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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 8

Analysis of secondary C57BL/6 recipients with transplanted bone marrow cells from treated CD46+/+/Hbbth-3 mice.

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Analysis of secondary C57BL/6 recipients with transplanted bone marrow c...
(A) Engraftment rates measured in the periphery based on the percentage of human CD46+ (hCD46+) cells in PBMCs after busulfan conditioning or total-body irradiation (TBI). (C57BL/6 recipients do not express hCD46.) (B) Percentage of human γ-globin–expressing peripheral blood RBCs. All mice received immunosuppression starting from week 4 after transplantation. (C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells. (C and D) γ-Globin/CD46 expression in secondary C57BL/6 recipients at week 20 after transplant (busulfan preconditioning). CD46+ cells were immunomagnetically separated from the chimeric bone marrow of 3 representative secondary mice and analyzed for γ-globin expression by flow cytometry. Notably, unlike humans, huCD46tg mice express CD46 on RBCs. (C) γ-Globin/CD46 marking rates of primary and secondary recipients at sacrifice. (D) γ-Globin expression in CD46+-selected cells from the hematopoietic tissues of secondary recipients (week 20). Each symbol represents an individual animal. (E) γ-Globin expression in secondary recipients that received a new (second) round of HSPC mobilization/in vivo transduction (n = 5). Secondary recipients (busulfan-preconditioned) were analyzed for γ-globin and CD46 expression at week 20 after transplantation (“Before in vivo transduction”). These mice were then mobilized and transduced in vivo with the HDAd-γ-globin plus HDAd-SB vectors. Four weeks after in vivo transduction, mice were sacrificed and analyzed (“Week 4 after in vivo transduction”). ***P ≤ 0.00003. Statistical analyses were performed using 1-way ANOVA.
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