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In vivo selection with lentiviral expression of Bcl2T69A/S70A/S87A mutant in hematopoietic stem cell-transplanted mice

Gene Therapy volume 25, pages 220–233 (2018)Cite this article

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Abstract

Current in vivo selections for hematopoietic stem cell (HSC)-based gene therapy are drug dependent and not without risk of cytotoxicity or tumorigenesis. We developed a new in vivo selection system with the non-phosphorylatable Bcl2 mutant Bcl2T69A/S70A/S87A (Bcl2AAA), which makes in vivo selection drug independent and without risk of cytotoxicity or tumorigenesis. We demonstrated in HSC-transplanted mice that Bcl2AAA facilitated efficient in vivo selection in the absence of any exogenously applied drugs under both myeloablative and non-myeloablative conditioning. In mice transplanted with retrovirally transduced sca-1-positive bone marrow cells, the marked cell level increased from 26.38% of input transduced cells to 92.61 ± 0.95% of peripheral blood cells for myeloablative transplantation or to 37.82 ± 6.35% for non-myeloablative transplantation 6 months after transplantation. Bcl2AAA did not induce tumorigenesis and does not influence hematopoiesis and the function of the reconstituted blood system. However, the high-level constitutive expression of Bcl2AAA mediated by retroviral vector induced exhaustion of the marked cells after tertiary transplantation. Fortunately, low-level constitutive expression of Bcl2AAA driven by an internal promoter in lentiviral vector could both maintain the marked cell level (24.13 ± 5.27%, 27.17 ± 5.51%, 24.33 ± 5.08%, and 22.07 ± 4.44% for primary, secondary, tertiary, and quaternary recipients) and avoid the exhaustion of the marked cells even in quaternary recipients. Importantly, the low-level constitutive expression of Bcl2AAA did not induce tumorigenesis. Thus, the in vivo selection employing the low-level constitutive expression of Bcl2AAA provides a general platform which is relevant for widespread applications of gene therapy.

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Acknowledgements

We thank Z.X. Hu for taking care of the animals. This work was supported by the start-up research fund from Hangzhou Dianzi University (KYS195612020) (to Y.-Y.W.) and the National Natural Science Foundation of China (30960443) (to Y.-Y. W.).

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Authors and Affiliations

  1. Department of Biomedical Engineering, College of Life Information Science and Instrument Engineering, Hangzhou Dianzi University, Hangzhou, China

    Yan-Yi Wang & Yong Yang

  2. Tonglu Experimental Junior Middle School, Hangzhou, China

    Shan Ma

  3. Department of Respiration, The 117th Hospital of PLA, Hangzhou, China

    Qingyong Chen & Demin Jiao

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  1. Yan-Yi Wang
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Correspondence to Yan-Yi Wang or Yong Yang.

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Wang, YY., Ma, S., Chen, Q. et al. In vivo selection with lentiviral expression of Bcl2T69A/S70A/S87A mutant in hematopoietic stem cell-transplanted mice. Gene Ther 25, 220–233 (2018). https://doi.org/10.1038/s41434-018-0008-9

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