Raav production methods

Abstract

Disclosed herein are improved methods of production of recombinant adeno- associated viruses (rAAV) for use in gene therapy. The improved methods comprise the addition of low doses of DMSO and valproic acid to cells transfected with rAAV-encoding plasmids and plasmids containing helper factors for the expression and / or assembly of rAAV capsids.

Description

RAAV PRODUCTION METHODSRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 442,608, filed February 1, 2023 and U.S. Provisional Application No. 63 / 448,872 filed February 28, 2023, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure provides improved methods of production of recombinant adeno- associated viruses (rAAV) for use in gene therapy.BACKGROUND

[0003] Therapeutics capable of providing functional gene replacements or means of editing defective genes promise relief from disorders caused by single gene mutations, many of which have no current hope of treatment other than palliative care.

[0004] Recombinant adeno-associated viruses (rAAV) are frequently proposed as vectors for gene therapies. However, rAAV also pose several problems that need to be solved for each therapeutic (Au 2022). Such problems include:- The limited size (~4.7 kb single-stranded DNA) of the rAAV genetic material, which must include inverted terminal repeats (ITR) necessary for packaging and replication of the genetic cassette, promoters, at least one gene of interest, and transcription terminators. Defects in large host genes and in multiple genes are thus difficult to treat with rAAV vectors.- Pre-existing antibodies to the rAAV capsid may prevent the therapeutic from transducing an effective number of cells in the afflicted tissue.- The rAAV may infect a variety of different cell types, not just cells in the afflicted tissue, leading to off-target effects. Liver pathology is often observed. Tissue-specific promoters may prevent such effects, but identification of the optimal promoters may be challenging.- Large numbers of rAAV particles are needed to deliver effective treatment to human, in the range of 1014to 1016viral genomes (vg) for systemic (e.g.intravenous) administration, yet rAAV manufacture is limited by current technologies.

[0005] There is a need for improved methods of rAAV production. Several media additives and culture conditions have been tested for effects on cell transfection and protein production, but with varying results. For example, sodium butyrate / butyric acid, a histone deacetylase inhibitor, has been reported to increase AAV capsid protein production when added 18 hours post transfection along with tryptone N1 (Zhao 2020). When using adenovirus infection to stimulate AAV expression from HEK293 cells, sodium butyrate with calcium fortification (using calcium chloride) increased virus yield, but addition of other factors such as dimethyl sulfoxide (DMSO), ethyl alcohol and N-acetyl-L-cysteine did not further increase virus production (Tsao 2001). Sodium butyrate was also shown to have a positive effect on transient transfection and protein production but with the addition of some other reagents it caused decreased cell viability (Cervera 2015). Other variables such as lithium acetate added prior to transfection and valproic acid added after transfection increased cell viability, transfection efficiency, and protein production, ( / t / .) Alternatively for another group, sodium butyrate demonstrated a neutral or negative effect when added at the time of transfection (Chahal 2014).

[0006] Ethylene glycol tetra-acetic acid (EGTA) added at 2 mM before transfection also increased transfection efficiency, although higher concentrations were cytotoxic (Feng 2007). Valproic acid added at the time of transfection or soon thereafter increased AAV titers (WO2018 / 226887, WO2020 / 172624). Sodium chloride supplementation after transfection has also been reported to increase AAV titers (Adamson-Small 2017). Caffeine increased the titer of lentiviruses but dramatically reduced the production of AAV (Ellis 2011).

[0007] A systematic approach to screen a small molecule library for rAAV production enhancers is described herein. The small molecules are from various classes of compounds including osmolality modifiers, volative organics, redox agents, chelating agents, epigenetic effectors, and amines. Surprisingly, a combination of low doses of dimethyl sulfoxide (DMSO) and valproic acid have a synergistic effect on rAAV production.SUMMARY

[0008] Disclosed herein is a method of producing a recombinant adeno-associated viral (rAAV) vector comprising transfecting cells with at least one nucleic acid capable of expressing a rAAV; adding a low dose of dimethyl sulfoxide (DMSO) and a low dose of valproic acid; culturing the transfected cells; and harvesting the rAAV. Example methods oftransfecting cells are given herein and 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 and expressing rAAV are given herein and 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 given herein and 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 given herein and other suitable cell culture methods are known in the art; the claimed invention is not limited to a particular cell culture method. Example methods of harvesting rAAV are given herein and other suitable rAAV harvesting methods are known in the art; the claimed invention is not limited to a particular rAAV harvesting method.

[0009] The low dose of DMSO could be about 1.6% volume / volume, or the low dose could be 1.2% to 2%, or the low dose could be 1.57% ± 0.4%. The low dose of DMSO is equal to or less than 2%. The low dose could be 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or a dose within ±0.05% of the preceding. A graphical representation of low doses of DMSO suitable for the claimed methods production is presented in Figure 4. The methods are suitable for the enhancement of rAAV production.

[0010] The low dose of valproic acid could be 0 mM to 2 mM, or the low dose could be 1.3 mM to 1.9 mM, or the low dose could be about 1.6 mM, or the low dose could be 1.59 mM ± 0.3 mM. The low dose could be 0.5 mM, 1 mM, 1.5 mM, or 2.0 mM, or a dose with ±0.25 mM of the preceding. In some examples, the effect of a dose of 1.6% DMSO was not statistically significant from the effect achieved with 1.6% DMSO / 1.6 mM valproic acid, indicating in some situations (e.g. rAAV, gene of interest, etc.) the low dose of valproic acid may not be needed for enhanced rAAV production. The low dose of valproic acid is equal to or less than 2 mM. A graphical representation of low doses of valproic acid suitable for the claimed methods is presented in Figure 4. The methods are suitable for the enhancement of rAAV production.

