Disrupting PCSK9 and ANGPTL3 genes with CRISPR gene editing technology for Hypercholesterolemia treatment

Homozygous familial hypercholesterolemia (HoFH) is a rare and severe condition resulting from genetic mutations and characterized by extremely high levels of low-density lipoprotein (LDL) cholesterol, thus increasing the risk of heart disease. Gene therapy provides the potential for lifelong therapeutic benefits with a single treatment. CRISPR/Cas9 systems based on Streptococcus pyogenes Cas9 (SpCas9) have been employed to disrupt the PCSK9 or ANGPTL3 genes, reducing circulating LDL cholesterol through LDL receptor (LDLR)-dependent and -independent mechanisms, respectively. However, the use of SpCas9 for multiplex gene editing remains largely limited in preclinical research due to suboptimal editing efficiency and delivery constraints. Recent studies suggest that Staphylococcus aureus Cas9 (SaCas9), a smaller ortholog with higher editing efficiency, may overcome these limitations. To explore a dual-pathway gene therapy strategy for HoFH, we hypothesized that CRISPR/SaCas9 could simultaneously disrupt ANGPTL3 and PCSK9 genes in mammalian cells. Using mouse N2a cells as a model, we first identified a highly efficient novel ANGPTL3 guide RNA; AB514. By delivering AB514 and a validated PCSK9 guide RNA with SaCas9 into N2a cells, we found that ANGPTL3 and PCSK9 genes could be edited simultaneously. Our findings demonstrated that SaCas9 mediated multiple gene editing in mammalian cells is feasible and the dual gene editing approach targeting PCSK9 and ANGPTL3 genes may represent a promising strategy for future HoFH therapy.