A highly scalable column-based manufacturing process for rAAV

Abstract

A scalable purification process suitable for industrial-scale production of rAAV vectors is provided. [Solution] The method includes the steps of harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest, lysing the harvest to produce a lysate, treating the lysate to produce a nucleic acid-reduced lysate, filtering the nucleic acid-reduced lysate to produce a clarified lysate, subjecting the clarified lysate to anion exchange column chromatography to produce a column eluate, subjecting the column eluate to size exclusion column chromatography to produce a second column eluate, subjecting the second column eluate to cation exchange column chromatography to produce a third column eluate consisting of rAAV vector particles, and filtering the third column eluate, thereby producing purified rAAV vector particles.

Description

[Background technology]

[0001]

[0002] Gene delivery is a promising method for the treatment of acquired and inherited diseases. Several virus-based systems for gene transfer have been described, including adeno-associated virus (AAV)-based systems.

[0002]

[0003] AAV is a helper-dependent DNA parvovirus belonging to the Dependovirus genus. AAV requires the function of a helper virus, such as adenovirus, herpesvirus, or vaccinia, to cause productive infection. In the absence of helper virus function, AAV establishes a latent state by inserting its genome into the chromosome of host cells. Subsequent infection with the helper virus rescues the integrated viral genome, which can then replicate to produce infectious AAV progeny.

[0003]

[0004] AAV has a broad host range and can replicate in cells from any species in the presence of a suitable helper virus. For example, human AAV can replicate in canine cells co-infected with a canine adenovirus. AAV has never been associated with any human or animal disease and does not appear to adversely affect the biology of host cells upon integration.

[0004]

[0005] AAV vectors can be engineered to carry heterologous nucleic acid sequences of interest (e.g., selected genes encoding therapeutic proteins, inhibitory nucleic acids, e.g., antisense molecules, ribozymes, miRNAs, etc.) by deleting the entire or partial internal portion of the AAV genome and inserting the nucleic acid sequence of interest between the ITRs. The ITRs still function in such vectors to allow replication and packaging of the rAAV containing the heterologous nucleic acid sequence of interest. The heterologous nucleic acid sequence is also typically linked to a promoter sequence that can drive expression of the nucleic acid in the target cells of the patient. Termination signals, such as polyadenylation sites, can also be included in the vector.

[0005]

[0006] The construction of infectious recombinant AAV (rAAV) vectors has been described in several publications, see, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; WO 92 / 01070 and WO 93 / 03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, RM (1994) Human Gene Therapy 5:793-801.

[0006]

[0007] Recombinant adeno-associated virus (AAV) vectors have shown good therapeutic promise in several early clinical trials by multiple groups. Development of this new class of biological product toward approval will involve improvements in vector characterization and quality control methods, including a better understanding of how vector design and manufacturing process parameters affect the impurity profile in clinical-grade vectors.

[0007]

[0008] An important objective in designing rAAV production and purification systems is to implement strategies to minimize / control the occurrence of production-related impurities, such as vector-related impurities including proteins, nucleic acids, and wild-type / pseudo-wild-type AAV species (wtAAV), as well as residual AAV encapsidated DNA impurities. Removal of impurities in AAV vectors is complicated due to the way in which rAAV vectors are produced. In one production process, rAAV vectors are produced by a transient transfection process using three plasmids. A significant amount of plasmid DNA is introduced into cells to produce rAAV vectors. In addition, when the rAAV vectors are released from the producing cells, the cellular proteins and nucleic acids are also released. Given that the rAAV vectors only account for about 1% of the biomass, it is a great challenge to purify rAAV vectors to a level of purity that can be used as a clinical human gene therapy product. (Smith PH Wright JF. Qu G. et al. 2003, Mo. Therapy, 7:8348; Chadeuf G. et al., Mo. Therapy 2005, 12:744. Report from the CHMP Gene Therapy Expert Group Meeting. European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0008]

[0009] Development of a manufacturing process to purify recombinant AAV as a product to treat human disease should achieve the following objectives: 1) consistent vector purity, titer, and safety; 2) feasibility of scalable manufacturing process; and 3) acceptable manufacturing costs. Current "industry standard" scalable AAV vector purification processes do not adequately achieve removal of impurities important to meet the first objective listed above (consistent vector purity, titer, and safety). Moreover, impurities cannot be adequately removed using current industry standard scalable purification processes because: 1) development of purification processes for viral products such as recombinant AAV for applications other than vaccines (where an immune response is typically sought rather than avoided) is relatively new; 2) many groups involved in developing scalable purification processes for AAV vectors have been unaware of high levels of vector-associated impurities and / or have assumed that such impurities do not contribute to clinically significant vector immunogenicity; and 3) it is technically challenging to develop a scalable purification process suitable for industrial-scale manufacturing of rAAV vectors.

[0009] [Summary of the invention]

[0010] In accordance with the invention, in some aspects, a method for purifying recombinant adeno-associated virus (rAAV) vector particles is provided, the method comprising the steps of: (a) harvesting cells and / or cell culture supernatant comprising the rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; and (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion or cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate. (g) subjecting the column eluate produced in step (f) or the concentrated column eluate to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate produced in step (g) or the diluted second column eluate to cation or anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from protein or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and (i) filtering the third column eluate produced in step (h) or the concentrated third column eluate, thereby producing purified rAAV vector particles. The following embodiments and aspects relate to one or more of steps (a) through (h) above.

[0010]

[0011] In certain embodiments, step (f) comprises subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange chromatography, and / or step (h) comprises subjecting the second column eluate or diluted second column eluate produced in step (g) to cation exchange column chromatography. In certain embodiments, step (f) comprises subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography, and / or step (h) comprises subjecting the second column eluate or diluted second column eluate produced in step (g) to anion exchange column chromatography.

[0011]

[0012] In some aspects, the concentration in step (b) and / or step (f) and / or step (h) is achieved by UF / DF, and optionally tangential flow filtration. In some embodiments, the concentration in step (b) reduces the volume of the harvested cells and cell culture supernatant by about 2-10 fold. In some embodiments, the concentration in step (f) reduces the volume of the column effluent by about 5-20 fold. In some aspects, the lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is achieved by microfluidization.

[0012]

[0013] In certain embodiments, after step (b) and before step (c), the method comprises the step (b)(i). In certain embodiments, step (b)(i) comprises reducing contaminating nucleic acids in the lysate. In some embodiments, step (b)(i) comprises treating the lysate with a nuclease, thereby reducing contaminating nucleic acids. In certain embodiments, the nuclease comprises benzonase.

[0013]

[0014] In some embodiments, the filtration of the clarified lysate or diluted clarified lysate in step (e) is through a filter having a pore diameter of between about 0.1 and 0.8 microns, inclusive. In certain embodiments, the dilution of the clarified lysate or diluted clarified lysate in step (e) is with an aqueous acetate buffer.

[0014]

[0015] In some embodiments, dilution of the second column eluate in step (g) is with an aqueous acetate buffer, hi certain embodiments, the aqueous acetate buffer has a pH between about 4.0 and 7.0, inclusive.

[0015]

[0016] In some embodiments, the rAAV vector particles are formulated with a surfactant to produce an AAV vector formulation. In certain embodiments, the rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation.

[0016]

[0017] In some embodiments, the cation or anion exchange column chromatography of step (f) comprises polyethylene glycol (PEG) conditioning column chromatography. In some embodiments, the cation or anion exchange column chromatography of step (f) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column. In some embodiments, the PEG in the PEG solution has an average molecular weight in the range of about 1,000 to 50,000, inclusive. In some embodiments, the cation or anion exchange column of step (e) comprises washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column. In some embodiments, the cation or anion exchange column of step (h) comprises washing the column with a detergent solution prior to elution of the rAAV vector particles from the column. In some embodiments, the PEG solution and / or detergent solution comprises an aqueous TrisCl / NaCl buffer or an aqueous phosphate / NaCl buffer. In some embodiments, the NaCl buffer comprises between about 20 and 250 mM NaCl, inclusive, or between about 50 and 200 mM NaCl, inclusive.

