of interest can encode a polypeptide (such as a therapeutic (e.g., for medical or veterinary use) or immunogenic (e.g., for a vaccine) polypeptide) or RNA. In some embodiments, the transgene is a cDNA sequence.

[0059] In some embodiments, the transgene encodes a therapeutic polypeptide. Examples of therapeutic polypeptides include cystic fibrosis transmembrane conductance regulator protein (CFTR), dystrophin (including minidystrophin and microdystrophin, for example Vincent et al., (1993) Nature Genetics 5:130; US Patent Publication No. 2003 / 017131; International Publication WO / 2008 / 088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al., Mol. Ther.See 16:657-64(2008), myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptide (IκB dominant mutation, etc.), sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-1-antitrypsin, adenosine deaminase, puroxanthine-guanine-phosphoribosyltransferase, β-glucosylceramidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, γ-interferon, interleukin 2, interleukin 4 granulocyte macrophage colony-stimulating factor, lymphotoxin, and the like), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors -3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial-derived growth factor, transforming growth factors -α and -β, and the like), lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis growth factor soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that regulate calcium handling (e.g., SERCA. 2A, inhibitors of PP1 and fragments thereof (e.g., WO2006 / 029319 and WO2007 / 100465), molecules that achieve knockdown of G protein-coupled receptor kinase type 2 (such as truncated constitutively active bARKct, etc.), anti-inflammatory factors (such as IRAP, etc.), anti-myostatin proteins, aspartoacylase, monoclonal antibodies (including single-chain monoclonal antibodies; an exemplary monoclonal antibody is the Herceptin® monoclonal antibody), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see U.S. 7,071,172)), angiogenesis inhibitors (such as vasohibin and other VEGF inhibitors, etc.) (e.g., vasohibin 2 [see WOJP2006 / 073052]), but not limited to these. Other exemplary therapeutic polypeptides include suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that promote or inhibit transcription of host factors (e.g., nuclease-inactive Cas9 linked to a transcriptional enhancer or inhibitor element, zinc finger protein linked to a transcriptional enhancer or inhibitor element, transcriptional activator-like (TAL) effector linked to a transcriptional enhancer or inhibitor element), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, frataxin (FXN), FAS ligand, and other polypeptides having therapeutic efficacy in a subject in need thereof. The transgene can also be a monoclonal antibody or antibody fragment (e.g., an antibody or antibody fragment directed against myostatin) (see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005)). Therapeutic polypeptides include those encoding a reporter polypeptide (e.g., an enzyme). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein, β-galactosidase, alkaline phosphatase, luciferase, and the chloramphenicol acetyltransferase gene.

[0060] Optionally, the transgene encodes a secreted polypeptide (e.g., a polypeptide secreted in its native state, or a polypeptide engineered to be secreted, e.g., by operable association with a secretion signal sequence known in the art).

[0061] Alternatively, in some embodiments, the transgene can encode antisense nucleic acids, ribozymes (e.g., as described in U.S. Patent No. 5,877,022), RNAs that achieve spliceosome-mediated / ram-splicing (Puttaraju et al., (1999) Nature Biotech. 17:246; see U.S. Patent No. 6,013,487; U.S. Patent No. 6,083,702), interfering RNAs (RNAi) (including siRNA, shRNA, or miRNA via gene silencing) (see Sharp et al., (2000) Science 287:2431), and other non-translated RNAs (such as "guide" RNAs), and the like. Exemplary non-translated RNAs include RNAi against multi-drug resistance (MDR) gene products (e.g., for treating and / or preventing tumors, and / or for administering to the heart to prevent damage by chemotherapy), RNAi against myostatin (e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., for treating and / or preventing tumors), RNAi against phospholamban (e.g., for treating cardiovascular diseases, see, e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory molecules or dominant negative molecules (such as phospholamban S16E) (e.g., for treating cardiovascular diseases, see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi directed against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0062] Furthermore, the transgene sequence can direct alternative splicing. As an illustration, a complementary antisense sequence (or other inhibitory sequence) to the 5' and / or 3' splice sites of dystrophin exon 51 can be delivered in combination with a U1 or U7 small nuclear (sn)RNA promoter to induce skipping of this exon. For example, a DNA sequence containing a U1 or U7 snRNA promoter located 5' to the antisense / inhibitory sequence(s) can be packaged in a cassette and delivered by an AAV vector of the present disclosure.

[0063] In some embodiments, the transgene can direct gene editing. For example, the transgene can encode a gene editing molecule (such as a guide RNA or nuclease). In some embodiments, the transgene can encode a zinc finger nuclease, a homing endonuclease, a TALEN (transcription activator-like effector nuclease), an NgAgo (Argonaute endonuclease), an SGN (structure-guided endonuclease), or an RGN (RNA-guided nuclease) (such as Cas9 nuclease or Cpf1 nuclease).

[0064] The transgene can share homology with a locus on the host chromosome and recombine with it. This approach can be utilized, for example, to correct gene deficiencies in host cells.

[0065] The transgene can be, for example, an immunogenic polypeptide for vaccination. The transgene can encode any immunogen of interest known in the art (including, but not limited to, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, an immunogen from the HIV or SIV gag protein, a tumor antigen, a cancer antigen, a bacterial antigen, a viral antigen, and the like).

[0066] The viral vectors described in the present disclosure serve as a means for delivering transgenes into a wide range of cells, including dividing and non-dividing cells. Using the viral vectors, transgenes can be delivered into cells in vitro, for example, to produce polypeptides in vitro or for ex vivo gene therapy. The viral vectors are additionally useful in methods of delivering transgenes to a subject in need thereof, for example, to express an immunogenic or therapeutic polypeptide, or a functional RNA. In this manner, the polypeptide or functional RNA can be produced in vivo in the subject. Since the subject has a deficiency in the polypeptide, the subject may require the polypeptide. Furthermore, since the production of the polypeptide or functional RNA in the subject can confer several beneficial effects, the method can be practiced.

[0067] Polypeptides or functional RNAs of interest can also be produced in cultured cells or in a subject using the viral vectors (e.g., to produce a polypeptide or, for example, in connection with screening methods, to use the subject as a bioreactor to observe the effect of a functional RNA on the subject).

