Proteolytically Cleavable Chimeric Polypeptides for Localized Gene Control
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Solution Overview
Problem
Existing inducible expression systems for controlling cellular behaviors and activities often require user-provided inputs and affect cells globally, lacking the ability to autonomously respond to specific environmental signals.
Innovation Solution
Development of chimeric polypeptides comprising an extracellular domain that binds to peptide-MHC, a proteolytically cleavable Notch receptor, and an intracellular domain that acts as a transcriptional activator or repressor, allowing for localized and specific modulation of cellular processes upon binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If conventional inducible expression systems are used to control cellular behaviors, then cellular activities can be modulated, but the systems require user-provided inputs and affect cells globally rather than providing autonomous localized control
Solution Approach 1:
The synNotch receptor system enables cells to autonomously detect environmental peptide-MHC signals and self-regulate gene expression without external user input. The chimeric polypeptide structure combines the binding member, Notch extracellular domain, and intracellular domain in a single autonomous unit that responds automatically to target cell signals
Solution Approach 2:
The invention merges the binding member, Notch extracellular domain, transmembrane domain, and intracellular domain into a single chimeric polypeptide structure. This consolidation creates an integrated system that simultaneously performs signal detection, signal transduction, and gene regulation functions
2Measurement precision
If conventional inducible expression systems are used, then cellular activities can be controlled, but the control is global rather than localized to specific cells or regions
Solution Approach 1:
The synNotch receptor system provides localized control by enabling individual cells to respond specifically to peptide-MHC signals present in their immediate environment. Each cell expressing the chimeric polypeptide autonomously detects and responds to local signals, creating spatially restricted gene expression patterns rather than global effects
Solution Approach 2:
The invention replaces conventional mechanical or chemical induction systems with a biological recognition system based on peptide-MHC binding. The specific binding member recognizes target peptide sequences presented by MHC molecules, enabling highly specific cell-to-cell communication and localized response activation
3Reliability
If chimeric polypeptides with proteolytically cleavable Notch receptor are used, then precise autonomous regulation of cellular functions is achieved, but the polypeptide structure becomes more complex
Solution Approach 1:
The chimeric polypeptide is segmented into distinct functional domains: an N-terminal binding member for signal recognition, a Notch extracellular domain with proteolytic cleavage sites for signal transduction, a transmembrane domain for membrane anchoring, and a C-terminal intracellular domain for gene regulation. This segmentation allows each domain to perform its specific function while maintaining overall system integration
Solution Approach 2:
The chimeric polypeptide is pre-configured with proteolytically cleavable sites in the Notch extracellular domain, preparing the system for rapid activation upon signal binding. The structure is designed in advance to undergo conformational changes and cleavage events that trigger immediate gene expression responses
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise and autonomous regulation of cellular functions, such as gene expression and immune responses, by using chimeric polypeptides that respond to specific binding partners, enhancing targeted cellular control and treatment efficacy.
Implementation Method 1
binding of the specific binding member to the peptide-MHC
Implementation Method 2
cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites
Data Source
AI summary
The instant disclosure provides chimeric polypeptides which modulate various cellular processes following a cleavage event induced upon binding of a specific binding member of the polypeptide with its binding partner. Methods of using chimeric polypeptides to modulate cellular functions, including e.g., induction of gene expression, are also provided. Nucleic acids encoding the subject chimeric polypeptides and associated expression cassettes and vectors as well as cells that contain such nucleic acids and/or expression cassettes and vectors are provided. Also provided, are methods of treating a subject using the described components and methods as well as kits for practicing the subject methods.