RU2772876C2 - Column-based methods for cleaning vector based on aav - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: in the present document, methods for cleaning vector particles based on a recombinant adeno associated virus (hereinafter – rAAV) are described. Methods include collection of cells, their lysis, lysate treatment with nuclease, at least 2 stages of column chromatography, and filtration of the resulting eluate. Column chromatography stages include cation-exchange chromatography, anion-exchange chromatography, exclusive chromatography and/or affine chromatography of AAV separately or in a combination and in any order. Proposed methods can be scaled to larger volumes and applied to a wide range of serotypes/capsid AAV options.

EFFECT: methods provide for the reduction in the formation of impurities during rAAV obtaining.

64 cl, 5 dwg, 3 tbl, 8 ex

Description

RELATED APPLICATIONS

This patent application claims priority of US Patent Application No. 62/527633, filed June 30, 2017; US patent application number 62/531744, filed July 12, 2017; and U.S. Patent Application No. 62/567,905, filed Oct. 4, 2017. The entire contents of the above applications are incorporated herein by reference, including the entire text, all tables, drawings, and sequences in their entirety.

INTRODUCTION

Gene delivery is a promising way to treat acquired and inherited diseases. A number of viral gene transfer systems have been described, including adeno-associated virus (AAV) systems.

AAV is a helper virus dependent DNA parvovirus belonging to the genus Dependovirus. A productive AAV infection requires functional elements of a helper virus, such as adenovirus, herpes virus, or vaccinia. In the absence of functional elements of the helper virus, AAV is in a latent state, integrating its genome into the chromosome of the host cell. Subsequent infection with the helper virus promotes replication of the integrated viral genome to form infectious AAV particles.

AAV has a wide host spectrum and is able to replicate in cells of any species in the presence of a suitable helper virus. For example, human AAV will replicate in dog cells co-infected with canine adenovirus. AAV is not associated with any human or animal disease and, when integrated, does not appear to adversely affect the biological properties of the host cell.

AAV-based vectors can be engineered to contain the desired heterologous nucleic acid sequence (e.g., a selected gene encoding a therapeutic protein, an inhibitory nucleic acid such as an antisense molecule, ribozyme, miRNA, etc.) by removing, in whole or in part, the interior of the AAV genome and insert the desired nucleic acid sequence between the ITRs. The ITRs remain functional in such vectors, whereby rAAV containing the desired heterologous nucleic acid sequence can be replicated and packaged. The heterologous nucleic acid sequence is also typically associated with a promoter sequence capable of directing expression of the nucleic acid in target cells of the patient. Termination signals such as polyadenylation sites can also be included in the vector.

The construction of infectious recombinant AAV vectors (rAAV) has been described in a number of publications. See, for example, US Pat. Nos. 5,173,414 and 5,139,941; international published patent applications number 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, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.

In the early phases of several multi-group clinical trials, recombinant adeno-associated virus (AAV) vectors have shown excellent therapeutic prospects. Development of this new class of biologicals for approval will include improvements in vector characterization and quality control, including a better understanding of how vector design and manufacturing process parameters affect the composition of impurities in clinical grade vectors.

An important goal in the design of rAAV production and purification systems is to implement strategies to minimize/control the formation of contaminants during rAAV production, such as proteins, nucleic acids, and vector-associated contaminants, including wild-type/pseudo-wild-type AAV (wtAAV) and residual contaminants. DNA encapsulated in AAV. The removal of contaminants in AAV-based vectors is difficult due to the way rAAV-based vectors are prepared. In one method, rAAV-based vectors are generated by transient transfection using three plasmids. To obtain vectors based on rAAV, a significant amount of plasmid DNA is introduced into cells. In addition, when rAAV-based vectors are released from producing cells, cellular proteins and nucleic acids are also released with them. Given that only about 1% of the biomass is in the rAAV vector, it is very difficult to purify rAAV-based vectors to a level of purity that can be used as a clinical drug for human gene therapy. (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).

The development of manufacturing processes for the purification of recombinant AAV as a drug for the treatment of human diseases should be carried out in the direction of the following goals: 1) stable indicators of purity, efficiency and safety of the vector; 2) stability of the production process; and 3) reasonable production cost. Current "industry standard" scalable AAV-based vector purification processes do not provide adequate removal of contaminants, which is essential to achieve the first goal above (consistent vector purity, efficacy, and safety). In addition, the inability to adequately remove contaminants using current standard scalable purification processes is due to: 1) the development of purification processes for viral preparations such as recombinant AAV for applications other than vaccines (in the case of vaccines, an immune response is usually desirable and does not require prevention) is a relatively new phenomenon; 2) many of the groups involved in the development of scalable AAV-based vector purification processes were unaware of the high levels of contaminants associated with the vector and/or assumed that such contaminants would not contribute to the clinically significant immunogenicity of the vector; and 3) it is technically difficult to develop scalable purification processes suitable for industrial production of rAAV vectors.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention provides methods for purifying and obtaining vector particles based on recombinant adeno-associated virus (rAAV). The methods of the present invention include at least 2 column chromatography steps.

In one embodiment, the method includes the steps of: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) to reduce nucleic acid contamination in the lysate and obtain a nucleic acid depleted lysate; (e) optionally filtering the nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate and optionally diluting the purified lysate to obtain a diluted purified lysate; (f) applying the nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e), or the diluted purified lysate obtained in step (e) to a cation exchange chromatography column to obtain an eluate from the column, containing rAAV vector particles, that is, to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally dilute the eluate from the column to obtain a dilute eluate from the column; (g) applying the column eluate or the diluted column eluate obtained in step (f) to an anion exchange chromatography column to obtain a second column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the second eluate from the column to obtain a concentrated second eluate from the column; (h) applying the second column eluate or the concentrated second column eluate obtained in step (g) to a size exclusion chromatography (SEC) column to obtain a third column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the third eluate from the column to obtain a concentrated third eluate from the column; and (i) filtering the third column eluate or concentrated third column eluate from step (h) to obtain purified rAAV vector particles.

In another embodiment, the method includes the steps of: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) to reduce nucleic acid contamination in the lysate and obtain a nucleic acid depleted lysate; (e) optionally filtering the nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate and optionally diluting the purified lysate to obtain a diluted purified lysate; (f) applying the nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e), or the diluted purified lysate obtained in step (e) to a cation exchange chromatography column to obtain an eluate from the column, containing rAAV vector particles, that is, for separating rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the eluate from the column to obtain a concentrated eluate from the column; (g) applying the column eluate or the concentrated column eluate obtained in step (f) to a size exclusion chromatography (SEC) column to obtain a second column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the second eluate from the column to obtain a concentrated second eluate from the column; (h) applying the second column eluate or the concentrated second column eluate obtained in step (g) to an anion exchange chromatography column to obtain a third column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein contaminants, or other impurities associated with the manufacturing process, and optionally diluting the third eluate from the column to obtain a diluted third eluate from the column; and (i) filtering the third column eluate or concentrated third column eluate from step (h) to obtain purified rAAV vector particles.

In yet another embodiment, the method includes the steps of: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) to reduce nucleic acid contamination in the lysate and obtain a nucleic acid depleted lysate; (e) optionally filtering the nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate and optionally diluting the purified lysate to obtain a diluted purified lysate; (f) applying the nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e), or the diluted purified lysate obtained in step (e) to a cation exchange chromatography column to obtain an eluate from the column, containing rAAV vector particles, that is, to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally dilute the eluate from the column to obtain a dilute eluate from the column; (g) applying the column eluate or the diluted column eluate obtained in step (f) to an anion exchange chromatography column to obtain a second column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from process-related impurities production, and optionally concentrating the second eluate from the column to obtain a concentrated second eluate from the column; (h) filtering the second column eluate or concentrated second column eluate from step (g) to obtain purified rAAV vector particles.

In a further embodiment, the method includes the steps of: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) to reduce nucleic acid contamination in the lysate and obtain a nucleic acid depleted lysate; (e) optionally filtering the nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate and optionally diluting the purified lysate to obtain a diluted purified lysate; (f) applying the nucleic acid-depleted lysate obtained in step (d) or the purified lysate or the diluted purified lysate obtained in step (e) to an AAV affinity chromatography column to obtain a column eluate containing rAAV vector particles, then is for separating rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally diluting the eluate from the column to obtain a dilute eluate from the column; (g) applying the column eluate or the diluted column eluate obtained in step (f) to an anion exchange chromatography column to obtain a second column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the second eluate from the column to obtain a concentrated second eluate from the column; (h) optionally applying the second column eluate or the concentrated second column eluate obtained in step (g) to a Size Exclusion Chromatography (SEC) column to obtain a third column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the third eluate from the column to obtain a concentrated third eluate from the column; and (i) filtering the second column eluate or the diluted second column eluate from step (g) or filtering the third column eluate or the concentrated third column eluate from step (h) to obtain purified rAAV vector particles.

In yet another embodiment, the method includes the steps of: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) to reduce nucleic acid contamination in the lysate and obtain a nucleic acid depleted lysate; (e) optionally filtering the nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate and optionally diluting the purified lysate to obtain a diluted purified lysate; (f) applying the nucleic acid-depleted lysate obtained in step (d) or the purified lysate or the diluted purified lysate obtained in step (e) to an AAV affinity chromatography column to obtain a column eluate containing rAAV vector particles, then is for separating rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally concentrating the eluate from the column to obtain a concentrated eluate from the column; (g) applying the column eluate or the concentrated column eluate obtained in step (f) to a size exclusion chromatography (SEC) column to obtain a second column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and optionally diluting the second eluate from the column to obtain a diluted second eluate from the column; (h) optionally applying a second column eluate or a concentrated second column eluate obtained in step (g) to an anion exchange chromatography column to obtain a third column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein contaminants, or other impurities associated with the manufacturing process, and optionally diluting the third eluate from the column to obtain a diluted third eluate from the column; and (i) filtering the second column eluate or the diluted second column eluate from step (g) or filtering the third column eluate or the concentrated third column eluate from step (h) to obtain purified rAAV vector particles.

In specific aspects of the processes of the present invention, the concentration in step (b) and/or step (f) and/or step (g) and/or step (h) is carried out by ultrafiltration/diafiltration, for example by tangential flow filtration ( TFF).

In specific aspects of the methods of the present invention, the concentration in step (b) results in a decrease in the volume of harvested cells and cell culture supernatant by about 2-20 times.

In specific aspects of the methods of the present invention, the concentration in step (f) and/or step (g) and/or step (h) results in a reduction in column eluate volume of about 5-20 times.

In specific aspects of the methods of the present invention, the lysis of the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b) is carried out by a physical or chemical means. Non-limiting examples of physical methods include microfluidization and homogenization. Non-limiting examples of chemical methods include detergents. Detergents include non-ionic and ionic detergents. Non-limiting examples of non-ionic detergents include Triton X-100. Non-limiting examples of detergent concentrations are concentrations ranging from about 0.1% to about 1.0%, inclusive.

In specific aspects of the methods of the present invention, step (d) comprises a nuclease treatment resulting in a reduction in nucleic acid contamination. Non-limiting examples of nucleases include benzonase.

In specific aspects of the methods of the present invention, the filtration of the purified lysate or the diluted purified lysate obtained in step (e) is carried out using a filter. Non-limiting examples of filters are filters having a pore diameter in the range of about 0.1 to 10 microns, inclusive.

In specific aspects of the methods of the present invention, dilution of the purified lysate obtained in step (e) is carried out with an aqueous phosphate, acetate or Tris buffer solution. Non-limiting examples of the pH of a solution is a pH in the range of about 4.0 to 7.4, inclusive. Non-limiting examples of the pH of a Tris solution is pH greater than 7.5, eg, from about 8.0 to 9.0, inclusive.

In specific aspects of the methods of the present invention, the dilution of the eluate from the column obtained in step (f), or the second eluate from the column obtained in step (g), is carried out with an aqueous phosphate, acetate or Tris buffer solution. Non-limiting examples of the pH of a solution is a pH in the range of about 4.0 to 7.4, inclusive. Non-limiting examples of the pH of a Tris solution is pH greater than 7.5, eg, from about 8.0 to 9.0, inclusive.

In specific aspects of the methods of the present invention, the rAAV vector particles obtained in step (i) are formulated with a surfactant to form an AAV-based vector composition.

In specific aspects of the methods of the present invention, the anion exchange column chromatography in step (f), (g) and/or (h) comprises polyethylene glycol (PEG) column chromatography.

