Truncated Complement Factor H Y402 Gene Therapy Cures C3 Glomerulonephritis

Abstract

Patients with both age-related macular degeneration (AMD) and C3 glomerulonephritis (C3G) are challenged by the absence of effective therapies to reverse and eliminate their disease burden. Capitalizing on complement dysregulation as both a significant risk factor for AMD and the known pathophysiology of C3G, we investigated the potential for adeno-associated virus (AAV) delivery of complement factor H (CFH) to rescue C3G in a Cfh-/- mouse model of C3G. While past efforts to treat C3G using exogenous human CFH resulted in limited success before immune rejection led to a foreign protein response, our findings demonstrate the capacity for long-term AAV-mediated delivery of truncated CFH (tCFH) to restore inhibition of the alternative pathway of complement and ultimately reverse C3G without immune rejection. Comparing results from the administration of several tCFH vectors also revealed significant differences in their relative efficiency and efficacy. These discoveries pave the way for subsequent development of AAV-mediated tCFH replacement therapy for patients with C3G, while simultaneously demonstrating proof of concept for a parallel AAV-mediated tCFH gene augmentation therapy for patients with AMD.

Keywords: C3 glomerulonephritis; C3 glomerulopathy; age-related macular degeneration; complement factor H; complement regulation; gene replacement; gene therapy.

Figure 1.. Schematic of CFH and truncated CFH (tCFH) proteins packaged into AAV targeting hepatocytes.

(A) Full-length CFH (CFH) consists of 20 short consensus repeats (SCRs) including the SCR spanning the AMD-risk variant CFH Y402H AA (red). Key binding domains and known roles are highlighted. The size (kDa) and retained SCRs for each tCFH protein is also shown. Line connecting SCRs denote missing SCRs compared to CFH. GAG, glycosaminoglycan (B) Each construct from inverted terminal repeat (ITR) to ITR, with its corresponding promoter (green arrow), tCFH cDNA (its size). TBG, thyroxine-binding globin; WPRE, Woodchuck Hepatitis B Post-Translational Response Element and poly A tail.

Figure 2.. Systemic, AAV-mediated replacement of CFH in Cfh−/− mice drives hepatic tCFH expression and reduces fluid phase C3b through restoration of the APC

(A) Representative Western blots showing circulating plasma levels of 12tCFH, 18tCFH, and FHL-1 in Cfh−/− mice treated by AAV-mediated CFH replacement. (B) Bar graph indicating normalized CFH expression in each cohort (± AAV treatment). The number of mice analyzed for each cohort is shown at the base of each bar. (C) Bar graph of normalized C3 fraction calculated as the C3 fraction of the total C3 + C3b for each animal and normalized against CFH-H/H, C3G-negative control mice. The number of mice analyzed for each cohort is shown at the base of each bar.

Figure 3.. AAV-mediated CFH replacement reduces C3 deposit accumulation and resolves C3G.

(A) Representative immunofluorescent confocal imaging of glomeruli from kidney sections of mice according to genotype indicated and AAV treatment (viral titer delivered) as shown. Scale bar is 100 microns. (B) Bar graph indicating the fraction of C3-positive (C3+) glomeruli out of the total glomeruli for which confocal imaging was obtained. The number of glomeruli analyzed for each cohort is shown at the base of each bar. Cfh−/− n=9; CFH-H/0 n=6; CFH-H/H n=9; Cfh−/− AAV12tCFH n=29; Cfh−/− AAV18tCFH n=9; and Cfh−/−AAVFHL-1 n=16. Unpaired t-test was used for comparison between treatment cohorts, *p<0.01. (C) Bar graph analyzing only AAV-treated Cfh−/− mice and assessing treatment as a function of the fraction of C3+ glomeruli out of the total glomeruli imaged. The number of mice in each treatment cohort is shown at the base of each bar. Unpaired t-test was used for comparison between treatment cohorts, *p<0.01.

Figure 4.. AAV-mediated CFH replacement minimizes hallmark histopathological findings in glomeruli and rescues C3G.

(A) Representative images following immunohistochemistry using Periodic-acid Schiff (PAS) staining to highlight features of the glomerulus of a kidney. Scale bar corresponds to 20 microns. Cfh−/− glomeruli had notable hypercellularity, some mesangial matrix expansion, and basement membrane (BM) thickening. CFH-H/0 mild hypercellularity, mild BM thickening, mild mesangial matrix expansion. CFH-H/H healthy glomeruli. Cfh−/−AAV12tCFH (1E13 vg), healthy glomeruli. Cfh−/−AAV18tCFH (1.5E13 vg), hypercellularity and mesangial matrix expansion. Cfh−/−AAVFHL-1 (1E13 vg), mild hypercellularity and mild mesangial matrix expansion. (B) Bar graph comparing masked scores assessing histopathology across mice with and without treatment. The number of mice in each cohort is shown at the base of each bar. Unpaired t-test was used for comparison between treatment cohorts, * p < 0.05, **p<0.01.