Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

Proc Natl Acad Sci U S A. 2016 Mar 15;113(11):2868-73. doi: 10.1073/pnas.1520244113. Epub 2016 Feb 29.

Abstract

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Keywords: CRISPR/Cas9; Cre recombinase; genome editing; lipid nanoparticle; protein delivery.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / administration & dosage
  • Bacterial Proteins / genetics
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems
  • Ceramides / chemistry
  • Cholesterol / chemistry
  • Drug Carriers
  • Endocytosis
  • Endonucleases / administration & dosage
  • Endonucleases / genetics
  • Endosomes / metabolism
  • Gene Knockout Techniques*
  • Genes, Reporter
  • Genes, Synthetic*
  • Genetic Engineering / methods*
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • HeLa Cells
  • Humans
  • Hypothalamus / metabolism
  • Integrases / administration & dosage
  • Integrases / genetics
  • Lipids / administration & dosage
  • Lipids / chemical synthesis
  • Lipids / chemistry*
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • Mice
  • Molecular Structure
  • Nanoparticles* / administration & dosage
  • Nanoparticles* / chemistry
  • Nanoparticles* / metabolism
  • Nanoparticles* / toxicity
  • Phosphatidylethanolamines / chemistry
  • RNA / genetics
  • Recombinant Proteins / biosynthesis
  • Recombination, Genetic
  • Static Electricity
  • Structure-Activity Relationship
  • Thalamus / metabolism

Substances

  • 1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine
  • Bacterial Proteins
  • Ceramides
  • Drug Carriers
  • Lipids
  • Lipofectamine
  • Luminescent Proteins
  • Phosphatidylethanolamines
  • Recombinant Proteins
  • fluorescent protein 583
  • Green Fluorescent Proteins
  • RNA
  • Cholesterol
  • Cre recombinase
  • Integrases
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases