Improved Retinal Structure and Function in Rho-/- Mice Following AAV-Delivered Rho Replacement

Abstract

Purpose: : Retinitis pigmentosa (RP), an incurable and debilitating inherited eye condition, often leads to blindness by mid-age and has an estimated prevalence of 1.5 million worldwide. Mutations in the rhodopsin gene (RHO), encoding the primary light-sensitive pigment of rod photoreceptors, are the most frequent and account for up to 25% of autosomal dominant RP cases. The Rho-/- mouse with a targeted disruption in the murine rhodopsin gene (Rho) has proved to be an immensely useful tool for development of gene-based therapeutic approaches for RHO-linked RP. For example, Rho-/- mice are ideal to test the efficacy of RHO replacement constructs, which will be necessary components of suppression and replacement therapies for RHO-linked dominant RP. A key question to be addressed in this study was whether expression of an AAV-RHO replacement in Rho-/- mice provided benefit at both histological and physiological levels, which to date have not been achieved.

Methods: : A number of recombinant 2/5 adeno-associated virus (AAV)-RHO replacement constructs have been generated in an attempt to reconstitute endogenous Rho levels.

Results: : The most efficient constructs achieved approximately 40% of wild type mouse Rho mRNA levels. Importantly, data from this study indicate that subretinal AAV-RHO delivery leads not only to RHO protein expression but also the generation of rod outer segments. This was confirmed by both light and transmission electron microscopy. Improvements at the structural level were accompanied by rod photoreceptor activity assessed by ERG. Notably, untreated Rho-/- mice neither elaborate rod outer segments nor have rod-derived electroretinograms (ERG).

Conclusions: : The most efficient AAV-RHO constructs evaluated in this study provide sufficient levels of RHO to be of therapeutic benefit in Rho-/- mice for the first time. These findings therefore represent important steps towards generating potent AAV-RHO replacement genes.

Keywords: gene transfer/gene therapy • opsins • photoreceptors