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Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutics, especially those that can correct gene mutations via homology-directed repair, have the potential to revolutionize the treatment of genetic diseases. However, it is challenging to develop homology-directed repair-based therapeutics because they require the simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

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Fig. 1: CRISPR–Gold can deliver Cas9 RNP and donor DNA in vivo and induce HDR.
Fig. 2: Synthesis and characterization of CRISPR–Gold.
Fig. 3: CRISPR–Gold induces HDR in vitro.
Fig. 4: CRISPR–Gold induces HDR and promotes the expression of dystrophin protein in primary myoblasts.
Fig. 5: CRISPR–Gold induces gene editing in the muscle tissue of Ai9 mice
Fig. 6: CRISPR–Gold promotes HDR in the dystrophin gene and dystrophin protein expression, and reduces muscle fibrosis in mdx mice, with CTX stimulation.
Fig. 7: CRISPR–Gold-induced dystrophin editing enhances muscle function with minimal off-target effects and without elevation of systemic inflammatory cytokines.

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References

  1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–822 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas system. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cho, S. W., Kim, S., Kim, J. M. & Kim, J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–190 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 201512503 (2015).

    Article  Google Scholar 

  8. Lin, S., Staahl, B., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3, 1–13 (2014).

    CAS  Google Scholar 

  9. Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lu, Q. L. et al. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc. Natl Acad. Sci. USA 102, 198–203 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Sun, W. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. Engl. 54, 12029–12033 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yu, X. et al. Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnol. Lett. 38, 919–929 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Miyata, K. et al. Polyplexes from poly(aspartamide) bearing 1,2-diaminoethane side chains induce pH-selective, endosomal membrane destabilization with amplified transfection and negligible cytotoxicity. J. Am. Chem. Soc. 130, 16287–16294 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Kim, H. J. et al. Introduction of stearoyl moieties into a biocompatible cationic polyaspartamide derivative, PAsp(DET), with endosomal escaping function for enhanced siRNA-mediated gene knockdown. J. Control. Release 145, 141–148 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Ding, Y. et al. Gold nanoparticles for nucleic acid delivery. Mol. Ther. 22, 1075–1083 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Singh, D., Sternberg, S. H., Fei, J., Ha, T. & Doudna, J. A. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat. Commun. 7, 1–8 (2016).

    CAS  Google Scholar 

  23. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Walkey, C. D. et al. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano 8, 2439–2455 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Liu, J. & Peng, Q. Protein-gold nanoparticle interactions and their possible impact on biomedical applications. Acta Biomater. 55, 13–27 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Chithrani, B. D., Ghazani, A. A. & Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662–668 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Chou, L. Y. T., Zagorovsky, K. & Chan, W. C. W. DNA assembly of nanoparticle superstructures for controlled biological delivery and elimination. Nat. Nanotechnol. 9, 148–155 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rouge, J. L., Hao, L., Wu, X. A., Briley, W. E. & Mirkin, C. A. Spherical nucleic acids as a divergent platform for synthesizing RNA–nanoparticle conjugates through enzymatic ligation. ACS Nano 8, 8837–8843 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Smith, R. C., Riollano, M., Leung, A. & Hammond, P. T. Layer-by-layer platform technology for small-molecule delivery. Angew. Chem. Int. Ed. Engl. 48, 8974–8977 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Lee, K. et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife 6, e25312 (2017).

    PubMed  PubMed Central  Google Scholar 

  33. Yang, L. et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 41, 9049–9061 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Hegde, M. R. et al. Microarray-based mutation detection in the dystrophin gene. Hum. Mutat. 29, 1091–1099 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nakamura, A. & Takeda, S. Exon-skipping therapy for Duchenne muscular dystrophy. Lancet 378, 546–547 (2011).

    Article  PubMed  Google Scholar 

  38. Mendell, J. R. et al. Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann. Neurol. 74, 637–647 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Kornegay, J. N. et al. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs. Mol. Ther. 18, 1501–1508 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol. Ther. 24, 564–569 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Pavlath, G. K. & Horsley, V. Cell fusion in skeletal muscle—central role of NFATC2 in regulating muscle cell size. Cell Cycle 2, 420–423 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Sy, J. C. et al. Sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction. Nat. Mater. 863–869 (2008).

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Acknowledgements

This work was supported by grants from the National Institutes of Health (U01 268201000043C-0-0-1 and R56 AI107116-01 to I.C. as well as grants from Calico, Roger’s and the Strategies for Engineered Negligible Senescence Research Foundation to I.C. This work was also supported by the W. M. Keck Foundation, Moore Foundation, Li Ka Shing Foundation and Center of Innovation programme of the Japan Science and Technology Agency. K.L. is a Siebel Fellow of the Siebel Scholars Foundation. F.J. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2201-14). M.A.D. is a California Institute for Regenerative Medicine (CIRM) post-doctoral fellow and is supported by CIRM training grant TG2-01164. J.A.D is a Howard Hughes Medical Institute Investigator. We thank M. West at the CIRM/QB3 Shared Stem Cell Facility and H. Nolla and T. Shovha at the Berkeley FACS facility for technical assistance, as well as D. Schaffer, L. S. Qi, B. Staahl, S. Lin and S. Yang for advice and technical support. This work used the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley, supported by National Institutes of Health S10 Instrumentation Grants S10RR029668 and S10RR027303.

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Contributions

K.L. planned and performed the experiments shown in all the figures, analysed and interpreted the data and wrote the manuscript. M.C. designed, planned and performed the in vivo studies shown in Figs. 5 and 6 and wrote the manuscript. H.M.P. and H.L. performed the in vivo experiments and analysed and interpreted the data. F.J. purified the Cas9 protein and analysed the gel data. H.J.K. and K.K. synthesized the PAsp(DET) polymer, performed the experiment shown in Supplementary Table 1 and contributed to the data interpretation. W.-c.H. and S.L. cultured the stem cells and performed the experiment shown in Supplementary Fig. 7. M.A.D. and J.E.C. generated the BFP-HEK cells and supported the deep sequencing analysis. V.A.M., K.C., H.M.P. and A.R. performed the in vitro and in vivo DNA analysis. C.S., M.M. and T.S. performed the muscle histology studies. F.L. and N.L.B. performed the deep sequencing analysis. J.E.C. and J.A.D. contributed to the design of the studies and data interpretation. I.C. and N.M. planned and integrated the work, interpreted the data and wrote the manuscript.

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Correspondence to Irina Conboy or Niren Murthy.

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K.L., H.M.P. and N.M. are co-founders of GenEdit. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine and Intellia Therapeutics.

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Lee, K., Conboy, M., Park, H.M. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 1, 889–901 (2017). https://doi.org/10.1038/s41551-017-0137-2

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