Unit

UNIT 14D.1 Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors

  1. Christian Mueller1,2,
  2. Dmitry Ratner1,2,
  3. Li Zhong1,3,
  4. Miguel Esteves-Sena1,4,
  5. Guangping Gao1,5,6

Published Online: 1 AUG 2012

DOI: 10.1002/9780471729259.mc14d01s26

Current Protocols in Microbiology

Current Protocols in Microbiology

How to Cite

Mueller, C., Ratner, D., Zhong, L., Esteves-Sena, M. and Gao, G. 2012. Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors. Current Protocols in Microbiology. 26:D:14D.1:14D.1.1–14D.1.21.

Author Information

  1. 1

    Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts

  2. 2

    Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts

  3. 3

    Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

  4. 4

    Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts

  5. 5

    Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, Massachusetts

  6. 6

    State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, People's Republic of China

Publication History

  1. Published Online: 1 AUG 2012

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. Curr. Protoc. Microbiol. 26:14D.1.1-14D.1.21. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • rAAV;
  • AAV production;
  • transfection;
  • AAV packaging;
  • cap;
  • cesium chloride