European Society of Gene & Cell Therapy location:Brugge, Belgium date:13-16 November 2008
Background: Recombinant adeno-associated viral (rAAV) vectors have recently been described to be efficient vehicles for gene transfer into the central nervous system. Previous studies using viral vectors have shown that production and purification methods greatly influence the immunogenicity and toxicity after stereotactic injection in the mouse brain.
Methods: Recombinant AAV vectors were produced under serum-free conditions in 293T cells using a CMV-eGFP-T2A-Fluc expression cassette and packaged with AAV7 capsid (rAAV2/7). Vectors were further purified and concentrated using ultracentrifugation, a iodyxanol gradient or size-exclusion concentrators. Each vector batch was stereotactically injected undiluted and a 20-fold dilution into the mouse striatum. Transduction efficiency and distribution of the vector was determined by in vivo bioluminescence imaging (BLI), immunohistochemical analysis of eGFP expression and Cavalieri measurements of transduced volume. Locoregional toxicity was assessed using a Chresyl-Violet staining procedure.
Results and Conclusion: All purification methods resulted in functional AAV2/7 vector under serum free conditions, showing efficient transduction of the mouse striatum. Dilution of the vector resulted in 20-fold decrease of the BLI signal and a smaller eGFP transduced volume. Co-localisation of eGFP and NeuN demonstrated efficient transduction of neuronal cells. Some undiluted vector preps transduced excessively outside the striatal area and resulted in mild toxicity. After dilution, no toxicity could be detected whereas the striatum was still efficiently transduced.
Our initial results underscore the relevance to study new rAAV production and purification methods for gene delivery in the brain.