In specific aspects of the methods of the present invention, the anion exchange chromatography column in step (g) and/or (h) is washed with a PEG solution prior to eluting the rAAV vector particles from the column.

In specific aspects of the methods of the present invention, the PEG has an average molecular weight in the range of from about 1000 to 80000 g/mol, inclusive.

In specific aspects of the methods of the present invention, the PEG has a concentration of from about 4% to about 10%, inclusive.

In specific aspects of the methods of the present invention, the anion exchange chromatography column in step (g) and/or (h) is washed with an aqueous surfactant solution prior to eluting the rAAV vector particles from the column.

In specific aspects of the methods of the present invention, the cation exchange chromatography column in step (f) is washed with a surfactant solution prior to eluting the rAAV vector particles from the column.

In specific aspects of the methods of the present invention, the PEG solution and/or the surfactant solution comprises an aqueous Tris-Cl/NaCl buffer, an aqueous phosphate/NaCl buffer, or an aqueous acetate/NaCl buffer.

In specific aspects of the methods of the present invention, the NaCl in the buffer or solution has a concentration of about 20-300 mM NaCl, inclusive, or about 50-250 mm NaCl, inclusive.

In specific aspects of the methods of the present invention, the surfactant includes a cationic or anionic surfactant.

In specific aspects of the methods of the present invention, the surfactant includes a surfactant with twelve carbons in the chain.

In specific aspects of the methods of the present invention, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarcosyl.

In specific aspects of the methods of the present invention, rAAV vector particles are eluted from the anion exchange column in steps (f), (g) and/or (h) with aqueous Tris-Cl/NaCl buffer.

In specific aspects of the methods of the present invention, the Tris-Cl/NaCl buffer contains 100-400 mM NaCl, inclusive, and optionally has a pH in the range of from about 7.5 to about 9.0, inclusive.

In specific aspects of the methods of the present invention, the anion exchange column in step (f), (g) and/or (h) is washed with an aqueous Tris-Cl/NaCl buffer.

In specific aspects of the methods of the present invention, NaCl in aqueous Tris-Cl/NaCl buffer has a concentration in the range of about 75-125 mm, inclusive.

In specific aspects of the methods of the present invention, the aqueous Tris-Cl/NaCl buffer has a pH of from about 7.5 to about 9.0, inclusive.

In specific aspects of the methods of the present invention, the anion exchange column in step (f), (g) and/or (h) is washed one or more times to reduce the amount of empty AAV capsids in the second or third eluate from the column.

In specific aspects of the methods of the present invention, washing the anion exchange column results in the removal of empty AAV capsids from the column prior to removal of rAAV and/or instead of rAAV, resulting in a reduction in the number of empty AAV capsids in the second or third eluate from the column.

In specific aspects of the methods of the present invention, washing the anion exchange column results in the removal of at least about 50% of the total empty AAV capsids from the column prior to removal of rAAV and/or instead of rAAV, resulting in a reduction in the number of empty AAV capsids in the second or third eluate from the column by about 50%.

In specific aspects of the methods of the present invention, NaCl in aqueous Tris-Cl/NaCl buffer has a concentration in the range of about 110-120 mM, inclusive.

In specific aspects of the methods of the present invention, the ratio and/or amount of eluted rAAV vector particles and empty AAV capsids is controlled by the wash buffer.

In specific aspects of the methods of the present invention, the vector particles are eluted from the cation exchange column in step (f) in aqueous phosphate/NaCl buffer or aqueous acetate/NaCl buffer. A non-limiting example of NaCl concentrations in buffer is a concentration range of about 125-500 mM NaCl, inclusive. A non-limiting example of a pH buffer is a pH range of about 5.5 to about 7.5, inclusive.

In specific aspects of the methods of the present invention, the anion exchange column in step (f), (g) and/or (h) contains a quaternary ammonium functional group, such as quaternized polyethyleneimine.

In specific aspects of the methods of the present invention, the separation/fractionation range (molecular weight) of the size exclusion (SEC) chromatography column in step (g) and/or (h) is from about 10,000 to about 600,000, inclusive.

In specific aspects of the methods of the present invention, the cation exchange column in step (f) contains a sulfonic acid functional group, such as a sulfopropyl group.

In specific aspects of the methods of the present invention, the AAV affinity chromatography column contains a protein or ligand that binds to the AAV capsid protein. Non-limiting examples of a protein include an antibody that binds to the AAV capsid protein. More specific non-limiting examples include the Llama single chain antibody (Camelid), which binds to the AAV capsid protein.

In specific aspects of the methods of the present invention, the method eliminates the step of ultracentrifugation in a cesium chloride gradient.

In specific aspects of the methods of the present invention, the rAAV vector particles comprise a transgene that encodes a nucleic acid selected from the group consisting of miRNA, antisense, microRNA, ribozyme, and small shRNA hairpin RNAs.

In specific aspects of the methods of the present invention, the rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRP), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, 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 α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF -II), TGFβ, activins, inhibins, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neuroturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), aphrins, noggin, sonic hedgehog and tyrosine hydroxylase.

In specific aspects of the methods of the present invention, the rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocyte chemoattractant 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.

In specific aspects of the methods of the present invention, the rAAV vector particles contain a transgene encoding a protein necessary for the correction of innate metabolic disorders, selected from the group consisting of 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, cystathion beta synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl β-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence.

In specific aspects of the methods of the present invention, the rAAV vector particles contain a transgene that encodes factor VIII or factor IX.

In specific aspects of the methods of the present invention, approximately 50-90% of the total rAAV vector particles are obtained by this method from the cell harvest obtained in step (a) or the concentrated cell harvest obtained in step (b).

In specific aspects of the methods of the present invention, higher purity rAAV vector particles are obtained by this method than rAAV vector particles produced or purified by a single purification on an AAV affinity column.

In specific aspects of the methods of the present invention, steps (c) and (d) are performed substantially simultaneously.

In specific aspects of the methods of the present invention, the NaCl concentration is adjusted to be in the range of about 100-400 mM NaCl, inclusive, or in the range of about 140-300 mM NaCl, inclusive, after step (c) but before step (f) .

In specific aspects of the methods of the present invention, the rAAV vector particles are based on an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10 and Rh74.

In specific aspects of the methods of the present invention, the rAAV vector particles contain a capsid sequence that is 70% or greater identical to the capsid sequence of SEQ ID NO:1 or SEQ ID NO:2 of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV10, Rh10, Rh74.

In specific aspects of the methods of the present invention, the rAAV vector particles contain an ITR sequence that is 70% or greater identical to the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, or Rh74.

In specific aspects of the methods of the present invention, the cells are suspension or adherent (adhesive) cells.

In specific aspects of the methods of the present invention, the cells are mammalian cells. Non-limiting examples include HEK cells such as HEK-293 cells.

In specific aspects of the methods of the present invention, the method is carried out in accordance with any of the columns, conditions, concentrations, molarity, volume, capacity, flow rate, pressure, material, temperature, pH, or step described in any of Examples 1-3.

In specific aspects of the methods of the present invention, cell lysis and/or preparation prior to column purification, as described herein, is carried out in accordance with any of the conditions, concentrations, molarity, volume, capacity, flow rate, pressure, material, temperature, pH or steps given in example 4.

DESCRIPTION OF THE DRAWINGS

Fig. 1 demonstrates CEX (Poros 50HS) → AEX (Poros 50HQ) → UF → SEC (Superdex 200 prep grade) column chromatography of an initial rAAV harvest volume, 500-600 ml, which can be scaled up to large volumes to significantly increase rAAV production ( e.g. 1.2 liters or more).

Fig. 2 shows CEX (Poros 50HS) → AEX (Poros 50HQ) column chromatography running an initial rAAV harvest volume, 500-600 ml, which can be scaled up to larger volumes to significantly increase rAAV production (e.g., 1.2 liters or more).

Fig. 3 shows CEX (Poros 50HS) → UF > SEC (Superdex 200 prep grade) > AEX (Poros 50HQ) column chromatography of an initial rAAV harvest volume, 500-600 ml, which can be scaled up to large volumes to significantly increase rAAV production ( e.g. 1.2 liters or more).

Fig. 4 demonstrates running affinity (AVB Sepharose HP) → AEX (Poros 50HQ) column chromatography of an initial rAAV harvest volume, 500-600 ml, which can be scaled up to large volumes to significantly increase rAAV production (e.g., 1.2 liters or more) . After elution from the low pH affinity column (about 7.5 ml of eluate), 4.5 ml of high pH neutralization solution was added followed by the addition of about 120 ml of 5 mM Tris-Cl pH 8.5/40 mM NaCl solution to reach the full volume loading, about 132 ml for an AEX column (Poros 50HQ).

Fig. 5 shows a process flow diagram.

DETAILED DESCRIPTION

The present invention provides methods for the purification and production of recombinant adeno-associated virus (rAAV) vectors that can be scaled up to large volumes. For example, a suspension culture of 5, 10, 10-20, 20-50, 50-100, 100-200 or more liters. The present invention provides methods for the purification and production of recombinant adeno-associated virus (rAAV) vectors that are also applicable to a wide range of AAV serotypes/capsid variants. The methods of the present invention used to purify or produce an rAAV vector involve the removal of contaminants associated with the manufacturing process. The methods of the present invention include a unique combination of chromatographic and process steps that provide scalability to purify many different serotypes/pseudotypes of rAAV vectors.

Impurities include impurities associated with the manufacturing process of the AAV vector, which include proteins, nucleic acids (eg DNA), cellular components such as intracellular and membrane components, which are impurities other than AAV vectors. The term "contaminants associated with the production process" refers to any components that are released during the purification and production of AAV, and are not actually particles of rAAV.

The term "proper rAAV vectors" refers to rAAV vector particles containing a heterologous nucleic acid (eg, transgene) capable of infecting target cells. The phrase excludes empty AAV capsids, AAV vectors without a full-length insert in the packaged genome, or AAV vectors containing contaminating host cell nucleic acids. In some embodiments, the term "proper rAAV vectors" refers to rAAV vector particles that also do not contain contaminating plasmid sequences in the packaged vector genome.

The terms "empty capsids" and "empty particles" refer to AAV particles or virions that contain the AAV capsid envelope, but lack, in whole or in part, a genome containing a heterologous nucleic acid sequence flanked on one or both sides of the AAV ITR. Such empty capsids do not function to transfer the heterologous nucleic acid sequence into the host cell or body cells.

The term "vector" refers to a small carrier of a nucleic acid molecule, a plasmid, a virus (eg, a rAAV vector), or other carrier that can be manipulated by inserting or incorporating a nucleic acid. Vectors can be used for genetic manipulation (ie "cloning vectors"), for introducing/transferring polynucleotides into cells, and for transcribing or translating the inserted polynucleotide into cells. An "expression vector" is a vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. A vector nucleic acid sequence typically 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. promoter, enhancer), intron, inverted terminal repeats (ITR), optional selectable marker, polyadenylation signal.

The rAAV vector is based on adeno-associated virus. AAV vectors are useful as gene therapy vectors because they can introduce nucleic acids/genetic material into cells so that the nucleic acids/genetic material can be retained in the cells. Because AAVs are not associated with pathogenic disease in humans, rAAV vectors can deliver heterologous nucleic acid sequences (eg, therapeutic proteins and agents) to patients without causing significant AAV pathogenesis or disease.

The term "recombinant" referring to a vector, such as rAAV vectors, as well as sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been prepared (ie, constructed) in a manner that is not normally found in nature. A specific example of a recombinant AAV vector would be a nucleic acid that is not normally present in the wild-type AAV genome and is inserted into the viral genome. An example would be a nucleic acid (eg, gene) encoding a therapeutic protein or polynucleotide sequence that is cloned into a vector with the 5', 3', and/or intron regions with which the gene is normally associated in the AAV genome. Although the term "recombinant" is not always used herein in relation to AAV vectors as well as sequences such as polynucleotides, recombinant forms are obviously also included, including AAV vectors, polynucleotides, etc., despite any such omission.

A "rAAV-based vector" or "rAAV vector" is generated from a wild-type virus genome, such as AAV, using molecular techniques, by removing the wild-type genome from the AAV genome and replacing it with a non-native (heterologous) nucleic acid, such as AAV nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, in the case of AAV, one or both of the inverted terminal repeat (ITR) sequences of the AAV genome are conserved in the rAAV vector. rAAV differs from the AAV genome because all or part of the AAV genome is replaced by a non-native sequence to an AAV genomic nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Therefore, the inclusion of a non-native sequence defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector".

