Efficient photoreceptor-targeted gene expression in vivo mediated by recombinant adeno-associated virus

Purpose. To develop a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Methods. Recombinant Adeno-associated Virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas by subretinal injection, employing a proximal murine rod opsin promoter (486 to -385) to drive expression. Results. Reporter gene product was detected in photoreceptors but not in any other retinal cell type or in the neighboring retinal pigmented epithelial cells. For the g/p-containing rAAV virus in the rat retina, both rod and cone photoreceptor transduction efficiency was nearly 100% in a region surrounding the injection site and typically encompassing 10-20% of the total retinal area. From this data we estimate 2.5 million photoreceptors were transduced as a result of a single 2 microliter subretinal inoculation. Expression persisted for at least 6 months. Conclusions. Extrapolating to the larger human retina, this level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The -containing rAAV stock used here was free of both Adenovinis and wild type AAV. Therefore, we also conclude that highly purified, helper virus-free AAV vectors can be used to achieve high frequency transduction of terminally differentiated, postmitotic photoreceptor cells.