[0011] In the claimed methods the addition of low dose DMSO and low dose valproic acid could be done 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 comprising a low dose of dimethyl sulfoxide (DMSO) and a low dose of valproic acid. The low dose of DMSO is equal to or less than 2% but greater than about 1%. Preferably the low dose of DMSO could be 1.2% to 2%,or the low dose could be 1.57% ± 0.4%. The low dose of valproic acid is equal to or less than 2 mM. The low dose of valproic acid or the low dose could be 1.3 mM to 1.9 mM, or the low dose could be about 1.6 mM, or the low dose could be 1.59 mM ± 0.3 mM.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 provides the initial screen of cell culture additives for the ability to enhance rAAV production. A. CTS VPC were seeded in Ambr 15 bioreactors, triply transfected with a helper plasmid, a plasmid encoding AAV rep and cap genes, and a plasmid containing a expression cassette encoding the anoctamin-5 wild type gene (ANO5 WT) and treated with additives according to the timing and concentrations in Table 1 (DMSO - 1%; sodium butyrate HIGH - 5 mM; sodium butyrate LOW - 1 mM; valproic acid HIGH - 3.36 mM). CTS LV-Max Production medium (ThermoFisher Scientific) is a chemically defined, serum- free, protein-free medium for growth and transfection of suspension-adapted HEK 293 cells. The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean. Control / CTS LV-Max, n = 4; all other conditions, n = 2. B. CTS VPC were seeded in Ambr 15 bioreactors, transfected with plasmids enabling production of anZVO5 WT rAAV and treated with additives according to the timing in Table 1. The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean. Control, n = 6; all other conditions, n = 3.

[0014] FIG. 2 provides a test of combinations of selected additives. CTS VPC were seeded in 125 mL shake flasks, transfected with plasmids enabling production of an ANO5 WT rAAV and treated with additives four hours post transfection (low dose: ethyl alcohol [EA] - 50 mM, valproic acid [VP A] - 1 mM, DMSO - 1%; high dose: EA - 100 mM, VPA - 2 mM, DMSO - 2%). The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR.

[0015] FIG. 3 provides an assessment of the proper timing for the addition of the selected additives and the effect of plating cell density. A. CTS VPC were seeded in Ambr 15 bioreactors, transfected with plasmids enabling production of an ANO5 WT rAAV and treated with additives according to the timing listed on the x-axis (hours). The total average day four titer in VG / mL of all runs is shown according to each experimental condition asdetermined by qPCR. Error bars, where applicable represent the standard error of the mean.B. Adherent HEK 293 cells were seeded at 7000 cells / cm2(Process C) or 14000 cells / cm2(Process B) and grown for four days before transfection with plasmids enabling production of a microdystrophin (jiDYS) or an ANO5 WT rAAV and treatment with additives. The total average day four titer in VG / cm2of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean.

[0016] FIG. 4 provides further refinement of the cell culture additive concentrations. A. CTS VPC were seeded in Ambr 15 bioreactors, transfected with plasmids enabling production of an ANO5 WT rAAV and treated with EA, VP A, and DMSO additives according to a 3 x 3 x3 full factorial DoE design. The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Polynomial regression analysis of D4 titer is determined by a method of least fit squares. A prediction profiler optimizes the response (i.e. D4 titer) of the input variables based on the regression model by maximizing desirability. The shaded areas represent the 95% confidence intervals. B. A contour profile map of the rAAV production enhancement by a concentration of DMSO (% v / v) and valproic acid (mM).

[0017] FIG. 5 provides the translation of the results with the selected additives to different cell lines, with different AAV capsids, and with different genes of interest. A. CTS VPC and Expi293 (Expi) cells were seeded in 125 mL shake flasks, transfected with plasmids enabling production of an ANO5 WT rAAV and treated with additives after 2 hours (VPA / DMSO) or4 hours (ethanol, VP A, DMSO). The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean. B. CTS VPC were seeded in Ambr 15 bioreactors, transfected with plasmids enabling production of an AAV1. ANO5 WT or AAV9ANO5 WT and treated with additives two hours post transfection. The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean.C. CTS VPC are seeded in Ambr 15 bioreactors, transfected with NT3, 5 ’ DYSF, or SaCas9 and treated with additives two hours post transfection. The total average day four titer in VG / mL of all runs is shown according to each experimental condition as determined by qPCR. Error bars, where applicable represent the standard error of the mean.DETAILED DESCRIPTION

[0018] Definitions

[0019] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. While not explicitly defined below, such terms should be interpreted according to their common meaning.

[0020] 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.

[0021] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0022] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0023] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0024] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2% and such ranges are included. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term“about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0025] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0026] As used herein, the terms “increased”, “decreased”, “high”, “low” or any grammatical variation thereof refer to a variation of about 90%, 80%, 50%, 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the reference composition, polypeptide, protein, etc. “Enhanced” or “enhancement” means an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (i.e. a doubling or 2-fold), 3-fold, 4-fold, 5-fold or more as compared to the reference method.

[0027] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0028] Also as used herein, “and / or” refers to and encompasses 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”).

[0029] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, antibody, polypeptide or nucleic acid, intends those having minimal sequence identity while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity across the length of the reference sequence and exhibits substantially equivalent biological activityto the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is in one aspect, a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement that in a further aspect, has the same or similar activity or function as the reference polynucleotide or its complement. One biological activity is a nucleotide encoding a protein, and, due to the degeneracy of the genetic code, nucleotides would be biologically equivalent if they encoded the same amino acid sequence even if different codons are used.

[0030] An equivalent of a protein or a polypeptide (referred to herein as the reference) shares at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity to the reference and retains the reference’s function and manufacturability.

[0031] As used herein, the terms “function,” “activity,” and “enzymatic activity” are used interchangeably.

[0032] As used herein the terms “purification”, “purifying”, or “separating” refer to the process of isolating one or more biomaterials (e.g., polynucleotides, polypeptides, or viral vectors) from a complex mixture, such as a cell lysate or a mixture of polypeptides. The purification, separation, or isolation need not be complete, i.e., some components of the complex mixture may remain with the one or more biomaterials (e.g., polynucleotides, polypeptides, or viral vectors) after the purification process. However, the product of purification should be enriched for the one or more biomaterials (e.g., polynucleotides, polypeptides, or viral vectors) relative to the complex mixture before purification and a significant portion of the other components initially present within the complex mixture should be removed by the purification process. Purification does not exclude subpopulations of the purified material.

[0033] The term “cell” as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. In some instances, the cell is a host cell, for example, a mammalian cell or a mammalian host cell. In some instances, the host cell is also referred to herein as a production cell or a packaging cell. In some cases, the cell line is a packaging cell line.

[0034] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples ofeukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, e.g., HEK293 cells, Chinese Hamster Ovary (CHO) cells, CHO-S cells, CH0-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.

[0035] “Prokaryotic cells” that usually 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 a circular loop called an episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0036] The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0037] As used herein, the terms “retain,” “similar,” and “same” are used interchangeably while describing a function, an activity or an functional activity of a polynucleotide, a protein and / or a peptide, referring 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 reference protein, polynucleotide and / or peptide.

[0038] As used herein, “expression” or “express” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0039] As used herein, the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.

[0040] 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, this term includes, but is not limited to,single-, double-, or multi -stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In certain embodiments, the polynucleotide comprises and / or encodes a messenger RNA (mRNA), a short hairpin RNA, and / or small hairpin RNA. In one embodiment, the polynucleotide is or encodes an mRNA. In certain embodiments, the polynucleotide is a double-strand (ds) DNA, such as an engineered double-stranded (ds) DNA or a ds cDNA synthesized from a single-stranded RNA.

[0041] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to 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. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0042] As used herein, the term “recombinant expression system” refers to a genetic construct or constructs for the expression of certain genetic material formed by recombination.

[0043] A “gene delivery vehicle” or “gene delivery vector” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; lipid nanoparticles; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as rabies virus, flavivirus, lentivirus, baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0044] A polynucleotide disclosed herein can be delivered to a cell or tissue using a gene delivery vehicle. “Gene delivery,” “gene transfer” “mRNA-based delivery”, “transducing,” and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection / transfection, or various otherprotein-based or lipid-based gene delivery complexes, including for example protamine complexes, lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, or combinations thereof) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide can be unmodified or can comprise one or more modifications; for example, a modified mRNA may comprise ARCA capping; enzymatic polyadenylation to add a tail of 100-250 adenosine residues; and substitution of one or both of cytidine with 5-methylcytidine and / or uridine with pseudouridine. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

[0045] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances.

[0046] “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene.

[0047] A “yeast artificial chromosome” or “YAC” refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed forreplication and preservation in yeast cells. Built using an initial circular plasmid, they are linearized by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. Yeast expression vectors, such as YACs, Yips (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than B ACs, producing chimeric effects.

[0048] As used herein, the term “viral capsid” or “capsid” refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and release into host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein (“capsid proteins”). As used herein, the term “encapsidated” means enclosed within a viral capsid.

[0049] As used herein, the term “helper” in reference to a virus or plasmid refers to a virus, a nucleic acid sequence, or a plasmid used to provide the additional components necessary for replication and packaging of a viral particle or recombinant viral particle, such as the modified AAV disclosed herein. The components encoded by a helper may include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus may encode necessary enzymes for the replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0050] As used herein, a “biological sample,” or a “sample,” can be obtained from a subject, cell line or cultured cell or tissue. Exemplary samples include, but are not limited to, cell sample, tissue sample, liquid samples such as blood and other liquid samples of biological origin (including, but not limited to, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / flushing, synovial fluid, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.

[0051] As used herein, the term “detectable marker” refers to at least one marker capable of directly or indirectly, producing a detectable signal. A non-exhaustive list of this marker includes enzymes which produce a detectable signal, for example by colorimetry,fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, P- galactosidase, glucose6 phosphate dehydrogenase, chromophores such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and / or chemical properties, such detection may be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation, the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as32P,35S ,89Zr or1251.

