Evaluation of recombinant complement factor H as therapy for orphan renal diseases Dense Deposit Disease & atypical HUS.

The human complement system provides defence against infections. It kills bacteria and viruses but in some circumstances can also cause injury to human tissue. Factor H (FH) is an important regulatory protein in the complement system which acts to prevent this injury. People who have certain mutations in their factor H gene may develop a serious kidney disease called atypical haemolytic uraemic syndrome. Other patients develop a rare kidney condition called dense deposit disease. Both sets of individuals have too little of the FH protein or it does not work properly. Many people (about a third of the UK population) have a common variant of the factor H gene that works well for most of their lives but malfunctions, in a poorly understood way, in later life predisposing them to blindness from age-related damage. It may be possible to treat some of these patients by providing more of a fully functional factor H protein. This is not currently possible. I am going to make large quantities of the pure protein using a unique protocol we have developed and will test if it can prevent or treat renal injury in animal models of disease prior to human trials.

Complement factor H (FH) is a 155kDa human plasma glycoprotein (500-800mg/L). Its major activities are limiting the amplification of the Alternative Pathway (AP) in both the fluid phase and on self surfaces. Mutations in FH are associated with the kidney diseases Dense Deposit Disease (DDD) and atypical Haemolytic Uraemic Syndrome (aHUS), The common polymorphic variants V62 and H402 are strongly associated with development of age related macular degeneration (AMD). Factor H replacement is a possible treatment strategy for such diseases. Plasma therapy has been effective in some cases of aHUS associated with heterozygous FH mutations. Secondly, combined liver-kidney transplantation appears to be an effective means of preventing recurrence of aHUS in renal transplants among individuals with renal failure due to FH-associated aHUS. Murine data shows that plasma purified murine and human FH can control C3 turnover in short-term studies in the only mouse model of DDD, the Factor H-deficient mouse strain (CFH-/-). The inability to generate large-scale quantities of murine FH and the rapid development of an immune response to human FH prevented long-term administration of both these agents.

Critically therefore, the efficacy of FH in preventing or ameliorating DDD in the CFH-/- mouse remains unexplored. Furthermore, the effects of exogenous FH in the only mouse model of aHUS (CFH-/- transgenic for a mutant mouse FH protein that functionally mimics human aHUS-associated FH mutations, CFH-/-.FH 16-20) also remain unknown. My supervisors have developed and applied for a patent for a method making FH protein using the yeast Pichia pastoris. The human protein is structurally and functionally fully active. The murine FH (rmFH) protein will be manufactured, purified and biophysically characterised using SDS-PAGE, dynamic light scattering and mass spectrometry. The binding affinity of rmFH for heparin, C3b and iC3b will be assessed by surface plasmon resonance (SPR) in comparison to plasma-derived murine FH. To assess complement regulatory functions of rmFH, AP cofactor assays on SDS-PAGE and Decay Acceleration Activity (DAA) measured by SPR will be performed. The rmFH protein will be used to perform in vitro studies examining rmFH binding to murine renal endothelium and tissue sections and to assess efficacy in extended studies in relevant animal models of DDD and aHUS. This work will advance the project to the point of being able to develop the product for human testing in renal disease and subsequently to evaluate FH as a therapy for the commonest cause of late-onset blindness AMD.