Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing

Abstract

Rationale Individuals with naturally occurring loss-of-function PCSK9 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels and protection against cardiovascular disease. Objective The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo. Methods and Results We used adenovirus to express Cas9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that within three to four days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic LDL receptor levels, and decreased plasma cholesterol levels (by 35%–40%) in the blood. No off-target mutagenesis was detected in 10 selected sites. Conclusions Genome editing with the CRISPR-Cas9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…Adenovirus-mediated delivery of Cas9 has been used to achieve in vivo genome editing in mouse lungs (Maddalo et al, 2014) and livers Ding et al, 2014;Wang et al, 2015a).…”

How Genome Editing could be used in the Treatment of Cardiovascular Diseases

“…NHEJ-mediated disruption of one or more of these genes should have a therapeutic effect. Proof-of-concept studies with PCSK9 in mouse models have demonstrated that CRISPR-Cas9 genome editing of the gene can occur with high efficiency in the adult liver, reducing blood PCSK9 levels by up to 90% and blood cholesterol levels up to 40% [5][6][7][8]. While there is concern for off-target effects, in other words, mutations occurring at sites other than the target gene, so far the proof-of-concept studies have found little evidence of this phenomenon in vivo, although the currently used techniques are not sensitive enough to detect mutations occurring in less than one in 10,000 cells.…”

Therapeutic editing of hepatocyte genome in vivo

“…and a 40% decrease in the levels of cholesterol in the blood. 78 Gene editing can also be used to eliminate or target viral genomes. In the context of the liver, the elimination of hepatitis B virus can be achieved in preclinical models by using CRISPRCas9, TALENs, and ZFNs nuclease platforms.…”