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.; 2:3RNA Molecular Biology, ULB-Cancer Research Center (U-CRC), Université Libre de Bruxelles; 3:Hospital del Niño Jesús, Madrid, Spain; 4:Hôpital Robert-Debre, Paris, France; 5:Hospital Sant Joan de Déu; Barcelona; 6:Hospital Gregorio Marañón, Madrid, Spain

Diamond Blackfan anemia (DBA) is a rare bone marrow failure syndrome (BMFS) with an estimated prevalence of 7 cases per million, in which the hallmark of the disease is macrocytic anemia. Allogenic hematopoietic stem cell transplantation (HSCT) represents the only curative treatment for the patients with DBA. Mutations in ribosomal protein (RP) gene RPS19 are present in around 25% of DBA patients. Therefore, here we aimed at the development of a clinically applicable lentiviral mediated ex vivo gene therapy to correct RPS19-haploinsuficient DBA HSCs. The therapeutic efficacy of PGK.CoRPS19.Wpre* and EF1α(s).CoRPS19.Wpre*-LVs was confirmed in primary CD34⁺ cells from RPS19-deficient patients. Transduction of CD34⁺ cells from these patients with the therapeutic LV was not toxic for the hematopoietic progenitor cells. Moreover, the therapeutic LV reverted the red blood cell differentiation defect characteristic of DBA CD34⁺ cells, increasing the output of CD71⁺/CD235⁺ mature erythroid cells, both in vitro and in vivo, and preserved the hematopoietic repopulationin NSG mice. Additionally, preliminary safety studies, showed that the ectopic expression of RPS19 did not produce evident changes in the repopulating function of healthy donors (HD) CD34⁺ cells. These studies were also confirmed by examining the healthy status of transplanted immunodeficient recipients, and also by investigating the clonal repertoire of transduced HSCs in transplanted mice. Taken together, the preclinical studies conducted in this work support that the proposed gene therapy appraoch should constitute an efficient and safe approach for the treatment of RPS19 DBA patients.