[0052] As used herein, the term “purification marker” refers to at least one marker useful for purification or identification. A non-exhaustive list of this marker 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 fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

[0053] As used herein, an “epitope tag” is a biological structure or sequence, such as a protein or carbohydrate, which acts as an antigen that is recognized by an antibody. In certain embodiments, an epitope tag is used interchangeably with a purification marker and / or an affinity tag.

[0054] A “composition” is intended to mean a combination of two or more compounds, such as a combination of an active polypeptide, polynucleotide, viral vector, or antibody and / or another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0055] A “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0056] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington’s Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0057] A “subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, non-human primates, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primates, particularly human. Besides being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or infant under the age of eighteen years of age.

[0058] The term “transduction” or “transduce” refers to viral particle-mediated transfer of genetic material into the cell (e.g., AAV-mediated gene transfer). See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

[0059] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. In one aspect, the term “treatment” excludes prevention or prophylaxis.

[0060] The term “suffering” as it related to the term “treatment” refers to a patient or individual who has been diagnosed with or is predisposed to a disease.

[0061] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. In one aspect, an effective amount is a therapeutically effective amount. Typically, dosageeffect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with theconcentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well 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 RNA virus replication ex vivo, in vitro or in vivo.

[0062] The term “administration” shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The invention is not limited by the route of administration, the formulation or dosing schedule.

[0063] As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Natural occurring adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus, while recombinant AAV (rAAV) can comprise both a single-stranded DNA or a self- complementary DNA. General information and reviews of AAV can be found in, for example, Carter, Handbook of Parvoviruses 1 : 169-228, 1989, and Berns, Virology 1743- 1764, 1999. However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, Parvoviruses and Human Disease 165-174, 1988, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61, 1974). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear 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 length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0064] 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 certain viral functions such as replication, virus packaging,integration and / or provirus rescue, and the like). The ITR can be an AAV ITR or a non-AAV ITR. For example, a non-AAV ITR sequence such as those of other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B- 19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and / or addition. In one embodiment, the ITR is partially or completely synthetic, such as the "double-D sequence" as described in United States 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 from any AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known or later discovered. An AAV ITR need not have the native terminal repeat sequence (e.g., a native AAV ITR sequence may be altered by insertion, deletion, truncation and / or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, persistence, and / or provirus rescue, and the like.

[0065] An “AAV expression cassette” as used herein refers to a nucleotide sequence comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV expression cassette can be replicated and packaged into infectious viral particles (e.g., AAV vectors) when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0066] 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. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a 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 AAV vector particle necessarily includes production of AAV expression cassette, as such a plasmid is contained within an AAV vector particle.

[0067] Natural occurring adeno- associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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 corrected 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_00 1862; at least portions of 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 relating to AAV-8); the AAV-9 genome is provided in Gao et 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). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). exacting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, pl 9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pl 9), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start 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).

[0068] Recombinant AAV genomes of the disclosure comprise nucleic acid molecule of the invention and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for 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. Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, for example rAAV 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 nucleotidesequences of the genomes of various AAV serotypes are known in the art. In some embodiments, to promote skeletal muscle specific expression, AAV1, AAV6, AAV8 or AAVrh.74 is used.

[0069] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0070] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0071] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments. The term “isolated” is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0072] The term “recombinant” as used herein with respect to polypeptides or polynucleotides, such as DNA or RNA, refers to molecules formed by laboratory methods of recombination, such as molecular cloning. Molecular cloning techniques are known in the art and may include, but is not limited to, PCR amplification of a polynucleotide, enzymaticdigestion of a polynucleotide, ligation of a polynucleotide into an expression cassette e.g., mammalian expression cassette), transformation, transfection or transduction of a cell with the polynucleotide, and expression of the polynucleotide to produce the polypeptide. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 2012. The term “recombinant polynucleotide” is meant to include fragments of protein-encoding polynucleotides. For instance, a recombinant polynucleotide may include a fragment of the polynucleotide that encodes for a human dysferlin protein. A recombinant polynucleotide may be produced by PCR amplification of a fragment of a protein-encoding polynucleotide. A recombinant polypeptide may be produced by expression of one or more recombinant polynucleotides. In one embodiment, the “recombinant” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0073] The term “Rep sequence” or "Rep coding sequence" means the nucleic acid sequences that encode the parvoviral or AAV non- structural proteins that mediate viral replication and the production of new virus particles. The parvovirus and AAV replication genes and proteins have been described in, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). The "Rep coding sequences" need not encode all of the parvoviral or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequences do not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52 and Rep40), in fact, it is believed that AAVS only expresses the spliced Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequences encode at least those replication proteins that are necessary for viral or vector genome replication and packaging into new virions. The Rep coding sequences will generally encode at least one large Rep protein (i.e., Rep78 / 68) and one small Rep protein (i.e., Rep52 / 40). In particular embodiments, the Rep coding sequences encode the AAV Rep78 protein and the AAV Rep52 and / or Rep40 proteins. In other embodiments, the Rep coding sequences encode the Rep68 and the Rep52 and / or Rep40 proteins. In a still further embodiment, the Rep coding sequences encode the Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins. In the native AAV genome, the different Rep proteins are encoded by a single gene through use of two different promoters and alternative splicing. For purposes of AAV vector production, however, Rep proteins can be expressed in producer cells from a single gene, or from distinct polynucleotides, one sequence for eachRep protein to be expressed. Thus, for example, a Rep encoding gene can be engineered to inactivate the p5 or pl9 promoter so that only small or only large Rep proteins are expressed.

