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Method Article
Els Verhoeyen
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; Université Côte d’Azur, Institut national de la santé et de la recherche médicale, Centre Méditerranéen de Médecine Moléculaire, Nice, France
Corresponding Author
Christian J. Buchholz
Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany; Division for Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Receptor-targeting of vector particles is a key technology for cell-type specific in vivo gene delivery. Lentiviral vectors (LVs) targeted to human T cell markers enable genetic modification of T cells directly in humanized mouse models. Illustrating the example of modifying T cells with chimeric antigen receptors (CARs), we provide detailed protocols for the generation and quality check of potent CD4- and CD8-targeted LVs, the humanization of immunodeficient mice as well as the administration of the vector stocks followed by monitoring for in vivo modified T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. A few weeks after administration, about 10% of T cells can be expected to convert to CAR T cells. The protocol can be easily adapted to LVs targeted to other types of receptors and/or delivering other genes of interest.
Keywords
T lymphocytes, genetic engineering, lentiviral vectors, T cells, chimeric antigen receptors, CARs, humanized mouse model, T cell targeting, in vivo gene delivery, receptor targeting
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Associated Publications
In vivo generation of human CD 19‐ CAR T cells results in B‐cell depletion and signs of cytokine release syndrome
by Anett Pfeiffer, Frederic B Thalheimer, Sylvia Hartmann, +9
EMBO Molecular Medicine (17 September, 2018)
In vivo generated human CAR T cells eradicate tumor cells
by Shiwani Agarwal, Tatjana Weidner, Frederic B. Thalheimer, +1
OncoImmunology (11 October, 2019)
In Vivo Generation of CAR T Cells Selectively in Human CD4+ Lymphocytes
by Shiwani Agarwal, Julia D.S. Hanauer, Annika M. Frank, +3
Molecular Therapy (15 May, 2020)
Method Article
Els Verhoeyen
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; Université Côte d’Azur, Institut national de la santé et de la recherche médicale, Centre Méditerranéen de Médecine Moléculaire, Nice, France
Corresponding Author
Christian J. Buchholz
Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany; Division for Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Dec, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Receptor-targeting of vector particles is a key technology for cell-type specific in vivo gene delivery. Lentiviral vectors (LVs) targeted to human T cell markers enable genetic modification of T cells directly in humanized mouse models. Illustrating the example of modifying T cells with chimeric antigen receptors (CARs), we provide detailed protocols for the generation and quality check of potent CD4- and CD8-targeted LVs, the humanization of immunodeficient mice as well as the administration of the vector stocks followed by monitoring for in vivo modified T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. A few weeks after administration, about 10% of T cells can be expected to convert to CAR T cells. The protocol can be easily adapted to LVs targeted to other types of receptors and/or delivering other genes of interest.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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Associated Publications
In vivo generation of human CD 19‐ CAR T cells results in B‐cell depletion and signs of cytokine release syndrome
by Anett Pfeiffer, Frederic B Thalheimer, Sylvia Hartmann, +9
EMBO Molecular Medicine (17 September, 2018)
In vivo generated human CAR T cells eradicate tumor cells
by Shiwani Agarwal, Tatjana Weidner, Frederic B. Thalheimer, +1
OncoImmunology (11 October, 2019)
In Vivo Generation of CAR T Cells Selectively in Human CD4+ Lymphocytes
by Shiwani Agarwal, Julia D.S. Hanauer, Annika M. Frank, +3
Molecular Therapy (15 May, 2020)