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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 7

Phenotypic correction of CD46+/+/Hbbth-3 mice by in vivo HSPC transduction/selection.

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Phenotypic correction of CD46+/+/Hbbth-3 mice by in vivo HSPC transducti...
(A) RBC analysis of healthy (CD46tg) mice, CD46+/+/Hbbth-3 mice prior to mobilization and in vivo transduction, and CD46+/+/Hbbth-3 mice that underwent in vivo transduction/selection (analyzed at week 29 after HDAd infusion) (n = 5). *P ≤ 0.05, **P ≤ 0.0002, ***P ≤ 0.00003. Statistical analysis was performed using 2-way ANOVA. (B) Supravital stain of peripheral blood smears with Brilliant cresyl blue for reticulocyte detection. Arrows indicate reticulocytes containing characteristic remnant RNA and micro-organelles. The percentages of positively stained reticulocytes in representative smears were: for CD46, 7%; for CD46+/+/Hbbth-3 before treatment, 31%; and for CD46+/+/Hbbth-3 after treatment, 12%. Scale bar: 20 μm. (C) Top: Blood smears. Scale bar: 20 μm. Middle: Bone marrow cytospins. Arrows indicate erythroblasts at different stages of maturation and a backshift in erythropoiesis with pro-erythroblast predominance in treated mice. Scale bar: 25 μm. Bottom: Tissue hemosiderosis by Perls’ stain. Iron deposition is shown as cytoplasmic blue pigments of hemosiderin in spleen tissue sections. The blood smear images for the control mice (CD46tg and CD46+/+/Hbbth-3, before transduction) in C and Figure 5D are from the same sample. (D) Macroscopic spleen images of 1 representative CD46tg and 1 untreated CD46+/+/Hbbth-3 mouse and 5 treated CD46+/+/Hbbth-3 mice. (E) At sacrifice, spleen size was determined as the ratio of spleen weight to total body weight (mg/g). Each symbol represents an individual animal. Data are presented as means ± SEM. *P ≤ 0.05. Statistical analysis was performed using 1-way ANOVA.
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