Pre-clinical development of a lentiviral vector expressing the anti-sickling βAS3 globin for gene therapy for sickle-cell disease

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin β-chain thatinduces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia andmultiple organ damage. Gene therapy is a potential alternative to HLA-matched allogeneichematopoietic stem cell transplantation, available to a minority of patients. We developed alentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16Dand 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 erythrocytesdifferentiated from transduced HSPCs from SCD patients. Transduced HSPCs weretransplanted in NSG immunodeficient mice to analyze biodistribution, chimerism andtransduction efficiency in bone marrow (BM), spleen, thymus and peripheral blood 12-14weeks after transplantation. Vector integration site analysis, performed in pre-transplantHSPCs and post-transplant BM cells from individual mice, showed a normal lentiviralintegration pattern and no evidence of clonal dominance. An in vitro immortalization assay(IVIM) showed the low genotoxic potential of GLOBE-AS3. This study enables a phase-I/IIclinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation ofautologous HSC transduced by GLOBE-AS3.