Modulating expression of polypeptides via new gene switch expression systems
Inventors
SHAH, Rutul R. • Reed, Thomas D. • BOLINGER, Cheryl G.
Assignees
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Publication Number
US-11946054-B2
Publication Date
2024-04-02
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 a method for integrating heterologous gene(s) into the genome of peripheral blood mononuclear cells. The method delivers a non-naturally occurring polynucleotide that comprises an AttP or AttB recombination sequence together with heterologous gene(s), and additionally delivers a polynucleotide encoding a serine recombinase selected from SF370, SPβc2, Bxb1, A118, or φRv1. This configuration supports targeted genome integration in PMBC using an AttP or AttB recombination sequence paired with a specific serine recombinase.
The heterologous gene(s) include genes encoding a chimeric antigen receptor and a cell tag. The cell tag is defined as an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 190-195 and 197-200. The chimeric antigen receptor therefore is provided as part of a heterologous gene set that includes both functional receptor activity and an associated cell tag for the engineered PMBC.
The disclosed embodiments specify refinements to the delivered nucleic acid components and to the payload composition. These refinements include delivering the AttP/AttB polynucleotide and the serine recombinase polynucleotide in a single vector and using constrained selections for downstream payload elements such as cytokines and defined fusion protein concepts. The described payload regulation concept uses ligand-inducible gene-switch components and inducible promoter control to enable ON-OFF cycling and dose-response behavior for heterologous expression.
Claims Coverage
The provided independent claim coverage centers on PMBC genome integration using an AttP or AttB-linked polynucleotide delivered together with a polynucleotide encoding one of specific serine recombinases, with payload-defined constraints for a chimeric antigen receptor and a cell tag. The inventive features include delivering AttP/AttB-linked heterologous genes and delivering a polynucleotide encoding a selected serine recombinase, while defining the cell tag by sequence-identity to specified SEQ IDs.
AttP or AttB recombination sequence with heterologous gene(s) for genome integration in PMBC
Delivering to the peripheral blood mononuclear cell a non-naturally occurring polynucleotide comprising an AttP or AttB recombination sequence and the heterologous gene(s).
Serine recombinase selected from SF370, SPβc2, Bxb1, A118, or φRv1
Delivering a polynucleotide encoding a SF370, SPβc2, Bxb1, A118, or φRv1 serine recombinase.
Heterologous genes encoding a chimeric antigen receptor and a cell tag
Wherein the heterologous gene(s) encode a chimeric antigen receptor and a cell tag comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 190-195 and 197-200.
Overall, the claim coverage centers on PMBC genome integration using an AttP or AttB-linked heterologous gene construct together with delivery of a specified serine recombinase, with the payload constrained to include a chimeric antigen receptor and a defined cell tag sequence identity threshold.
Stated Advantages
Improved sensitivity for non-steroidal diacylhydrazine ligands, with higher transcription at lower ligand concentration.
Reduced side effects versus unmodified RXR.
Documented Applications
Engineered cells where a ligand-inducible gene switch modulates expression of heterologous genes including cytokines, interleukins, and cell tag concepts.
CAR/TCR-related payload context including chimeric antigen receptor and kill-switch cell tag concepts such as HER1t and CD20 tag concepts.
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