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Publication Number
US-10669549-B2
Publication Date
2020-06-02
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 provides a method of controlling a transgene in a primary T-cell comprising an anti-CD19 chimeric antigen receptor. A heterologous nucleic acid includes a promoter operably linked to the transgene and operably linked to an RNA degradation element, where the RNA degradation element is an AU rich element.
The method exposes the primary T-cell to CD19, where binding of CD19 by the chimeric antigen receptor activates the primary T-cell and changes a metabolic state of the primary T-cell. After the change in metabolic state, expressing the transgene results in an increased amount of polypeptide made from the transgene.
The document also describes RNA destabilizing elements in transcript regions, including 3UTR and 5UTR contexts, and multicistronic or bicistronic vector designs for expressing a CAR and transgene payloads under RDE or DE control. The transgene payloads described include cytokines, antibodies, checkpoint inhibitors, cytotoxics, and imaging reporters.
Claims Coverage
The provided partial claim set includes one independent claim. The independent claim centers on AU rich RDE-mediated control of transgene expression in an anti-CD19 CAR primary T-cell linked to activation-associated metabolic state changes, with increased payload expression after CD19 binding.
AU rich RDE-linked transgene in anti-CD19 CAR primary T-cell
A method of controlling a transgene using a primary T-cell comprising an anti-CD19 chimeric antigen receptor and a heterologous nucleic acid that includes a polynucleotide encoding a promoter operably linked to the transgene and operably linked to a RNA degradation element, where the RNA degradation element is an AU rich element and the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RNA degradation element.
CD19 binding activates metabolic state to increase transgene polypeptide expression
Exposing the primary T-cell to CD19 such that binding of CD19 by the chimeric antigen receptor activates the primary T-cell and changes a metabolic state of the primary T-cell, and expressing the transgene such that the amount of polypeptide made from the transgene is increased after the change in metabolic state.
The main inventive concept is AU rich RDE-mediated coupling of anti-CD19 CAR activation to a change in metabolic state, producing increased transgene polypeptide expression after CD19 binding.
Stated Advantages
Increases the amount of polypeptide made from the transgene after CD19 binding changes the metabolic state of the primary T-cell.
Achieves state-dependent transgene expression linked to CAR activation through an AU rich element RDE in the transgene transcript.
Documented Applications
Controlling transgene expression in anti-CD19 CAR primary T-cells using CD19 exposure to increase transgene-derived polypeptide expression after metabolic state changes.
Broad therapeutic use of RDE-controlled transgene payloads in immune-cell therapeutics.
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