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 10 3 and 10 5 DNA-containing particles per cell depending on the cell type targeted. Moreover, based on large animal studies, a clinical dose in humans will require 10 12 –10 14 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. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.