[0068] Generally, in order to treat and / or prevent any disease state in which it is beneficial to deliver a therapeutic polypeptide or functional RNA using the nucleic acids and viral vectors of the present disclosure, a transgene encoding the polypeptide or functional RNA can be delivered. Exemplary disease states include cystic fibrosis (cystic fibrosis transmembrane conductance regulator protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin), and other blood diseases; Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS ligand, cytokines (including interferon);RNAi (including RNAi against VEGF or multidrug resistance gene products), mir-26a [e.g., for hepatocellular carcinoma]), diabetes (insulin), Duchenne type (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycan [e.g., a, β, γ], RNAi against myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptide (IκB dominant mutation, etc.), sarcospan, utrophin, mini-utrophin, antisense or RNAi against splice sites in the dystrophin gene that induce exon skipping [see, e.g., WO / 2003 / 095647]), antisense against U7 snRNA that induces exon skipping [see, e.g., WO / 2006 / 021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker type, including muscular dystrophy, Gaucher disease (glucocerebrosidase), Hurler disease (L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogenosis (e.g., Fabry disease [a-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α-1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina;For example, PDGF for macular degeneration, and / or other inhibitors of vasohibin or VEGF or other angiogenesis inhibitors that treat / prevent retinal disorders in, for example, type I diabetes, diseases of solid organs such as the brain (Parkinson's disease [GDNF], astrocytoma [endostatin, angiostatin, and / or RNAi against VEGF], glioblastoma [endostatin, angiostatin, and / or RNAi against VEGF]), diseases of the liver, kidney, heart (including congestive heart failure) or peripheral artery (PAD) (for example, protein phosphatase inhibitor I (I-1) and its fragments (for example, IIC), serca2a, zinc finger protein that regulates the phospholamban gene, Barkct, [32 - adrenergic receptor, 2 - adrenergic receptor kinase (BARK), phosphoinositide - 3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that achieve knockdown of G - protein - coupled receptor kinase type 2 (short - form constitutively active bARKct, etc.); calsarcin, RNAi against phospholamban;By delivery of inhibitory phospholamban or dominant negative molecules (such as phospholamban S16E, etc.), arthritis (insulin-like growth factor), joint diseases (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivery of endothelial NOS, inducible NOS), improvement of heart transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), renal failure (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors (such as IRAP and TNFa soluble receptor, etc.)), hepatitis (interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, Friedreich's ataxia (FRDA), spinocerebellar ataxia (including SCA1, SCA2, and SCA3), phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, and the like, but not limited to these. The present disclosure can be further used after organ transplantation to increase the success of transplantation and / or reduce the negative side effects of organ transplantation or adjuvant therapy (e.g., by administering immunosuppressants or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenetic proteins (such as BNP 2, 7, etc., RANKL and / or VEGF) can be administered together with allogeneic bone grafts, for example, after resection or surgical removal in cancer patients.;

[0069] In some embodiments, the viral vectors of the present disclosure can be used to deliver a transgene encoding a polypeptide or a functional RNA for treating and / or preventing liver diseases or disorders. Liver diseases or disorders include, for example, primary biliary cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, hepatitis B, hepatitis C, alcoholic liver disease, fibrosis, jaundice, primary sclerosing cholangitis (PSC), Budd-Chiari syndrome, hemochromatosis, Wilson's disease, alcoholic fibrosis, non-alcoholic fibrosis, hepatic steatosis, Gilbert's syndrome, biliary atresia, α1-antitrypsin deficiency, Alagille syndrome, progressive familial intrahepatic cholestasis, hemophilia B, hereditary angioedema (HAE), homozygous familial hypercholesterolemia (HoFH), heterozygous familial hypercholesterolemia (HeFH), von Gierke's disease (GSD I), hemophilia A, methylmalonic acidemia, propionic academia, homocystinuria, phenylketonuria (PKU), tyrosinemia type 1, arginase 1 deficiency, argininosuccinate lyase deficiency, carbamoyl phosphate synthetase 1 deficiency, citrullinemia type 1, citrin deficiency, Crigler-Najjar syndrome type 1, cystinosis, Fabry disease, glycogenosis 1b, LPL deficiency, N-acetylglutamate synthase deficiency, ornithine transcarbamylase deficiency, ornithine translocase deficiency, primary oxaluria type 1, or ADA SCID.

[0070] Using the viral vectors of the present disclosure, transgenes used to produce induced pluripotent stem cells (iPS) can be delivered. For example, using the viral vectors of the present disclosure, stem cell-related nucleic acid(s) can be delivered into non-pluripotent cells (such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, heart cells, nerve cells, epithelial cells, endothelial cells, and the like). Transgenes encoding factors related to stem cells are known in the art. Non-limiting examples of such factors related to stem cells and pluripotency include Oct-3 / 4, the SOX family (such as SOX1, SOX2, SOX3, and / or SOX15), the Klf family (such as Klf1, Klf2, Klf4, and / or Klf5), the Myc family (such as C-myc, L-myc, and / or N-myc), NANOG, and / or LIN28.

[0071] Using the viral vectors of the present disclosure, transgenes can be delivered for the treatment and / or prevention of metabolic disorders (such as diabetes) (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), lysosomal storage disorders (such as mucopolysaccharidosis disorders) (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [α-L-iduronidase], Scheie syndrome [α-L-iduronidase], Hurler-Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilippo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:α-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfatase], B [β-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (e.g., Pompe disease; lysosomal acid α-glucosidase).

[0072] In some embodiments, the transgene is useful for the treatment of Friedreich's ataxia. In some embodiments, the transgene encodes frataxin (FXN) protein. The frataxin protein can be, for example, a human frataxin protein. An exemplary human frataxin protein sequence is provided below (SEQ ID NO: 65). MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA See also Uniprot accession number Q16595, which is hereby incorporated by reference in its entirety. In some embodiments, the frataxin protein has a sequence that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence of the human frataxin protein. In some embodiments, the frataxin protein has a sequence that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the sequence of SEQ ID NO: 65. In some embodiments, the human frataxin protein is an isoform, variant (e.g., alternative splice variant), or mutant of frataxin. In some embodiments, the mutant frataxin has one or more of the substitutions shown in Table 3.

Table 3

[0073] In some embodiments, the transgene comprises frataxin cDNA codon-optimized compared to the wild-type sequence. For example, the cDNA can be modified to remove hidden splice acceptor / donor sites, reduce the frequency of rare codon usage, remove ribosome entry sites, and the like. In some embodiments, the transgene comprises CpG-optimized frataxin cDNA. For example, the cDNA can be modified to reduce the number of CpG dinucleotides.

