Genetically-modified immune cells comprising a microrna-adapted shrna (shrnamir)
AssigneePrecision BioSciences, Inc.
InventorsAaron Martin, Jon E. Chatterton, Michelle Brenda Pires
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Abstract
The present invention encompasses genetically-modified immune cells (and populations thereof) expressing a microRNA-adapted shRNA (shRNAmiR) that reduces the expression of a target endogenous protein. Methods for reducing the expression of an endogenous protein in an immune cell are also provided wherein the method comprises introducing a shRNAmiR that targets the endogenous protein. Using shRNAmiRs for knocking down the expression of a target protein allows for stable knockdown of expression of endogenous proteins in immune cells.
Claims372
A genetically-modified immune cell comprising in its genome a nucleic acid sequence encoding a microRNA-adapted shRNA (shRNAmiR), wherein said shRNAmiR is expressed in said genetically-modified immune cell, and wherein said shRNAmiR reduces expression of a target protein in said genetically-modified immune cell.
The genetically-modified immune cell of any one of claims 83-85, wherein said nucleic acid sequence encoding said shRNAmiR is in a reverse orientation as said nucleic acid sequence encoding said CAR or exogenous TCR.
The genetically-modified immune cell of any one of claims 42-442, wherein said genetically-modified immune cell comprises within its genome a cassette comprising, from 5′ to 3′:
- (a) a first promoter;
- (b) said nucleic acid sequence encoding said CAR or said exogenous TCR, wherein an intron sequence is positioned within said nucleic acid sequence encoding said CAR or said exogenous TCR, and wherein said nucleic acid sequence encoding said shRNAmiR is positioned within said intron sequence;
- (c) optionally a first termination signal;
- (d) a second promoter;
- (e) said nucleic acid sequence encoding said HLA-E fusion protein;
- and
- (f) optionally a second termination signal;
- wherein said nucleic acid sequence encoding said CAR or said exogenous TCR and said nucleic acid sequence encoding said shRNAmiR are operably linked to said first promoter, and wherein said nucleic acid sequence encoding said HLA-E fusion protein is operably linked to said second promoter.
A method for reducing the expression of an endogenous protein in an immune cell, said method comprising introducing into said immune cell a template nucleic acid comprising a nucleic acid sequence encoding a shRNAmiR, wherein said template nucleic acid is inserted into the genome of said immune cell and said shRNAmiR is expressed, and wherein said shRNAmiR reduces expression of an endogenous target protein in said immune cell.
Dependencies are inferred from the claim text.