title: Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.
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.
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155;
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
authors.forename: John A
authors.initials: JA
authors.lastname: Zuris
authors.affiliation: Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: Fantao
authors.initials: F
authors.lastname: Meng
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
authors.forename: Holly
authors.initials: H
authors.lastname: Rees
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155;
authors.forename: Shuo
authors.initials: S
authors.lastname: Sun
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155;
authors.forename: Pu
authors.initials: P
authors.lastname: Deng
authors.affiliation: Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: Yong
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138;
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155;
authors.forename: Dimitra
authors.initials: D
authors.lastname: Pouli
authors.affiliation: Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155;
authors.forename: Irene
authors.initials: I
authors.lastname: Georgakoudi
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138; drliu@fas.harvard.edu qiaobing.xu@tufts.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA 02155; drliu@fas.harvard.edu qiaobing.xu@tufts.edu.
authors.forename: Qiaobing
authors.initials: Q
authors.lastname: Xu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002518:Ceramides / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D004337:Drug Carriers; D004705:Endocytosis; D004720:Endonucleases / Q000008:administration & dosage / Q000235:genetics; D011992:Endosomes / Q000378:metabolism; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D019426:Integrases / Q000008:administration & dosage / Q000235:genetics; D008055:Lipids / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D015394:Molecular Structure; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D010714:Phosphatidylethanolamines / Q000737:chemistry; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013788:Thalamus / Q000378:metabolism
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D001426:Bacterial Proteins; D002518:Ceramides; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D012313:RNA; D002784:Cholesterol; C045073:Cre recombinase; D019426:Integrases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9; Cre recombinase; genome editing; lipid nanoparticle; protein delivery