Large commercial scale lentiviral vector production system and vectors produced thereby

Abstract

In accordance with the present invention, a method for increasing the yield of rLV vector particles comprising a trans gene encoding a therapeutic protein or fragment thereof is disclosed. In one approach, cells are transfected with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix at pH 7.1 wherein the calcium chloride and plasmids form a complex which is added to the cells at a constant speed. The cells are then incubated for a suitable time period wherein virus particle media is collected at least twice during the incubation period and stored in a cold storage unit, thereby reducing virus inactivation.

What is claimed is: 
     
         1 . A method for increasing the production of recombinant lentivirus (rLV) comprising a therapeutically beneficial transgene, comprising
 a) transfecting cells with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix, said calcium chloride and said plasmids forming a complex, said complex being added to said cells at a uniform constant speed;   b) incubating said cells for 56 hours, wherein virus particle containing media is collected at 32 hours, replaced with fresh media which is collected at 56 hours, said collected media being placed in a cold storage unit, thereby reducing virus inactivation;   c) concentrating the viral particles in said media via tangential flow filtration;   d) treating said particles with an enzyme which reduces nucleic acid contamination;   e) centrifuging the treated particles of step d) to further concentrate said rLV; and   f) layering the concentrated rLV of step e) over a sucrose gradient, thereby producing rLV at high yield.   
     
     
         2 . The method of  claim 1 , wherein said rLV is produced at approximately 3×10 8 /ml. 
     
     
         3 . An rLV vector formulation comprising rLV particles purified using the method of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         4 . 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. 
     
     
         5 . 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. 
     
     
         6 . 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, 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 7 , wherein the gene product is Factor VIII or Factor IX. 
     
     
         9 . The method of  claim 1 , wherein said calcium chloride mix is at pH 7.1.