Diamond-Blackfan anemia is a bone marrow failure syndrome primarily characterized by erythroblastopenia and associated with an increased incidence of cancer and congenital abnormalities. The most frequently mutated genes in DBA cases are RPS19 (25%) and RPL5 (11%). A lentiviral gene therapy approach for the treatment of RPS19-deficient patients is under development in our lab. Due to its direct interaction with MDM2, P53 master regulator, RPL5 might require strict endogenous regulation. Therefore, we have developed a homologous recombination (HR) gene editing strategy for the treatment of RPL5-deficient patients based on CRISPR/Cas9 system and adeno-associated viral vectors harboring a codon-optimized sequence of RPL5-cDNA, in both single-stranded (ssAAV6) and self-complementary (scAAV6) configurations. The efficacy of the HDR-mediated gene editing was optimized using healthy donor CD34⁺ cells from bone marrow (BM) and cord blood (CB) and the two vectors at different multiplicities of infection (MOIs). The scAAV yielded higher efficacy and lower toxicity than the ssAAV at 10-30 times lower MOI. In CB-CD34⁺ cells, HR analysis of the scAAV showed a mean of 79% edited alleles detected by ddPCR and 73% of edited colonies (CFCs) at MOI of 3x10³ genome-copies per cell (GC/cell). In BM-CD34+ cells, the toxicity of ssAAV was unbearable but further optimization based on the use of scAAV and variable pre-stimulation protocol allowed to achieve a mean of 50% edited alleles detected by ddPCR at MOI of 3x10² GC/cell. In addition, the effect in toxicity and efficacy of transient inhibition of P53 is under evaluation.