The recombinant AAV vector sequence can be packaged into a "particle" referred to herein for subsequent infection (transduction) of a cell ex vivo , in vitro or in vivo . When a recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as "rAAV" or "rAAV particle" or "rAAV virion". Such rAAVs, rAAV particles, and rAAV virions include proteins that encapsulate or contain the vector genome. Specific examples in the case of AAV include capsid proteins.

The term "genome" of a vector refers to that portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated to form the rAAV particle. In cases where recombinant plasmids are used to construct or produce recombinant AAV vectors, the genome of the AAV vector does not include a "plasmid" portion that does not match the vector genomic sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid genome is referred to as the "plasmid backbone", which is essential for cloning and amplification of the plasmid, a process necessary for propagation and production of the recombinant virus, but which is not itself packaged or encapsulated into rAAV particles. Thus, the term "genome" of a vector refers to a nucleic acid that is packaged or encapsulated in rAAV.

The term "accessory functional elements for AAV" refers to coding sequences (proteins) derived from AAV that can be expressed to produce AAV gene products and AAV vectors, which in turn function in trans and to allow for productive replication and packaging. AAV. Thus, auxiliary functional elements of AAV include AAV open reading frames (ORFs), including rep, cap, and others, such as AAP for certain AAV serotypes. Rep expression products have been shown to have many functions, including but not limited to: recognition, binding, and nicking of the AAV DNA origin of replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. Cap expression products (capsids) provide the necessary functions for packaging. AAV accessory functional elements are used in trans to complement the AAV functional elements that are absent from the AAV vector genomes.

The term "AAV helper construct" generally refers to a nucleic acid sequence that includes nucleotide sequences conferring AAV functions removed from an AAV vector to be used, for example, to generate an AVV transducing vector for gene therapy delivery to a subject of a nucleic acid sequence of interest. AAV helper constructs are commonly used to provide transient expression of the AAV rep and/or cap genes to fill in missing AAV functions required for AAV vector replication. In general, helper constructs do not have AAV ITRs and cannot self-replicate and self-package. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the plasmids pAAV/Ad and pIM29+45, which encode both the Rep expression product and the Cap expression product (See, for example, Samulski et al. (1989) J. Virol. 63:3822 -3828 and McCarty et al (1991) J. Virol 65:2936-2945). A number of other vectors have been described that encode Rep and/or Cap expression products (See, for example, US Pat. Nos. 5,139,941 and 6,376,237).

The term "helper functions" refers to viral (non-AAV related) and/or cellular functions on which AAV replication depends. The term includes proteins and RNAs that are essential for AAV replication, including fragments involved in AAV gene transcription activation, AAV mRNA stage-specific splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid packaging. Viral accessory functional elements can be derived from any of the known helper viruses such as adenovirus, herpesvirus (except herpes simplex type 1), and vaccinia virus.

The term "helper vector" generally refers to a nucleic acid molecule that includes polynucleotide sequences that provide helper functions. Such sequences can be in a helper vector and transfected into a suitable host cell. The helper vector is capable of supporting the production of the rAAV virion in the host cell. Accessory vectors may be in the form of a plasmid, phage, transposon, or cosmid. In addition, the complete set of adenovirus genes is not required for the implementation of auxiliary functions. For example, adenoviral mutants incapable of DNA replication and late gene synthesis have been reported to allow 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 required to provide accessory functions (Carter et al., (1983) Virology 126:505) . Adenoviruses that are defective in the E1 region or have an E4 region removed cannot support AAV replication. Thus, the E1A and E4 regions appear to be, directly or indirectly, required for AAV replication (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 adenoviral 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 Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)). Studies on the accessory functions provided by adenoviruses having mutations in the E1B coding region have yielded conflicting results, but E1B55k may be required for AAV virion production, while E1B19k is not (Samulski et al., (1988) J. Virol. 62:206-210 ). In addition, International Publication Number WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945 describe helper vectors encoding various adenovirus genes. Examples of accessory vectors include an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus 72 kD E2A coding region, an adenovirus E1A coding region, and an adenovirus E1B region that lacks an intact E1B55k coding region. Such helper vectors are described, for example, in international publication number WO 01/83797.

As used herein, the term "serotype" means a distinguishing feature used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological features are determined by the absence of cross-reactivity between antibodies to different AAVs. Differences in cross-reactivity are usually due to differences in the sequences of the capsid proteins/antigenic determinants (eg, due to differences in the VP1, VP2 and/or VP3 sequences of the AAV serotypes).

According to the traditional definition, serotype means that the virus of interest has been tested for the presence of a neutralizing

and activity in serum specific to all existing and characterized serotypes, and no antibodies were found that neutralize the virus of interest. When naturally occurring virus isolates are found and/or when capsid mutants are obtained, they may or may not be serologically distinct from any of the currently existing serotypes. Thus, in cases where a new virus (eg AAV) is serologically distinct, the new virus (eg AAV) will be a subgroup or variant of the corresponding serotype. For many mutant viruses with modifications to the capsid sequence, serological testing for neutralizing activity has yet to be performed to determine if they have a different serotype according to the traditional definition of serotype. Accordingly, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that are not serologically distinct and may represent a subgroup or variant of this serotype.

rAAV vectors include any viral strain or serotype. As a non-limiting example, the rAAV vector plasmid or genome or particle (capsid) can be based on any AAV serotype, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11. Such vectors may be based on the same strain or serotype (or subset or variant) or may differ from one another. As a non-limiting example, a plasmid or vector genome or rAAV particle (capsid) based on the genome of a single serotype may be identical to one or more capsid proteins packaging the vector. In addition, the rAAV vector plasmid or genome may be based on an AAV serotype (e.g., AAV2) genome that differs in one or more capsid proteins that package the vector genome, in which case at least one of the three capsid proteins may have, for example, the sequence AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1 or SEQ ID NO:2 or variants thereof. Therefore, rAAV vectors include gene/protein sequences that are identical to gene/protein sequences specific to a particular serotype as well as to mixed serotypes.

In various exemplary embodiments, the rAAV vector includes or contains a capsid sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, 99.5%, etc.) is identical to one or more sequences of the capsid proteins AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1 or SEQ ID NO:2. In various exemplary embodiments, the rAAV vector includes or contains a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 %, 99.5%, etc.) is identical to the ITR of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74.

rAAV such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, SEQ ID NO:1 and SEQ ID NO:2 and their variants, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art to include one or more heterologous polynucleotide sequences (transgenes) flanked by one or more functional AAV ITR sequences. Such vectors have one or more wild-type AAV genes completely or partially deleted, but retain at least one functional ITR flanking sequence necessary for survival, replication and packaging of the recombinant vector into an rAAV vector particle. Therefore, the rAAV vector genome will include, in cis, sequences necessary for replication and packaging (eg, functional ITR sequences).

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, as well as spliced or unspliced mRNA, rRNA, tRNA, and inhibitory DNA or RNA (interfering RNA, e.g., small or short hairpin (sh)RNA, microRNA, small interfering(s) RNA, trans-splicing RNA or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded or triple-stranded, linear or circular, and can be of any length. When discussing nucleic acids, the sequence or structure of a particular polynucleotide is written herein, as is customary, in the 5' to 3' direction.

The term "heterologous" nucleic acid sequence refers to a polynucleotide inserted into an AAV plasmid or vector for vector-mediated transfer/delivery of the polynucleotide into a cell. Heterologous nucleic acid sequences are different from the AAV nucleic acid, ie are not native to the AAV nucleic acid. After transfer/delivery into a cell, the heterologous nucleic acid sequence contained in the vector can be expressed (eg, transcribed and translated if necessary). Alternatively, the heterologous polynucleotide contained in the vector and transferred/delivered into the cell need not be expressed. Although the term "heterologous" in relation to nucleic acid sequences and polynucleotides is not always used herein, reference to a nucleic acid sequence or polynucleotide even in the absence of this modifier "heterologous" is meant to include heterologous nucleic acid sequences and polynucleotides.

"Polypeptides", "proteins" and "peptides" encoded by a "nucleic acid sequence" include full-length native sequences, whether in the case of naturally occurring proteins or functional subsequences, modified forms or sequence variants, provided that a subsequence, modified form, or variant retains some degree of functionality of the native full-length protein. Such polypeptides, proteins and peptides encoded by nucleic acid sequences may, but need not be, identical to an endogenous protein that is defective or deficient or deficient in the mammal being treated.

The term "transgene" is used herein for convenience to refer to a nucleic acid (eg, heterologous) that is intended to be introduced or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence.

Into the cell containing the transgene, the transgene has been introduced/transferred via an AAV plasmid or vector, "transduction" or "transfection" of the cell. The terms "transduce" and "transfect" refer to the introduction of a molecule, such as a nucleic acid, into a host cell (eg, HEK293) or body cells. 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 a recipient cell or organism, it can be stably present in that cell or organism and further transmitted or inherited by progeny cells or progeny organisms of the recipient cells or cells of the organism.

The term "host cell" means, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be used or have been used as recipients of an AAV vector plasmid, AAV helper construct, helper vector, or other transfer DNA. The term includes the progeny of the original cell that has been transfected. Thus, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of one parent cell may not necessarily be completely identical in morphology or genome with the original parent cell due to natural, accidental or intentional mutation. Examples of host cells include human embryonic kidney (HEK) cells such as HEK293.

A "transduced cell" is a cell into which a transgene has been introduced. Accordingly, a "transduced" cell means a genetic change in a cell following the incorporation of an exogenous molecule, such as a nucleic acid (eg, a transgene) into the cell. Thus, a "transduced" cell is a cell, or progeny thereof, into which an exogenous nucleic acid has been introduced. The cell(s) may be propagated (cultured) while the introduced protein is expressed or the nucleic acid is transcribed, or a vector such as rAAV is produced by the cell. For gene therapy methods and uses, the transduced cell may reside in the subject.

As used herein, the term "stable" in relation to a cell or "stably integrated" means that nucleic acid sequences, such as a selectable marker or a heterologous nucleic acid sequence, or a plasmid, or a vector, have been inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.) or persist in the recipient cell or host extrachromosomally, remaining on the chromosome or persisting extrachromosomally for a certain period of time. In cell culture, nucleic acid sequences such as a heterologous nucleic acid sequence or plasmid or vector inserted into a chromosome can be maintained in the chromosome over a large number of cell passages.

The term "cell line" or "cell line" refers to a population of cells capable of continuous or sustained growth and division in vitro under appropriate culture conditions. The cell lines may, but need not be, clones of a single progenitor cell. In cell lines, spontaneous or induced changes in the karyotype can occur during the maintenance of a cell line or during the transfer of such clones, as well as during long passage in tissue culture. Thus, progeny cells derived from a cell line may not be exactly identical to progenitor cells or cultures. An example of a cell line applicable to the purification methods of the present invention is HEK293.

The term "expression control element" refers to a nucleic acid sequence(s) that affect the expression of an operably linked nucleic acid. Control elements, including expression control elements referred to herein, such as promoters and enhancers. rAAV vectors may include one or more "expression control elements". Typically, such elements are included to ensure correct transcription of the heterologous polynucleotide and, if necessary, translation (e.g., promoter, enhancer, splicing signal for introns to help maintain the correct reading frame of the gene and ensure correct translation of the mRNA, stop codons, etc. ). Such elements typically act in the cis-position and are referred to as "cis-acting" elements, but may also act in the trans-position.

Expression can be controlled at the level of transcription, translation, splicing, messenger RNA stability, etc. Typically, an expression control element that modulates transcription is located near the 5' end (ie, "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (ie "after") of the transcribed sequence or within the transcript (eg, in an intron). The expression control elements can be located near or at a distance from the transcribed sequence (eg, 1-10, 10-25, 25-50, 50-100, 100-500 or more nucleotides from the polynucleotide), even at a considerable distance. However, due to length limitations of rAAV vectors, expression control elements are typically within 1-1000 nucleotides of the transcribed nucleic acid.

Functionally, the expression of an operably linked nucleic acid is at least partially controlled by an element (eg, a promoter) such that the element modulates transcription of the nucleic acid and, if necessary, translation of the transcript. A specific example of an expression control element is a promoter, which is typically located 5' to the left of the transcribed sequence. A promoter generally increases the number of transcripts expressed from an operably linked nucleic acid compared to the number of transcripts expressed in the absence of a promoter.

