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Molecular therapy. Methods & clinical development
Jun 08, 2023;29 58-69.

An improved medium formulation for efficient gene editing, expansion and engraftment of hematopoietic stem and progenitor cells.

Rajeev Rai, Asma Naseem, Winston Vetharoy, Zohar Steinberg, Adrian J Thrasher, Giorgia Santilli, Alessia Cavazza
Author Information
  1. Rajeev Rai: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  2. Asma Naseem: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  3. Winston Vetharoy: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  4. Zohar Steinberg: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  5. Adrian J Thrasher: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  6. Giorgia Santilli: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
  7. Alessia Cavazza: Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
PMID: 36950452 DOI: 10.1016/j.omtm.2023.02.014

Abstract

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR-Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now under way. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology-directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both and , with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized HSPC culture protocol can improve the efficacy, feasibility, and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology.

Keywords

cell culture culture medium gene editing hematopoietic stem cells

References

  1. Stem Cell Reports. 2017 Apr 11;8(4):977-990 [PMID: 28330619]
  2. Mol Ther Nucleic Acids. 2018 Sep 7;12:89-104 [PMID: 30195800]
  3. Sci Rep. 2019 Mar 28;9(1):5300 [PMID: 30923342]
  4. Trends Biotechnol. 2015 Feb;33(2):132-40 [PMID: 25595557]
  5. Science. 2013 Aug 23;341(6148):1233151 [PMID: 23845947]
  6. Cell Rep. 2020 Sep 1;32(9):108093 [PMID: 32877675]
  7. Nucleic Acids Res. 2020 Jan 24;48(2):517-532 [PMID: 31799598]
  8. Cell Stem Cell. 2016 Jul 7;19(1):107-19 [PMID: 27237736]
  9. Blood. 2000 Dec 15;96(13):4185-93 [PMID: 11110690]
  10. J Clin Invest. 2014 Jun;124(6):2378-95 [PMID: 24762436]
  11. Nat Commun. 2019 Apr 9;10(1):1634 [PMID: 30967552]
  12. Nat Commun. 2020 Aug 12;11(1):4034 [PMID: 32788576]
  13. Nat Rev Mol Cell Biol. 2019 Nov;20(11):698-714 [PMID: 31263220]
  14. Mol Ther. 2021 Nov 3;29(11):3205-3218 [PMID: 34509667]
  15. Nat Med. 2022 Jan;28(1):71-80 [PMID: 35075289]
  16. EMBO Mol Med. 2017 Sep;9(9):1198-1211 [PMID: 28667090]
  17. Cell Stem Cell. 2018 Dec 6;23(6):820-832.e9 [PMID: 30416070]
  18. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4040-4 [PMID: 8633013]
  19. Science. 2011 Jul 8;333(6039):218-21 [PMID: 21737740]
  20. Nucleic Acids Res. 2021 Jan 25;49(2):969-985 [PMID: 33398341]
  21. Hum Gene Ther. 2021 Jan;32(1-2):43-51 [PMID: 32935622]
  22. Nat Med. 2018 Aug;24(8):1216-1224 [PMID: 30082871]
  23. N Engl J Med. 2021 Jan 21;384(3):252-260 [PMID: 33283989]
  24. Science. 2014 Sep 19;345(6203):1509-12 [PMID: 25237102]
  25. Br J Haematol. 2012 Sep;158(6):778-87 [PMID: 22816563]
  26. Sci Transl Med. 2021 Jun 16;13(598): [PMID: 34135108]
  27. Exp Hematol. 2016 Jul;44(7):635-40 [PMID: 27090409]
  28. Nature. 2014 Jun 12;510(7504):235-240 [PMID: 24870228]
  29. Cell Rep. 2018 May 29;23(9):2606-2616 [PMID: 29847792]
  30. Nat Biotechnol. 2020 Nov;38(11):1298-1308 [PMID: 32601433]
  31. Mol Ther Nucleic Acids. 2022 Jun 02;29:36-46 [PMID: 35784015]
  32. Bone Marrow Transplant. 1997 Jun;19(11):1095-101 [PMID: 9193752]
  33. Blood. 1998 Aug 15;92(4):1131-41 [PMID: 9694700]
  34. Blood. 2004 Jan 15;103(2):545-52 [PMID: 14504079]
  35. Sci Transl Med. 2017 Oct 11;9(411): [PMID: 29021165]
  36. Sci Transl Med. 2017 Nov 1;9(414): [PMID: 29093179]
  37. EMBO Mol Med. 2019 Mar;11(3): [PMID: 30670463]
  38. Science. 2010 Sep 10;329(5997):1345-8 [PMID: 20688981]
  39. Stem Cells. 2003;21(2):236-44 [PMID: 12634420]
  40. Front Genome Ed. 2021 Mar 31;3:618378 [PMID: 34713250]
  41. Cell Stem Cell. 2007 Dec 13;1(6):635-45 [PMID: 18371405]
  42. J Clin Invest. 2020 Dec 1;130(12):6677-6687 [PMID: 32897878]
  43. Cell Prolif. 2012 Jun;45(3):266-78 [PMID: 22429797]
  44. N Engl J Med. 2021 May 27;384(21):2002-2013 [PMID: 33974366]
  45. Blood. 2021 May 13;137(19):2598-2608 [PMID: 33623984]
  46. Nat Commun. 2018 Jul 16;9(1):2741 [PMID: 30013077]
  47. Stem Cells Transl Med. 2021 Jul;10(7):996-1007 [PMID: 33666363]
  48. Nat Rev Immunol. 2010 Mar;10(3):182-92 [PMID: 20182458]
  49. EMBO Mol Med. 2021 Mar 5;13(3):e13545 [PMID: 33475257]
  50. Nat Med. 2018 Nov;24(11):1683-1690 [PMID: 30275570]
  51. Blood. 2004 Jun 1;103(11):4102-10 [PMID: 14976039]

Grants

  1. /Wellcome Trust
Journal Article
Article has an altmetric score of 1

Word Cloud

Created with Highcharts 10.0.0culturegeneeditingstemHSPCsmediumhematopoieticcellsexpansioncellpowerfultherapeuticprogenitorclinicaltargetinglong-termrepopulatingstemnessGeneemergedtoolcorrectionmonogenicdiseasesCRISPR-Cas9appliedshowngreatpromiseproof-of-principlepreclinicalstudiestreathematologicaldisorderstrialsusingtoolsnowwayNonethelessremainimportantchallengesneedaddressedefficiencyprimitiveapplicationstudyassessedeffectdifferentcompositionsabilityproliferateundergohomology-directedrepair-mediatedknock-inreporterpreservingfeaturesdemonstratedsupplementingagonistsfine-tuningcytokinecompositionpossibleachievehighlevelsbeneficialbalancepreservationOverallimplementationoptimizedHSPCprotocolcanimproveefficacyfeasibilityapplicabilitykeystepunlockingfullpotentialtechnologyimprovedformulationefficientengraftment

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