Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 157, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/60400
Keywords
Bioengineering; Issue 157; CAR-T cells; transduction; expansion; retrovirus; PBMC; central memory
Categories
Funding
- NIH [5R01AI096966-06S1, 1UM1AI26617, P51OD011106/P51RR000167]
- MN REACH grant [5U01HL127479-03, 1R01A143380-01, 1UM14126617]
- NIAID Division of Intramural Research
- NIH Intramural AIDS Targeted Antiviral Program
- NIH Nonhuman Primate Reagent Resource [R24 OD010976, U24 AI126683]
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Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.
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