31. AAV Gene Therapy for Choroideremia: Dose Determination Analyses
Authors: Irena Ignatova, Paolo Falabella, Katherine A. High, Linda B. Couto
Journal: Molecular Therapy (2016)

Choroideremia (CHM) is a degenerative, X-linked retinal disease characterized by deletions or mutations in the choroideremia (CHM) gene, resulting in defective or absent Rab escort protein-1 (REP-1). In normal retinas, the CHM gene is expressed in multiple cell types, including retinal pigment epithelium, photoreceptors, and choroidal cells, though there is evidence that the RPE cell is the primary disease-causing cell type. Absence or deficiency of REP-1 leads to cellular apoptosis and degeneration of the retinal pigment epithelium (RPE), choroid, and retinal photoreceptors. Choroideremia is an excellent disease candidate for gene transfer as it shares several key features with other inherited retinal degenerative diseases that are also candidates for AAV-mediated vector delivery. These features include: a) the small and localized nature of the target area, namely the retina; b) the tissue boundaries surrounding the area to be injected, which minimizes exposure of other cells to the vector and facilitates treatment with a relatively small vector dose; and c) the favorable immune status of the eye with respect to viral vector-mediated gene transfer. We are conducting a Phase 1 clinical trial to treat CHM subjects using either 5×1010 or 1×1011 vg/eye of an AAV2-hRep1 vector, and delivering the vector via a subretinal injection. Due to the lack of suitable animal models that mimic CHM disease in humans, we took a different approach to assess the potential therapeutic dose. A molecular analysis of non-human primate retinas was performed following subretinal injection of AAV2-hCHM to determine the relative amount of human Rep1 compared to endogenous monkey Rep1. Cynomolgus monkey eyes were injected subretinally with either 5×1011, 7.5×1011 or 2×1012 vg of AAV2-hRep1. Fundus images were taken following the injection of 300 ul of vector, and these images were used at the time of sacrifice to dissect the tissue associated with the injection bleb. The remaining retina was also collected and analyzed separately. RNA was isolated and absolute quantitative RT-PCR was performed using both cynomolgus monkey-specific Rep1 primers/probe and human Rep1-specific primers/probe. No human Rep1 mRNA was observed in non-injected eyes, and the amount of human Rep1 mRNA in the bleb area was determined to be at least 50 fold higher than cynomolgus Rep1 mRNA following a dose of 5×1011 vg/eye. At the two higher doses, similar levels of hRep1 mRNA, ranging from 270-380 times more than cynomolgus Rep1 mRNA, were observed. These data suggest that in the Phase 1 clinical trial, the low dose subjects (5×1010 vg/eye) will express hRep1 at a level approximately five fold higher than normal and the high dose subjects will express at least 10 fold higher levels of hRep1 than normal. Ongoing studies are evaluating the level of hRep1 in normal human retinas to confirm these ratios. Thus, the results from this non-conventional pharmacology model provide an estimate of the potential therapeutic dose of AAV2-hCHM for use in clinical studies.