SCALABLE MANUFACTURING PLATFORM FOR VIRAL VECTOR PURIFICATION AND VIRAL VECTORS SO PURIFIED FOR USE IN GENE THERAPY

• US 20190321463A1
• Filed: 05/23/2019
• Published: 10/24/2019
• Est. Priority Date: 01/28/2010
• Status: Abandoned Application

• Current Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Original Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Inventors: Wright, John Fraser, Qu, Guang, Hauck, Bernd, High, Katherine A.
• Application Number:
  US16/421,397
• Publication Number:
  US 20190321463A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 39/23 Parvoviridae, e.g. feline panleukopenia virus
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0091 Purification or manufacturing processes for gene therapy compositions
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/864 Parvoviral vectors , e.g. parvovirus, densovirus
• CPC Class Codes: C12N 15/8645 Adeno-associated virus
• CPC Class Codes: C12N 2750/14011 Parvoviridae
• CPC Class Codes: C12N 2750/14051 Methods of production or purification of viral material
• CPC Class Codes: C12N 2750/14111 Dependovirus, e.g. adenoassociated viruses
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Y 301/01064 Retinoid isomerohydrolase (3.1.1.64)

Abstract

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

Claims

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 10¹⁵ particles per mL.

4. The method of claim 1, wherein said AAV particles of step i) are present at a concentration of 10¹⁶ particles per mL.

5. The method of claim 1, wherein said AAV particles of step i) are present at a concentration of 10¹⁷ 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.