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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 Therapeutics

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 6

Analysis of γ-globin expression in in vivo–transduced CD46+/+/Hbbth-3 mice after in vivo selection.

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Analysis of γ-globin expression in in vivo–transduced CD46+/+/Hbbth-3 mi...
(A) Percentage of human γ-globin in peripheral RBCs measured by flow cytometry. Red arrows indicate the time points of O6-BG/BCNU treatment. Different symbols represent 3 independent experiments. The data up to week 16 are identical to those in Figure 5A. (B) Percentage of γ-globin–expressing cells in hematopoietic tissues at sacrifice (week 29) analyzed by flow cytometry. *P ≤ 0.05, **P ≤ 0.0002, ***P ≤0.00003. (C) γ-Globin expression in MACS-purified Ter119 cells. Bone marrow cells from primary recipients at week 29 were immunomagnetically selected for Ter119+ cells. γ-Globin expression was measured in Ter119+ and Ter119– cells by flow cytometry. ***P ≤ 0.0002. (D) Fold enrichment of γ-globin+ erythroid (Ter119+) and nonerythroid (Ter119–) cells in peripheral blood, bone marrow, and spleen before versus after in vivo selection (week 16 vs. week 29). n = 5, **P ≤ 0.0002. (E) Percentage of human γ-globin protein compared with mouse α-globin protein, measured by HPLC in RBCs. Statistical analyses were done with the nonparametric Kruskal-Wallis test. (F) Level of human γ-globin mRNA over adult mouse β-major globin mRNA measured by RT-qPCR in peripheral blood cells. Untreated CD46+/+/Hbbth-3 mice were used as control. Each symbol represents an individual animal.
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