An in vivo CRISPR base editing therapy to inactivate the ANGPTL3 gene: nomination of a development candidate for VERVE-201

Abstract Background Lowering cumulative exposure to low-density lipoprotein cholesterol (LDL-C) is the primary treatment for patients with homozygous familial hypercholesterolemia (HoFH) and established atherosclerotic cardiovascular disease. Due to access, adherence, and healthcare infrastructure limitations, an important fraction of such patients fail to achieve adequate lowering of LDL-C. Durable inactivation in the liver of a cholesterol-raising gene with a one-time therapy offers potential to address this unmet need. Purpose Both human genetic and pharmacologic studies have validated inactivation of the angiopoietin-like protein 3 gene (ANGPTL3) as an approach to lower LDL-C and triglyceride levels, particularly when ANGPTL3 reductions >80% can be achieved. Here, we outline a series of preclinical activities to optimize “VERVE-201”, a CRISPR base editing therapy targeting ANGPTL3. Methods Preclinical development efforts prioritized: (i) identification of a DNA site where editing of a single base pair inactivates ANGPTL3; and (ii) selection of a guide RNA and adenine base editor combination that precisely and specifically inactivates ANGPTL3; and (iii) a delivery approach suitable for all patients, including those with HoFH who lack sufficient low-density lipoprotein receptors (LDLR) needed for hepatic uptake of traditional lipid nanoparticles. Results Bioinformatic and in vitro screening of target sites in the ANGPTL3 gene identified a location where a single A•T to G•C DNA base pair edit leads to disruption of a splice donor and read through into a premature stop codon. To maximize editing of the ANGPTL3 gene while minimizing “off-target” editing elsewhere in the genome, >200 rationally engineered and chemically modified base editing and gRNA configurations were evaluated. Lead candidates were evaluated in primary human hepatocytes to quantify ANGPTL3 editing as well as any “off-target” editing at >600 candidate sites. In a cynomolgus monkey non-human primate model, a single dose of a drug product precursor that used a lipid nanoparticle delivery mechanism achieved potent and durable effects, with a 96% decrease from baseline in circulating ANGPTL3 616 days following administration. The non-human primate homologue to VERVE-201 (“VERVE-201cyno”) incorporates a GalNAc targeting ligand into the lipid nanoparticle, which bypasses LDLR to enable uptake via the liver-specific asialoglycoprotein receptor. VERVE-201cyno led to robust suppression of circulating ANGPTL3 in both a non-human primate model of HoFH and wild-type monkeys, with an average reduction of 89% and 88% respectively 90 days following administration. Conclusions These preclinical data provide the scientific foundation for nomination of a development candidate for VERVE-201, a “once-and-done” gene editing therapy intended to precisely, potently, and durably inactivate hepatic ANGPTL3 and thereby lower LDL-C and triglyceride concentrations permanently. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Verve Therapeutics