[0017]

[0018] In some embodiments, the rAAV vector particles are eluted from the cation or anion exchange column of step (f) into an aqueous TrisCl / NaCl buffer. In some embodiments, the TrisCl / NaCl buffer comprises between 50 and 200 mM NaCl. In some embodiments, the rAAV vector particles are eluted from the cation or anion exchange column of step (h) into an aqueous phosphate / NaCl buffer. In some embodiments, the phosphate / NaCl buffer comprises between about 100 and 500 mM NaCl, inclusive. In some embodiments, the cation exchange column of step (f) comprises a quaternary ammonium functional group. In some embodiments, the quaternary ammonium functional group comprises a quaternized polyethyleneimine.

[0018]

[0019] In certain embodiments, for example, the size exclusion column of step (g) has a separation range (molecular weight) of between about 10,000 and 600,000, inclusive. In some embodiments, the cation exchange column of step (h) comprises a sulfonic acid. In some embodiments, the cation exchange column of step (h) comprises a sulfopropyl.

[0019]

[0020] In certain embodiments, the methods disclosed herein exclude the step of cesium chloride gradient ultracentrifugation.

[0020]

[0021] In some embodiments, the rAAV vector particle comprises a transgene encoding a nucleic acid selected from the group consisting of an siRNA, an antisense molecule, an miRNA, a ribozyme, and an shRNA. In some embodiments, the rAAV vector particle comprises a transgene encoding a nucleic acid selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα). , platelet-derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0021]

[0022] In some embodiments, the rAAV vector particle comprises a transgene encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1-IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta, and gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[0022]

[0023] In certain embodiments, the rAAV vector particle is capable of detecting any of the following enzymes: carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl, ribozyme, ribozyme, ribozyme-binding protein 1 (RIRP1), ribozyme-binding protein 2 (RIRP2), ribozyme-binding protein 3 (RIRP3), ribozyme-binding protein 4 (RIRP4), ribozyme-binding protein 5 (RIRP5), ribozyme-binding protein 6 (RIRP6), ribozyme-binding protein 7 (RIRP7), ribozyme-binding protein 8 (RIRP8), ribozyme-binding protein 9 (RIRP9), ribozyme-binding protein 10 (RIRP1), ribozyme-binding protein 11 (RIRP1), ribozyme-binding protein 12 (RIRP1), ribozyme-binding protein 13 (RIRP1), ribozyme-binding protein 14 (RIRP1), ribozyme-binding protein 15 (RIRP1), ribozyme-binding protein 16 (RIRP1), ribozyme-binding protein 17 (RIRP1), ribozyme-binding protein 18 (RIRP1), ribozyme-binding protein 19 (RIRP1), ribozyme-binding protein 20 (RIRP1), ribozyme-binding protein 21 (RIRP1), ribozyme-binding protein In some embodiments, the rAAV vector particle comprises a transgene encoding a protein useful for correcting an inborn error of metabolism selected from the group consisting of acetyl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence. In some embodiments, the rAAV vector particle comprises a transgene encoding factor VIII or factor IX.

[0023]

[0024] In some embodiments, the methods described herein recover about 40-70% of the total rAAV vector particles from the harvest produced in step (a) or the concentrated harvest produced in step (b). In some embodiments, the methods described herein produce rAAV vector particles having a higher purity than rAAV vector particles produced or purified by AAV affinity column purification. In some embodiments, the methods described herein produce rAAV vector particles having a higher purity than rAAV vector particles produced or purified by AAV affinity column combined with anion exchange column purification. In some embodiments, the methods described herein produce rAAV vector particles having a higher purity than rAAV vector particles produced or purified by AAV affinity column combined with anion exchange column and cation exchange purification.

[0024]

[0025] In some aspects, the rAAV vector particles (e.g., authentic rAAV vector particles) include any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10, AAVtyr -3 The peptide, such as a cell-targeting peptide, (3YAF, see, e.g., U.S. Pat. No. 8,445,267), is derived from an AAV selected from the group consisting of modified AAV capsids.

[0025]

[0026] In some embodiments, the authentic rAAV vector particles are present at a concentration of about 100 mg / mL at the end (e.g., in the third column eluate of step (h)). In some embodiments, the authentic rAAV vector particles are present at a concentration of about 10 mg / mL at the end (e.g., in the third column eluate of step (h)). 15 10 particles or more per mL 16 particles or more, e.g., 10 per mL 17 It is present in concentrations of particles or greater. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows a representative all-column AAV purification scheme using anion, gel filtration (size exclusion) and cation chromatography columns: AIEX - anion exchange chromatography; UF / DF - ultrafiltration / diafiltration; SEC - size exclusion chromatography; and CIEX, cation exchange chromatography. The scheme can also be performed in the reverse order. [Diagram 2] FIG. 1 shows various design options for developing a column-based AAV purification process. [Diagram 3]Figure 1 shows comparative results of AAV purification using four (1-4) different purification schemes: (1) AVB (antibody-based AAV affinity column); and different column combinations: (2) AVB (AAV affinity column) combined with AIEX and CIEX; (3) AVB (AAV affinity column) combined with AIEX; and (4) AIEX combined with SEC-size exclusion and CIEX chromatography. The results show that purification scheme (4) produces fewer impurities than schemes (1 and 2), and potentially even fewer than scheme (3). As indicated, UF / DF-ultrafiltration / diafiltration was used for concentration, but is optionally included in the column AAV purification scheme. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention provides a vector purification platform for recombinant adeno-associated virus (AAV) vectors (rAAV) that contains unique features that distinguish it from current "industry standard" scalable AAV vector purification processes: 1) a platform process assembled with standardized units that can be used for the purification of different AAV vector serotypes / capsid variants, including removal of process impurities and manufacturing related impurities, and 2) a unique combination of chromatography and process steps that offers unexpected scalability to purify rAAV vectors of many different serotypes / pseudotypes.

[0028]

[0028] Impurities include AAV vector production-related impurities, including proteins, nucleic acids (e.g., DNA), cellular components, e.g., intracellular and membrane components, that are impurities distinct from the AAV vector. The term "vector-production-related impurities" refers to any components released during the AAV production process.

[0029]

[0029] A bona fide AAV vector refers to an AAV vector particle that contains a heterologous nucleic acid (e.g., a transgene) that can infect a target cell. The phrase excludes empty AAV capsids, AAV vectors that lack a complete insert in the packaged genome, or AAV vectors that contain contaminating host cell nucleic acid. In one embodiment, a bona fide AAV vector refers to an AAV vector particle that also lacks contaminating plasmid sequences in the packaged vector genome.

[0030] "Empty capsid" and "empty particle" refer to an AAV particle or virion that contains an AAV capsid shell but lacks, in whole or in part, a genome containing a heterologous nucleic acid sequence flanked on one or both sides by AAV ITRs. Such empty capsids do not function to transfer heterologous nucleic acid sequences into a host cell or cells within an organism.

[0031]

[0031] The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., AAV vector), or other vehicle that can be manipulated by inserting or incorporating a nucleic acid. A vector can be used for genetic engineering (i.e., "cloning a vector") to introduce / transfer a polynucleotide into a cell and to transcribe or translate the inserted polynucleotide in the cell. An "expression vector" is a vector that contains a gene or nucleic acid sequence with regulatory regions necessary for expression in a host cell. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, an expression control element (e.g., a promoter, an enhancer), an intron, an inverted terminal repeat (ITR), an optional selectable marker, a polyadenylation signal.

