rAAV production method

Abstract

Disclosed herein is an improved method for producing recombinant adeno-associated viruses (rAAVs) intended for use in gene therapy. The improved method involves adding low doses of DMSO and valproic acid to cells transfected with a plasmid encoding the rAAV and a plasmid containing helper factors for the expression and / or assembly of the rAAV capsid. While exemplary methods for transfecting cells are provided herein, other transfection methods are known in the art. The claimed invention is not limited to any particular transfection method.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 442,608, filed February 1, 2023, and U.S. Provisional Patent Application No. 63 / 448,872, filed February 28, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure provides improved methods for producing recombinant adeno-associated viruses (rAAV) for use in gene therapy. [Background technology]

[0003] Therapies that can provide functional gene replacements or provide a means to edit defective genes have the potential to alleviate the disorders caused by single gene mutations, many of which currently have no hope of treatment other than palliative care.

[0004] Recombinant adeno-associated viruses (rAAVs) have been frequently proposed as vectors for gene therapy. However, rAAVs present several challenges that must be overcome for each therapy (Au 2022). These challenges include: The genetic material of rAAV is limited in size (approximately 4.7 kb of single-stranded DNA), and must contain inverted terminal repeats (ITRs), a promoter, at least one gene of interest, and a transcription terminator, all of which are required for packaging and replication of the gene cassette. Therefore, large host gene deletions or deletions of multiple genes are difficult to treat with rAAV vectors. -Pre-existing antibodies against the rAAV capsid may prevent the treatment from transducing an effective number of cells in the affected tissue. - rAAV can infect not only cells within the affected tissue but also a variety of different cell types, potentially resulting in off-target effects. Liver lesions have been frequently observed. Tissue-specific promoters may prevent such effects. However, identifying optimal promoters can be challenging. - For effective treatment in humans, the doses required for systemic (e.g., intravenous) administration are 10 14 ~10 16 A large number of rAAV particles, ranging from 100 to 100 viral genomes (vg), are required, but current technology is limited in its production.

[0005] Improved rAAV production methods are needed. Several media additives and culture conditions have been tested for their effects on cell transfection and protein production, with mixed results. For example, the histone deacetylase inhibitor sodium butyrate / butyric acid has been reported to increase AAV capsid protein production when added together with tryptone N1 18 hours after transfection (Zhao 2020). When stimulating AAV expression from HEK293 cells by adenovirus infection, calcium enrichment (using calcium chloride) and sodium butyrate increased virus yield, but the addition of other factors, such as dimethyl sulfoxide (DMSO), ethyl alcohol, and N-acetyl-L-cysteine, did not further increase virus production (Tsao 2001). Furthermore, sodium butyrate was shown to have a positive effect on transient transfection and protein production, but the addition of several other reagents decreased cell viability (Cervera 2015). For example, other variables such as lithium acetate added before transfection and valproic acid added after transfection increased cell viability, transfection efficiency, and protein production. (Ibid.) Alternatively, sodium butyrate had a neutral or negative effect when added at the time of transfection (Chahal 2014).

[0006] Adding 2 mM ethylene glycol tetraacetic acid (EGTA) prior to transfection also increased transfection efficiency, but higher concentrations were cytotoxic (Feng 2007). Adding valproic acid at the time of transfection or immediately thereafter increased AAV titers (WO2018 / 226887, WO2020 / 172624). Supplementing with sodium chloride after transfection has also been reported to increase AAV titers (Adamson-Small 2017). Caffeine increased lentiviral titers but dramatically reduced AAV production (Ellis 2011).

[0007] A systematic small molecule library screening method for rAAV production enhancers is described herein. The small molecules are derived from various types of compounds, including osmotic agents, volatile organic compounds, redox agents, chelators, epigenetic effectors, and amines. Surprisingly, the combination of low doses of dimethyl sulfoxide (DMSO) and valproic acid had a synergistic effect on rAAV production. Summary of the Invention

[0008] Disclosed herein are methods for producing recombinant adeno-associated virus (rAAV) vectors, including transfecting cells with at least one nucleic acid capable of expressing rAAV, adding a low dose of dimethyl sulfoxide (DMSO) and a low dose of valproic acid, culturing the transfected cells, and harvesting the rAAV. Examples of methods for transfecting cells are provided herein, although other transfection methods are known in the art. The claimed invention is not limited to a particular transfection method. Examples of cells capable of being transfected with rAAV and expressing rAAV are provided herein, although other suitable cells are known in the art. The claimed invention is not limited to a particular cell or cell line. Examples of nucleic acids capable of expressing rAAV are provided herein, although other suitable nucleic acids are known in the art. The claimed invention is not limited to a particular nucleic acid or rAAV type. Examples of cell culture methods are provided herein, although other suitable cell culture methods are known in the art. The claimed invention is not limited to a particular cell culture method. Although examples of methods for harvesting rAAV are provided herein, other suitable methods for harvesting rAAV are known in the art, and the claimed invention is not limited to any particular method for harvesting rAAV.

[0009] A low dose of DMSO may be about 1.6% volume / volume, or a low dose may be 1.2% to 2%, or a low dose may be 1.57% ± 0.4%. A low dose of DMSO is 2% or less. A low dose may be 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or may be within ± 0.05% of the aforementioned doses. A graphical representation of low doses of DMSO suitable for the claimed manufacturing method is shown in Figure 4. The method is suitable for enhancing rAAV production.

[0010] A low dose of valproic acid may be 0 mM to 2 mM, or 1.3 mM to 1.9 mM, or about 1.6 mM, or 1.59 mM ± 0.3 mM. A low dose may be 0.5 mM, 1 mM, 1.5 mM, or 2.0 mM, or within ± 0.25 mM of the aforementioned doses. In some cases, the effect of a 1.6% DMSO dose was not statistically significant compared to the effect obtained with 1.6% DMSO / 1.6 mM valproic acid. This suggests that in some cases (e.g., rAAV, gene of interest, etc.), a low dose of valproic acid may not be necessary for enhanced rAAV production. A low dose of valproic acid is 2 mM or less. A graphical representation of a low dose of valproic acid suitable for the claimed method is shown in Figure 4. This method is suitable for enhanced rAAV production.

[0011] In the claimed methods, the addition of the low dose of DMSO and the low dose of valproic acid may occur 0 to 6 hours after transfection, or 0.5 to 5 hours after transfection, or 1 to 4 hours after transfection, or 2 to 4 hours after transfection.

[0012] Further disclosed herein is a cell culture medium containing low-dose dimethyl sulfoxide (DMSO) and low-dose valproic acid. The low-dose DMSO is 2% or less but greater than about 1%. The low-dose DMSO is preferably 1.2% to 2%, or the low dose may be 1.57% ± 0.4%. The low-dose valproic acid is 2 mM or less. The low-dose valproic acid may be 1.3 mM to 1.9 mM, or may be about 1.6 mM, or may be 1.59 mM ± 0.3 mM. [Brief explanation of the drawings]

