Measurement of a complement inhibitory protein following AAV.CR2-CR1 administration in a retinal degradation model of geographic atrophy (GA)

Abstract

Purpose : The role of complement dysfunction in GA pathogenesis is supported and evidenced by increases in complement C3, C5, and activated membrane attack complex in drusen extracted from human donor eyes. This finding has been further validated by recent approvals of C3 and C5 inhibitors for the treatment of GA. Given the apparent link between GA and complement overactivation, inhibition of complement is a focus for the treatment of GA. To support the evaluation of an AAV vector expressing a novel complement receptor 2-complement receptor 1 fusion protein (CR2-CR1) designed to block the function of C3 and C5 at the site of complement fragment deposition at the cell surface in an acute retinal degeneration model, we developed an immunoassay to measure the presence of this complement inhibitory protein and evaluated changes in complement fragment accumulation by Western analysis following gene therapy treatment.

Methods : C57BL/6 mice were randomly assigned to treatment groups (naïve, vehicle-treated, and AAV.CR2-CR1) and treatments administered bilaterally by intraocular injection followed 4 weeks later by intraperitoneal sodium iodate (NaIO₃) to induce retinal damage. To measure concentrations of CR2-CR1, an immunoassay was generated using a biotinylated polyclonal antibody (pAb) specific for the c-terminal end of CR2-CR1 captured on a streptavidin MSD plate followed by detection with a ruthenylated pAb specific for the n-terminal portion of CR2-CR1. This assay was qualified to measure CR2-CR1 in both ocular homogenate and serum. C3/C3b/iC3b/C3d fragment levels were determined by Western analysis.

Results : CR2-CR1 was detectable in ocular homogenate in AAV.CR2-CR1 treated mice following NaIO₃ at levels ranging between 30 to 20,000 pg/mg of total protein. No systemic escape of CR2-CR1 was detectable. The levels of complement fragments present between gene therapy treated and vehicle treated animals following NaIO₃ treatment will be presented.

Conclusions : The described methods will facilitate the ability to correlate biodistribution of CR2-CR1 protein, complement fragment generation, and efficacy. A robust assay for the measurement of this complement inhibitory protein encoded by AAV.CR2-CR1 vector is now available to support definitive pharmacology studies in a model of acute retinal degradation, toxicology studies and future clinical analyses.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.