As used herein, the term "enhancer" may refer to a sequence that is adjacent to a nucleic acid sequence, such as a selectable marker, or a heterologous nucleic acid sequence. Enhancer elements are usually located before the promoter element, but also function and can be located after or within the sequence. Therefore, the enhancer element may be located before or after, for example, within 100 base pairs, 200 base pairs, or 300 base pairs or more of a nucleic acid sequence, such as a selectable marker, and/or a heterologous nucleic acid sequence encoding a therapeutic protein or polynucleotide sequence. Enhancer elements typically increase the expression of an operably linked nucleic acid over that provided by a promoter element.

The term "operably linked" means that the regulatory sequences necessary for the expression of the nucleic acid sequence are located in suitable positions relative to the sequence to influence the expression of the nucleic acid sequence. The same definition is sometimes applied to the location of nucleic acid sequences and transcription control elements (eg, promoters, enhancers, and terminators) in an expression vector, such as the rAAV vector.

In an example of an expression control element, it is operably linked to the nucleic acid such that the control element modulates the expression of the nucleic acid. More specifically, for example, that two DNA sequences are operably linked means that the two DNAs are arranged (cis or trans) such that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

Accordingly, additional elements for vectors include, without limitation, an expression control element (e.g., a promoter/enhancer), a transcription termination signal or stop codon, 5' or 3' untranslated regions (e.g., polyadenylation sequences (polyA)), which flank the sequence, for example, one or more copies of the AAV ITR sequence, or an intron.

Additional elements include, for example, polynucleotide sequence fillers or spacers, for example, providing better packaging and reducing the amount of contaminating nucleic acid. AAV vectors typically include DNA inserts ranging in size from about 4 kb to about 5.2 kb. or a little more. Thus, for shorter sequences, the inclusion of a filler or spacer is necessary to adjust the length to approximately the normal size of a viral genomic sequence suitable for packaging the vector into an rAAV particle. In various embodiments, the filler/spacer nucleic acid sequence is a non-translated (non-coding protein) nucleic acid segment. For a nucleic acid sequence less than 4.7 kb, the filler or spacer polynucleotide sequence will be of such length that the total (for example, after insertion into a vector) sequence is about 3.0-5.5 kb or about 4.0-5, 0 KB, or about 4.3-4.8 KB.

A “therapeutic protein”, in one embodiment, is a peptide or protein that can alleviate or reduce symptoms resulting from an insufficient amount, absence, or defect of a protein in a cell or subject. The "therapeutic" protein encoded by the transgene may provide a benefit to the subject, such as correcting a genetic defect, correcting a deficiency (expression or functionality) of a gene, and the like.

Non-limiting examples of heterologous nucleic acids encoding gene products (e.g., therapeutic proteins) used in accordance with the present invention include those that can be used to treat a disease or disorder, including, but not limited to, "hemostasis" or bleeding disorders. such as hemophilia A, patients with hemophilia A and 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, deficiency vitamin K epoxide reductase C1, gamma carboxylase deficiency; anemia, trauma-related bleeding, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation syndrome (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (ie, FXa inhibitors); and platelet disorders such as Bernard-Soulier syndrome, Glanzmann's thrombasthenia and platelet pool deficiency.

Nucleic acid molecules, vectors such as cloning vectors, expression vectors (eg vector genomes), and plasmids can be produced using recombinant DNA techniques. The availability of information about nucleotide sequences makes it possible to obtain nucleic acid molecules in various ways. For example, a heterologous nucleic acid encoding Factor IX (FIX) containing a vector or plasmid can be obtained by various standard cloning methods, by recombinant DNA technology, by cell expression or in vitro translation, and by chemical synthesis. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like. ways. For example, nucleic acids can be isolated using hybridization methods or database screening computer methods. Such methods include, but are not limited to: (1) hybridizing genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) screening using antibodies to detect polypeptides that have common structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers that anneal to the nucleic acid sequence of interest; (4) computer search of related sequences in databases of sequences; and (5) differential screening of the library of subtractable nucleic acids.

The term "isolated" when used as a composition modifier means that the compositions are human-made or isolated, in whole or at least in part, from their natural environment in vivo . Typically, isolated compositions are substantially free of one or more materials with which they are normally associated in nature, eg, one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

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

The term "isolated" does not exclude a combination produced by the human hand, such as a recombinant rAAV and a pharmaceutical composition. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), or derivatized forms, or forms expressed in human hand-derived host cells.

The term "substantially pure" refers to a formulation containing at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). The formulation may contain at least 75% by weight, or about 90-99% by weight, of the compound of interest. Purity is assessed by methods appropriate to the compound of interest (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

The phrase "consisting primarily of" when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of that sequence. For example, when used in relation to an amino acid sequence, the phrase includes the sequence as such and molecular modifications that do not affect the basic and novel characteristics of the sequence.

Methods known in the art to produce rAAV virions: for example, transfection with an AAV vector and AAV accessory sequences in combination with co-infection with a single AAV helper virus (e.g., adenovirus, herpes virus, or vaccinia virus) or transfection with a recombinant AAV vector, AAV helper vector and helper vector. Non-limiting methods for producing rAAV virions are described, for example, in US Patent Nos. 6,001,650 and 6,004,797; Once a recombinant rAAV vector has been formed (ie, vector production in cell culture systems), rAAV virions can be obtained from host cells and cell culture supernatant and purified as described herein.

In the first step, host cells that produce rAAV virions are typically harvested, optionally in conjunction with the collection of cell culture supernatant (medium) in which host cells (suspension or adhesive) that produce rAAV virions are cultured. In the methods described herein, the harvested cells and optionally the cell culture supernatant may be used as is, as appropriate, or may be concentrated. In addition, if the infection is carried out to express accessory functional elements, the residual helper virus may be inactivated. For example, an adenovirus can be inactivated by heating to about 60° C. for, for example, 20 minutes or more, only the helper virus is inactivated because AAV is thermostable while the helper adenovirus is thermolabile.

Cells and/or harvest supernatant are lysed by disrupting the cells, for example by chemical or physical means such as detergent treatment, microfluidization and/or homogenization, to release rAAV particles. During cell lysis or after cell lysis, a nuclease, such as benzonase, may be added to destroy contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtration, centrifugation, to obtain a clarified cell lysate. In a specific example, the lysate is filtered through a micron diameter filter (such as a filter with a pore size of 0.1-10.0 µm, for example, a filter with a pore size of 0.45 µm and/or a pore size of 0.2 µm) to receive CLARIFIED LYSATE.

The lysate (optionally clarified) contains AAV particles (rAAV vectors proper and AAV empty capsids) and impurities associated with the AAV vector manufacturing process, such as soluble cellular components from host cells, which may include, among others, cellular proteins, lipids and/ or nucleic acids, and components of the cell culture medium. The optionally clarified lysate is then subjected to additional purification steps to purify the AAV particles (including the rAAV vectors themselves) of impurities by chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

As disclosed herein, after cell lysis, optional clarification, and optional dilution or concentration, a plurality of sequential chromatographic steps are used to purify rAAV particles. Such methods generally omit the cesium chloride gradient ultracentrifugation step.

As disclosed herein, the first chromatography step may be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step may be an anion exchange chromatography or size exclusion chromatography (SEC). Thus, in one method of purifying rAAV, purification is performed by cation exchange chromatography followed by purification by anion exchange chromatography.

Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step may be size exclusion chromatography (SEC). Thus, in another method for purifying rAAV, purification is by cation exchange chromatography followed by purification by size exclusion chromatography (SEC).

As also disclosed herein, the first chromatography step may be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in yet another method for purifying rAAV, purification is by affinity chromatography followed by purification by anion exchange chromatography.

Optionally, a third chromatography may be added to the above chromatography steps. Typically, an optional third chromatographic step follows the cation exchange, anion exchange, size exclusion or affinity chromatography steps.

Thus, in a further purification method for rAAV, purification is by cation exchange chromatography followed by purification by anion exchange chromatography followed by size exclusion chromatography (SEC) purification. And in yet another additional method for purifying rAAV, purification is by cation exchange chromatography followed by purification by size exclusion chromatography (SEC) followed by purification by anion exchange chromatography.

In yet another additional purification method for rAAV, purification is by affinity chromatography followed by purification by anion exchange chromatography followed by size exclusion chromatography (SEC) purification. In yet another method of rAAV purification, purification is by affinity chromatography followed by size exclusion chromatography (SEC) purification followed by anion exchange chromatography.

Cation exchange chromatography serves to separate AAV particles from cellular and other components present in the clarified lysate and/or column eluate after size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV particles over a wide pH range include, without limitation, any sulfonic acid resins as indicated by the presence of a sulfonate functional group, including aryl and alkyl substituted sulfonates such as sulfopropyl or sulfoethyl. Typical matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers from Thermo Fisher Scientific, Inc., Waltham, MA). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers from GE Healthcare, Marlborough, MA) and commercial resin series ^DOWEX® , AMBERLITE® and ^AMBERLYST® from Aldrich Chemical Company ( ^Milliwaukee , WI). Weak cation exchange resins include, without limitation, any resins based on carboxylic acids. Examples of cation exchange resins also include carboxymethyl (CM), phospho (based on a phosphate functional group), methylsulfonate (S), and sulfopropyl (SP) resins.

Anion exchange chromatography serves to separate AAV particles from proteins, cellular and other components present in the clarified lysate and/or column eluate after size exclusion chromatography. Anion exchange chromatography can also be used to control the amount of empty AAV capsids in the eluate. For example, an anion exchange column with an associated rAAV vector can be washed with an average concentration of NaCl (eg, about 100-125 mM, eg, 110-115 mM) and elute some of the empty capsids without eluting the rAAV vectors. Subsequently, the rAAV vector bound to the anion exchange column can be eluted with a higher concentration of NaCl (e.g., about 130-300 mM NaCl), and thus obtain a column eluate with a reduced amount of empty AAV capsids and proportionally increased amounts of rAAV.

Examples of anion exchange resins include, without limitation, resins based on polyamine resins and other resins. Examples of strong anion exchange resins include resins based primarily on a quaternized nitrogen atom, including, without limitation, resins based on quaternary ammonium salts such as trialkylbenzylammonium resins. Suitable exchange chromatography resins include, without limitation, MACRO PREP Q (strong anion exchanger from BioRad, Hercules, Calif.); UNOSPHERE Q (strong anion exchanger from BioRad, Hercules, Calif.); POROS 50HQ (strong anion exchanger from Applied Biosystems, Foster City, Calif.); POROS XQ (strong anion exchanger from Applied Biosystems, Foster City, Calif.); POROS 50D (weak anion exchanger from Applied Biosystems, Foster City, Calif.); POROS 50PI (weak anion exchanger from Applied Biosystems, Foster City, Calif.); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchanger from GE healthcare, Marlborough, MA); DEAE SEPHAROSE (weak anion exchanger from Amersham Biosciences, Piscataway, N.J.); Q SEPHAROSE (strong anion exchanger from Amersham Biosciences, Piscataway, N.J.). Additional examples of anion exchange resins include resins with aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE) groups.

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

The cation exchange chromatography column can be equilibrated with standard buffers and according to manufacturer's specifications. For example, the chromatography medium can be equilibrated with 5-100 mM or 10-50 mM phosphate buffer, eg 10-30 mM, and sodium chloride. After equilibration, a sample is applied to the column. The chromatographic medium is then washed at least once or more, for example 2-10 times. The chromatographic medium is eluted with a high salt buffer at least once, but can also be eluted 2 or more times with the same buffer or a buffer with a higher salt content.

Typical equilibration buffers, washings and eluents for cation exchange chromatography have a suitable pH in the range of about 3 to 8, more typically 4 to 7.5, such as 6.0-6.5, 6.5-7.0, 7.0-7.5 or any pH within the indicated ranges, for example 7.0, 7.1, 7.2, 7.3 or 7.4.

Suitable equilibration buffers, washings and eluents for cation exchange columns are known in the art and are usually anionic. Such buffers include, without limitation, buffers containing the following buffer ions: phosphate, acetate, citrate, borate, or sulfate.

In one embodiment, the cation exchange chromatography medium is first equilibrated, sampled and washed with a low salt concentration, e.g. 60-125 mm or any concentration within these ranges, for example, 100 mm.

After the first wash, the chromatography medium can be treated with a higher salt concentration to elute impurities, for example, with a higher NaCl concentration, or with another buffer of higher ionic strength. After eluting additional contaminants from the column, to elute the rAAV particles, the ionic strength of the buffer can be increased with a salt such as NaCl, KCl, sulfate, formate, or acetate, and recovered. In one possible embodiment, the elution is carried out with a high salt concentration, for example, 200-500 mm NaCl, or any concentration within these ranges, for example, 250 mm, 300 mm, 350 mm or 400 mm.