[0013] [Figure 1]Figure 1 shows the results of an initial screening of cell culture additives for their ability to enhance rAAV production. A. CTS VPCs were seeded in Ambr 15 bioreactors and transfected in triplicate with a helper plasmid, a plasmid encoding the AAV rep and cap genes, and a plasmid containing an expression cassette encoding the anoctamin-5 wild-type gene (ANO5 WT). They were treated with additives (DMSO 1%, high sodium butyrate 5 mM, low sodium butyrate 1 mM, high valproic acid 3.36 mM) according to the timing and concentrations shown in Table 1. CTS LV-Max Production Medium (ThermoFisher Scientific) is a chemically defined, serum-free, protein-free medium used for the growth and transfection of suspension-adapted HEK 293 cells. The total mean titer (in VG / mL) for day 4 of all runs was determined by qPCR and is shown for each experimental condition. Error bars indicate the standard error of the mean, where applicable. Control / CTS LV-Max, n=4; all other conditions, n=2. B. CTS VPCs were seeded into Ambr 15 bioreactors, transfected with plasmids to produce ANO5 WT rAAV, and treated with additives according to the timing in Table 1. The total mean titer (in VG / mL) for day 4 of all runs was determined by qPCR and is shown for each experimental condition. Error bars indicate the standard error of the mean, where applicable. Control, n=6; all other conditions, n=3. [Figure 2] Figure 2 shows validation of selected additive combinations. CTS VPCs were seeded into 125 mL shake flasks, transfected with plasmids to produce ANO5 WT rAAV, and treated with additives 4 hours post-transfection (low dose: ethyl alcohol [EA] 50 mM, valproic acid [VPA] 1 mM, DMSO 1%; high dose: EA 100 mM, VPA 2 mM, DMSO 2%). The combined mean titers (in VG / mL) for day 4 of all runs were determined by qPCR and are shown for each experimental condition. [Figure 3]Figure 3 shows the appropriate timing of additive addition and the evaluation of the effect of seeding cell density. A. CTS VPCs were seeded into Ambr 15 bioreactors, transfected with plasmids to produce ANO5 WT rAAV, and treated with additives according to the timing listed on the x-axis (hours). The total mean titer (in VG / mL) for all runs on day 4 was determined by qPCR and is shown for each experimental condition. Error bars indicate the standard error of the mean, where applicable. B. Adherent HEK 293 cells were seeded at 7,000 cells / cm² (Process C) or 14,000 cells / cm² (Process B) and grown for 4 days, after which they were transfected with plasmids to produce microdystrophin (µDYS) or ANO5 WT rAAV and treated with additives. The total mean titer (in VG / cm²) for all runs on day 4 was determined by qPCR and is shown for each experimental condition. Error bars indicate the standard error of the mean, where applicable. [Figure 4] Figure 4 shows further refinement of cell culture additive concentrations. A. CTS VPCs were seeded into Ambr 15 bioreactors, transfected with plasmids to produce ANO5 WT rAAV, and treated with EA, VPA, and DMSO additives according to a 3x3x3 full-factorial DoE design. The combined mean titer (in VG / mL) for day 4 of all runs was determined by qPCR and is shown for each experimental condition. Polynomial regression analysis of D4 titers was determined by least-squares fit. The predictive profiler optimizes the response of the input variables (i.e., D4 titer) based on a regression model that maximizes desirability. Shaded areas represent 95% confidence intervals. B. Contour profile map of rAAV production enhancement with DMSO (% v / v) and valproic acid (mM) concentrations. [Figure 5]Figure 5 shows the results for various cell lines using selected additives, various AAV capsids, and interpretation of the results for various genes of interest. A. CTS VPCs and Expi293 (Expi) cells were seeded into 125 mL shake flasks, transfected with plasmids to produce ANO5 WT rAAV, and treated with additives after 2 hours (VPA / DMSO) or 4 hours (ethanol, VPA, DMSO). The combined average titer (in VG / mL) for all runs on day 4 was determined by qPCR and is shown for each experimental condition. Error bars indicate the standard error of the mean, where applicable. B. CTS VPCs were seeded into Ambr 15 bioreactors, transfected with plasmids to produce AAV1.ANO5 WT or AAV9.ANO5 WT, and treated with additives 2 hours after transfection. The combined average titer (in VG / mL) for all runs on day 4 was determined by qPCR and is shown for each experimental condition. Error bars indicate standard error of the mean, where applicable. C.CTS VPCs were seeded into Ambr 15 bioreactors, transfected with NT3, 5'DYSF, or SaCas9, and treated with additives 2 hours post-transfection. The total mean titer (in VG / mL) for day 4 of all runs was determined by qPCR and is shown for each experimental condition. Error bars indicate standard error of the mean, where applicable. DETAILED DESCRIPTION OF THE INVENTION

[0014] definition

[0015] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. For example, terms such as those defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related fields, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Unless expressly defined below, such terms should be interpreted according to their ordinary meaning.

[0016] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0017] The practice of the present invention will employ, unless otherwise indicated, standard techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0018] Unless otherwise indicated by context, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. By way of example, if a composite is described herein as comprising components A, B, and C, it is specifically intended that any or combination of A, B, or C may be omitted and may not be claimed in the singular or in any combination.

[0019] Unless expressly indicated otherwise, all specific embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and their biological equivalents.

[0020] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations and may vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by changes of + / - 15%, or + / - 10%, or + / - 5%, or + / - 2%, including those ranges. It is understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0021] The practice of the present invention will employ, unless otherwise indicated, standard techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2002. nd edition(1989);Current Protocols In Molecular Biology(FMAusubel,et al.eds.,(1987));the series Methods in Enzymology(Academic Press, Inc.):PCR2:A Practical Approach(MJMacPherson,BDHames and GRTaylor eds.(1995)),Harlow and Lane,eds.(1988)Antibodies,a Laboratory Manual,and See Animal Cell Culture (RI Freshney, ed. (1987)).

[0022] As used herein, the terms "increased," "decreased," "high," "low," or any grammatical variation thereof, refer to about a 90%, 80%, 50%, 20%, 10%, 5%, 1%, 0.5%, or even 0.1% variation in a reference composition, polypeptide, protein, etc. "Enhanced" or "enhancement" refers to a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (i.e., a 2-fold or 2-fold), 3-fold, 4-fold, 5-fold or greater increase compared to the reference method.

[0023] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" are intended to mean that the component, range, dosage form, etc. is suitable for the purposes of the present disclosure.

[0024] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0025] Unless explicitly recited and intended otherwise, when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, equivalents, or biological equivalents thereof, are implied to be within the scope of the present disclosure. As used herein, the term "biologically equivalent thereof" when referring to a reference protein, antibody, polypeptide, or nucleic acid is intended to be synonymous with "equivalent thereof" and is intended to have minimal sequence identity while maintaining the desired structure or functionality. Unless specifically recited herein, all polynucleotides, polypeptides, or proteins referred to herein are expected to also include their equivalents. For example, a reference to a polynucleotide, polypeptide, or protein is intended to have at least about 70% homology or identity, or at least 80% homology or identity, or at least about 85%, or at least about 90%, or at least about 95%, or 98% homology or identity over the entire length of the reference sequence, exhibiting substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent, in one embodiment, is a polynucleotide that hybridizes to a reference polynucleotide or its complementary strand under stringent conditions, and in a further embodiment, is a polynucleotide that has the same or equivalent activity or function as the reference polynucleotide or its complementary strand. One biological activity is a nucleotide that encodes a protein. Nucleotides are biologically equivalent if they encode the same amino acid sequence, even if different codons are used due to the degeneracy of the genetic code.

[0026] An equivalent of a protein or polypeptide (referred to herein as a reference) shares at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity with the reference and retains the function and manufacturability of the reference.

[0027] As used herein, the terms "function," "activity," and "enzymatic activity" are used interchangeably.

