title: Production and Characterization of Vectors Based on the Cardiotropic AAV Serotype 9.
abstract: Vectors based on adeno-associated virus serotype 9 (AAV9) efficiently transduce cardiomyocytes in both rodents and large animal models upon either systemic or regional vector delivery. In this chapter, we describe the most widely used production and purification method of AAV9. This production approach does not depend on the use of a helpervirus but instead on transient transfection of HEK293T cells with a plasmid containing the recombinant AAV genome and a second plasmid encoding the AAV9 capsid proteins, the AAV Rep proteins and the adenoviral helper functions. The recombinant AAV is then purified by iodixanol density gradient centrifugation. This chapter also describes in detail the characterization and quality control methods required for assuring high quality vector preparations, which is of particular importance for experiments in large animal models.
authors.affiliation: Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1030, New York, NY, 10029-6574, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Kohlbrenner
authors.affiliation: Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1030, New York, NY, 10029-6574, USA. thomas.weber@mssm.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weber
mesh_terms: D002213:Capsid / Q000378:metabolism; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics* / Q000648:ultrastructure; D003956:Dialysis; D005822:Genetic Vectors / Q000378:metabolism*; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D065288:Serogroup; D014162:Transfection; D014461:Ultracentrifugation; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure
keywords: AAV; AAV characterization; AAV production; AAV9; Adeno-associated virus; Cardiotropic; Gene therapy; Serotype