Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3

Proc Natl Acad Sci U S A. 2021 Mar 9;118(10):e2020401118. doi: 10.1073/pnas.2020401118.

Abstract

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9-based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

Keywords: Angptl3; CRISPR-Cas9 mRNA delivery; genome editing; lipid nanoparticles.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Angiopoietin-Like Protein 3
  • Angiopoietin-like Proteins* / genetics
  • Angiopoietin-like Proteins* / metabolism
  • Animals
  • CRISPR-Associated Protein 9 / genetics*
  • Drug Carriers* / chemistry
  • Drug Carriers* / pharmacokinetics
  • Drug Carriers* / pharmacology
  • Female
  • Gene Editing*
  • Lipids* / chemistry
  • Lipids* / pharmacokinetics
  • Lipids* / pharmacology
  • Liver / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • Nanoparticles / chemistry*
  • Organ Specificity
  • RNA, Guide* / chemistry
  • RNA, Guide* / genetics
  • RNA, Guide* / pharmacokinetics
  • RNA, Guide* / pharmacology
  • RNA, Messenger* / chemistry
  • RNA, Messenger* / genetics
  • RNA, Messenger* / pharmacokinetics
  • RNA, Messenger* / pharmacology

Substances

  • Angiopoietin-Like Protein 3
  • Angiopoietin-like Proteins
  • Angptl3 protein, mouse
  • Drug Carriers
  • Lipids
  • RNA, Guide
  • RNA, Messenger
  • CRISPR-Associated Protein 9