Conclusion et perspectives

Les essais in vivo réalisés avec les myoblastes obtenus de la différenciation des hiPSCs corrigées génétiquement démontrent un potentiel de fusion. Le protocole de différenciation employé doit être optimisé pour augmenter la quantité de fibres positives exprimant la dystrophine humaine. Cliniquement, dans le cadre d’une thérapie cellulaire, il sera nécessaire d’éliminer les séquences codant pour le gène rapporteur eGFP afin d’éviter une réaction immunitaire chez le patient et réduire les risques générés par l’utilisation de vecteurs viraux. Finalement, les hiPSCs différenciées en cellules myogéniques selon le protocole développé précédemment par notre laboratoire ont une capacité de fusion moindre que les myoblastes provenant d’une culture primaire [67]. Cependant, les résultats présentés permettent d’affirmer qu’il est possible d’obtenir des fibres exprimant la dystrophine résultant d’une fusion des myoblastes dérivées d’hiPSCs corrigées génétiquement avec les fibres musculaires de souris.

Plusieurs études effectuées par d'autres groupes proposent des méthodes alternatives pour différencier les hiPSCs en myoblastes, comme l’utilisation de petites molécules telle CHIR99021. Cette molécule inhibe certaines voies de signalisation impliqués dans la différenciation des cellules souches embryonnaires et favorise le développement de précurseurs myogéniques exprimant PAX7 [70-72]. Toutefois, dans l’ensemble, peu de méthodes sont efficaces et reproductibles pour produire une population cellulaire aux capacités semblables à celles des myoblastes.

Dans le cadre de futurs travaux, il serait pertinent d’utiliser comme outil moléculaire un lentivirus non intégratif qui peut infecter un large spectre de types cellulaires, tout en exprimant de façon transitoire les transgènes qu’il contient. De plus, l’emploi des hiPSCs jumelé avec un lentivirus codant pour les facteurs de transcription initiaux de la myogenèse, tels les facteurs de transcription PAX3 et PAX7, pourrait être une avenue intéressante. Ceux-ci sont essentiels au niveau du chevauchement des rôles dans la myogenèse et permettent la formation des précurseurs myogéniques et du bassin de cellules satellites. [73]. Finalement, le présent projet de recherche démontre qu’une combinaison des outils moléculaires employés pour réparer le gène DMD et appliqués dans des cellules hiPSCs dystrophiques permet l’expression de la dystrophine humaine in vivo.

55

Bibliographie

1. Jr., C.H., Larry Roberts, Allan Larson, Helen I'Anson, David Eisenhour, Integrated Principles of Zoology, in Integrated Principles of Zoology, M.-H.H. Education, Editor. 2005. p. 872.

2. Alexander Aulehla, O.P., On periodicity and directionality of somitogenesis. Anatomy and Embryology, 2006. 211(1 supplement).

3. C.Florian Bentzinger, Y.X.W.a.M.A.R., Building Muscle : Molecular Regulation of Myogenesis. Cold Spring Harbor Laboratory Press, 2012. Cold Spring Harbor

Perspectives in Biology.

4. Rudnicki, S.B.P.C.a.M.A., Cellular and Molecular Regulation of Muscle Regeneration. Physiological Reviews, 2004. 84(1): p. 209-238.

5. Robert L. Davis, H.W., Andrew B. Lassar, Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell, 1987. 51(6): p. 987-1000.

6. H Weintraub, S.J.T., R L Davis, M J Thayer, M A Adam, A B Lassar, and A D Miller, Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. PNAS, 1989: p. 5434-5438.

7. Simon P Quenneville, P.C., Daniel Skuk, Matin Paradis, Marlyne Goulet, Joël Rousseau, Xiao Xiao, Luis Garcia and Jacques P Tremblay, Autologous Transplantation of Muscle Precursor Cells Modified with a Lentivirus for Muscular Dystrophy: Human Cells and Primate Models. Molecular Therapy, 2007. 15(2): p. 431-438.

