Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo

Abstract

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Strategy for delivering proteins into mammalian cells by fusion or noncovalent complexation with polyanionic macromolecules and complexation with cationic lipids.
Figure 2: Delivery of Cre recombinase to cultured human cells.
Figure 3: Delivery of TALE transcriptional activators into cultured human cells.
Figure 4: Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcription activators to cultured human cells.
Figure 5: DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid–mediated protein:sgRNA delivery using 1.6 μl RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA.
Figure 6: In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear.

Accession codes

Primary accessions

Sequence Read Archive

References

  1. 1

    Putney, S.D. & Burke, P.A. Improving protein therapeutics with sustained-release formulations. Nat. Biotechnol. 16, 153–157 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Mullen, L. et al. Latent cytokines for targeted therapy of inflammatory disorders. Expert Opin. Drug Deliv. 11, 101–110 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Leader, B., Baca, Q.J. & Golan, D.E. Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Discov. 7, 21–39 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Hartung, S.D. et al. Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human α-L-iduronidase gene. Mol. Ther. 9, 866–875 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Wang, J. et al. Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment. Nat. Biotechnol. 26, 901–908 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Midoux, P., Pichon, C., Yaouanc, J.-J. & Jaffrès, P.-A. Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers. Br. J. Pharmacol. 157, 166–178 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Bodles-Brakhop, A.M., Heller, R. & Draghia-Akli, R. Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments. Mol. Ther. 17, 585–592 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Kay, M.A., Glorioso, J.C. & Naldini, L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat. Med. 7, 33–40 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Zangi, L. et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat. Biotechnol. 31, 898–907 (2013).

    CAS  Article  Google Scholar 

  13. 13

    Wadia, J.S., Stan, R.V. & Dowdy, S.F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Daniels, D.S. & Schepartz, A. Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif. J. Am. Chem. Soc. 129, 14578–14579 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Cronican, J.J. et al. Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem. Biol. 5, 747–752 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Thompson, D.B., Cronican, J.J. & Liu, D.R. Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells. Methods Enzymol. 503, 293–319 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Thompson, D.B., Villaseñor, R., Dorr, B.M., Zerial, M. & Liu, D.R. Cellular uptake mechanisms and endosomal trafficking of supercharged proteins. Chem. Biol. 19, 831–843 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Allen, T.M. & Cullis, P.R. Liposomal drug delivery systems: from concept to clinical applications. Adv. Drug Deliv. Rev. 65, 36–48 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Shete, H.K., Prabhu, R.H. & Patravale, V.B. Endosomal escape: a bottleneck in intracellular delivery. J. Nanosci. Nanotechnol. 14, 460–474 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Aguilera, T.A., Olson, E.S., Timmers, M.M., Jiang, T. & Tsien, R.Y. Systemic in vivo distribution of activatable cell penetrating peptides is superior to that of cell penetrating peptides. Integr. Biol. (Camb.) 1, 371–381 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013).

    CAS  Article  Google Scholar 

  22. 22

    Judge, A.D., Bola, G., Lee, A.C.H. & MacLachlan, I. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494–505 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Basha, G. et al. Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells. Mol. Ther. 19, 2186–2200 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Zelphati, O. et al. Intracellular delivery of proteins with a new lipid-mediated delivery system. J. Biol. Chem. 276, 35103–35110 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Adrian, J.E. et al. Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells. J. Control. Release 144, 341–349 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Morris, M.C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173–1176 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Colletier, J.-P., Chaize, B., Winterhalter, M. & Fournier, D. Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer. BMC Biotechnol. 2, 9 (2002).

    Article  Google Scholar 

  28. 28

    Lawrence, M.S., Phillips, K.J. & Liu, D.R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129, 10110–10112 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Liu, J., Gaj, T., Patterson, J.T., Sirk, S.J. & Barbas, C.F. III. Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS ONE 9, e85755 (2014).

