Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases
C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the “Loss of a primer binding site” technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The “Loss of a primer binding site” technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.
1. Blanpain C, Migeotte I, Lee B, Vakili J, Doranz BJ, Govaerts C, et al. CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist. Blood. 1999;94(6):1899-905.
2. Joshi A, Punke EB, Sedano M, Beauchamp B, Patel R, Hossenlopp C, et al. CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis. Scientific Reports. 2017;7(1):232.
3. Hossein K, Elham H, Gholamhossein H, Mohammadtaghi R, Hossein S, Kazemi AM. CCR5 Δ 32 mutation is not prevalent in Iranians with chronic HBV infection. Journal of Medical Virology. 2013;85(6):964-8.
4. Ghorban K, Dadmanesh M, Hassanshahi G, Momeni M, Zare-Bidaki M, Kazemi Arababadi M, et al. Is the CCR5 Δ 32 Mutation Associated with Immune System-Related Diseases?2012.
5. Pokorny V, McQueen F, Yeoman S, Merriman M, Merriman A, Harrison A, et al. Evidence for negative association of the chemokine receptor CCR5 d32 polymorphism with rheumatoid arthritis. Annals of the rheumatic diseases. 2005;64(3):487-90.
6. Moy RH, Huffman AP, Richman LP, Crisalli L, Wang XK, Hoxie JA, et al. Clinical and immunologic impact of CCR5 blockade in graft-versus-host disease prophylaxis. Blood. 2017.
7. Palmer LA, Sale GE, Balogun JI, Li D, Jones D, Molldrem JJ, et al. Chemokine Receptor CCR5 Mediates Allo-Immune Responses in Graft-vs-Host Disease. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2010;16(3):311-9.
8. Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong-Staal F. Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature. 1984;312(5990):166.
9. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996;381(6584):661.
10. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382(6593):722-5.
11. de Silva E, Stumpf MP. HIV and the CCR5-Delta32 resistance allele. FEMS microbiology letters. 2004;241(1):1-12.
12. Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. The New England journal of medicine. 2009;360(7):692-8.
13. Martinson JJ, Chapman NH, Rees DC, Liu Y-T, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nature Genetics. 1997;16:100.
14. Zare-Bidaki M, Karimi-Googheri M, Hassanshahi G, Zainodini N, Arababadi MK. The frequency of CCR5 promoter polymorphisms and CCR5 Δ 32 mutation in Iranian populations. Iranian Journal of Basic Medical Sciences. 2015;18(4):312-6.
15. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-72.
16. Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, et al. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation. 2010;121(9):1113-23.
17. Kaufman DS. Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. Blood. 2009;114(17):3513-23.
18. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-Guided Human Genome Engineering via Cas9. Science. 2013;339(6121):823-6.
19. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339(6121):819-23.
20. Choi YS, Park Y-B, Ha C-W, Kim JA, Heo J-C, Han W-J, et al. Different characteristics of mesenchymal stem cells isolated from different layers of full term placenta. PLOS ONE. 2017;12(2):e0172642.
21. Iseli C, Ambrosini G, Bucher P, Jongeneel CV. Indexing strategies for rapid searches of short words in genome sequences. PLoS One. 2007;2(6):e579.
22. Yu C, Zhang Y, Yao S, Wei Y. A PCR Based Protocol for Detecting Indel Mutations Induced by TALENs and CRISPR/Cas9 in Zebrafish. PLOS ONE. 2014;9(6):e98282.
23. Luther SA, Cyster JG. Chemokines as regulators of T cell differentiation. Nature immunology. 2001;2(2):102-7.
24. Laurichesse JJ, Persoz A, Theodorou I, Rouzioux C, Delfraissy JF, Meyer L. Improved virological response to highly active antiretroviral therapy in HIV-1-infected patients carrying the CCR5 Delta32 deletion. HIV medicine. 2007;8(4):213-9.
25. Arenzana-Seisdedos F, Parmentier M. Genetics of resistance to HIV infection: Role of co-receptors and co-receptor ligands. Seminars in immunology. 2006;18(6):387-403.