8. Tariq Maqbool, K.J., Genetic control of muscle development: learning from Drosophila. Journal of Muscle Research and Cell Motility, 2007. 28(7-8): p. 397-407.

9. David A. Hutcheson, J.Z., Allyson Merrell, Malay Haldar, and Gabrielle Kardon, Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for b-catenin. GENES & DEVELOPMENT, 2009. 23: p. 997-1013.

10. Duchenne, G.-B.-A., De l’électrisation localisée et de son application à la physiologie, à la pathologie et à la thérapeutique. 1861: p. 353-356.

11. E.Davies, K.J.N.K., Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment. European Molecular Biology Organization, 2004. 5: p. 872- 876.

12. JS Chamberlain, J.P., DM Muzny, RA Gibbs, JE Ranier, CT Caskey, AA Reeves, Expression of the murine Duchenne muscular dystrophy gene in muscle and brain. Science, 1988. 239(4846): p. 1416-1418.

13. Buckle, Y.B.a.J., Cytogenetic heterogeneity of translocations associated with Duchenne muscular dystrophy. Clinical Genetics, 2008. 29(2): p. 108-115.

14. Coffey AJ, R.R., Green ED, Cole CG, Butler R, Anand R, Giannelli F, Bentley DR., Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach. Genomics, 1992. 12(3): p. 474-484. 15. Koenig M, H.E., Bertelson CJ, Monaco AP, Feener C, Kunkel LM., Complete cloning of

the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell Press, 1987. 50(3): p. 509-517. 16. Anthony P. Monaco, A.P.W., Iona Millwood, Zoia Larin, Hans Lehrach, A yeast artificial

chromosome contig containing the complete Duchenne muscular dystrophy gene. Genomics, 1992. 12(3): p. 465-473.

56

17. Derek J. Blake, A.W., Sarah E. Newey and Kay E. Davies, Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle. Physiological Reviews, 2002.

82(2): p. 291-329.

18. Joe W. McGreevy, C.H.H., Mark A. McIntosh and Dongsheng Duan, Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Disease Models & Mechanisms, 2015. 8(3): p. 195-213.

19. Guiraud S, A.-R.A., Vieira NM, Davies KE, van Ommen GJ, Kunkel LM., Pathogenesis and Therapy of Duchenne and Becker Muscular Annual review of genomics and human genetics, 2015. 16: p. 281-308.

20. Constantin, B., Dystrophin complex functions as a scaffold for signalling proteins. Biochim. Biophys. Acta, Biomembranes, 2014. 1838(2): p. 635-642.

21. M. Koenig, A.P.M., L.M. Kunkel, The complete sequence of dystrophin predicts a rod- shaped cytoskeletal protein. Cell Press, 1988. 53(2): p. 219-228.

22. Sicinski P, G.Y., Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ., The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science, 1989. 244: p. 1578-1580.

23. Jachinta E. Rooney, J.V.W., Melissa A. Dechert, Nichole L. Flintoff-Dye, Stephen J. Kaufman, Dean J. Burkin, Severe muscular dystrophy in mice that lack dystrophin and α7 integrin. Cell Science, 2006: p. 2185-2195.

24. Jennifer V. Welser, J.E.R., Nicolette C. Cohen,Praveen B. Gurpur, Cherie A. Singer, Rebecca A. Evans, Bryan A. Haines, and Dean J. Burkin, Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack 7 Integrin

and Utrophin. The American Journal of Pathology, 2009. 175(4): p. 1545-1554.

25. Morgan, C.A.C.a.J.E., Duchenne’s muscular dystrophy: animal models usedto investigate pathogenesis and develop therapeutic strategies. International Journal of Experimental Pathology, 2003. 84(4): p. 165-172.

26. Peter Mombaerts, J.I., Randall S. Johnson, Karl Herrup‡, Susumu Tonegawa, Virginia E. Papaioannou., RAG-1-deficient mice have no mature B and T lymphocytes. Cell Press, 1992. 68(5): p. 869-877.

