A naturally occurring loss-of-function mutation associated with reduced blood lipid levels can be introduced by artificial means—a form of genetic engineering called base editing. Whether the mutation occurs by itself or is deliberately introduced, the consequences are the same. Besides lowering the levels of triglycerides and low-density lipoprotein cholesterol in the blood, the mutation provides protection against coronary heart disease.
These findings come from mouse research conducted by scientists based at the University of Pennsylvania. The scientists, led by Kiran Musunuru, M.D., Ph.D., an associate professor of cardiovascular medicine, assessed whether base editing—a variation of CRISPR/Cas9 genome editing that does not require breaks in the double-strand of DNA—might be used to introduce the protective mutation into a gene called ANGPTL3 (angiopoietin-like 3).
Earlier studies at Penn found that single copies of inactivating mutations in ANGPTL3 are found in about one in every 250 people of European heritage; however, people with mutations in both copies of the gene are rarer. Among people who have one inactivated copy of ANGPTL3, the benefits do not appear to be accompanied by any adverse health consequences. Consequently, the ANGPTL3 protein is an attractive target for new heart disease drugs.
In the current study, Penn scientists demonstrated that they could use base editing in vivo to introduce loss-of-function mutations into ANGPTL3 and reduce blood lipid levels. Details of the scientists’ work appeared February 26 in the journal Circulation, in an article entitled “Reduced Blood Lipid Levels With In Vivo CRISPR-Cas9 Base Editing of ANGPTL3.”
“Base editor 3 (BE3),” the article’s authors wrote, “can introduce cytosine-to-thymine changes at desired sites in the genome, eg, nonsense mutations, into Pcsk9 (proprotein convertase subtilisin/kexin type 9) in mice.” To implement the cytosine-to-thymine changes, the Penn scientists took advantage of base editing’s ability to directly and irreversible convert a specific DNA base into another at a targeted genomic locus.
“This proof-of-principle study showed that base editing of ANGPTL3 is a potential way to permanently treat patients with harmful blood lipid levels,” Musunuru said. “It would be especially useful in patients with a rare condition called homozygous familial hypercholesterolemia, which causes sky-high cholesterol levels and dramatically increased risk of heart attack. They are very difficult to treat with today's medications, and a one-time CRISPR 'vaccination' might be ready to use in these patients within five years.”
The study took a three-part approach. First, the team injected normal mice with the base-editing treatment for the ANGPTL3 gene. After a week, sequencing of the ANGPTL3 target site in liver samples from the mice revealed a median 35% percent editing rate in the target gene and no off-target mutations. In addition, the mean levels of blood lipids were significantly lower in the treated mice by up to 30% compared to untreated mice.
Second, the researchers compared mice with the modified ANGPTL3 gene to those injected with a base-editing treatment for another liver gene, Pcsk9, for plasma cholesterol and triglycerides. After a week, ANGPTL3 targeting caused a similar reduction in cholesterol but a much greater decline in triglycerides compared to targeting Pcsk9. The PCSK9 protein is the target of currently available medications, including evinacumab, which has been shown to reduce cholesterol (but not triglycerides) as well as the risk of heart attack and stroke.
Third, they looked at how base editing of the ANGPTL3 gene performed in a mouse model of homozygous familial hypercholesterolemia (in which knocking out Pcsk9 had little effect). After two weeks, the treated mice showed substantially reduced triglycerides (56%) and cholesterol (51%) compared to untreated mice.
Dr. Musunuru's lab is now preparing to test CRISPR-based treatments against the human ANGPTL3 gene in human liver cells transplanted into mice. This will provide important information on efficacy and safety that will be needed before human trials can move forward.