Development of Gene Therapy for Age-­related Exudative Macular Degeneration.

Age-related macular degeneration (AMD) became the commonest cause of legal blindness in developed countries by 1990, compounded by the ageing of the population, the absence of any useful therapy and a dearth of knowledge as to the molecular biological underlying abnormalities. The publications embodied in this thesis extend over the following 28 years and contribute original knowledge by way of laboratory experimentation into the pathogenesis and then novel animal and human clinical trials. The programme lead to international pharmaceutical development of unique viral gene therapy for AMD. Tissue culture models for phagocytosis and lysozymal degradation of retinal receptor outer segments showed that excess receptor load or inhibition of the key degradative enzymes, Cathepsin D and Cathepsin S, each resulted in accumulation of intracellular debris and lipofuscin in retinal pigment epithelial cells. Specific Inhibition of cat D or cat S with targeted novel antisense oligonucleotides also lead to the development in small animals of many of the changes seen in AMD. Neovascular AMD caused most of the blindness at that time. Laser photocoagulation could obliterate subretinal blood vessels, but at the expense of loss of remaining central vision. Early studies in this thesis showed a remarkable panretinal activation of the retinal metabolic marker, glial fibrillary acidic protein. Laser also upregulated fibroblastic growth factor, with a therapeutic benefit in the RCS rat model of retinal degeneration, but no FGF abnormality was found in the AMD animal models. Extensive phenotyping of my clinical AMD population allowed haplotype analysis of newly discovered genetic variations in this disease. No differences were found in wet compared to dry AMD, nor in response to photodynamic therapy. This data was contributed to two large multinational publications which reported rare AMD variants and compared the AMD genomic variations to those found with glaucoma. The effect of focal laser on the upregulation of VEGF was described and this finding lead to the creation of novel animal models of VEGF modulated subretinal and intraretinal neovascularization. These models were made with intense laser applications to the retina or by creation of VEGF transgenic mice. The models proved indispensable in assessing potential therapy for wet AMD, first with RNA inhibition with our own antisense oligonucleotide and later with viral gene therapy. The first successful anti VEGF gene therapy in these publications used Adenovirus to transfer the gene for sFlt-1 in order to block corneal neovascularization in animals. When Adeno-associated viral gene transfer became a possibility, these studies confirmed successful RPE- 65 replacement in rodents and then dogs. Corneal vascularization was again inhibited in animals, this time using AAV-sFLT-1 formulated in our laboratories. The efficacy of upregulating sFlt-1 to block VEGF-induced choroidal neovascularization was confirmed in laser-induced and VEGF transgenic models. The next step was the evaluation of this AAV-sFLT-1 construct in non-human primates for local and systemic safety, toxicity and efficacy in blocking laser-induced CNV. This work proved successful and lead to the first investigator sponsored application (CTX) for ocular gene therapy in adults in Australia and in the world for this disease. Phase 1 and randomized phase 2 trials were undertaken on 40 patients with persisting retinal or subretinal fluid from wet AMD despite extensive prior humanised anti-VEGF antibody therapy. These showed excellent reversal of fluid on OCT scans in 50%, but no better response than controls in the others. It was postulated that selection of advanced non-responsive cases, possible under-transfection of the aged retina and insufficient sFLT-1 production may have explained the outcome. The partial success rate was not yet equivalent to that of the current gold standard treatment with VEGF antibodies, but was sufficient to stimulate other groups to design improved AAV gene therapy constructs and to accelerate further development. This pioneering human and preclinical scientific data formed the basis of a major corporate fund-raising on the NASDAQ exchange in New York, which assets have pushed further clinical trials of gene therapy for neovascular AMD by way of intravitreal injection. My latest publication in this series established in non-human primates the value of prior surgical removal of the internal limiting membrane of the retina. This greatly enhanced reporter gene transfection after intravitreal injection and may be the key to therapeutic success. The studies reported here were the first ocular viral gene therapy trials in adults and they have stimulated a large amount of further research and development to better control this major cause of blindness