[0032]

[0032] AAV vectors are derived from adeno-associated viruses. AAV vectors are useful as gene therapy vectors because they can enter cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained in the cell. In addition, these viruses can introduce nucleic acid / genetic material to specific sites, such as specific sites on chromosome 19. Because AAV is not associated with pathogenic disease in humans, AAV vectors can deliver heterologous nucleic acid sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0033]

[0033] The term "recombinant" as modified genes of vectors, such as rAAV vectors, and as sequences of modified genes, such as recombinant polynucleotides and polypeptides, generally means that the composition has been manipulated (i.e., altered) in a manner that does not occur in nature. A particular example of a recombinant AAV vector would be when a nucleic acid not normally present in the wild-type AAV genome is inserted into the genome of the virus. An example would be when a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector with or without the 5', 3' and / or intron regions with which the gene is normally associated in the AAV genome. As used herein, the term "recombinant" is not necessarily used with respect to sequences of AAV vectors, polynucleotides, etc., although recombinant forms including AAV vectors, polynucleotides, etc. are expressly included despite any such omissions.

[0034]

[0034] A "rAAV vector" is derived from the wild-type genome of a virus such as AAV by using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the rAAV vector. rAAV is clearly distinguished from the AAV genome because all or part of the AAV genome has been replaced with a non-native sequence, e.g., a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence, with respect to the nucleic acid of the AAV genome. The incorporation of a non-native sequence therefore defines the AAV as a "recombinant" AAV vector, which can be referred to as a "rAAV vector."

[0035]

[0035] Recombinant AAV vector sequences can be packaged and are referred to herein as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo. When recombinant vector sequences are encapsidated or packaged in an AAV particle, the particle can also be referred to as "rAAV" or "rAAV particle" or "rAAV virion." Such rAAV, rAAV particles and rAAV virions include proteins that encapsidate or package the vector genome. As a specific example, in the case of AAV, capsid proteins are included.

[0036]

[0036] Vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form the rAAV particle. When a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone", which is important for the cloning and amplification of the plasmid, processes necessary for propagation and recombinant virus production, but is not itself packaged or encapsidated into the rAAV particle. Thus, vector "genome" refers to the nucleic acid that is packaged or encapsidated by the rAAV.

[0037]

[0037] "AAV helper functions" refers to coding sequences (proteins) from AAV that can be expressed to provide AAV gene products and AAV vectors that function automatically in trans for productive AAV replication and packaging in a stepwise manner. Thus, AAV helper functions include both major AAV open reading frames (ORFs), rep and cap. The rep expression product has been shown to have many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap expression product (capsid) provides the necessary packaging functions. AAV helper functions are used in trans to compensate for AAV functions that are missing from the AAV vector genome.

[0038]

[0038] "AAV helper construct" generally refers to a nucleic acid sequence that includes nucleotide sequences that provide AAV functions deleted from an AAV vector to be used to produce a transducing AAV vector for delivery of a nucleic acid sequence of interest, for example, for gene therapy to a subject. AAV helper constructs are typically used to transiently express AAV rep and / or cap genes to compensate for missing AAV functions required for AAV vector replication. Helper constructs generally lack AAV ITRs and cannot replicate or package by themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. Several AAV helper constructs have been described, such as plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products (see, e.g., Samulsk et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945). Many other vectors have been described that encode Rep and / or Cap expression products (see, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237).

[0039]

[0039] The term "auxiliary functions" refers to non-AAV derived viral and / or cellular functions that AAV depends on for replication. The term includes proteins and RNAs required for AAV replication, including portions involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and packaging of AAV capsids. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0040]

[0040] "Accessory function vector" generally refers to a nucleic acid molecule that includes a polynucleotide sequence that provides accessory functions. Such sequences can be on the accessory function vector and transfected into a suitable host cell. The accessory function vector can support rAAV virion production in the host cell. The accessory function vector can be in the form of a plasmid, phage, transposon, or cosmid. In addition, not all adenovirus genes are required for accessory functions. For example, adenovirus mutants that are incapable of DNA replication and late gene synthesis have been reported to be permissive for AAV replication (Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al., (1971) Virology 45:317). Similarly, mutants in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions (Carter et al., (1983) Virology 126:505). Adenoviruses defective in the E1 region or lacking the E4 region are unable to support AAV replication. Thus, the E1A and E4 regions appear to be required for AAV replication, either directly or indirectly (Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505).Other characterized adenovirus mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al. (1980) Virology 104:502); E2A (Handa et al. (1975) J. Gen. Virol. 29:239; Strauss et al. (1976) J. Virol. 17:140; Myers et al. (1980) J. Virol. 35:665; Jay et al. (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al. (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper in I CRC Handbook of Parvoviruses (ed. P. Tijssen, 1990) Functions); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, although E1B55k may be required for AAV virion production, while E1B19k is not (Samulski et al., (1988) J. Virol. 62:206-210). In addition, WO 97 / 17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe accessory function vectors encoding various adenoviral genes. Exemplary accessory function vectors include the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72 kD coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the complete E1B55k coding region. Such accessory function vectors are described, for example, in WO 01 / 83797.

[0041]

[0041] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined on the basis of a lack of cross-reactivity between antibodies comparing one AAV to another. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in AAV serotypes in VP1, VP2, and / or VP3 sequences).

[0042]

[0042] Based on the traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity against sera specific for all existing and characterized serotypes, and no antibodies were found to neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants arise, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, the new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity must be further performed on mutant viruses with altered capsid sequences to determine whether they are of another serotype according to the traditional definition of serotype. Thus, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) and non-serologically distinct viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0043]

[0043] rAAV vectors include any virus strain or serotype. As a non-limiting example, a rAAV plasmid or vector genome or particle (capsid) can be based on any AAV serotype, e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, etc. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from each other. As a non-limiting example, a rAAV plasmid or vector genome or particle (capsid) based on the genome of one serotype can be identical in one or more of the capsid proteins that package the vector. In addition, a rAAV plasmid or vector genome can be based on a serotype genome of AAV (e.g., AAV2) that is distinct from one or more of the capsid proteins that package the vector genome, where at least one of the three capsid proteins can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or variants thereof. rAAV vectors therefore contain gene / protein sequences identical to those characteristic of particular serotypes, as well as mixed serotypes.

[0044]

[0044] In various exemplary embodiments, the rAAV vector comprises or consists of a capsid sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to a capsid protein of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In various exemplary embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to an ITR(s) of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

[0045]

[0045] rAAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as mutant, hybrid, and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to contain one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such vectors have one or more of the wild-type AAV genes deleted in whole or in part, but retain at least one functional adjacent ITR sequence(s) as necessary for rescuing, replicating, and packaging the recombinant vector into the rAAV vector particle. The rAAV vector genome therefore contains the sequences necessary for replication and packaging (e.g., functional ITR sequences) in cis configuration.

[0046]

[0046] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-, double-, or triplexed, linear or circular, and of any length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0047]

[0047] A "heterologous" nucleic acid sequence refers to a polynucleotide inserted into an AAV plasmid or vector for the purpose of vector-mediated transfer / delivery of the polynucleotide into a cell. A heterologous nucleic acid sequence is distinct from, i.e., non-native with respect to, the AAV nucleic acid. Once transferred / delivered into a cell, a heterologous nucleic acid sequence contained within a vector may be expressed (e.g., transcribed and translated, if appropriate). Alternatively, a heterologous polynucleotide contained within a transferred / delivered vector in a cell may not be expressed. Although the term "heterologous" is not necessarily used herein in reference to nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide is intended to include heterologous nucleic acid sequences and polynucleotides, despite the omission, even in the absence of a "heterologous" modifying gene.

[0048]

[0048] "Polypeptides," "proteins," and "peptides" encoded by a "nucleic acid sequence" consequently include the naturally occurring protein, as well as functional subsequences, modified forms, or sequence variants, so long as the modified forms or variants retain some degree of functionality of the native full-length protein. Such polypeptides, proteins, and peptides encoded by nucleic acid sequences can, but need not, be identical to endogenous proteins that are defective or poorly or incompletely expressed in the mammal being treated.

