Reversible Epigenome Editing of <i>PCSK9</i> as a Therapeutic Strategy

Among the best-established causal risk factors for cardiovascular disease is the blood concentration of low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), an antagonist to the LDL receptor, has emerged as a promising therapeutic target for the prevention of coronary heart disease. Current PCSK9 inhibitors are administered via subcutaneous injection, and their effects are short-lived. An alternative “one-and-done” strategy using genome editing to disrupt PCSK9 at the DNA level has been demonstrated in preclinical animal models, including non-human primates. However, concerns about the permanence and irreversibility of the genomic changes have been raised and might limit the acceptance of the therapies. Recently, a set of CRISPR-based epigenome editing tools, CRISPRoff and CRISPRon, were reported to regulate gene expression via site-directed methylation and demethylation of gene promoters, respectively. We hypothesized that these epigenome editing tools could durably and reversibly induce methylation changes in the PCSK9 promoter and thereby modulate its expression. We first screened CRISPRoff guide RNAs (gRNAs) targeting the PCSK9 promoter, individually and in dual combinations, for their ability to reduce PCSK9 expression in the human HuH-7 hepatoma cell line. We then performed long-term experiments with the lead candidate gRNAs. We found that these CRISPRoff gRNAs induced profound increases in methylation at CpG dinucleotides in the PCSK9 promoter, with up to 80% decreases in PCSK9 expression. These methylation increases and gene expression decreases have endured through >56 cell divisions so far, with only mild attenuation over time. Using the same gRNAs with CRISPRon in cells previously treated with CRISPRoff, we observed moderate decreases in methylation and increases in PCSK9 expression. Having established these effects in vitro , we are similarly assessing the durability and reversibility of epigenome editing using the lead gRNAs in a PCSK9 -humanized mouse model. Overall, this work provides a proof of concept of precise gene regulation via methylation and demethylation and suggests a potential new therapeutic approach for protection against coronary heart disease.