Highly Purified Recombinant Adeno-Associated Virus Vectors: Preparation and Quantitation

Adeno-associated virus (AAV) is a nonpathogenic, replication-defective parvovirus that is being developed as a vector for human gene transfer. The recent interest in recombinant (r)AAV has been driven by the unexpected finding that these simple vectors can efficiently transduce a variety of postmitotic cells in vivo, resulting in robust long-term gene expression. Efficient in vivo gene transfer via rAAV transduction requires reasonably high multiplicities of infection, estimated to be between 103 and 105 DNA-containing particles per cell depending on the cell type targeted. Moreover, based on large animal studies, a clinical dose in humans will require 1012–1014 rAAV vector particles, depending on the level of therapeutic protein expression needed for treatment efficacy (1). Therefore, the ability to produce pure, high-titer rAAV is critical for clinical applications. This chapter discusses the isolation of stable rAAV producer cell lines and the associated downstream methods of vector purification and quantitation.