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Hamostaseologie 2017; 37(01): 45-52
DOI: 10.5482/HAMO-16-09-0035
Plenary lecture
Schattauer GmbH

Therapeutic genome editing with engineered nucleases

Therapeutische Genomchirurgie mit Designer-Nukleasen
Simone A. Haas   *
1   Institute for Cell and Gene Therapy, Medical Center – University of Freiburg, Germany
2   Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Germany
3   Graduate Program in Molecular Medicine, Faculty of Biology, University of Freiburg, Freiburg, Germany
,
Viviane Dettmer   *
1   Institute for Cell and Gene Therapy, Medical Center – University of Freiburg, Germany
2   Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Germany
4   Graduate Program in Biology, Faculty of Biology, University of Freiburg, Freiburg, Germany
,
Toni Cathomen
1   Institute for Cell and Gene Therapy, Medical Center – University of Freiburg, Germany
2   Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Germany
5   Faculty of Medicine, University of Freiburg, Freiburg, Germany
› Author Affiliations Fundings Research in our lab is currently supported by grants of the German Federal Ministry of Education and Research (BMBF-01EO0803), the German Research Foundation (SFB1160-TP17), the German Academic Exchange Service (DAAD–91526643), and the European Commission (SCIDNET–666908, CARAT–667980).
Further Information

Publication History

received: 25 September 2016

accepted in revised form: 20 January 2016

Publication Date:
28 December 2017 (online)

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Summary

Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

Zusammenfassung

Die zielgerichtete Genombearbeitung mit Zinkfinger-Nukleasen, TALE-Nukleasen und CRISPR/Cas-Nukleasen hat eine neue Ära in der Gentherapie eingeläutet. Angeborene Erkrankungen, die mit einem herkömmlichen Gentherapieansatz nicht behandelt werden können, wie zum Beispiel Erbkrankheiten mit dominanter Vererbung oder Krankheiten, die durch Mutationen in eng regulierten Genen hervorgerufen werden, können nun durch präzise Genomchirurgie behandelt werden. Darüber hinaus ermöglichen Designer-Nukleasen neue genetische Interventionsmöglichkeiten, um Infektionskrankheiten oder Krebs zu bekämpfen. In diesem Aufsatz beschreiben wir die Entwicklung der verschiedenen Klassen von programmierbaren Nukleasen, diskutieren die Herausforderungen bei der Translation von Gen-Editing in die klinische Anwendung und geben einen Ausblick darauf, welche Anwendungen in naher Zukunft in die Klinik gelangen werden.

Keywords

clinical application - clinical trial - designer nuclease - gene editing - gene therapy - genetic disorder

Schlüsselwörter

klinische Anwendung - klinische Studie - Designer-Nuklease - Gen-Editing - Gentherapie - genetische Erkrankung

* these authors contributed equally.


 