[0049]

[0049] "Transgene" is used herein for convenience to refer to a nucleic acid (e.g., heterologous) that is intended or introduced into a cell or organism. A transgene includes any nucleic acid, such as a heterologous nucleic acid that encodes a therapeutic protein or polynucleotide sequence.

[0050]

[0050] In a cell carrying a transgene, the transgene has been introduced / transferred by a plasmid or AAV vector, "transduction" or "transfection" of the cell. The terms "transduction" and "transfect" refer to the introduction of a molecule, such as a nucleic acid, into a host cell (e.g., HEK293) or cell of an organism. The transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, the introduced nucleic acid can be stably maintained in that cell or organism and further passed on to or inherited by the progeny cells or organisms of the recipient cell or organism's cells.

[0051]

[0051] "Host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of AAV vector plasmids, AAV helper constructs, accessory function vectors, or other transfer DNA. The term includes the progeny of the original transfected cell. Thus, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations. Exemplary host cells include human embryonic kidney (HEK) cells, such as HEK293.

[0052]

[0052] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell refers to the genetic change in a cell following incorporation of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene), into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid has been introduced, or its progeny. The cell(s) can be propagated (cultured) and the cell expresses the introduced protein or transcribes the nucleic acid, or produces a vector such as rAAV. For gene therapy uses and methods, the transduced cell can be present in a subject.

[0053]

[0053] As used herein, the term "stable" or "stably integrated" with respect to cells means that a nucleic acid sequence, such as a selectable marker or heterologous nucleic acid sequence, or a plasmid or vector, is inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.) or is maintained extrachromosomally in a recipient cell or host organism and remains in the chromosome or is maintained extrachromosomally for a period of time. In the case of cultured cells, a nucleic acid sequence, such as a heterologous nucleic acid sequence, inserted into a chromosome, or a plasmid or vector, can be maintained over the course of multiple cell passages.

[0054]

[0054] "Cell line" refers to a population of cells capable of continuous or long-term growth and division under suitable culture conditions in vitro. A cell line can be, but need not be, a population of clones derived from a single progenitor cell. In cell lines, spontaneous or induced changes can occur in the karyotype during storage or transfer of such clonal populations, as well as during long-term passaging in tissue culture. Thus, progeny cells derived from a cell line may not be strictly identical to the ancestral cells or cultures. An exemplary cell line applicable to the purification method of the present invention is HEK293.

[0055]

[0055] "Expression control element" refers to a nucleic acid sequence(s) that influences the expression of an operably linked nucleic acid. Control elements include expression control elements such as promoters and enhancers described herein. An rAAV vector can include one or more "expression control elements". Typically, such elements are included to facilitate transcription and, if appropriate, translation of the appropriate heterologous polynucleotide (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, and stop codons, etc.). Such elements typically act in a cis fashion and are referred to as "cis-acting" elements, although they may also act in a trans fashion.

[0056] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream"). Expression control elements can also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., in an intron). Expression control elements can be located adjacent to the transcribed sequence or even at a significant distance (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide). Nevertheless, due to the length limitations of the rAAV vector, expression control elements are typically within 1-1000 nucleotides of the transcribed nucleic acid.

[0057]

[0057] Functionally, expression of an operably linked nucleic acid can be controlled, at least in part, by an element (e.g., a promoter) such that the element regulates transcription of the nucleic acid and, where appropriate, translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' to the sequence to be transcribed. A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0058]

[0058] As used herein, "enhancer" can refer to a sequence located adjacent to a nucleic acid sequence, such as a selectable marker or a heterologous nucleic acid sequence. Enhancer elements are typically located upstream of a promoter element, but can also function and be located downstream or internal to the sequence. Thus, enhancer elements can be located upstream or downstream, for example, within 100, 200, or 300 or more base pairs of a selectable marker and / or a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Enhancer elements typically increase expression of an operably linked nucleic acid beyond that caused by a promoter element.

[0059]

[0059] The term "operably linked" means that regulatory sequences required for expression of a nucleic acid sequence are positioned in an appropriate position relative to the sequence so as to effect expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, e.g., a rAAV vector.

[0060] In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. More specifically, for example, two DNA sequences that are operably linked means that the two DNA sequences are positioned in such a relationship (either cis or trans) that at least one of the DNA sequences can have a physiological effect on the other sequence.

[0061]

[0061] Thus, additional elements for a vector include, but are not limited to, expression control elements (e.g., promoters / enhancers), transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) present adjacent to sequences such as the AAV ITR sequences, or one or more copies of an intron.

[0062]

[0062] Additional elements include, for example, filler or stuffer polynucleotide sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept DNA insertions, generally having a size range of about 4 kb to about 5.2 kb, or slightly more. Thus, for shorter sequences, a stuffer or filler is included to adjust the length to near or to the normal size of the viral genomic sequence that is acceptable for packaging of the vector into rAAV particles. In various embodiments, the filler / stuffer nucleic acid sequence is a non-translated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 Kb, the filler or stuffer polynucleotide sequence has a length that, when combined with the sequence (e.g., inserted into a vector), has a total length of between about 3.0-5.5 Kb, or between about 4.0-5.0 Kb, or between about 4.3-4.8 Kb.

[0063] A "therapeutic protein" is, in one embodiment, a peptide or protein that can alleviate or relieve a symptom resulting from an insufficient amount, absence, or deficiency of a protein in a cell or subject. A "therapeutic" protein encoded by a transgene can provide a benefit to a subject, e.g., correct a genetic defect, correct a deficiency (of expression or function) of a gene, etc.

[0064]

[0064] Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) useful according to the present invention include those that may be used in the treatment of diseases or disorders including, but not limited to, "blood congestion" or blood clotting disorders, such as hemophilia A, hemophilia A patients with inhibitory antibodies, hemophilia B, deficiency of coagulation factors VII, VIII, IX and X, XI, V, XII, II, von Willebrand factor, combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleeding associated with anemia, trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC); hyperanticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors); and platelet disorders, such as Bernard-Lesurie syndrome, Glanzmann thrombasthenia, and storage pool deficiency.

[0065] Nucleic acid molecules, vectors, such as cloning vectors, expression vectors (e.g., vector genomes), and plasmids can be prepared using recombinant DNA techniques. The availability of nucleotide sequence information allows for the preparation of nucleic acid molecules by various means. For example, heterologous nucleic acid encoding factor IX (FIX), including vectors or plasmids, can be produced by cell expression or in vitro translation and chemical synthesis techniques using a variety of standard cloning, recombinant DNA techniques. The purity of the polynucleotide can be determined by sequencing, gel electrophoresis, and the like. For example, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to, (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) antibody screening to detect polypeptides with shared structural features, e.g., using expression libraries; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers that can anneal to the nucleic acid sequence of interest; (4) computer searches of sequence databases for related sequences; and (5) differential screening of subtracted nucleic acid libraries.

[0066]

[0066] The term "isolated," when used as a modifier of a composition, means that the composition has been produced by the hand of man or has been completely or at least partially separated from its naturally occurring in vivo environment. Generally, an isolated composition is substantially free of one or more materials that are normally associated with it in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0067]

[0067] With respect to proteins, the terms "isolated protein" or "isolated and purified protein" are sometimes used herein. This term primarily refers to a protein produced by expression of a nucleic acid molecule. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it is naturally associated, such that it exists in "substantially pure" form.

[0068]

[0068] The term "isolated" does not exclude combinations produced by the hand of man, such as recombinant rAAV and pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipid-modified) or derivatized forms, or forms expressed in a host cell produced by the hand of man.

