Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
LuxturnaTM is a retinal gene therapy treatment for an inherited retinal degeneration (IRD) caused by mutations in RPE65, a gene that encodes for an enzyme involved in the visual cycle. RPE65 cDNA is delivered subretinally using recombinant adeno-associated virus (rAAV) and successfully improves visual function in patients diagnosed with Leber’s congenital amaurosis (LCA), a group of severe hereditary retinal degenerations. Despite the success of LuxturnaTM, there remain several challenges to the development of new retinal gene therapies. Here, I’ve focused on solving several of these limitations, including evaluating the safety of same eye AAV readministration, manipulation of the gene transfer vector, and development of an in vitro IRD model. Readministration of AAV to the same retina may be desirable in order to treat additional areas not targeted during the initial injections or to boost transgene expression levels at a later timepoint in areas of the retina that have already been transduced. However, safety and efficacy concerns regarding an inflammatory immune response to the viral capsid upon readministration have kept us from exploring the possibility of successive injections. We administered bilateral subretinal injections of 1E11 vg rAAV2-hRPE65v2 to three unaffected non-human primates and then delivered repeat subretinal injections in those same eyes two months later. Serial ophthalmic exams were carried out during the course of the study. Peripheral blood monocytes and serum samples were collected at baseline and post-injection timepoints to characterize the cellular and humoral immune responses. In addition, we have identified an AAV8BP2 capsid variant through rational mutagenesis of a tyrosine residue that is able to transduce retinal bipolar cells more efficiently than the parent serotype. Our ability to do so may be integral for development of optogenetic treatment options for cases of severe photoreceptor degeneration in which the usual machinery must be bypassed. Additionally, we assessed the utility of patient-specific induced pluripotent stem cells to model early CRB1-associated LCA. All three of these findings allow us to confront the current assumptions in the field and expand our toolbox for developing new and modifying existing AAV retinal gene therapies.
Weed, Lindsey, "Addressing Aav-Mediated Gene Therapy Challenges In The Retina: Readministration, Aav Capsid Optimization, And In Vitro Disease Modeling" (2019). Publicly Accessible Penn Dissertations. 3396.