[0074] In some embodiments, the transgene comprises frataxin cDNA comprising the sequence of SEQ ID NO: 19, or a sequence that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical thereto. In some embodiments, the transgene comprises frataxin cDNA comprising the sequence of SEQ ID NO: 20, or a sequence that is at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical thereto.

[0075] Polyadenylation (polyA) signal The polyadenylation signal is a nucleotide sequence found in almost all mammalian genes and controls the addition of a stretch of approximately 200 adenosine residues (poly(A) tail) to the 3' end of the gene transcript. The poly(A) tail contributes to the stability of the mRNA, and mRNAs lacking the poly(A) tail are rapidly degraded. There is also evidence that the presence of the poly(A) tail positively contributes to the translatability of the mRNA by affecting the initiation of translation.

[0076] In some embodiments, the nucleic acids and AAV transfer cassettes of the present disclosure include one or more polyadenylation signals. In some embodiments, the nucleic acids and AAV transfer cassettes include two, three, four, or more polyadenylation signals. The polyadenylation signal can be a polyadenylation signal of simian virus 40 (SV40), α-globin (such as human α-globin, mouse α-globin, or rabbit α-globin), β-globin (such as human β-globin, mouse β-globin, or rabbit β-globin), human collagen, polyomavirus, human growth hormone (hGH) or bovine growth hormone (bGH), or a variant thereof.

[0077] In some embodiments, the polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal (such as the bGH polyadenylation signal having the sequence of SEQ ID NO: 21). In some embodiments, the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal (such as the hGH polyadenylation signal having the sequence of SEQ ID NO: 22). In some embodiments, the polyadenylation signal is a human β-globin polyadenylation signal (such as the human β-globin polyadenylation signal having the sequence of SEQ ID NO: 23). In some embodiments, the polyadenylation signal is a rabbit β-globin polyadenylation signal (such as the rabbit β-globin polyadenylation signal having the sequence of SEQ ID NO: 24). In some embodiments, the polyadenylation signal includes any one of the sequences of SEQ ID NOs: 21-24, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0078] In some embodiments, the polyadenylation signal can be present in the reverse orientation in the nucleic acid or cassette. In the reverse orientation, the polyadenylation signal can act as a safety factor. For example, the reverse polyadenylation signal can prevent significant transcription in the reverse direction from the promoter.

[0079] In some embodiments, the nucleic acid or AAV transfer cassette comprises two polyadenylation signals (such as the polyadenylation signals of SEQ ID NOs: 21 and 22). In embodiments where the nucleic acid or AAV transfer cassette comprises two polyadenylation signals, one of the signals may be present in the reverse orientation.

[0080] Stuffer sequence AAV vectors typically tolerate DNA inserts having a defined size range generally of about 4 kb to about 5.2 kb or slightly larger. Thus, for shorter sequences, it may be necessary to include additional nucleic acids in the insert fragment to achieve the required length that is acceptable for the AAV vector. The stuffer sequence may be isolated from or derived from non-coding regions (such as intronic regions) of known genes or nucleic acid sequences. The stuffer sequence can be, for example, a sequence having a length between 1 and 10, 10 and 20, 20 and 30, 30 and 40, 40 and 50, 50 and 60, 60 and 75, 75 and 100, 100 and 150, 150 and 200, 200 and 250, 250 and 300, 300 and 400, 400 and 500, 500 and 750, 750 and 1,000, 1,000 and 1,500, 1,500 and 2,000, 2,000 and 2,500, 2,500 and 3,000, 3,000 and 3,500, 3,500 and 4,000, 4,000 and 4,500, 4,500 and 5,000, 5,500 and 6,000, 6,000 and 7,000, 7,000 and 8,000, or 8,000 and 9,000 nucleotides. The stuffer sequence can be located in the nucleic acid or cassette at any desired position such that it does not interfere with function or activity.

[0081] In some embodiments, the nucleic acid or AAV transfer cassette of the present disclosure includes a spacer sequence. In some embodiments, the spacer sequence includes an intronic sequence or a sequence derived therefrom. In some embodiments, the spacer sequence is a chimeric sequence. In some embodiments, the spacer sequence is isolated from or derived from a gene such as α1 - antitrypsin or albumin. In some embodiments, the spacer sequence is selected from any one of the sequences of SEQ ID NOs: 25 - 27, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0082] Intronic sequence In some embodiments, the nucleic acid and / or transfer cassette of the present disclosure may include an intronic sequence. By including an intronic sequence, factors important for efficient nuclear export and translation can be recruited to the transcribed mRNA. Thus, including an intronic sequence can promote expression as compared to expression in the absence of the intronic sequence.

[0083] In some embodiments, the intronic sequence is a hybrid sequence or a chimeric sequence. In some embodiments, the intronic sequence is isolated from or derived from one or more intronic sequences of β - globin, triβ - actin, murine minute virus (MVM), factor IX, SV40, and / or human IgG (heavy or light chain). In some embodiments, the intronic sequence includes any one of the sequences of SEQ ID NOs: 13 - 16, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0084] Kozak sequence The Kozak sequence is a short sequence centered around the translation start site of eukaryotic mRNA that enables efficient initiation of mRNA translation. The ribosomal translation apparatus recognizes the AUG start codon within the context of the Kozak sequence.

[0085] In some embodiments, the AAV transfer cassette of the present disclosure may include a Kozak sequence. The Kozak sequence may promote the translation efficiency and overall expression of the transgene. The Kozak sequence may be located immediately 5' to the transgene sequence or may overlap with the transgene sequence.

[0086] The Kozak sequence in the nucleic acid or AAV transfer cassette of the present disclosure may be a consensus sequence or a modified version thereof. The Kozak sequence may include any one of the sequences of SEQ ID NOs: 17-18 or 66-70, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0087] Nucleic Acids and AAV Transfer Cassettes In some embodiments, a nucleic acid or adeno-associated virus (AAV) transfer cassette includes one or more of an enhancer, a promoter, an intronic sequence, a Kozak sequence, a transgene sequence, a polyadenylation signal, and / or a suffer sequence. In some embodiments, a nucleic acid or adeno-associated virus (AAV) transfer cassette includes any combination of an enhancer, a promoter, an intronic sequence, a Kozak sequence, a transgene sequence, a polyadenylation signal, and / or a suffer sequence.