[0069]

[0069] The term "substantially pure" refers to a preparation that contains at least 50-60% by weight of the compound of interest (e.g., a nucleic acid, oligonucleotide, protein, etc.). The preparation may contain at least 75%, or about 90-99%, by weight of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0070]

[0070] The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid sequence, means a sequence having the properties of the given sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel properties of the sequence.

[0071] Methods for generating rAAV virions are known in the art, such as: transfection using an AAV vector and AAV helper sequences in conjunction with co-infection with an AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus), or transfection using a recombinant AAV vector, an AAV helper vector, and an accessory function vector. Non-limiting methods for generating rAAV virions are described, for example, in U.S. Patent Nos. 6,001,650 and 6,004,797. After production of recombinant rAAV vectors (i.e., generation of vectors in cell culture systems), rAAV virions can be obtained from host cells and cell culture supernatants and purified as described herein.

[0072]

[0072] As an initial step, typically, the host cells producing rAAV virions can be harvested, optionally in combination with harvesting the cell culture supernatant in which the host cells producing rAAV virions were cultured. In the methods herein, the harvested cells and optionally the cell culture supernatant can be used as is or purified, as appropriate. Furthermore, if infection is used to express accessory functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60°C, for example, for 20 minutes or more, thereby inactivating only the helper virus, since AAV is heat stable but the helper adenovirus is heat labile.

[0073]

[0073] The harvested supernatant and cells are lysed by disrupting the cells, e.g., by microfluidization to release the rAAV particles. A nuclease, such as benzonase, may then be added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cellular debris by filtration, centrifugation, etc., resulting in a clarified cell lysate. In certain instances, the lysate is filtered using micron-sized pore filters (e.g., through 0.45 μm and / or 0.2 μm filters) to obtain a clarified lysate.

[0074]

[0074] The clarified lysate contains AAV particles (authentic rAAV vectors and AAV empty capsids) and soluble cellular components from the host cells, which may include impurities associated with AAV vector production, such as cellular proteins, lipids, and / or nucleic acids, as well as cell culture media components. The clarified lysate is then subjected to an additional purification step using chromatography to purify the AAV particles (including the authentic rAAV vectors) from impurities. Prior to the first step of chromatography, the clarified lysate may be diluted with an appropriate buffer.

[0075]

[0075] Multiple sequential chromatographic steps are typically used to purify rAAV particles. Such methods typically exclude a cesium chloride gradient ultracentrifugation step.

[0076] As disclosed herein, the first chromatography step can be anion exchange chromatography or cation exchange chromatography. If the first chromatography step is anion exchange chromatography, the third chromatography step can be cation exchange chromatography. Thus, in one rAAV purification method, purification is by anion exchange chromatography, followed by size exclusion chromatography, and then by cation exchange chromatography.

[0077]

[0077] If the first chromatography step is cation exchange chromatography, the third chromatography step can be anion exchange chromatography. Thus, in another rAAV purification method, purification is by cation exchange chromatography, then by size exclusion chromatography, and then by anion exchange chromatography.

[0078] Anion exchange chromatography serves to separate AAV particles from proteins, cells and other components present in the clarified lysate and / or column eluate from size exclusion chromatography. Anion exchange resins include, but are not limited to, polyamine resin-based resins and other resins. Examples of strong anion exchange resins include resins generally based on quaternized nitrogen atoms, including, but not limited to, quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography includes MACRO PREP Q (strong anion exchanger, available from BioRad, Hercules, CA); UNOSPHERE Q (strong anion exchanger, available from BioRad, Hercules, CA); POROS 50HQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA); POROS 50D (weak anion exchanger, available from Applied Biosystems, Foster City, CA); POROS 50PI (weak anion exchanger, available from Applied Biosystems, Foster City, CA); SOURCE 30Q (strong anion exchanger, available from Amersham Biosciences, Piscataway, NJ); DEAE SEPHAROSE (weak anion exchanger, available from Amersham Biosciences, Piscataway, NJ); Q SEPHAROSE (strong anion exchanger, available from Amersham Exemplary anion exchange resins include, but are not limited to, aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0079] Cation exchange chromatography serves to further separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV virions over a wide pH range include, but are not limited to, any sulfonic acid-based resin as indicated by the presence of sulfonate functional groups, including aryl and alkyl substituted sulfonates, e.g., sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS50, POROS SP, POROS S (strong cation exchangers, available from Applied Biosystems, Foster City, CA). Additional examples include the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® families of resins, available from Aldrich Chemical Company (Milliwaukee, WI). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on phosphate functional groups), methylsulfonate (S) and sulfopropyl (SP) resins.

[0080] Chromatographic media such as cation exchange, anion exchange and size exclusion can be equilibrated, washed and eluted with various buffers under various conditions such as pH and buffer volume. The following is intended to describe certain non-limiting examples, but is not intended to limit the invention.

[0081]

[0081] Cation exchange chromatography can be equilibrated using standard buffers according to the manufacturer's specifications. For example, the chromatography medium can be equilibrated with 5-50 mM, or 10-40 mM, e.g., 10-30 mM acetate buffer, and sodium chloride. After equilibration, the sample is then loaded. The chromatography medium is then washed at least once or more, e.g., 2-5 times. Elution from the chromatography medium is at least once with a high salt buffer, but elution can also be performed two or more times with a buffer of the same or higher salt concentration.

[0082]

[0082] Typical equilibration buffers and solutions for washing and elution for cation exchange chromatography have a suitable pH of about pH 3 to pH 8, more typically about pH 4 to pH 6, for example pH 4.1, 4.2, 4.3, 4.4, 4.5 to 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or any pH within or between the above ranges.

[0083] Suitable equilibration buffers and solutions for washing and elution for cation exchange columns are known in the art and are generally anionic. Such buffers include, but are not limited to, buffers using the following buffering ions: phosphate, acetate, citrate, borate, or sulfate.

[0084] In one embodiment, the cation exchange chromatography medium is first equilibrated, the sample is applied, and washed with a low salt concentration, e.g., 10-100 mM NaCl, e.g., 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 60-100 mM, or any concentration in or between these ranges. Following sample application, the chromatography medium can also be treated with another buffer with a higher salt concentration, e.g., a higher NaCl concentration, or a stronger ionic strength, to elute impurities. One example for use as the second buffer is an acetate buffer with a NaCl concentration of 100 mM-200 mM, or any concentration in or between these aforementioned ranges. After additional impurities have been eluted from the column, the ionic strength of the buffer can be increased using salts such as NaCl, KCl, sulfate, formate, or acetate to elute the AAV particles, and then recovered.

[0085]

[0085] The wash solution for the anion exchange chromatography medium may contain polyethylene glycol (PEG). This is referred to as polyethylene glycol (PEG) conditioned column chromatography. The PEG wash solution can be applied to the anion exchange chromatography medium prior to elution of the AAV vector particles.

[0086]

[0086] Typical equilibration buffers and solutions for washing and elution for anion exchange chromatography are suitable at a pH of about pH 5 to pH 12, more typically about pH 6 to pH 10, and even more typically about pH 7 to pH 9.5, for example, a pH of 7.1, 7.2, 7.3, 7.4 to 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 to 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or any pH in or between the above ranges.

[0087]

[0087] Equilibration buffers and solutions suitable for washing and elution for anion exchange columns are known in the art and are generally cationic or zwitterionic in nature. Such buffers include, but are not limited to, buffers with the following buffer ions: N-methylpiperazine; piperazine; bis-tris; bis-trispropane; triethanolamine; tris; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetate, etc. To elute the sample, the ionic strength of the starting buffer is increased using salts such as NaCl, KCl, sulfates, formates, or acetates.

[0088] In one embodiment, the anion exchange chromatography medium is first equilibrated, the sample is applied, and washed with a low salt concentration, e.g., 10-100 mM NaCl, e.g., 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 60-100 mM, or any concentration in or between these ranges. Following sample application, the chromatography medium can also be treated with a higher salt concentration, e.g., a higher NaCl concentration, or another buffer of stronger ionic strength, to elute impurities. One example for use as the second buffer is a Tris-based buffer with a NaCl concentration of 100 mM-200 mM, or any concentration in or between the ranges mentioned above. After additional impurities have been eluted from the column, the AAV particles can be recovered using a higher concentration of salt.

