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Genetic in vivo engineering of human T lymphocytes in mouse models

Nature Protocols volume 16, pages 3210–3240 (2021)Cite this article

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Abstract

Receptor targeting of vector particles is a key technology to enable cell type–specific in vivo gene delivery. For example, T cells in humanized mouse models can be modified by lentiviral vectors (LVs) targeted to human T-cell markers to enable them to express chimeric antigen receptors (CARs). Here, we provide detailed protocols for the generation of CD4- and CD8-targeted LVs (which takes ~9 d in total). We also describe how to humanize immunodeficient mice with hematopoietic stem cells (which takes 12–16 weeks) and precondition (over 5 d) and administer the vector stocks. Conversion of the targeted cell type is monitored by PCR and flow cytometry of blood samples. A few weeks after administration, ~10% of the targeted T-cell subtype can be expected to have converted to CAR T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10–15 humanized mice is obtained. We also discuss how the protocol can be easily adapted to use LVs targeted to other types of receptors and/or for delivery of other genes of interest.

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Fig. 1: Overview of procedure.
Fig. 2: Alternative mouse models that can be used for in vivo CAR T cell generation.
Fig. 3: Plasmids for LV production.
Fig. 4: Characterization of target cells and vector stocks.
Fig. 5: Follow-up of humanization of NSG mice.
Fig. 6: Detection of in vivo–generated CAR T cells by flow cytometry.

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Data availability

Source data for Figs. 4 and 5 are provided. Data shown in Fig. 6 were previously published in ref. 12.

Code availability

Two NTA software scripts, the continuous flow script and the stop flow script, have been deposited in figshare (10.6084/m9.figshare.13221602) and are available as Supplementary Information.

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Acknowledgements

This work was supported by grants from the NIH (grant number 1 R01 AI145045-01) and Health Holland (LentiPace II) to C.J.B. The authors acknowledge continous support by the Federal Ministry of Health (03292364) to C.J.B. The authors thank the staff of the Plateau de Biologie Expérimentale de la Souris (PBES) animal care facility at the Ecole Normale Supérieure de Lyon and the flow cytometry platform (SFR BioSciences Lyon) (UMS3444/US8), Lyon, France.

Author information

Authors and Affiliations

  1. Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany

    Tatjana Weidner, Shiwani Agarwal, Gundula Braun & Christian J. Buchholz

  2. International Center for Infectiology, research team Enveloped Viruses, Vectors and Innate Responses, Institut national de la santé et de la recherche médicale, Unité 1111, Centre national de la recherche scientifique, Unité mixte de recherche 5308, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, University of Lyon, Lyon, France

    Séverine Perian, Floriane Fusil & Els Verhoeyen

  3. Division for Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany

    Jessica Hartmann & Christian J. Buchholz

  4. Université Côte d’Azur, Institut national de la santé et de la recherche médicale, Centre Méditerranéen de Médecine Moléculaire, Nice, France

    Els Verhoeyen

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  1. Tatjana Weidner
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Contributions

T.W. drafted the protocol on LV vector stock generation and designed figures. S.A. drafted the protocol on CAR T cell detection, and S.P. and F.F. drafted the protocol on mouse humanization. G.B. and J.H. contributed to the writing of the manuscript. C.J.B. and E.V. supervised work, drafted the general parts and revised the manuscript.

Corresponding authors

Correspondence to Els Verhoeyen or Christian J. Buchholz.

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Competing interests

E.V. and C.J.B are listed as inventors on patents on receptor-targeted LVs that have been licensed out. All other authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Bryan Strauss and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Additional initial assessment was performed by informal referee Stephen Gottschalk.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Pfeiffer, A. et al. EMBO Mol. Med. 10, e9158 (2018): https://doi.org/10.15252/emmm.201809158

Agarwal, S., Weidner, T., Thalheimer, F. B. & Buchholz, C. J. Oncoimmunology 8, e1671761 (2019): https://doi.org/10.1080/2162402X.2019.1671761

Agarwal, S. et al. Mol. Ther. 28, 1783–1794 (2020): https://doi.org/10.1016/j.ymthe.2020.05.005

Supplementary information

Reporting Summary

Supplementary Data 1

These scripts can be copied into the script panel of the NTA software. There are two options. The continuous flow script should be used when a syringe pump is in place (‘NTA_Script_Continous_Flow’), whereas the stop flow script (‘NTA_Script_Stop_Flow’) should be used in its absence.

Source data

Source Data Fig. 4

Data provided in Fig. 4D displayed as individual data points, showing titers (t.u./ml) of each LV stock obtained by incubation with target cells.

Source Data Fig. 5

Data shown in Fig. 5, B and C displayed as individual data points, showing the percentage of hCD45+ cells (B) and the percentage of CD19+ or CD3+ of hCD45+ cells (C) for each mouse and time point, respectively.

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Weidner, T., Agarwal, S., Perian, S. et al. Genetic in vivo engineering of human T lymphocytes in mouse models. Nat Protoc 16, 3210–3240 (2021). https://doi.org/10.1038/s41596-021-00510-8

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