[0074] As used herein, the term “cap sequence” or “cap coding sequences” refers to the structural proteins that form a functional capsid (i.e., can package DNA and infect target cells), e.g., parvovirus or AAV capsids. In one embodiment, the cap coding sequences encode all of the parvovirus or AAV capsid subunits, but less than all of the capsid subunits may be encoded as long as a functional capsid is produced. In another embodiment, the cap sequences are present on a single nucleic acid molecule. The capsid structure of autonomous parvoviruses and AAV are described in more 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 encoding both the Rep and Cap genes of one or more AAV.

[0075] In some embodiments, an adeno-associated viral (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.

[0076] Further disclosed herein are adeno-associated viral (AAV) vectors. In some embodiments, the AAV vectors comprise any gene or gene fragment.

[0077] 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), promoter, intron, selection marker, or origin of replication (ORI).

[0078] 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), selection marker, origin of replication (ORI), untranslated region (UTR), or polyadenylation (polyA) signal.

[0079] Further disclosed herein are uses 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.

[0080] Inverted Terminal Repeats

[0081] 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, the two or more ITRs are the same. In some embodiments, the two or more ITRs are different.

[0082] In some embodiments, the recombinant polynucleotide is flanked by the 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 the pair of ITRs. In some embodiments, the ITRs in the first pair of ITRs are the same. In some embodiments, the ITRs in the first pair of ITRs are different. In some embodiments, the ITRs in the second pair of ITRs are the same. In some embodiments, the ITRs in the second pair of ITRs are different. In some embodiments, the ITRs in the first pair of ITRs are the same as the ITRs in the second pair of ITRs. In some embodiments, at least one ITR in the first pair of ITRs is the same as at least one ITR in the second pair of ITRs. In some embodiments, the ITRs in the first pair of ITRs are different from the ITRs in the second pair of ITRs. In some embodiments, at least one ITR in the first pair of ITRs is different from at least one ITR in the second pair of ITRs.

[0083] In some embodiments, the ITR is a viral ITR. In some embodiments, the ITR is an AAV ITR. In some embodiments, the AAV ITR is selected from an ITR from at least one of 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 ITR is an AAV2 ITR. In some embodiments, the AAV ITR is an AAV5 ITR. The ITR sequences for AAV1-6 can be found, for example, in Grimm et al.. J. Virol.80(l):426-39, 2006, which is incorporated by reference in its entirety.

[0084] In some embodiments, the recombinant polynucleotide does not comprise an AAV sequence other than an inverted terminal repeat (ITR).

[0085] In some embodiments, the recombinant polynucleotide does not comprise a viral sequence other than an inverted terminal repeat (ITR).

[0086] Promoters

[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 promoters. In some embodiments, the promoter is a eukaryotic promoter. Examples of eukaryotic promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EFla) promoter, CAG promoter, phospholy cerate kinase gene (PGK) promoter, tetracycline response element (TRE) promoter, human U6 nuclear (U6) promoter, and 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.

[0088] In some embodiments, the promoter is a tissue-specific promoter. Examples of tissues include, but are not limited to, muscle, epithelial, connective, and nervous 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-P 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.

[0089] 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 another embodiment, 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 myocytespecific 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 a-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7) promoter, a C5-12 promoter, a murine 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, hypoxia- inducible nuclear factor.

[0090] Polyadenylation signal

[0091] 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 (poly A) signals. In some embodiments, the polyA signal is an artificial polyA signal.

[0092] Expression Cassettes and Packaging Systems

[0093] Further disclosed herein are adeno-associated viral (AAV) expression cassettes. In some embodiments, the AAV expression cassette comprises: (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any of the ITRs disclosed herein; (b) any gene or gene fragment disclosed herein; and (c) a second ITR, wherein the second ITR comprises any of the ITRs disclosed herein, wherein the gene or gene fragment of (b) is flanked by the first and second ITRs of (a) and (c).

[0094] Further disclosed herein are adeno-associated viral (AAV) plasmids. In some embodiments, the AAV expression cassette comprises: (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any of the ITRs disclosed herein; (b) any gene or gene fragment disclosed herein; and (c) a second ITR, wherein the second ITR comprises any of the ITRs disclosed herein, wherein the gene or gene fragment of (b) is flanked by the first and second ITRs of (a) and (c).

[0095] Further disclosed herein are adeno-associated viral (AAV) packaging systems. In some embodiments, the AAV packaging systems comprise: (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 from an adenovirus. In some embodiments, the one or more genes from the adenovirus mediate AAV replication. In some embodiments, the one or more genes from the adenovirus are selected from E4, E2a, and VA. In some embodiments, the rep-cap plasmid comprises one or more polynucleotides encoding the adeno-associated virus rep and cap genes. In some embodiments, the rep gene encodes for one or more of life cycle proteins selected from Rep78, Rep68, Rep62, and Rep40. In some embodiments, the cap gene encodes for one or more of capsid proteins selected from VP1, VP2, and VP3. In some embodiments, the 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 repgene 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.

[0096] In some embodiments, the AAV packaging systems comprise: (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 from an adenovirus. In some embodiments, the one or more genes from the adenovirus mediate AAV replication. In some embodiments, the one or more genes from the adenovirus are selected from E4, E2a, and VA. In some embodiments, the rep-cap plasmid comprises one or more polynucleotides encoding the adeno- associated virus rep and cap genes. In some embodiments, the rep gene encodes for one or more of life cycle proteins selected from Rep78, Rep68, Rep62, and Rep40. In some embodiments, the cap gene encodes for one or more of 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.

