Adenovirus-mediated delivery of Factor H attenuates complement C3 induced pathology in the murine retina: a potential gene therapy for age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly, with no therapy available for 90% of patients. Recent genetic evidence implicates activation of complement in the pathogenesis of AMD. We have recently discovered that adenovirus (Ad)-mediated expression of complement component C3 (AdCMVC3) in the murine retina recapitulates many of the pathological features found in human AMD. In the present study, utilizing a gene therapy approach, we examine whether Ad-mediated expression of complement Factor H (AdCAGfH) attenuates AdCMVC3-mediated retinal pathology. METHODS: AdCMVC3 was co-injected with either AdCAGfH or a negative control virus expressing green fluorescent protein (AdCMVGFP) into the subretinal space of adult mice. The resulting retinal pathology was analyzed by histology and immunocytochemistry and retinal function was quantified by electroretinography. RESULTS: Morphological and functional analyses indicated that AdCMVC3-mediated retinal pathology could be attenuated by AdCAGfH. Specifically, endothelial cell proliferation was reduced by 91% and atrophy of retinal pigment epithelium (RPE) could be attenuated by 69%. AdCAGfH injected eyes exhibited 90-150% greater A-wave and 120-180% greater B-wave amplitudes relative to control eyes. Immunocytochemical analysis of rhodopsin and RPE65 was consistent with the rescue of photoreceptors and RPE in AdCAGfH injected eyes. CONCLUSIONS: C3-induced pathology in murine retina can be attenuated by Ad-mediated expression of Factor H. Expression of Factor H is worthy of further study as a potential gene therapy for AMD.

The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

Ex vivo biocompatibility evaluation of a new modified cellulose membrane.

To evaluate membrane biocompatibility, an open loop ex vivo model was designed simulating the hemodialysis procedure. Blood was withdrawn continuously from healthy nonuremic donors, heparinized, and pumped through a module containing the membrane to be studied. C3a generation in the module was determined at various time points comparing the cuprammonium cellulose (CC) membrane and four types of modified cellulose (MC) membrane, each with a different degree of hydroxyl (OH-) group substitution. In other studies, C3a generation in the ex vivo mode was compared with that during in vivo dialysis. In the ex vivo model, C3a generation with MC membranes was reduced by 70% compared with CC. However, within the MC group, the degree of C3a generation did not correlate with the degree of OH-group substitution. In vivo studies confirmed the reduced degree of C3a generation with the MC membrane compared with CC. Additionally, validation studies using the CC membrane showed excellent agreement between C3a generation during ex vivo perfusion and in vivo dialysis. The results suggest that a group of new MC membranes causes substantially less complement activation than the CC membrane but that the degree of complement activation with various subtypes of MC membranes is not related to the degree of OH-group substitution.