[0089]

[0089] The wash solution for the anion exchange chromatography medium may contain polyethylene glycol (PEG). This is referred to as polyethylene glycol (PEG) conditioned column chromatography. The PEG wash solution can be applied to the anion exchange chromatography medium prior to elution of the AAV vector particles.

[0090] Typically, the PEG in such cleaning solutions has an average molecular weight in the range of about 1,000 to 50,000, inclusive. Typical amounts of PEG in such cleaning solutions range from about 0.1% to about 20% PEG or the ranges mentioned above or any amount therein, or from about 1% to about 10% PEG or the ranges mentioned above or any amount therein.

[0091]

[0091] Size exclusion chromatography media can be equilibrated using standard buffers according to the manufacturer's specifications. For example, chromatography media can be equilibrated with phosphate buffers, e.g., 1-5 mM, 5-50 mM, or 5-25 mM, and phosphate buffers, e.g., 25-50 mM, 50-100 mM, 100-150 mM, or 125-175 mM sodium chloride.

[0092] After equilibration, the sample is then loaded. The flow-through containing the AAV particles is then collected. An additional volume of buffer (e.g., phosphate buffer) can be added for AAV particle recovery based on the amount of chromatography medium and / or column size.

[0093] In certain embodiments, the size exclusion chromatography media has a separation range (molecular weight) of between about 10,000 and 600,000, inclusive. Specific resins (media) suitable for size exclusion chromatography include, but are not limited to, porous cellulose particles or beads, cross-linked agarose (Sepharose), cross-linked dextran (Sephadex), styrene-divinylbenzene (Dianon HP-20), polyacrylamide (Bio Gel), methacrylics (Toyopearl), and controlled pore glass.

[0094]

[0094] The volume of buffer for elution can be based on the amount of chromatography media and / or column size to achieve AAV particle recovery, with typical volumes being 1-10 times the volume of the column.

[0095]

[0095] The column eluate is collected after elution(s) / passage through each of the chromatographic steps. AAV can be detected in the fractions using standard techniques, such as monitoring UV absorbance at 260 and 280 nm.

[0096] The use of cation or anion exchange chromatography media, the nature of the media used (i.e., strong or weak ion exchanger) and the salt concentration, buffer used, and pH conditions can vary based on the AAV capsid (i.e., serotype or pseudotype of the AAV capsid). Although AAV capsid structures typically share characteristics such as size and shape, capsids can have different amino acid sequences, resulting in subtle differences in molecular topology and surface charge distribution. Thus, capsid sequence variants are expected to be amenable to purification by the methods of the present invention, and the relevant methods can be determined in a systematic manner using chromatography media and buffer screening studies to determine if different conditions are used for AAV capsid variants for AAV particle purification.

[0097]

[0097] The eluate containing AAV particles from any of the anion, size exclusion, or cation exchange chromatography steps described herein can be efficiently concentrated by UF / DF, if desired. The volume reduction can be controlled by one of skill in the art. In certain non-limiting examples, the volume reduction achieved is between about 1-20 fold, inclusive. Thus, a 1-fold reduction reduces the volume by half, e.g., 1000 ml is concentrated to 500 ml. A 10-fold reduction reduces the volume by a factor of 10, e.g., 2000 ml is concentrated to 200 ml. A 20-fold reduction reduces the volume by a factor of 20, e.g., 2000 ml is concentrated to 100 ml.

[0098] A non-limiting example of UF / DF is tangential flow filtration (TFF), e.g., hollow fiber membranes with a nominal pore size corresponding to a molecular weight cutoff of 100 kDa, which allows preparation when large amounts of AAV vector are present in the larger volume of eluate.

[0099]

[0099] The methods of the invention achieve substantial recovery of AAV particles. For example, the methods of the invention achieve recovery of AAV particles of about 40-70% of the total rAAV vector particles from the harvested host cells and host cell culture supernatant. In another example, the AAV particles are present in the final (e.g., third column) eluate at a concentration of about 100 mg / mL. The AAV vector particles are present in the final (e.g., third column) eluate at a concentration of about 10 per mL. 15 More than 10 particles per mL 16 Particles, 10 per mL 17 The concentration of the particles may be present.

[0100] Alternatively, if the AAV vector particle concentration is lower, the purified AAV particles can be concentrated. For example, the purified AAV particles can be concentrated to 10 per mL by ultrafiltration / diafiltration (e.g., TFF). 15 If a higher concentration of vector is desired, the purified AAV particles can be concentrated to 10 per mL by ultrafiltration / diafiltration (e.g., TFF). 16 It can be concentrated into particles or even higher.

[0101]

[0101] The combination of purification of AAV particles from clarified cell lysate by an entire column chromatography process, and concentration (if necessary) of the purified AAV particles by ultrafiltration / diafiltration (e.g., TFF) provides large quantities of highly purified recombinant rAAV vector.

[0102] In other embodiments, rAAV virions having a packaged genome (i.e., authentic rAAV vector particles) are "substantially free" of AAV encapsidated nucleic acid impurities when at least about 60% or more of the virions present are rAAV virions having a packaged genome (i.e., authentic rAAV vector particles). Production of rAAV virions having a packaged genome (i.e., authentic rAAV vector particles) that are substantially free of AAV encapsidated nucleic acid impurities can be about 40% to about 20% or less, about 20% to about 10% or less, about 10% to about 5% or less, about 5% to about 1% or less, or less of the product that contains AAV encapsidated nucleic acid impurities.

[0103] Methods for determining the infectious titer of an AAV vector containing a transgene are known in the art (see, e.g., Zhen et al. (2004) Hum. Gene Ther. (2004) 15:709). Methods for assaying empty capsids and AAV vector particles with packaged genomes are known (see, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128).

[0104] To determine the disassembled / denatured capsid, the purified AAV can be subjected to SDS-polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel, and then run the gel until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein (see, for example, Wobus et al., J. Virol. (2000) 74:9281-9293). The secondary antibody that binds to the primary antibody contains a means for detecting the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsid.

[0105]

[0105] 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0106]

[0106] All patent applications, publications, patents, and other references, GenBank citations, and ATCC citations cited herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

[0107]

[0107] All features disclosed herein may be combined in any combination. Each feature disclosed herein may also be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed features (e.g., nucleic acid sequences, vectors, rAAV vectors, etc.) are examples of a class of equivalent or similar features.

[0108]

[0108] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "an AAV vector" or "AAV particle" includes a plurality of such AAV vectors and AAV particles, and a reference to "a cell" or "host cell" includes a plurality of cells and host cells.

[0109]

[0109] As used herein, the term "about" means a value that is within 10% (plus or minus) of a reference value.

[0110]

[0110] As used herein, all numerical values ​​or ranges of numbers include integers within such ranges and fractions or integers of values ​​within the ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to 80% or greater identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0111]

[0111] References to integers with greater than or less than include any number greater than or less than the referenced number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., all the way down to the number 1; less than 10 includes 9, 8, 7, etc., all the way down to the number 1.

[0112]

[0112] As used herein, all numerical values ​​or ranges are inclusive. Moreover, all numerical values ​​or ranges include fractions of values ​​and integers within such ranges, and fractions of integers within such ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to a numerical range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, reference to a range of 1-50 includes up to and including 50, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth.

[0113]

[0113] Reference to a series of ranges includes ranges combining the boundary values ​​of the different ranges within that series. Thus, by way of example, a series of ranges, e.g., 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, 1,000 to 1,500, 1,500 to 2,000, 2,000 to 2,500, References to 0, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include ranges such as 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000 etc.

