Abstract
Purpose: :
Accumulation of autofluorescent A2E-lipofuscin pigments within retinal pigment epithelium (RPE) cells is seen in several forms of macular degeneration, including age-related-macular degeneration (AMD) and Stargardt’ disease. In a recent study, using the Stargardt’ disease-mouse model, the abca4-/- mouse, we observed for the first time increased oxidative stress and complement activation in vivo due to accumulation of A2E-lipofuscin fluorophores. Paradoxically, this was accompanied by reduced expression of negative complement regulatory protein genes (CRPs) in the RPE cells. In the current study, we over-expressed one or more CRPs in the RPE of the abca4-/- mouse. We hypothesized that these regulatory proteins will protect RPE cells from inappropriate attack by the complement system, and thereby prevent photoreceptor loss.
Methods: :
We prepared mouse and human recombinant adeno-associated viruses (AAV) expressing various complement- and inflammatory-related protein genes. The AAV-CRP genes were delivered to the subretinal space of the Balb/C (WT) and albino abca4-/- (KO) mice, via a trans-scleral/trans-choroidal approach under direct visualization. Control injections were performed with AAV-null or AAV-GFP viruses. Fundus photographs were taken before, immediate and at different time points following the subretinal injection. The expression levels for CRPs genes were measured by qRT-PCR and complement activation was evaluated by immunocytochemistry. Visual retinoids and lipofuscin pigments were quantitated by high-performance liquid chromatography.
Results: :
Both WT and KO AAV-CRRY-injected mice showed several-fold increase in CRRY expression levels compared to control eyes. The expression of the targeted protein was confined to the RPE based on immunofluorescence analysis. Surprisingly, modulating the CRRY expression levels in the KO RPE cells lead to two-fold increase of other CRP genes such DAF1, CD59a and CD59b. More importantly, over-expression of CRRY significantly reduces the C3 break-down fragment (C3b) accumulation in the RPE cells by immunohistochemistry.
Conclusions: :
Preliminary data suggest that by modulating the ocular immune response via CRP-gene-based therapy, we can enhance the RPE defensive mechanisms against aberrant complement attack and chronic inflammation. Ongoing analysis of the AAV-CRP-injected mice are focusing on retina histology and photoreceptor function.
Keywords: retinal pigment epithelium • gene/expression • inflammation