[0028] As used herein, the terms "purification," "purifying," or "separating" refer to the process of isolating one or more biological materials (e.g., polynucleotides, polypeptides, or viral vectors) from a complex mixture, such as a cell lysate or a polypeptide mixture. Purification, separation, or isolation need not be complete; that is, some components of a complex mixture may remain with one or more biological materials (e.g., polynucleotides, polypeptides, or viral vectors) after a purification process. However, the purified product must be enriched for one or more biological materials (e.g., polynucleotides, polypeptides, or viral vectors) compared to the complex mixture prior to purification; a substantial portion of other components initially present in the complex mixture must be removed by the purification process. Purification does not exclude subpopulations of purified material.

[0029] As used herein, the term "cell" can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or commercially available. In some examples, the cell is a host cell, e.g., a mammalian cell or a mammalian host cell. In some examples, the host cell is also referred to herein as a producer cell or packaging cell. In some examples, the cell line is a packaging cell line.

[0030] "Eukaryotic cells" include all kingdoms of life except Monera. They are easily distinguishable by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotes, or organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most distinctive membrane-bound structure is the nucleus. Unless specifically recited, the term "host" includes eukaryotic cellular hosts, such as yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkey, bovine, porcine, mouse, rat, avian, reptile, and human cells, such as HEK293 cells, Chinese hamster ovary (CHO) cells, CHO-S cells, CHO-K1 cells, 293T cells, HeLa cells, baby hamster kidney (BHK) cells, Sf9 cells, yeast cells, stem cells, satellite cells, and muscle cells. Examples of muscle cells include, but are not limited to, skeletal muscle cells, cardiac muscle cells, and smooth muscle cells.

[0031] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and spiral. Rather than the complex replication process of eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, Escherichia coli, and Salmonella.

[0032] The term "encoding," when applied to a nucleic acid sequence, refers to a polynucleotide that, in its natural state or when manipulated by methods known to those of skill in the art, is said to "encode" a polypeptide that can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid, and the coding sequence can be deduced from the complementary strand.

[0033] As used herein, the terms "retain," "similar," and "same" are used interchangeably and while describing a function, activity, or functional activity of a polynucleotide, protein, and / or peptide, refer to a functional activity of at least about 20% (including but not limited to, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or about 100%) of the activity of the referenced protein, polynucleotide, and / or peptide.

[0034] As used herein, "expression" or "expressing" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA.

[0035] As used herein, the term "functional" can be used to modify any molecular, biological, or cellular material to denote achieving a particular, specific effect.

[0036] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural bases, or chemically or biochemically modified non-natural bases, or derivatized nucleotide bases. In certain embodiments, a polynucleotide comprises and / or encodes messenger RNA (mRNA), short hairpin RNA, and / or small hairpin RNA. In one embodiment, the polynucleotide is mRNA or encodes mRNA. In certain embodiments, the polynucleotide is double-stranded (ds) DNA, e.g., engineered double-stranded (ds) DNA or ds cDNA synthesized from single-stranded RNA.

[0037] The terms "protein," "peptide," and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. Alternatively, the subunits may be linked by other bonds, such as, for example, ester bonds, ether bonds, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up the sequence of a protein or peptide. As used herein, the term "amino acid" refers to natural amino acids and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0038] As used herein, the term "recombinant expression system" refers to a genetic construct directed to the expression of specific genetic material formed by recombinant means.

[0039] "Gene delivery vehicle" or "gene delivery vector" is defined as any molecule capable of carrying an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers (including natural and synthetic polymers), lipoproteins, lipid nanoparticles, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, bacteria, or viruses such as rabies virus, flavivirus, lentivirus, baculovirus, adenovirus, and retrovirus, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art that have been reported for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy and simple protein expression.

[0040] The polynucleotides disclosed herein can be delivered to cells or tissues using a gene delivery vehicle. As used herein, "gene transfer," "mRNA-based delivery," "transducing," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include various known techniques, such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein- or lipid-based gene delivery complexes, including, for example, protamine complexes, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, or combinations thereof), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used for introducing polynucleotides). The introduced polynucleotide may be unmodified or may contain one or more modifications. For example, the modified mRNA may include ARCA capping, enzymatic polyadenylation to add a tail of 100-250 adenosine residues, and substitution of one or both of cytidine for 5-methylcytidine and / or uridine for pseudouridine. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance often requires that the introduced polynucleotide contain an origin of replication compatible with the host cell or be integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors have been found to be capable of mediating gene transfer into mammalian cells.

[0041] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of chromosomal DNA. Plasmids are often circular or double-stranded. Plasmids provide a mechanism for horizontal gene transfer within microbial populations and often confer a selective advantage under given environmental conditions. Plasmids may carry genes that provide resistance to natural antibiotics in competitive environmental niches, or the proteins produced may act as toxins under similar circumstances.

[0042] Plasmids used in genetic engineering are called "plasmid vectors." Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that confers resistance to a specific antibiotic and a multiple cloning site (MCS, or polylinker). The MCS is a short region containing several commonly used restriction enzyme sites, allowing DNA fragments to be easily inserted into that location. Another major use of plasmids is the mass production of proteins. In this case, researchers grow bacteria containing a plasmid carrying the gene of interest. The bacteria can be induced to produce large amounts of the protein from the inserted gene, as well as to produce proteins that confer antibiotic resistance.

[0043] "Yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (greater than 100 kb, up to 3000 kb). YACs are artificially constructed chromosomes containing telomere, centromere, and origin of replication sequences necessary for yeast cell replication and storage. When assembled using an initial circular plasmid, YACs are linearized using restriction enzymes, and then DNA ligase can be used to add a gene or sequence of interest to the linear molecule using the cohesive ends. Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), are very useful. Because yeast itself is a eukaryotic cell, eukaryotic protein products can be obtained with post-translational modifications, but YACs are known to be more unstable than BACs and produce chimeric effects.

[0044] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of protein oligomeric structural subunits ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within a viral capsid.

[0045] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus, nucleic acid sequence, or plasmid used to provide additional components necessary for replication and packaging of a viral particle or recombinant viral particle, such as, for example, the modified AAV disclosed herein. Helper-encoded components can include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, a helper virus can encode enzymes required for viral genome replication. Non-limiting examples of helper viruses and helper plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0046] As used herein, a "biological sample" or "sample" can be obtained from a subject, a cell line, or a cultured cell or tissue. Examples of samples include, but are not limited to, cell samples, tissue samples, liquid samples such as blood, and other liquid samples of biological origin (including, but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's or pre-ejaculatory fluid, female semen, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / vaginal washings, synovial fluid, mucosal secretions, stool water, pancreatic juice, sinus lavage, bronchopulmonary aspirate, blastocyst fluid, or umbilical cord blood).

[0047] As used herein, the term "detectable marker" refers to at least one marker capable of generating a detectable signal, either directly or indirectly. This non-exhaustive list of markers includes enzymes that generate a signal detectable, for example, by colorimetry, fluorescence, luminescence, etc., e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, e.g., fluorescent, luminescent dyes; groups having electron density that are detected by electron microscopy or by electrical properties, e.g., conductivity, amperometry, voltammetry, electrical resistance, e.g., the molecule is large enough to induce a detectable modification in a physical and / or chemical property, and such detection may be performed by optical methods, e.g., diffraction, surface plasmon resonance, surface alteration, contact angle change, or by optical methods, e.g., atomic force spectroscopy, tunneling effect, or the like. 32 P, 35 S, 89 Zr, or 125 This may be achieved by physical methods such as by using radioactive molecules such as I.

[0048] As used herein, the term "purification marker" refers to at least one marker useful for purification or identification. A non-exhaustive list of markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC, or any other fluorescent dye or hapten.

[0049] As used herein, an "epitope tag" is a biological structure or sequence, e.g., a protein or carbohydrate, that acts as an antigen recognized by an antibody. In certain embodiments, epitope tags are used interchangeably with purification markers and / or affinity tags.