27. Katharine Bushby, R.F., David J Birnkrant, Laura E Case, Paula R Clemens, Linda Cripe, Ajay Kaul, Kathi Kinnett, Craig McDonald, Shree Pandya, James Poysky, Frederic Shapiro, Jean Tomezsko, Carolyn Constantin,, Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care. THE LANCET Neurology, 2010. 9(2): p. 177-189.

28. Annexstad EJ, L.-P.I., Rasmussen M., Duchenne muscular dystrophy. Journal of the Norwegian Medical Association, 2014. 14: p. 1361-1364.

29. Ellen M. Welch, E.R.B., Jin Zhuo, Yuki Tomizawa, Westley J. Friesen, Panayiota Trifillis,Sergey Paushkin, Meenal Patel, Christopher R. Trotta, Seongwoo Hwang,

Richard G. Wilde, Gary Karp,James Takasugi, Guangming Chen, Stephen Jones, Hongyu Ren, Young-Choon Moon, Donald Corson,Anthony A. Turpoff, Jeffrey A. Campbell, M. Morgan Conn, Atiyya Khan, Neil G. Almstead, Jean Hedrick,Anna Mollin, Nicole Risher, Marla Weetall, Shirley Yeh, Arthur A. Branstrom, Joseph M. Colacino, and W.D.J. John Babiak, Samit Hirawat, Valerie J. Northcutt, Langdon L. Miller, Phyllis Spatrick, Feng He,Masataka Kawana, Huisheng Feng, Allan Jacobson, Stuart W. Peltz & H. Lee

Sweeney, PTC124 targets genetic disorders caused by nonsense mutations. Nature, 2007.

57

30. Richard S. Finkel, K.M.F., Brenda Wong, Carsten Bo¨nnemann, Jacinda Sampson,H. Lee Sweeney, Allen Reha, Valerie J. Northcutt, Gary Elfring, Jay Barth, Stuart W. Peltz, Phase 2a Study of Ataluren-Mediated Dystrophin Production in Patients with Nonsense Mutation Duchenne Muscular Dystrophy. PLoS One, 2013. 8(12).

31. Rebecca J. Fairclough, M.J.W.a.K.E.D., Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nature, 2013. 14: p. 373-378.

32. Qi-Long Lu, T.Y., Shin’ichi Takeda, Luis Garcia, Francesco Muntoni and Terence

Partridge, The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy. Molecular Therapy, 2011. 19(1): p. 9-15.

33. Sander, J.K.J.a.J.D., TALENs: a widely applicable technology for targeted genome editing. Nature Review Molecular Cell Biology, 2013. 14: p. 49-55.

34. Carroll, D., Genome Engineering with Targetable Nucleases. Annual Review of Biochemistry, 2014. 83: p. 409-439.

35. Tomas Cermak, E.L.D., Michelle Christian, Li Wang, Yong Zhang, Clarice Schmidt, Joshua A. Baller, Nikunj V. Somia, Adam J. Bogdanove and Daniel F. Voytas, Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011. 39(12): p. e82.

36. Musunuru, R.M.G.a.K., Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. The Journal of Clinical Investigation, 2014. 124(10): p. 4155-4161. 37. Victoria M. Bedell, Y.W., Jarryd M. Campbell, Tanya L. Poshusta, Colby G. Starker,

Randall G. Krug, Wenfang Tan, Sumedha G. Penheiter, Alvin C. Ma, Anskar Y.H. Leung, Scott C. Fahrenkrug, Daniel F. Carlson, Daniel F. Voytas, Karl J. Clark, Jeffrey J. Essner and Stephen C. Ekker, In vivo Genome Editing Using High Efficiency TALENs. Nature, 2012. 491: p. 114-118.

38. Angrand, B.D.a.P.-O., Targeted genome modifications using TALEN. M/S Revues, 2014.

30(2): p. 186-193.

39. Jacques P Tremblay, D.S., Benjamino Palmieri and David M Rothstein, A Case for Immunosuppression for Myoblast Transplantation in Duchenne Muscular Dystrophy. the American Society of Gene Therapy. 17(7): p. 1122–1124.

