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 controlling expression of a transgene, comprising the steps of:
obtaining a primary T-cell comprising a chimeric antigen receptor, and a heterologous nucleic acid comprising a polynucleotide encoding the transgene that is operably linked to a polynucleotide encoding a RNA degradation element (RDE), wherein the RDE is an AU101 (Interferon gamma or IFNg) or an AU14 (IL6), wherein the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RDE, wherein an RDE binding protein binds to the RDE and regulates expression of the transgene;
exposing the primary T-cell to a ligand for the chimeric antigen receptor wherein binding of the ligand by the chimeric antigen receptor activates the primary T-cell and the primary T-cell proliferates, and the activation of the primary T-cell changes binding of the RDE by the RDE binding protein; and
expressing the transgene wherein the transgene has increased levels of expression after the activation of the primary T-cell compared to the level of expression of the transgene before activation of the primary T-cell.
2. The method of claim 1 wherein the 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, or a checkpoint inhibitor.
3. The method of claim 1 , wherein the transgene encodes an IL-2, and IL-12, an IL-15, an IL-18 or a TNF-α.
4. The method of claim 3 , wherein the transgene encodes an IL-2.
5. The method of claim 3 , wherein the transgene encodes an IL-12.
6. The method of claim 3 , wherein the transgene encodes an IL-15.
7. The method of claim 3 , wherein the transgene encodes an IL-18.
8. The method of claim 3 , wherein the transgene encodes a TNF-α.
9. The method of claim 2 , wherein the transgene encodes an anti-4-1BB antibody.
10. The method of claim 1 , wherein the transgene encodes a CD40L.
11. The method of claim 1 , wherein the transgene encodes a Hsp60 or a Hsp70.
12. The method of claim 11 , wherein the transgene encodes a Hsp60.
13. The method of claim 11 , wherein the transgene encodes a Hsp70.
14. The method of claim 1 , wherein the chimeric antigen receptor is an anti-DLL3 chimeric antigen receptor.
15. The method of claim 14 , wherein the ligand is a DLL3 found on a target cell.
16. The method of claim 15 , wherein the target cell is a tumor cell.
17. The method of claim 16 , wherein the tumor cell is a small cell lung cancer cell.
18. The method of claim 16 , wherein the tumor cell is a melanoma cell.
19. The method of claim 16 , wherein the tumor cell is an IDH1 mutant glioma cell.