Modulating expression of polypeptides via new gene switch expression systems
Inventors
SHAH, Rutul R. • Reed, Thomas D. • BOLINGER, Cheryl G.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11692194-B2
Publication Date
2023-07-04
Expiration Date
Abstract
Disclosed herein are polynucleotides encoding ligand-inducible gene switch polypeptides, and systems comprising gene switch polypeptides for modulating the expression of a heterologous gene and an interleukin in a host cell. The compositions, methods and systems described herein facilitate ligand dependent expression of polypeptides including but not limited to cytokines and antigen binding polypeptides.
Core Innovation
The invention provides ligand-inducible gene switch systems in which transcriptional control is achieved by split nuclear receptor-based transactivation and DNA-binding polypeptides. A first gene switch polypeptide includes a transactivation domain fused to a first ligand binding domain, and a second gene switch polypeptide includes a DNA binding domain fused to a second ligand binding domain. The first and second gene switch polypeptides are connected by a polypeptide linker.
The systems are configured so that the ligand binding domains control the activity of the transactivation domain and DNA binding domain, enabling ligand-inducible regulation of expression of a heterologous gene. The disclosed context includes ligand-inducible ON/OFF control with low basal expression, expression cassettes and promoters that can be ligand-inducible, and co-expression of heterologous genes including cytokines in host or effector cell contexts.
The invention is also described as a modified ecdysone two-hybrid gene switch system that provides improved sensitivity to non-steroidal ligands, including diacylhydrazines, compared with steroidal ligands. The systems are framed to increase transcription at lower ligand concentrations and to reduce side effects by eliminating native DNA-binding and transactivation domains and avoiding problems associated with overexpressed RXR.
Claims Coverage
Independent claim coverage is centered on a vector encoding two linked gene switch polypeptides with distinct transcriptional functions, plus sequence identity constraints to specified SEQ ID NOs. The inventive features present across the inputs are the dual-part fusion architecture, the polypeptide linker, and the at-least-90% identity requirements.
Dual-part gene switch polypeptide fusion architecture
A first gene switch polypeptide comprising a transactivation domain fused to a first ligand binding domain and a second gene switch polypeptide comprising a DNA binding domain fused to a second ligand binding domain.
Polypeptide linker connecting the gene switch polypeptides
The first and second gene switch polypeptides are connected by a polypeptide linker.
Sequence identity constraints to specified SEQ ID NOs
The first gene switch polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 183 and/or the second gene switch polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 187 or 188.
The claim scope focuses on a vector that encodes two gene switch polypeptides with a transactivation domain and a DNA binding domain, each fused to ligand binding domains, linked by a polypeptide linker, and constrained by at least 90% amino-acid identity to specified SEQ ID NOs.
Stated Advantages
Ligand-inducible ON/OFF control with tight low basal expression.
Improved sensitivity to non-steroidal ligands versus steroidal ligands.
Enables higher transcription at lower ligand concentrations.
Reduces side effects by eliminating native DNA-binding/transactivation domains and avoiding issues from overexpressed RXR.
Enhanced engineered cell survival upon ligand-induced mbIL-15 expression in the described context.
Repeatable ON→OFF→ON ligand cycling with low background.
Ligand-induced IL-12 expression in the described context to support engineered immune effector functionality.
Documented Applications
Gene-switch regulated immune effector cell engineering, including ligand-inducible regulation of transgenes using VELEDIMEX.
Co-expression systems in which a heterologous gene and cytokines are controlled using the ligand-inducible gene switch in response to ligand.
Cytokine payloads, including IL-15 and membrane-bound chimeric IL-15, expressed as heterologous genes in response to ligand.
Engineering of immune effector cells to express cytokines such as mbIL-15 and RTS/RTS-IL-12 under ligand-inducible control in the described context.
Use of the system context for repeated ligand-inducible cycling (ON→OFF→ON) with low background.
Engineered-cell functions, including CAR/TCR-associated payload regulation and dose-dependent behavior with ligand veledimex and ligand withdrawal effects.
Interested in licensing this patent?