Актуальные направления и риски применения препаратов на основе технологий редактирования генома

Актуальность. В настоящее время разработано множество различных подходов к редактированию генома, основанных на применении разных систем редактирования, осуществлении модификаций генома с образованием одноцепочечных или двухцепочечных разрывов ДНК, in vivo или ex vivo, с восстановлением последовательности генома с помощью гомологичной рекомбинации или негомологичного соединения концов ДНК. Однако применение систем редактирования генома сопряжено с возможным возникновением целого ряда рисков, вследствие сложной биологии таких препаратов и фундаментального значения цели их воздействия – молекулы ДНК.

Цель. Анализ актуальных направлений и рисков, связанных с применением препаратов на основе систем редактирования генома, способов снижения рисков и методов их исследования, используемых для выявления и контроля возникновения нежелательных эффектов.

Обсуждение. Анализ данных литературы показал, что нежелательные эффекты от применения препаратов на основе систем редактирования генома могут быть связаны как со способами доставки компонентов системы в клетку, так и с функциональной активностью самой системы (недостаточное целевое или нежелательное нецелевое действия). В обзоре обозначены основные риски при использовании систем редактирования генома. Установлено, что для снижения рисков применения систем редактирования генома предпочтительно проведение репарации разрывов ДНК путем гомологичной рекомбинации, использование обладающих большей специфичностью и точностью рестрикции систем редактирования генома и эндонуклеаз в их составе, увеличение специфичности гРНК (для CRISPR/Cas), контролируемая коррекция активности элементов системы регуляции клеточного цикла и апоптоза, регуляция продолжительности экспрессии и персистенции компонентов систем редактирования генома в клетках и др.

Заключение. Освещение основных рисков, связанных с применением этой группы препаратов, является актуальным в связи с необходимостью предоставления данных в регистрационном досье на высокотехнологичный лекарственный препарат, касающихся оценки качества, эффективности и безопасности.

1. Ребриков ДВ. Редактирование генома человека. Вестник РГМУ. 2016;(3):4–15. EDN: WFQBMX

2. Uddin F, Rudin CM, Sen T. CRISPR gene therapy: applications, limitations, and implications for the future. Front Oncol. 2020;10:1387. https://doi.org/10.3389/fonc.2020.01387

3. Cyranoski D. The CRISPR-baby scandal: what’s next for human gene-editing. Nature. 2019;566(7745):440–2. https://doi.org/10.1038/d41586-019-00673-1

4. Cohen J. Did CRISPR help—or harm—the first-ever gene-edited babies? Science. 2019. https://doi.org/10.1126/science.aay9569

5. Cox D, Platt R, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21(2):121–31. https://doi.org/10.1038/nm.3793

6. Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism and regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol. 2003;4(9):712–20. https://doi.org/10.1038/nrm1202

7. Guirouilh-Barbat J, Lambert S, Bertrand P, Lopez BS. Is homologous recombination really an error-free process? Front Genet. 2014;5:175. https://doi.org/10.3389/fgene.2014.00175

8. Choi EH, Yoon S, Koh YE, Seo Y-J, Kim KP. Maintenance of genome integrity and active homologous recombination in embryonic stem cells. Exp Mol Med. 2020;52:1220–9. https://doi.org/10.1038/s12276-020-0481-2

9. Creeden JF, Nanavaty NS, Einloth KR, Gillman CE, Stanbery L, Hamouda DM, et al. Homologous recombination proficiency in ovarian and breast cancer patients. BMC Cancer. 2021;21(1):1154. https://doi.org/10.1186/s12885-021-08863-9

10. Lai JKH, Toh PJY, Cognart HA, Chouhan G, Saunders TE. DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells. Elife. 2022;11:e72302. https://doi.org/10.7554/eLife.72302

11. Yamaguchi T, Uchida E, Okada T, Ozawa K, Onodera M, Kume A, et al. Aspects of gene therapy products using gene editing technology in Japan. Hum Gene Ther. 2020;31(19–20):1043–53. https://doi.org/10.1089/hum.2020.156

12. Richardson C, Ray G, DeWitt M, Curie G, Corn J. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016;34(3):339–44. https://doi.org/10.1038/nbt.3481

