A novel method for real-time analysis of the complement C3b:FH:FI complex reveals dominant negative CFI variants in age-related macular degeneration.

Age-related macular degeneration (AMD) is linked to 2 main disparate genetic pathways: a chromosome 10 risk locus and the alternative pathway (AP) of complement. Rare genetic variants in complement factor H ( CFH; FH ) and factor I ( CFI; FI ) are associated with AMD. FH acts as a soluble cofactor to facilitate FI's cleavage and inactivation of the central molecule of the AP, C3b. For personalised treatment, sensitive assays are required to define the functional significance of individual AP genetic variants. Generation of recombinant FI for functional analysis has thus far been constrained by incomplete processing resulting in a preparation of active and inactive protein. Using an internal ribosomal entry site (IRES)-Furin- CFI expression vector, fully processed FI was generated with activity equivalent to serum purified FI. By generating FI with an inactivated serine protease domain (S525A FI), a real-time surface plasmon resonance assay of C3b:FH:FI complex formation for characterising variants in CFH and CFI was developed and correlated well with standard assays. Using these methods, we further demonstrate that patient-associated rare genetic variants lacking enzymatic activity (e.g. CFI I340T) may competitively inhibit the wild-type FI protein. The dominant negative effect identified in inactive factor I variants could impact on the pharmacological replacement of FI currently being investigated for the treatment of dry AMD.
Competing Interests: TH has received employment income and equity from Gyroscope Therapeutics, Novartis. EW has received consultancy income from Alexion Pharmaceuticals, Biocryst and Novartis. AL has received consultancy income and equity from Gyroscope Therapeutics and consultancy income from Novartis and Alexion Pharmaceuticals. DS has received consultancy income or funding from Alcon, Bayer Pharmaceuticals, Boehringer Ingelheim, BVI Medical, the Dutch Ophthalmic Research Centre, Gyroscope Therapeutics, Roche, and Alcon. KM has received consultancy income from Freeline Therapeutics and MPM Capital as well as grant income from Gemini Therapeutics and Catalyst Biosciences. CH has recently consulted for Roche, Gyroscope Therapeutics, Q32 Bio, Freeline Therapeutics, Biocryst and Chinook Therapeutics; all income was donated to Newcastle University. CH has received grant income from Ra Pharmaceuticals, and employment income and equity from Gyroscope Therapeutics. DK has received consultancy income and equity from Gyroscope Therapeutics, and consultancy income from Alexion Pharmaceuticals, Novartis, Apellis and Sarepta. DK, KM, CH, TH and TC are authors of patent applications referencing recombinant complement factor I production or formation of the C3b/FH/FI trimolecular complex. TH and CH are employees of Gyroscope Therapeutics, a Novartis company; their contributions to this work were solely at Newcastle University, the opinions are entirely their own and not necessarily those of Gyroscope Therapeutics nor Novartis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.
(Copyright © 2022 Hallam, Cox, Smith-Jackson, Brocklebank, Baral, Tzoumas, Steel, Wong, Shuttleworth, Lotery, Harris, Marchbank and Kavanagh.)