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, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, 28040. Spain.

Dyskeratosis congenita (DC) is a very low prevalence disease included within inherited bone marrow failure (BMF) syndromes. It is caused by mutations in genes involved in telomere maintenance, leading to premature telomere shortening. One of the most prevalent mutated gene in DC is TERC, which encodes the telomerase long non-coding RNA component (TERC) and exhibits autosomal dominant inheritance (AD-DC). The allogeneic transplant of hematopoietic stem cells (HSCs) is the only curative treatment of the BMF. However, there are several limitations that hamper the success of allogeneic transplantation in a high percentage of DC patients. In order to develop an alternative curative treatment for BMF related to DC, we propose a gene therapy approach consisting of an autologous transplant of ex vivo corrected HSCs using CRISPR/Cas9 technology and adeno-associated vectors (AAVs). CRISPR/Cas9 system produced a double strand break (DSB) that would be repaired by homologous recombination (HR) using the template delivered by the AAV which contains the corrected TERC gene and the homology arms. HR efficiency has been analysed in healthy CD34⁺ total cells and in colony forming units. RNA expression and maturation patterns have been studied to demonstrate that were not affected as consequence of designed editing approach. Next steps will be to demonstrate phenotypic correction on TERC-deficient models and HSC from TERC-deficient patients.