Lentiviral Gene Therapy of the Retinal Pigment Epithelium in Mice Lacking MYO7A, the Usher 1B Protein

Abstract

Purpose: : Usher syndrome is a combined blindness and deafness disorder. Usher type 1B is due to mutations in the MYO7A gene that encodes an unconventional myosin expressed in the retinal pigment epithelium (RPE) and photoreceptor cells. The aim of this study is to develop lentiviral vectors that express the wild type human MYO7A cDNA in order to rescue cellular phenotypes in the Myo7a–null mutant mice.

Methods: : The 6.8–kb cDNA of human MYO7A was cloned into the HIV1–derived third generation, SIN (self–inactivating) lentiviral vector down stream of different promoters, and VSV.G protein pseudotyped viral stocks were produced. Lentiviruses were used to infect primary RPE cell cultures as well as the RPE in vivo. The extent of viral MYO7A expression, and the correction of mutant phenotypes were then assessed.

Results: : Virally–encoded CMV promoter drove excessive expression of MYO7A in primary mouse RPE cells, and caused cell death. A chimeric promoter, which contains a partial CMV promoter and a 160 bp MYO7A gene sequence, resulted in weak MYO7A expression in HEK293T cells but produced a normal level of MYO7A protein in Myo7a–null mutant RPE cells. Treatment of Myo7a–null RPE cells with the lentivirus encoding the chimeric promoter and MYO7A cDNA corrected defective phagocytosis and abnormal melanosome motility. Subretinal delivery of MYO7A–expressing lentiviruses also restored melanosome localization in mutant RPE cells.

Conclusions: : Recombinant lentiviral vectors are effective vehicles for delivering and expressing large transgenes of interest to the RPE. The expression levels of MYO7A are critical for achieving therapeutic effects. These studies suggest a strategy for treating retinal degeneration in Usher 1B.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • retinal degenerations: hereditary