Abstract Body (Do not enter title and authors here): Background: CRISPR-based genetic medicines targeting PCSK9 have shown unparalleled potential for sustained LDLc reduction, addressing a critical challenge of patient nonadherence. Yet, the risk of off-target effects and toxicities in current Cas9-based approaches, like CRISPR-base editing, threatens their broad application. Targeted epigenetic silencing, which enables durable gene repression without modifying the underlying DNA code, offers a promising solution by dramatically minimizing off-target risks. Despite this promise, existing technologies have yet to prove effective at therapeutic doses (<1mg/kg). Here, we present the first disclosure of an optimized CRISPR-CasX epigenetic silencing technology capable of demonstrating highly potent LDLc lowering in non-human primates (NHPs).

Methods: We developed a novel investigational treatment, STX1150, composed of an mRNA encoding a highly engineered CRISPR-CasX epigenetic repressor and PCSK9-targeting gRNA encapsulated into lipid nanoparticles. The efficacy of STX1150 was evaluated extensively in vitro and in vivo.

Results: High-throughput in vitro screening identified guide RNAs that potently decreased PCSK9 mRNA, and treatment of primary human hepatocytes with lead molecules reduced PCSK9 mRNA and secreted protein by >95%. Notably, leads exhibited higher potency than Cas9-based PCSK9-lowering molecules, highlighting the potential for more safe and effective LDLc-lowering. A single dose of STX1150 reduced serum hPCSK9 by >95%, with repression persisting for ≥20 weeks in hPCSK9 transgenic mice. Intravenous infusion of STX1150 in NHPs across a dose range durably reduced serum PCSK9 by up to 90% and LDLc by up to 65% for ≥12 weeks. At 0.75 mg/kg, the lowest effective dose demonstrated by any PCSK9 epigenetic silencer to date, LDLc was reduced by ~50%, and liver enzyme profiles were comparable to controls.

Conclusion: We engineered the first CRISPR epigenetic silencing therapy that potently and durably reduces PCSK9 and LDLc after therapeutic relevant dosing in NHPs, suggesting the possibility of a greater therapeutic index when compared to existing Cas9-based approaches, including base editors. Our findings mark the field’s first demonstration of a broadly applicable CRISPR based LDLc-lowering agent that is on par with, if not exceeding, standard-of-care approaches. This innovative approach shows promise for safe and durable LDLc reduction in high-risk ASCVD patients and will be further developed.