Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Gonzalo Martinez-Navajas; Jorge Ceron-Hernandez; Iris Simon; Pablo Lupiañez; Sofia Diaz-McLynn; Sonia Perales; Ute Modlich; Jose A Guerrero; Francisco Martin; Teresa Sevivas; Maria L Lozano; Jose Rivera; Veronica Ramos-Mejia; Claudia Tersteeg; Pedro J Real

doi:10.1016/j.omtn.2023.06.008

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Mol Ther Nucleic Acids. 2023 Jun 12:33:75-92. doi: 10.1016/j.omtn.2023.06.008. eCollection 2023 Sep 12.

Authors

Gonzalo Martinez-Navajas^{1

2}, Jorge Ceron-Hernandez^{1

2}, Iris Simon^{1

2}, Pablo Lupiañez^{1

2}, Sofia Diaz-McLynn¹, Sonia Perales^{1

2

3}, Ute Modlich⁴, Jose A Guerrero^{5

6}, Francisco Martin^{1

7}, Teresa Sevivas⁸, Maria L Lozano⁹, Jose Rivera^{9

10}, Veronica Ramos-Mejia¹, Claudia Tersteeg¹¹, Pedro J Real^{1

2

3}

Affiliations

¹ GENyO, Pfizer-Universidad de Granada-Junta de Andalucia Centre for Genomics and Oncological Research, PTS, Granada, Avenida de la Ilustracion 114, 18016 Granada, Spain.
² University of Granada, Department of Biochemistry and Molecular Biology I, Faculty of Science, Avenida Fuentenueva S/n, 18071 Granada, Spain.
³ Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain.
⁴ Department of Gene and Cell Therapy, Institute of Regenerative Medicine, University of Zürich, Wagistrasse 12, 8952 Schlieren-Zürich, Switzerland.
⁵ Department of Haematology, University of Cambridge, Cambridge, UK.
⁶ National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK.
⁷ University of Granada, Department of Biochemistry and Molecular Biology III and Immunology, Faculty of Medicine, Avenida Ilustracion S/n, 18016 Granada, Spain.
⁸ Serviço de Sangue, Medicina Transfusional e Imunohemoterapia Do Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.
⁹ Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII, U765 Murcia, Spain.
¹⁰ Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC), Madrid, Spain.
¹¹ Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Abstract

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbβ, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C.

Keywords: Bernard-Soulier syndrome; CRISPR-Cas9; GPIb-V-IX receptor; MT: RNA/DNA Editing; disease models; gene therapy; hGP9; hematopoietic stem cells; induced pluripotent stem cells; lentiviral vector; von Willebrand factor.