40. Sara Benedetti, H.H.a.F.S.T., Repair or replace? Exploiting novel gene and cell therapy strategies for muscular dystrophies. The FEBS journal, 2013. 280(17): p. 4263-4280. 41. Lafreniere JF, M.P., Tremblay JP, El Fahime E., Growth factors improve the in vivo

migration of human skeletal myoblasts by modulating their endogenous proteolytic activity. Transplantation, 2004. 77(11): p. 1741-1747.

42. Li, C.-J.L.a.L., Tacrolimus in preventing transplant rejection in Chinese patients – optimizing use. Drug Design, Development and Therapy, 2015. 9: p. 473-485. 43. Daniel Skuk, M.G., Brigitte Roy, Jacques P. Tremblay, Efficacy of Myoblast

Transplantation in Nonhuman Primates Following Simple Intramuscular Cell Injections: Toward Defining Strategies Applicable to Humans. Experimental Neurology, 2002.

175(1): p. 112-126.

44. Tremblay, D.S.a.J.P., "Engineering" Myoblast Transplantation. Graft, 2001. 4(8): p. 558- 570.

45. Vannucci L, L.M., Chiuppesi F, Ceccherini-Nelli L, Pistello M., Viral vectors: a look back and ahead on gene transfer technology. NEW MICROBIOLOGICA. 36(1): p. 1-22. 46. Richard Mann, R.C.M., David Baltimore, Construction of a retrovirus packaging mutant

and its use to produce helper-free defective retrovirus. Cell Press, 1983. 33(1): p. 153- 159.

58

47. J. Vargas Jr, G.L.G., V. Najfeld, M.E Klotman and A. Cara, Novel Integrase-Defective Lentiviral Episomal Vectors for Gene Transfer. HUMAN GENE THERAPY, 2004.

15(4): p. 361-372.

48. Cornetta, A.S.a.K., Design and Potential of Non-Integrating Lentiviral Vectors. Biomedicines, 2014. 2: p. 14-35.

49. Gerard J. McGarrity, G.H., April Winemiller, Kris Andre, David Stein, Gary Blick, Richard N. Greenberg, Clifford Kinder, Andrew Zolopa, Gwen Binder-Scholl, Pablo Tebas, Carl H. June, Laurent M. Humeau andTessio Rebello, Patient monitoring and follow-up in lentiviral clinical trials. Journal of Gene Medicine, 2013. 15(2): p. 78-82. 50. Sharon Gerecht-Nira, J.I.-E., Cell therapy using human embryonic stem cells. Transplant

Immunology, 2004. 12(3-4): p. 203-209.

51. Kaufman, M.J.E.a.M.H., Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981. 292: p. 154-156.

52. Thomas C. Doetschman, H.E., Margot Katz, Werner Schmidt, Rolf Kemler, The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Developmental Biology, 1985. 87: p. 27-45. 53. Kazutoshi Takahashi, K.T., Mari Ohnuki, Megumi Narita, Tomoko Ichisaka, Kiichiro

Tomoda, and Shinya Yamanaka, Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell, 2007. 131(5): p. 861-872.

54. Kazutoshi Takahashi, S.Y., Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 2006. 126(4): p. 663-676. 55. Leonardo M.R. Ferreira, M.A.M.-R., How induced pluripotent stem cells are redefining

personalized medicine. Gene, 2013. 520(1): p. 1-6.

56. Yu J, V.M., Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-1920.

57. Fusaki N, B.H., Nishiyama A, Saeki K, Hasegawa M., Efficient induction of transgene- free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy, Ser. B, Physical and Biological Sciences, 2009. 85(8): p. 348-362.

58. Vimal K. Singh, M.K., Neeraj Kumar, Abhishek Saini and Ramesh Chandra, Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Cell and Developmental Biology, 2015. 3(2): p. 1-18.

59. Grivennikov, E.V.N.a.I.A., Induced Pluripotent Stem Cells: From Derivation to Application in Biochemical and Biomedical Research. Biochemistry, 2014. 79(13): p. 1425-1441.

