UC Berkeley

Current retinal gene therapies using adeno-associated viral (AAV) vectors are limited in their efficacy due to their dependence on an invasive subretinal delivery route to achieve sufficient gene delivery. Additionally, the genetic heterogeneity of inherited retinal degenerative diseases makes gene replacement and antisense therapeutic strategies impractical for treating a broad array of patients. This dissertation sought to address these limitations by creating intravitreally-permissive AAV vectors through directed evolution and examining their therapeutic potential using a mutation-independent approach.

Firstly, we examined natural barriers to intravitreal vector delivery. We identified the inner limiting membrane (ILM) as the main obstacle to efficient intravitreal gene delivery and found mild digestion with a nonspecific protease, Pronase E, substantially enhanced retinal transduction for several AAV serotypes, most notably for AAV serotype 5 (AAV5). Secondly, we evolved more efficient glial-permissive AAV vectors in the CNS and retina using an in vitro selection methodology. We isolated an AAV variant, ShH10, closely related to AAV6, capable of efficient, selective Müller cell infection through intravitreal injection. Thirdly, we utilized this variant to examine the efficacy of intravitreally targeting Müller cells to secrete glial-derived neurotrophic factor (GDNF) as a mutation-independent therapy for delaying photoreceptor degeneration in a rat model of retinitis pigmentosa (RP). We achieved functional and histological rescue in treated animals at five months post injection, with an average approximate rescue of 50% in ERG a and b-wave amplitudes. Lastly, we evolved AAV variants to intravitreally transduce photoreceptors using an in vivo selection approach in a transgenic line of mice bearing a rhodopsin-GFP fusion. We isolated an AAV variant, 7M8, derived from an AAV2 peptide display library, capable of significant intravitreal photoreceptor transduction relative to AAV2.

The creation of intravitreally-permissive AAV vectors as well as the development of mutation-independent gene therapies will enable safer and more effective therapies for treating inherited retinal degenerative diseases.