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Publication Number
US-10323249-B2
Publication Date
2019-06-18
Expiration Date
Abstract
Control Devices are disclosed including RNA destabilizing elements (RDE), RNA control devices, and destabilizing elements (DE) combined with Chimeric Antigen Receptors (CARs) or other transgenes in eukaryotic cells. Multicistronic vectors are also disclosed for use in engineering host eukaryotic cells with the CARs and transgenes under the control of the control devices. These control devices can be used to optimize expression of 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 after stimulation of the CAR on the eukaryotic cell.
Core Innovation
The invention relates to a method for expressing a transgene in a primary T-cell that includes a chimeric antigen receptor. The method uses a heterologous nucleic acid encoding the transgene operably linked to a RNA destabilizing element (RDE), where the RDE is an AU rich element, and the RDE is bound by an RDE binding protein that regulates expression of the transgene transcript.
A primary T-cell is bound to a target ligand for the chimeric antigen receptor at a target site in a subject, such that ligand binding activates the primary T-cell. Primary T-cell activation changes a metabolic state and changes binding of the RDE by the RDE binding protein. The resulting expression program increases the amount of polypeptide made from the transgene after activation.
In described embodiments, RDE binding is connected to glycolysis, including regulation involving a glycolytic enzyme such as glyceraldehyde phosphate dehydrogenase. Activation of the primary T-cell is described as changing glycolysis and reducing binding of the RDE in the transcript by the RDE binding protein, with regulation of transcript half-life and/or translation through RDE binding protein effects, including optional use of a second RDE binding protein.
Claims Coverage
The only explicitly identified independent claim is clm-00001, which defines a method for expressing a transgene in a primary T-cell using a RNA destabilizing element (RDE) whose binding by an RDE binding protein is altered upon chimeric antigen receptor activation. Three inventive features are present.
RDE-linked transgene expression regulated by RDE binding protein
A primary T-cell comprising a chimeric antigen receptor and a heterologous nucleic acid encoding the transgene operably linked to an AU rich RNA destabilizing element (RDE), wherein the RDE binding protein binds to the RDE and regulates expression of the transgene.
CAR activation-driven metabolic state changes RDE binding
Binding the primary T-cell to a target ligand for the chimeric antigen receptor at a target site wherein activation changes a metabolic state of the primary T-cell and this changes binding of the RDE by the RDE binding protein.
Post-activation increase in transgene polypeptide output
Expressing the transgene so that the amount of polypeptide made from the transgene is increased after activation of the cell by the chimeric antigen receptor.
Across the claim set, coverage centers on RDE-mediated control of transgene expression in a CAR-bearing primary T-cell, where target-ligand activation changes metabolic state and alters RDE binding by an RDE binding protein to increase transgene polypeptide output after activation.
Stated Advantages
Increases the amount of polypeptide made from the transgene after activation of the cell by the chimeric antigen receptor.
Enables regulation of transgene expression via an RDE binding protein whose binding is changed by CAR-mediated metabolic state changes.
Documented Applications
Targeting and killing of AML cells using the method that includes killing an AML cell.
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