Gene Therapy for MAK-associated RP

Abstract

Purpose: One in 55 people with Ashkenazi Jewish ancestry harbor an Alu repeat in exon 9 of the MAK gene. Homozygosity of this founder mutation results in a loss of normal MAK transcript, abnormal elongation of the photoreceptor connecting cilia and development of autosomal recessive retinitis pigmentosa (RP). The purpose of this study was to develop a viral gene transfer approach to treat MAK-associated RP.

Methods: Dermal fibroblasts were obtained from patients with MAK-associated RP. These cells were used to both investigate the pathophysiology of the disease and as a cell source for derivation of patient specific induced pluripotent stem cells (iPSCs). These iPSCs were differentiated into photoreceptor precursor cells using our previously published protocol consisting of a stepwise addition of neurotrophic growth factors, and inhibitors of Wnt, Notch and BMP. Patient specific cell lines carrying the MAK mutation and Mak-/- mice were used as gene transfer recipients. A combination of rt-PCR, western blotting, immunocytochemistry, confocal microscopy, electron microscopy, fundus photography, OCT and ERG were used to evaluate the efficacy of the gene transfer approach.

Results: Full-length retinal MAK cDNA, generated via rt-PCR amplification from total human retinal RNA, was TA cloned and subsequently ligated into both lentiviral and AAV vectors for in vitro and in vivo gene transfer experiments respectively. One week following transduction of patient specific fibroblasts and iPSC derived photoreceptor precursor cells, full length MAK could be detected via rt-PCR and western blotting. Confocal microscopy revealed that restoration of wild type MAK protein resulted in a significant shortening of primary cilia. Subretinal injection of AAV-MAK into Mak-/- mice resulted in a significant slowing of disease progression.

Conclusions: We have demonstrated that viral-mediated gene replacement of MAK can mitigate the pathogenic effects of disease-causing mutations in this gene in mice and in patient-specific human cell lines. This is an important step toward a clinical trial of gene replacement therapy for patients affected with MAK-associated RP.

Keywords: 721 stem cells • 538 gene transfer/gene therapy • 688 retina