Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses

Gene transfer vectors based on the replication-defective human parvovirus, adeno-associated virus type 2 (AAV-2), are viable candidates for irt vivo a nd ex vivo human use. However, widespread testing of AAV vectors has been l imited by difficulties in generating pure, high-titer vector stocks that ar e fully characterized. To address these issues, we have developed a single- step purification scheme using heparin affinity chromatography, Recovery fr om the crude lysate starting material exceeds 70%, and the end product rAAV vector is highly purified and appears to be free of adenovirus and cellula r contaminates. Importantly, purified vectors retain predicted in vivo biol ogic activity. Concurrently, we have developed simple and rapid approaches for vector quantification using real-time PCR, These new methods, combined with the use of stable producer cell lines for rAAV production, make the co mmercial production of rAAV vectors for human use truly viable and pragmati c.