Genetic in vivo engineering of human T lymphocytes in mouse models

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. This protocol describes production of lentiviral vectors targeted to receptors present on specific cell types, humanization of mice, administration of the lentiviral vectors and detection of the presence of transduced cell types.