Permanent alteration of PCSK9 in vivo genome editing

项目总结

PROJECT SUMMARY Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the prevention of coronary heart disease (CHD). A gene specifically expressed in and secreted from the liver, and believed to function primarily as an antagonist to the low-density lipoprotein receptor (LDLR), PCSK9 was originally identified as the cause of autosomal dominant hypercholesterolemia in some families, with gain-of- function mutations in the gene driving highly elevated LDL cholesterol (LDL-C) levels and premature CHD. In subsequent studies, individuals with single loss-of-function mutations in PCSK9 were found to experience a significant reduction of both LDL-C levels (~30%–40%) as well as CHD risk (88%). Notably, even individuals with two loss-of-function mutations in PCSK9—resulting in ~80% reduction in LDL-C levels—appear to suffer no adverse clinical consequences. The ability to permanently alter the human genome has been made possible by the technology now commonly known as genome editing. Recently published clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems use Streptococcus pyogenes Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence (protospacer) immediately preceding an NGG motif (PAM, or protospacer-adjacent motif) recognized by Cas9. CRISPR-Cas9 generates a double-strand break (DSB) that is usually repaired by non-homologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations resulting in gene knock-out. A newer version of Cas9 termed a “base editor” selectively edits cytosine bases to thymine, without the need for DSBs, and thus may represent a safer means by which to introduce knock-out nonsense mutations. In light of the observed high efficiencies of CRISPR-Cas9 in mammalian cells in vitro, we seek to assess whether a one-time delivery of CRISPR-Cas9 can be used to permanently disrupt the human PCSK9 gene in vivo efficiently and safely and, if so, the optimal CRISPR-Cas9 system to use for this purpose. Success in completing this translational project will provide critical information on the feasibility of an in vivo genome- editing approach that could ultimately yield a one-shot, long-term therapy that permanently reduces blood LDL- C levels and thus serves as a “vaccination” against cardiovascular disease.