The biochemistry of AAV DNA replication and the development of AAV vectors for gene therapy and delivery
The biochemistry of AAV DNA replication and the development of AAV vectors for gene therapy and delivery are the focus of Dr. Muzyczka's laboratory. His laboratory has developed a complete in vitro AAV DNA replication assay. The reaction relies on the Rep protein and a crude extract from uninfected cells. This assay should be useful for identifying the cellular DNA plymerase(s) and accessory proteins used for AAV DNA replication. Furthermore, because AAV DNA replication involves only strand displacement DNA synthesis, an in vitro AAV DNA replication system should be useful for identifying the cellular enzymes that are involved uniquely in leading strand cellular DNA synthesis. The goal, therefore, is to identify the cellular enzymes required for AAV DNA replication. In addition, Dr. Muzyczka's group continue to work on AAV vectors because they appear to be promising for gene therapy. Aside from the high integration frequency and the potential for site-specific integration, AAV is non pathogenic, has a broad host range, and appears to be able to infect a variety of tissue types. Finally, AAV sequences appear to have little effect on the expression of the foreign gene. His current experiments with AAV vectors focus on three issues that need to be resolved if AAV vectors are to be useful. First, there is relatively little information available about the behavior of AAV in primary cells or whole animals.
To obtain more information Muzyczka's laboratory is collaborating with a number of laboratories who are in a position to test AAV vectors in primary cells by supplying them with AAV recombinant stocks carrying appropriate marker genes. Second, they are constructing vectors carrying a variety of promoters to determine if AAV proviruses can supply sufficient expression to correct genetic defects. Third, a number of strategies for improving the methods currently used to grow recombinant AAV viruses are being tested. DR. Muzyczka is also working on the problem of defining the mechanism of AAV integration.
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