  • References

  • 1 Naldini L. Gene therapy returns to centre stage. Nature 2015; 526 (7573): 351-360.
    CrossrefPubMedGoogle Scholar
  • 2 Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 1996; 93 (03) 1156-1160.
    CrossrefPubMedGoogle Scholar
  • 3 Smith J, Bibikova M, Whitby FG. et al. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 2000; 28 (17) 3361-3369.
    CrossrefPubMedGoogle Scholar
  • 4 Urnov FD, Miller JC, Lee YL. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435 (7042): 646-651.
    CrossrefPubMedGoogle Scholar
  • 5 Christian M, Cermak T, Doyle EL. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 2010; 186 (02) 757-761.
    CrossrefPubMedGoogle Scholar
  • 6 Li T, Huang S, Jiang WZ. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 2011; 39 (01) 359-372.
    CrossrefPubMedGoogle Scholar
  • 7 Miller JC, Tan S, Qiao G. et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 2011; 29 (02) 143-148.
    CrossrefPubMedGoogle Scholar
  • 8 Mussolino C, Morbitzer R, Lütge F. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011; 39 (21) 9283-9293.
    CrossrefPubMedGoogle Scholar
  • 9 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 (39) E2579-2586.
    CrossrefPubMedGoogle Scholar
  • 10 Jinek M, Chylinski K, Fonfara I. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337 (6096): 816-821.
    CrossrefPubMedGoogle Scholar
  • 11 Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPRCas9. Science 2014; 346 (6213): 1258096.
    PubMedGoogle Scholar
  • 12 Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem 2014; 83: 409-439.
    CrossrefPubMedGoogle Scholar
  • 13 Mussolino C, Mlambo T, Cathomen T. Proven and novel strategies for efficient editing of the human genome. Curr Opin Pharmacol 2015; 24: 105-112.
    CrossrefPubMedGoogle Scholar
  • 14 Zetsche B, Gootenberg JS, Abudayyeh OO. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015; 163 (03) 759-771.
    CrossrefPubMedGoogle Scholar
  • 15 Boissel S, Jarjour J, Astrakhan A. et al. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 2014; 42 (04) 2591-2601.
    CrossrefPubMedGoogle Scholar
  • 16 Sadelain M, Papapetrou EP, Bushman FD. Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer 2012; 12 (01) 51-58.
    CrossrefPubMedGoogle Scholar
  • 17 Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet 2016; 17 (05) 300-312.
    CrossrefPubMedGoogle Scholar
  • 18 Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 1991; 252 (5007): 809-817.
    CrossrefPubMedGoogle Scholar
  • 19 Kim CA, Berg JM. A 2.2 A resolution crystal structure of a designed zinc finger protein bound to DNA. Nat Struct Biol 1996; 03 (11) 940-945.
    CrossrefPubMedGoogle Scholar
  • 20 Jamieson AC, Miller JC, Pabo CO. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2003; 02 (05) 361-368.
    CrossrefPubMedGoogle Scholar
  • 21 Boch J, Scholze H, Schornack S. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009; 326 (5959): 1509-1512.
    CrossrefPubMedGoogle Scholar
  • 22 Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science 2009; 326 (5959): 1501.
    CrossrefPubMedGoogle Scholar
  • 23 Boch J, Bonas U. Xanthomonas AvrBs3 familytype III effectors: discovery and function. Ann Rev Phytopathol 2010; 48: 419-436.
    CrossrefPubMedGoogle Scholar
  • 24 Mak AN, Bradley P, Cernadas RA. et al. The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 2012; 335 (6069): 716-719.
    CrossrefPubMedGoogle Scholar
  • 25 Makarova KS, Wolf YI, Alkhnbashi OS. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 2015; 13 (11) 722-736.
    CrossrefPubMedGoogle Scholar
  • 26 Gao F, Shen XZ, Jiang F. et al. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol 2016; 34 (07) 768-773.
    CrossrefPubMedGoogle Scholar
  • 27 Lee SH, Turchiano G, Ata H. et al. Failure to detect DNA-guided genome editing using Natronobacterium gregoryi Argonaute. Nat Biotechnol. 2016 DOI: 10.1038/nbt.3753 [Epub ahead of print].
    PubMedGoogle Scholar
  • 28 Weiner A, Zauberman N, Minsky A. Recombinational DNA repair in a cellular context: a search for the homology search. Nat Rev Microbiol 2009; 07 (10) 748-755.
    CrossrefPubMedGoogle Scholar
  • 29 Höher T, Wallace L, Khan K. et al. Highly efficient zinc-finger nuclease-mediated disruption of an eGFP transgene in keratinocyte stem cells without impairment of stem cell properties. Stem Cell Rev 2012; 08 (02) 426-434.