    Article  Google Scholar 

  30. 30

    Tessarollo, L., Vogel, K.S., Palko, M.E., Reid, S.W. & Parada, L.F. Targeted mutation in the neurotrophin-3 gene results in loss of muscle sensory neurons. Proc. Natl. Acad. Sci. USA 91, 11844–11848 (1994).

    CAS  Article  Google Scholar 

  31. 31

    Maeder, M.L. et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243–245 (2013).

    CAS  Article  Google Scholar 

  32. 32

    Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    CAS  Article  Google Scholar 

  33. 33

    McNaughton, B.R., Cronican, J.J., Thompson, D.B. & Liu, D.R. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc. Natl. Acad. Sci. USA 106, 6111–6116 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

    CAS  Article  Google Scholar 

  35. 35

    Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  38. 38

    Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  Article  Google Scholar 

  39. 39

    Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    CAS  Article  Google Scholar 

  40. 40

    Guilinger, J.P., Thompson, D.B. & Liu, D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    CAS  Article  Google Scholar 

  41. 41

    Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    CAS  Article  Google Scholar 

  42. 42

    Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638–646 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Lumpkin, E.A. et al. Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene Expr. Patterns 3, 389–395 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Sojung Kim, D.K. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 10.1101/gr.171322.113 (2014).

  45. 45

    Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    CAS  Article  Google Scholar 

  46. 46

    Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  Article  Google Scholar 

  47. 47

    Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Sage, C. et al. Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 307, 1114–1118 (2005).

    CAS  Article  Google Scholar 

  49. 49

    Sander, J.D. et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 41, e181 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

J.A.Z. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 106601-2) D.B.T., J.P.G. and J.L.B. were supported by US National Institutes of Health (NIH) R01 GM095501 (to D.R.L.), Defense Advanced Research Projects Agency HR0011-11-2-0003 (to D.R.L.) and N66001-12-C-4207 (to D.R.L.), and the Howard Hughes Medical Institute (HHMI). D.R.L. was supported as a HHMI Investigator. Z.-Y. C. was supported by US National Institutes of Health (R01 DC006908), the Bertarelli Foundation, and the David-Shulsky Foundation. Y.S. was supported by the Frederick and Ines Yeatts Hair Cell Regeneration grant and by The National Nature Science Foundation of China NSFC81300824. J.H.H. was supported by National Science Foundation Graduate Research Fellowship Program (DGE1144152). M.L.M. and J.K.J. were supported by an NIH Director's Pioneer Award (DP1 GM105378). We thank A. Lawson, M. Sonnett, R. Xiao, S. Wang and J. Gehrke for technical assistance. We thank A. Edge, Massachusetts Eye & Ear Infirmary, Boston, for mouse embryonic stem cell (ES) line Tau-GFP and J. Johnson, Southwestern Medical Center, University of Texas, for floxP-tdTomato mice (The Jackson Laboratory).

Author information

Affiliations

Authors

Contributions

J.A.Z., D.B.T., Y.S., Z.-Y.C. and D.R.L. designed the research and analyzed the data. J.A.Z., D.B.T., Y.S., J.P.G. and J.L.B. generated research materials and performed the experiments. M.L.M. designed and constructed TALEs and dCas9 activator sgRNAs. J.P.G., J.A.Z. and J.H.H. analyzed DNA sequencing data. J.K.J., Z.-Y.C. and D.R.L. supervised the research. All authors wrote the manuscript.

Corresponding author

Correspondence to David R Liu.

Ethics declarations

Competing interests

The co-authors have filed a provisional patent application related to this work. J.K.J. and D.R.L. are cofounders of consultants for Editas Medicine, a company that applies genome-editing technologies. J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.L.M. is currently an employee of Editas Medicine.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1 and 2, Supplementary Notes and Supplementary Results (PDF 9148 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zuris, J., Thompson, D., Shu, Y. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33, 73–80 (2015). https://doi.org/10.1038/nbt.3081

Download citation

Further reading

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing