5th Australasian Gene Therapy Society Meeting : Date April 18–20,2007 Venue Shine Dome Academy of Science, Canberra, ACT, Australia
- Book ID
- 102337755
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 269 KB
- Volume
- 9
- Category
- Article
- ISSN
- 1099-498X
- DOI
- 10.1002/jgm.1059
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✦ Synopsis
Large vector doses are necessary for developing successful therapies employing non-replicating viral vectors. This can be especially important in the treatment of many catastrophic diseases, such as cancer or the muscular dystrophies. Among non-replicating viral vectors, recombinant adeno-associated virus (rAAV) has numerous advantages. Typically, rAAV is produced by co-transfection of adherent HEK 293 cells.
Although relatively efficient at small scale, expansion of adherent cells is not feasible beyond a certain size. In contrast, volumetric expansion of suspension cells provides a means for increasing cell number over several orders of magnitude, limited by the available vessel volume. To produce rAAV in suspension cells, we engineered three recombinant baculovirus expression vectors (BEVs) providing the cis and trans elements required for efficient rAAV production in Sf9 insect cells. Improved culture conditions increased the yield of rAAV recovered from a bioreactor to approximately 2 × 10 14 DNase-resistant particles per liter. Our advances in large-scale production of rAAV have allowed us to address the treatment of solid tumors and Duchenne muscular dystrophy (DMD). For cancer therapy, we have used shRNA-expressing rAAV to target Hec1, an essential kinetochore protein. Hec1 depletion prevents chromatid attachment to microtubules; as a result, the affected cells arrest in mitosis and die, whereas non-dividing cells are unaffected. To treat DMD, we are investigating an exon skipping strategy to ameliorate the disease via restoration of functional dystrophin. We believe that rAAV production in insect cells will provide the scalable, cost-effective, and current good manufacturing practice (cGMP)-compliant process required to support clinical trials in these, and other, important areas.