Genome-editing technologies, such as CRISPR–Cas9, are promising for treating otherwise incurable genetic diseases. Great progress has been made for ex vivo genome editing; however, major bottlenecks exist in the development of efficient, safe and targetable in vivo delivery systems, which are needed for the treatment of many diseases. To achieve high efficacy and safety in therapeutic, in vivo genome editing, editing activities must be controlled spatially and temporally in the body, which requires novel materials, delivery strategies and control mechanisms. Thus, there is currently a tremendous opportunity for the biomaterials research community to develop in vivo delivery systems that overcome the problems of low editing efficiency, off-targeting effect, safety, and cell and tissue specificity. In this Review, we summarize delivery approaches and provide perspectives on the challenges and possible solutions, aiming to stimulate further development of engineered materials for in vivo delivery of genome-editing machinery.
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Gaj, T., Gersbach, C. A. & Barbas, C. F. 3rd ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).
Miller, J., McLachlan, A. D. & Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, 1609–1614 (1985).
Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).
Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).
Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 16, 387–399 (2017).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).
Mojica, F. J., Diez-Villaseñor, C., García-Martínez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).
Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005).
Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).
Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Lee, C. M., Cradick, T. J. & Bao, G. The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol. Ther. 24, 645–654 (2016).
Lee, C. M., Cradick, T. J., Fine, E. J. & Bao, G. Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol Ther. 24, 475–487 (2016).
Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).
Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).
Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl. Acad. Sci. USA 110, 15644–15649 (2013).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, (289–292 (2019).
Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, (292–295 (2019).
Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).
Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).
Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).
Abudayyeh, O. O. et al. RNA targeting with CRISPR–Cas13. Nature 550, 280–284 (2017).
Qu, L. et al. Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol. 37, 1059–1069 (2019).
Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).
Martin, R. M. et al. Highly efficient and marker-free genome editing of human pluripotent stem cells by CRISPR-Cas9 RNP and AAV6 donor-mediated homologous recombination. Cell Stem Cell 24, 821–828 (2019).
Walkey, C. D., Olsen, J. B., Guo, H., Emili, A. & Chan, W. C. Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J. Am. Chem. Soc. 134, 2139–2147 (2012).
Walkey, C. D. et al. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano 8, 2439–2455 (2014).
Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019).
Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).
Komarova, Y. & Malik, A. B. Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu. Rev. Physiol. 72, 463–493 (2010).
Dreher, M. R. et al. Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J. Natl. Cancer Inst. 98, 335–344 (2006).
Yuan, F., Krol, A. & Tong, S. Available space and extracellular transport of macromolecules: effects of pore size and connectedness. Ann. Biomed. Eng. 29, 1150–1158 (2001).
Orsi, M., Sanderson, W. E. & Essex, J. W. Permeability of small molecules through a lipid bilayer: a multiscale simulation study. J. Phys. Chem. B 113, 12019–12029 (2009).
Zhang, S., Gao, H. & Bao, G. Physical principles of nanoparticle cellular endocytosis. ACS Nano 9, 8655–8671 (2015).
Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).
Ginn, S. L., Amaya, A. K., Alexander, I. E., Edelstein, M. & Abedi, M. R. Gene therapy clinical trials worldwide to 2017: An update. J. Gene Med. 20, e3015 (2018).
Yanik, M. et al. In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies. Prog. Retin. Eye Res. 56, 1–18 (2017).
Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015).
Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).
Nelson, C. E. et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat. Med. 25, 427–432 (2019).
Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).
Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).
Hakim, C. H. et al. AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice. JCI Insight 3, e124297 (2018).
Bak, R. O. & Porteus, M. H. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep. 20, 750–756 (2017).
Maddalena, A. et al. Triple vectors expand AAV transfer capacity in the retina. Mol Ther 26, 524–541 (2018).
Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).
Baruteau, J., Waddington, S. N., Alexander, I. E. & Gissen, P. Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects. J. Inherit. Metab. Dis. 40, 497–517 (2017).
Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).
Chen, X. & Goncalves, M. A. Engineered viruses as genome editing devices. Mol. Ther. 24, 447–457 (2016).
Dai, X. et al. One-step generation of modular CAR-T cells with AAV-Cpf1. Nat. Methods 16, 247–254 (2019).
Sun, L., Li, J. & Xiao, X. Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat. Med. 6, 599–602 (2000).
Chew, W. L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods 13, 868–874 (2016).
Schucht, R. et al. A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors. Mol. Ther. 14, 285–292 (2006).
Follenzi, A., Sabatino, G., Lombardo, A., Boccaccio, C. & Naldini, L. Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors. Hum. Gene Ther. 13, 243–260 (2002).
Blömer, U. et al. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71, 6641–6649 (1997).
Abordo-Adesida, E. et al. Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses. Hum. Gene Ther. 16, 741–751 (2005).
Wanisch, K. & Yanez-Munoz, R. J. Integration-deficient lentiviral vectors: a slow coming of age. Mol. Ther. 17, 1316–1332 (2009).
Heckl, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat. Biotechnol. 32, 941–946 (2014).
Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
LaFleur, M. W. et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nat. Commun. 10, 1668 (2019).
Mangeot, P. E. et al. Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Nat Commun 10, 45 (2019).
Candolfi, M. et al. Effective high-capacity gutless adenoviral vectors mediate transgene expression in human glioma cells. Mol. Ther. 14, 371–381 (2006).
Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488–492 (2014).
