1. Establish an AAV vector production laboratory: The vector production laboratory has been established with a minimal staff. As such, the vector lab is capable of producing 2 research grade AAV vector preparations per week and has produced 14 this year.? ? 2a and 2b. Vectors with improved immune parameters and transcytosis activity: AAV vectors are derived from a non-pathogenic family of viruses. The icosahedral protein capsid from these viruses is used as a delivery vehicle to deliver therapeutic genes. The majority of the capsids currently used for gene therapy vectors are derived from AAVs that infect humans. Because these viruses are prevalent, 80-90% of the human population has neutralizing antibodies to these capsids that render them inactive. In order to create AAV vectors that are not neutralized by the human immune system, the laboratory is currently screening AAV capsids that have low preexisting immunity in humans for transduction of retinal cell types. These capsids are derived from viruses that do not infect humans, but instead use non-human primates and non-primates as hosts. Reporter vectors employing several non-human primate and non-primate capsids with low preexisting immunity in humans have been produced and will be tested for retinal transduction in mice. A capsid with transcytosis activity in the lung was identified by a research group that I directed previously at a biotechnology company. We are currently examining this capsid, and other AAV capsids for transcytosis activity in the retina. The vectors will be injected both intravitreally and subretinally. ? ? 2c. Small molecule-regulated gene expression: A research plan has been formulated and a staff member is being recruited to carry it out.? ? 3. Preclinical studies for a retinoschisis clinical trial: We would like to design an AAV retinoschisin construct that will establish a wild type expression pattern in the retina with respect to cell type and expression level. To do this, we are mapping various elements of the retinoschisis gene that may confer wild type expression by installing them into reporter vectors and examining the expression patterns in the eyes of transduced animals. The critical factors will be distilled into a clinical AAV retinoschisin construct. Once the AAV retinoschisin construct is established, preclinical efficacy and toxicity studies will follow.? ? 4a. Improved AMD therapeutics: A collaboration has been established with Dr. Carmen Clapp of the Universidad Nacional Autonoma de Mexico, Juriquilla campus, to examine a novel anti-angiogenic fragment of prolactin, called vasoinhibin, in the context of AAV vectors. AAV vectors encoding vasoinhibin and sFlt-1 (a soluble VEGF receptor fragment that acts as a competitive inhibitor) will be compared in an animal model of AMD. The vasoinhibin and sFlt-1 vectors are currently being built and produced. ? ? 4b. Improved AMD animal models: AAV VEGF and reporter vectors employing various AAV serotypes are being provided to the Dr. Miller's group for optimization of the assay.