[0088] In some embodiments, a nucleic acid or adeno-associated virus (AAV) transfer cassette includes, from 5' to 3', a 5' inverted terminal repeat (ITR), a promoter, a transgene sequence, a polyadenylation signal, and a 3' ITR.

[0089] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, an enhancer, a promoter, a transgene sequence, a polyadenylation signal, and a 3' ITR.

[0090] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, an enhancer, a promoter, an intronic sequence, a transgene sequence, a polyadenylation signal, and a 3' ITR.

[0091] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, a promoter, an intronic sequence, a transgene sequence, a polyadenylation signal, and a 3' ITR.

[0092] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, a polyA signal (in reverse), a promoter, an intronic sequence, a transgene sequence, a polyadenylation signal, a stuffer sequence, and a 3' ITR.

[0093] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, a stuffer sequence, a polyadenylation signal (in reverse), a promoter, an intronic sequence, a transgene sequence, a polyadenylation signal, a stuffer sequence, and a 3' ITR.

[0094] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, a stuffer sequence, a polyadenylation signal (in reverse), a promoter, a transgene sequence, a polyadenylation signal, a stuffer sequence, and a 3' ITR.

[0095] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', a 5' ITR, a promoter, an intronic sequence, a transgene sequence, a polyadenylation signal, a suffer sequence, and a 3' ITR.

[0096] In any of the above embodiments, the nucleic acid or AAV transfer cassette may further comprise a Kozak sequence. The Kozak sequence may be located immediately 5' to the transgene sequence. The Kozak sequence may have any one of the sequences of SEQ ID NOs: 17-18.

[0097] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', the elements shown in Table 4, or any subset thereof. Different exemplary nucleic acids or AAV transfer cassettes are shown in each row of the table. "x" indicates that the indicated element is included in the nucleic acid or AAV transfer cassette.

Table 4

[0098] In any of the above embodiments, the transgene sequence may encode a frataxin (FXN) protein. The transgene sequence may have, for example, the sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the transgene may encode an FXN protein having the sequence of SEQ ID NO: 65.

[0099] In any of the above embodiments, the 5' ITR has the sequence of SEQ ID NO: 1, and the 3' ITR may have the sequence of SEQ ID NO: 2 or 3.

[0100] In any of the above embodiments, the enhancer may have any one of the sequences of SEQ ID NOs: 4-5.

[0101] In any of the above embodiments, the promoter may have any one of the sequences of SEQ ID NOs: 6 to 12.

[0102] In any of the above embodiments, the intronic sequence may have any one of the sequences of SEQ ID NOs: 13 to 16.

[0103] In any of the above embodiments, the polyadenylation signal may include any one of the sequences of SEQ ID NOs: 21 to 24.

[0104] In any of the above embodiments, the stuffer sequence may include any one of the sequences of SEQ ID NOs: 25 to 27.

[0105] In some embodiments, the nucleic acid or AAV transfer cassette comprises, from 5' to 3', the elements and sequences shown in Table 5, or any subset thereof. Different exemplary nucleic acids or AAV transfer cassettes are shown in each row of the table. The numbers provided in the table correspond to SEQ ID numbers.

Table 5-1

Table 5-2

[0106] In some embodiments, the nucleic acid or AAV transfer cassette comprises any one of the sequences of SEQ ID NOs: 28 to 64, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0107] The nucleic acids and AAV transfer cassettes described herein can be incorporated into a vector (e.g., a plasmid or a bacmid) using standard molecular biology techniques. The vector (e.g., a plasmid or a bacmid) can further contain one or more gene elements (e.g., the AAV rep gene and the cap gene) and helper virus protein sequences that are used during the production of AAV.

[0108] Recombinant AAV and AAV production methods Recombinant AAV vectors can be produced using nucleic acids and AAV transfer cassettes, and vectors (e.g., plasmids) containing the nucleic acids and AAV transfer cassettes described herein. The AAV vector can contain a single-stranded genome or a double-stranded genome (i.e., scAAV). High-titer AAV preparations can be produced using techniques known in the art (such as standard triple transfection or production methods using baculovirus).

[0109] Typically, methods for the production of AAV vectors involve four components, where the plasmids act in trans and the transgenes act in cis. These components include: 1) a plasmid containing the AAV Rep and Cap genes for capsid formation and replication; 2) a plasmid containing adenovirus helper genes; 3) a cassette containing the transgene flanked by two inverted terminal repeats (ITRs); and 4) a viral packaging cell line. Since AAV is highly infectious and naturally present in a large percentage of the human population, cell cultures and all materials can be fully tested for transient wild-type AAV infection prior to use.

[0110] In some embodiments, a method of producing a recombinant AAV vector comprises contacting an AAV-producing cell (e.g., a HEK293 cell) with a nucleic acid, an AAV transfer cassette, or a vector (e.g., a plasmid) of the present disclosure. In some embodiments, the method further comprises contacting the AAV-producing cell with one or more additional vectors (e.g., plasmids) encoding, for example, the AAV rep and cap genes and helper virus protein sequences. In some embodiments, the method further comprises maintaining the AAV-producing cell under conditions such that AAV is produced.

[0111] In some embodiments, a method of producing a recombinant AAV vector comprises contacting an AAV-producing cell (e.g., an insect cell such as an Sf9 cell) with at least one insect cell-compatible vector comprising a nucleic acid of the present disclosure or an AAV transfer cassette. An "insect cell-compatible vector" is any biological or chemical compound or formulation that facilitates the transformation or transfection of an insect cell by a nucleic acid. In some embodiments, the insect cell-compatible vector is a baculovirus vector. In some embodiments, the method further comprises maintaining the insect cell under conditions such that AAV is produced.

[0112] In some embodiments, the AAV-producing cell is transfected (e.g., using a transfection reagent) with three plasmids: (1) a first plasmid comprising a nucleic acid of the present disclosure or an AAV transfer cassette, (2) a second plasmid comprising the sequences of the AAV rep and cap genes, and (3) a third plasmid comprising helper virus protein sequences. See, for example, FIG. 5. The AAV-producing cell can be any of the cells listed in Table 2. The AAV-producing cell can be maintained under conditions such that AAV is subsequently produced. The AAV can then be purified using standard techniques such as cesium chloride (CsCl) gradient centrifugation or column chromatography techniques.

