Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway.

The complement alternative pathway (AP) is implicated in numerous diseases affecting many organs, ranging from the rare hematological disease paroxysmal nocturnal hemoglobinuria (PNH), to the common blinding disease age-related macular degeneration (AMD). Critically, the AP amplifies any activating trigger driving a downstream inflammatory response; thus, components of the pathway have become targets for drugs of varying modality. Recent validation from clinical trials using drug modalities such as inhibitory antibodies has paved the path for gene targeting of the AP or downstream effectors. Gene targeting in the complement field currently focuses on supplementation or suppression of complement regulators in AMD and PNH, largely because the eye and liver are highly amenable to drug delivery through local (eye) or systemic (liver) routes. Targeting the liver could facilitate treatment of numerous diseases as this organ generates most of the systemic complement pool. This review explains key concepts of RNA and DNA targeting and discusses assets in clinical development for the treatment of diseases driven by the alternative pathway, including the RNA-targeting therapeutics ALN-CC5, ARO-C3, and IONIS-FB-LRX, and the gene therapies GT005 and HMR59. These therapies are but the spearhead of potential drug candidates that might revolutionize the field in coming years.

Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort.

Rare variants (RVs) in the gene encoding the regulatory enzyme complement factor I (CFI; FI) that reduce protein function or levels increase age-related macular degeneration risk. A total of 3357 subjects underwent screening in the SCOPE natural history study for geographic atrophy secondary to age-related macular degeneration, including CFI sequencing and serum FI measurement. Eleven CFI RV genotypes that were challenging to categorize as type I (low serum level) or type II (normal serum level, reduced enzymatic function) were characterized in the context of pure FI protein in C3b and C4b fluid phase cleavage assays and a novel bead-based functional assay (BBFA) of C3b cleavage. Four variants predicted or previously characterized as benign were analyzed by BBFA for comparison. In all, three variants (W51S, C67R, and I370T) resulted in low expression. Furthermore, four variants (P64L, R339Q, G527V, and P528T) were identified as being highly deleterious with IC50s for C3b breakdown >1 log increased versus the WT protein, while two variants (K476E and R474Q) were ∼1 log reduced in function. Meanwhile, six variants (P50A, T203I, K441R, E548Q, P553S, and S570T) had IC50s similar to WT. Odds ratios and BBFA IC50s were positively correlated (r = 0.76, p < 0.01), while odds ratios versus combined annotation dependent depletion (CADD) scores were not (r = 0.43, p = 0.16). Overall, 15 CFI RVs were functionally characterized which may aid future patient stratification for complement-targeted therapies. Pure protein in vitro analysis remains the gold standard for determining the functional consequence of CFI RVs.

Atypical hemolytic uremic syndrome in the era of terminal complement inhibition: an observational cohort study.

Historically, the majority of patients with complement-mediated atypical hemolytic uremic syndrome (CaHUS) progress to end-stage kidney disease (ESKD). Single-arm trials of eculizumab with a short follow-up suggested efficacy. We prove, for the first time to our knowledge, in a genotype matched CaHUS cohort that the 5-year cumulative estimate of ESKD-free survival improved from 39.5% in a control cohort to 85.5% in the eculizumab-treated cohort (hazard ratio, 4.95; 95% confidence interval [CI], 2.75-8.90; P = .000; number needed to treat, 2.17 [95% CI, 1.81-2.73]). The outcome of eculizumab treatment is associated with the underlying genotype. Lower serum creatinine, lower platelet count, lower blood pressure, and younger age at presentation as well as shorter time between presentation and the first dose of eculizumab were associated with estimated glomerular filtration rate >60 ml/min at 6 months in multivariate analysis. The rate of meningococcal infection in the treated cohort was 550 times greater than the background rate in the general population. The relapse rate upon eculizumab withdrawal was 1 per 9.5 person years for patients with a pathogenic mutation and 1 per 10.8 person years for those with a variant of uncertain significance. No relapses were recorded in 67.3 person years off eculizumab in those with no rare genetic variants. Eculizumab was restarted in 6 individuals with functioning kidneys in whom it had been stopped, with no individual progressing to ESKD. We demonstrated that biallelic pathogenic mutations in RNA-processing genes, including EXOSC3, encoding an essential part of the RNA exosome, cause eculizumab nonresponsive aHUS. Recessive HSD11B2 mutations causing apparent mineralocorticoid excess may also present with thrombotic microangiopathy.

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.

Homodimeric Minimal Factor H: In Vivo Tracking and Extended Dosing Studies in Factor H Deficient Mice.

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.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

Rare Genetic Variants in Complement Factor I Lead to Low FI Plasma Levels Resulting in Increased Risk of Age-Related Macular Degeneration.

