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S. Cashman, K. Ramo, R. Kumar-Singh; Protection Against Laser-Induced Choroidal Neovascularization Following Delivery of an Adenovirus Expressing a Soluble Form of Human CD59 to Murine Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2772.
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Polymorphisms in a number of complement genes have been shown to be either strongly predictive of or protective against Age-related Macular Degeneration (AMD). In patients with AMD, deposition of the Membrane Attack Complex (MAC) on the retinal pigment epithelium (RPE), Bruch’s membrane, and choroidal blood vessels has been observed. Choroidal neovascularization (CNV) in the classic laser-induced mouse model of wet AMD has been shown to be dependent on the formation of MAC. We have previously shown that in vivo delivery of an adenovirus vector expressing human membrane-associated CD59 protects against human MAC deposition on murine ocular tissues, including the RPE. Protection against the effects of MAC was limited to those cells expressing the membrane associated CD59. We now wished to test the hypothesis that delivery of an adenovirus vector expressing a soluble form of human CD59 (sCD59) can block the formation of laser-induced CNV, and can do so also when vector is administered distal from the site of laser burn.
Murine hepatocytes (Hepa1c1c7) cells were incubated in 1% normal human serum (NHS) diluted in media collected from human ARPE-19 cells infected with either an adenovirus expressing sCD59 or GFP (AdCAGsCD59, AdCAGGFP). The resulting lysis was measured by fluorescence activated cell sorting (FACS) analysis of propidium iodide uptake. Either an AdCAGsCD59 or a control virus (AdCAGpA) was injected into the subretinal space of C57Bl6/J mice, and 72h post-injection the mice were subjected to argon laser photocoagulation. Seven days post-laser, eyes were harvested, and size of CNV measured following staining of RPE/choroid/sclera with FITC-conjugated GSL I isolectin B4.
Hepa1c1c7 cells incubated in 1% NHS pre-conditioned with supernatant from AdCAGsCD59-infected ARPE-19 cells exhibited 38.2% lysis, relative to 77.6% lysis when the NHS was pre-conditioned with supernatant from AdCAGGFP infected cells. Subretinal delivery of an adenovirus expressing human sCD59 to C57Bl6/J mice reduced the size of laser-induced CNV by 39% relative to control (AdCAGpA)-injected mice.
A soluble human CD59 expressed from an adenovirus delivered to murine RPE efficiently protects against laser-induced CNV in vivo, and does so beyond the site of vector administration. Adenovirus has been shown to be safe and effective in several ocular gene therapy clinical trials; hence the approach of preventing choroidal neovascularization in patients with an increased risk of wet AMD may be envisaged.
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