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Therapeutic Genome Editing and In Vivo Delivery

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Abstract

Improvements in the understanding of human genetics and its roles in disease development and prevention have led to an increased interest in therapeutic genome editing via the use of engineered nucleases. Various approaches have been explored in the past focusing on the development of an effective and safe system for sequence-specific editing. Compared to earlier nucleases such as zinc finger nuclease and transcription activator-like effector nuclease, the relatively low cost and ease of producing clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) systems have made therapeutic genome editing significantly more feasible. CRISPR/Cas9 genome editing has shown great potential to correct genetic mutations implicated in monogenic diseases and to eradicate latent or chronic viral infections in preclinical studies. Several CRISPR/Cas9-based therapeutics have reached the clinical stage, including treatments for inherited red blood cell disorders and Leber Congenital Amaurosis 10, as well as CRISPR/Cas9-edited T cells designed to target and destroy cancer cells. Further advances in therapeutic genome editing will rely on a safe and more efficient method of in vivo CRISPR/Cas9 delivery and improved efficiency of homology-directed repair for site-specific gene insertion or replacement. While other reviews have focused on one or two aspects of CRISPR/Cas9 genome editing, this review aims to provide a summary of the mechanisms of genome editing, the reasons for the emerging interest in CRISPR/Cas9 compared to other engineered nucleases, the current progress in developing CRISPR/Cas9 delivery systems, and the current preclinical and clinical applications of CRISPR/Cas9 genome editing.

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References

  1. Willyard C. New human gene tally reignites debate. Nature. 2018;558:354–5.

    Article  CAS  PubMed  Google Scholar 

  2. Prakash V, Moore M, Yáñez-Muñoz R. Current progress in therapeutic gene editing for monogenic diseases. Mol Ther. 2016;24:465–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chang H, Pannunzio N, Adachi N, Lieber M. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol. 2017;18:495–506.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Liu M, Rehman S, Tang X, Gu K, Fan Q, Chen D, et al. Methodologies for improving HDR efficiency. Front Genet. 2019;9. https://doi.org/10.3389/fgene.2018.00691.

  5. Cassandri M, Smirnov A, Novelli F, Pitolli C, Agostini M, Malewicz M et al. Zinc-finger proteins in health and disease. Cell Death Discov. 2017;3(1). https://doi.org/10.1038/cddiscovery.2017.71.

  6. Gupta R, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest. 2014;124:4154–61.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ramirez C, Foley J, Wright D, Müller-Lerch F, Rahman S, Cornu T, et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods. 2008;5:374–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moore R, Chandrahas A, Bleris L. Transcription activator-like effectors: a toolkit for synthetic biology. ACS Synth Biol. 2014;3:708–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nishimasu H, Ran F, Hsu P, Konermann S, Shehata S, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156:935–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie. 2015;117:119–28.

    Article  CAS  PubMed  Google Scholar 

  11. Cameron P, Coons M, Klompe S, Lied A, Smith S, Vidal B, et al. Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nat Biotechnol. 2019;37:1471–7.

    Article  CAS  PubMed  Google Scholar 

  12. McMahon S, Zhu W, Graham S, Rambo R, White M, Gloster T. Structure and mechanism of a type III CRISPR defense DNA nuclease activated by cyclic oligoadenylate. Nat Commun. 2020;11(1). https://doi.org/10.1038/s41467-019-14222-x.

  13. Paul B, Montoya G. CRISPR-Cas12a: functional overview and applications. Biom J. 2020;43(1):8–17.

    Google Scholar 

  14. O’Connell M. Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR–Cas systems. J Mol Biol. 2019;431:66–87.

    Article  PubMed  CAS  Google Scholar 

  15. Naeem M, Majeed S, Hoque M, Ahmad I. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells. 2020;9:1608. https://doi.org/10.3390/cells9071608.

    Article  CAS  PubMed Central  Google Scholar 

  16. Gough V, Gersbach C. Immunity to Cas9 as an obstacle to persistent genome editing. Mol Ther. 2020;28:1389–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Liu C, Zhang L, Liu H, Cheng K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release. 2017;266:17–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xu X, Wan T, Xin H, Li D, Pan H, Wu J et al. Delivery of CRISPR/Cas9 for therapeutic genome editing. J Gene Med. 2019;21(7). https://doi.org/10.1002/jgm.3107

  19. Modzelewski A, Chen S, Willis B, Lloyd K, Wood J, He L. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc. 2018;13:1253–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Alghadban S, Bouchareb A, Hinch R, Hernandez-Pliego P, Biggs D, Preece C, et al. Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes. Sci Rep-UK. 2020;10(1). https://doi.org/10.1038/s41598-020-74960-7.

