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John H. Sweigard, Kasmir Ramo, Kelly N. Ma, Siobhan M. Cashman, Rajendra Kumar-Singh; CD46, CD55, And CD59-mediated Attenuation Of Complement-induced Damage On Murine RPE Cells In-vitro And In-vivo: Therapeutic Implications For Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4012.
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Activation of the complement system has been implicated in the pathogenesis of Age-related Macular Degeneration (AMD). The Bruch’s membrane and retinal pigment epithelium (RPE) of AMD patients contain membrane attack complex (MAC) and C3b on their surface. Membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) regulate complement at the level of C3b, C3 convertase, and MAC assembly, respectively. The aim of this study was to evaluate the potential of increasing CD46, CD55, or CD59 expression on RPE cells in-vitro and in-vivo as an approach to reduce complement mediated damage to RPE cells.
We generated recombinant adenovirus vectors expressing human CD46, CD55, or CD59. We infected murine cells with each adenovirus followed by incubation in human serum using conditions that allow either classical and alternative pathway complement activation or alternative activity alone. We measured complement-mediated damage to murine cells by quantifying lysis and MAC formation. Adenovirus expressing CD46, CD55, or CD59 was delivered to the subretinal space of mice and one week later, ocular flat mounts were challenged with human serum and levels of complement mediated MAC damage quantified. CD59 was also tested in a laser-induced model of choroidal neovascularization (CNV). Size of CNV was quantified by lectin staining.
Adenovirus-mediated delivery of CD55 and CD59 protect murine cells from classical and alternative mediated MAC damage (94% and 96% reduction in lysis) while CD46 protects murine cells from alternative pathway (40% reduction in lysis), but not classical, mediated MAC damage. Subretinal adenoviral delivery of CD46, CD55, or CD59 renders efficient transduction of RPE cells. Levels of CD46, CD55, and CD59 in RPE cells following subretinal injection offer protection from human MAC deposition (55%, 56%, and 23% reduction in MAC). CD59 expression in the laser model of CNV formation reduces CNV size by 60%.
Transgenes encoding CD46, CD55, and CD59 are efficiently expressed in RPE cells when delivered via an adenovirus vector. Expression of CD46 offers protection from alternative pathway MAC mediated damage, but not classical. CD55 and CD59 offer protection from alternative and classical MAC mediated damage. Protection from CD59 has been extended into the in-vivo laser induced murine model of CNV formation.
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