[0050] A "composition" is intended to mean a combination of two or more compounds, such as, for example, a combination of active polypeptides, polynucleotides, viral vectors, or antibodies, and / or another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0051] A "pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active, e.g., a solid support, to make the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0052] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0053] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, non-human primates, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans. In addition to being useful for treating humans, the present invention is also useful for veterinary treatment of companion mammals, exotic animals, and farm animals, including mammals, rodents, and the like. In one embodiment, mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or child under the age of 18.

[0054] The term "transduction" or "transducing" refers to viral particle-mediated introduction of genetic material into a cell (e.g., AAV-mediated gene transfer). See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

[0055] "Treating" a disease or "treatment" of a disease includes (1) preventing the disease, i.e., preventing the clinical symptoms of the disease from developing in a patient who may be susceptible to the disease but who has not yet experienced or exhibited disease symptoms, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) palliating the disease, i.e., causing a reduction in the disease or its clinical symptoms. In one embodiment, the term "treatment" excludes prevention or prophylaxis.

[0056] The term "suffering" when associated with the term "treatment" refers to a patient or individual who has been diagnosed with or is susceptible to a disease.

[0057] An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or doses. Such delivery depends on many variables, including the duration for which individual dosage units are used, the bioavailability of the therapeutic agent, and the route of administration. However, it is understood that the specific dosage level of the therapeutic agent of the present invention for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the subject's age, weight, general health, sex, and diet, the time of administration, the rate of excretion, the drug combination, and the severity and administration form of the particular disorder being treated. Therapeutic doses are generally titrated to optimize safety and efficacy. In one embodiment, an effective amount is a therapeutically effective amount. Typically, dose-effect relationships from initial in vitro and / or in vivo testing can provide useful guidance regarding appropriate dosages for patient administration. It is generally desirable to administer an amount of compound effective to achieve serum levels corresponding to concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulation and administration procedures, are known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to inhibit the replication of an RNA virus ex vivo, in vitro, or in vivo.

[0058] The term "administration" is intended to include, but is not limited to, oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal or infusion administration, subcutaneous injection or implantation), administration by inhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), which may be formulated, alone or together, in suitable dosage unit formulations containing standard non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each administration route. The present invention is not limited by the route of administration, formulation, or dosing schedule.

[0059] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. While native adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells in which certain functions are provided by a co-infected helper virus, recombinant AAV (rAAV) can contain both single-stranded and self-complementary DNA. General information and reviews on AAV can be found, for example, in Carter, Handbook of Parvoviruses 1:169-228, 1989, and Berns, Virology 1743-1764, 1999. However, since various serotypes are known to be very closely related structurally, functionally, and even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, Parvoviruses and Human Disease 165-174, 1988, J.R.P.Tattison, ed.; and Rose, Comprehensive Virology 3:1-61, 1974.) For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all produce three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the entire length of the genome, and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns suggest that the replication functions in each serotype are under similar regulatory control.

[0060] The term "inverted terminal repeat" or "ITR" includes any palindromic viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a specific viral function, such as replication, viral packaging, integration, and / or proviral rescue). ITRs can be AAV or non-AAV ITRs. For example, non-AAV ITR sequences, such as those of other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19, etc.), or the SV40 hairpin that functions as the SV40 origin of replication, can also be used as ITRs, and can be further modified by truncation, substitution, deletion, insertion, and / or addition. In one embodiment, the ITRs are partially or completely synthetic, such as the "double-D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al. See also FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). An "AAV inverted terminal repeat" or "AAV ITR" may be derived from any AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, and may be derived from snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, or any other AAV now known or later discovered. The AAV ITRs do not necessarily have to be native long terminal repeat sequences (e.g., native AAV ITR sequences may be modified by insertions, deletions, truncations, and / or missense mutations), so long as they mediate the desired function, such as replication, viral packaging, persistence, and / or proviral rescue.

[0061] "AAV expression cassette," as used herein, refers to a nucleotide sequence comprising one or more polynucleotides of interest (or transgenes) flanked by AAV inverted terminal repeats (ITRs). Such an AAV expression cassette, when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products, can be replicated and packaged into infectious viral particles (e.g., AAV vectors).

[0062] An "AAV virion" or "AAV vector" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV expression cassette. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV vector particle necessarily includes production of an AAV expression cassette, and thus, a plasmid, is contained within the AAV vector particle.

[0063] Native adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is described in Gao et al. al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) couple to alternative splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), resulting in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately drive viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0064] The recombinant AAV genome of the present disclosure comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA of the rAAV genome may be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In some embodiments, AAV1, AAV6, AAV8, or AAVrh.74 are used to promote specific expression in skeletal muscle.

[0065] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0066] As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, means excluding other elements that are of any essential significance to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined herein would not exclude trace contaminants from isolation and purification methods, and would not exclude, for example, pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace elements of other components, and excluding more than substantial method steps for administering a composition of the invention, or more than process steps for producing a composition, or more than process steps for achieving an intended result. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0067] As used herein with respect to nucleic acids, e.g., DNA or RNA, the term "isolated" refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the polymer. The term "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments. The term "isolated" also refers to polypeptides, proteins, and / or host cells that have been isolated from other cellular proteins, and is intended to encompass both purified and recombinant polypeptides. In another embodiment, the term "isolated" refers to a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, separated from cellular or other components with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. As will be apparent to those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to be distinguished from its naturally occurring counterpart.

[0068] As used herein with reference to a polypeptide or a polynucleotide, such as DNA or RNA, the term "recombinant" refers to a molecule formed by recombinant laboratory methods, such as molecular cloning. Molecular cloning techniques are known in the art and include, but are not limited to, PCR amplification of a polynucleotide, enzymatic digestion of a polynucleotide, ligation of a polynucleotide into an expression cassette (e.g., a mammalian expression cassette), transformation, transfection, or transduction of a cell with a polynucleotide, and expression of the polynucleotide to produce a polypeptide. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 2012. The term "recombinant polynucleotide" is intended to include a fragment of a polynucleotide that encodes a protein. For example, a recombinant polynucleotide may include a fragment of a polynucleotide encoding human dysferlin protein. A recombinant polynucleotide may be generated by PCR amplification of a fragment of a polynucleotide that encodes a protein. A recombinant polypeptide may be generated by expression of one or more recombinant polynucleotides. In one embodiment, a "recombinant" polypeptide refers to a polypeptide that is separated from or substantially free of other components of the organism or virus in which it occurs in nature, e.g., structural components of a cell or virus, or at least some of the other polypeptides or nucleic acids that are normally associated with the polypeptide.

[0069] The term "Rep sequence" or "Rep coding sequence" refers to a nucleic acid sequence encoding a parvovirus or AAV nonstructural protein that mediates viral replication and the production of new virus particles. Parvovirus and AAV replication genes and proteins are described, for example, in FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). A "Rep coding sequence" need not encode all of the parvovirus or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequence need not encode all four AAV Rep proteins (Rep78, Rep68, Rep52, and Rep40). In fact, AAVs are believed to express only the spliced ​​Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequence encodes at least those replication proteins required for replication of the virus or vector genome and packaging into new virions. The Rep coding sequence generally encodes at least one large Rep protein (i.e., Rep78 / 68) and one small Rep protein (i.e., Rep52 / 40). In specific embodiments, the Rep coding sequence encodes the AAV Rep78 protein and the AAV Rep52 and / or Rep40 protein. In other embodiments, the Rep coding sequence encodes the Rep68 protein and the Rep52 and / or Rep40 protein. In further embodiments, the Rep coding sequence encodes the Rep68 and Rep52 proteins, the Rep68 and Rep40 proteins, the Rep78 and Rep52 proteins, or the Rep78 and Rep40 proteins. In the native AAV genome, different Rep proteins are encoded by a single gene through the use of two different promoters and alternative splicing.However, for purposes of AAV vector production, the Rep proteins may be expressed in producer cells from a single gene or from separate polynucleotides, one sequence for each Rep protein being expressed. Thus, for example, the Rep-encoding gene may be engineered to inactivate the p5 or p19 promoter so that only the small or large Rep protein is expressed.

