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.

Description

[0001] The present application claims the priority of U.S. Provisional Patent Application Serial No. 60 / 086,898 filed May 27, 1998, the entire disclosure of which is incorporated herein by reference without disclaimer. The government may have certain rights in the present invention pursuant to grant numbers PO1 HL59412 and PO1 NS36302 from the National Institutes of Health.1.0 BACKGROUND OF THE INVENTION[0002] 1.1 Field of the Invention[0003] The present invention relates generally to the field of virology, and in particular, to methods for preparing highly-purified, high-titer recombinant adeno-associated virus compositions. In certain embodiments, the invention concerns the use of equilibrium density centrifugation techniques, affinity chromatographic media, and in certain embodiments anion- and cation-exchange resins, to remove rAAV particles from solution and to prepare highly purified viral stocks for use in a variety of investigative, diagnostic and therapeutic regimens. Metho...

AI Technical Summary

Benefits of technology

[0017] In certain embodiments, to improve the yield and / or recovery of virus particles from such a gradient, one may add to one or more steps of the gradient one or more salts to reduce or prevent aggregation of the virus and any cellular debris or proteins, polypeptides, etc. which may be present in the crude sample. In an exemplary embodiment, the inventors have shown that the addition of salt to the 15% iodixanol step in a discontinuous gradient improves the recovery of virus particles from an iodixanol gradient. As an example, the addition of NaCl to a final concentration of about 1 M in the 15% step was found by the inventors to be particularly advantageous in recovery of purified rAAV particles from the 40% step of such a gradient. While addition of one or more salts to one or more of the other steps in the gradient may be performed as required, in most instances, the inventors have shown that the presence of salt in other steps were either unnecessary or unwarranted. In situations where one or more salts are added to a layer which comprises the rAAV particles, following centrifugation it may be desirable to remove or reduce the concentration of salt in such a fraction prior to use of, or further purification of, the rAAV. Such removal may readily be achieved by dialysis, microconcentration, ultrafiltration, and the like.

[0031] A method for reducing the concentration of adenovirus in a recombinant adeno-associated virus composition is also provided that generally involves centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient, collecting from the iodixanol gradient at least a first fraction comprising the recombinant adeno-associated virus, contacting the at least a first fraction comprising the recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, removing any non-bound species from the matrix, and eluting the virus from the matrix.

Problems solved by technology

While addition of one or more salts to one or more of the other steps in the gradient may be performed as required, in most instances, the inventors have shown that the presence of salt in other steps were either unnecessary or unwarranted.

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