Large-scale combined car transduction and crispr gene editing of t cells
AssigneeBoard of Regents, The University of Texas System
InventorsKaty REZVANI, Rafet BASAR, Elif GOKDEMIR, Mayra SHANLEY
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Abstract
Embodiments of the disclosure encompass methods and compositions for producing engineered T cells. The disclosure concerns large-scale processes for producing T cells that may be engineered to have disruption of expression of one or more genes using CRISPR and also express at least one heterologous antigen receptor. Specific embodiments include particular parameters for the process. The T cells may or may not be viral-specific.
Claims41
An in vitro method of producing engineered T cells, comprising the steps of, in any order:
- (a) optionally exposing T cells from a mixture of cells to negative or positive selection to enrich the T cells;
- (b) expanding for a first time period T cells with an effective amount of one or more of interleukin (IL)-2, IL-4, IL-7, IL-12, IL-15, IL-21 and/or one or more other T-cell tropic cytokines (s);
- (c) delivering to the T cells an effective amount of Cas9 or CpF1, and one or more guide RNAs to disrupt expression of one or more endogenous genes in the T cells;
- (d) modifying the T cells to express one or more heterologous antigen receptors and/or one or more heterologous cytokines;
- and
- (e) expanding the T cells in the presence of one or more viral antigens and optionally one or more cytokines to produce viral-specific cells.
An in vitro method of producing engineered viral-specific T cells, comprising the steps of:
- (a) exposing peripheral blood mononuclear cells to first virus-specific mixture of peptide antigens;
- (b) stimulating for a first time period the cells with one or more cytokines specific for stimulation of T cells for the first virus to produce stimulated viral-specific T cells;
- and
- (c) delivering to the stimulated viral-specific T cells an effective amount of Cas9 and one or more guide RNAs to disrupt expression of one or more endogenous genes in the cells, thereby producing gene edited viral-specific T cells.
Dependencies are inferred from the claim text.