Coordinating gene expression using RNA destabilizing elements
Inventors
Wang, Benjamin • Zeiner, Gusti • McNally, Krista
Assignees
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Publication Number
US-10688132-B2
Publication Date
2020-06-23
Expiration Date
Abstract
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.
Core Innovation
The invention relates to expressing a plurality of transgenes in a primary T-cell that comprises a chimeric antigen receptor and ligand-triggerable activation. Separate heterologous nucleic acids encode each transgene operably linked to a polynucleotide encoding an RNA destabilizing element (RDE). The RDEs are specified as AU 101 (Interferon gamma or IFNg) or an AU14 (IL6), and each RDE is bound by an RDE binding protein to regulate expression of its corresponding transgene.
Upon binding of the target ligand by the chimeric antigen receptor, the primary T-cell is activated and the T-cell proliferates. Activation changes binding of the RDE binding protein to each AU 101 or AU14 RDE, thereby changing regulation of transgene expression. The method provides increased levels of expression of the transgenes after activation compared to the level of expression before activation.
The approach supports control of multiple transgenes using different RDEs and RDE binding proteins within the same T-cell, thereby enabling coordinated expression of more than one payload after ligand-triggered CAR activation. The disclosed constructs and regulation are described as ligand- and/or cell-state–dependent RNA control devices that tune expression dynamics in eukaryotic immune cells, including CAR T cells, using AU-rich RDEs and their associated RDE binding proteins.
Claims Coverage
The independent claim is clm-00001. It covers a ligand-triggered CAR T-cell transgene expression method using two distinct RDE-containing transgene transcripts and RDE binding proteins whose binding changes upon CAR activation, resulting in increased transgene expression after activation.
Ligand-activated CAR T cells with AU 101 or AU14 RDE-controlled transgenes
A method that obtains a primary T-cell comprising a chimeric antigen receptor, a first heterologous nucleic acid encoding a first transgene operably linked to a first RNA destabilizing element (RDE) that is an AU 101 (IFNg) or an AU14 (IL6), and a second heterologous nucleic acid encoding a second transgene operably linked to a second RDE that is an AU 101 (IFNg) or an AU14 (IL6), wherein each RDE binding protein binds to its respective RDE and regulates expression.
CAR ligand binding changes RDE binding by RDE binding proteins
Binding the primary T-cell to a target ligand for the chimeric antigen receptor at a target site in a subject activates the primary T-cell and the T-cell proliferates, and wherein the activation changes binding of the first RDE by the first RDE binding protein and binding of the second RDE by the second RDE binding protein.
Increased transgene expression after CAR activation
Expressing the first and second transgenes such that the first and second transgenes have increased levels of expression after activation of the primary T-cell compared to the level of expression before activation of the primary T-cell.
Independent claim clm-00001 is focused on ligand-triggered CAR activation of a primary T-cell containing multiple transgenes, each operably linked to AU 101 (IFNg) or AU14 (IL6) RNA destabilizing elements, where RDE binding proteins change their binding after CAR activation to produce increased post-activation transgene expression.
Stated Advantages
Increased levels of expression of the first and second transgenes after activation of the primary T-cell compared to before activation.
Documented Applications
Therapeutic use of RDE-controlled CAR T cells (and related CAR T-cell configurations) with therapeutic payload/transgene expression tied to receptor-triggered metabolic state, including expression of viral payloads/oncolytic viruses.
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