  21. Nüssing S, House I, Kearney C, Chen A, Vervoort S, Beavis P, et al. Efficient CRISPR/Cas9 gene editing in uncultured naive mouse T cells for in vivo studies. J Immunol. 2020;204:2308–15.

    Article  PubMed  CAS  Google Scholar 

  22. Yu J. Electroporation of CRISPR-Cas9 into malignant B cells for loss-of-function studies of target gene via knockout. Methods Mol Biol. 2020;2050:85–90.

    Article  CAS  PubMed  Google Scholar 

  23. Dean D. Microinjection. In Brenner’s Encyclopedia of Genetics (2nd etition), Maloy S. Hughe K. edit., Academic Press, 2013; pp409-10.

  24. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider P. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1:841–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lissandrello C, Santos J, Hsi P, Welch M, Mott V, Kim E, et al. High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing. Sci Rep-UK. 2020;10(1). https://doi.org/10.1038/s41598-020-73755-0.

  26. Xu C, Ruan M, Mahajan V, Tsang S. Viral delivery systems for CRISPR. Viruses. 2019;11(1):28. https://doi.org/10.3390/v11010028.

    Article  CAS  PubMed Central  Google Scholar 

  27. Chung S, Mollhoff I, Nguyen U, Nguyen A, Stucka N, Tieu E, et al. Factors impacting efficacy of AAV-mediated CRISPR-based genome editing for treatment of choroidal neovascularization. Mol Ther Methods Clin. 2020;17:409–17.

    Article  CAS  Google Scholar 

  28. Muruve D. The innate immune response to adenovirus vectors. Hum Gene Ther. 2004;15:1157–66.

    Article  CAS  PubMed  Google Scholar 

  29. Ricobaraza A, Gonzalez-Aparicio M, Mora-Jimenez L, Lumbreras S, Hernandez-Alcoceba R. High-capacity adenoviral vectors: expanding the scope of gene therapy. Int J Mol Sci. 2020;21(10):3643. https://doi.org/10.3390/ijms21103643.

    Article  CAS  PubMed Central  Google Scholar 

  30. Schiwon M, Ehrke-Schulz E, Oswald A, Bergmann T, Michler T, Protzer U, et al. One-vector system for multiplexed CRISPR/Cas9 against hepatitis B virus cccDNA utilizing high-capacity adenoviral vectors. Mol Ther-Nucl Acids. 2018;12:242–53.

    Article  CAS  Google Scholar 

  31. Ehrke-Schulz E, Heinemann S, Schulte L, Schiwon M, Ehrhardt A. Adenoviral vectors armed with papillomavirus oncogene specific CRISPR/Cas9 kill human-papillomavirus-induced cervical cancer cells. Cancers. 2020;12(7):1934. https://doi.org/10.3390/cancers12071934.

    Article  CAS  PubMed Central  Google Scholar 

  32. White M, Whittaker R, Gándara C, Stoll E. A guide to approaching regulatory considerations for lentiviral-mediated gene therapies. Hum Gene Ther Method. 2017;28:163–76.

    Article  CAS  Google Scholar 

  33. Joglekar A, Sandoval S. Pseudotyped lentiviral vectors: one vector, many guises. Hum Gene Ther Method. 2017;28:291–301.

    Article  CAS  Google Scholar 

  34. Milone M, O’Doherty U. Clinical use of lentiviral vectors. Leukemia. 2018;32:1529–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Aregger M, Chandrashekhar M, Tong A, Chan K, Moffat J. Pooled lentiviral CRISPR-Cas9 screens for functional genomics in mammalian cells. Methods Mol Biol. 2018; pp169-88.

  36. Ortinski P, O’Donovan B, Dong X, Kantor B. Integrase-deficient lentiviral vector as an all-in-one platform for highly efficient CRISPR/Cas9-mediated gene editing. Mol Ther Methods Clin. 2017;5:153–64.

    Article  CAS  Google Scholar 

  37. Daya S, Berns K. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev. 2008;21:583–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol. 2016;21:75–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mitani K, Kubo S. Adenovirus as an integrating vector. Curr Gene Ther. 2002;2:135–44.

