1:CIEMAT/CIBERER; 2:IIS-Fundación Jimenez Diaz (IIS-FJD, UAM); 3:CIMA; 4:Hôpital Robert-Debre; 5:Hospital Gregorio Marañón

Diamond-Blackfan Anemia (DBA) is an inherited bone marrow failure (IBMF) syndrome mainly characterized by red cell hypoplasia, congenital abnormalities and increased risk of cancer. Among the genes associated with DBA, RPS19 is the most frequently mutated (25%) one, followed by RPL5 (11%). Our laboratory has developed a lentiviral gene therapy approach for the treatment of RPS19-deficient patients. Here, we propose a homologous recombination (HR) gene editing strategy for the treatment of RPL5-deficient patients, since this protein might require a tight endogenous regulation due to its direct interaction with the MDM2, P53 master regulator. In particular, we have developed a single-stranded adeno-associated viral vector 6 (ssAAV6) harboring a codon optimized sequence of the RPL5 cDNA. Designed sgRNAs were delivered as ribonucleoprotein, which facilitated the achievement of around 90% indels in CD34⁺ cells, either from healthy donor cord blood (CB) or RPL5-deficient patient bone marrow (BM). To optimize the transduction, and thus the efficacy of HDR-mediated gene editing, healthy donor CD34⁺ cells from BM and CB were transduced with CoRPL5-AAV6 at different multiplicities of infection (MOIs). RT-qPCR assay targeting CoRPL5 sequence allowed us to detect the AAV sequence in up to 36% and 14% of CB-CD34⁺ cells transduced at MOIs of 10⁴ and 3x10³ genome copies per cell, respectively. Consistently, the efficacy of homologous recombination was 31% and 14% in these samples, respectively. Different strategies are under development to reduce toxicity and increase the gene editing efficacy in BM CD34⁺ cells.