[0097] In some embodiments, the adeno-associated viral 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 from an adenovirus. In some embodiments, the one or more genes from the adenovirus mediate AAV replication. In some embodiments, the one or more genes from the adenovirus are selected from E4, E2a, and VA.

[0098] In some embodiments, the adeno-associated viral 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 from an adenovirus. In some embodiments, the one or more genes from the adenovirus mediate AAV replication. In some embodiments, the one or more genes from the adenovirus are selected from E4, E2a, and VA.

[0099] Viral Vectors

[0100] Further disclosed herein are adeno-associated viral (AAV) vectors (e.g., AAV viruses or AAV particles). In some embodiments, the AAV vectors comprise, consist of, or consist essentially of any of the polynucleotides disclosed herein.

[0101] 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.

[0102] Further disclosed herein are dual adeno-associated viral (AAV) vector systems 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 gene or gene fragment disclosed herein; and (b) a second AAV vector, wherein the second AAV vector comprises any of the 3’ hDYSF polynucleotides disclosed herein.

[0103] 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.

[0104] Compositions

[0105] Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the dual AAV vector systems disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the AAV vectors disclosed herein.

[0106] Further disclosed herein is a composition comprising, consisting of, or consisting essentially of: (a) a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises, consists of, or consists essentially of any of the gene or gene fragment disclosed herein; and (b) a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0107] Further disclosed herein is a composition 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 gene or gene fragment disclosed herein; and (b) a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0108] In some embodiments, any of the compositions disclosed herein further comprise at least one of a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed 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.

[0109] The pharmaceutically acceptable carrier, diluent, or excipient 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 must be 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 actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The 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 a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0110] Sterile injectable solutions are prepared by incorporating the polynucleotides, plasmids, viral vectors, or dual vector systems disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0111] Methods for Producing AAV Vectors

[0112] Disclosed herein are methods of producing an adeno-associated viral (AAV) vector (e.g., virus or viral particle). Methods of producing AAV vectors are known in the art. For instance, such methods are disclosed in, for example, WO 01 / 83692, which is incorporated by reference herein in its entirety. 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 are incorporated by reference in their entirety. Various approaches for producing AAVs are described in Ratschin et al., Mol. Cell. Biol. 4:2072, 1984; Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466, 1984; Tratschin et al., Mol. Cell. Biol. 5:3251, 1985; McLaughlin et al., J. Virol., 62: 1963, 1988; and Lebkowski et al., Mol. Cell. Biol., 7:349, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989; U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658.776 ; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 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 which are incorporated by reference in their entirety.

[0113] In some embodiments, the method for producing an adeno-associated viral (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 that stably expresses 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 the 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 from cellular debris and proteins. Alternatively, or additionally, the method further comprises lysing the population of transduced cells to produce a cellular lysate. In some embodiments, the method further comprises subjecting the cellular lysate to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free from cellular debrisand proteins. In some embodiments, the purity of the purified AAV vector sample is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure. However, even a purified AAV may contain subpopulations having capsid variations, including post-translational modifications, or various amounts and compositions of nucleic acids.

[0114] Cells

[0115] Further disclosed herein are cells comprising any of the plasmids, expression cassettes or AAV vectors disclosed herein. The cells can be prokaryotic or eukaryotic cells. Nonlimiting examples of eukaryotic cells include mammalian, e.g., hamster, murine, rat, canine, ovine or human cells.

[0116] Any of the cells disclosed herein may be packaging cells that produce infectious rAAV. In some embodiments, the packaging cells are stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells). Non-limiting examples of prokaryotic cells comprise bacterial cells (e.g., Escherichia coli) and archaeal cells. The cells of the disclosure can be used to produce a cell bank, e.g., an Accession Cell Banks (ACB) for non-GMP purpose or GMP Master Cell Bank (MCB). The aliquote of the cells, in one embodiment, are expanded from an original inoculum to a larger volume before culture in the bioreactor for the production.

[0117] Viral Titers

[0118] Titers of AAV vectors to be administered in methods of the invention will vary depending, for example, on the particular AAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of AAV may range from at least about IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about IxlO13to about IxlO14or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg). For instance, dosages of AAV may range from at least about IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about 2xl012, about 3xl012, about 4xl012, about 5xl012, about 6xl012, about 7xl012, about 8xl012, about 9xl012, about IxlO13to about IxlO14viral genomes.

[0119] AAV dosage can be determined by multiple methods, which include but are not limited to ELISA, assessment of the reverse transcriptase activity, FACS, transduction assays northern blotting (e.g., semi -quantitative northern), dot blot analysis or PCR (e.g., qPCR). It is well known that the AAV doses can be determined by measuring AAV vector genomes with quantitative real-time PCR (qPCR). Such qPCR methods overcome the inconsistency or arbitrary results from conventional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. The forms of the plasmids can impact the dosage results from the qPCR methods. In one embodiment, the circular or supercoiled DNA or plasmids are used as a quantification standard.