Additional components may be included in buffers for equilibrating, washing and elution solutions. For example, a cation exchange chromatography wash buffer may include an anionic surfactant such as sarcosyl (eg 1-10 mM), an anion exchange chromatography wash buffer may include a cationic surfactant such as dodecyltrimethylammonium chloride (eg 1-10 mM).

Typical equilibration buffers, anion exchange chromatography washes and eluents have a pH in the range of about 7.5-12, more typically about 8.0-10, and even more typically about 8.0-9.0, e.g. pH 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

Suitable equilibration buffers, rinses, and eluents for anion exchange columns are typically cationic or zwitterionic in nature. Such buffers include, without limitation, buffers containing the following buffer agents: N-methylpiperazine; piperazine; Bis Tris; Bis Tris propane; triethanolamine; Tris; N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid and the like. To elute the sample, the ionic strength of the starting buffer is increased using a salt such as NaCl, KCl, sulfate, formate, or acetate. Such equilibration buffers, wash and elution solutions may contain the above buffering agents at a concentration of from about 5 to 100 mm, more typically from about 10 to 50 mm.

In one embodiment, the anion exchange chromatography medium is first equilibrated, sampled and washed with a low salt concentration, e.g. 50-150 mM NaCl such as 50-60, 60-70, 70-80, 80-90, 90-100, 100-100 mM or any concentration within these ranges. After the first wash, the chromatography medium can be treated with a higher salt concentration to elute impurities such as empty AAV capsids, for example with a higher concentration of NaCl, or another buffer with higher ionic strength. One example of a buffer for use as a second buffer is a Tris buffer with a NaCl concentration of about 110 mM-125 mM, or any concentration within the specified range.

After eluting additional contaminants from the column, the rAAV particles can be recovered by elution with a buffer with a higher salt concentration. One example of an eluting buffer is a Tris buffer with a NaCl concentration of 125 mM or more, such as 125-150 mM, 150-200 mM or 200-250 mM NaCl, or any concentration within the indicated range.

Wash solutions for anion exchange columns may include polyethylene glycol (PEG). This chromatography is called polyethylene glycol (PEG) column chromatography. PEG wash solutions can be applied to an anion exchange chromatography column prior to eluting the AAV vector particles.

Typically, the PEG in such wash solutions has an average molecular weight in the range of about 1,000 to 80,000 g/mol, inclusive. Typical amounts of PEG in such wash solutions range from about 0.1% to about 20% PEG, or any amount within these stated ranges, or from about 1% to about 10% PEG, or any amount within these ranges.

Size Exclusion Chromatography (SEC) medium can be equilibrated with standard buffers and according to manufacturer's specifications. For example, the chromatography medium can be equilibrated with phosphate buffer at a concentration of, for example, about 1-5 mM, 5-50 mM or 5-25 mM, and NaCl at a concentration of, for example, about 50-100 mM, 100-150 mM, 150 -200 mM, 200-250 mM, 250-300 mM or 300-400 mM, or any concentration within the specified range.

After balancing, the sample is applied. Next, an eluate containing rAAV particles is collected. For more complete recovery of rAAV particles, additional volumes of buffer (eg, phosphate buffer) may be used based on the amount of chromatography medium and/or column size.

In particular embodiments, the size exclusion chromatography medium has a separation range (molecular weight) of 10,000 to 600,000 inclusive. Specific resins (media) suitable for size exclusion chromatography include, without limitation, porous cellulose particles or beads, cross-linked agarose (Sepharose, GE Healthcare, Marlborough, MA), cross-linked dextran (Sephadex, GE Healthcare, Marlborough, MA) , styrene-divinylbenzene (Dianon HP-20), polyacrylamide (Bio Gel), methacrylate (Toyopearl), and custom pore glass.

Affinity columns usually consist of a ligand bound or conjugated to a substrate. Specific examples of ligands include AAV binding antibodies. Such substrates include Sepharose and other materials commonly used in such affinity purification applications and can be manufactured or are commercially available (eg, AVB Sepharose™ High Performance, GE Healthcare, Marlborough, MA).

Suitable equilibration buffers, washings and eluting solutions for affinity columns are known to typically contain Tris or acetate. For example, the affinity chromatography medium can be equilibrated with Tris buffer at a concentration of, for example, about 1-5 mM, 5-50 mM, or 5-20 mM, and NaCl at a concentration, for example, about 50-100 mM, 100-150 mM, 150-200 mM, 200-250 mM, 250-300 mM, or any concentration within the specified range.

Typical affinity chromatography equilibration buffers have a pH in the range of about 7.5-9, more typically about 8.0-8.5, and even more typically about 8.0, 8.1, 8.2, 8, 3, 8.4 or 8.5.

After balancing, the sample is applied. The rAAV particles are then eluted from the affinity column by lowering the pH of the buffer below 7.0. The elution buffer may contain acetate and the pH of the buffer is typically less than 5.0, more typically less than 4.0, such as less than 3.0, more specifically between about 2.0 and 3.0, or any pH within the indicated ranges.

The volumes of equilibration buffers, wash and elution solutions may vary depending on the amount of chromatography medium and/or column size for more complete recovery of rAAV particles. Typical volumes are 1-10 column volumes.

The eluate from the column is collected after passing the eluate/filtrate through each of the chromatography steps. AAVs can be detected in fractions using standard methods such as UV absorbance monitoring at 260 and 280 nm.

The use of a cationic or anion exchange medium for chromatography, the nature of the medium used (i.e., strong or weak ion exchangers), salt concentration, buffer used, and pH may vary depending on the AAV capsid (i.e., AAV capsid serotype or pseudotype). While the structure of AAV capsids may not vary greatly in size and shape, capsids may 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 invention, and suitable methods can be determined by methodically selecting the chromatography medium and screening buffers to identify the various AAV capsid variant conditions that can be used to purify rAAV particles.

Eluates containing rAAV particles from any of the cation exchange, anion exchange, size exclusion and/or affinity chromatography steps described herein can be efficiently concentrated by ultrafiltration/diafiltration if necessary. Volume reduction can be controlled by a person skilled in the art. In specific non-limiting examples, a volume reduction of about 1-30 times, inclusive, is achieved. Thus, a 1-fold reduction reduces the volume by half, for example, 1000 ml is concentrated to 500 ml. A 10-fold reduction means a 10-fold reduction in volume, for example 2000 ml is concentrated to 200 ml. A 20-fold reduction means a 20-fold reduction in volume, for example 2000 ml is concentrated to 100 ml. A 30-fold reduction means a 30-fold decrease in volume, for example, 2000 ml is concentrated to 66.67 ml.

A non-limiting example of ultrafiltration/diafiltration is tangential flow filtration (TFF). For example, a hollow fiber membrane with a nominal pore size corresponding to a molecular weight of 100 kDa, so that the AAV vector can be isolated in large quantities from a large volume of eluate.

Cell lysate and column eluates containing rAAV particles after any of the cation exchange, anion exchange, size exclusion or affinity chromatography steps described herein may be diluted if necessary. Typical dilutions are 25-100%, 1-2 times, 2-5 times, or to any volume, or any number of times within the ranges indicated.

The methods of the present invention provide significant recovery of rAAV particles. For example, the methods of the present invention recover approximately 40-70% of rAAV particles based on the total amount of rAAV vector particles from host cells and the harvested supernatant from the host cell culture. In another example, rAAV particles are present in the final (eg, after the third column) eluate at a concentration of about 100 mg/mL. rAAV vector particles may be present in the final (eg, after the third column) eluate at a concentration of about 1010-1011 particles per ml or greater, 1011-1012 particles per ml, 1012-1013 particles per ml.

Alternatively, if concentrations of rAAV vector particles are lower, purified rAAV particles can be concentrated. For example, purified AAV particles can be concentrated by ultrafiltration/diafiltration (eg TFF). If higher vector concentrations are needed, the purified AAV particles can be concentrated to 1012-1013 particles per ml or more, 1013-1014 particles per ml or more using ultrafiltration/diafiltration (eg TFF) or even more.

In other embodiments, genome-packed rAAV particles (i.e., rAAV vector particles themselves) are “substantially free of “AAV-encapsulated nucleic acid contaminants” when at least about 30% or more of the virions present are particles. rAAV with packaged genomes (i.e., rAAV vector particles themselves). In the production of genome-packed rAAV particles (i.e., rAAV vector particles proper) substantially free of AAV-encapsulated nucleic acid contaminants, the amount of product containing AAV-encapsulated nucleic acid contaminants can range from 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 than 1%.

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

To determine the presence or amount of degraded/denatured capsid, purified AAV can be subjected to SDS polyacrylamide gel electrophoresis, any gel that can separate the three capsid proteins can be used, e.g. transferred to a nylon or nitrocellulose membrane. Next, antibodies against AAV capsid proteins are used as primary antibodies that bind to denatured capsid proteins (see, for example, Wobus et al., J. Virol. (2000) 74:9281-9293). To detect the primary antibody, a secondary antibody is used that binds to the primary antibody. Binding between primary and secondary antibodies is detected semi-quantitatively and thus the number of capsids is determined. Another method may be analytical HPLC with a SEC column or analytical ultracentrifugation.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described herein.

All applications, publications, patents and other references, references to GenBank and ATCC mentioned in this document are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will be used for verification.

All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed characteristics (eg, nucleic acid sequences, vectors, rAAV vectors, etc.) are one example of equivalent or similar characteristics.

In this document, the singular forms also include plural referents, unless the context clearly indicates otherwise. Thus, for example, reference to "AAV vector" or "AAV particle" includes a plurality of such AAV vectors and AAV particles, and reference to a "cell" or "host cell" includes a plurality of cells and host cells.

As used herein, the term "about" means values that are within 10% (plus or minus) of a given value.

As used herein, all numeric values or numeric ranges include integers within such ranges and fractions of values or fractional numbers within the ranges, unless the context clearly indicates otherwise. Thus, by way of illustration, reference to an identity of 80% or more 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., etc.

A reference to an integer greater than or less than includes any number greater than or less than the given number, respectively. So, for example, a reference to a number less than 100 includes 99, 98, 97, and so on. up to the number one (1); and a reference to a number less than 10 includes 9, 8, 7, and so on. up to the number one (1).

In this document, all numerical values or ranges are included. In addition, all numerical values or ranges include fractions of values and integers within such ranges and fractional numbers within such ranges, unless the context clearly indicates otherwise. Thus, by way of illustration, 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., etc. The reference to the range 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and etc., up to 50 inclusive, 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., etc.

A reference to a set of ranges includes ranges that combine the limit values of different ranges. Thus, by way of illustration, reference to a number of ranges, e.g., 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 150-200 200-250 250-300 300-400 400-500 500-750 750-1000 1000-1500 1500-2000 2000-2500 2500-3000 3000-3500 3500- 4000, 4000-4500, 4500-5000, 5500-6000, 6000-7000, 7000-8000 or 8000-9000, includes ranges 10-50, 50-100, 100-1000, 1000-3000, 2000-4000, etc. P.

The invention as a whole is disclosed herein using affirmative language to describe numerous embodiments and aspects. The invention also specifically includes embodiments that exclude a particular subject, in whole or in part, such as substances or materials, process steps and conditions, protocols or techniques. For example, in some embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even if the invention as a whole is not described herein in terms that the invention does not include, aspects that are not expressly excluded in the present invention, however, are disclosed in this document.

A number of embodiments of the invention have been described. However, a person skilled in the art, without departing from the essence and scope of the invention, can make various changes and modifications to the invention in order to adapt it to various applications and conditions. Accordingly, the following examples are intended to illustrate and not to limit the scope of the claimed invention.

EXAMPLES

Example 1

Examples of first column, cation exchange or affinity

1.1. Cation exchange column, Poros50 HS (or Poros XS from ThermoFisher). Preconditioning, equilibration, application of material, washing and elution

1.1.1. Buffer examples

1.1.1.1. Buffer, pH, Conductivity

Preconditioning: WFI

3x dilution buffer: 60mM Na-phosphate, pH 7.3

Equilibration buffer: 20mM Na-phosphate, pH 7.3, 100mM NaCl

Wash buffer 1 and 3: 20mM Na-phosphate, pH 7.3, 100mM NaCl

Wash buffer 2: 20 mM Na-phosphate, pH 7.3, 100 mM NaCl, surfactant (5 mM) such as sarcosyl

Elution buffer: 20mM Na-phosphate, pH 7.3, 300mM NaCl (may be more than 300mM, eg 300-400, eg 400mM NaCl).

