Gene Correction of Wiskott–Aldrich syndrome iPS Cells Rescues Proplatelet Defects and Improves Platelet Size

Abstract

Wiskott–Aldrich syndrome (WAS) is a severe X-linked disorder caused by loss-of-function mutations in the WAS gene, responsible for encoding WAS protein (WASP), a key regulator of the actin cytoskeleton in all hematopoietic cells, except red blood cells. The mechanism underlying microthrombocytopenia, a distinctive feature of WAS and a major contributor to mortality, remains not fully elucidated. In this study, using different gene-editing strategies, we corrected mutations in patient-derived WAS-induced pluripotent stem cell (iPSC) lines, generating isogeneic WAS-iPSC lines. These included lines with direct mutation-specific correction and lines incorporating a WASP transgene cassette regulated by the MND or WAS1.6 kb promoter integrated at the safe harbor AAV1 site. Our results demonstrated that direct mutation correction successfully restored WASP levels to the equivalent of the wild-type in iPSC-derived megakaryocytes (MKs). In contrast, the AAV1-targeted strategy using the MND and WAS1.6 promoters yielded a lower level of WASP. Notably, only the mutation-specific correction lines exhibited improvements in proplatelet structures and generated larger-sized platelets. Our findings underscore the crucial roles of WASP during human thrombopoiesis and suggest that therapeutic approaches, such as direct gene correction, which can achieve physiologic levels of WASP in MKs, hold promise for ameliorating platelet defects in individuals with WAS.

Keywords

thrombocytopenia - Wiskott–Aldrich syndrome - gene targeting

Authors' Contribution

Conceptualization: N.I. and K.S. Designed the experiments: P.I. and N.I. Investigation: P.I., S.P., P.A., P.M., P.P., and N.S. Funding acquisition: K.S., N.I., and V.S. Writing original draft: N.I., P.I., and K.S. Review and editing: K.S., N.I., and V.S. Project administration: K.S. and N.I. Supervision: V.S.

