Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

Gene Ther. 2001 Sep;8(17):1323-32. doi: 10.1038/sj.gt.3301529.


This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.

MeSH terms

  • Animals
  • Blotting, Southern / methods
  • Blotting, Western / methods
  • Brain / enzymology*
  • Cells, Cultured
  • Dependovirus / genetics*
  • Gene Expression
  • Genetic Therapy / methods*
  • Genetic Vectors / administration & dosage
  • Genetic Vectors / genetics*
  • Injections
  • Luciferases / genetics*
  • Rats
  • Rats, Wistar


  • Luciferases