Fusion Polypeptide Architecture for Sustained Gene Expression Control
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Solution Overview
Problem
Existing gene expression modulation technologies face challenges in achieving precise and sustained regulation of target gene activity, particularly in terms of duration and magnitude of expression changes.
Innovation Solution
Development of polypeptide molecules comprising specific polypeptide domains linked by a chain, where at least one domain is an endonuclease or gene modulator, such as Cas protein or transcriptional regulators like KRAB, DNMT, and EZH2, to form complexes that enhance target gene expression modulation, achieving significant and prolonged changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If conventional gene modulators are used to regulate target gene expression, then gene expression modulation is achieved, but the duration and magnitude of expression changes are insufficient
Solution Approach 1:
The patent combines multiple functional domains into a single polypeptide molecule: a guide sequence domain for target recognition, a Cas protein domain for DNA binding, and a gene modulator domain (such as KRAB, DNMT, EZH2, or ZNF689) for transcriptional regulation. This multi-domain fusion creates a unified molecular system that simultaneously achieves precise target localization and sustained epigenetic modification, resolving the contradiction between duration and precision by integrating both functions in one molecule.
Solution Approach 2:
The invention employs composite polypeptide structures that integrate different functional components with distinct properties. The guide sequence provides sequence-specific targeting, the Cas protein provides stable DNA binding capability, and the epigenetic modifier domains provide sustained transcriptional regulation. This composite architecture enables the system to maintain precise target recognition while achieving prolonged gene expression modulation through epigenetic mechanisms.
2Productivity
If gene modulators are used alone, then some expression change is achieved, but the magnitude and persistence of effect are limited compared to polypeptide complexes
Solution Approach 1:
The patent merges multiple gene modulator domains (such as KRAB for transcriptional repression, DNMT for DNA methylation, EZH2 for histone modification, and ZNF689 for chromatin remodeling) with the Cas protein and guide sequence into a single fused polypeptide. This integration allows the system to achieve greater magnitude of gene expression change through cooperative epigenetic mechanisms while maintaining a unified molecular structure that simplifies delivery and expression compared to using multiple separate components.
3Stability of the object's composition
If short polypeptide chains are used to link domains, then structural simplicity is maintained, but stability and sustained activity are reduced
Solution Approach 1:
The patent optimizes parameters of the polypeptide chain linking domains, specifically using extended linkers with lengths of 20-100 amino acids containing specific sequences (such as (GGGS)4 to (GGGS)10 repeats). This parameter optimization provides sufficient flexibility and spacing between functional domains while maintaining structural stability, allowing each domain to function independently yet cooperatively within the fused polypeptide structure.
Data Source
AI summary
The present disclosure provides polypeptide molecules comprising one or more polypeptide chains and systems, compositions, and methods of use thereof, wherein the polypeptide molecule comprising one or more polypeptide chains (or engineered polypeptide chain) can be used to effect regulation of a target gene in a cell (e.g., an endogenous target gene in a cell).