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In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia
Hongjie Wang, … , Evangelia Yannaki, André Lieber
Hongjie Wang, … , Evangelia Yannaki, André Lieber
Published November 13, 2018
Citation Information: J Clin Invest. 2019;129(2):598-615. https://doi.org/10.1172/JCI122836.
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Research Article Hematology

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

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Abstract

Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myeloablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries, where the greatest demand for a β-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involves HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow, where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase–based integration machinery, a micro-LCR–driven γ-globin gene, and an MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose O6-benzylguanine plus bis-chloroethylnitrosourea. We showed in “healthy” human CD46–transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable γ-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near-complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and “portable” approach could permit a broader clinical application of thalassemia gene therapy.

Authors

Hongjie Wang, Aphrodite Georgakopoulou, Nikoletta Psatha, Chang Li, Chrysi Capsali, Himanshu Bhusan Samal, Achilles Anagnostopoulos, Anja Ehrhardt, Zsuzsanna Izsvák, Thalia Papayannopoulou, Evangelia Yannaki, André Lieber

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Figure 5

Analysis of in vivo–transduced CD46+/+/Hbbth-3 mice that did not receive O6BG/BCNU treatment.

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Analysis of in vivo–transduced CD46+/+/Hbbth-3 mice that did not receive...
(A) Percentage of human γ-globin in peripheral RBCs measured by flow cytometry. The experiment was performed 3 times, indicated by different symbol shapes. (B) γ-Globin expression in erythroid (Ter119+) and nonerythroid (Ter119–) blood cells. ***P ≤ 0.00003 by 1-way ANOVA test. (C) RBC analysis of healthy (CD46tg) mice (n = 3), CD46+/+/Hbbth-3 mice prior to mobilization and in vivo transduction (n = 14), and CD46+/+/Hbbth-3 mice that underwent in vivo transduction and were analyzed at week 16 (n = 8). *P ≤ 0.05. Statistical analysis was performed using 2-way ANOVA. (D) Histological phenotype. Top: Blood smears. Middle: Supravital stain of peripheral blood smears with Brilliant cresyl blue for reticulocyte detection. The percentages of positively stained reticulocytes in representative smears were: for CD46tg, 8% ± 0.8%; for CD46+/+/Hbbth-3 before transduction, 39% ± 1.3%; and for CD46+/+/Hbbth-3 week 16 after transduction, 26% ± 0.45%. Bottom: Extramedullary hemopoiesis. Scale bars: 20 μm. (E and F) Analysis of secondary recipients. Total bone marrow from week 16 in vivo–transduced mice was transplanted into C57BL/6 mice that received sublethal busulfan preconditioning. Mice received immunosuppression during the period of observation. (E) Engraftment based on the percentage of human CD46+ (hCD46+) PBMCs. (C57BL/6 recipients do not express hCD46.) (F) Percentage of human γ-globin+ RBCs. Each symbol represents an individual animal.

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