702. Successful correction of human beta thalassemia major using a self-inactivating lentiviral vector

Beta thalassemias are the most common single gene disorders that are potentially amenable to gene therapy. Unlike oncoretroviral vectors, lentiviral vectors (LV) have recently shown remarkable stability in transmitting the human (Hu) β-globin cassette. Thus far, LV carrying the Hu β/γ globin genes show variable expression in mice, an increase murine (Mu) hemoglobin (Hb) by 1–4 gm/dL/LV copy, leading to correction of heterozygous thalassemia mice and variable, often incomplete correction of a thalassemia major mouse model. We sought to develop a LV with high Hu β-globin expression, reduced position effects and improved biosafety, for effective gene therapy of Hu thalassemia major (TM), where it is ultimately relevant. A self-inactivating LV, BGI, carrying the chicken hypersensitive site-4 element (known for its enhancer blocking and chromatin barrier function), the Hu β-globin gene and its regulatory elements was used to transduce CD34+ cells (twice in 24 hrs; MOI 1–7) from bone marrow (BM) of 4 patients with TM. BGI/mock-transduced TM or normal (NL) BM CD34+ cells were: 1. Placed in unilineage erythroid differentiation cultures (Cx) and analyzed for cell expansion, differentiation, apoptotic cells and β-globin production (by FACS and HPLC). 2. Plated in CFU-assay and gene transfer efficiency analyzed in individual BFUe. 3. Transplanted into sublethally irradiated NOD/SCID-β2Mnull mice, long-term expression analyzed in blood and BM and Hu CD34+ cells and erythroid precursors isolated from Mu BM for CFU-assay. Erythroid liquid Cx showed that differentiation and expansion of TM-BGI CD34+ cells were indistinguishable from NL. While TM Cx arrested at the polychromatophil normoblast stage and formed rare poorly hemoglobinized RBC, NL and TM-BGI Cx underwent normal erythroid differentiation to mature well-hemoglobinized RBC. Apoptotic cells comprised 1 ± 0.5% of NL, 2 ± 1% of TM-BGI, and 49 ± 12% of TM Cx. There were 70 ± 11% HbA+ cells in NL, 60 ± 5% in TM-BGI and 16 ± 6% in TM Cx; β-globin chains comprised 70 ± 5% of all β-like globins in NL, 75 ± 14% in TM-BGI and 7 ± 7% in TM Cx. CFU assay showed all BFUe from NL, 86 ± 5% from TM-BGI and 5 ± 2.5% from TM CD34+ cells were HbA+. In all these data, NL vs TM-BGI were not different (P > .3) while NL vs. TM were significantly different (P < .05–.001). These results were confirmed in vivo: 10–16 wks after transplant (n = 24), there was multilineage human cell engraftment; HbA+ erythroid cells were seen only in NL and TM-BGI xenografts while apoptotic cells were present only in TM xenografts. 60% of erythroid colonies from NL, 55% from TM-BGI and none from the TM group of mice were HbA+ (n = 43). In blood of these mice, small quantities of HbA+ cells were seen only in NL and TM-BGI xenografts. The xenograft data reflect that the genetic correction extended to primitive TM progenitor cells. The mean HbA fluorescence intensity and its variability in NL was similar to that in TM-BGI in liquid Cx, in BFUe or in xenografts. Taken together, these data comprise the first report of complete phenotypic and functional correction of human TM, both in vitro and in a xenograft model in vivo, with normal β-globin production, reversal of apoptosis and effective erythropoiesis.