Efficient delivery and expression of foreign genes into the mammalian retina, particularly photoreceptor cells, will provide new understanding of photoreceptor function at the molecular and clinical level. It is proposed to thoroughly test the Adeno-Asociated Virus (AAV) vector, with the ultimate goal of gene therapy for human retinal disease. Fundamental aspects of adapting the AAV vector system to the retina will be addressed. AAV delivery will be examined by intravitreal and subretinal injection, cell specificity of gene expression, efficiency of gene transfer, and the stability of expression. It will also be determined if AAV is effective for retinal gene therapy and is free of pathologic side effects in two animal models.
Aim 1 examines the basics of AAV- mediated transgene expression in the normal mouse and rabbit eye. The time course of retinal expression following single and successive subretinal and intravitreal injections will be examined. The feasibility of using retinoblastoma cells in culture for the initial testing of new gene constructs before their use in animals will be examined. Initially, short regions of proximal murine, bovine and human opsin promoter regions known to drive expression in the mammalian retina will be linked to the reporter gene beta-galactosidase or c-myc-tagged neurotrophin genes.
Aim 2 addresses the problems of controlling expression levels and cell-specificity of the transgene product with longer, more complete opsin promoters. Regulatory regions include a putative silencer region and an upstream tissue-specific promoter element. The applicant will also employ the murine beta- phosphodiesterase (PDEb) and heterologous test genes in preparation for therapeutic testing in the rd mouse.
Aim 3 determines if intraocular injection of AAV is safe. For ocular injection of AAV to be suitable for human therapy, it cannot generate inflammatory response, ocular pathology or inappropriate delivery and expression of the transgene product in extra-retinal or extra-ocular tissues.
Aims 4 and 5 establish the therapeutic potential of AAV vectors in two well-documented animal models: (1) somatic cell gene therapy in the rd mouse by introduction of AAV-murine PDEb constructs into neonataI mice; and (2) prophylactic introduction of neurotrophin genes (CNTF, BDNF, NT4) into rat photoreceptors to afford resistance to light damage upon constant light exposure. These studies will be used to develop viable and safe strategies for human retinal gene therapy.
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