Production and discovery of novel recombinant adeno-associated viral vectors

Curr Protoc Microbiol. 2012 Aug;Chapter 14:Unit14D.1. doi: 10.1002/9780471729259.mc14d01s26.

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.

MeSH terms

  • Cell Line
  • Dependovirus / genetics*
  • Dependovirus / isolation & purification
  • Dependovirus / metabolism
  • Genetic Vectors / genetics*
  • Genetic Vectors / isolation & purification
  • Genetic Vectors / metabolism
  • Humans
  • Real-Time Polymerase Chain Reaction / methods
  • Transfection / methods*
  • Ultracentrifugation / methods
  • Virus Cultivation / methods*