[0070] As used herein, the term "cap sequence" or "cap coding sequence" refers to structural proteins that form functional capsids (i.e., capable of packaging DNA and infecting target cells), such as parvovirus or AAV capsids. In one embodiment, the cap coding sequence encodes all of the parvovirus or AAV capsid subunits, although not all capsid subunits need to be encoded as long as a functional capsid is produced. In another embodiment, the cap sequence is present on a single nucleic acid molecule. The structure of autonomously replicating parvovirus and AAV capsids is described in detail in Bernard N. Fields et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). A rep-cap plasmid is a plasmid that encodes both the Rep gene and the Cap gene of one or more AAVs.

[0071] In some embodiments, an adeno-associated virus (AAV) vector comprises (a) a first inverted terminal repeat (ITR), (b) a polynucleotide encoding a gene or gene fragment, and (c) a second ITR, wherein the polynucleotide is flanked by the first and second ITRs. The polynucleotide encoding a gene is often complexed with regulatory elements, such as a promoter, an enhancer, an internal ribosome entry site (IRES), an intron, a transcription terminator, and / or a polyA tail signal sequence.

[0072] Further disclosed herein is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector comprises any gene or gene fragment.

[0073] In some embodiments, the polynucleotides, plasmids, viral vectors (e.g., viruses or viral particles), vector systems, viral packaging systems, cells, and compositions further comprise one or more nucleotide sequences comprising, consisting of, or consisting essentially of an inverted terminal repeat (ITR), a promoter, an intron, a selectable marker, or an origin of replication (ORI).

[0074] In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise one or more additional nucleotide sequences comprising an inverted terminal repeat (ITR), a selectable marker, an origin of replication (ORI), an untranslated region (UTR), or a polyadenylation (polyA) signal.

[0075] Disclosed herein is the use of any of the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder.

[0076] Inverted terminal sequence

[0077] In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise a nucleotide sequence comprising, consisting of, or consisting essentially of one or more inverted terminal repeats (ITRS). In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise two, three, four, five, or six or more nucleotide sequences comprising, consisting of, or consisting essentially of two, three, four, five, or six or more ITRs. In some embodiments, two or more ITRs are identical. In some embodiments, two or more ITRs are different.

[0078] In some embodiments, the recombinant polynucleotide is flanked by two or more ITRs. In some embodiments, the gene or gene fragment is flanked by a pair of ITRs. In some embodiments, the expression cassette sequence is flanked by a pair of ITRs. In some embodiments, the ITRs of a first pair of ITRs are identical. In some embodiments, the ITRs of a first pair of ITRs are different. In some embodiments, the ITRs of a second pair of ITRs are identical. In some embodiments, the ITRs of a second pair of ITRs are different. In some embodiments, an ITR of a first pair of ITRs is identical to an ITR of a second pair of ITRs. In some embodiments, at least one ITR of a first pair of ITRs is identical to at least one ITR of a second pair of ITRs. In some embodiments, an ITR of a first pair of ITRs is different from an ITR of a second pair of ITRs. In some embodiments, at least one ITR of a first pair of ITRs is different from at least one ITR of a second pair of ITRs.

[0079] In some embodiments, the ITRs are viral ITRs. In some embodiments, the ITRs are AAV ITRs. In some embodiments, the AAV ITRs are selected from ITRs from at least one of the AAV serotypes AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAVrh.10, AAV-11, AAV-12, and AAV-13. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the AAV ITRs are AAV5 ITRs. ITR sequences for AAV1-6 can be found, for example, in Grimm et al., J. Virol. 80(1):426-39, 2006, which is incorporated by reference in its entirety.

[0080] In some embodiments, the recombinant polynucleotide does not include AAV sequences other than the inverted terminal repeats (ITRs).

[0081] In some embodiments, the recombinant polynucleotide does not include any viral sequences other than the inverted terminal repeats (ITRs).

[0082] promoter

[0083] In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise a nucleotide sequence comprising, consisting of, or consisting essentially of one or more promoters. In some embodiments, the promoter is a eukaryotic promoter. Examples of eukaryotic promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the elongation factor 1 alpha (EF1a) promoter, the CAG promoter, the phosphoglycerate kinase gene (PGK) promoter, the tetracycline response element (TRE) promoter, the human U6 nuclear promoter, and the UAS promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

[0084] In some embodiments, the promoter is a tissue-specific promoter. Examples of tissues include, but are not limited to, muscle, epithelium, connective, and nerve tissue. Examples of tissue-specific promoters include, but are not limited to, B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / bAlb promoter, SV40 / hAlb promoter, SV40 / CD43 promoter, SV40 / CD45 promoter, and NSE / RU5' promoter.

[0085] In some embodiments, the promoter is a recombinant promoter. In some embodiments, the recombinant promoter is a recombinant muscle-specific promoter. In some embodiments, the recombinant muscle-specific promoter is a recombinant myosin heavy chain-creatine kinase muscle-specific promoter. In other embodiments, the muscle-specific promoter comprises a human skeletal actin gene element, a cardiac actin gene element, a desmin promoter, a skeletal alpha-actin (ASKA) promoter, a troponin I (TNNI2) promoter, a muscle cell-specific enhancer-binding factor mef-binding element, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, a hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, a C5-12 promoter, a mouse creatine kinase enhancer element, a skeletal fast-twitch troponin c gene element, a slow-twitch cardiac troponin c gene element, a slow-twitch troponin I gene element, or a hypoxia-inducible nuclear factor.

[0086] Polyadenylation signal

[0087] In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise a nucleotide sequence comprising, consisting of, or consisting essentially of one or more polyadenylation (polyA) signals. In some embodiments, the polyA signals are artificial polyA signals.

[0088] Expression cassettes and packaging systems

[0089] Further disclosed herein is an adeno-associated virus (AAV) expression cassette. In some embodiments, the AAV expression cassette comprises: (a) a first inverted terminal repeat (ITR), the first ITR comprising any of the ITRs disclosed herein; (b) any gene or gene fragment disclosed herein; and (c) a second ITR, the second ITR comprising any of the ITRs disclosed herein, wherein the gene or gene fragment of (b) is adjacent to the first and second ITRs of (a) and (c).

[0090] Further disclosed herein are adeno-associated virus (AAV) plasmids. In some embodiments, the AAV expression cassette comprises: (a) a first inverted terminal repeat (ITR), the first ITR comprising any of the ITRs disclosed herein; (b) any gene or gene fragment disclosed herein; and (c) a second ITR, the second ITR comprising any of the ITRs disclosed herein, wherein the gene or gene fragment of (b) is adjacent to the first and second ITRs of (a) and (c).

