Abstract 4140609: Engineering and Development of Optimal CRISPR Based Genome Modification Molecules for Safe and Effective LDL-C Lowering
Authors: Christopher Duncan-Lewis, Matthew Harms, Maroof Adil, Raniel Alcantra-Lee, Farah Bardai, Kristina Oresic Bender, Shyam Sundhar Bale, Emeric Charles, Kai-Yuan Chen, Isabel Colin, Lana Corbo, Fred Deiter, Benjamin Demaree, Sarah Denny, Jason Fernandes, Matthew Gardner, Piotr Hadaczek, Myles Hochman, Bernadette Hsu, Santhosh Karanth, Maitreyee Karmarkar, Steven Keller, Heather Kozy, Oleh Krupa, Dena Leeman, Yuexuan Li, Maria Mirotsou, Amanda Mok, Anna Mrak, Lokesh Narsineni, Sterling Ripley-Phipps, Kirsten Reimer, Keith Szulwach, Shilpa Shroff, Vanessa Tran, Kuei-Ling Tung, Addison Wright, Benjamin Oakes, Aarif Khakoo
Journal: Circulation (2024)

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 &gt;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 &gt;90% in vivo, with repression lasting &gt;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.