[0113] The produced recombinant AAV vector may contain a capsid of any serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV). In some embodiments, the produced recombinant AAV vector may contain a capsid protein with one or more amino acid modifications (e.g., substitutions and / or deletions) compared to the original AAV capsid. For example, the recombinant AAV vector may contain a modified AAV capsid derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. In some embodiments, the produced AAV vector is AAV9. In some embodiments, the produced AAV vector is AAV1. In some embodiments, the produced AAV vector is AAV4.

[0114] Using the recombinant AAV vector, the target cells can be transduced with the transgene sequence by contacting the recombinant AAV vector with the target cells.

[0115] Composition Compositions are also provided that contain the nucleic acids, AAV transfer cassettes, plasmids, cells, or recombinant AAV vectors of the present disclosure. In some embodiments, the composition is a liquid composition. In some embodiments, the composition is a solid composition.

[0116] In some embodiments, pharmaceutical compositions are provided that include the nucleic acids, AAV transfer cassettes, plasmids, cells, or recombinant AAV vectors of the present disclosure. Pharmaceutical compositions for use as described in and according to the present disclosure, in addition to the nucleic acids, AAV transfer cassettes, plasmids, cells, or recombinant AAV vectors, may include pharmaceutically acceptable excipients, carriers, buffers, stabilizing substances, or other materials (e.g., diluents, adjuvants, fillers, preservatives, antioxidants, lubricants, solubilizing agents, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents). Such materials must preferably be non-toxic. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington’s Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. The exact nature of the carrier or other material depends on the route of administration, which can be oral or by injection (e.g., intradermal, subcutaneous, or intravenous).

[0117] Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powders, or liquids. Tablets can include solid carriers or adjuvants. Liquid pharmaceutical compositions generally include a liquid carrier (such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oils, etc.). Physiological saline, dextrose solutions or other sugar solutions, or glycols (such as ethylene glycol, propylene glycol, or polyethylene glycol, etc.) can be included. Capsules can include solid carriers such as gelatin.

[0118] For intravenous injection, intradermal injection, subcutaneous injection, or injection at the affected site, the pharmaceutical composition can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Suitable solutions can include, for example, isotonic vehicles (such as sodium chloride, Ringer's solution, and / or lactated Ringer's solution, etc.). Antiseptic substances, stabilizing substances, buffers, antioxidants, and / or other additives can be included as required.

[0119] Method of treatment Using the AAV vectors of the present disclosure (including AAV vectors prepared using the nucleic acids or AAV transfer cassettes of the present disclosure), the diseases, disorders, or other conditions of a subject in need of treatment or prevention of a disease, disorder, or other condition can be treated or prevented. The subject can be a mammal or a bird. In some embodiments, the mammal is a cat, dog, mouse, rat, horse, cow, pig, guinea pig, or non-human primate. In some embodiments, the subject is a human. The human can be a pediatric subject, an adult subject, or an elderly subject.

[0120] The AAV vectors of the present disclosure or compositions containing them can be contacted with cells in vivo or ex vivo. The cells can then be maintained under conditions sufficient for expression of the transgene in the cells.

[0121] The AAV vectors of the present disclosure or compositions containing them can be administered to a subject in need thereof. The administration can be by any means known in the art. Optionally, the viral vector and / or composition is delivered in a therapeutically effective dose in a pharmaceutically acceptable carrier. In some embodiments, a therapeutically effective dose of the viral vector and / or composition is delivered.

[0122] Administration of the viral vector and / or composition to be administered to a subject may be determined in a routine manner depending on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular viral vector or composition, the nucleic acid to be delivered, and the like. Exemplary dosages for achieving therapeutic efficacy are at least about 10 5 at least about 10 6 at least about 10 7 at least about 10 8 at least about 10 9 at least about 10 10 at least about 10 11 at least about 10 12 at least about 10 13 at least about 10 14 at least about 10 15 transducing units, optionally at a titer of about 10 8 to about 10 13 transducing units.

[0123] In certain embodiments, multiple administrations (e.g., 2, 3, 4 or more administrations) can be used to achieve the desired level of gene expression over various intervals of time (e.g., 1 day, 1 week, 1 month, 1 year, etc.).

[0124] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovarian), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle), intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., including airway surface and transdermal administration, to both skin and mucosal surfaces), intralymphatic, and the like, and direct injection into a tissue or organ (e.g., to the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration can also be to a tumor (e.g., within or near a tumor or lymph node). The most suitable route in a given case will depend on the nature and severity of the condition being treated and / or prevented, as well as the nature of the particular vector being used.

[0125] In some embodiments, the AAV vector or composition comprising the vector can be administered by direct injection into cardiac tissue or central nervous system (CNS) tissue. In some embodiments, the AAV vector or composition comprising the vector can be delivered intracranially (including intrathecal, intraneural, intracerebral, or intraventricular administration). In some embodiments, the AAV vector or composition comprising the vector can be delivered to the heart by direct administration to the myocardium by epicardial injection followed by minithoracotomy, by intracoronary injection, or by endocardial myocardial injection.

[0126] Delivery to the target tissue can also be achieved by delivery of a depot comprising the viral vector and / or capsid. In representative embodiments, the depot comprising the viral vector and / or capsid is implanted into skeletal muscle, myocardial, and / or diaphragmatic muscle tissue, or the tissue can be contacted with a film or other matrix comprising the viral vector and / or capsid. Such implantable matrices or substrates are described in U.S. Patent No. 7,201,898.

[0127] Administration of AAV can result in robust and persistent transgene expression in the target cells or tissues. For example, transgene expression can persist for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, or longer.

[0128] In some embodiments, a method of treating a subject in need of treatment comprises administering to the subject a therapeutically effective amount of a nucleic acid, AAV transfer cassette, plasmid, cell, or recombinant AAV of the present disclosure. In some embodiments, the subject is a human subject. In some embodiments, the subject has Friedreich's ataxia. Administration can result in the expression of a therapeutically effective amount of the FXN protein in the subject's CNS tissue (e.g., neural tissue) or heart tissue.

