Scalable manufacturing platform for viral vector purification and viral vectors so purified for use in gene therapy

Abstract

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.

What is claimed is: 
     
         1 . A method for purifying bona fide AAV vector particles comprising a transgene encoding a therapeutic protein or fragment thereof from an AAV preparation comprising AAV vector particles, empty capsids and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids, said method comprising:
 a) harvesting cells comprising recombinant AAV;   b) concentrating said cells via Tangential Flow Filtration   c) lysing said cells by microfluidization to form a lysate;   d) filtering, thereby clarifying the lysate of step c);   e) purifying AAV particles by Ion Exchange Column Chromatography and optionally concentrating column eluate by Tangential Flow Filtration;   f) mixing said eluate with cesium chloride and subjecting said mixture to centrifugation, thereby forming a gradient;   g) collecting viral particles separated in step f) and subjecting the same buffer exchange by Tangential Flow Filtration;   h) formulating purified AAV particles with surfactant to provide an AAV particle formulation;   i) filtering said formulation to remove any remaining impurities, wherein said bona fide AAV vector particles are present in said AAV product in an amount of at least 95%.   
     
     
         2 . The method of  claim 1 , wherein said AAV vector particles are present at a concentration of 100 mg/mL. 
     
     
         3 . The method of  claim 1 , wherein said AAV particles of step i) are present at a concentration of 10 15  particles per mL. 
     
     
         4 . The method of  claim 1 , wherein said AAV particles of step i) are present at a concentration of 10 16  particles per mL. 
     
     
         5 . The method of  claim 1 , wherein said AAV particles of step i) are present at a concentration of 10 17  particles per mL. 
     
     
         6 . The method of  claim 1 , wherein said AAV vector particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9. 
     
     
         7 . An AAV vector formulation comprising AAV particles purified using the method of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         8 . The method of  claim 1 , wherein said transgene encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, and a miRNA a ribozyme and a shRNA. 
     
     
         9 . The method of  claim 1  wherein said transgene encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), 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 growth factors I and II (IGF-I and IGF-II), TGFβ, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. 
     
     
         10 . The method of  claim 1 , wherein said transgene encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through 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, 
     
     
         11 . The method of  claim 1 , wherein said transgene comprises a nucleic acid encoding a protein useful for correction of in born errors of metabolism selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence. 
     
     
         12 . The method of  claim 11 , wherein the gene product is Factor VIII or Factor IX. 
     
     
         13 . The method of  claim 1 , comprising collecting the empty capsid fraction separated in step f). 
     
     
         14 . A highly purified empty capsid preparation obtained from the method of  claim 13 .