Adeno-Associated Virus 8-Mediated Gene Therapy for Choroideremia: Preclinical Studies in in Vitro ind in Vivo Models

Choroideremia (CHM) is a slowly progressive X-linked retinal degeneration that results ultimately in total blindness due to loss of photoreceptors, retinal pigment epithelium, and choroid. CHM, the gene implicated in choroideremia, encodes Rab escort protein-1 (REP-1), which is involved in the post-translational activation via prenylation of Rab proteins. We evaluated AAV8.CBA.hCHM, a human CHM encoding recombinant adeno-associated virus serotype 8 (rAAV8) vector, which targets retinal cells efficiently, for therapeutic effect and safety in vitro and in vivo in a murine model of CHM. In vitro studies assayed the ability of the vector to produce functional REP-1 protein in established cell lines and in CHM patient derived primary fibroblasts. Assays included Western blots, immunofluorescent labeling, and a REP-1 functional assay which measured the ability of exogenous REP-1 to prenylate Rab proteins. The in vivo work used unilateral subretinal delivery of AAV8.hCHM to treat a murine model of CHM, with the effects of treatment evaluated with pupillometry, ophthalmoscopy, histology, and immunofluorescence analysis. The contralateral eye was treated with an AAV8.EGFP control. The results of the in vitro analysis demonstrated that the AAV8.CBA.hCHM vector was capable of inducing robust expression of REP-1 protein in a dose-responsive fashion in cultured cells, with the prenylation assay indicating that the exogenous REP-1 protein produced was capable of assisting in the activation of Rab proteins. This functional assay was also applied to CHM patient fibroblasts transduced with an AAV serotype 2 version of the vector and demonstrated that exogenous REP-1 produced in these cells was functional. The in vivo subretinal treatment of CHM mice with AAV8.CBA.hCHM resulted in the improvement of pupillary response in the treated eyes of some animals, as well as slowing of progression as judged by ophthalmoscopy. Histology of treated animals showed properly localized expression of human REP-1 and a significant improvement in outer retina health as determined by an increase in the thickness of the outer nuclear layer in treated eyes compared to controls. Combined, these results indicate that transduction with AAV8.CBA.hCHM reduces the biochemical and pathogenic defects in CHM both in vitro and in vivo and supports the application of AAV8 vectors in CHM gene therapy. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Jean Bennett Second Advisor Katherine A. High This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/1014