[0129] In some embodiments, administration can result in the alleviation of one or more symptoms of Friedreich's ataxia. For example, administration can (1) improve coordination (ataxia) in the subject's arms and legs, (2) increase the subject's energy levels and / or decrease fatigue and loss of muscle strength, (3) improve vision, hearing loss, or speech in the subject, (3) decrease scoliosis or its rate of progression, (4) improve symptoms of diabetes (such as insulin sensitivity), or (5) remit heart pathologies (such as hypertrophic cardiomyopathy or arrhythmia). The improvement in the subject due to treatment can be an improvement compared to the subject prior to treatment or compared to a typical subject with Friedreich's ataxia.

[0130] In some embodiments, administration can result in the extension of the subject's lifespan. For example, administration can extend the lifespan of the subject by about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 5 - 10 years, or more than 10 years compared to a typical subject with Friedreich's ataxia.

Examples

[0131] The following examples are included herein for illustrative purposes only and are not intended to be limiting.

[0132] Example 1: Preparation of Recombinant AAV Vectors in Mammalian Cells Three plasmids are provided. The first plasmid contains a transfer cassette encoding human frataxin with two adjacent ITRs (SEQ ID NO: 1, SEQ ID NO: 2 or 3) (cDNA (SEQ ID NO: 19 or 20)), and has any one of the sequences of SEQ ID NOs: 28 to 64. The second plasmid contains sequences encoding the Rep gene and the Cap gene. The third plasmid contains various "helper" sequences (E4, E2a, and VA) required for AAV production.

[0133] The three plasmids are transfected into virus-producing cells (such as HEK293) using an appropriate transfection reagent (such as Lipofectamine (trademark)). After incubation for a predetermined period of time at 37°C, AAV particles are collected from the culture medium or the cells are lysed to release the AAV particles. The AAV particles are then purified, titered, and can be stored at -80°C for later use.

[0134] Example 2: Preparation of Recombinant AAV Vectors in Insect Cells A first recombinant baculovirus vector is provided. The first recombinant baculovirus vector contains a transfer cassette sequence encoding human frataxin with two adjacent ITRs (SEQ ID NO: 1, SEQ ID NO: 2 or 3) (cDNA (SEQ ID NO: 19 or 20)), and the transfer cassette has any one of the sequences of SEQ ID NOs: 28 to 64.

[0135] Insect cells (such as Sf9) are co-infected in suspension culture with the first recombinant baculovirus vector and at least one additional recombinant baculovirus vector containing sequences encoding the AAV Rep gene and the Cap protein. After incubation for a predetermined period of time at 28°C, AAV particles are collected from the culture medium or the cells are lysed to release the AAV particles. The AAV particles are then purified, titered, and can be stored at -80°C for later use.

[0136] Example 3: The recombinant AAV packaging FXN transgene transduces cardiac cells in vivo and extends the lifespan of FXN-deficient mice. A plasmid containing an AAV transfer cassette (SEQ ID NO: 32) containing the human FXN transgene was prepared using standard cloning techniques (FXN plasmid). A composition comprising the FXN plasmid, a second plasmid containing sequences encoding the AAV Rep gene and the Cap (AAV9) gene, and a third plasmid containing sequences encoding the AAV helper sequences was prepared and used to transfect HEK293 cells using a standard "triple transfection" protocol. HEK293 cells were maintained under standard culture conditions (37 °C, 5% CO2) to allow production of the recombinant self-complementary AAV9 vector. This procedure was repeated multiple times, and the AAV9 vector yield was quantified using ddPCR®. As shown in Figure 1, the yield of AAV9 packaging the FXN transgene (AAV9-FXN) in each run was between 10 13 ~10 14 vector genomes.

[0137] Lec2 cells in culture were transduced with recombinant AAV9-FXN. Figure 4 shows the expression of human FXN (ng / mg) in cultured Lec2 cells transduced with various doses of AAV9-FXN. Higher expression of hFXN was observed with the use of higher doses of the vector.

[0138] Mice lacking FXN in the heart and skeletal muscle (FXN flox / flox MCKCre + ) were also infected. Mice were treated at 3 weeks of age with either saline or AAV9-FXN (5×10 13 vg / kg) and survival was monitored. As shown in Figure 2, treatment with AAV9-FXN significantly increased lifespan. The median survival of saline-injected mice was 64 days, while the median survival of AAV9-FXN-injected mice was 138.5 days.

[0139] In separate experiments, FXN-deficient mice were treated at 3 weeks of age with either saline or low or high doses of AAV9-FXN (1×10 13 or 5×10 13 vg / kg, respectively). Mice were sacrificed 3 weeks after treatment and heart tissue was analyzed. As shown in Figure 3A, human FXN (hFXN) DNA was detectable in heart tissue from AAV9-FXN-treated mice. hFXN DNA was transcribed into RNA (Figure 3B) and translated into protein (Figure 3C). Higher doses of AAV9-FXN led to higher levels of FXN DNA, RNA, and protein in heart samples.

[0140] Collectively, these data show that recombinant AAV vectors can be produced using the AAV transfer cassettes of the present disclosure that contain the FXN transgene, and that target cells can be transduced in vivo.

[0141] Numbered embodiments Notwithstanding the appended claims, the present disclosure describes the following numbered embodiments of the present disclosure.

[0142] 1. A nucleic acid comprising, from 5’ to 3’, a 5’ inverted terminal repeat (ITR); a promoter; a transgene sequence; a polyadenylation signal; and a 3’ ITR, wherein the transgene sequence encodes a frataxin (FXN) protein.

[0143] 2. The nucleic acid of embodiment 1, wherein at least one of the 5’ ITR and the 3’ ITR is about 110 to about 160 nucleotides in length.

[0144] 3. The nucleic acid of embodiment 1 or 2, wherein the 5’ ITR is the same length as the 3’ ITR.

[0145] 4. The nucleic acid of embodiment 1 or 2, wherein the 5’ ITR and the 3’ ITR have different lengths.

[0146] 5. At least one of the 5’ ITR and the 3’ ITR is a nucleic acid of any one of Embodiments 1-4 isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

[0147] 6. The nucleic acid of Embodiment 1, wherein the 5’ ITR comprises the sequence of SEQ ID NO: 1 or a sequence that is at least 95% identical thereto.

[0148] 7. The nucleic acid of any one of Embodiments 1-6, wherein the 3’ ITR comprises the sequence of SEQ ID NO: 2 or a sequence that is at least 95% identical thereto.

[0149] 8. The nucleic acid of any one of Embodiments 1-7, wherein the 3’ ITR comprises the sequence of SEQ ID NO: 3 or a sequence that is at least 95% identical thereto.