[0120] In some embodiment, dosages may be expressed in the units of vg / kg, based on a supercoiled DNA or plasmid as the quantitation standard. For example, dosages of AAV is about 1X106-1X1016vg / kg, about 1X108-1X1015vg / kg, or about lxlOlo-lxlO14vg / kg, based on a supercoiled DNA or plasmid as the quantitation standard. In another embodiment, the dosages are about at least IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about 2xl012, about 4xl012, about 6xl012, about 8xl012, about IxlO13, about 2xl013, about 2.4xl013, about 3xl013, about 4xl013, about 5xl013, about 6xl013, about 7xl013, about 8xl013, about 9xl013, about IxlO14, about IxlO15, or at least about IxlO16vg / kg. In one embodiment, the dosage is at least 2xl012, 4xl012, 6xl012, 8xl012, IxlO13, 2xl013, 2.4xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, or 8xl013vg / kg, based on a supercoiled DNA or plasmid as the quantitation standard.

[0121] In some embodiments, the methods disclosed herein comprise administering at least about IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about 2xl012, about 3xl012, about 4xl012, about 5xl012, about 6xl012, about 7xl012, about 8xl012, about 9xl012, about IxlO13vg in a total volume of 1.5 ml per injection. In some embodiments, the methods disclosed herein comprise administering a total daily dose of at least about IxlO6, about IxlO7, about IxlO8, about IxlO9, about IxlO10, about IxlO11, about IxlO12, about 2xl012, about 3xl012, about 4xl012, about 5xl012, about 6xl012, about 7xl012, about 8xl012, about 9xl012, about IxlO13, about 2xl013, about 5xl013, about 7xl013, about IxlO14vg. One exemplary method of determining encapsidated vector genome titer uses quantitative PCR, such as the methods described in Pozsgai et al., Mol. Ther. 25(4): 855-869, 2017, which is incorporated by reference in its entirety.EXAMPLES

[0122] 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.

[0123] The present technology 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, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0124] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0125] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0126] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0127] 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 incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0128] Other aspects are set forth within the following claims.

[0129] Example 1: HEK293 Adherent Cell Transfection

[0130] Allow freshly thawed cells to recover in culture for three or more passages post-thaw before transfecting. Passage cell cultures every 3 or 4 days. The cell viability of the cell culture should not exceed 5.5xl06cells / mL. Four days prior to transfection, seed T-175 flasks at 7000 to 14000 cells / cm2in 35 mL complete DMEM.

[0131] On the day of transfection prepare serum-free DMEM medium by adding 100X GlutaMAX at a IX concentration (5 mL) and 450 g / L glucose to a total of 6 g / L (1.66 mL). Prepare transfection complexes using prewarmed serum-free DMEM medium as the diluent. Prepare polyethylenimine (PEI) mixture in 50 mL conical tube for each condition and incubate for 5 minutes. Prepare DNA mixture in 50 mL conical tube for each condition. Add PEI mixture to DNA mixture and mix well. After ten minutes pipette mixture into flasks, 3.5 mL for each flask. Return flask to incubator shaker. Two hours to 18 hours post transfection add additives (Table I) to flasks. The day after transfection, aspirate medium and replace with 35 mL of prewarmed serum -free medium containing additives. Four days after transfection add 10% v / v fresh lysis buffer (500 mM Tris-HCl pH 8.0, 20 mM MgCh, 1% TWEEN-20) to flasks and incubated at 37°C for two hours. Spin down lysate 300 x g at room temperature for ten minutes. Transfer supernatant to a conical tube and filter using a 0.45 pm filter. Make two 200 pl aliquots and four 1.5 mL aliquots of each flask and store at -80°C.Table 1. Additives tested for ability to enhance AAV production.

[0132] Four cell culture additives are identified as enhancers of rAAV production (Fig. 1 A).Ethyl alcohol, DMSO, valproic acid and tryptone N1 are further tested at various concentrations to determine an approximate optimal amount of each additive (Fig. IB).Because tryptone N1 is a mixture of compounds, it is not well defined to a degree that would be required for pharmaceutical grade production, and it was thus excluded from further consideration.

[0133] Next, the selected additives are tested for their ability to synergize with one another. A combination of low dose DMSO and low dose valproic acid appeared to provide the best enhancement of rAAV production (Fig. 2).

[0134] Example 2. Refinement of conditions for cell culture additives.

[0135] Although cell culture additives are identified and an approximate concentration of each is identified, many variables still exist. One such variable is the timing for the addition of each additive.

[0136] Transfections are performed as in Example 1, but the additives are provided to the cell culture either 1 hour prior to transfection or one, two, four or eight hours post transfection. A combination of DMSO and valproic acid added one to two hours post transfection provides the best enhancement of rAAV production (Fig. 3 A).

[0137] Density of the cells to be transfected is also tested on adherent HEK 293 cells to determine whether they were as effective. These cells are anchorage-dependent and require the use of serum to grow. Cells are seeded 96 hours before transfection at two different densities (Process B and Process C) and transfect with rAAV constructs containing AN05 WT or pDystrophin genes in the expression cassette. After transfection with these two transgenes, the cells are treated with either ddH2O or 1.6 mM VPA / 1.6% DMSO two hours post transfection. At 96 hours the cells are chemically lysed and clarified before analysis for VG titer by qPCR. For both AN05 WT and microdystrophin the two additives result in a five-fold increase in VG / cm2 titer (Figure 3B). Furthermore, seeding cells at a lower density (Process C) improve AAV production as the control from Process B was below the assay limit of detection.

