Engineered Polypeptides for EV Protein Loading
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Solution Overview
Problem
Current methods for bioengineering extracellular vesicles (EVs) face challenges in efficiently incorporating bioengineered proteins into EVs, leading to heterogeneous sorting efficiencies and low loading densities, which hampers their therapeutic and diagnostic applications.
Innovation Solution
Development of polypeptides comprising an ephrin receptor cysteine-rich domain, fibronectin type III domains, and a transmembrane domain, lacking ephrin binding or kinase activity, which can be used to create targeted extracellular vesicles or hybridosomes for enhanced cargo loading and delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If endogenous EV-marker proteins or commonly used sorting proteins are overexpressed to enable protein sorting into EVs, then protein loading into EVs is achieved, but sorting efficiency becomes heterogeneous and loading density remains low
Solution Approach 1:
The patent modifies the sorting protein by fusing it to a transmembrane anchor domain and signal sequence, fundamentally changing its localization and sorting parameters. This engineered construct directs the protein to the plasma membrane and enables its incorporation into EVs during budding, achieving both high loading density and homogeneous sorting efficiency that endogenous markers cannot provide
2Adaptability or versatility
If EV surface is customized with tissue targeting ligands and pharmaceutical agents are loaded, then therapeutic targeting capability is improved, but the complexity of EV production and purification increases
Solution Approach 1:
The patent divides the sorting protein into functional modules: a signal sequence for ER localization, a transmembrane anchor domain for membrane insertion, and a cargo-binding domain for protein recruitment. This modular design enables independent optimization of each function and simplifies the engineering of targeted EVs with specific targeting ligands and cargo combinations
Solution Approach 2:
The engineered transmembrane sorting protein serves multiple functions simultaneously: it acts as a membrane anchor, a sorting signal, and a cargo-recruitment platform. This multi-functionality reduces the need for multiple separate components, thereby simplifying the overall EV production system while maintaining high targeting capability
3Measurement precision
If GFP-fusion proteins of EV markers are overexpressed to track protein sorting, then sorting visualization is achieved, but sorting efficiency becomes highly heterogeneous despite similar expression levels
Solution Approach 1:
The patent introduces an engineered transmembrane sorting protein as an intermediary that mediates cargo sorting into EVs. This intermediary protein, with its defined transmembrane anchor and cargo-binding domain, provides a consistent and controllable sorting mechanism that eliminates the heterogeneous sorting observed with endogenous markers, while still allowing GFP-fusion proteins to serve as effective visualization tools
Data Source
AI summary
The present disclosure pertains to polypeptides (in particular, polypeptides comprising Eph receptor domain(s), i.e., Eph receptor-derived polypeptides), nanovesicles (e.g., extracellular vesicles (EVs) and hybridosomes) comprising such polypeptides. Said polypeptides can act as membrane bound protein scaffolds to which molecules of interest can be attached. The polypeptides and nanovesicles can be used in targeting, therapeutic and/or diagnostic applications. Also provided are nucleic acids and expression vectors encoding such polypeptides as well as cells expressing said polypeptides. Further provided are methods for producing nanovesicles comprising such polypeptides. Compositions comprising such polypeptides or nanovesicles as well as their uses are also described.