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Publication Number
US-10323248-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 controlling a transgene in a primary T-cell by coupling transgene expression to cellular metabolic state changes. A primary T-cell is obtained comprising a receptor and a heterologous nucleic acid that contains a polynucleotide encoding the transgene operably linked to a polynucleotide encoding an RNA degradation element (RDE), and the RDE is an AU rich element. The heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RDE.
The primary T-cell is exposed to a ligand for the receptor, and binding of the ligand by the receptor activates the primary T-cell and thereby changes a metabolic state of the primary T-cell. The system is configured so that expressing the transgene results in an amount of polypeptide made from the transgene that is increased after the change in metabolic state of the primary T-cell.
The disclosed framework links AU-rich RDE-mediated RNA degradation to metabolic activation and regulation of transcript stability and effector output, in connection with controlled regulation of transgene expression. Related concepts include RNA destabilizing elements localized within RNA features such as UTRs or introns, engineered to alter binding of RNA-binding proteins such as HuR or glycolytic/energy enzymes such as GAPDH.
Claims Coverage
The provided claims center on one independent claim covering a primary T-cell system with an AU rich RDE coupled to ligand-activated metabolic state changes, and increased transgene polypeptide expression after that change. The broader claim material also mentions dependent refinements for receptor type, payload classes, reporter imaging, and RDE regulation by additional binding proteins.
Au-rich rde operably linked to transgene transcript in a primary t-cell
A primary T-cell comprising a receptor is used with a heterologous nucleic acid comprising a polynucleotide encoding the transgene operably linked to a polynucleotide encoding an RNA degradation element (RDE), where the RDE is an AU rich element, and the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RDE.
Ligand-induced receptor activation that changes metabolic state
The primary T-cell is exposed to a ligand for the receptor, wherein binding of the ligand by the receptor activates the primary T-cell and thereby changes a metabolic state of the primary T-cell.
Transgene polypeptide expression increases after metabolic state change
The transgene is expressed such that the amount of polypeptide made from the transgene is increased after the change in metabolic state of the primary T-cell.
T-cell receptor activation in the primary t-cell
The receptor is a T-cell receptor.
Therapeutic transgene payload classes
The heterologous nucleic acid encoding the transgene includes specified therapeutic protein types, including cytokines, FasL, antibodies, growth factors, chemokines, polypeptide- or polysaccharide-cleaving enzymes, granzymes, perforin, or checkpoint inhibitors.
Reporter imaging at a ligand-expressing target site
A reporter is imaged at a target site expressing the ligand.
Rde regulation by binding glycolytic enzyme
The RDE in the transcript is bound by a glycolytic enzyme selected from the listed group.
Second rde-binding protein increases transcript half-life
A second RDE binding protein increases the half-life of the transcript associated with the RDE.
The claim coverage combines an AU rich RDE linked to a transgene-encoding transcript in a primary T-cell, ligand-dependent receptor activation that changes metabolic state, and increased transgene polypeptide output after that change. Dependent features add T-cell receptor use, therapeutic payload classes, reporter imaging, and RDE regulation through glycolytic enzyme binding or a second RDE-binding protein that increases transcript half-life.
Stated Advantages
Increases the amount of polypeptide made from the transgene after the change in metabolic state of the primary T-cell.
Mitigates toxicity while maintaining effective target-cell killing.
Documented Applications
Engineering primary T-cells with a CAR and transgene payload for disease targeting, including antigens for AML/DLBCL (e.g., CD33, CD19).
Use of reporters that can be imaged at a target site expressing the ligand.
Payload delivery and controlled transgene expression are described, including RDE-controlled second transgenes.
Therapeutic uses across cancer, inflammation, and infection are described.
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