1. Vectors with transcytosis activity and low preexisting immunity in humans: Reporter vectors employing several non-human primate and non-primate capsids with low preexisting immunity in humans have been produced and are being tested for transcytosis activity after intravitreal injection into mice and rats. We are also assessing the tropisms of these capsids in a mouse model of retinoschisis. This has allowed us to select an optimal capsid for AAV vectors designed to treat this disease.? ? 2. Small molecule regulation of transgenes: A postdoctoral fellow has been recently hired for this project and is exploring 2 complementary strategies for achieving small molecule control of gene expression.? ? 3. Preclinical studies for a retinoschisis clinical trial: Studies were carried out to develop an optimized AAV retinoschisin vector for clinical evaluation. Several AAV capsid types and vector genome configurations were evaluated in order to optimize the efficiency of gene transfer while restricting the expression of retinoschisin to the cells that produce the protein in wild type animals. AAV serotype 8 was shown to mediate robust expression in all of the relevant retinal cells types but also transduced nonretinal cell types (ciliary bodies and corneal epithelium) that do not normally produce retinoschisin. Retinoschisin expression was restricted to the relevant cell types by the use of the human retinoschisin promoter. This promoter had not been previously characterized. The promoter sequences that confer high level expression and tissue specificity were identified in the context of AAV8 retinoschisin vectors. The vectors carrying various promoter constructs were tested for specific retinoschisin expression by intravitreal injection into a mouse model of retinoschisis. End points were retinoschisin expression, structural improvement of the retina, and ERG b-wave restoration. This work resulted in an optimize vector construct that we would like to move to the clinic. We are using this vector to complete the necessary preclinical efficacy and toxicity studies for investigative new drug (IND) filing.? ? 4. Improved AMD therapeutics: In collaboration with Dr. Carmen Clapp of the Universidad Nacional Autonoma de Mexico, Juriquilla campus, we are examining a novel anti-angiogenic fragment of prolactin, called vasoinhibin, in the context of AAV vectors. AAV vectors encoding vasoinhibin, prolactin, and sFlt-1 (a soluble VEGF receptor fragment that acts as a competitive inhibitor) have been produced and are currently being tested in an animal model of diabetic retinopathy.? ? 5. CEP290: Vectors have been prepared for this project and will be tested in vitro for expression and in vivo for efficacy.
