Regulatable complement inhibition of the alternative pathway mitigates age-related macular degeneration pathology

Abstract

Purpose : Age-related macular degeneration (AMD) occurs after age 65. AMD has two forms, dry (atrophic) and wet (angiogenic). AMD is a multifactorial disease; risk factors include aging, smoking, and complement dysregulation. The amplification loop of the complement alternative pathway (AP) is responsible for the majority of complement activation on cell surfaces and extracellular membranes in animal models of this disease. The AP is inhibited naturally by circulating complement protein factor H (fH). Complement component C3 (C3) protein increases and decreases respective to complement activation. In wet AMD, complement effector molecules increase angiogenesis, activate microglia and recruit immune cells, amplifying the complement-microglia inflammatory feedback loop, leading to tissue damage.

Methods : The C3 promoter (−1005 to +251), pC3, was cloned into a pTR backbone and plasmids (pC3-mCherry/CR2-fH) were synthesized and utilized to generate adeno-associated virus serotype 5 (AAV5) vectors. pC3 activation was determined in transiently transfected ARPE19 cells stimulated with H₂O₂ and normal human serum (+/- TT30 or NAC). Laser-induced choroidal neovascularization (CNV) was analyzed in mice treated with AAV5-pC3-mCherry/CR2-fH, using imaging (optical coherence tomography), functional (electroretinography, ERG), molecular (western blotting, complement activation) and histologic (microglia activation and migration) readouts.

Results : Proof of concept was provided that pC3 is modulated in a complement and oxidative stress-dependent manner in human ARPE19 cells, examining mCherry fluorescence. Safe concentrations of AAV5-pC3-CR2-fH, when injected subretinally, were identified using ERG and OCT (10¹⁰ - 10¹¹ vg/mL). The expression of CR2-fH significantly reduced CNV in a dose dependent manner, with a maximum reduction of lesion size of ~35% when compared to mCherry-treated animals. CR2-fH expression reduced CNV-associated ocular C3 activation as assessed by western-blotting for the C3a breakdown products C3d/C3dg. We expect to document a reduction in microglia migration into the subretinal space.

Conclusions : Here we demonstrated that regulating AP inhibition in a complement-dependent manner can ameliorate pathology, and hypothesize this effect to be due, in part, by reducing microglial activation and the proinflammatory microenvironment.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.