    Article  CAS  PubMed  Google Scholar 

  40. Follenzi A, Santambrogio L, Annoni A. Immune responses to lentiviral vectors. Curr Gene Ther. 2007;7(5):306–15.

    Article  CAS  PubMed  Google Scholar 

  41. Tong S, Moyo B, Lee C, Leong K, Bao G. Engineered materials for in vivo delivery of genome-editing machinery. Nature Reviews Materials. 2019;4(11):726–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li L, Hu S, Chen X. Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities. Biomaterials. 2018;171:207–18. https://doi.org/10.1016/j.biomaterials.2018.04.031.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Cui S, Wang Y, Gong Y, Lin X, Zhao Y, Zhi D, et al. Correlation of the cytotoxic effects of cationic lipids with their headgroups. Toxicol Res-UK. 2018;7:473–9.

    Article  CAS  Google Scholar 

  44. Givens B, Naguib Y, Geary S, Devor E, Salem A. Nanoparticle-based delivery of CRISPR/Cas9 genome-editing therapeutics. AAPS J. 2018;20(6). https://doi.org/10.1208/s12248-018-0267-9.

  45. Guo A, Wang Y, Xu S, Zhang X, Li M, Liu Q, et al. Preparation and evaluation of pH-responsive charge-convertible ternary complex FA-PEI-CCA/PEI/DNA with low cytotoxicity and efficient gene delivery. Colloid Surface B. 2017;152:58–67.

    Article  CAS  Google Scholar 

  46. Strojan K, Lojk J, Bregar V, Veranič P, Pavlin M. Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in CHO cells. Toxicol in Vitro. 2017;41:12–20.

    Article  CAS  PubMed  Google Scholar 

  47. Huang Q, Li S, Ding Y, Yin H, Wang L, Wang R. Macrocycle-wrapped polyethylenimine for gene delivery with reduced cytotoxicity. Biomater Sci-UK. 2018;6:1031–9. https://doi.org/10.1039/C8BM00022K.

    Article  CAS  Google Scholar 

  48. Chou S, Yang P, Ban Q, Yang Y, Wang M, Chien C, et al. Dual supramolecular nanoparticle vectors enable CRISPR/Cas9-mediated knockin of retinoschisin 1 gene: a potential nonviral therapeutic solution for X-linked juvenile retinoschisis. Adv Sci. 2020;7(10):1903432.

    Article  CAS  Google Scholar 

  49. Venault A, Huang Y, Lo J, Chou C, Chinnathambi A, Higuchi A, et al. Tunable PEGylation of branch-type PEI/DNA polyplexes with a compromise of low cytotoxicity and high transgene expression: in vitro and in vivo gene delivery. J Mater Chem B. 2017;5:4732–44.

    Article  CAS  PubMed  Google Scholar 

  50. Fan Y, Sahdev P, Ochyl LJ, Akerberg J, Moon J. Cationic liposome–hyaluronic acid hybrid nanoparticles for intranasal vaccination with subunit antigens. J Control Release. 2015;208:121–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Qian Y, Liang X, Yang J, Zhao C, Nie W, Liu L, et al. Hyaluronan reduces cationic liposome-induced toxicity and enhances the antitumor effect of targeted gene delivery in mice. ACS Appl Mater Interfaces. 2018;10:32006–16.

    Article  CAS  PubMed  Google Scholar 

  52. Liu Y, Cao Z, Xu C, Lu Z, Luo Y, Wang J. Optimization of lipid-assisted nanoparticle for disturbing neutrophils-related inflammation. Biomaterials. 2018;172:92–104.

    Article  CAS  PubMed  Google Scholar 

  53. Luo Y, Xu C, Li H, Cao Z, Liu J, Wang J, et al. Macrophage-specific in vivo gene editing using cationic lipid-assisted polymeric nanoparticles. ACS Nano. 2018;12(2):994–1005.

    Article  CAS  PubMed  Google Scholar 

  54. Luo Y, Liang L, Gan Y, Liu J, Zhang Y, Fan Y, et al. An all-in-one nanomedicine consisting of CRISPR-Cas9 and an autoantigen peptide for restoring specific immune tolerance. ACS Appl Mater Interfaces. 2020;12:48259–71.

    Article  CAS  PubMed  Google Scholar 

  55. Alsaiari S, Patil S, Alyami M, Alamoudi K, Aleisa F, Merzaban J, et al. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. J Am Chem Soc. 2017;140:143–6.

