Naturally occurring loss-of-function mutations in ANGPTL3 (angiopoietin-like 3) are associated with reduced blood triglycerides, low-density lipoprotein cholesterol, and risk of coronary heart disease, with no apparent adverse health consequences, making ANGPTL3 a compelling therapeutic target. 1 We assessed whether base editing, a variation on CRISPR-Cas9 genome editing that does not require DNA double-strand breaks, could be used in vivo to introduce loss-of-function mutations into ANGPTL3 and reduce blood lipid levels. Base editor 3 (BE3) can introduce cytosine-to-thymine changes at desired sites in the genome, 2 eg, nonsense mutations, into Pcsk9 (proprotein convertase subtilisin/kexin type 9) in mice. 3 Animal studies and other procedures were performed as previously described 3 in accordance with University of Pennsylvania guidelines.

We screened potential sites of base-edited nonsense mutations in Angptl3 in Neuro-2a cells, identifying robust BE3 activity at the codon Gln-135 site with protospacer sequence AGCCCTTCAACACAAGGTCA. We produced adenoviral vectors encoding BE3 with no guide RNA (BE3-control), guide RNA targeting Angptl3 Gln-135 (BE3-Angptl3), or guide RNA targeting Pcsk9 Trp-159 (BE3-Pcsk9). First, we injected BE3-control or BE3-Angptl3 into 5-week-old male C57BL/6J mice. At day 7, deep sequencing of the Angptl3 target site in liver samples from euthanized BE3-Angptl3–treated mice revealed a median editing rate of 35%; deep sequencing of 10 top predicted sites of off-target mutagenesis (from the CRISPOR website 4) showed no evidence of editing (Figure [A and B]). Pretreatment plasma ANGPTL3, triglycerides, and cholesterol (R&D Systems MANL30; Thermo Fisher TR22421, TR13421) were similar between the groups (2.0%, 0.1%, 0.5% differences), whereas at day 7, the mean analyte levels were all significantly lower in BE3-Angptl3–treated mice (49%, 31%, 19%)(Figure [A]).