[0091] Further disclosed herein are adeno-associated virus (AAV) packaging systems. In some embodiments, the AAV packaging system comprises (a) any of the expression cassettes disclosed herein, (b) an adenovirus helper plasmid, and (c) a rep-cap plasmid. In some embodiments, the adenovirus helper plasmid comprises one or more genes derived from adenovirus. In some embodiments, the one or more genes derived from adenovirus mediate AAV replication. In some embodiments, the one or more genes derived from adenovirus are selected from E4, E2a, and VA. In some embodiments, the rep-cap plasmid comprises one or more polynucleotides encoding the rep and cap genes of an adeno-associated virus. In some embodiments, the rep gene encodes one or more life cycle proteins selected from Rep78, Rep68, Rep62, and Rep40. In some embodiments, the cap gene encodes one or more capsid proteins selected from VP1, VP2, and VP3. In some embodiments, the expression cassette comprises one or more ITRs. In some embodiments, the ITR is an AAV ITR. In some embodiments, the serotype of the AAV ITR is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV ITR is different from the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is different from the serotype of the AAV capsid protein.

[0092] In some embodiments, the AAV packaging system comprises (a) any of the expression cassettes disclosed herein, (b) an adenovirus helper plasmid, and (c) a rep-cap plasmid. In some embodiments, the adenovirus helper plasmid comprises one or more genes derived from adenovirus. In some embodiments, the one or more genes derived from adenovirus mediate AAV replication. In some embodiments, the one or more genes derived from adenovirus are selected from E4, E2a, and VA. In some embodiments, the rep-cap plasmid comprises one or more polynucleotides encoding the rep and cap genes of adeno-associated virus. In some embodiments, the rep gene encodes one or more life cycle proteins selected from Rep78, Rep68, Rep62, and Rep40. In some embodiments, the cap gene encodes one or more capsid proteins selected from VP1, VP2, and VP3. In some embodiments, the 3' hDYSF AAV expression cassette comprises one or more ITRs. In some embodiments, the ITRs are AAV ITRs. In some embodiments, the serotype of the AAV ITRs is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV ITRs is different from the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is different from the serotype of the AAV capsid protein.

[0093] In some embodiments, the adeno-associated virus packaging system comprises (a) any of the expression cassettes disclosed herein and (b) an adenovirus helper plasmid. In some embodiments, the adenovirus helper plasmid comprises one or more genes derived from adenovirus. In some embodiments, the one or more genes derived from adenovirus mediate AAV replication. In some embodiments, the one or more genes derived from adenovirus are selected from E4, E2a, and VA.

[0094] In some embodiments, the adeno-associated virus packaging system comprises (a) any of the expression cassettes disclosed herein and (b) an adenovirus helper plasmid. In some embodiments, the adenovirus helper plasmid comprises one or more genes derived from adenovirus. In some embodiments, the one or more genes derived from adenovirus mediate AAV replication. In some embodiments, the one or more genes derived from adenovirus are selected from E4, E2a, and VA.

[0095] viral vectors

[0096] Further disclosed herein is an adeno-associated virus (AAV) vector (for example, AAV virus or AAV particle).In some embodiments, the AAV vector comprises, consists of, or essentially consists of any of the polynucleotides disclosed herein.

[0097] In some embodiments, the AAV vector is an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh.10, rh.20, or rh.74. In some embodiments, the AAV vector is an AAV of serotype rh.74.

[0098] Further disclosed herein is a dual adeno-associated virus (AAV) vector system comprising two or more of the AAV vectors disclosed herein. In some embodiments, the dual AAV vector system comprises (a) a first AAV vector, wherein the first AAV vector comprises any of the genes or gene fragments disclosed herein, and (b) a second AAV vector, wherein the second AAV vector comprises any of the 3' hDYSF polynucleotides disclosed herein.

[0099] In some embodiments, the dual AAV vector system comprises, consists of, or consists essentially of (a) a first AAV vector, wherein the first AAV vector comprises, consists of, or consists essentially of any of the AAV vectors disclosed herein, and (b) a second AAV vector.

[0100] composition

[0101] Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the dual AAV vector systems disclosed herein. Further disclosed herein are compositions comprising, consisting of, or essentially consisting of any of the AAV vectors disclosed herein.

[0102] Further disclosed herein are (a) recombinant adeno-associated virus (rAAV) vectors, which comprise, consist of, or consist essentially of any of the genes or gene fragments disclosed herein, and (b) compositions comprising, consisting of, or consisting essentially of a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0103] Further disclosed herein are compositions comprising, consisting of, or consisting essentially of (a) a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV comprises, consists of, or consists essentially of any of the genes or gene fragments disclosed herein, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0104] In some embodiments, any of the compositions disclosed herein further comprises at least one pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients, preferably inert at the dosages and concentrations used, and include buffers and surfactants such as Pluronics. Examples of acceptable carriers include, but are not limited to, phosphate-buffered saline, preservatives, and the like.

[0105] Pharmaceutically acceptable carriers, diluents, or excipients may be suitable for injectable use. Examples of pharmaceutically acceptable carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0106] Sterile injectable solutions are prepared by incorporating the polynucleotide, plasmid, viral vector, or dual vector system disclosed herein in the required amount in an appropriate solvent with various other ingredients listed above, and then filter sterilization, as needed. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from the previously filter-sterilized solution.

[0107] AAV vector manufacturing method

[0108] Disclosed herein are methods for producing adeno-associated virus (AAV) vectors (e.g., viruses or viral particles). Methods for producing AAV vectors are known in the art. For example, such methods are disclosed in, for example, WO01 / 83692, which is incorporated herein by reference in its entirety. The general principles of AAV production are reviewed in, for example, Carter, Current Opinions in Biotechnology 1533-1539, 1992; and Muzyczka, Curr. Topics in Microbial. and Immunol. 158:97-129, 1992, each of which is incorporated herein by reference in its entirety. Various AAV production methods have been described by Ratschin et al.,Mol.Cell.Biol.4:2072,1984;Hermonat et al.,Proc.Natl.Acad.Sci.USA,81:6466,1984;Tratschin et al.,Mo1.Cell.Biol.5:3251,1985;McLaughlin et al. al., J. Virol., 62:1963, 1988; and Lebkowski et al., Mol. Cell. Biol., 7:349, 1988; Samulski et al. al., J. Virol., 63:3822-3828, 1989; U.S. Patent No. 5,173,414; WO95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al., Vaccine 13:1244-1250, 1995; Paul et al., Human Gene Therapy 4:609-615, 1993; Clark et al., Gene Therapy 3:1124-1132, 1996; U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595.Each of these documents is incorporated by reference in its entirety.

[0109] In some embodiments, a method for producing an adeno-associated virus (AAV) vector comprises transducing a cell with any of the AAV packaging systems disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a recombinant cell stably expressing the adeno-associated virus rep and cap genes. In some embodiments, the method further comprises culturing the cell to produce a population of transduced cells. In some embodiments, the method further comprises collecting a supernatant from the population of transduced cells. In some embodiments, the method further comprises subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and proteins. Alternatively, the method further comprises lysing the population of transduced cells to produce a cell lysate. In some embodiments, the method further comprises subjecting the cell lysate to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and proteins. In some embodiments, purified AAV vector samples are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure. However, even purified AAV may contain subpopulations with varying capsids containing post-translational modifications or nucleic acids of varying amounts and compositions.

[0110] cell

[0111] Also disclosed herein is a cell that comprises any of the plasmids, expression cassettes or AAV vectors disclosed herein.Cells can be prokaryotic or eukaryotic cells.Non-limiting examples of eukaryotic cells include mammalian cells, such as hamster, mouse, rat, dog, sheep or human cells.