13. DeWitt MA, Magis W, Bray NL, Wang T, Berman JR, Urbinati F, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med. 2016;8(360):360ra134. https://doi.org/10.1126/scitranslmed.aaf9336

14. Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn J, Murthy N. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife. 2017;6:e25312. https://doi.org/10.7554/eLife.25312

15. Горяев АА, Савкина МВ, Мефед КМ, Бондарев ВП, Меркулов ВА, Тарасов ВВ. Редактирование генома и биомедицинские клеточные продукты: современное состояние, безопасность и эффективность. БИОпрепараты. Профилактика, диагностика, лечение. 2018;18(3):140–9. https://doi.org/10.30895/2221-996X-2018-18-3-140-149

16. Kim M-S, Kini AG. Engineering and application of zinc finger proteins and TALEs for biomedical research. Mol Cells. 2017;40(8):533–41. https://doi.org/10.14348/molcells.2017.0139

17. Yuanyuan X., Zhanjun Li. CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. 2020;18:2401–15. https://doi.org/10.1016/j.csbj.2020.08.031

18. You L, Tong R, Li M, Liu Y, Xue J, Lu Y. Advancements and obstacles of CRISPR-Cas9 technology in translational research. Mol Ther Methods Clin Dev. 2019;13:359–70. https://doi.org/10.1016/j.omtm.2019.02.008

19. Pinjala P, Tryphena KP, Prasad R, Khatri DK, Sun W, Singh SB, et al. CRISPR/Cas9 assisted stem cell therapy in Parkinson’s disease. Biomater Res. 2023;27(1):46. https://doi.org/10.1186/s40824-023-00381-y

20. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med. 2021;384(3):252–60. https://doi.org/10.1056/NEJMoa2031054

21. Erkut E, Yokota T. CRISPR therapeutics for Duchenne muscular dystrophy. Int J Mol Sci. 2022;23(3):1832. https://doi.org/10.3390/ijms23031832

22. Graham C, Hart S. CRISPR/Cas9 gene editing therapies for cystic fibrosis. Expert Opin Biol Ther. 2021;21(6):767–80. https://doi.org/10.1080/14712598.2021.1869208

23. Porteus MH. A new class of medicines through DNA editing. N Engl J Med. 2019;380(10):947–59. https://doi.org/10.1056/NEJMra1800729

24. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403–7. https://doi.org/10.1126/science.aad5143

25. Amoasii L, Hildyard JCW, 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(6410):86–91. https://doi.org/10.1126/science.aau1549

26. Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, 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. 2018;24(8):1216–24. https://doi.org/10.1038/s41591-018-0137-0

27. Chandrasekaran AP, Song M, Kim KS, Ramakrishna S. Different methods of delivering CRISPR/Cas9 into cells. Prog Mol Biol Transl Sci. 2018;159:157–76. https://doi.org/10.1016/bs.pmbts.2018.05.001

28. Chen F, Alphonse M, Liu Q. Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2020;12(3):e1609. https://doi.org/10.1002/wnan.1609

29. 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. https://doi.org/10.1016/j.jconrel.2017.09.012

30. Fu Y, Foden J, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31(9):822–6. https://doi.org/10.1038/nbt.2623

31. Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids. 2015;4(11):e264. https://doi.org/10.1038/mtna.2015.37

32. Davies B. The technical risks of human gene editing. Hum Reprod. 2019;34(11):2104–11. https://doi.org/10.1093/humrep/dez162

33. Zuccaro MV, Xu J, Mitchell C, Marin D, Zimmerman R, Rana B, et al. Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell. 2020;183(6):1650-64.e15. https://doi.org/10.1016/j.cell.2020.10.025

34. Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013;12(4):393–4. https://doi.org/10.1016/j.stem.2013.03.006

35. Obermeier M, Vadolas J, Verhulst S, Goossens E, Baert Y. Lipofection of non-integrative CRISPR/Cas9 ribonucleoproteins in male germline stem cells: a simple and effective knockout tool for germline genome engineering. Front Cell Dev Biol. 2022;10:891173. https://doi.org/10.3389/fcell.2022.891173

36. Bittlinger M, Hoffmann D, Sierawska AK, Mertz M, Schambach A, Strech D. Risk assessment in gene therapy and somatic genome-editing: An expert interview study. Gene and Genome Editing. 2022;3–4:100011. https://doi.org/10.1016/j.ggedit.2022.100011

37. Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B, Kinner A, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med. 2010;16(2):198–204. https://doi.org/10.1038/nm.2088

38. Taheri-Ghahfarokhi A, Taylor BJM, Nitsch R, Lundin A, Cavallo AL, Madeyski-Bengtson K, et al. Decoding non-random mutational signatures at Cas9 targeted sites. Nucleic Acids Res. 2018;46(16):8417–34. https://doi.org/10.1093/nar/gky653

39. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765–71. https://doi.org/10.1038/nbt.4192

40. Boroviak K, Fu B, Yang F, Doe B, Bradley A. Revealing hidden complexities of genomic rearrangements generated with Cas9. Sci Rep. 2017;7(1):12867. https://doi.org/10.1038/s41598-017-12740-6

41. Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016;34(3):334–8. https://doi.org/10.1038/nbt.3469

42. Breese EH, Buechele C, Dawson C, Cleary ML, Porteus MH. Use of genome engineering to create patient specific MLL translocations in primary human hematopoietic stem and progenitor cells. PLoS One. 2015;10(9):e0136644. https://doi.org/10.1371/journal.pone.0136644

43. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24(7):927–30. https://doi.org/10.1038/s41591-018-0049-z

44. Otto T, Sicinski P. Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer. 2017;17(2):93–115. https://doi.org/10.1038/nrc.2016.138

45. Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med. 2018;24(7):939–46. https://doi.org/10.1038/s41591-018-0050-6

46. Anderson KR, Haeussler M, Watanabe C, Janakiraman V, Lund J, Modrusan Z, et al. CRISPR off-target analysis in genetically engineered rats and mice. Nat Methods. 2018;15(7):512–4. https://doi.org/10.1038/s41592-018-0011-5

47. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9. https://doi.org/10.1016/j.cell.2013.08.021

48. Tycko J, Myer VE, Hsu PD. Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell. 2016;63(3):355–70. https://doi.org/10.1016/j.molcel.2016.07.004

49. Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, Kwon JB, Gersbach CA. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol. 2019;37(6):657–66. https://doi.org/10.1038/s41587-019-0095-1

50. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351(6268):84–8. https://doi.org/10.1126/science.aad5227

51. Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 2018;4:63. https://doi.org/10.1038/s41421-018-0069-3

52. Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, 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. 2018;24(8):1216–24. https://doi.org/10.1038/s41591-018-0137-0

53. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016;34(8):863–8. https://doi.org/10.1038/nbt.3609

54. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015;33(1):73–80. https://doi.org/10.1038/nbt.3081

55. Shen C-C, Hsu M-N, Chang C-W, Lin M-W, Hwu J-R, Tu Y, Hu Y-C. Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation. Nucleic Acids Res. 2019;47(3):e13. https://doi.org/10.1093/nar/gky1165

56. Tu Z, Yang W, Yan S, Yin A, Gao J, Liu X, et al. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci Rep. 2017;7:42081. https://doi.org/10.1038/srep42081

57. Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC, Iyer V. WGE: a CRISPR database for genome engineering. Bioinformatics. 2015;31(18):3078–80. https://doi.org/10.1093/bioinformatics/btv308

58. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaudet J-B, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17(1):148. https://doi.org/10.1186/s13059-016-1012-2

59. Lessard S, Francioli L, Alfoldi J, Tardif JC, Ellinor PT, MacArthur DG, et al. Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci. Proc Natl Acad Sci USA. 2017;114(52):E11257-E11266. https://doi.org/10.1073/pnas.1714640114

60. Miller NA, Farrow EG, Gibson M, Willig LK, Twist G, Yoo B, et al. A 26-hour system of highly sensitive whole genome sequencing for emergency management of genetic diseases. Genome Med. 2015;7:100. https://doi.org/10.1186/s13073-015-0221-8

61. Рачинская ОА, Меркулов ВА. Применение методов цитогенетического анализа при оценке качества клеточных линий в составе биомедицинских клеточных продуктов. БИОпрепараты. Профилактика, диагностика, лечение. 2018;18(1):25–32. https://doi.org/10.30895/2221-996X-2018-18-1-25-32