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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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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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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