Method of preparing recombinant adeno-associated virus compositions

Abstract

Disclosed are methods for the isolation and purification of high-titer recombinant adeno-associated virus (rAAV) compositions. Also disclosed are methods for reducing or eliminating the concentration of helper adenovirus in rAAV samples. Methods are disclosed that provide highly-purified rAAV stocks having titers up to about 10 13 particles/ml at particle-to-infectivity ratios of less than 100 in processes that are accomplished about 24 hours or less.

What is claimed is:  
     
         1 . A method of isolating a recombinant adeno-associated virus, comprising applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting said recombinant adeno-associated virus from said gradient.  
     
     
         2 . The method of  claim 1 , wherein said iodixanol gradient is a discontinuous gradient.  
     
     
         3 . The method of  claim 2 , wherein said iodixanol gradient comprises an about 15% iodixanol step, an about 25% iodixanol step, an about 40% iodixanol step, and an about 60% iodixanol step.  
     
     
         4 . The method of  claim 3 , wherein said recombinant adeno-associated virus is collected from said 40% iodixanol step.  
     
     
         5 . The method of  claim 3 , wherein said 15% iodixanol step further comprises about 1 M NaCl.  
     
     
         6 . The method of  claim 1 , wherein said iodixanol gradient is subjected to centrifugation after applying said sample.  
     
     
         7 . The method of  claim 1 , further comprising contacting said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix, removing non-bound species from said matrix, and eluting said virus from said matrix.  
     
     
         8 . The method of  claim 7 , wherein said matrix comprises heparin agarose type I or heparin agarose type II-S.  
     
     
         9 . The method of  claim 7 , wherein said matrix is comprised within an HPLC column.  
     
     
         10 . The method of  claim 7 , wherein said virus is eluted from said matrix with a solution comprising about 1 M NaCl.  
     
     
         11 . The method of  claim 1 , further comprising contacting said recombinant adeno-associated virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix, and collecting the non-interacting virus from said hydrophobic matrix.  
     
     
         12 . The method of  claim 11 , wherein said hydrophobic matrix comprises phenyl groups.  
     
     
         13 . The method of  claim 12 , wherein said hydrophobic matrix is phenyl-sepharose.  
     
     
         14 . The method of  claim 1 , further comprising applying said recombinant adeno-associated virus to a cesium chloride equilibrium density gradient, and collecting said recombinant adeno-associated virus from said gradient.  
     
     
         15 . The method of  claim 1 , further comprising contacting said recombinant adeno-associated virus with at least a first ion exchange chromatography medium, under conditions effective to permit interaction of said virus with said medium, removing non-interacting species from said medium, and eluting said virus from said medium.  
     
     
         16 . The method of  claim 1 , wherein said sample further comprises a virus.  
     
     
         17 . The method of  claim 16 , wherein said sample further comprises an adenovirus.  
     
     
         18 . The method of  claim 1 , wherein said sample further comprises at least a first polypeptide or protein.  
     
     
         19 . The method of  claim 1 , wherein said sample further comprises a cell extract or a growth medium.  
     
     
         20 . A method of isolating a recombinant adeno-associated virus, comprising the steps of: 
 a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;    b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;    c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;    d) removing non-bound species from said matrix; and    e) eluting said virus from said matrix.    
     
     
         21 . A method of isolating a recombinant adeno-associated virus, comprising the steps of: 
 a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;    b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;    c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;    d) removing non-bound species from said matrix;    e) eluting said virus from said matrix;    f) contacting the eluted virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix; and    g) collecting the non-interacting virus from said hydrophobic matrix.    
     
     
         22 . A method for reducing or eliminating adenovirus from a recombinant adeno-associated virus composition contaminated with adenovirus, comprising applying a sample containing recombinant adeno-associated virus and adenovirus to an iodixanol gradient, and collecting from said gradient at least a first fraction comprising said recombinant adeno-associated virus.  
     
     
         23 . A method of producing a recombinant adeno-associated virus having a particle-to-infectivity ratio of less than about 100 to 1, comprising the steps of: 
 a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient;    b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus;    c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix;    d) removing non-bound species from said matrix; and    e) eluting said virus from said matrix.    
     
     
         24 . Recombinant adeno-associated virus, prepared by applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting said recombinant adeno-associated virus from said gradient.  
     
     
         25 . A kit comprising, in a suitable container, iodixanol, a matrix comprising heparin and instructions for isolating recombinant adeno-associated virus.  
     
     
         26 . The kit of  claim 25 , wherein said iodixanol is formulated as an iodixanol gradient.  
     
     
         27 . The kit of  claim 25 , wherein said matrix comprises heparin agarose type I or heparin agarose type II-S.  
     
     
         28 . The kit of  claim 25 , further comprising a hydrophobic matrix.  
     
     
         29 . The kit of  claim 28 , wherein said hydrophobic matrix comprises phenyl groups.  
     
     
         30 . The kit of  claim 29 , wherein said hydrophobic matrix is phenyl-sepharose.