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.

Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

Adeno-Associated Virus Vector Gene Delivery Elevates Factor I Levels and Downregulates the Complement Alternative Pathway In Vivo.

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.