A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver

Adv Mater. 2020 Nov;32(46):e2003537. doi: 10.1002/adma.202003537. Epub 2020 Oct 14.

Abstract

Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.

Keywords: CRISPR/Cas9; cardiovascular disease; gene therapy; multiplex gene editing; nanoparticles.

MeSH terms

  • Angiopoietin-like Proteins / genetics
  • CRISPR-Cas Systems / genetics
  • Drug Carriers / chemistry*
  • Gene Editing*
  • Gene Transfer Techniques
  • Humans
  • Lipids / chemistry
  • Liver / metabolism*
  • Nanoparticles / chemistry
  • Proprotein Convertase 9 / genetics

Substances

  • ANGPTL3 protein, human
  • Angiopoietin-like Proteins
  • Drug Carriers
  • Lipids
  • Lipofectamine
  • Proprotein Convertase 9