Correction of Sickle Cell Anemia with γ-Globin Gene Delivered by Lentivirus Vector in the Setting of Myeloablative or Reduced Intensity Conditioning, and Establishing Critical Determinants for Successful Gene Therapy for Sickle Cell Disease

While genetic delivery of recombinant anti-sickling β-globin genes have been shown to correct murine sickle cell anemia (SCA), correction of SCA by delivery of a natural hemoglobin, fetal hemoglobin (HbF), the proportion of genetically modified hematopoietic stem cells (HSC), or amount of HbF necessary to correct the disease is unknown. We designed a lentivirus vector carrying γ-globin exons with β-globin regulatory elements and non-coding sequences, GbG. First, GbG or mock transduced Berkeley sickle HSC were transplanted using a myeloablative (lethal irradiation) transplant model, to acheive full donor chimerism. GbG mice showed high HbF expression (HbF 41 ± 5% measured by HPLC) that was sustained in primary (6 mo) and secondary (7.5 mo) transplant recipients, and resulted in effective correction of hematological and functional RBC parameters, and reduction of inflammation that results from sickle cell disease. We found significantly reduced irreversibly sickled cells (2.3 ± 0.7% in GbG versus 10.2 ± 0.3% in mock mice; p30% HbF/F-cell and >60% F-cells consistently corrected SCA. The mean HSC transduction (assessed by secondary HbF+ CFU-S at 6 months post transplant) was 50% and 30% in the myeloablative and reduced intensity transplant models, respectively, with 1–3 GbG copies/ cell. Furthermore, three GbG mice showed correction of SCA with 20% HSC transduction, a clinically achievable goal. Taken together, this study is the first demonstration of correction of SCA with gene therapy using γ-globin, and defines critical determinants for effective gene therapy of this disease. Mouse Model Hb (g/dl) RBC 106/ul) Reticulocyte (%) WBC (K/ul) *p