P 375 Bietti's crystalline retinopathy

Pupose:In order to develop a treatment for recessive retinitis pigmentosa the ultimate objective is to introduce a normal copy of the abnormal gene into photoreceptors. The post-mitotic status of photoreceptor cells make them unsuitable targets for existing retroviral vectors. One of the strategies that we have been exploiting is adenoviral (AV) mediated gene transfer. Here we report successful transduction of ocular tissues including neuroretina and retinal pigment epithelium (RPE) with a IacZ reporter gene using an adenoviral vector. Methods:The recombinant adenovirus, pXCXRBb. contains an E.Coli B-galactosidase gene driven by a Rous Sarcoma virus promotor and with SV40 polyadenylation signals. Approximately 0.4~1 of viral suspension containing pXCXRBb at 109 pfo/ml was injected with a 1.5 cm, 34-gauge hypodermic needle into the vitreous and into the subretinal space of HsdOla:MFl mice. Control MFI and BALB/cOlaHsd eyes were injected with an equivalent volume of PBS. Animals were perfused 4,7 and 14 days postinjection with 2% pamformaldehyde and 0.05 46 glutaraldehyde in O.lM phosphate buffer. The eye cups were incubated at room temperature with X-gal overnight to detect lacZ activity (blue staining) in transduced cells. The tissue was paraffin embedded and sectioned at 5-10 pm thickness. Sections were counterstained with nuclear fast red and examined by light microscopy. Results and Conclusion:Fine blue staining of the RPE and of iris-and ciliary body epithelium was observed in BALB/c controls indicating endogenous B-gal activity which makes evaluation of transduction after intravitreal injections difficult in these mice. This was not seen in control MFI mice which we therefore used in our subsequent experiments. Distinct B-gal staining of RPE cells, photoreceptor cells and optic nerve glia cells was only detected in injected eyes and abundant near the injection site. Over time. the percentage of IacZ-positive cells decreased and is highest during the first week post injection. Whether thts is due to an immune response against cell transduced by the AV vector remains to be determined by ongoing experiments. Our results to date suggest that the current generation of replication deficient adenoviral vectors may be a tool of limited use for gene transfer into the retina. However, this work will serve as a baseline for further development of gene therapy of RF' for which application of a non pathogenic, integrating DNA vector such as adeno associated virus with a photoreceptor cell specific promotor may have higher potential.