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R. Kumar-Singh, K. Ramo, S. Cashman; Humanized Model of Membrane Attack Complex (MAC) Formation on Murine Retina and Protection Against MAC by an Adenovirus-Delivered Human CD59. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3871.
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© ARVO (1962-2015); The Authors (2016-present)
Age related macular degeneration (AMD) is the leading cause of blindness in the elderly. Polymorphisms in several complement regulatory proteins including Factor H have recently implicated over-active complement as a key player in the pathogenesis of AMD. Immunohistochemical analysis of drusen and retinal pigment epithelium (RPE) from AMD patients indicates the presence of a variety of complement proteins including the membrane attack complex (MAC). We wished to test the hypothesis that local over-expression of human complement regulatory protein CD59 may protect against human MAC deposition in vivo. Cross-species differences between human and murine complement systems limit testing the efficacy of human complement regulatory proteins in non human systems in vivo. Hence, we developed a humanized murine model of measuring human MAC deposition in vitro and in vivo and used this model to measure protection of murine RPE against human MAC by adenovirus delivered human CD59.
Murine hepatocytes were exposed to normal human serum and the resulting lysis was measured by the uptake of propidium iodide and fluorescence activated cell sorting (FACS). MAC deposition on murine retina and RPE was measured by immunohistochemistry against human MAC. In order to measure the potential of protecting murine cells in culture or retina and RPE in vivo against human MAC deposition, we pre infected those cells or tissues with a recombinant adenovirus vector expressing human CD59 prior to exposure to normal human serum.
A total of 96.1 % of murine hepatocytes were lysed after exposure to normal human serum. However, if murine hepatocytes were pre infected with adenovirus expressing human CD59, only 12.3 % of cells were lysed. In contrast, pre infection of hepatocytes with adenovirus expressing a non specific transgene (GFP) resulted in 95.3% lysis of these cells. Subretinal delivery of adenovirus expressing human CD59 in vivo in mice subsequently protected those tissues from human MAC deposition. In a similar experiment, a recombinant adenovirus vector expressing a non specific protein (GFP), failed to protect murine retina against human MAC.
The humanized model of MAC deposition on murine retina enables the safe and rapid testing of human complement proteins in vivo. Human CD59 efficiently protects against human MAC deposition on murine retinal tissues. Since adenovirus has been previously proven to be efficacious and safe in ocular clinical gene therapy trials, the approach of inhibiting MAC deposition on RPE by local over-expression of CD59 may be envisaged in AMD patients.
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