CTx001 for Geographic Atrophy: A Gene Therapy Expressing Soluble, Truncated Complement Receptor 1 (Mini-CR1)

Purpose: Preclinical evaluation of a novel gene therapy called CTx001 for treating geographic atrophy (GA). CTx001 encodes a protein called mini-CR1, which is a soluble fragment of complement receptor 1. Design: CTx001 was used in vitro and in vivo to analyze expression and complement-modulating activity. Mini-CR1 was used in vitro to analyze complement-modulating activity, and its ability to cross human Bruch's membrane was evaluated ex vivo. Participants: CTx001, which is a self-complementary rAAV2 gene therapy vector expressing mini-CR1. Recombinant mini-CR1 protein, retinal pigment epithelium (RPE) cell lines, serum, human donor Bruch's membrane, and a rat model. Methods: Recombinant mini-CR1 protein was produced in mammalian cells and purified. C3b and C4b breakdown assays were performed. Wieslab assays measured complement regulatory activity in serum. Mini-CR1 binding to C3b was measured using biolayer interferometry. The diffusion of mini-CR1 across human Bruch's membrane was assessed using an Ussing chamber. Retinal pigment epithelium cell lines were transduced with CTx001 to assess expression, including directionality and complement modulatory activity. In vivo efficacy of CTx001 was tested using a rat laser-induced choroidal neovascularization (CNV) model. Main Outcome Measures: C3b/iC3b/C4b degradation, inhibition of membrane attack complex (MAC) formation in human serum, mini-CR1 binding to C3b, vector transduction efficiency, protein secretion and localization, and complement inhibition in vivo. Results: < 0.01). Conclusions: CTx001 is a potent inhibitor of complement. It efficiently transduces RPE cells, resulting in apical and basolateral secretion and crosses Bruch's membrane, so it is expected to deliver mini-CR1 to the retina and choroid. These findings support its further development as a 1-time gene therapy for addressing complement overactivation in GA. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.