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Molecular therapy. Methods & clinical development
Sep 10, 2021;22 237-248.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Sara Fañanas-Baquero, Oscar Quintana-Bustamante, Daniel P Dever, Omaira Alberquilla, Rebeca Sanchez-Dominguez, Joab Camarena, Isabel Ojeda-Perez, Mercedes Dessy-Rodriguez, Rolf Turk, Mollie S Schubert, Annalisa Lattanzi, Liwen Xu, Jose L Lopez-Lorenzo, Paola Bianchi, Juan A Bueren, Mark A Behlke, Matthew Porteus, Jose-Carlos Segovia
Author Information
  1. Sara Fañanas-Baquero: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  2. Oscar Quintana-Bustamante: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  3. Daniel P Dever: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  4. Omaira Alberquilla: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  5. Rebeca Sanchez-Dominguez: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  6. Joab Camarena: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  7. Isabel Ojeda-Perez: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  8. Mercedes Dessy-Rodriguez: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  9. Rolf Turk: Integrated DNA Technologies, Coralville, IA 52241, USA.
  10. Mollie S Schubert: Integrated DNA Technologies, Coralville, IA 52241, USA.
  11. Annalisa Lattanzi: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  12. Liwen Xu: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  13. Jose L Lopez-Lorenzo: Hospital Universitario Fundación Jiménez Díaz, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28015, Spain.
  14. Paola Bianchi: UOC Ematologia, Fisiopatologia delle Anemie, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Milan 20122, Italy.
  15. Juan A Bueren: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
  16. Mark A Behlke: Integrated DNA Technologies, Coralville, IA 52241, USA.
  17. Matthew Porteus: Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  18. Jose-Carlos Segovia: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain.
PMID: 34485608 DOI: 10.1016/j.omtm.2021.05.001

Abstract

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell ( ) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Keywords

AAV6 CRISPR-Cas9 gene editing gene therapy hematopoietic stem cell hemolytic anemia pyruvate kinase deficiency

References

  1. Am J Hum Genet. 1984 May;36(3):634-9 [PMID: 6731438]
  2. Cell Stem Cell. 2019 Apr 4;24(4):551-565.e8 [PMID: 30905619]
  3. Br J Haematol. 2016 Sep;174(6):952-61 [PMID: 27470218]
  4. Gene Ther. 2021 Jun;28(6):373-390 [PMID: 33712802]
  5. Nat Biotechnol. 2016 Apr;34(4):424-9 [PMID: 26950749]
  6. Nat Commun. 2018 Dec 21;9(1):5454 [PMID: 30575740]
  7. Blood Rev. 2007 Jul;21(4):217-31 [PMID: 17360088]
  8. EMBO Mol Med. 2014 May 23;6(6):835-48 [PMID: 24859981]
  9. Clin Genet. 2020 Jan;97(1):89-102 [PMID: 31231794]
  10. Science. 2013 Oct 11;342(6155):253-7 [PMID: 24115442]
  11. Mol Ther Methods Clin Dev. 2020 May 04;17:1097-1107 [PMID: 32478125]
  12. N Engl J Med. 2018 Apr 19;378(16):1479-1493 [PMID: 29669226]
  13. Nat Med. 2018 Aug;24(8):1216-1224 [PMID: 30082871]
  14. N Engl J Med. 2021 Jan 21;384(3):252-260 [PMID: 33283989]
  15. Nat Commun. 2016 Jan 28;7:10548 [PMID: 26817820]
  16. J Perinatol. 2010 Mar;30(3):233-6 [PMID: 20182430]
  17. Sci Transl Med. 2017 Jan 11;9(372): [PMID: 28077679]
  18. Stem Cell Reports. 2015 Dec 8;5(6):1053-1066 [PMID: 26549847]
  19. Science. 2018 Jan 12;359(6372): [PMID: 29326244]
  20. Nat Commun. 2017 Jun 23;8:16007 [PMID: 28643790]
  21. Nature. 2016 Nov 17;539(7629):384-389 [PMID: 27820943]
  22. Rambam Maimonides Med J. 2014 Oct 29;5(4):e0028 [PMID: 25386344]
  23. EMBO Mol Med. 2017 Nov;9(11):1574-1588 [PMID: 28899930]
  24. Haematologica. 2020 Jan 23;106(1):238-249 [PMID: 31974203]
  25. N Engl J Med. 2017 Mar 2;376(9):848-855 [PMID: 28249145]
  26. Nat Med. 2019 Feb;25(2):234-241 [PMID: 30664781]
  27. Nucleic Acids Res. 2014 Jan;42(2):1365-78 [PMID: 24157834]
  28. Gene Ther. 2011 May;18(5):479-87 [PMID: 21160533]
  29. Blood. 2020 Sep 10;136(11):1241-1249 [PMID: 32702739]
  30. PLoS One. 2019 Oct 16;14(10):e0223775 [PMID: 31618280]
  31. Blood. 2018 May 17;131(20):2183-2192 [PMID: 29549173]
  32. Mol Ther. 2019 May 8;27(5):986-998 [PMID: 30930113]
  33. Nature. 2014 Jun 12;510(7504):235-240 [PMID: 24870228]
  34. Nature. 2010 Sep 16;467(7313):318-22 [PMID: 20844535]
  35. Blood. 2016 May 26;127(21):2513-22 [PMID: 26903548]
  36. Sci Rep. 2016 Oct 07;6:34531 [PMID: 27713537]
  37. Mol Ther. 2016 Aug;24(7):1187-98 [PMID: 27138040]
  38. Mol Ther. 2009 Dec;17(12):2000-9 [PMID: 19755962]
  39. Nature. 2018 Jan 11;553(7687):171-177 [PMID: 29323295]
  40. Nat Med. 2018 Jul;24(7):927-930 [PMID: 29892067]
  41. Blood Cells Mol Dis. 2016 Mar;57:100-9 [PMID: 26832193]
  42. Nat Biotechnol. 2015 May;33(5):538-42 [PMID: 25798939]
  43. Blood. 2021 May 13;137(19):2598-2608 [PMID: 33623984]
  44. Nat Med. 2019 May;25(5):776-783 [PMID: 30911135]
  45. Mol Ther Methods Clin Dev. 2017 Jan 11;4:137-148 [PMID: 28344999]
  46. Haematologica. 2018 Feb;103(2):e82-e86 [PMID: 29242305]
  47. Mol Ther Methods Clin Dev. 2018 Aug 14;10:313-326 [PMID: 30182035]
Journal Article
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Word Cloud

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