In vivo Analysis of Cone Specific Transgene Expression Using Lentiviral Vectors

Abstract

Purpose: : Lentiviral vectors have become attractive delivery vehicles since they readily transfect non-dividing cells and provide stable and long-term gene expression in vivo. Clinical applications require a gene transfer vector capable of accurately targeting particular cell types in the retina. To develop such a vector, we compared the expression of the green fluorescent protein (GFP) gene after subretinal injections of lentiviral constructs with the transgene expression driven by unspecific and cone specific promoters.

Methods: : Lentiviral vectors were constructed expressing GFP under the transcriptional control of the murine cytomegalovirus (CMV), arrestin3 (arr3), middle-wavelength-sensitive (MWS) opsin and short-wavelength-sensitive (SWS) opsin promoters. Viral vectors were injected into the subretinal space of wild type (SV129) and knockout mice (CNGA3-/-). GFP expression was analyzed by confocal scanning laser ophthalmoscopy (cSLO) in vivo immediately after injection and at later time points. Cell specific transgene expression was confirmed by examination of GFP fluorescence in vertical retinal slices.

Results: : Use of the constitutive CMV promoter yielded unspecific GFP expression surrounding the injection site. First results revealed efficient and specific expression of GFP in cone photoreceptors when using constructs containing one of the cone-specific promoters. In vivo imaging showed confined retinal detachment immediately following subretinal injection. Fluorescence could be detected within one week post procedure.

Conclusions: : Confocal SLO imaging is a useful tool to document and follow up effects of subretinal injection, especially when using GFP as reporter gene. Cone specific gene expression can be achieved by using lentiviral vectors that contain regulatory elements of cone specific genes. This allows for precise delivery of transgenes to cone photoreceptors within the retinal ultrastructure. These observations have significance in devising gene therapy strategies for various cone and cone-rod dystrophies.

Keywords: gene transfer/gene therapy • photoreceptors • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)