[0138] To fine tune the results of the pilot studies, a full factorial Design of Experiments (DoE) study is conducted. DoE methodology has been used to optimize the production of a wide range of biologies, including AAV. The benefit of DoE is that it is a systematic approach to investigate several variables in a product or process at one time and predict the optimal conditions for maximum output. The higher the number of runs for a given design, the higher the statistical Power will be, increasing the ability to distinguish signal from noise. The 48-way Ambr 15 microbioreactor system (Sartorius) is well-suited to perform DoE studies and therefore the constraints of this study are designed around it.

[0139] A 3 x 3 x 3 full factorial design with a minimum of 27 total runs is used, however to increase the statistical Power two replicates are added at the four edges and center of the cube for a total of 45 runs. The three levels of each input variable are as follows: ethanol - 0 mM, 50 mM, 100 mM; valproic acid - 0 mM, 1.25 mM, 2.50 mM; and DMSO - 0%, 1%, 2%. Samples are collected at 96 hours post transfection and submitted for qPCR. A polynomial regression model is generated in JMP based on D4 titers using mixture linear, interaction, and response surface terms. Overall, the fit of the data in the reduced model is good with an r2value of 0.75 indicating that 75% of the variation in data is explained by the model. The Press r2also demonstrates that 50% of the variability in the model could be explained by removing one data point. A desirability algorithm in the prediction profiler is used to optimize the response, which is D4 titer. The optimum settings for predicted maximum titer are ethyl alcohol at 0 mM, valproic acid at 1.59 mM ± 0.3 mM and DMSO at 1.57% ± 0.4% (Fig. 4A). The predicted titer is 4.40E+11 vg / mL with a 95% confidence interval of 3.8E+11 to 5.0E+11 vg / mL. The predicted relationship of varying concentrations of DMSO and valproic acid are graphically presented in Fig. 4B.

[0140] Example 3. Translation to other cells, AAV capsids and constructs.

[0141] Next, the additives are tested work on several other cell lines, AAV serotypes, and genes-of-interest. Both CTS VPC and Expi293 cells are commercial cell lines from Thermo Fisher Scientific. CTS VPC and Expi293 cells are derived from the same parental suspension HEK 293F cell line and are closely related subclones. Expi293 cells are tested against CTS VPCs in 125 mL shake flasks with 100 mM ethanol, 2 mM VP A, 2% DMSO, and 1.6 mM VPA / 1.6% DMSO. Expi293 cells performed comparably to CTS VPC under all conditions (Figure 5A). As single agents, both VPA and DMSO show significantly higher titers after 96 hours as compared with the control, and together they have a synergistic effect on increasing AAV production. While not statistically significant, ethanol also has a modest effect on increasing VG titer in Expi293 cells.

[0142] To rule out any capsid-specific effects of cell culture additives, CTS VPCs are transfected with a plasmid having a transfer cassette containing the ANO5 WT transgene and plasmids containing either AAV1 or AAV9. Overall, the production of AAV1. ANO5 WT is lower than AAVrh74..4 5 WT. However, the presence of 1.6 mM VPA / 1.6% DMSO did increase AAV production by approximately two-fold (Figure 5B). These two additives have a more drastic effect on AAV9.4M 5 WT, increasing VG titer approximately eight-fold. In thecase of both AAV1 and AAV9 in this experiment, valproic acid did not have a significant impact on overall yield. In fact, 1.6% DMSO is just as effective as 1.6 mM VPA / 1.6% DMSO.

[0143] Further, the effect of cell culture additives is tested on AAV production in CTS VPC using different ITR-containing transgenes. The size and structure of different transgenes may have a dramatic impact on AAV production. A few different transgenes are chosen for testing, some part of a dual vector approach (5 ’ DYSF a self-complementary vector (NT3), or one specific for gene-editing (saCas ). In all cases 1.6 mM VPA / 1.6% improved vector yield ranging from 2- to 4-fold (Figure 5C). Like the findings with different serotypes, it appears that 1.6 mM VPA alone is not as effective in improving AAV production with these transgenes. Though unlike the capsid test, the combination of VPA / DMSO using NT3 and saCas9 did have a synergistic effect as the titer achieved using both is much greater than DMSO alone.

[0144] In summary, three cell culture additives that improve AAV production are characterized in a HEK 293 triple transfection system. These additives are effective on 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 just contribute to a greater percentage of fully encapsidated vectors. More studies will be needed to determine the potency, infectivity, and stability of AAV vectors produced in the presence of cell culture additives. Regardless, an average of a five-fold increase of VG titer during production is achieved using low doses of VPA and DMSO. These data will improve upstream processes to become more efficient, producing larger amounts of drug product using less starting material. This in turn will lower the cost of AAV production and improve patient access to gene therapies.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of producing a recombinant adeno-associated viral (rAAV) vector comprising:(a) transfecting cells with at least one nucleic acid capable of expressing a 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. The method of Claim 1, wherein the low dose of DMSO is about 1.6% volume / volume, 1.2% to 2%, or 1.57% ± 0.4%.

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

4. The method of Claim 1, wherein step (b) is done 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. The method of Claim 1, wherein production of the rAAV is enhanced compared to a method without step (b).

6. A cell culture medium comprising a low dose of dimethyl sulfoxide (DMSO) and a low dose of valproic acid.

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

8. 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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