Lentiviral Vectors as Gene Therapy Vehicles of Usher Syndrome 1B

Abstract

Abstract: : Purpose: Usher syndrome is a combined blindness and deafness disease. Usher type 1B is due to mutations in the MYO7A gene that encodes an unconventional myosin expressed in the retinal pigmented epithelium (RPE) and photoreceptor cells. The aim of this study is to develop viral vectors that express the wild type human MYO7A cDNA, for use in subsequent attempts to rescue cellular phenotypes in the Myo7a–null mutant mice. Methods: The cDNA of MYO7A is relatively large (7 Kb); therefore the "third generation", self–inactivating (SIN) recombinant lentiviral vector was chosen as the gene transfer vehicle. The full–length human MYO7A cDNA was cloned down stream of either the CMV promoter or a modified CMV promoter fused to a MYO7A enhancer. Lentiviral particles were produced by co–transfection of HEK293T cells with the viral vector and three separate helper plasmids expressing the necessary GAG, POL, Rev, and VSV G proteins. Lentiviral stocks were concentrated by ultracentrifugation of culture media, and viral titers were determined by immunostaining of infected HEK cells with MYO7A antibodies. Concentrated viruses were delivered to the subretinal space of neonatal and mature eyes of the shaker1 mice that carry a null mutation in the Myo7a gene. Injected eyes were analyzed at various times after infection. Results: Both versions of lentiviral vectors express the human MYO7A protein in infected HEK cells in vitro. Furthermore, in vivo infection shows that the CMV promoter effectively drives transgene expression in the RPE of both neonatal and adult eyes.Conclusions:Recombinant lentiviral vectors are effective vehicles in expressing and delivering large transgenes of interest to the RPE. * Supported by grants from the Research to Prevent Blindness Foundation, Foundation Fighting blindness, and National Eye Institute.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • retinal degenerations: cell biology