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

Abstract

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.

Keywords: RNAi; antisense oligonucleotide; clinical trials; complement; gene therapy; preclinical models.

FIGURE 1

RNA targeting via RNA interference (RNAi) or antisense oligonucleotides (ASO). (a) In RNAi, a double‐stranded small interfering (si) RNA is delivered to the target cell where it is taken up by the RNA‐induced silencing complex (RISC). The RNA strands are dissociated, and only the antisense strand remains bound to RISC and functions as a guide to align the complex to target sequences and promote subsequent degradation. (b) Gene knockdown via antisense oligonucleotide works in a similar manner to RNAi; however, a gene‐specific single‐stranded RNA is directly delivered to the target cells where it binds to its target sequence and promotes RNA degradation via RNaseH1. Figure was created with BioRender.com

FIGURE 2

Gene targeting via adeno‐associated virus (AAV) gene therapy. (a) The recombinant (r) AAV genome is packaged in an icosahedral capsid and comprises a promoter, the protein‐encoding sequence and a polyadenylation site (as well as other cis‐regulatory elements required for expression) with two inverted terminal repeats at either end required for efficient packaging of the genome into its capsid. The genome elements are modular and are often optimized specifically for each gene therapy, although almost all have the inverted terminal repeats (ITRs) of AAV2. The route of administration coupled with the capsid serotype determines tropism of the viral vector to specific cell types to reach the target cells. (b) On binding to its cell surface receptor, the AAV vector is taken up by receptor‐mediated endocytosis and resides in the endosomes until it escapes and enters the nucleus. The viral vector uncoats and releases it DNA cargo into the nucleus where it exists as extrachromosomal episomes. The single‐stranded AAV genome is converted to double‐stranded DNA via second strand synthesis. The rAAV promoter drives transgene expression which then follows default transcription and translation pathways, that is, export to the cytoplasm, translation into protein, post‐translational modifications and secreted in the presence of a signal peptide. Figure was created with BioRender.com

FIGURE 3

Gene targeting via CRISPR. (a) Components of the CRISPR system, Cas9 and guide RNAs, can either be delivered via a single plasmid or on separate plasmids. (b) The guide RNA binds upstream of so‐called protospacer adjacent motif (PAM) sequences which are recognized by Cas9. If the guide RNA matches the sequence, then Cas9 unwinds the DNA and introduces a double‐strand break into the DNA backbone. These breaks can either be repaired via non‐homologous end joining, resulting in a gene knockout if the break is at the start of the gene, or via homology‐directed repair if a donor DNA is provided to replace the original DNA. Abbreviations: sgRNA, single guide RNA; PAM, protospacer adjacent motifs. Figure was created with BioRender.com

FIGURE 4

Alternative pathway gene‐targeting therapies currently in clinical development or preclinical characterization. The diagram illustrates RNA therapies, CRISPR and AAV gene therapies discussed in this review and demonstrates that most of the development is in the treatment of ocular models using AAV gene therapy. Abbreviations: AAV, adeno‐associated virus; ASO, antisense oligonucleotides; CRISPR, clustered regularly interspaced short palindromic repeats; RNAi, RNA interference. Figure was created with BioRender.com