    Article  PubMed  CAS  Google Scholar 

  56. Wang Y, Shahi P, Xie R, Zhang H, Abdeen A, Yodsanit N, et al. A pH-responsive silica–metal–organic framework hybrid nanoparticle for the delivery of hydrophilic drugs, nucleic acids, and CRISPR-Cas9 genome-editing machineries. J Control Release. 2020;324:194–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhang B, Wu P, Zou J, Jiang J, Zhao R, Luo B, et al. Efficient CRISPR/Cas9 gene-chemo synergistic cancer therapy via a stimuli-responsive chitosan-based nanocomplex elicits anti-tumorigenic pathway effect. Chem Eng J. 2020;393:124688. https://doi.org/10.1016/j.cej.2020.124688.

    Article  CAS  Google Scholar 

  58. Gao X, Jin Z, Tan X, Zhang C, Zou C, Zhang W, et al. Hyperbranched poly(β-amino ester) based polyplex nanoparticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer. J Control Release. 2020;321:654–68.

    Article  CAS  PubMed  Google Scholar 

  59. Zhao X, Glass Z, Chen J, Yang L, Kaplan D, Xu Q. mRNA delivery using bioreducible lipidoid nanoparticles facilitates neural differentiation of human mesenchymal stem cells. Adv Healthc Mater. 2020;2000938:2000938. https://doi.org/10.1002/adhm.202000938.

    Article  CAS  Google Scholar 

  60. Lee K, Conboy M, Park H, Jiang F, Kim H, Dewitt M, et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng. 2017;1:889–901.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Ray M, Lee Y, Hardie J, Mout R, Yeşilbag Tonga G, Farkas M, et al. CRISPRed macrophages for cell-based cancer immunotherapy. Bioconjug Chem. 2018;29:445–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Wang P, Zhang L, Xie Y, Wang N, Tang R, Zheng W, et al. Genome editing for cancer therapy: delivery of Cas9 protein/sgRNA plasmid via a gold nanocluster/lipid core-shell nanocarrier. Adv Sci. 2017;4(11):1700175. https://doi.org/10.1002/advs.201700175.

    Article  CAS  Google Scholar 

  63. Zhang L, Wang L, Xie Y, Wang P, Deng S, Qin A, et al. Triple-targeting delivery of CRISPR/Cas9 to reduce the risk of cardiovascular diseases. Angew Chem Int Edit. 2019;58:12404–8.

    Article  CAS  Google Scholar 

  64. Chen X, Chen Y, Xin H, Wan T, Ping Y. Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing. Proc Natl Acad Sci U S A. 2020;117:2395–405.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Peng H, Le C, Wu J, Li X, Zhang H, Le X. A Genome-editing nanomachine constructed with a clustered regularly interspaced short palindromic repeats system and activated by near-infrared illumination. ACS Nano. 2020;14:2817–26.

    Article  CAS  PubMed  Google Scholar 

  66. Pan Y, Yang J, Luan X, Liu X, Li X, Yang J, et al. Near-infrared upconversion–activated CRISPR-Cas9 system: a remote-controlled gene editing platform. Sci Adv. 2019;5(4):eaav7199. https://doi.org/10.1126/sciadv.aav7199.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Wu Y, Zheng J, Zeng Q, Zhang T, Xing D. Light-responsive charge-reversal nanovector for high-efficiency in vivo CRISPR/Cas9 gene editing with controllable location and time. Nano Res. 2020;13:2399–406.

    Article  CAS  Google Scholar 

  68. Kaushik A, Yndart A, Atluri V, Tiwari S, Tomitaka A, Gupta P, et al. Magnetically guided non-invasive CRISPR-Cas9/gRNA delivery across blood-brain barrier to eradicate latent HIV-1 infection. Sci Rep-UK. 2019;9(1). https://doi.org/10.1038/s41598-019-40222-4.

  69. Ryu J, Won E, Lee H, Kim J, Hui E, Kim H, et al. Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy. Biomaterials. 2020;232:119736. https://doi.org/10.1016/j.biomaterials.2019.119736.

    Article  CAS  PubMed  Google Scholar 

  70. Trabulo S, Cardoso A, Düzgüneş N, Jurado A, Pedroso de Lima M. Cell-penetrating peptide-based systems for nucleic acid delivery: a biological and biophysical approach. Method Enzymol. 2012;509:277–300. https://doi.org/10.1016/B978-0-12-391858-1.00014-9.