Based on eluate collection based on either the volume of elution buffer (e.g., eluate is collected after collection of the volume of the peak located before the main peak, set in advance), or an increase in the absorbance of A280, for example, an increase of 1 mEOP compared to the base absorbance of A280.

Stripping solution: high salt concentration, e.g. 1M NaCl

Sanitizing solution: 1M NaCl, 1N NaOH

Storage solution: approximately 22.5% ethanol (e.g. 18-22.5%)

1.1.2. Column preparation example

1.1.2.1. Downflow preconditioning (4 column volumes) of WFI

1.1.2.2. Downstream equilibration (4 column volumes) of equilibration buffer

1.1.3. Cleaning example

1.1.3.1. Column application of sample material in downflow mode (25 column volumes)

1.1.3.2. Wash 1 downstream (4 column volumes) wash buffer 1

1.1.3.3. Wash 2 downstream (6 column volumes) wash buffer 2

1.1.3.4. Wash 3 downstream (8 column volumes) wash buffer 3

1.1.4. An example of a blocking (flushing) volume (volumes) determined depending on the scale, column size

1.1.5. Binding capacity: 2.5-3x load (Poros50 HS) with or without ultrafiltration/diafiltration

1.1.6. Approximate flow rate: 150 cm/h - 300 cm/h

1.1.7. Permissible load: 10L or more

1.1.8. Expected recovery: 60-90%

1.2. Affinity column, AVB-Sepharose HP

1.2.1. buffer list

1.2.1.1. Buffer (pH, conductivity), each blocking volume

1.2.1.2. Examples of buffers/solutions

Equilibration buffer: 20mM Tris, pH 8.0, 250mM NaCl

Elution buffer: 10mM Na-acetate, pH 2.5, 250mM NaCl

Neutralizing buffer: 1M Tris-Cl, pH 10.5

Sanitizing solution: PAB (120mM phosphoric acid, 167mM acetic acid, 2.2% benzyl alcohol)

Storage solution: approximately 22.5% ethanol (e.g. 18-22.5%)

1.2.2. Approximate flow rate: approximately 150 cm/h or more

1.2.3. Expected recovery: 70-90% (AAV virion capsid), 15-70% (vector genomes)

Example 2

Examples of a second column, anion exchange or size exclusion

1.3. Anion exchange column, Poros 50HQ (Poros XQ from ThermoFisher). Preconditioning, equilibration, application of material, washing and elution:

performs a double function (further cleanup & control of the ratio of empty and non-empty vectors)

1.3.1. Buffer examples

1.3.1.1. Buffer, pH, conductivity, each blocking volume

Preconditioning: WFI

3x (or 4x) dilution buffer: ca. 60mM (or ca. 80mM) Tris, pH 8.5 (e.g., after dilution, material is applied in 10-50mM Tris, pH 8.0-8.5, 100mM NaCl)

Equilibration Buffer (and Wash Buffer 1): 20mM Tris, pH 8.5, 100mM NaCl

Wash buffer 2 (to remove empty AAV capsids): 20mM Tris, pH 8.5, 115mM NaCl

Elution buffer: 20mM Tris, pH 8.5, NaCl ≥ 120mM, e.g. 200mM NaCl

Column Strip Buffer: 1M NaCl

Sanitizing solution: 1N. NaOH

Storage solution: approximately 22.5% ethanol (e.g. 18-22.5%)

1.3.2. Column preparation example

1.3.2.1. Downflow preconditioning (5 column volumes) of WFI

1.3.2.2. Downstream equilibration (4 column volumes) of equilibration buffer

1.3.3. Cleaning example

1.3.3.1. Applying cation exchange eluate & dilute downstream eluate to the column (50 column volumes)

1.3.3.2. 1 downstream wash (5 column volumes) of equilibration buffer

1.3.3.3. Downstream elution 1 (removal of empty capsids) (3 column volumes) of elution buffer 1

1.3.3.4. Downstream elution 2 (retrieval of non-empty AAV vectors) (3 column volumes) of elution buffer 2

1.3.4. Approximate flow rate: 150 cm/h - 300 cm/h

1.4. SEC column, SEC (e.g. Superdex 200 prep grade from GE healthcare), optional

1.4.1. Buffer examples

1.4.1.1. Buffer, pH, conductivity, each blocking volume

Preconditioning: WFI

Equilibration Buffer, Wash Buffer & Elution Buffer: 10mM Na-phosphate, pH 7.2, 150-300mM NaCl (higher concentrations of NaCl, eg 300mM, should improve recovery of the AAV vector (vector genomes).

Based on eluate collection based on either the volume of elution buffer (e.g., eluate is collected after collection of the volume of the peak located before the main peak, set in advance), or an increase in the absorbance of A280, for example, an increase of 1 mEOP compared to the base absorbance of A280.

Sanitizing solution: 0.5 N NaOH

Storage solution: 22.5% ethanol (e.g. 18-22.5%)

1.4.2. Column preparation example

1.4.2.1. Downflow preconditioning (2 column volumes) of WFI

1.4.2.2. Downflow equilibration (2 column volumes) of equilibration buffer (PBS 300)

1.4.3. Cleaning example

1.4.3.1. Downflow column application (0.05 column volumes)

1.4.4. Downstream elution (1.5 column volumes) of equilibration buffer (dPBS)

1.4.5. Load capacity example: ≤5% column volume

1.4.6. Approximate flow rate: 45 cm/h

1.4.7. When collecting the eluate, base either on the volume of elution buffer or on the optical density of A280

Example 3

Example of a third column, size exclusion or anion exchange. The third column is optional and may not be required if the first column is an affinity column (eg AVB-Sepharose HP). The use of the third column also depends on which column was used as the second column (SEC > HQ or HQ > SEC, etc.).

1.5. SEC column, SEC (e.g. Superdex 200 prep grade from GE healthcare), optional

1.5.1. Buffer examples

1.5.1.1. Buffer, pH, conductivity, each blocking volume

Preconditioning: WFI

Equilibration buffer and running buffer: 10mM Na-P, pH 7.2, 150-300mM NaCl (higher concentrations of NaCl, e.g. 300mM, should improve recovery of the AAV vector (vector genomes)

Based on eluate collection based on either the volume of elution buffer (e.g. eluate is collected after collecting the pre-main peak volume predetermined) or an increase in absorbance of A280, e.g. 1 mAu increase over baseline absorbance of A280.

Sanitizing solution: 0.5 N NaOH

Storage solution: 22.5% ethanol

1.5.2. Column preparation example

1.5.2.1. Downflow preconditioning (2 column volumes) of WFI

1.5.2.2. Downflow equilibration (2 column volumes) of equilibration buffer (PBS 300)

1.5.3. Cleaning example

1.5.3.1. Downflow column application (0.05 column volumes)

1.5.3.2. Downstream elution (1.5 column volumes) of equilibration buffer (dPBS)

1.5.4. Load capacity example: ≤5% column volume

1.5.5. Approximate flow rate: 45 cm/h

1.5.6. When collecting the eluate, base either on the volume of elution buffer or on the optical density of A280

1.6. The anion exchange column, Poros 50HQ (Poros XQ from ThermoFisher), performs a dual function (further purification of rAAV & control of the ratio of empty to non-empty rAAV vectors). Preconditioning, equilibration, application of material, washing and elution

1.6.1. Buffer examples

1.6.1.1. Buffer, pH, conductivity, each blocking volume

Preconditioning: WFI

3x (or 4x) dilution buffer: 60mM (or 80mM) Tris, pH 8.5 (e.g. after dilution, material is applied in 10-50mM Tris, pH 8.0-8.5, 100mM NaCl)

Equilibration Buffer (and Wash Buffer 1): 20mM Tris, pH 8.5, 100mM NaCl

Wash buffer 2 (to remove empty AAV capsids): 20mM Tris, pH 8.5, 115mM NaCl

Elution buffer: 20mM Tris, pH 8.5, NaCl ≥ 120mM, e.g. 200mM NaCl

Column Strip Buffer: 1M NaCl

Sanitizing solution: 1N. NaOH

Storage solution: approximately 22.5% ethanol (e.g. 18-22.5%)

1.6.2. Column preparation example

1.6.2.1. Downflow preconditioning (5 column volumes) of WFI

1.6.2.2. Downstream equilibration (4 column volumes) of equilibration buffer

1.6.3. Cleaning example

1.6.3.1. Applying CEC Eluate & Dilute Downflow Eluate to Column (50 Column Volumes)

1.6.3.2. 1 downstream wash (5 column volumes) of equilibration buffer

1.6.3.3. Downstream elution 1 (removal of empty capsids) (3 column volumes) of elution buffer 1

1.6.3.4. Downstream elution 2 (retrieval of non-empty AAV vectors) (3 column volumes) of elution buffer 2

1.6.4. Flow rate example: 150 cm/h - 300 cm/h

Example 4

An example of cell lysis and preparation prior to column purification.

1. Cell lysis, chemical or physical

1.1 Example of a chemical lysis method

1.6.5. Triton X-100 or equivalent non-ionic surfactant

Final concentration, 0.1%-1%

Incubation time, up to 1 hour

Rotor agitation speed is usually about 400~600 rpm (or more)

Incubation temperature is approximately 25-37°C (e.g. 37°C)

1.6.6. Benzonase

Final concentration, ≥50 U/ml, e.g. 100 U/ml

The treatment may be carried out simultaneously or after the surfactant.

The concentration of MgCl ₂ is 1.0-5.0 mm (for example, 2 mm)

1.1.3. Additional salt for better isolation of the AAV vector during or after the filtration step

The final concentration of NaCl is 200-400mM (e.g. 300mM)

NaCl, other NaCl equivalents may also be used

1.7. An example of a physical lysis method (microfluidization, homogenization, etc.)

1.7.1. Operating conditions (based on 10 L of adherent cell culture, culture volume can be increased)

Pressure approximately < 5000 psi

Temperature approximately 18-25°C

1.7.2. Preprocessing Buffer & Chase Buffer

Buffer, pH, Conductivity

It all depends on the conditions of application to the first column; in most cases, the DF buffer is the same or equivalent buffer used to equilibrate the first column. If the first column is HS, PBS may be a common buffer.

When treated with benzonase, the pH and conductivity values should be within the operating pH and conductivity range for benzonase (eg pH approx. 6.5-8.5, conductivity >15 mS/cm). A typical amount of benzonase for DNA digestion is 100-200 U/ml. For proper cleavage, 2 mM MgCl ₂ must be added.

Volume

: The volume of the pretreatment buffer should be larger than the retention volume of the system (in most cases, this volume is three times the retention volume).

: The volume of the chase buffer is 5-10% of the sample volume.

2. Tangential Flow Filtration (TFF, also known as UF/DF), optional

2.1. TFF Hollow Fiber Cartridges

2.1.1. In the case of 10 l of cell culture

TFF is performed prior to cell lysis

Filters such as GE hollow fiber cartridges UFP-100-C-9A (1.2 m ² ) 10 liters

Capacity. Preliminary data for 20 l also fit UFP-100-C-9A hollow fiber cartridges.

TFF is performed before cell lysis or after cell lysis. TFR is performed prior to cell lysis

Maximum pressure. TMP. ≥5 psig if administered before cell lysis (5-10 psi if administered after cell lysis)

Preprocessing buffer or chase buffer. (In case of cation exchange HS) 20mM phosphate buffer, pH 7-7.5/ 100-150mM NaCl as first column is acceptable buffer (In case of anion exchange HQ) as first column 20mM Tris, pH 7.5-8 is acceptable buffer ,5/ 100-150mM NaCl.

2.1.2. In the case of ultrafiltration, to concentrate the purified AAV vector intermediate before loading onto the SEC column

TFF to concentrate the eluate after the anion exchange column (Poros 50 HQ) to about 5% of the volume of the SEC column

Material: eluate after Poros 50HQ with a volume of 0.75 column volumes

Configuration: UFP-C-100-4MA with hollow fibers (in case of initial volume 10 l)

Wash volume (retention volume): ~45 ml

Equilibration buffer: PBS300 (10mM Na-P, pH 7.2/300mM NaCl)

Sanitization buffer: 1 N NaOH

The same conditions apply for the culture of adherent cells.

TFF can be performed before cell lysis or after cell lysis.