60. E.Davis, K.J.n.K., Duchenne muscular dystrophy and dystrophin : pathogenesis and opportunities for treatment. EMBO Reports, 2004: p. 872-876.

61. Yamanaka, S., Induced Pluripotent Stem Cells: Past,Present, and Future. Cell Press, 2012. 10(6): p. 678-684.

62. Hongmei Lisa Li, N.F., Noriko Sasakawa, Saya Shirai, Tokiko Ohkame, Tetsushi Sakuma,, N.A. Michihiro Tanaka, Akira Watanabe, Hidetoshi Sakurai, Takashi

Yamamoto,, and S.Y.a.A. Hotta, Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR- Cas9. Stem Cell report, 2015. 4(1): p. 143-154.

63. EJ, P.S., Brown EJ, Baltimore D, Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 2002. 295.

59

64. Andrea L. Szymczak-Workman, K.M.V.a.D.A.A.V., Design and Construction of 2A Peptide-Linked Multicistronic Vectors. Cold Spring Harbor Laboratory Press, 2012. 65. Davis HE, M.J., Yarmush ML., Polybrene increases retrovirus gene transfer efficiency by

enhancing receptor-independent virus adsorption on target cell membranes. Biophysical Chemistry, 2002. 97(2-3): p. 159-172.

66. Keisuke Okita, Y.M., Yoshiko Sato,Aki Okada, Asuka Morizane, Satoshi Okamoto, Hyenjong Hong, Masato Nakagawa, Koji Tanabe,Ken-ichi Tezuka, Toshiyuki Shibata, Takahiro Kunisada,Masayo Takahashi, Jun Takahashi, Hiroh Saji & Shinya Yamanaka, A more efficient method to generate integration-free human iPS cells. Nature Methods, 2011. 8(5): p. 409-412.

67. Sébastien Goudenege, C.L., Nicolas B Huot, Christine Dufour, Isao Fujii, Jean Gekas, Joël Rousseau1 and Jacques P Tremblay1, Myoblasts Derived From Normal hESCs and Dystrophic hiPSCs Efficiently Fuse With Existing Muscle Fibers Following

Transplantation. Molecular Therapy, 2012. 20(11): p. 2153-2167.

68. Gussoni E, P.G., Lanctot AM, Sharma KR, Miller RG, Steinman L, Blau HM., Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature, 1992. 356: p. 435-438.

69. Daniel Skuk, M.G., Brigitte Roy, Vincent Piette, Claude H. Côté, Pierre Chapdelaine, Jean-Yves Hogrel, Martin Paradis, Jean-Pierre Bouchard, Michel Sylvain, Jean-Guy Lachance, Jacques P. Tremblay, First test of a “high-density injection” protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up. Neuromuscular Disorders, 2007.

17(1): p. 38-46.

70. Akihito Tanaka, K.W., Katsuya Miyake, Akitsu Hotta, Makoto Ikeya, Takuya Yamamoto,Tokiko Nishino, Emi Shoji, Atsuko Sehara-Fujisawa, Yasuko Manabe, Nobuharu Fujii,Kazunori Hanaoka, Takumi Era, Satoshi Yamashita, Ken-ichi Isobe, En Kimura, Hidetoshi Sakurai, Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro. PLoS One, 2013.

8(4): p. 1-14.

71. Tiziano Barberi, M.B., Zehra Dincer, Georgia Panagiotakos, Nicholas D Socci and Lorenz Studer, Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nature Medicine, 2007. 13(5): p. 642-648.

72. Michael Shelton, J.M., Jun Liu,1 Richard L. Carpenedo,Simon-Pierre Demers,William L. Stanford,and Ilona S. Skerjanc, Derivation and Expansion of PAX7-Positive Muscle Progenitors from Human and Mouse Embryonic Stem Cells. Stem Cell report, 2014. 3: p. 516-529.

73. A. Rudnicki, Y.X.W.a.M., Satellite cells, the engines of muscle repair. Nature Review Molecular Cell Biology, 2012. 13(2): p. 127-133.