[0150] 9. The nucleic acid of any one of Embodiments 1-8, wherein the promoter drives the expression of the transgene.

[0151] 10. The nucleic acid of any one of Embodiments 1-9, wherein the promoter is a constitutive promoter.

[0152] 11. The nucleic acid of any one of Embodiments 1-9, wherein the promoter is an inducible promoter.

[0153] 12. The nucleic acid of any one of Embodiments 1-11, wherein the promoter is a tissue-specific promoter.

[0154] 13. The nucleic acid according to any one of Embodiments 1 to 12, wherein the promoter is selected from the group consisting of a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus (MMTV) promoter, a Rous sarcoma virus (RSV) promoter, a polyhedrin promoter, a chicken β-actin (CBA) promoter, an EF-1α promoter, an EF-1α short promoter, an EF-1α core promoter, a dihydrofolate reductase (DHFR) promoter, a GUSB240 promoter, a GUSB379 promoter, and a phosphoglycerate kinase (PGK) promoter.

[0155] 14. The nucleic acid according to Embodiment 13, wherein the promoter is a chicken β-actin (CBA) promoter.

[0156] 15. The nucleic acid according to Embodiment 13, wherein the promoter is an EF-1α promoter, an EF-1α short promoter, or an EF-1α core promoter.

[0157] 16. The nucleic acid according to Embodiment 13, wherein the promoter is a GUSB240 promoter.

[0158] 17. The nucleic acid according to Embodiment 13, wherein the promoter is a GUSB379 promoter.

[0159] 18. The nucleic acid according to Embodiment 13, wherein the promoter is a PGK promoter.

[0160] 19. The nucleic acid according to any one of Embodiments 1 to 12, wherein the promoter comprises a sequence selected from any one of SEQ ID NOs: 6 to 12, or a sequence that is at least 95% identical thereto.

[0161] 20. The nucleic acid according to any one of Embodiments 1 to 19, wherein the FXN protein is a human FXN protein.

[0162] 21. The nucleic acid of any one of Embodiments 1 to 20, wherein the FXN protein has the sequence of SEQ ID NO: 65 or a sequence that is at least 95% identical thereto.

[0163] 22. The nucleic acid of any one of Embodiments 1 to 21, wherein the transgene sequence is CpG-optimized.

[0164] 23. The nucleic acid of any one of Embodiments 1 to 21, wherein the transgene sequence comprises the sequence of SEQ ID NO: 19 or 20, or a sequence that is at least 95% identical thereto.

[0165] 24. The nucleic acid of any one of Embodiments 1 to 24, wherein the nucleic acid comprises a Kozak sequence immediately 5' of the transgene sequence.

[0166] 25. The nucleic acid of Embodiment 24, wherein the Kozak sequence comprises the sequence of SEQ ID NO: 17 or 18, or a sequence that is at least 95% identical thereto.

[0167] 26. The nucleic acid of any one of Embodiments 1 to 25, wherein the polyadenylation signal is selected from the polyadenylation signals of simian virus 40 (SV40), human α-globin, rabbit α-globin, human β-globin, rabbit β-globin, human collagen, polyomavirus, human growth hormone (hGH), and bovine growth hormone (bGH).

[0168] 27. The nucleic acid of Embodiment 26, wherein the polyadenylation signal is the bovine growth hormone polyadenylation signal.

[0169] 28. The nucleic acid of Embodiment 26, wherein the polyadenylation signal is the human growth hormone polyadenylation signal.

[0170] 29. The nucleic acid of Embodiment 26, wherein the polyadenylation signal is the human β-globin polyadenylation signal.

[0171] 30. The nucleic acid according to embodiment 26, wherein the polyadenylation signal is a rabbit β-globin polyadenylation signal.

[0172] 31. The nucleic acid according to any one of embodiments 1 to 25, wherein the polyadenylation signal comprises any one of the sequences of SEQ ID NOs: 21 to 24, or a sequence that is at least 95% identical thereto.

[0173] 32. The nucleic acid according to any one of embodiments 1 to 31, wherein the nucleic acid further comprises an enhancer.

[0174] 33. The nucleic acid according to embodiment 32, wherein the enhancer is a CMV enhancer.

[0175] 34. The nucleic acid according to embodiment 32, wherein the enhancer comprises the sequence of SEQ ID NO: 4 or 5, or a sequence that is at least 95% identical thereto.

[0176] 35. The nucleic acid according to any one of embodiments 1 to 34, wherein the cassette further comprises an intronic sequence.

[0177] 36. The nucleic acid according to embodiment 35, wherein the intronic sequence is a chimeric sequence.

[0178] 37. The nucleic acid according to embodiment 35, wherein the intronic sequence is a hybrid sequence.

[0179] 38. The nucleic acid according to embodiment 35, wherein the intronic sequence comprises a sequence isolated from or derived from one or more intronic sequences of β-globin, chicken β-actin, murine minute virus, and human IgG.

[0180] 39. The nucleic acid according to embodiment 35, wherein the intronic sequence comprises any one of the sequences of SEQ ID NOs: 13 to 16, or a sequence that is at least 95% identical thereto.

[0181] 40. The nucleic acid according to any one of Embodiments 1 to 39, further comprising at least one stuffer sequence.

[0182] 41. The nucleic acid according to Embodiment 40, wherein the nucleic acid comprises two stuffer sequences.

[0183] 42. The nucleic acid according to Embodiment 40, wherein the at least one stuffer sequence comprises any one of the sequences of SEQ ID NOs: 25 to 27, or a sequence that is at least 95% identical thereto.

[0184] 43. The nucleic acid according to Embodiment 1, wherein the nucleic acid comprises any one of the sequences of SEQ ID NOs: 28 to 64, or a sequence that is at least 95% identical thereto.

[0185] 44. A plasmid comprising the nucleic acid according to any one of Embodiments 1 to 43.

[0186] 45. A cell comprising the nucleic acid according to any one of Embodiments 1 to 43 or the plasmid according to Embodiment 44.

[0187] 46. A method for producing a recombinant AAV vector, the method comprising contacting an AAV-producing cell with the nucleic acid according to any one of Embodiments 1 to 43 or the plasmid according to Embodiment 44.

[0188] 47. A recombinant AAV vector produced by the method according to Embodiment 46.

