Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders

Abstract

Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein -glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial culture after a multistep purification process. This glycosylation-optimized factor H showed full complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface–selective regulation of complement activation

“…A previously reported careful comparison between plasma-purified versions of V62-CFH and I62-CFH provided a benchmarking opportunity ( 22 ). Our yields of recombinant V62-CFH and I62-CFH exceeded those of previous attempts to produce CFH in mammalian ( 33 ), insect ( 34 ), and moss cells ( 35 , 55 ). Our versions of V62-CFH and I62-CFH differ in sequence at position 936 as well as at position 62 ( i.e.…”

An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy

“…74 Data on recombinant glycosylation-optimized full-length FH produced in moss and injected intravenously suggested serum t 1/2 of minutes rather than hours. 75 In this context, both homodimeric mini-FH constructs possess markedly improved serum t 1/2 compared with FH 1-5^18-20 ( Figure 5A) and ability to restore plasma C3 levels. Thus, as with the in vitro fluid-phase assay, FH 1-5^18-20^R1-2 was most effective in restoring normal complement function in vivo.…”

Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms

“…In order to test whether heterosplicing is a common phenomenon in P. patens upon transgene expression, we analyzed already existing recombinant human complement factor H (FH)-producing moss lines 40 . Our RT-PCR analysis revealed that expression of the human FH CDS in moss generated two transcript variants, the full-length FH transcript of 3,639 bp and a 1,215 bp FH variant, which could lead to a truncated FH isoform with a molecular mass of 45.6 kDa ( Supplementary Fig.…”

“…The first moss-made drug candidate, moss-aGal for enzyme replacement therapy of Fabry disease, has successfully completed phase I clinical trials 38 . Additionally, a variety of other potential biopharmaceuticals is being produced in moss [39][40][41] .…”