Purpose: Specific mutations of the retinal guanylyl cyclase-1 (retGC1) gene have been linked to Leber congenital amaurosis type 1 (LCA1) and cone-rod dystrophies in humans, diseases that are amenable to treatments using molecular based therapies. As a step towards developing a therapeutic transgene for LCA1, we analyzed the cell specific and developmental activity profiles of fragments of the human retGC1 5' flanking region in vivo.
Methods: We generated self inactivating lentiviral vector constructs carrying three different fragments of the human retGC1 promoter fused to a nuclear localized beta-galactosidase reporter gene (nlacZ). The transgenes were packaged into lentiviral vectors, which were then used to transduce retinal progenitor cells of the developing chick. We monitored the expression of nlacZ in the retina over the course of development and in the retina, brain and pineal gland just prior to hatching.
Results: A 1.8 kb fragment of the retGC1 5' flanking region upstream of Exon 2 was capable of targeting nlacZ expression to photoreceptor cells in vivo and its activity was augmented by the presence of intron 1. We also demonstrated that the cell specific activity of this fragment arises, at least in part, by silencing expression in non-photoreceptor cells during the final stages of retinal development.
Conclusions: We have identified a human retGC1 promoter fragment that exhibits photoreceptor cell specific activity in vivo. Our results suggest that an element located in the proximal promoter may play a role in silencing expression of this gene in non-photoreceptor cells, thereby by shaping the restricted expression pattern of GC1 in the retina.