    Article  CAS  Google Scholar 

  71. Hoffner G, Soues S, Djian P. Aggregation of expanded huntingtin in the brains of patients with Huntington disease. Prion. 2007;1:26–31.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Shin J, Kim K, Chao M, Atwal R, Gillis T, MacDonald M, et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet. 2016:ddw286. https://doi.org/10.1093/hmg/ddw286.

  73. Monteys A, Ebanks S, Keiser M, Davidson B. CRISPR/Cas9 editing of the mutant Huntingtin allele in vitro and in vivo. Mol Ther. 2017;25:12–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109:E2579–86. https://doi.org/10.1073/pnas.1208507109.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Dabrowska M, Juzwa W, Krzyzosiak W, Olejniczak M. Precise excision of the CAG tract from the Huntingtin gene by Cas9 nickases. Front Neurosci-Switz. 2018;12. https://doi.org/10.3389/fnins.2018.00075.

  76. Jiang H, Mankodi A, Swanson M, Moxley R, Thornton C. Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet. 2004;13:3079–88.

    Article  CAS  PubMed  Google Scholar 

  77. Lin X, Miller J, Mankodi A, Kanadia R, Yuan Y, Moxley R, et al. Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet. 2006;15:2087–97.

    Article  CAS  PubMed  Google Scholar 

  78. Dastidar S, Ardui S, Singh K, Majumdar D, Nair N, Fu Y, et al. Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells. Nucleic Acids Res. 2018;46:8275–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Lo Scrudato M, Poulard K, Sourd C, Tomé S, Klein A, Corre G, et al. Genome editing of expanded CTG repeats within the human DMPK gene reduces nuclear RNA foci in the muscle of DM1 mice. Mol Ther. 2019;27:1372–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Wang Y, Hao L, Wang H, Santostefano K, Thapa A, Cleary J, et al. Therapeutic genome editing for myotonic dystrophy type 1 using CRISPR/Cas9. Mol Ther. 2018;26:2617–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Ikeda M, Taniguchi-Ikeda M, Kato T, Shinkai Y, Tanaka S, Hagiwara H, et al. Unexpected mutations by CRISPR-Cas9 CTG repeat excision in myotonic dystrophy and use of CRISPR interference as an alternative approach. Mol Ther Methods Clin. 2020;18:131–44.

    Article  CAS  Google Scholar 

  82. Batra R, Nelles D, Pirie E, Blue S, Marina R, Wang H, et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell. 2017;170(5):899–912.e10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Batra R, Nelles D, Roth D, Krach F, Nutter C, Tadokoro T, et al. The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1. Nat Biomed Eng. 2020;5:157–68. https://doi.org/10.1038/s41551-020-00607-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Castaman G, Matino D. Hemophilia A and B: molecular and clinical similarities and differences. Haematologica. 2019;104:1702–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Sung J, Park C, Leem J, Cho M, Kim D. Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs. Exp Mol Med. 2019;51:1–9.

    Article  CAS  PubMed  Google Scholar 

  86. Chen H, Shi M, Gilam A, Zheng Q, Zhang Y, Afrikanova I, et al. Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII. Sci Rep-UK. 2019;9(1). https://doi.org/10.1038/s41598-019-53198-y.

  87. Park C, Sung J, Cho S, Kim J, Kim D. Universal correction of blood coagulation factor VIII in patient-derived induced pluripotent stem cells using CRISPR/Cas9. Stem Cell Rep. 2019;12:1242–9.

    Article  CAS  Google Scholar 

  88. Ohmori T, Nagao Y, Mizukami H, Sakata A, Muramatsu S, Ozawa K, et al. CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures hemophilia B mice. Sci Rep-UK. 2017;7(1). https://doi.org/10.1038/s41598-017-04625-5.

  89. Wang L, Yang Y, Breton C, White J, Zhang J, Che Y, et al. CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX–knockout mice. Blood. 2019;133:2745–52.

    Article  CAS  PubMed  Google Scholar 

  90. Babačić H, Mehta A, Merkel O, Schoser B. CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: a systematic review. PLoS One. 2019;14(2):e0212198. https://doi.org/10.1371/journal.pone.0212198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Shimo T, Maruyama R, Yokota T. Designing effective antisense oligonucleotides for exon skipping. Methods Mol Biol. 2017:143–55.

