Abstract
Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%-80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12-14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.