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.
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Acknowledgments
This work is supported by NIH P50 HL112324, R01 HL119046, R01 HL117505, R01 HL128099, R01 HL129814, R01 HL131404 and Trans- Atlantic Network of Excellence grants 13CVD01 and 14CVD03 from the Leducq Foundation. We would also like to acknowledge the Gene Therapy Resource Program (GTRP) of the National Heart, Lung, and Blood Institute, National Institutes of Health for providing some of the gene vectors used in these studies.
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Kohlbrenner, E., Weber, T. (2017). Production and Characterization of Vectors Based on the Cardiotropic AAV Serotype 9. In: Ishikawa, K. (eds) Cardiac Gene Therapy. Methods in Molecular Biology, vol 1521. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6588-5_6
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DOI: https://doi.org/10.1007/978-1-4939-6588-5_6
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