[0114]

[0114] The invention has been generally disclosed herein using positive language and numerous embodiments and aspects have been described. The invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, are excluded in whole or in part. For example, in certain embodiments or aspects of the invention, materials and / or method steps are excluded. Thus, even if the invention is not generally expressed herein as not including, aspects of the invention that are not expressly excluded are nevertheless disclosed herein.

[0115]

[0115] Several embodiments of the present invention have been described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Thus, the following examples are intended to illustrate but not to limit the scope of the invention as claimed.

[0116] [Related Applications]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 316,252, filed March 31, 2016. The entire contents of the aforementioned application are incorporated herein by reference, including all text, tables, sequence listings, and figures.

[0117]

[0116] The present invention may be as follows. [1] 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating the rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes: [2] The method according to [1], wherein the concentration in step (b) and / or step (f) and / or step (h) is achieved by ultrafiltration / diafiltration, optionally by tangential flow filtration. [3] The method according to [1], wherein the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2 to 10 times. [4] The method according to claim 1, wherein the concentration in step (f) reduces the volume of the column eluate by about 5 to 20 times. [5] The method of claim 1, wherein the lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is by microfluidization. [6] The method of claim 1, wherein step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids. [7] The method according to [6], wherein the nuclease comprises benzonase. [8] 2. The method of claim 1, wherein the filtering of the clarified lysate or the diluted clarified lysate in step (e) is with a filter having a pore diameter of between about 0.1 and 0.8 microns, inclusive. [9] 2. The method of claim 1, wherein the dilution of the clarified lysate or the diluted clarified lysate in step (e) is with an aqueous acetate buffer.

[10] The method of claim 1, wherein the dilution of the second column eluate in step (g) is with an aqueous acetate buffer.

[11] The method according to

[10] , wherein the aqueous acetate buffer solution has a pH between about 4.0 and 7.0, inclusive.

[12] The method of claim 1, wherein step (i) and / or the rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation.

[13] The method of claim 1, wherein the anion exchange column chromatography in step (f) comprises polyethylene glycol (PEG)-conditioned column chromatography.

[14] The method of claim 13, wherein the anion exchange column chromatography in step (f) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column.

[15] The method according to

[14] , wherein the PEG in the PEG solution has an average molecular weight in the range of about 1,000 to 50,000, inclusive.

[16] The method of claim 1, wherein the anion exchange column in step (f) comprises washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column.

[17] The method of claim 1, wherein the cation exchange column in step (h) comprises washing the column with a detergent solution prior to elution of the rAAV vector particles from the column.

[18] The method according to any one of

[14] to

[17] , wherein the PEG solution and / or the surfactant solution comprises a TrisCl / NaCl aqueous buffer solution or a phosphate / NaCl aqueous buffer solution.

[19] The method according to

[18] , wherein the NaCl buffer contains between about 20 and 250 mM NaCl, inclusive, or between about 50 and 200 mM NaCl, inclusive.

[20] The method of claim 1, wherein the rAAV vector particles are eluted from the anion exchange column in step (f) into an aqueous TrisCl / NaCl buffer. [twenty one] The method according to

[20] , wherein the TrisCl / NaCl buffer contains 50 to 200 mM NaCl. [twenty two] The method of claim 1, wherein the rAAV vector particles are eluted from the cation exchange column in step (h) into an aqueous phosphate / NaCl buffer. [twenty three] The method according to

[22] , wherein the phosphate / NaCl buffer contains between about 100 and 500 mM NaCl, inclusive. [twenty four] The method of claim 1, wherein the anion exchange column in step (f) contains a quaternary ammonium functional group, such as a quaternized polyethyleneimine. [twenty five] The method of claim 1, wherein the size exclusion column in step (g) has a separation range (molecular weight) of between about 10,000 and 600,000, inclusive.

[26] The method of claim 1, wherein the cation exchange column in step (h) contains a functional group, such as sulfonic acid or sulfopropyl.

[27] The method according to any one of [1] to

[26] , wherein the cesium chloride gradient ultracentrifugation step is excluded.

[28] The method according to any one of [1] to

[27] , wherein the rAAV vector particle comprises an introduced gene encoding a nucleic acid selected from the group consisting of siRNA, an antisense molecule, miRNA, a ribozyme, and shRNA.

[29] The rAAV vector particle is capable of expressing any of the following hormones: insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet derived growth factor (PDGF), , insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[30] The method of any one of [1] to

[28] , wherein the rAAV vector particle comprises a transgene encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[31] The rAAV vector particle may comprise any one of the following enzymes: carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-coA dehydrogenase, The method according to any one of [1] to

[28] , comprising an introduced gene encoding a protein useful for correcting an inborn error of metabolism selected from the group consisting of propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[32] The method according to any one of [1] to

[28] , wherein the rAAV vector particle contains an introduced gene encoding factor VIII or factor IX.

[33] The method described in any one of [1] to

[32] , wherein approximately 40 to 70% of all rAAV vector particles are recovered from the harvest produced in step (a) or the concentrated harvest produced in step (b).

[34] The method according to any one of [1] to

[33] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by AAV affinity column purification.

[35] The method according to any one of [1] to

[34] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by an AAV affinity column combined with anion exchange column purification.

[36] The method according to any one of [1] to

[34] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by an AAV affinity column in combination with anion exchange column and cation exchange purification.

[37] The method according to any one of [1] to

[37] , wherein the rAAV vector particle is derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10.

[38] 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles. The method includes:

[39] The method according to

[38] , wherein the concentration in step (b) and / or step (f) and / or step (h) is achieved by ultrafiltration / diafiltration, optionally by tangential flow filtration.

[40] The method according to

[38] , wherein the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2 to 10 times.

[41] The method of claim 38, wherein the concentration in step (f) reduces the volume of the column eluate by about 5 to 20 times.

[42] The method of claim 38, wherein the lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is by microfluidization.

[43] The method of claim 38, wherein step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids.

[44] The method according to

[43] , wherein the nuclease comprises benzonase.

[45] 38. The method of claim 38, wherein the filtering of the clarified lysate or the diluted clarified lysate in step (e) is through a filter having a pore diameter of between about 0.1 and 0.8 microns, inclusive.

[46] 38. The method of claim 38, wherein the dilution of the clarified lysate or the diluted clarified lysate in step (e) is with an aqueous acetate buffer.

[47] 38. The method of claim 38, wherein the dilution of the second column eluate in step (g) is with an aqueous acetate buffer.

[48] 47. The method of claim 47, wherein the aqueous acetate buffer solution has a pH between about 4.0 and 7.0, inclusive.

[49] The method of claim 38, wherein the rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation.

[50] The method of claim 38, wherein the anion exchange column chromatography in step (h) comprises polyethylene glycol (PEG)-conditioned column chromatography.

[51] 38. The method of claim 38, wherein the anion exchange column chromatography in step (h) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column.

[52] The method according to

[51] , wherein the PEG in the PEG solution has an average molecular weight in the range of about 1,000 to 50,000, inclusive.

[53] 38. The method of claim 38, wherein the anion exchange column in step (h) comprises washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column.

[54] 38. The method of claim 38, wherein the cation exchange column in step (f) comprises washing the column with a detergent solution prior to elution of the rAAV vector particles from the column.

[55] The method according to any one of

[50] to

[54] , wherein the PEG solution and / or the surfactant solution comprises a TrisCl / NaCl aqueous buffer solution or a phosphate / NaCl aqueous buffer solution.

[56] 55. The method according to claim 55, wherein the NaCl buffer contains between about 20 and 250 mM NaCl, inclusive, or between about 50 and 200 mM NaCl, inclusive.

[57] The method of claim 38, wherein the rAAV vector particles are eluted from the anion exchange column in step (h) into an aqueous TrisCl / NaCl buffer solution.

[58] The method according to

[57] , wherein the TrisCl / NaCl buffer contains 50 to 200 mM NaCl.

