Genetic rescue of a human model of Bernard-Soulier syndrome based on CRISPR-Cas9 gene therapy

J Cerón(1,2) G Martínez-Navajas(1,2) L López-Onieva(1,2) P Real(1,2)

1:Universidad de Granada; 2:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia

Bernard Soulier syndrome (BSS) is an autosomal recessive platelet disorder characterized by thrombocytopenia, large platelets and frequent bleeding. It is caused by mutations in one of the three genes coding for the membrane glycoprotein complex in platelets GPIb-IX-V. The GPIb-IX-V complex is a receptor for Von Willebrand Factor and it is involved in platelet adhesion and aggregation. When severe bleeding occurs, platelet transfusion is the only available treatment for these patients and recurrent transfusions can induce platelet refractoriness. Restoration of the mutated gene in hematopoietic stem cells by gene therapy might represent a powerful treatment for BBS patients.

Induced pluripotent stem cells (iPSCs) are an excellent tool for human disease modeling and a platform where new therapies can be tested. We used an induce pluripotent stem cell line derived from a BSS patient containing a mutation c.259T>C in the GP9 gene (hiPSC-BSS) to assess the potential of the CRISPR-Cas9 system to target and repair the endogenous gene by homologous recombination.

We started from a genetically heterogeneous pool of hiPSC-BSS cells transduced with CRISPR/Cas9. We used a single cell cloning system to obtain colonies from a single cell (clones), whose GP9 locus was analyzed by sequencing. We achieved a homologous recombination (HR) efficiency of 2.3% in iPSCs (2 out of 75 clones were rescued). The two rescued clones were heterozygous for GP9, meaning that only one of the two alleles had been correctly edited. This condition should be sufficient to achieve the rescue of BSS disease.