  92. Aartsma-Rus A, Straub V, Hemmings R, Haas M, Schlosser-Weber G, Stoyanova-Beninska V, et al. Development of exon skipping therapies for duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues. Nucleic Acid Ther. 2017;27:251–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Amoasii L, Long C, Li H, Mireault A, Shelton J, Sanchez-Ortiz E, et al. Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Sci Transl Med. 2017;9(418):eaan8081. https://doi.org/10.1126/scitranslmed.aan8081.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Amoasii L, Hildyard J, Li H, Sanchez-Ortiz E, Mireault A, Caballero D, et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science. 2018;362:86–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Ifuku M, Iwabuchi K, Tanaka M, Lung M, Hotta A. Restoration of dystrophin protein expression by exon skipping utilizing CRISPR-Cas9 in myoblasts derived from DMD patient iPS Cells. Methods Mol Biol. 1828;2018:191–217.

    Google Scholar 

  96. Min Y, Li H, Rodriguez-Caycedo C, Mireault A, Huang J, Shelton J, et al. CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells. Sci Adv. 2019;5(3):eaav4324. https://doi.org/10.1126/sciadv.aav4324.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis. 2006;1(1). https://doi.org/10.1186/1750-1172-1-40.

  98. Daiger S, Sullivan L, Bowne S. Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013;84:132–41.

    Article  CAS  PubMed  Google Scholar 

  99. Athanasiou D, Aguila M, Bellingham J, Li W, McCulley C, Reeves P, et al. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy. Prog Retin Eye Res. 2018;62:1–23.

    Article  CAS  PubMed  Google Scholar 

  100. Tsai Y, Wu W, Lee T, Wu W, Xu C, Park K, et al. Clustered regularly interspaced short palindromic repeats-based genome surgery for the treatment of autosomal dominant retinitis pigmentosa. Ophthalmology. 2018;125:1421–30.

    Article  PubMed  Google Scholar 

  101. Vagni P, Perlini L, Chenais N, Marchetti T, Parrini M, Contestabile A, et al. Gene editing preserves visual functions in a mouse model of retinal degeneration. Front Neurosci-Switz. 2019;13. https://doi.org/10.3389/fnins.2019.00945.

  102. Cai Y, Cheng T, Yao Y, Li X, Ma Y, Li L, et al. In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway. Sci Adv. 2019;5(4):eaav3335. https://doi.org/10.1126/sciadv.aav3335.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Cremers F. Molecular genetics of Leber congenital amaurosis. Hum Mol Genet. 2002;11:1169–76.

    Article  CAS  PubMed  Google Scholar 

  104. Maeder M, Shen S, Burnight E, Gloskowski S, Mepani R, Friedland A, et al. 687. Therapeutic correction of an LCA-causing splice defect in the CEP290 gene by CRISPR/Cas-mediated genome editing. Mol Ther. 2015;23:S273–4.

    Article  Google Scholar 

  105. Burnight E, Wiley L, Drack A, et al. CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype. Gene Ther. 2014;21:662–72. https://doi.org/10.1038/gt.2014.39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Maeder M, Stefanidakis M, Wilson C, Baral R, Barrera L, Bounoutas G, et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019;25:229–33.

    Article  CAS  PubMed  Google Scholar 

  107. MA B. Sickle Cell Disease [Internet]. GeneReviews®. 2003 [cited 27 February 2021]. Available from: https://pubmed.ncbi.nlm.nih.gov/20301551/

  108. Ashley-Koch A, Yang Q, Olney R. Sickle hemoglobin (Hb S) allele and sickle Cell disease: a HuGE review. Am J Epidemiol. 2000;151:839–45.

    Article  CAS  PubMed  Google Scholar 

  109. Thein S. The molecular basis of β-thalassemia. CSH Perspect Med. 2013;3(5):a011700–0.

    Google Scholar 

  110. Cai L, Bai H, Mahairaki V, Gao Y, He C, Wen Y, et al. A universal approach to correct various HBB gene mutations in human stem cells for gene therapy of ®-thalassemia and sickle cell disease. Stem Cells Transl Med. 2017;7:87–97.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  111. Basak A, Hancarova M, Ulirsch J, Balci T, Trkova M, Pelisek M, et al. BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations. J Clin Invest. 2015;125:2363–8.