Bjursell, M. et al. Therapeutic genome editing with CRISPR/Cas9 in a humanized mouse model ameliorates α1-antitrypsin deficiency phenotype. EBioMedicine 29, 104–111 (2018).
Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 (2017).
Li, A. et al. A self-deleting AAV-CRISPR system for in vivo genome editing. Mol. Ther. Methods Clin. Dev. 12, 111–122 (2019).
Boutin, S. et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum. Gene Ther. 21, 704–712 (2010).
Colella, P., Ronzitti, G. & Mingozzi, F. Emerging issues in AAV-mediated in vivo gene therapy. Mol. Ther. Methods Clin. Dev. 8, 87–104 (2018).
Meliani, A. et al. Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system. Hum. Gene Ther. Methods 26, 45–53 (2015).
Mendell, J. R. et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl. J. Med. 377, 1713–1722 (2017).
Vandamme, C., Adjali, O. & Mingozzi, F. Unraveling the complex story of immune responses to AAV vectors trial after trial. Hum. Gene Ther. 28, 1061–1074 (2017).
Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).
van der Loo, J. C. & Wright, J. F. Progress and challenges in viral vector manufacturing. Hum. Mol. Genet. 25, R42–R52 (2016).
Getts, D. R., Shea, L. D., Miller, S. D. & King, N. J. Harnessing nanoparticles for immune modulation. Trends Immunol. 36, 419–427 (2015).
Wang, H. X. et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906 (2017).
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 (2015).
Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).
Chen, Z. et al. Targeted delivery of CRISPR/Cas9-mediated cancer gene therapy via liposome-templated hydrogel nanoparticles. Adv. Funct. Mater. 27, 1703036 (2017).
Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).
Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497–507 (2018).
Pan, Y. et al. Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform. Sci. Adv. 5, eaav7199 (2019).
Lao, Y. H. et al. HPV oncogene manipulation using nonvirally delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute. Adv. Sci. 5, 1700540 (2018).
Wang, H. X. et al. Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide. Proc. Natl. Acad. Sci. USA 115, 4903–4908 (2018).
Jiang, C. et al. A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res 27, 440–443 (2017).
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).
Zhu, H. et al. Spatial control of in vivo CRISPR-Cas9 genome editing via nanomagnets. Nat. Biomed. Eng. 3, 126–136 (2019).
Nesargikar, P. N., Spiller, B. & Chavez, R. The complement system: history, pathways, cascade and inhibitors. Eur. J. Microbiol. Immunol. (Bp) 2, 103–111 (2012).
Oh, P. et al. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat. Biotechnol. 25, 327–337 (2007).
Qiu, Y. et al. Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions. Nat. Commun. 8, 15594 (2017).
Rapoport, S. I. Advances in osmotic opening of the blood-brain barrier to enhance CNS chemotherapy. Expert. Opin. Investig. Drugs 10, 1809–1818 (2001).
Timbie, K. F., Mead, B. P. & Price, R. J. Drug and gene delivery across the blood–brain barrier with focused ultrasound. J. Control. Release 219, 61–75 (2015).
Monsky, W. L. et al. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial growth factor. Cancer Res. 59, 4129–4135 (1999).
Wong, K. M., Horton, K. J., Coveler, A. L., Hingorani, S. R. & Harris, W. P. Targeting the tumor stroma: the biology and clinical development of pegylated recombinant human hyaluronidase (PEGPH20). Curr. Oncol. Rep. 19, 47 (2017).
Senturk, S. et al. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. Nat. Commun. 8, 14370 (2017).
Maji, B. et al. A high-throughput platform to identify small-molecule inhibitors of CRISPR-Cas9. Cell 177, 1067–1079 (2019).
Zhou, X. X. et al. A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription. ACS Chem. Biol. 13, 443–448 (2018).
Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).
Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).
Joshi, M., Pathak, S., Sharma, S. & Patravale, V. Design and in vivo pharmacodynamic evaluation of nanostructured lipid carriers for parenteral delivery of artemether: Nanoject. Int. J. Pharm. 364, 119–126 (2008).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
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).
DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016).
Capecchi, M. R. Altering the genome by homologous recombination. Science 244, 1288–1292 (1989).
Rivera-Torres, N., Banas, K., Bialk, P., Bloh, K. M. & Kmiec, E. B. Insertional mutagenesis by CRISPR/Cas9 ribonucleoprotein gene editing in cells targeted for point mutation repair directed by short single-stranded DNA oligonucleotides. PLoS One 12, e0169350 (2017).
Park, S. H. et al. Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease. Nucleic Acids Res. 47, 7955–7972 (2019).
Byrne, S. M., Ortiz, L., Mali, P., Aach, J. & Church, G. M. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res. 43, e21 (2015).
Eoh, J. & Gu, L. Biomaterials as vectors for the delivery of CRISPR-Cas9. Biomater. Sci. 7, 1240–1261 (2019).
Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).
Lu, B. et al. Delivering SaCas9 mRNA by lentivirus-like bionanoparticles for transient expression and efficient genome editing. Nucleic Acids Res. 47, e44 (2019).
This work was supported by the Cancer Prevention and Research Institute of Texas (RR140081 to G.B.), the National Institutes of Health (R01EB026893 to S.T., UG3TR002863 to K.L. and UG3HL151545 to G.B.) and Defense Advanced Research Projects Agency (HR0011-19-2-0009 to K.L.).
The authors declare no competing interests.
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