Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing
Abstract
Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.
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Copyright 2010, Mary Ann Liebert, Inc.
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Published in print: October 2010
Published ahead of print: 16 September 2010
Published online: 14 September 2010
Published ahead of production: 22 July 2010
Accepted: 19 July 2010
Received: 25 May 2010
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L.H.V. and M.L. are inventors on patents licensed to various biopharmaceutical companies, including ReGenX. J.M.W. is a consultant to ReGenX Holdings, and is a founder of, holds equity in, and receives a grant from affiliates of ReGenX Holdings; in addition, he is an inventor on patents licensed to various biopharmaceutical companies, including affiliates of ReGenX Holdings.
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