P71

Editing of human Dyskerin Pseudouridine Synthase 1 gene in hematopoietic stem cells: A new tool to address the treatment of bone marrow failure in patients with X-linked dyskeratosis congenita

N W Meza(1) D Llorente-Arroyo(1) M L Lozano(1) C Carrascoso-Rubio(1,2) R Murillas(3) L Sastre(2) R Perona(2) J A Bueren(1) G Guenechea(1)

1:Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040. Spain.; 2:Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, 28029. Spain.; 3:Division of Epithelial Biomedicine - Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, 28040. Spain.

Telomere biology disorders (TBDs) are caused by impaired telomere maintenance. Critically short telomeres limit the replicative cell capacity, leading to reduced tissue renewal. TBDs are variably characterized by bone marrow failure (BMF), cancer predisposition and multiorgan system complications, including organ failure and/or fibrosis. Progressive BMF is the most common life-threatening complication of TBD, affecting up to 80% of patients with classical dyskeratosis congenita (DC). X-linked dyskeratosis congenita (X-DC) is a TBD inherited BMF syndrome caused by mutations in the DKC1 gene, which plays an essential role for telomerase function and rRNA pseudourydilation. The only curative treatment for the BMF of these patients is hematopoietic stem and progenitor cell transplantation (HSPCT). However, the poor outcomes of HSPCT, make necessary the development of alternative treatments. Here, we developed a gene editing approach based on the use of a rAAV donor vector and sgRNA/Cas9 ribonucleoprotein to generate specific DKC1 cDNA transgene integration and functional expression in healthy cord blood derived CD34+ progenitors, as proof of concept. Using specific sgRNAs, stable genome editing and ex vivo expansion rates have been analysed, either in liquid or in clonogenic cultures. These findings would propose a strategy that could be used as a therapy in patients. Next steps will aim the genetic editing of HSPCs from X-DC patients.