[0112] Any of the cells disclosed herein may be packaging cells that produce infectious rAAV. In some embodiments, the packaging cells are stably transformed avian cells, such as HeLa cells, 293 cells, and PerC.6 cells (293 lineages). In other embodiments, the packaging cells are non-transformed cancer cells, such as low-passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey lung cells). Non-limiting examples of prokaryotic cells include bacterial cells (e.g., Escherichia coli) and archaeal cells. The cells of the present disclosure can be used to generate cell banks, such as Accession Cell Banks (ACBs) for non-GMP purposes or Master Cell Banks (MCBs) for GMP purposes. In one embodiment, an aliquot of cells from the original inoculum is expanded to a larger volume and then cultured in a production bioreactor.

[0113] Viral titer

[0114] The titer of the AAV vector administered in the methods of the invention will vary depending, for example, on the particular AAV, the mode of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of the AAV should be at least about 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 or more DNase-resistant particles (DRP). Doses may also be expressed in units of viral genomes (vg). For example, the dose of AAV is at least about 1 x 10 6 , approximately 1x10 7 , approximately 1x108 , approximately 1x10 9 , approximately 1x10 10 , approximately 1x10 11 , approximately 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , approximately 7x10 12 , about 8x10 12 , approximately 9x10 12 , approximately 1x10 13 ~approx. 1x10 14 It may be the extent of a viral genome.

[0115] The dosage of AAV can be determined by several methods, including, but not limited to, ELISA, reverse transcriptase activity assessment, FACS, transduction assays, Northern blotting (e.g., semi-quantitative Northern), dot blot analysis, or PCR (e.g., qPCR). It is known that the dosage of AAV can be determined by measuring the AAV vector genome using quantitative real-time PCR (qPCR). Such qPCR methods overcome the problems of inconsistent or arbitrary results from conventional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. The morphology of the plasmid can affect the dosage results from the qPCR method. In one embodiment, circular or supercoiled DNA or plasmid is used as the quantitative standard.

[0116] In some embodiments, the dosage may be expressed in units of vg / kg, based on supercoiled DNA or a plasmid as a quantitative standard. For example, the dosage of AAV may be about 1x10 based on supercoiled DNA or a plasmid as a quantitative standard. 6 ~1x10 16 vg / kg, approx. 1x10 8 ~1x10 15 vg / kg, or approximately 1x10 10 ~1x10 14In another embodiment, the dosage is at least about 1x10 6 , approximately 1x10 7 , approximately 1x10 8 , approximately 1x10 9 , approximately 1x10 10 , approximately 1x10 11 , approximately 1x10 12 , about 2x10 12 , about 4x10 12 , about 6x10 12 , about 8x10 12 , approximately 1x10 13 , about 2x10 13 , about 2.4x10 13 , about 3x10 13 , about 4x10 13 , about 5x10 13 , about 6x10 13 , approximately 7x10 13 , about 8x10 13 , approximately 9x10 13 , approximately 1x10 14 , approximately 1x10 15 , or at least about 1x10 16 In one embodiment, the dosage is at least 2 x 10 vg / kg, based on supercoiled DNA or plasmid as a quantitative standard. 12 , 4x10 12 , 6x10 12 , 8x10 12 , 1x10 13 , 2x10 13 , 2.4x10 13 , 3x10 13 , 4x10 13 , 5x10 13 , 6x10 13 , 7x10 13 , or 8x10 13 vg / kg.

[0117] In some embodiments, the methods disclosed herein provide for the administration of at least about 1 x 10 mAb in a total volume of 1.5 ml per injection. 6 , approximately 1x10 7 , approximately 1x10 8 , approximately 1x10 9 , approximately 1x10 10 , approximately 1x10 11, approximately 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , approximately 7x10 12 , about 8x10 12 , approximately 9x10 12 , approximately 1x10 13 In some embodiments, the methods disclosed herein comprise administering at least about 1 x 10 vg. 6 , approximately 1x10 7 , approximately 1x10 8 , approximately 1x10 9 , approximately 1x10 10 , approximately 1x10 11 , approximately 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , approximately 7x10 12 , about 8x10 12 , approximately 9x10 12 , approximately 1x10 13 , about 2x10 13 , about 5x10 13 , approximately 7x10 13 , approximately 1x10 14 vg. One example of a method for determining the titer of encapsidated vector genomes uses quantitative PCR, e.g., the method described in Pozsgai et al., Mol. Ther. 25(4):855-869, 2017, which is incorporated by reference in its entirety. [Example]

[0118] 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 technology belongs.

[0119] The technology of the present disclosure illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" shall be read expansively and without limitation. Furthermore, the terms and expressions employed herein are used as terms of description and not of limitation. The use of such terms and expressions is not intended to exclude any equivalents of the properties shown and described or portions thereof. However, it is recognized that various modifications are possible within the scope of the technology of the present disclosure as claimed.

[0120] Accordingly, it should be understood that the materials, methods, and examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present technology.

[0121] The present technology has been described broadly and generically herein. Each narrower embodiment, sub-generic group within the generic disclosure, also forms part of the disclosed technology. This includes the generic description of the disclosed technology with a condition or negative limitation that removes any subject matter from the category, regardless of whether the excluded material is specifically recited herein.

[0122] Furthermore, when features or aspects of the disclosed technology are described in terms of a Markush group, one of skill in the art will recognize that the disclosed technology is also thereby described in terms of any individual member or subgroup member of the Markush group.

[0123] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0124] Other aspects are within the scope of the following claims.

[0125] Example 1: Transfection of HEK293 adherent cells

[0126] Freshly thawed cells were allowed to recover for at least three passages after thawing before transfection. Cell cultures were passaged every three or four days. The cell viability of the cell cultures was 5.5 x 10 6 Do not exceed 7,000–14,000 cells / cm in 35 mL of complete DMEM. Four days before transfection, a T-175 flask was cultured at 7,000–14,000 cells / cm. 2 Sow seeds in.

[0127] On the day of transfection, prepare serum-free DMEM medium by adding 100X GlutaMAX at a 1X concentration (5 mL) and 450 g / L glucose to a total of 6 g / L (1.66 mL). Prepare the transfection complexes using pre-warmed serum-free DMEM medium as a diluent. Prepare the polyethyleneimine (PEI) mixture in a 50 mL conical tube for each condition and incubate for 5 minutes. Prepare the DNA mixture in a 50 mL conical tube for each condition. Add the PEI mixture to the DNA mixture and mix well. After 10 minutes, pipette the mixture into flasks, making 3.5 mL for each flask. Return the flasks to the incubator shaker. 2 to 18 hours after transfection, add the additives (Table I) to the flasks. The day after transfection, aspirate the medium and replace with 35 mL of pre-warmed serum-free medium containing the additives. Four days after transfection, 10% v / v fresh lysis buffer (500 mM Tris-HCl pH 8.0, 20 mM MgCl, 1% TWEEN®-20) is added to the flask and incubated at 37°C for 2 hours. The lysate is spun down at 300 x g for 10 minutes at room temperature. The supernatant is transferred to a conical tube and filtered using a 0.45 μm filter. For each flask, two 200 μl and four 1.5 mL aliquots are prepared and stored at -80°C. [Table 1]

[0128] Four cell culture additives were identified as enhancers of rAAV production (Figure 1A). Ethyl alcohol, DMSO, valproic acid, and tryptone N1 were further tested at various concentrations to determine the approximate optimal amount of each additive (Figure 1B). Tryptone N1, being a mixture of compounds, was not clearly defined to the extent required for pharmaceutical-grade production and was therefore excluded from further consideration.

[0129] Selected additives were then tested for their ability to synergize with each other, and the combination of low-dose DMSO and low-dose valproic acid appeared to provide the best improvement in rAAV production (Figure 2).