[0189] 48. The recombinant AAV vector according to Embodiment 47, wherein the recombinant AAV vector is single-stranded AAV (ssAAV).

[0190] 49. The recombinant AAV vector according to Embodiment 47, wherein the recombinant AAV vector is self-complementary AAV (scAAV).

[0191] 50. The recombinant AAV vector according to any one of embodiments 47-49, wherein the recombinant AAV vector comprises a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

[0192] 51. The recombinant AAV vector according to any one of embodiments 47-49, wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to the wild-type AAV capsid protein.

[0193] 52. A composition comprising the nucleic acid according to any one of embodiments 1-43, the plasmid according to embodiment 44, the cell according to embodiment 45, or the recombinant AAV vector according to any one of embodiments 47-51.

[0194] 53. A method of treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of the nucleic acid according to any one of embodiments 1-43, the plasmid according to embodiment 44, the cell according to embodiment 45, or the recombinant AAV vector according to any one of embodiments 47-41.

[0195] 54. The method of embodiment 53, wherein the subject has Friedreich's ataxia.

[0196] 55. The method of embodiment 53 or 54, wherein the subject is a human subject.

[0197] 56. The method according to any one of embodiments 53-55, wherein the nucleic acid, the plasmid, the cell, or the recombinant AAV vector is administered by direct injection into the central nervous system.

[0198] The foregoing are examples of the present invention and should not be construed as limiting thereof. The present invention is defined by the following claims, together with the equivalents of the claims contained therein.

Claims

1. From 5' to 3', a 5' inverted terminal repeat (ITR) containing SEQ ID NO: 1; a constitutive promoter containing any one of the sequences of SEQ ID NOs: 6-12, or a sequence that is at least 95% identical thereto; a transgene containing a sequence that is at least 95% identical to SEQ ID NO: 19 or 20, encoding frataxin (FXN) protein; one or more polyadenylation signals containing any one of the sequences of SEQ ID NOs: 21-24, or a sequence that is at least 95% identical thereto; and a 3' ITR containing SEQ ID NO: 2 or 3 A nucleic acid comprising.

2. An intronic sequence, wherein the intronic sequence contains any one of the sequences of SEQ ID NOs: 13-16, or a sequence that is at least 95% identical thereto; A Kozak sequence immediately 5' of the transgene sequence, wherein the Kozak sequence contains the sequence of SEQ ID NO: 17 or 18, or a sequence that is at least 95% identical thereto; The promoter contains a sequence selected from any one of the sequences of SEQ ID NOs: 6-12, or a sequence that is at least 95% identical thereto; The one or more polyadenylation signals contain any one or more of the sequences of SEQ ID NOs: 21-24, or a sequence that is at least 95% identical thereto; and / or Any combination of these The nucleic acid according to claim 1, comprising.

3. From 5' to 3', A 5' inverted terminal repeat (ITR); A promoter, wherein the promoter contains a sequence selected from any one of the sequences of SEQ ID NOs: 6-12; An intronic sequence, wherein the intronic sequence contains any one of the sequences of SEQ ID NOs: 13-16, or a sequence that is at least 95% identical thereto; A Kozak sequence immediately 5' of the transgene sequence, wherein the Kozak sequence contains the sequence of SEQ ID NO: 17 or 18, or a sequence that is at least 95% identical thereto; One or more polyadenylation signals, wherein the one or more polyadenylation signals contain any one or more of the sequences of SEQ ID NOs: 21-24; and A 3' ITR The nucleic acid according to claim 1 or 2, comprising.

4. The nucleic acid according to any one of claims 1-3, wherein the transgene sequence is CpG-optimized.

5. The nucleic acid according to any one of claims 1-4, wherein the nucleic acid contains an enhancer.

6. The nucleic acid according to claim 5, wherein the enhancer comprises the sequence of SEQ ID NO: 4 or 5, or a sequence that is at least 95% identical thereto.

7. The nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid comprises any one of the sequences of SEQ ID NOs: 28 to 64, or a sequence that is at least 85% identical thereto.

8. The nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid comprises any one of the sequences of SEQ ID NOs: 28 to 64, or a sequence that is at least 90% identical thereto.

9. The nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid comprises any one of the sequences of SEQ ID NOs: 28 to 64, or a sequence that is at least 95% identical thereto.

10. The nucleic acid according to any one of claims 7 to 9, wherein the nucleic acid comprises the sequence of SEQ ID NO: 51, or consists of the sequence of SEQ ID NO:

51.

11. The nucleic acid according to any one of claims 7 to 9, wherein the nucleic acid comprises the sequence of SEQ ID NO: 49, or consists of the sequence of SEQ ID NO:

49.

12. The nucleic acid according to any one of claims 7 to 9, wherein the nucleic acid comprises the sequence of SEQ ID NO: 32, or consists of the sequence of SEQ ID NO:

32.

13. A plasmid comprising the nucleic acid according to any one of claims 1 to 12.

14. A cell comprising the nucleic acid according to any one of claims 1 to 12 or the plasmid according to claim 13.

15. A method for producing a recombinant AAV vector, comprising contacting an AAV-producing cell with the nucleic acid according to any one of claims 1 to 12 or the plasmid according to claim 13.

16. A recombinant AAV vector produced by the method according to claim 15.

17. The recombinant AAV vector according to claim 16, wherein the recombinant AAV vector is single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

18. The recombinant AAV vector according to claim 16, wherein the recombinant AAV vector comprises a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

19. The recombinant AAV vector according to any one of claims 16 to 18, wherein the AAV vector contains a capsid protein from AAV9.

20. A composition comprising the nucleic acid according to any one of claims 1 to 12, the plasmid according to claim 13, the cell according to claim 14, or the recombinant AAV vector according to any one of claims 16 to 19.

21. A recombinant AAV vector for use as a medicament, comprising a therapeutically effective amount of the nucleic acid according to any one of claims 1 to 12, or the plasmid according to claim 13.

22. The recombinant AAV vector for use according to claim 21, wherein the recombinant AAV vector is administered by direct injection into the central nervous system (CNS) of a subject.

23. The recombinant AAV vector for use according to claim 22, wherein the administration results in the expression of frataxin (FXN) protein in the CNS and / or heart tissue of the subject.

24. The recombinant AAV vector for use according to claim 22 or 23, wherein the subject has Friedreich's ataxia.

25. The recombinant AAV vector for use according to claim 24, wherein the subject is a human subject.

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