Modular EV Protein Loading Construct Against ADAM10 Cleavage
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for loading proteins into engineered extracellular vesicles (EVs) face limitations in loading density and are susceptible to enzymatic cleavage, particularly by ADAM10, which affects the efficiency and stability of the loaded proteins.
Innovation Solution
A nucleic acid construct comprising an effector module, scaffold module, transmembrane domain module, and intracellular domain anchoring module, connected by a linker peptide, which enhances protein loading efficiency and stability by utilizing specific transmembrane domain sequences and scaffold sequences to protect against enzyme cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods are used to load proteins into EVs, then the loading process is simple, but the loading density is low and the proteins are susceptible to enzymatic cleavage
Solution Approach 1:
The nucleic acid construct is divided into distinct functional modules: effector module, scaffold module, transmembrane domain module, and intracellular domain anchoring module. Each module performs a specific function, allowing independent optimization of protein loading density and protection against enzymatic cleavage while maintaining overall system functionality.
Solution Approach 2:
The scaffold module is nested within the nucleic acid construct structure, with the scaffold sequence positioned to protect the effector module from enzymatic cleavage. The transmembrane domain module is nested within the membrane structure, enabling integrated protection and loading functions.
2Reliability
If scaffold sequences are used to protect against ADAM10 cleavage, then protein stability is improved, but the construct complexity increases
Solution Approach 1:
The scaffold module is designed with specific sequences that preemptively protect the effector module from ADAM10 cleavage. The scaffold sequence is positioned and structured to block enzyme access before cleavage can occur, providing protective action in advance rather than reacting after damage occurs.
Solution Approach 2:
The scaffold module acts as an intermediary element between the effector module and the external environment. It provides a protective interface that prevents direct exposure of the effector module to enzymatic cleavage while maintaining the functional integrity of the loaded protein.
Data Source
AI summary
Provided are a nucleic acid construct for achieving modular loading of an engineered EVs functional protein and use of said construct. A modular design principle is adopted, specific modules are selected for combination, a mutant sequence is optimized and screened to obtain an improved nucleic acid construct, and finally, the engineered EVs modularly loaded with functional protein is converted and expressed. The product has the following advantages that: I) the modular loading of the engineered EVs functional protein is achieved; 2) the loading density and the loading efficiency of the protein inside and outside an EVs membrane are improved; and 3) a specific support sequence module is selected to avoid enzyme digestion. The construct is used to prepare the engineered EVs, so that the modular loading of the functional protein can be achieved, the loading efficiency is improved, and the construct has a wide clinical application value and market prospect.