[0130] Example 2. Improving cell culture additive conditions.

[0131] Although the cell culture additives have been identified and the approximate concentrations of each specified, many variables still exist, one of which is the timing of addition of each additive.

[0132] Transfection was performed as in Example 1, except that additives were added to the cell culture either 1 hour before transfection or 1, 2, 4, or 8 hours after transfection. The combination of DMSO and valproic acid added 1 to 2 hours after transfection resulted in the best improvement in rAAV production (Figure 3A).

[0133] We also tested different transfection cell densities for adherent HEK293 cells to determine whether they were equally effective. These cells are anchorage-dependent and require the use of serum to grow. Ninety-six hours prior to transfection, cells were seeded at two different densities (Process B and Process C) and transfected with rAAV constructs containing the ANO5 WT or m-dystrophin gene within an expression cassette. Following transfection with these two transgenes, cells were treated with either ddH2O or 1.6 mM VPA / 1.6% DMSO 2 hours post-transfection. At 96 hours, cells were chemically lysed and purified, followed by analysis of VG titers by qPCR. For both ANO5 WT and micro-dystrophin, the two additives resulted in a 5-fold increase in VG / cm2 titers (Figure 3B). Furthermore, the control from Process B was below the detection limit of the assay, and seeding cells at a lower density (Process C) improved AAV production.

[0134] A full-factorial design of experiments (DoE) study was conducted to fine-tune the results of the pilot study. DoE techniques have been used to optimize the manufacturing of a wide range of biologics, including AAV. The advantage of DoE is that it is a systematic approach that investigates several variables in a product or process at once and predicts the optimal conditions for maximum output. The more runs a given design has, the higher its statistical power and the greater its ability to distinguish signal from noise. The 48-way Ambr 15 microbioreactor system (Sartorius) is well suited to conducting DoE studies, and the limitations of this study are designed around them.

[0135] A 3 × 3 × 3 full factorial design was used, with a minimum of 27 total runs. To increase statistical power, two replicates were added at the four tangents and center of the cube, for a total of 45 runs. Three levels of each input variable were used: ethanol—0 mM, 50 mM, 100 mM; valproic acid—0 mM, 1.25 mM, 2.50 mM; and DMSO—0%, 1%, 2%. Samples were taken 96 hours post-transfection and qPCR was performed. A polynomial regression model was generated in JMP based on the D4 titers using linear mixture, interaction, and response surface terms. Overall, the fit of the data to the reduced model yielded an r of 0.75, indicating that 75% of the variability in the data was explained by the model. 2 The value is good. 2 demonstrates that 50% of the model's variability can be explained by removing a single data point. A desirability algorithm was used in the predictive profiler to optimize the response resulting in D4 titer. The optimal settings for predicted maximum titer were 0 mM ethyl alcohol, 1.59 mM ± 0.3 mM valproic acid, and 1.57% ± 0.4% DMSO (Figure 4A). The predicted titer was 4.40E+11 vg / mL with a 95% confidence interval of 3.8E+11 to 5.0E+11 vg / mL. The predicted relationship between various concentrations of DMSO and valproic acid is illustrated in Figure 4B.

[0136] Example 3. Interpretation for other cells, AAV capsids, and constructs.

[0137] We next tested the additives against several other cell lines, AAV serotypes, and genes of interest. Both CTS VPC and Expi293 cells are commercially available from Thermo Fisher Scientific. CTS VPC and Expi293 cells are derived from the same suspension-type parent HEK 293F cell line and are closely related subclones. Expi293 cells were tested in 125 mL shake flasks containing 100 mM ethanol, 2 mM VPA, 2% DMSO, and 1.6 mM VPA / 1.6% DMSO, compared with CTS VPC cells. Expi293 cells performed comparably to CTS VPC cells under all conditions (Figure 5A). As single agents, both VPA and DMSO demonstrated significantly higher titers after 96 hours compared to the control. They exerted a synergistic effect in increasing AAV production. Although not statistically significant, ethanol also had a moderate effect on increasing VPA titers in Expi293 cells.

[0138] To rule out any capsid-specific effects of cell culture additives, a plasmid carrying a transfer cassette containing the ANO5 WT transgene and a plasmid containing either AAV1 or AAV9 were transfected into CTS VPCs. Overall, production of AAV1.ANO5 WT was lower than that of AAVrh74.ANO5 WT. However, the presence of 1.6 mM VPA / 1.6% DMSO increased AAV production approximately twofold (Figure 5B). These two additives had a more dramatic effect on AAV9.ANO5 WT, increasing VG titers approximately eightfold. In this experiment, valproic acid had no significant effect on overall yield for either AAV1 or AAV9. In fact, 1.6% DMSO was just as effective as 1.6 mM VPA / 1.6% DMSO.

[0139] We further tested the effect of cell culture additives on AAV production in CTS VPCs using different ITR-containing transgenes. Different transgene sizes and structures can dramatically affect AAV production. A small number of different transgenes were selected for testing, including those part of a dual-vector approach (5'DYSF), a self-complementary vector (NT3), or one specific for gene editing (saCas9). In all cases, 1.6 mM VPA / 1.6% improved vector yields by a factor of 2-4 (Figure 5C). Similar to the results with different serotypes, 1.6 mM VPA alone did not appear to be very effective in improving AAV production with these transgenes. Unlike the capsid studies, the VPA / DMSO combination with NT3 and saCas9 had a synergistic effect, with titers achieved using both transgenes being significantly greater than DMSO alone.

[0140] In summary, three cell culture additives that improve AAV production were characterized in the HEK 293 triple transfection system. These additives were effective across several different cell lines, AAV serotypes, and genes of interest. It is unclear whether VPA and DMSO increase the total amount of capsids produced or contribute to a higher proportion of fully encapsidated vectors. Further studies are needed to determine the potency, infectivity, and stability of AAV vectors produced in the presence of cell culture additives. In any case, an average 5-fold increase in VG titer was achieved during production by using low doses of VPA and DMSO. These data will lead to more efficient upstream processes, thereby producing more medicines using less starting material. This, in turn, will lower the cost of AAV production and improve patient access to gene therapy.

Claims

1. 1. A method for producing a recombinant adeno-associated virus (rAAV) vector, comprising: (a) transfecting a cell with at least one nucleic acid capable of expressing an rAAV; (b) adding a low dose of dimethyl sulfoxide (DMSO) and a low dose of valproic acid; (c) culturing the transfected cells; and (d) harvesting the rAAV.

2. 10. The method of claim 1, wherein the low dose of DMSO is about 1.6% v / v, 1.2%-2%, or 1.57%±0.4%.

3. 2. The method of claim 1, wherein the low dose of valproic acid is between 0 mM and 2 mM, between 1.3 mM and 1.9 mM, about 1.6 mM, or 1.59 mM±0.3 mM.

4. 2. The method of claim 1, wherein step (b) is performed 0 to 6 hours after step (a), 0.5 to 5 hours after step (a), 1 to 4 hours after step (a), or 2 to 4 hours after step (a).

5. 2. The method of claim 1, wherein production of the rAAV is improved compared to a method without step (b).

6. Cell culture medium containing low doses of dimethyl sulfoxide (DMSO) and low doses of valproic acid.

7. 7. The medium of claim 6, wherein the low dose of DMSO is about 1.6% v / v, 1.2% to 2%, or 1.57% ± 0.4%.

8. 7. The medium of claim 6, wherein the low dose of valproic acid is 1.3 mM to 1.9 mM, about 1.6 mM, or 1.59 mM±0.3 mM.

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