189. Therapeutic Gene Transfer as a Treatment Option for Age-Related Macular Degeneration
Authors: Erik Wielechowski, Angelica Medina-Jaszek, Omua Ahonkhai, Muhammad Arif, Jean Bennett, Albert Maguire, Anna Tretiakova, James M. Wilson
Journal: Molecular Therapy (2016)

Age-related macular degeneration, otherwise known as AMD, is a progressive, irreversible eye condition in which the retina is permanently damaged by either atrophy of its pigment epithelial layer (dry AMD) or abnormal blood vessel growth in the retina's membrane (wet AMD). Wet AMD, the more advanced, severe version of the disease, is induced by overexposure to vascular endothelial growth factor (VEGF) in the eye. This VEGF-induced blood vessel growth leads to rupture and leaking of the retina's membrane, endangering the integrity of the macula, which is vital for acute central vision. Once the macula is scarred and central vision is lost, it cannot be recovered. Current treatments for wet AMD include several drugs (i.e. lucentis) that inhibit VEGF, yielding a reduction in retinal thickness and moderate increases in visual acuity. While these treatments succeed in prohibiting disease progression, they require frequent intravitreal injections. In the future, the goal is to prevent disease progression while avoiding repeated injections with the use of therapeutic gene transfer. To develop this new generation of AMD therapeutic, we sought to create an efficacious AAV vector expressing anti-VEGF antibody via systematic optimization of all vector elements. Six promoters, 12 coding sequence variants, an F2A linker, and 3 internal ribosome entry sites (IRES) were evaluated for tissue-specific expression in mouse eye. Mean expression from the 2 best promoters, UbC and CB7, was 400 and 450 ng/mg of eye lysate, respectively, compared with the CMV promoter at 10 ng/mg. In the context of a CMV promoter, mean expression for best-performing IRES (EMCV) was 2 ng/mg of tissue lysate, which was 5-fold lower than that from the construct containing an F2A linker (10 ng/mg). The best-expressing coding sequence variants, named AMD11 and AMD42, yielded 12 and 13 ng/mg of tissue lysate, respectively. In order to identify a clinical candidate vector, the best-performing elements were combined to generate 4 additional vectors, which were evaluated in both eyes of 8 rhesus macaques for each vector. Following subretinal injection with 1×1011 GC/eye, anterior chamber fluid was sampled on days 15 and 29. Among the 16 injected eyes, average expression of AAV-UbC-AMD11 was 473.6 ng/ml (ranging from 123.5 to 933 ng/ml) of anterior chamber fluid on day 15 after vector administration and 546.2 ng/ml (262 to 1263 ng/ml) on day 29. Average expression of AAV-UbC-AMD42 was 360.6 ng/ml (144.6 to 646.8 ng/ml) and 536.8 ng/ml (238.3 to 920.7 ng/ml) on days 15 and 29, respectively. For AAV-CB7-AMD11, average expression on days 15 and 29 was 972.4 ng/ml (495.9 to 1684.7 ng/ml) and 1100.3 ng/ml (422.3 to 2187.8 ng/ml). The final vector, AAV-CB7-AMD42, expressed at 1922.2 ng/ml (707.9 to 2892.7 ng/ml) on day 15 and 1396 ng/ml (431.2 to 2346.4 ng/ml) on day 29. After final selection, the clinical candidate vector will be evaluated in formal nonclinical studies prior to a clinical trial. Selection criteria will include expression levels and vector yields.