Abstract

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.