26. Thio CL, Astemborski J, Bashirova A, Mosbruger T, Greer S, Witt MD, et al. Genetic protection against hepatitis B virus conferred by CCR5Delta32: Evidence that CCR5 contributes to viral persistence. Journal of virology. 2007;81(2):441-5.
27. Belnoue E, Kayibanda M, Deschemin JC, Viguier M, Mack M, Kuziel WA, et al. CCR5 deficiency decreases susceptibility to experimental cerebral malaria. Blood. 2003;101(11):4253-9.
28. Patel DD, Zachariah JP, Whichard LP. CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clinical immunology. 2001;98(1):39-45.
29. Mack M, Brühl H, Gruber R, Jaeger C, Cihak J, Eiter V, et al. Predominance of mononuclear cells expressing the chemokine receptor CCR5 in synovial effusions of patients with different forms of arthritis. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology. 1999;42(5):981-8.
30. Barcellos LF, Schito AM, Rimmler JB, Vittinghoff E, Shih A, Lincoln R, et al. CC-chemokine receptor 5 polymorphism and age of onset in familial multiple sclerosis. Multiple Sclerosis Genetics Group. Immunogenetics. 2000;51(4-5):281-8.
31. O'Shea JJ, Ma A, Lipsky P. Cytokines and autoimmunity. Nature Reviews Immunology. 2002;2(1):37.
32. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140(6):883-99.
33. Lei F, Haque R, Xiong X, Song J. Modulation of autoimmune diseases by iPS cells. Methods in molecular biology (Clifton, NJ). 2014;1213:365-77.
34. Haque R, Lei F, Xiong X, Bian Y, Zhao B, Wu Y, et al. Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity. Journal of immunology (Baltimore, Md : 1950). 2012;189(3):1228-36.
35. Hew M, O'Connor K, Edel MJ, Lucas M. The possible future roles for iPSC-derived therapy for autoimmune diseases. Journal of clinical medicine. 2015;4(6):1193-206.
36. Svejgaard A. The immunogenetics of multiple sclerosis. Immunogenetics. 2008;60(6):275.
37. Ebers GC. Environmental factors and multiple sclerosis. The Lancet Neurology. 2008;7(3):268-77.
38. Goldberg P. Multiple sclerosis: vitamin D and calcium as environmental determinants of prevalence: (A viewpoint) part 2. biochemical and genetic factors. International Journal of Environmental Studies. 1974;6(2-3):121-9.
39. Willis SN, Stadelmann C, Rodig SJ, Caron T, Gattenloehner S, Mallozzi SS, et al. Epstein–Barr virus infection is not a characteristic feature of multiple sclerosis brain. Brain. 2009;132(12):3318-28.
40. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007;318(5858):1920-3.
41. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell. 2007;131(5):861-72.
42. Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006;126(4):663-76.
43. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet (London, England). 2009;373(9674):1550-61.
44. Murai M, Yoneyama H, Ezaki T, Suematsu M, Terashima Y, Harada A, et al. Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nature immunology. 2003;4(2):154-60.
45. Wysocki CA, Burkett SB, Panoskaltsis-Mortari A, Kirby SL, Luster AD, McKinnon K, et al. Differential roles for CCR5 expression on donor T cells during graft-versus-host disease based on pretransplant conditioning. Journal of immunology (Baltimore, Md : 1950). 2004;173(2):845-54.
46. Fischereder M, Luckow B, Hocher B, Wuthrich RP, Rothenpieler U, Schneeberger H, et al. CC chemokine receptor 5 and renal-transplant survival. Lancet (London, England). 2001;357(9270):1758-61.
47. Hütter G, Neumann M, Nowak D, Klein S, Klüter H, Hofmann W-K. The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation. Journal of Inflammation. 2011;8(1):29.
48. Telenti A. Safety concerns about CCR5 as an antiviral target. Current opinion in HIV and AIDS. 2009;4(2):131-5.
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