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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In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

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 1

Integrating HDAd5/35++ vector for HSPC gene therapy of hemoglobinopathies.

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Integrating HDAd5/35++ vector for HSPC gene therapy of hemoglobinopathie...
(A) Vector structure. In HDAd-γ-globin/mgmt, the 11.8-kb transposon is flanked by inverted transposon repeats (IR) and FRT sites for integration through a hyperactive Sleeping Beauty transposase (SB100X) provided from the HDAd-SB vector (right panel). The γ-globin expression cassette contains a 4.3-kb version of the β-globin LCR consisting of 4 DNase hypersensitivity (HS) regions and the 0.7-kb β-globin promoter. The 76-Ile HBG1 gene including the 3′-UTR (for mRNA stabilization in erythrocytes) was used. To avoid interference between the LCR/β-promoter and EF1A promoter, a 1.2-kb chicken HS4 chromatin insulator (Ins) was inserted between the cassettes. The HDAd-SB vector contains the gene for the activity-enhanced SB100X transposase and Flpe recombinase under the control of the ubiquitously active PGK and EF1A promoters, respectively. (B) In vivo transduction of mobilized CD46tg mice. HSPCs were mobilized by s.c. injections of human recombinant G-CSF for 4 days followed by 1 s.c. injection of AMD3100. Thirty and 60 minutes after AMD3100 injection, animals were injected i.v. with a 1:1 mixture of HDAd-γ-globin/mgmt plus HDAd-SB (2 injections, each 4 × 1010 viral particles). Mice were treated with immunosuppressive (IS) drugs for the next 4 weeks to avoid immune responses against the human γ-globin and MGMT(P140K). O6-BG/BCNU treatment was started at week 4 and repeated every 2 weeks 3 times. With each cycle the BCNU concentration was increased, from 5 to 7.5 to 10 mg/kg. Immunosuppression was resumed 2 weeks after the last O6-BG/BCNU injection. (C) Percentage of human γ-globin+ peripheral RBCs measured by flow cytometry. (D) Percentage of human γ-globin+ cells in peripheral blood mononuclear cells (MNC), total cells, erythroid Ter119+ cells, and nonerythroid Ter119– cells. (E) Percentage of human γ-globin protein compared with adult mouse globin chains (α, β-major, β-minor) measured by HPLC in RBCs at week 18. (F) Percentage of human γ-globin mRNA compared with adult mouse β-major globin mRNA measured by RT-qPCR in total in peripheral blood cells at week 18. Mice that did not receive any treatment were used as a control. In C–F, each symbol represents an individual animal.