Hook Protein System for Intracellular Trafficking Control
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Solution Overview
Problem
Current methods for controlling the secretion or cell membrane-anchoring of proteins, particularly therapeutic proteins like cytokines, face challenges in implementation, especially in primary cells, leading to difficulties in vivo and potential systemic side effects.
Innovation Solution
A polynucleotide system comprising a gene encoding a hook protein and a protein of interest, where the hook protein is fused with a cellular compartment-retention peptide and the protein of interest is fused with a hook protein-binding domain, allowing for controlled secretion or cell membrane-anchoring through independent transcription signals and a competing molecule interaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the RUSH system uses independent expression of hook and protein of interest with CMV promoter and IVS-IRES signal, then proper expression is achieved in various cell lines, but implementation fails in primary cells
Solution Approach 1:
The invention divides the expression control into two separate transcriptional units: one for the hook protein and another for the protein of interest. Each gene is under the control of its own transcription-activating signal, eliminating the dependency on IVS-IRES elements that fail in primary cells. This segmentation allows independent optimization of expression control for each component.
Solution Approach 2:
The invention changes the transcriptional control parameters by replacing the CMV promoter with tissue-specific or inducible promoters (such as albumin promoter for liver-specific expression, or tet-responsive promoter for doxycycline-inducible expression). This parameter change adapts the system to different cell types and experimental conditions, including primary cells where the original system failed.
2Quantity of substance
If protein of interest is systemically administered for therapeutic purposes, then therapeutic coverage is achieved, but systemic side effects occur
Solution Approach 1:
The invention implements local quality control by using tissue-specific promoters (e.g., albumin promoter for hepatocytes, keratin promoters for epithelial cells) to restrict protein of interest expression to specific target tissues. This ensures therapeutic proteins are produced only where needed, eliminating systemic circulation and associated side effects while maintaining effective local concentration.
Solution Approach 2:
The invention performs preliminary action by using inducible promoters (such as tet-responsive or steroid-responsive promoters) that allow precise temporal control of protein expression. The protein of interest is only expressed when the inducer is administered, enabling on-demand therapeutic production and immediate cessation by stopping the inducer, thus preventing unwanted systemic exposure and side effects.
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 modulation of protein secretion or anchoring, reducing systemic side effects and improving therapeutic efficacy by targeting specific cellular compartments.
Implementation Method 1
the hook protein has specific binding affinity for the hook protein-binding domain
Data Source
AI summary
The present invention relates to a polynucleotide comprising a gene encoding a hook protein and a gene encoding a protein of interest, said protein of interest being either a secretory protein or a cell membrane-anchored protein, wherein:said gene encoding the hook protein is under the control of a first transcription-activating signal,said gene encoding the protein of interest is under the control of a second transcription-activating signal, said second transcription-activating signal allowing a lower rate or frequency of transcription initiation than the first transcription-activating signal,said hook protein is fused to a cellular compartment-retention peptide, andsaid protein of interest is fused to a hook protein-binding domainIt also related to vectors comprising the polynucleotide, cells comprising the polynucleotide or the vector and compositions comprising the same. It further relates to methods and uses for modulating the secretion or cell membrane-anchorage of a protein of interest, or for preventing and/or treating a disease in a subject in need thereof.