    Article  PubMed  PubMed Central  Google Scholar 

  112. GlobeNewswire: CRISPR Therapeutics and Vertex Announce New Clinical Data for Investigational Gene-Editing Therapy CTX001TM in Severe Hemoglobinopathies at the 25th Annual European Hematology Association (EHA) Congress [Internet]. Globenewswire.com. 2020 [cited 30 August 2020]. Available from: https://www.globenewswire.com/news-release/2020/06/12/2047260/0/en/CRISPR-Therapeutics-and-Vertex-Announce-New-Clinical-Data-for-Investigational-Gene-Editing-Therapy-CTX001-in-Severe-Hemoglobinopathies-at-the-25th-Annual-European-Hematology-Associ.html

  113. Chen Y, Sheng J, Trang P, Liu F. Potential application of the CRISPR/Cas9 system against herpesvirus infections. Viruses. 2018;10(6):291. https://doi.org/10.3390/v10060291.

    Article  CAS  PubMed Central  Google Scholar 

  114. van Diemen F, Kruse E, Hooykaas M, Bruggeling C, Schürch A, van Ham P, et al. CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. PLoS Pathog. 2016;12(6):e1005701. https://doi.org/10.1371/journal.ppat.1005701.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Yin D, Ling S, Wang D, Dai Y, Jiang H, Zhou X, et al. Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice. Nat Biotechnol. 2021. https://doi.org/10.1038/s41587-020-00781-8.

  116. Okoye A, Picker L. CD4+T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev. 2013;254:54–64.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  117. Dahabieh M, Battivelli E, Verdin E. Understanding HIV latency: the road to an HIV cure. Annu Rev Med. 2015;66:407–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Liao H, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat Commun. 2015;6(1). https://doi.org/10.1038/ncomms7413.

  119. Zhu W, Lei R, Le Duff Y, Li J, Guo F, Wainberg M, et al. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA. Retrovirology. 2015;12(1). https://doi.org/10.1186/s12977-015-0150-z.

  120. Wang G, Zhao N, Berkhout B, Das A. A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes all infectious provirus in infected T cell cultures. Cell Rep. 2016;17:2819–26.

    Article  CAS  PubMed  Google Scholar 

  121. Lebbink R, de Jong D, Wolters F, Kruse E, van Ham P, Wiertz E, et al. A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape. Sci Rep-UK. 2017;7(1). https://doi.org/10.1038/srep41968.

  122. Ophinni Y, Inoue M, Kotaki T, Kameoka M. CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures. Sci Rep-UK. 2018;8(1). https://doi.org/10.1038/s41598-018-26190-1.

  123. Zhao N, Wang G, Das A, Berkhout B. Combinatorial CRISPR-Cas9 and RNA interference attack on HIV-1 DNA and RNA can lead to cross-resistance. Antimicrob Agents Ch. 2017;61(12). doi:https://doi.org/10.1128/AAC.01486-17.

  124. Dash P, Kaminski R, Bella R, Su H, Mathews S, Ahooyi T et al. Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nat Commun. 2019;10(1). https://doi.org/10.1038/s41467-019-10366-y.

  125. Münger K, Baldwin A, Edwards K, Hayakawa H, Nguyen C, Owens M, et al. Mechanisms of human papillomavirus-Induced oncogenesis. J Virol. 2004;78:11451–60. https://doi.org/10.1128/JVI.78.21.11451-11460.2004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Zhen S, Lu J, Wang L, Sun X, Zhang J, Li X, et al. In vitro and in vivo synergistic therapeutic effect of cisplatin with human papillomavirus16 E6/E7 CRISPR/Cas9 on cervical cancer cell line. Transl Oncol. 2016;9:498–504.

    Article  PubMed  PubMed Central  Google Scholar 

  127. Zhen S, Lu J, Liu Y, Chen W, Li X. Synergistic antitumor effect on cervical cancer by rational combination of PD1 blockade and CRISPR-Cas9-mediated HPV knockout. Cancer Gene Ther. 2019;27:168–78.

    Article  PubMed  CAS  Google Scholar 

  128. Ling K, Yang L, Yang N, Chen M, Wang Y, Liang S, et al. Gene targeting of HPV18 E6 and E7 synchronously by nonviral transfection of CRISPR/Cas9 system in cervical cancer. Hum Gene Ther. 2020;31:297–308.

    Article  CAS  PubMed  Google Scholar 

  129. Tu T, Budzinska M, Vondran F, Shackel N, Urban S. Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via sodium taurocholate cotransporting polypeptide-dependent uptake of enveloped virus particles. J Virol. 2018;92(11):e02007–17. https://doi.org/10.1128/JVI.02007-17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology. 2004;126:1750–8. https://doi.org/10.1053/j.gastro.2004.03.018.