Purpose: Rare genetic variants in complement factor I (CFI) that cause low systemic levels of the protein (FI) have been reported as a strong risk factor for advanced age-related macular degeneration (AMD). This study set out to replicate these findings. Methods: FI levels were measured by sandwich ELISA in an independent cohort of 276 patients with AMD and 205 elderly controls. Single-nucleotide polymorphism genotyping and Sanger sequencing were used to assess genetic variability. Results: The median FI level was significantly lower in those individuals with AMD and a rare CFI variant (28.3 µg/mL) compared to those with AMD without a rare CFI variant (38.8 µg/mL, P = 0.004) or the control population with (41.7 µg/mL, P = 0.0085) or without (41.5 µg/mL, P < 0.0001) a rare CFI variant. Thirty-six percent of patients with AMD with a rare CFI variant had levels below the fifth percentile, compared to 6% in controls with CFI variants. Multiple regression analyses revealed a decreased FI level associated with a rare CFI variant was a risk factor for AMD (early or late AMD: odds ratio [OR] 12.05, P = 0.03; early AMD: OR 30.3, P = 0.02; late AMD: OR 10.64, P < 0.01). Additionally, measurement of FI in aqueous humor revealed a large FI concentration gradient between systemic circulation and the eye (∼286-fold). Conclusions: Rare genetic variants in CFI causing low systemic FI levels are strongly associated with AMD. The impermeability of the Bruch's membrane to FI will have implications for therapeutic replacement of FI in individuals with CFI variants and low FI levels at risk of AMD.

Functional Characterization of Rare Genetic Variants in the N-Terminus of Complement Factor H in aHUS, C3G, and AMD.

Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD) have all been strongly linked with dysfunction of the alternative pathway (AP) of complement. A significant proportion of individuals with MPGN, C3G, aHUS and AMD carry rare genetic variants in the CFH gene that cause functional or quantitative deficiencies in the factor H (FH) protein, an important regulator of the AP. In silico analysis of the deleteriousness of rare genetic variants in CFH is not reliable and careful biochemical assessment remains the gold standard. Six N-terminal variants of uncertain significance in CFH were identified in patients with these diseases of the AP and selected for analysis. The variants were produced in Pichia Pastoris in the setting of FH CCPs 1-4, purified by nickel affinity chromatography and size exclusion and characterized by surface plasmon resonance and haemolytic assays as well as by cofactor assays in the fluid phase. A single variant, Q81P demonstrated a profound loss of binding to C3b with consequent loss of cofactor and decay accelerating activity. A further 2 variants, G69E and D130N, demonstrated only subtle defects which could conceivably over time lead to disease progression of more chronic AP diseases such as C3G and AMD. In the variants S159N, A161S, and M162V any functional defect was below the capacity of the experimental assays to reliably detect. This study further underlines the importance of careful biochemical assessment when assigning functional consequences to rare genetic variants that may alter clinical decisions for patients.

Comparison of behavioral deficits and acute neuronal degeneration in rat lateral fluid percussion and weight-drop brain injury models.

The behavioral and histological effects of the lateral fluid percussion (LFP) brain injury model were compared with the weight drop impact-acceleration model with 10 min of secondary hypoxia (WDIA + H). LFP injury resulted in significant motor deficits on the beam walk and inclined plane, and memory deficits on the radial arm maze and Morris water maze. Motor deficits following LFP remained throughout 6 weeks of behavioral testing. WDIA + H injury produced significant motor deficits on the beam walk and inclined plane immediately following injury, but these effects were transient and recovered by 14 days post-injury. In contrast to the LFP injury, the WDIA + H injured animals showed no memory deficits on the radial arm maze and Morris water maze. In order to determine if the differences in behavioral outcome between models were due to differences in injury mechanism or injury severity, 10 LFP-injured animals were matched with 10 WDIA-injured animals based on injury severity (i.e., time to regain righting reflex after brain injury). The LFP-matched injury group showed greater impairment than the WDIA + H matched injury group on the radial arm maze and Morris water maze. Histological examination of LFP-injured brains with Fluoro-Jade staining 24 h, 48 h, and 7 days post-injury revealed degenerating neurons in the cortex, thalamus, hippocampus, caudate-putamen, brainstem, and cerebellum, with degenerating fibers tracts in the corpus callosum and other major tracts throughout the brain. Fluoro-Jade staining following WDIA+H injury revealed damage to fibers in the optic tract, lateral olfactory tract, corpus callosum, anterior commissure, caudate-putamen, brain stem, and cerebellum. While both models produce reliable and characteristic behavioral and neuronal pathologies, their differences are important to consider when choosing a brain injury model.