Abstract
C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH1–5^18–20^R1–2)], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH KD=505 nM; mFH KD=1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH-/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH-/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH-/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G.
Highlights
C3 glomerulopathy (C3G) is a rare kidney disease associated with abnormal complement regulation [1]
Using analytical size exclusion gel filtration, the new mHDM-Factor H (FH) construct eluted at approximately the same volume as the Mouse Homodimeric Minimal FH Construct (mHDM-FH) v associated virus (AAV)-hHDM-FH
We further demonstrate the efficacy of homodimeric minimal FH constructs (HDM-FH, FH1-5^18-20^R1-2) in the treatment of experimental C3G through the generation of a murine analog and delivery of the human version of the drug via gene therapy
Summary
C3 glomerulopathy (C3G) is a rare kidney disease associated with abnormal complement regulation [1]. There are 3 activating pathways: the classical, lectin and alternative pathway (AP) each playing integral roles in immune [4, 5] and inflammatory responses [5, 6]. These pathways lead to activation of C3 and C5, cascading into the common terminal pathway which terminates with the formation of the membrane attack complex (MAC) [7]. The AP has two properties that make it highly dangerous to self, if not properly regulated: first, it is constantly activated through low levels of spontaneous C3 hydrolysis ( known as a tickover) providing the ability to rapidly respond to invading pathogens [11, 12] and/or label necrotic or apoptotic cells for removal [5] and second, it contains an amplification loop that in certain conditions can be responsible for greater than 80% of the lytic potential of the complement system [13]
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