Coordinating gene expression using RNA destabilizing elements
Control Devices are disclosed including RNA destabilizing elements (RDE), and RNA control devices, combined with transgenes, including Chimeric Antigen Receptors (CARs) in eukaryotic cells. RDEs can be combined with RNA control devices to make RDEs that include ligand mediated control. These smart RDEs and other RDEs can be used to optimize expression of transgenes, e.g., CARs, in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered at desired times from the eukaryotic cell.
1. A method of expressing a plurality of transgenes, comprising the steps of: obtaining a primary T-cell comprising a receptor, a first heterologous nucleic acid comprising a polynucleotide encoding a first transgene, and a polynucleotide encoding a first RNA destabilizing element (RDE), wherein the first RDE is an AU rich element, a second heterologous nucleic acid comprising a polynucleotide encoding a second transgene, and a polynucleotide encoding a second RDE, wherein the second RDE is an AU rich element, wherein the first heterologous nucleic acid is transcribed to make a first transcript encoding the first transgene operably linked to the first RDE, wherein a glycolytic enzyme with RDE binding activity-binds to the first RDE and regulates expression of the first transgene, and the second heterologous nucleic acid is transcribed to make a second transcript encoding the second transgene operably linked to the second RDE, wherein the glycolytic enzyme with RDE binding activity-binds to the second RDE and regulates expression of the second transgene;
and binding the receptor on the primary T-cell to a ligand wherein binding of the ligand by the receptor activates the primary T-cell and the primary T-cell increases glycolytic activity whereby the glycolytic enzyme with RDE binding activity catalyzes a step of glycolysis, wherein activation of glycolysis in the primary T-cell reduces the amount of the glycolytic enzyme with RDE binding activity available to bind the first RDE and reduces the amount of the glycolytic enzyme with RDE binding activity available to bind the second RDE which produces increased expression of the first and second transgenes.
2. The method of claim 1 , wherein the first transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a reporter, or a checkpoint inhibitor.
3. The method of claim 2 , wherein the second transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a reporter, or a checkpoint inhibitor.
4. The method of claim 1 , wherein a polypeptide encoded by the first transgene is secreted from the primary T-cell.
5. The method of claim 4 , wherein the secreted polypeptide acts on a target cell.
6. The method of claim 4 , wherein a polypeptide encoded by the second transgene is secreted from the primary T-cell.
7. The method of claim 6 , wherein the secreted polypeptide acts on a target cell.
8. The method of claim 6 , wherein the target cell is a cancer cell or a bacterium.
9. The method of claim 8 , further comprising the step of killing the target cell.
10. The method of claim 1 , wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 5 or an AU 21.
11. The method of claim 1 , wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 19 or an AU 22.
12. The method of claim 1 , wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 3, an AU 20, an AU 10, an AU 7, or an AU 23.
13. The method of claim 1 , wherein the receptor is a chimeric antigen receptor or a T-cell receptor.