[59] The method of claim 38, wherein the rAAV vector particles are eluted from the cation exchange column in step (f) into an aqueous phosphate / NaCl buffer.

[60] The method of claim 59, wherein the phosphate / NaCl buffer contains between about 100 and 500 mM NaCl, inclusive.

[61] The method of claim 38, wherein the anion exchange column in step (h) contains quaternized polyethyleneimine functional groups.

[62] The method of claim 38, wherein the size exclusion column in step (g) has a separation range (molecular weight) of between about 10,000 and 600,000, inclusive.

[63] The method of claim 38, wherein the cation exchange column in step (f) contains sulfopropyl functional groups.

[64] The method according to any one of

[38] to

[63] , wherein the cesium chloride gradient ultracentrifugation step is excluded.

[65] The method according to any one of

[38] to

[64] , wherein the rAAV vector particle comprises an introduced gene encoding a nucleic acid selected from the group consisting of siRNA, an antisense molecule, miRNA, a ribozyme and an shRNA.

[66] The rAAV vector particle is capable of expressing any of the following hormones: insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet derived growth factor (PDGF), , insulin-like growth factors I and II (IGF-I and IGF-II), TGFβ, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[67] The method of any one of

[38] to

[64] , wherein the rAAV vector particle comprises a transgene encoding a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

[68] The rAAV vector particle is further characterized in that it is capable of expressing carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystatin beta-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-coA dehydrogenase, prote ... The method according to any one of

[38] to

[64] , further comprising an introduced gene encoding a protein useful for correcting an inborn error of metabolism selected from the group consisting of: ropionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin cDNA sequence.

[69] The method described in any one of

[38] to

[64] , wherein the rAAV vector particle contains an introduced gene encoding factor VIII or factor IX.

[70] The method described in any one of

[38] to

[64] , wherein approximately 40 to 70% of the total rAAV vector particles are recovered from the harvest produced in step (a) or the concentrated harvest produced in step (b).

[71] The method according to any one of

[38] to

[64] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by AAV affinity column purification.

[72] The method according to any one of

[38] to

[64] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by an AAV affinity column combined with anion exchange column purification.

[73] The method according to any one of

[38] to

[64] , which produces rAAV vector particles having a higher purity than rAAV vector particles produced or purified by an AAV affinity column in combination with anion exchange column and cation exchange purification.

[74] The method described in any one of

[38] to

[64] , wherein the rAAV vector particle is derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10.

Claims

1. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method, wherein said concentrating in step (b) reduces the volume of said harvested cells and cell culture supernatant by 2-10 fold.

2. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the concentrating in step (f) reduces the volume of the column eluate by a factor of 5 to 20.

3. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: A method wherein step (d) comprises treating with a nuclease to thereby reduce contaminating nucleic acids, said nuclease comprising benzonase.

4. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein said dilution of said second column eluate in step (g) is with an aqueous acetate buffer, said aqueous acetate buffer having a pH between 4.0 and 7.0, inclusive.

5. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the anion exchange column chromatography in step (f) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column.

6. 6. The method of claim 5, wherein the PEG in the PEG solution has an average molecular weight ranging from 1,000 to 50,000 inclusive.

7. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The anion exchange column chromatography of step (f) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column, and optionally, the PEG in the PEG solution has an average molecular weight in the range of 1,000 to 50,000, inclusive; or the anion exchange column of step (f) comprises washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column; the cation exchange column of step (h) comprising washing the column with a detergent solution prior to elution of the rAAV vector particles from the column; The method, wherein the PEG solution and / or the surfactant solution comprises an aqueous TrisCl / NaCl buffer or an aqueous phosphate / NaCl buffer.

8. 8. The method of claim 7, wherein the aqueous NaCl buffer contains between 20 and 250 mM NaCl, inclusive.

9. 9. The method of claim 8, wherein the aqueous NaCl buffer contains between 50 and 200 mM NaCl, inclusive.

10. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the rAAV vector particles are eluted from the anion exchange column in step (f) into an aqueous TrisCl / NaCl buffer solution, the aqueous TrisCl / NaCl buffer solution comprising 50-200 mM NaCl.

11. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: wherein the rAAV vector particles are eluted from the cation exchange column in step (h) into an aqueous phosphate / NaCl buffer solution, the aqueous phosphate / NaCl buffer solution comprising between 100 and 500 mM NaCl, inclusive.

12. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the anion exchange column of step (f) comprises a quaternary ammonium functional group.

13. The method of claim 12 , wherein the quaternary ammonium functional group comprises a quaternized polyethyleneimine.

14. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein said size exclusion column in step (g) has a separation range (molecular weight) of between 10,000 and 600,000, inclusive.

15. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the cation exchange column of step (h) comprises sulfonic acid groups or sulfopropyl groups.

16. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) lysing the concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method, wherein said concentrating in step (b) reduces the volume of said harvested cells and cell culture supernatant by 2-10 fold.

17. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the concentrating in step (f) reduces the volume of the column eluate by a factor of 5 to 20.

18. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: A method wherein step (d) comprises treating with a nuclease to thereby reduce contaminating nucleic acids, said nuclease comprising benzonase.

19. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein said dilution of said second column eluate in step (g) is with an aqueous acetate buffer, said aqueous acetate buffer having a pH between 4.0 and 7.0, inclusive.

20. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: the anion exchange column chromatography of step (h) comprising washing the column with a PEG solution prior to elution of the rAAV vector particles from the column; The method wherein the PEG in said PEG solution has an average molecular weight in the range of 1,000 to 50,000 inclusive.

21. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The anion exchange column chromatography of step (h) comprises washing the column with a PEG solution prior to elution of the rAAV vector particles from the column, and optionally, the PEG in the PEG solution has an average molecular weight in the range of 1,000 to 50,000, inclusive; the anion exchange column in step (h) comprising washing the column with an aqueous detergent solution prior to elution of the rAAV vector particles from the column; or the cation exchange column in step (f) comprising washing the column with a detergent solution prior to elution of the rAAV vector particles from the column; The method, wherein the PEG solution and / or the surfactant solution comprises an aqueous TrisCl / NaCl buffer or an aqueous phosphate / NaCl buffer.

22. 22. The method of claim 21, wherein the aqueous NaCl buffer solution contains between 20 and 250 mM NaCl, inclusive.

23. 23. The method of claim 22, wherein the aqueous NaCl buffer solution contains between 50 and 200 mM NaCl, inclusive.

24. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: the rAAV vector particles are eluted from the anion exchange column in step (h) into an aqueous TrisCl / NaCl buffer solution, the aqueous TrisCl / NaCl buffer solution comprising 50-200 mM NaCl.

25. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The rAAV vector particles are eluted from the cation exchange column in step (f) into an aqueous phosphate / NaCl buffer; The method of claim 1, wherein said aqueous phosphate / NaCl buffer solution contains between 100 and 500 mM NaCl, inclusive.

26. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the anion exchange column of step (h) comprises quaternized polyethyleneimine functional groups.

27. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein said size exclusion column in step (g) has a separation range (molecular weight) of between 10,000 and 600,000, inclusive.

28. 1. A method for purifying recombinant adeno-associated virus (rAAV) vector particles, comprising: (a) harvesting cells and cell culture supernatant containing rAAV vector particles to produce a harvest; (b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest; (c) dissolving the harvested material produced in step (a) or the concentrated harvested material produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; (e) filtering the nucleic acid-depleted lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; (f) subjecting the clarified lysate or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from proteins or other production impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; (g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector particles, thereby separating rAAV vector particles from protein impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; (h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange column chromatography to produce a third column eluate comprised of rAAV vector particles, and optionally concentrating the third column eluate to produce a concentrated third column eluate; (i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles; A method comprising: The method of claim 1, wherein the cation exchange column of step (f) comprises sulfopropyl functional groups.

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