Abstract Body (Do not enter title and authors here): Background
Elevated low-density lipoprotein cholesterol (LDL-C) is a key risk factor for atherosclerotic cardiovascular disease (ASCVD), and reducing LDL-C lowers major adverse cardiovascular event risk. Heterozygous familial hypercholesterolemia (HeFH) is a genetic disease characterized by high LDL-C and ASCVD risk, with ~90% of genetically confirmed cases due to autosomal dominant, loss-of-function mutations in the LDLR gene. Despite multiple treatment options, many patients fail to reach target LDL-C levels. Furthermore, evidence suggests a favorable benefit and safety profile with extremely low LDL-C (10–40 mg/dL) and supports aggressively lowering LDL-C in ASCVD.
Hypothesis
We have developed a novel CRISPR-based one-time treatment strategy to edit the LDLR gene to achieve LDLR upregulation and significant LDL-C lowering in patients with HeFH and ASCVD. Our strategy was informed by a naturally occurring gain-of-function deletion in the 3′ untranslated region (UTR) of LDLR that led to a mean 74% lower LDL-C compared with non-carriers, and no adverse effects. We employ dual gRNAs to delete negative regulatory regions of the LDLR 3′ UTR to upregulate mRNA and protein expression.
Methods and Results
A potent human dual gRNA pair was identified through primary human hepatocyte screening. To demonstrate in vivo proof of concept, Ldlr^+/− mice were injected with lipid nanoparticles (LNPs) containing a CRISPR nuclease mRNA, and a surrogate gRNA pair targeting the orthologous genomic region. Mice treated with LNPs demonstrated >15-fold increase in LDLR protein in the liver and >80% biomarker reduction. Administration of two different LNP formulations (Genevant Sciences) to non-human primates was well tolerated as indicated by maintenance of body weight, temperature, and clinical presentation, and an acceptable clinical chemistry, hematology, and coagulation profile. Terminal liver samples demonstrated productive editing, LDLR mRNA upregulation, and increased LDLR protein. Notably, treatment induced a remarkable >90% mean decrease in serum LDL-C.
Conclusions
Our findings demonstrate preclinical validation of an alternate mechanism for robust LDL-C lowering by deleting 3′ UTR regulatory elements in the LDLR gene resulting in potent LDLR upregulation. This in vivo strategy represents a potentially transformative one-time treatment for lowering LDL-C in patients with HeFH and ASCVD to significantly reduce cardiovascular risk.