2.1.3. TFF for final formulation and removal of possible low molecular weight impurities

Material: Poros50 HQ eluate (or SEC peak corresponding to vector)

Configuration: UFP-C-100-4MA with hollow fibers (in case of initial volume 10 l)

Wash volume (retention volume): ~45 ml

Equilibration buffer: PBS180 (10mM Na-P, pH 7.3/ 180mM NaCl)

Diafiltration buffer: PBS180 (10mM Na-P, pH 7.3/ 180mM NaCl)

Sanitization buffer: 1 N NaOH

Target concentration: 1.0E+13 vector genomes/ml

Diafiltration: Volume 12 times the target volume UF (5.0E+12 vector genomes/mL)

2.2. Variable tangential flow filtration (ATF) or TFF with coiled membrane or flat membrane module is an alternative to TFF with hollow fiber cartridge

3. Lightening & Filtering

3.1. Depth filter

Filter options include Clarisolve 20MS (0.5-20 µM), Sartoclear DL series filters (10-1 µM) or Millistak C0HC or D0HC (0.65-8 µM)

The capacity can be 1 l/25 cm ² .

Maximum flow rate, approximately 250 ml/min/25 cm ² (10 ml/min/cm ² )

Maximum operating pressure approximately ~32psig

A suitable conditioning buffer may be PBS300

Number of filtration stages (one or two, coarse filter, then fine filter): One stage possible: coarse filter

3.2. Millipore SHC (0.5/0.2 µM) or Sartopore 2 (0.45/0.2 µM, Sartorius)

Capacity approx. 500 ml/500 cm2 ⁽ 0.2% Triton X-100 helps a little, 10-20% increase)

Maximum flow rate approx. 1000 ml/min/500 cm ² (2 ml/min/cm ² )

Maximum operating pressure approximately ~35psig

An example of a conditioning buffer is PBS300

In the last step, the capacity can be smaller (about 55 maxi Sartopore 2 (1.8 m ² ) is required); if it is the 2nd stage of filtration, the capacity can be increased

3.3. For 10 L of adherent cell culture as an example

For a culture volume of 10 L, Sartopore 2 MaxiCaps (1.2 m ² ) can be used.

The same filter can be used for a culture volume of 20 liters.

The conductivity of the pre-treatment & chase buffer can be about 15-30 mS/cm to prevent potential interaction between the AAV vector and the membrane.

Example 5

1.0 Desirable criteria applied for 1.2 L and high volume rAAV vector production process designed for 500-600 ml rAAV harvest volume

1.1 rAAV vector (eg, LK03-FVIII) harvested from 1.2 L bioreactor suspension culture

Process Criteria Units Methods used for analysis Production (titre) for harvest ≥ 5×1010 vector genomes (vg)/ml qPCR

1.2 Purification of rAAV vector from 1.2 L bioreactor suspension culture

Process Criteria Units Methods used for analysis Vector extraction ≥ 30% purified vector qPCR Title of the final composition of the vector ≥ 5×10 ¹² vg/ml qPCR The ratio of empty/non-empty particles in the final composition of the vector ≤ 5 OD ₂₆₀ / OD ₂₈₀ Appearance of the cleaned vector Clear, colorless solution without visible particles visual inspection Admixture of cellular DNA in the final composition of the vector Analysis result, ≤ 100 pg/10 ⁹ vg qPCR Impurity of plasmid DNA in the final composition of the vector Analysis result, ≤ 100 pg/10 ⁹ vg qPCR Cellular protein admixture in final vector composition The result of the analysis, < 30 ng / ml ELISA Benzonase admixture in final vector composition Analysis result, < 0.10 ng/ml ELISA AdE1 admixture in final vector composition Analysis result, ≤ 100 pg/10 ⁹ vg qPCR pH of the final composition of the vector 7.3±0.5 pH measurement of solutions on USP<791>

Example 6

2.0 Production of rAAV vector (eg LK03-FVIII) from 1.2 L bioreactor suspension culture

2.1 The flow chart is shown in Figure 5, which is an example flow chart for the cell harvest portion of the rAAV vector isolation and purification process.

Example 7

As disclosed herein, the lysis methods, the number of columns, and the type of columns can be selected and used in a different order.

4.0 Chromatographic Process for 1.2 L Bioreactor Suspension Culture

4.1 Variables during chromatographic purification of the rAAV vector (eg LK03-FVIII)

4.1.1 The following parameters are designed for the chromatographic part of the rAAV vector isolation and purification process:

Stage Process parameters 1st chromatographic column POROS 50HS (CEX) - cation exchange AVB Sepharose High Performance (affinity) 2nd chromatographic column resin POROS 50HQ (AEX) - anion exchange Superdex 200 Prep Grade (SEC) 3rd chromatographic column resin POROS 50HQ (AEX) - anion exchange Superdex 200 Prep Grade (SEC) Not necessary

4.2 Non-limiting examples of the order of column purification steps (A-E):

1st column, cation exchange (Poros 50HS) → 2nd column, anion exchange (e.g. Poros 50HQ)

1st column, cation exchange (Poros 50HS) → 2nd column, anion exchange (e.g. Poros 50HQ) → 3rd column, size exclusion (e.g. Superdex 200 PG),

1st column, cation exchange (Poros 50HS) → 2nd column, size exclusion (e.g. Superdex 200 PG) → 3rd column, anion exchange (e.g. Poros 50HQ)

1st column, affinity (AVB Sepharose HP) → 2nd column, anion exchange (e.g. Poros 50HQ) → 3rd column (optional), size exclusion (e.g. Superdex 200 PG)

1st column, affinity (AVB Sepharose HP) → 2nd column, size exclusion (e.g. Superdex 200 PG) → 3rd column (optional), anion exchange (e.g. Poros 50HQ)

Example 8

Typical capsid proteins (VP1) of AAV.

AAV-SPK VP1 capsid protein (SEQ ID NO:1)

1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLD

61 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ

121 AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDS

181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV

241 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ

301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA

361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFED

421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW

481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSS

541 GVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS

601 QGALPGMMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADP

661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE

721 GTYSERPIGTRYLTRNL

AAV-LK03 VP1 capsid protein (SEQ ID NO:2)

MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

--->

SEQUENCE LIST

<110> SPARK THERAPEUTICS, INC.

Oh, Younghoon

Qu, Guang

<120> COLUMN METHODS OF VECTOR PURIFICATION BASED ON AAV

<130> 023637-0460469

<140> PCT/US2018/040430

<141> 2018-06-29

<150> 62/527.633

<151> 2017-06-30

<150> 62/531.744

<151> 2017-07-12

<150> 62/567.905

<151> 2017-10-04

<160> 2

<170>PatentIn version 3.5

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Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

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Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

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Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

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Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

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Asn Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

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Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

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Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

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Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

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Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

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Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

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Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

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Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

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<---

Claims (108)

