222. The LCR-Free Y-Globin Lentiviral Vector Combining Two HPFH Activating Elements Corrects Murine Thalassemic Phenotype In Vivo

Abstract

The β-thalassemias result from reduced or absent expression of the β-chain of hemoglobin (α2β2; HbA) causing precipitation of excess α-chains and eventually apoptosis. Thus, factors that reduce the degree of chain imbalance such as an innate ability to increase fetal hemoglobin (HbF;α2γ2), as in the HPFH phenotype, have an ameliorating effect on the disease. Hence, gene therapy of β-thalassemia based on γ-globin addition via viral vectors, displays a considerable advantage. In this study, we assessed the efficiency of our previously generated LCR-free γ-globin self inactivating vector GGHI (Papanikolaou et al. Hum Gene Ther 2012) to correct the thalassemic phenotype in vivo in the Hbth3/+ C57BL/6J mouse model (thal3 model). Recipient mice aged about 12 weeks, were treated with the myelosuppresant factor busulfan, while donor mice, of the same age and of the opposite gender, were treated with 5-fluorouracil, prior to transplantation. Total bone marrow was isolated from 5-fluorouracil-treated donors and was transduced with GGHI in X-VIVO™ medium containing cytokines (mIL-1a, mIL-3, mIL-6 and mSCF) at an MOI=30, employing the spinoculation method. The transduced cells were then transplanted via tail vein intravenous injection to the recipients. To evaluate the therapeutic effect of GGHI, blood was collected from recipient mice prior and post transplantation for 4 months and, hemoglobin levels (g/dl), hematocrit and total red blood cell count were assessed by a hematological analyzer. The expression of human γ-globin in peripheral blood was assessed by flow cytometry using an anti-HbF monoclonal antibody. Our results documented that transplanted thalassemic mice (n=4) with GGHI-corrected hemopoietic stem cells, exhibited an increase in hematocrit values by 22.3% (ranging from 24.5% to 35.7%, p=0.02) with a concomitant increase in hemoglobin levels, reaching an average of 11.1 g/dl in transplanted mice vs 8.8 g/dl to those prior to transplantation, which corresponds to a 25.5% increase (p=0.008). Human γ-globin was detected in the peripheral blood of all transplanted animals by flow cytometry and ranged from 20 to 45%. Transduction efficiency in these experiments was estimated to be 35-50% as assessed in vitro in CFUs by PCR for vector-specific sequences. In summary, these results demonstrate for the first time that viral-mediated globin gene transfer via an LCR-free γ-globin lentiviral vector in hemopoietic stem cells effectively corrects a severe hemoglobin disorder.