Abstract

Background: CRISPR-based genetic medicines offer a promising “one and done” approach to improve cardiovascular health by lowering LDL-C. We performed a comparative analysis of different gene editing approaches to targeting PCSK9 for potent and safe lowering of LDL-C. Based on our findings, we engineered two investigational products using a novel CRISPR-CasX platform: a gene editor and an epigenetic editor. We demonstrated the efficacy and safety of these agents in vivo, including in non-human primates (NHPs). Methods: We engineered two novel CasX-based molecules. STX-1100 is a CasX-based gene editor designed to knock out PCSK9 in hepatocytes and consists of an engineered CasX mRNA and a PCSK9-targeting gRNA encapsulated into lipid nanoparticles (LNPs). STX-1150 is a CasX-based epigenetic editor engineered to silence PCSK9 expression in hepatocytes. It consists of an mRNA encoding a catalytically-inactive modified CasX with a DNA methyltransferase domain and a chromatin effector domain, plus a PCSK9-targeting gRNA encapsulated into LNPs. The efficacy and selectivity of STX-1100, STX-1150, and a Cas9-based adenine base editor were assessed in vitro. Both editing and epigenetic editing approaches were advanced for testing in vivo. Results: STX-1100 achieved 95% editing of the PCSK9 gene in vitro, reducing secreted PCSK9 levels by 95%. Off-target editing was not observed in vitro at 10x EC90. In NHPs, a single dose of STX-1100 resulted in dose-dependent PCSK9 editing, saturating at 1.5 mg/kg, and leading to an LDL-C reduction of up to 54% for ≥300 days. Doses up to 3.0 mg/kg were well-tolerated. STX-1150 achieved >95% reduction in PCSK9 mRNA and protein in vitro. RNA-seq in PHHs confirmed high specificity, silencing only the PCSK9 transcript. A single dose reduced serum PCSK9 levels by >90% in vivo, with repression lasting >24 weeks. On-target CpG methylation near the PCSK9 promoter was observed, corroborating serum PCSK9 reduction. Conclusion: Through CasX engineering, we developed two agents that lower PCSK9 levels in vivo with high selectivity and durability after a single dose in mice and NHPs. Comparing different modalities suggests that gene editing and epigenetic editing may offer a greater therapeutic index and fewer off-target effects compared to existing Cas9-based approaches such as base editing. These innovative molecules show great potential for safe and durable LDL-C lowering in high-risk ASCVD patients and support advancement to clinical testing.