Genetic in vivo engineering of human T lymphocytes in mouse models

Abstract

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.

Early induction of cytokine release syndrome by rapidly generated CAR T cells in a preclinical mouse model

“…The protocol for production and characterization of VSV-LV stocks was adapted from previously. 31 VSV-LV contained the transfer plasmid pS-CD19-CAR-W encoding a second generation CD19-CAR with a CD28 and CD3z signaling domain under control of the SFFV promotor. In brief, 2.5 × 10 7 β2M -/- , CD47 high HEK293T cells were seeded in a T175 flask 24 h before transfection with 17.5 μg of packaging plasmid pCMVdR8.9, 11.4 μg of transfer plasmid pSEW-mycCD19.CAR(28z) and 6.1 μg of envelope plasmid pMDG-2.…”

Potential roles of hyaluronic acid inin vivoCAR T cell reprogramming for cancer immunotherapy

“…Conjugation of anti-CD3 to vectors can prime circulating T cells, but in much lower activity than pre-administration of soluble anti-CD3 antibody [102]. Pretreatment can be an injection of anti-CD3 or CD28 monoclonal antibodies or exogenous IL-2, IL-7 or IL -15 [102,105,115]. Furthermore, the expression of CD44 receptors in T cells after activation increases [85], which could be beneficial for the uptake of HA-decorated nanoparticles.…”

“…102 Pretreatment can be an injection of anti-CD3 or CD28 monoclonal antibodies or exogenous IL-2, IL-7 or IL-15. 102,105,115 Furthermore, the expression of CD44 receptors in T cells after activation increases, 85 which could be beneficial for the uptake of HAdecorated nanoparticles.…”