Development of Novel Promoters That Drive Photoreceptor Specific Expression in an EIAV Lentiviral Vector

Abstract

Purpose:: We wanted to develop an EIAV based lentiviral vector that displays photoreceptor specific gene expression with strong expression in the target photoreceptor cell and minimal expression in non-target cell types.

Methods:: Towards this aim we firstly used a transient luciferase reporter assay and utilised various cell types including the human Y-79 retinoblastoma cell line to compare different truncated photoreceptor specific promoters. The promoters derived from rhodopsin (Rho), ß- phosphodiesterase 6b (PDE 6b) and retinitis pigmentosa 1 (RP1) genes were evaluated alone or in combination with an enhancer element derived from the inter-photoreceptor retinoid-binding protein gene (IRBP).

Results:: We found that the PDE 6b promoter in combination with three multiple upstream copies of the IRBP enhancer showed the greatest reporter gene expression in the Y-79 cell line, with minimal expression in the non-photoreceptor cell lines, HT1080, D407 and ARPE-19. Next, the different promoter/reporter constructs were transferred in to an EIAV (Equine Infectious Anemia Virus) lentiviral vector. These recombinant EIAV vectors maintained the photoreceptor-specific expression profile seen within the reporter plasmids in vitro. Following subretinal delivery of these EIAV vectors in to mice we noted that the hPDE 6b promoter in combination with a single IRBP enhancer gave the strongest photoreceptor specific expression in vivo.

Conclusions:: In conclusion, we have developed a series of novel photoreceptor specific promoters that demonstrate strong expression in the target photoreceptor cell whilst maintaining minimal expression in non-target cells and we have shown that these promoters maintain this expression profile in vivo when transferred to the EIAV lentiviral vector platform. These promoters would be useful to drive the expression of photoreceptor specific gene therapies to treat a range of ocular disorders where the photoreceptor cell is the primary disease target, such as Stargardt disease.

Keywords: gene transfer/gene therapy • photoreceptors • degenerations/dystrophies