Fig. 1

Higher vector titers with 100-fold concentration and more efficient transduction in xenograft mice for the optimized forward-oriented vector for globin expression. a We produced the forward-oriented globin-expressing vector with 100-fold concentration, which was optimized by the use of a short β-globin locus control region (LCR) composed of hypersensitive sites 2, 3, and 4 (HS2, HS3, and HS4), a large size of the β-globin promoter (LP), and a β-globin downstream region (DR) lacking the polyA signal. Vector titers of the forward-oriented vectors were evaluated, as compared with the optimized vector in the reverse orientation and a standard GFP-marking vector (n = 3–13). **p < 0.01, *p < 0.05 evaluated by t-test. b We transduced human CD34⁺ cells with reverse-oriented vector, forward-oriented vector, and standard marking vector with a GFP marker gene, and the transduced cells were transplanted into NOD/SCID/IL2Rγ^null mice (n = 3–5). Four months after transplantation, xenograft bone marrow cells were collected and cultured for human erythroid cells with human specific cytokines. c Human cell engraftment and average vector copy numbers (VCNs) in peripheral blood cells were evaluated for 4 months post transplant. **p < 0.01 and *p < 0.05 evaluated by Dunnett’s test, compared with the forward-oriented vector. d, e After human erythroid cell differentiation from xenograft bone marrow cells (n = 3), we evaluated GFP expression using fluorescence microscopy (d) as well as GFP-positive percentages in erythroid cells by flow cytometry (e). Scale bars represent 100 μm. *p < 0.05 evaluated by t-test between forward-oriented and reverse-oriented vectors. EPO: erythropoietin, FL: fms-related tyrosine kinase 3 ligand, GFP: enhanced green fluorescent protein, IL3: interleukin 3, MOI: multiplicity of infection, Mp: murine stem cell virus promoter, P: β-globin promoter, PCR: polymerase chain reaction, SCF: stem cell factor, TPO: thrombopoietin, YFP: enhanced yellow fluorescent protein