    Article  CAS  PubMed  Google Scholar 

  131. Liu X, Hao R, Chen S, Guo D, Chen Y. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. J Gen Virol. 2015;96(8):2252–61. https://doi.org/10.1099/vir.0.000159.

    Article  CAS  PubMed  Google Scholar 

  132. Karimova M, Beschorner N, Dammermann W, Chemnitz J, Indenbirken D, Bockmann J et al. CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep-UK. 2015;5(1). https://doi.org/10.1038/srep13734.

  133. Dong C, Qu L, Wang H, Wei L, Dong Y, Xiong S. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antivir Res. 2015;118:110–7. https://doi.org/10.1016/j.antiviral.2015.03.015.

    Article  CAS  PubMed  Google Scholar 

  134. Sakuma T, Masaki K, Abe-Chayama H, Mochida K, Yamamoto T, Chayama K. Highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vectors for inactivation of hepatitis B virus. Genes Cells. 2016;21:1253–62. https://doi.org/10.1111/gtc.12437.

    Article  CAS  PubMed  Google Scholar 

  135. Li H, Sheng C, Liu H, Wang S, Zhao J, Yang L, et al. Inhibition of HBV expression in HBV transgenic mice using AAV-delivered CRISPR-SaCas9. Front Immunol. 2018;9. https://doi.org/10.3389/fimmu.2018.02080.

  136. Liu Y, Zhao M, Gong M, Xu Y, Xie C, Deng H, et al. Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus. Antivir Res. 2018;152:58–67.

    Article  CAS  PubMed  Google Scholar 

  137. Stone D, Long K, Loprieno M, De Silva FH, Kenkel E, Liley R, et al. CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice. Mol Ther Methods Clin. 2020;20:258–75.

    Article  CAS  Google Scholar 

  138. Kayesh M, Amako Y, Hashem M, Murakami S, Ogawa S, Yamamoto N, et al. Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA. Virus Res. 2020;290:198191. https://doi.org/10.1016/j.virusres.2020.198191.

    Article  CAS  PubMed  Google Scholar 

  139. Zhen S, Qiang R, Lu J, Tuo X, Yang X, Li X. Enhanced antiviral benefit of combination therapy with anti-HBV and anti-PD1 gRNA/cas9 produces a synergistic antiviral effect in HBV infection. Mol Immunol. 2021;130:7–13.

    Article  CAS  PubMed  Google Scholar 

  140. Lu Y, Xue J, Deng T, Zhou X, Yu K, Deng L, et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med. 2020;26:732–40.

    Article  CAS  PubMed  Google Scholar 

  141. Wang X, Rivière I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther-Oncolytics. 2016;3:16015. https://doi.org/10.1038/mto.2016.15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Depil S, Duchateau P, Grupp S, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19:185–99. https://doi.org/10.1038/s41573-019-0051-2.

    Article  CAS  PubMed  Google Scholar 

  143. CRISPR Therapeutics Reports Positive Top-Line Results from Its Phase 1 CARBON Trial of CTX110™ in Relapsed or Refractory CD19+ B-cell Malignancies. GlobeNewswire News Room. https://www.globenewswire.com/news-release/2020/10/21/2111729/0/en/CRISPR-Therapeutics-Reports-Positive-Top-Line-Results-from-Its-Phase-1-CARBON-Trial-of-CTX110-in-Relapsed-or-Refractory-CD19-B-cell-Malignancies.html. Published 2021. Accessed March 14, 2021.

  144. Ayanoglu F, Elcin A, Elcin Y. Bioethical issues in genome editing by CRISPR-Cas9 technology. Turk J Biol. 2020;44(2):110–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Yang H, Ren S, Yu S, Pan H, Li T, Ge S, et al. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. Int J Mol Sci. 2020;21(18):6461. https://doi.org/10.3390/ijms21186461.

    Article  CAS  PubMed Central  Google Scholar 

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  1. Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia, 30602, USA

    Amanda Catalina Ramirez-Phillips & Dexi Liu

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Ramirez-Phillips, A.C., Liu, D. Therapeutic Genome Editing and In Vivo Delivery. AAPS J 23, 80 (2021). https://doi.org/10.1208/s12248-021-00613-w

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