    CrossrefPubMedGoogle Scholar
  • 30 Pattanayak V, Ramirez CL, Joung JK, Liu DR. Revealing off-target cleavage specificities of zincfinger nucleases by in vitro selection. Nat Methods 2011; 08 (09) 765-770.
    CrossrefPubMedGoogle Scholar
  • 31 Guilinger JP, Pattanayak V, Reyon D. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 2014; 11 (04) 429-435.
    CrossrefPubMedGoogle Scholar
  • 32 Mussolino C, Alzubi J, Fine EJ. et al. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res 2014; 42 (10) 6762-6773.
    CrossrefPubMedGoogle Scholar
  • 33 Miller JC, Zhang L, Xia DF. et al. Improved specificity of TALE-based genome editing using an expanded RVD repertoire. Nat Methods 2015; 12 (05) 465-471.
    CrossrefPubMedGoogle Scholar
  • 34 Juillerat A, Pessereau C, Dubois G. et al. Optimized tuning of TALEN specificity using non-conventional RVDs. Sci Rep 2015; 05: 8150.
    CrossrefPubMedGoogle Scholar
  • 35 Bibikova M, Carroll D, Segal DJ. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 2001; 21 (01) 289-297.
    CrossrefPubMedGoogle Scholar
  • 36 Händel EM, Alwin S, Cathomen T. Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol Ther 2009; 17 (01) 104-111.
    CrossrefPubMedGoogle Scholar
  • 37 Doyon Y, Vo TD, Mendel MC. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 2011; 08 (01) 74-79.
    CrossrefPubMedGoogle Scholar
  • 38 Kandavelou K, Ramalingam S, London V. et al. Targeted manipulation of mammalian genomes using designed zinc finger nucleases. Biochem Biophys Res Commun 2009; 388 (01) 56-61.
    CrossrefPubMedGoogle Scholar
  • 39 Szczepek M, Brondani V, Buchel J. et al. Structurebased redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 2007; 25 (07) 786-793.
    CrossrefPubMedGoogle Scholar
  • 40 Miller JC, Holmes MC, Wang J. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 2007; 25 (07) 778-785.
    CrossrefPubMedGoogle Scholar
  • 41 Fu Y, Sander JD, Reyon D. et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 2014; 32 (03) 279-284.
    CrossrefPubMedGoogle Scholar
  • 42 Cho SW, Kim S, Kim Y. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 2014; 24 (01) 132-141.
    CrossrefPubMedGoogle Scholar
  • 43 Kleinstiver BP, Pattanayak V, Prew MS. et al. Highfidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016; 529 (7587): 490-495.
    CrossrefPubMedGoogle Scholar
  • 44 Slaymaker IM, Gao L, Zetsche B. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 2016; 351 (6268): 84-88.
    CrossrefPubMedGoogle Scholar
  • 45 Müller M, Lee CM, Gasiunas G. et al. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. Mol Ther 2016; 24 (03) 636-644.
    CrossrefPubMedGoogle Scholar
  • 46 Lee CM, Cradick TJ, Bao G. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells. Mol Ther 2016; 24 (03) 645-654.
    CrossrefPubMedGoogle Scholar
  • 47 Kim D, Kim J, Hur JK. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 2016; 34 (08) 863-868.
    CrossrefPubMedGoogle Scholar
  • 48 Kleinstiver BP, Tsai SQ, Prew MS. et al. Genomewide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 2016; 34 (08) 869-874.
    CrossrefPubMedGoogle Scholar
  • 49 Ran FA, Hsu PD, Lin CY. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013; 154 (06) 1380-1389.
    CrossrefPubMedGoogle Scholar
  • 50 Tsai SQ, Wyvekens N, Khayter C. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 2014; 32 (06) 569-576.
    CrossrefPubMedGoogle Scholar
  • 51 Wang J, Exline CM, DeClercq JJ. et al. Homologydriven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 2015; 33 (12) 1256-1263.
    CrossrefPubMedGoogle Scholar
  • 52 Mock U, Machowicz R, Hauber I. et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV coreceptor CCR5. Nucleic Acids Res 2015; 43 (11) 5560-5571.
    CrossrefPubMedGoogle Scholar
  • 53 Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 2014; 24 (06) 1012-1019.
    CrossrefPubMedGoogle Scholar
  • 54 Sather BD, Romano GSIbarra, Sommer K. et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med 2015; 07 (307): 307ra156.
    CrossrefPubMedGoogle Scholar
  • 55 De Ravin SS, Reik A, Liu PQ. et al. Targeted gene addition in human CD34 hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 2016; 34 (04) 424-429.
    CrossrefPubMedGoogle Scholar
  • 56 Hubbard N, Hagin D, Sommer K. et al. Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome. Blood 2016; 127 (21) 2513-2522.