Publikationsverlauf

Eingereicht: 09. Juli 2024

Angenommen: 20. Dezember 2024

Accepted Manuscript online:
24. Dezember 2024

Artikel online veröffentlicht:
25. Februar 2025

• References

• 1 Candotti F. Clinical manifestations and pathophysiological mechanisms of the Wiskott-Aldrich syndrome. J Clin Immunol 2018; 38 (01) 13-27
• 2 Thrasher AJ, Burns SO. WASP: a key immunological multitasker. Nat Rev Immunol 2010; 10 (03) 182-192
• 3 Vieira RC, Pinho LG, Westerberg LS. Understanding immunoactinopathies: a decade of research on WAS gene defects. Pediatr Allergy Immunol 2023; 34 (04) e13951
• 4 Albert MH, Slatter MA, Gennery AR. et al. Hematopoietic stem cell transplantation for Wiskott-Aldrich syndrome: an EBMT Inborn Errors Working Party analysis. Blood 2022; 139 (13) 2066-2079
• 5 Braun CJ, Boztug K, Paruzynski A. et al. Gene therapy for Wiskott-Aldrich syndrome. –long-term efficacy and genotoxicity. Sci Transl Med 2014; 6 (227) 227ra33
• 6 Ferrua F, Cicalese MP, Galimberti S. et al. Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study. Lancet Haematol 2019; 6 (05) e239 –e253
• 7 Magnani A, Semeraro M, Adam F. et al. Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome. Nat Med 2022; 28 (01) 71-80
• 8 Sabri S, Foudi A, Boukour S. et al. Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment. Blood 2006; 108 (01) 134-140
• 9 Bender M, Stritt S, Nurden P. et al. Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott-Aldrich syndrome-like platelet defect. Nat Commun 2014; 5: 4746
• 10 Paul DS, Casari C, Wu C. et al. Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice. Blood Adv 2017; 1 (18) 1398-1408
• 11 Kajiwara M, Nonoyama S, Eguchi M. et al. WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro. Br J Haematol 1999; 107 (02) 254-262
• 12 Ingrungruanglert P, Amarinthnukrowh P, Rungsiwiwut R. et al. Wiskott-Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation. Thromb Haemost 2015; 113 (04) 792-805
• 13 Amarinthnukrowh P, Ittiporn S, Tongkobpetch S. et al. Clinical and molecular characterization of Thai patients with Wiskott-Aldrich syndrome. Scand J Immunol 2013; 77 (01) 69-74
• 14 Haddad E, Cramer E, Rivière C. et al. The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation. Blood 1999; 94 (02) 509-518
• 15 Palazzo A, Bluteau O, Messaoudi K. et al. The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation. J Thromb Haemost 2016; 14 (12) 2524-2535
• 16 Prislovsky A, Zeng X, Sokolic RA. et al. Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis. Platelets 2013; 24 (04) 288-296
• 17 Prislovsky A, Marathe B, Hosni A. et al. Rapid platelet turnover in WASP(-) mice correlates with increased ex vivo phagocytosis of opsonized WASP(-) platelets. Exp Hematol 2008; 36 (05) 609-623
• 18 Okita K, Matsumura Y, Sato Y. et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 2011; 8 (05) 409-412
• 19 Takayama N, Nishikii H, Usui J. et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood 2008; 111 (11) 5298-5306
• 20 Miller JC, Holmes MC, Wang J. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 2007; 25 (07) 778-785
• 21 Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 2013; 14 (01) 49-55
• 22 Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F. A transcription activator-like effector toolbox for genome engineering. Nat Protoc 2012; 7 (01) 171-192
• 23 Astrakhan A, Sather BD, Ryu BY. et al. Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome. Blood 2012; 119 (19) 4395-4407
• 24 Thon JN, Montalvo A, Patel-Hett S. et al. Cytoskeletal mechanics of proplatelet maturation and platelet release. J Cell Biol 2010; 191 (04) 861-874
• 25 Eckly A, Scandola C, Oprescu A. et al. Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids. J Thromb Haemost 2020; 18 (11) 2987-3001
• 26 Wang JY, Doudna JA. CRISPR technology: a decade of genome editing is only the beginning. Science 2023; 379 (6629) eadd8643
• 27 Wahlster L, Daley GQ. Progress towards generation of human haematopoietic stem cells. Nat Cell Biol 2016; 18 (11) 1111-1117
• 28 Dever DP, Bak RO, Reinisch A. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 2016; 539 (7629) 384-389
• 29 Ferrari G, Thrasher AJ, Aiuti A. Gene therapy using haematopoietic stem and progenitor cells. Nat Rev Genet 2021; 22 (04) 216-234
• 30 Haltalli MLR, Wilkinson AC, Rodriguez-Fraticelli A, Porteus M. Hematopoietic stem cell gene editing and expansion: state-of-the-art technologies and recent applications. Exp Hematol 2022; 107: 9-13
• 31 Laskowski TJ, Van Caeneghem Y, Pourebrahim R. et al. Gene correction of iPSCs from a Wiskott-Aldrich syndrome patient normalizes the lymphoid developmental and functional defects. Stem Cell Rep 2016; 7 (02) 139-148
• 32 Rai R, Romito M, Rivers E. et al. Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott-Aldrich syndrome. Nat Commun 2020; 11 (01) 4034
• 33 Ferrari S, Valeri E, Conti A. et al. Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy. Cell Stem Cell 2023; 30 (05) 549-570
• 34 Vavassori V, Ferrari S, Beretta S. et al. Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells. Blood 2023; 142 (09) 812-826
• 35 Wimberger S, Akrap N, Firth M. et al. Simultaneous inhibition of DNA-PK and PolΘ improves integration efficiency and precision of genome editing. Nat Commun 2023; 14 (01) 4761
• 36 Ferrari S, Jacob A, Cesana D. et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell 2022; 29 (10) 1428-1444 .e9
• 37 Chen PJ, Liu DR. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet 2023; 24 (03) 161-177
• 38 Fiumara M, Ferrari S, Omer-Javed A. et al. Genotoxic effects of base and prime editing in human hematopoietic stem cells. Nat Biotechnol 2024; 42 (06) 877-891
• 39 Zou J, Mali P, Huang X, Dowey SN, Cheng L. Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 2011; 118 (17) 4599-4608
• 40 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8 (11) 2281-2308
• 41 Hockemeyer D, Soldner F, Beard C. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 2009; 27 (09) 851-857