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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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.
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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