1. A method for purifying vector particles based on recombinant adeno-associated virus (rAAV), where the method excludes ultracentrifugation in a cesium chloride gradient, where this method includes the steps: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the aforementioned cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) with a nuclease to reduce nucleic acid contamination in the lysate, thereby obtaining a lysate depleted in nucleic acids; (e)(i) optionally filtering the aforementioned nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate, and (e)(ii) optionally diluting the aforementioned purified lysate to obtain a diluted purified lysate; (f) applying the above nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e)(i), or the diluted purified lysate obtained in step (e)(ii)-(f)( i), on a cation exchange chromatography column to obtain a column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (f)(ii) optionally diluting the above eluate with columns to obtain a dilute eluate from the column; (g) applying the above eluate from the column from step (f)(i) or the above diluted eluate from the column obtained from step (f)(ii) to (g)(i) onto an anion exchange chromatography column to obtain a second eluate with a column containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (g)(ii) optionally concentrating the aforementioned second column eluate to obtain a concentrated second column eluate; (h) applying the aforementioned second column eluate from step (g)(i) or the aforementioned concentrated second column eluate obtained from step (g)(ii) to (h)(i) to a size exclusion chromatography (SEC) column to obtain a third eluate from the column containing rAAV vector particles, i.e. to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (h)(ii) optionally concentrating the aforementioned third eluate from the column to obtain a concentrated third eluate from the column; and (i) filtering the aforementioned third column eluate from step (h)(i) or the aforementioned concentrated third column eluate obtained from step (h)(ii), thereby obtaining purified rAAV vector particles. 2. A method for purifying vector particles based on recombinant adeno-associated virus (rAAV), where the method excludes ultracentrifugation in a cesium chloride gradient, where this method includes the steps: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the aforementioned cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) with a nuclease to reduce nucleic acid contamination in the lysate, thereby obtaining a lysate depleted in nucleic acids; (e)(i) optionally filtering the aforementioned nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate, and (e)(ii) optionally diluting the aforementioned purified lysate to obtain a diluted purified lysate; (f) applying the above nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e)(i), or the diluted purified lysate obtained in step (e)(ii)-(f)( i), on a cation exchange chromatography column to obtain a column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (f)(ii) optionally concentrating the above eluate with columns for obtaining a concentrated eluate from the column; (g) applying the above eluate from the column from step (f)(i) or the above concentrated eluate from the column obtained from step (f)(ii) to (g)(i) onto a size exclusion chromatography (SEC) column to obtain a second column eluate containing rAAV vector particles, i.e. to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (g)(ii) optionally diluting the aforementioned second column eluate to obtain a diluted second column eluate ; (h) applying the aforementioned second column eluate from step (g)(i) or the aforementioned diluted second column eluate obtained from step (g)(ii) to (h)(i) to an anion exchange chromatography column to obtain a third a column eluate containing rAAV vector particles, that is, to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (h)(ii) optionally diluting the aforementioned third column eluate to obtain a diluted third column eluate; and (i) filtering the aforementioned third column eluate from step (h)(i) or the aforementioned diluted third column eluate obtained from step (h)(ii), thereby obtaining purified rAAV vector particles. 3. A method for purifying vector particles based on recombinant adeno-associated virus (rAAV), where the method excludes ultracentrifugation in a cesium chloride gradient, where this method includes the steps: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the aforementioned cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) with a nuclease to reduce nucleic acid contamination in the lysate, thereby obtaining a lysate depleted in nucleic acids; (e)(i) optionally filtering the aforementioned nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate, and (e)(ii) optionally diluting the aforementioned purified lysate to obtain a diluted purified lysate; (f) applying the above nucleic acid-depleted lysate obtained in step (d), the purified lysate obtained in step (e)(i), or the diluted purified lysate obtained in step (e)(ii)-(f)( i), on a cation exchange chromatography column to obtain a column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (f)(ii) optionally diluting the above eluate with columns to obtain a dilute eluate from the column; (g) applying the above eluate from the column from step (f)(i) or the above diluted eluate from the column obtained from step (f)(ii) to (g)(i) onto an anion exchange chromatography column to obtain a second eluate with a column containing rAAV vector particles, that is, to separate rAAV vector particles from impurities associated with the manufacturing process, and (g)(ii) optionally concentrating the above second column eluate to obtain a concentrated second column eluate; (h) filtering the above second column eluate from step (g)(i) or the above concentrated second column eluate obtained from step (g)(ii), thereby obtaining purified rAAV vector particles. 4. A method for purifying vector particles based on recombinant adeno-associated virus (rAAV), where the method excludes ultracentrifugation in a cesium chloride gradient, where this method includes the steps: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the aforementioned cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) with a nuclease to reduce nucleic acid contamination in the lysate, thereby obtaining a lysate depleted in nucleic acids; (e)(i) optionally filtering the aforementioned nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate, and (e)(ii) optionally diluting the aforementioned purified lysate to obtain a diluted purified lysate; (f) applying the aforementioned nucleic acid-depleted lysate obtained in step (d) or the purified lysate from step (e)(i) or the diluted purified lysate obtained in step (e)(ii)-(f)(i ), to an AAV affinity chromatography column to obtain a column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (f)(ii) optionally diluting the above eluate with columns to obtain a dilute eluate from the column; (g) applying the above eluate from the column from step (f)(i) or the above diluted eluate from the column obtained from step (f)(ii) to (g)(i) onto an anion exchange chromatography column to obtain a second eluate with a column containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (g)(ii) optionally concentrating the aforementioned second column eluate to obtain a concentrated second column eluate; (h) optionally applying the aforementioned second column eluate from step (g)(i) or the aforementioned concentrated second column eluate obtained from step (g)(ii)-(h)(i) to a size exclusion chromatography (SEC) column ) to obtain a third column eluate containing rAAV vector particles, i.e. to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (h)(ii) optionally concentrating the aforementioned third column eluate to obtain a concentrated third eluate from the column; and (i) filtering the aforementioned second eluate from the column from step (g)(i) or the aforementioned concentrated second eluate from the column obtained from step (g)(ii), or filtering the aforementioned third eluate from the column from step (h)(i) or the aforementioned concentrated third column eluate obtained in step (h)(ii), thus obtaining purified rAAV vector particles. 5. A method for purifying vector particles based on recombinant adeno-associated virus (rAAV), where the method excludes ultracentrifugation in a cesium chloride gradient, where this method includes the steps: (a) harvesting cells and/or cell culture supernatant containing rAAV-based vector particles to harvest cells; (b) optionally concentrating the aforementioned cell harvest obtained in step (a) to obtain a concentrated cell harvest; (c) lysing the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) to obtain a lysate; (d) treating the lysate obtained in step (c) with a nuclease to reduce nucleic acid contamination in the lysate, thereby obtaining a lysate depleted in nucleic acids; (e)(i) optionally filtering the aforementioned nucleic acid-depleted lysate obtained in step (d) to obtain a purified lysate, and (e)(ii) optionally diluting the aforementioned purified lysate to obtain a diluted purified lysate; (f) applying the above nucleic acid-depleted lysate from step (d) or the purified lysate from step (e)(i) or the diluted purified lysate from step (e)(ii)-(f)(i) , to an AAV affinity chromatography column to obtain a column eluate containing rAAV vector particles, i.e. to separate rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (f)(ii) optionally concentrating the above eluate from the column to obtain a concentrated eluate from the column; (g) applying the above eluate from the column from step (f)(i) or the above concentrated eluate from the column obtained from step (f)(ii) to (g)(i) onto a size exclusion chromatography (SEC) column to obtain a second column eluate containing rAAV vector particles, i.e. to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (g)(ii) optionally diluting the aforementioned second column eluate to obtain a diluted second column eluate ; (h) optionally applying the aforementioned second column eluate from step (g)(i) or the aforementioned diluted second column eluate obtained from step (g)(ii) to (h)(i) to an anion exchange chromatography column to obtain a third column eluate containing rAAV vector particles, i.e. to separate the rAAV vector particles from protein impurities or other impurities associated with the manufacturing process, and (h)(ii) optionally diluting the aforementioned third column eluate to obtain a diluted third column eluate ; and (i) filtering the aforementioned second eluate from the column from step (g)(i) or the aforementioned diluted second eluate from the column obtained from step (g)(ii), or filtering the aforementioned third eluate from step (h)(i) from the column or the aforementioned diluted third column eluate obtained in step (h)(ii), thus obtaining purified rAAV vector particles. 6. The method according to any one of paragraphs. 1-5, in which the aforementioned concentration in step (b) and/or step (f) and/or step (g) and/or step (h) is carried out by ultrafiltration/diafiltration, optionally by tangential flow filtration (TFF). 7. The method according to any one of paragraphs. 1-6, wherein the aforementioned concentration in step (b) results in a volume reduction of the aforementioned harvested cells and cell culture supernatant by about 2-20 times. 8. The method according to any one of paragraphs. 1-7, in which the aforementioned concentration in step (f) and/or in step (g) and/or in step (h) leads to a decrease in the volume of the aforementioned eluate from the column by about 5-20 times. 9. The method according to any one of paragraphs. 1-8, in which the aforementioned lysis of the aforementioned cell harvest obtained in step (a) or the aforementioned concentrated cell harvest obtained in step (b) is carried out by a physical or chemical method. 10. The method of claim 9 wherein the physical method comprises microfluidization and homogenization. 11. The method of claim 9, wherein the chemical method comprises a detergent, optionally a non-ionic detergent such as Triton X-100, optionally at a concentration in the range of from about 0.1% to 1.0%, inclusive. 12. The method according to any one of paragraphs. 1-11, in which the nuclease includes benzonase. 13. The method according to any one of paragraphs. 1-12, in which the aforementioned filtration of the aforementioned purified lysate or the aforementioned diluted purified lysate obtained in step (e) is carried out with a filter having a pore diameter in the range of about 0.1 to 10 microns, inclusive. 14. The method according to any one of paragraphs. 1-13, in which the aforementioned dilution of the aforementioned purified lysate obtained in step (e) is carried out with an aqueous phosphate, acetate or Tris buffer solution. 15. The method according to any one of paragraphs. 1-14, in which the above dilution of the above column eluate obtained in step (f) or the above second column eluate obtained in step (g) is carried out with an aqueous phosphate, acetate or Tris buffer solution. 16. The method of claim 14 or 15, wherein said aqueous phosphate or acetate buffer solution has a pH in the range of about 4.0 to 7.4, inclusive. 17. The method of claim 14 or 15, wherein the aforementioned aqueous Tris buffer solution has a pH greater than 7.5, optionally from about 8.0 to 9.0, inclusive. 18. The method according to any one of paragraphs. 1-17, wherein the aforementioned rAAV vector particles obtained in step (i) are formulated with a surfactant to obtain an rAAV vector composition. 19. The method according to any one of paragraphs. 1-18, wherein the aforementioned anion exchange column chromatography in step (f), (g) and/or (h) comprises polyethylene glycol (PEG) column chromatography. 20. The method of claim 19 wherein said anion exchange column chromatography in step (g) and/or (h) comprises washing said column with a PEG solution before eluting said rAAV vector particles from the column. 21. The method of claim 19 or 20, wherein the PEG has an average molecular weight in the range of about 1,000 to 80,000 g/mol, inclusive. 22. The method according to any one of paragraphs. 19-21, in which the PEG has a concentration of from about 4% to about 10%, inclusive. 23. The method according to any one of paragraphs. 1-22, wherein said anion exchange column chromatography in step (g) and/or (h) comprises washing said column with an aqueous surfactant solution before eluting said rAAV vector particles from the column. 24. The method according to any one of paragraphs. 1-23, wherein the aforementioned cation exchange column chromatography in step (f) comprises washing the aforementioned column with an aqueous surfactant solution before eluting the aforementioned rAAV vector particles from the column. 25. The method according to any one of paragraphs. 20-24, wherein said PEG solution and/or said surfactant solution comprises an aqueous Tris-Cl/NaCl buffer, an aqueous phosphate/NaCl buffer, or an aqueous acetate/NaCl buffer. 26. The method of claim 25, wherein said NaCl buffer contains about 20-300 mM NaCl, inclusive, or about 50-250 mM NaCl, inclusive. 27. The method according to any one of paragraphs. 23-25, wherein said surfactant includes a cationic or anionic surfactant. 28. The method according to any one of paragraphs. 23-27, in which the aforementioned surfactant includes a surfactant with twelve carbons in the chain. 29. The method according to any one of paragraphs. 23-28, wherein said surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarcosyl. 30. The method according to any one of paragraphs. 1-29, in which the above rAAV vector particles are eluted from the above anion exchange column in step (f), (g) and/or (h) with aqueous Tris-Cl/NaCl buffer. 31. The method of claim 30, wherein the aforementioned Tris-Cl/NaCl buffer contains 100-400 mM NaCl, inclusive, and optionally has a pH in the range of about 7.5 to about 9.0, inclusive. 32. The method according to any one of paragraphs. 1-31, in which the above anion exchange column in step (f), (g) and/or (h) is washed with an aqueous Tris-Cl/NaCl buffer. 33. The method of claim 32 wherein said NaCl in said aqueous Tris-Cl/NaCl buffer has a concentration in the range of about 75-125 mM, inclusive. 34. The method according to any one of paragraphs. 30-32, wherein the aforementioned aqueous Tris-Cl/NaCl buffer has a pH of about 7.5 to about 9.0, inclusive. 35. The method according to any one of paragraphs. 1-34, wherein the above anion exchange column in step (f), (g) and/or (h) is washed one or more times to reduce the amount of empty AAV capsids in the second or third eluate from the column. 36. The method according to claim 32 or 35, wherein washing the above anion exchange column results in the removal of empty AAV capsids from the column prior to removal of rAAV and/or instead of rAAV, which results in a decrease in the number of empty AAV capsids in the second or third eluate from the column. 37. The method of claim 32 or 35, wherein washing the above anion exchange column results in the removal of at least about 50% of the total empty AAV capsids from the column prior to removal of rAAV and/or instead of rAAV, resulting in a reduction in empty AAV capsids in the second or third eluate from the column by about 50%. 38. The method according to any one of paragraphs. 32 or 35-37, wherein the aforementioned NaCl in the aforementioned aqueous Tris-Cl/NaCl buffer has a concentration in the range of about 110-120 mM, inclusive. 39. The method according to any one of paragraphs. 32-38, wherein the ratio and/or amount of the aforementioned elutable rAAV vector particles and empty AAV capsids is controlled by the aforementioned wash buffer. 40. The method according to any one of paragraphs. 1-39, wherein the aforementioned rAAV vector particles are eluted from the aforementioned cation exchange column in step (f) in aqueous phosphate/NaCl buffer or aqueous acetate/NaCl buffer. 41. The method of claim 40, wherein said phosphate/NaCl buffer or aqueous acetate/NaCl buffer contains about 125-500 mM NaCl, inclusive. 42. The method of claim 40, wherein said phosphate/NaCl buffer or aqueous acetate/NaCl buffer has a pH in the range of about 5.5 to about 7.5, inclusive. 43. The method according to any one of paragraphs. 1-42, wherein said anion exchange column in step (f), (g) and/or (h) contains a quaternary ammonium functional group such as quaternized polyethyleneimine. 44. The method according to any one of paragraphs. 1-43, in which the separation/fractionation range (molecular weight) of the above column for size exclusion (SEC) chromatography in stage (g) and/or (h) is from about 10,000 to about 600,000 inclusive. 45. The method according to any one of paragraphs. 1-44, wherein the aforementioned cation exchange column in step (f) contains a sulfonic acid functional group such as a sulfopropyl group. 46. The method according to any one of paragraphs. 3-45, wherein the AAV affinity chromatography column contains a protein or ligand that binds to the AAV capsid protein. 47. The method of claim 46, wherein the protein comprises an antibody that binds to the AAV capsid protein. 48. The method of claim 47 wherein the antibody that binds to the AAV capsid protein comprises a Llama single chain antibody (Camelid). 49. The method according to any one of paragraphs. 1-48, in which the method eliminates the step of ultracentrifugation in a cesium chloride gradient. 50. The method according to any one of paragraphs. 1-49, wherein the aforementioned rAAV vector particles contain a transgene that encodes a nucleic acid selected from the group consisting of miRNA, antisense molecule, microRNA, ribozyme, and small shRNA hairpin RNAs. 51. The method according to any one of paragraphs. 1-49, wherein the aforementioned rAAV vector particles contain a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRP), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, 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 α (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF -II), TGFβ, activins, inhibins, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary n neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neuroturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. 52. The method according to any one of paragraphs. 1-49, wherein the aforementioned rAAV vector particles contain a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 to IL-17), monocytic chemoattractant 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. 53. The method according to any one of paragraphs. 1-49, in which the above-mentioned rAAV vector particles contain a transgene encoding a protein necessary for the correction of congenital metabolic disorders, selected from the group consisting of 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, cystathion beta synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl β-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence. 54. The method according to any one of paragraphs. 1-53, wherein the aforementioned rAAV vector particles contain a transgene that encodes factor VIII or factor IX. 55. The method according to any one of paragraphs. 1-54, wherein approximately 50-90% of the total rAAV vector particles are obtained by this method from the harvest obtained in step (a) or the concentrated harvest of cells obtained in step (b). 56. The method according to any one of paragraphs. 1-55, in which this method produces rAAV vector particles of higher purity than rAAV vector particles obtained or purified by a single purification on an AAV affinity column. 57. The method according to any one of paragraphs. 1-56, in which steps (c) and (d) are carried out essentially simultaneously. 58. The method according to any one of paragraphs. 25-57, wherein the NaCl concentration is adjusted to be in the range of about 100-400 mM NaCl, inclusive, or in the range of about 140-300 mM NaCl, inclusive, after step (c) but before step (f). 59. The method according to any one of paragraphs. 1-58, wherein the above rAAV vector particles are based on an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10 and Rh74. 60. The method according to any one of paragraphs. 1-59, wherein the above rAAV vector particles contain a capsid sequence 70% or more identical to the capsid sequence of SEQ ID NO:1 or SEQ ID NO:2 of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV10, Rh10, Rh74. 61. The method according to any one of paragraphs. 1-60, wherein the above rAAV vector particles contain an ITR sequence that is 70% or more identical to the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, or Rh74. 62. The method according to any one of paragraphs. 1-61, in which the above cells are a suspension or attached cells. 63. The method according to any one of paragraphs. 1-62, wherein said cells are mammalian cells. 64. The method according to any one of paragraphs. 1-63, wherein said cells are HEK-293 cells.

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