    CrossrefPubMedGoogle Scholar
  • 57 Hoban MD, Cost GJ, Mendel MC. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 2015; 125 (17) 2597-2604.
    CrossrefPubMedGoogle Scholar
  • 58 Genovese P, Schiroli G, Escobar G. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 2014; 510 (7504): 235-240.
    CrossrefPubMedGoogle Scholar
  • 59 Provasi E, Genovese P, Lombardo A. et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med 2012; 18 (05) 807-815.
    CrossrefPubMedGoogle Scholar
  • 60 Lombardo A, Genovese P, Beausejour CM. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 2007; 25 (11) 1298-1306.
    CrossrefPubMedGoogle Scholar
  • 61 Traxler EA, Yao Y, Wang YD. et al. A genome-editing strategy to treat beta-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 2016; 22 (09) 987-990.
    CrossrefPubMedGoogle Scholar
  • 62 Dever DP, Bak RO, Reinisch A. et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 2016; 539 (7629): 384-389.
    CrossrefPubMedGoogle Scholar
  • 63 Li H, Haurigot V, Doyon Y. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 2011; 475 (7355): 217-221.
    CrossrefPubMedGoogle Scholar
  • 64 Sharma R, Anguela XM, Doyon Y. et al. In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 2015; 126 (15) 1777-1784.
    CrossrefPubMedGoogle Scholar
  • 65 Yang Y, Wang L, Bell P. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 2016; 34 (03) 334-338.
    CrossrefPubMedGoogle Scholar
  • 66 Guan Y, Ma Y, Li Q. et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 2016; 08 (05) 477-488.
    CrossrefPubMedGoogle Scholar
  • 67 Yin H, Song CQ, Dorkin JR. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 2016; 34 (03) 328-333.
    CrossrefPubMedGoogle Scholar
  • 68 Zuris JA, Thompson DB, Shu Y. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 2015; 33 (01) 73-80.
    CrossrefPubMedGoogle Scholar
  • 69 Hoban MD, Lumaquin D, Kuo CY. et al. CRISPR/ Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. Mol Ther 2016; 24 (09) 1561-1569.
    CrossrefPubMedGoogle Scholar
  • 70 Pankowicz FP, Barzi M, Legras X. et al. Reprogramming metabolic pathways in vivo with CRISPR/ Cas9 genome editing to treat hereditary tyrosinaemia. Nat Commun 2016; 07: 12642.
    CrossrefPubMedGoogle Scholar
  • 71 Ding W, Hu Z, Zhu D. et al. Zinc finger nucleases targeting the human papillomavirus E7 oncogene induce E7 disruption and a transformed phenotype in HPV16/18-positive cervical cancer cells. Clin Cancer Res 2014; 20 (24) 6495-6503.
    CrossrefPubMedGoogle Scholar
  • 72 Bloom K, Ely A, Mussolino C. et al. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol Ther 2013; 21 (10) 1889-1897.
    CrossrefPubMedGoogle Scholar
  • 73 Holt N, Wang J, Kim K. et al. Human hematopoietic stem/progenitor cells modified by zincfinger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 2010; 28 (08) 839-847.
    CrossrefPubMedGoogle Scholar
  • 74 Perez EE, Wang J, Miller JC. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 2008; 26 (07) 808-816.
    CrossrefPubMedGoogle Scholar
  • 75 Su S, Hu B, Shao J, Shen B, Du J, Du Y. et al. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci Rep 2016; 06: 20070.
    CrossrefPubMedGoogle Scholar
  • 76 Valton J, Guyot V, Marechal A. et al. A Multidrugresistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy. Mol Ther 2015; 23 (09) 1507-1518.
    CrossrefPubMedGoogle Scholar
  • 77 Poirot L, Philip B, Schiffer-Mannioui C. et al. Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf “ Adoptive T-cell Immunotherapies. Cancer Res 2015; 75 (18) 3853-3864.
    CrossrefPubMedGoogle Scholar
  • 78 Tebas P, Stein D, Tang WW. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014; 370 (10) 901-910.
    CrossrefPubMedGoogle Scholar
  • 79 Karpinski J, Hauber I, Chemnitz J. et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotech 2016; 34 (04) 401-409.
    CrossrefPubMedGoogle Scholar
  • 80 Komor AC, Kim YB, Packer MS. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016; 533 (7603): 420-424.
    CrossrefPubMedGoogle Scholar
  • 81 Amabile A, Migliara A, Capasso P. et al. Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing. Cell 2016; 167 (01) 219-232. e14.
    CrossrefPubMedGoogle Scholar
  • 82 Corrigan-Curay J, O’Reilly M, Kohn DB. et al. Genome editing technologies: defining a path to clinic. Mol Ther 2015; 23 (05) 796-806.
    CrossrefPubMedGoogle Scholar