Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles
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 an...
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| Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 11; pp. 2868 - 2873 |
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| Main Authors | , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
National Academy of Sciences
15.03.2016
National Acad Sciences |
| Subjects | |
| Online Access | Get full text |
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| 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. |
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| AbstractList | 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. The therapeutic potential of protein-based genome editing is dependent on the delivery of proteins to appropriate intracellular targets. Here we report that combining bioreducible lipid nanoparticles and negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the self-assembly of nanoparticles for potent protein delivery and genome editing. The design of bioreducible lipids facilitates the degradation of nanoparticles inside cells in response to the reductive intracellular environment, enhancing the endosome escape of protein. In addition, modulation of protein charge through either genetic fusion of supercharged protein or complexation of Cas9 with its inherently anionic sgRNA allows highly efficient protein delivery and effective genome editing in mammalian cells and functional recombinase delivery in the rodent brain. 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. 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.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. |
| Author | Zuris, John A. Pouli, Dimitra Rees, Holly Liu, David R. Sun, Shuo Deng, Pu Gao, Xue Georgakoudi, Irene Meng, Fantao Wang, Ming Wu, Qi Xu, Qiaobing Han, Yong |
| Author_xml | – sequence: 1 givenname: Ming surname: Wang fullname: Wang, Ming organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 – sequence: 2 givenname: John A. surname: Zuris fullname: Zuris, John A. organization: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138 – sequence: 3 givenname: Fantao surname: Meng fullname: Meng, Fantao organization: Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 – sequence: 4 givenname: Holly surname: Rees fullname: Rees, Holly organization: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138 – sequence: 5 givenname: Shuo surname: Sun fullname: Sun, Shuo organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 – sequence: 6 givenname: Pu surname: Deng fullname: Deng, Pu organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 – sequence: 7 givenname: Yong surname: Han fullname: Han, Yong organization: Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 – sequence: 8 givenname: Xue surname: Gao fullname: Gao, Xue organization: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138 – sequence: 9 givenname: Dimitra surname: Pouli fullname: Pouli, Dimitra organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 – sequence: 10 givenname: Qi surname: Wu fullname: Wu, Qi organization: Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 – sequence: 11 givenname: Irene surname: Georgakoudi fullname: Georgakoudi, Irene organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 – sequence: 12 givenname: David R. surname: Liu fullname: Liu, David R. organization: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138 – sequence: 13 givenname: Qiaobing surname: Xu fullname: Xu, Qiaobing organization: Department of Biomedical Engineering, Tufts University, Medford, MA 02155 |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/26929348$$D View this record in MEDLINE/PubMed |
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| DocumentTitleAlternate | Delivery of genome-editing proteins using lipids |
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| Notes | SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 14 ObjectType-Article-1 ObjectType-Feature-2 content type line 23 Author contributions: M.W., Q.W., D.R.L., and Q.X. designed research; M.W., J.A.Z., F.M., S.S., Y.H., and D.P. performed research; M.W., J.A.Z., F.M., H.R., P.D., and X.G. contributed new reagents/analytic tools; M.W., J.A.Z., F.M., S.S., Y.H., D.P., Q.W., I.G., D.R.L., and Q.X. analyzed data; and M.W., J.A.Z., F.M., H.R., D.P., Q.W., I.G., D.R.L., and Q.X. wrote the paper. Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved February 5, 2016 (received for review October 12, 2015) |
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| Snippet | A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane,... The therapeutic potential of protein-based genome editing is dependent on the delivery of proteins to appropriate intracellular targets. Here we report that... |
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| SubjectTerms | Animals Bacterial Proteins - administration & dosage Bacterial Proteins - genetics Ceramides - chemistry Cholesterol - chemistry CRISPR-Cas Systems Drug Carriers Endocytosis Endonucleases - administration & dosage Endonucleases - genetics Endosomes - metabolism Gene Knockout Techniques Genes, Reporter Genes, Synthetic Genetic Engineering - methods Genomes Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - genetics HeLa Cells Humans Hypothalamus - metabolism Integrases - administration & dosage Integrases - genetics Lipids Lipids - administration & dosage Lipids - chemical synthesis Lipids - chemistry Luminescent Proteins - biosynthesis Luminescent Proteins - genetics Mice Molecular Structure Nanoparticles Nanoparticles - administration & dosage Nanoparticles - chemistry Nanoparticles - metabolism Nanoparticles - toxicity Phosphatidylethanolamines - chemistry Physical Sciences Proteins Recombinant Proteins - biosynthesis Recombination, Genetic Ribonucleic acid RNA RNA - genetics Static Electricity Structure-Activity Relationship Thalamus - metabolism |
| Title | Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles |
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