One-shot, one cure with genome editing for dyslipidemia.

One of the most inspiring examples of rapid bench-to-bedside translation has been the development of proprotein conver-tase subtilisin/kexin 9 (PCSK9) inhibitory antibodies for the reduction of serum low-density lipoprotein and prevention of coronary artery disease. Astute genetic analysis of gain-of-function mutations in patients with elevated low-density lipo-protein (LDL) levels 1 built the case for the role of PCSK9 in LDL receptor binding and lysosomal destruction. 2 Now the prospect of lipid-lowering therapy delivered monthly via injection is on the horizon with successful clinical trials for the 2 leading monoclonal antibodies. 3 The logical next expectation would be for a permanent disruption of the PCSK9 gene, rendering continued therapy unnecessary because the naturally occurring and protective loss-of-function mutation could be given as a 1-time treatment. Ding et al 4 present evidence that the technology for this now exists with genome editing and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease. CRISPR/ Cas9 is the latest nuclease with demonstrated ability to generate double-strand breaks in mammalian cells at a specified target site, and thereby create frame-shift mutations when error-prone repair processes insert or delete nucleotides at the break site. The authors demonstrate the ease with which a PCSK9-specific nuclease system can be designed, validated for editing efficiency, and delivered to hepatocytes with an adenoviral vector. Ding et al 4 designed synthetic guide RNAs (gRNA) that hybridize a 20-nucleotide DNA sequence in exons 1 and 2 of the PCSK9 gene and assessed the ability to generate mutations using a Surveyor assay in 293 cells. This assay uses a mis-match-specific DNA endonuclease to cleave DNA at sites of base-substitution and other distortions. It makes for a rapid and simple tool to evaluate the editing efficiency of a designed gRNA. The authors then injected adenoviruses expressing Cas9, the CRISPR-associated nuclease derived from Strep-tococcus pyogenes, and an active gRNA into mice via the tail vein. The efficiency of in vivo mutagenesis of Pcsk9 was assessed in genomic DNA harvested from mouse livers 3 to 4 days after injection with the Surveyor assay. Finally, a test for clinical efficacy was conducted with 5 mice receiving the CRISPR-Pcsk9 virus, and 5 mice each receiving either no virus or GFP virus as negative controls. PCSK9, triglyceride, cholesterol, and alanine transaminase levels were measured in all mice to evaluate the response to treatment. The efficacy of CRISPR/Cas9-mediated generation of mutations in Pcsk9 was ≈50% both in vitro …