Proteolytically Cleavable Chimeric Polypeptides for Local Gene Control
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Solution Overview
Problem
Existing inducible expression systems for controlling cellular behaviors and activities are often global and require user-input, lacking the ability to autonomously respond to environmental signals and modulate cellular processes with precision.
Innovation Solution
Development of chimeric polypeptides comprising an extracellular domain that binds to a peptide-MHC, a proteolytically cleavable Notch receptor polypeptide, and an intracellular domain that acts as a transcriptional activator or repressor, allowing for localized and signal-induced cleavage and release of the intracellular domain to modulate gene expression or cellular functions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional inducible expression systems are used to control cellular behaviors, then cellular activities can be modulated, but the systems affect cells globally and require user-provided input, lacking precision and autonomy
Solution Approach 1:
The chimeric polypeptide is divided into distinct functional domains: an extracellular domain for signal detection, a proteolytically cleavable Notch receptor polypeptide for signal transduction, and an intracellular domain for transcriptional activation. This segmentation allows the system to respond locally to specific environmental cues rather than globally, achieving precise control of cellular processes in target cells only.
Solution Approach 2:
The chimeric polypeptide system is autonomous in detecting environmental signals and transducing them into desired behavioral outputs without requiring continuous user input. The extracellular domain autonomously detects specific ligands, triggering proteolytic cleavage of the Notch receptor domain, which automatically releases the intracellular domain to modulate gene expression, creating a self-regulating system.
2Adaptability or versatility
If inducible expression systems require user-provided input to control cellular activities, then system activation can be controlled, but the ability to autonomously respond to environmental signals is lost
Solution Approach 1:
The extracellular domain of the chimeric polypeptide acts as an intermediary that detects specific environmental ligands and translates them into intracellular signaling events through proteolytic cleavage of the Notch receptor domain. This intermediary mechanism enables the system to autonomously respond to environmental cues while maintaining ease of operation through natural ligand binding events.
3Reliability
If global inducible systems are used to modulate cellular behaviors, then broad cellular control is achieved, but specificity to particular cells or processes is reduced
Solution Approach 1:
The chimeric polypeptide system exhibits local quality by confining its activity to specific cells that express the construct and encounter its ligand in the environment. The proteolytic cleavage and subsequent transcriptional activation occur only in these localized contexts, providing high specificity for particular cell types and processes while maintaining efficiency through targeted action rather than global modulation.
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, localized modulation of cellular processes and gene expression in response to specific environmental cues, enhancing the control and specificity of cellular engineering applications.
Implementation Method 1
a proteolytically cleavable Notch receptor polypeptide comprising one or more proteolytic cleavage sites; wherein binding of the specific binding member to the peptide-MHC induces 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.