996. Direct Comparison of Adeno-Associated Virus (AAV) Serotypes for Muscle Expression of Soluble Anti-Angiogenic Proteins

Skeletal muscle has been considered an attractive target for gene delivery because of its large size, good capacity for protein synthesis, and easy accessibility for intramuscular injection. Direct injection of viral vectors into muscles was widely used not only for the treatment of muscular disorders but also for expression of secreted proteins to treat variety of diseases such as hemophilia, lysosomal storage disorders, and cancer. Adeno-associated viral (AAV) vectors have been successfully used for long-term expression of transgene products in skeletal muscle. Recently, a number of novel AAV serotypes were isolated from nonhuman primates. In the present work, we attempted to determine which AAV serotype is suitable for muscle expression of anti-angiogenic proteins for systemic cancer gene therapy. We constructed AAV vectors (AAV2/1, AAV2/2, AAV2/ 7, and AAV2/8) expressing secretable murine endostatin (mEnd) or human soluble Flk-1 (hsFlk-1) and examined the secreted therapeutic protein levels in vitro and in vivo. High level of secreted proteins was observed in supernatant of AAV transduced murine myoblast cell line (C2C12) by ELISA analysis. The highest protein level of both mEnd and hsFlk-1 was obtained from AAV2/1 transduced C2C12 cells (1>>8>7>2). On the other hand, when these vectors (5x1010 particle each) were injected into a quadriceps muscle of immunocompetent C57BL/6 mice, the highest serum level of therapeutic protein was observed in AAV2/8 injected mice (8>7>1>2) one week after injection. Sustained expression of mEnd was detected at least for four months. In contrast, the serum level of hsFlk-1 was rapidly decreased to the background level two weeks after injection, although ELISA analysis demonstrated continuous expression of hsFLK-1 in the muscle. There is an apparent discrepancy between the transduction efficiency (1>7>8>2) and the serum levels of secreted protein (8>7>1>2). When we injected AAV vector expressing hsFlk-1 into muscle of immunodeficient mice (CB17scid), sustained expression of serum hsFlk-1 was detected for four months, indicating that secreted hsFlk-1 protein may elicit a strong immune response in immunocompetent mice with rapid elimination of secreted proteins by neutralizing antibody against hsFlk-1. AAV mediated expression of secreted protein seems to be influenced by various factors. AAV2/8 vector is the most efficient for expression of anti-angiogenic proteins. Control of antigenicity of secreted protein is the key element for achiving long-term secretion from muscle of immunocompetent animals