title: Production and discovery of novel recombinant adeno-associated viral vectors.
abstract: In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.
authors.affiliation: Gene Therapy Center, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Mueller
authors.forename: Dmitry
authors.initials: D
authors.lastname: Ratner
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.forename: Miguel
authors.initials: M
authors.lastname: Esteves-Sena
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
mesh_terms: D002460:Cell Line; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods*; D014461:Ultracentrifugation / Q000379:methods; D014776:Virus Cultivation / Q000379:methods*