Redox-Responsive Peptide Coacervates for Enhanced mRNA Delivery and Intracellular Release.

Area of Science:

• Biotechnology
• Materials Science
• Molecular Biology

Background:

• Efficient messenger RNA (mRNA) delivery is crucial for nucleic acid therapeutics but faces challenges with current lipid nanoparticle technology, including biosafety concerns and limited endosomal escape.
• Phase-separating peptides (PSPs) present a biocompatible alternative, yet often struggle with structural stability and controlled intracellular release.

Purpose of the Study:

• To develop a chemically defined, single-component coacervate system (HBpep-SS4) for enhanced mRNA delivery with intrinsic redox-responsiveness.
• To evaluate the system's capacity for encapsulating diverse RNA cargos, achieving efficient transfection, and enabling functional applications like genome editing.

Main Methods:

• Development of HBpep-SS4, a peptide designed with tandem cysteines for redox-responsiveness and coacervate formation.
• Encapsulation efficiency assessment for various RNA types, including self-amplifying RNAs.
• Transfection efficiency testing across multiple cell lines and functional validation through genome editing assays (EGFP disruption, HBB locus editing).
• Mechanistic studies on cellular uptake pathways and endosomal escape.

Main Results:

• HBpep-SS4 forms stable coacervates encapsulating >95% mRNA and exhibits glutathione-triggered cytosolic release.
• High transfection efficiency was achieved for linear, circular, and large self-amplifying RNAs across multiple cell lines.
• Successful genome editing was demonstrated, with 86.0% EGFP disruption and 72.5% HBB locus editing.
• Cellular uptake via phagocytosis and bypass of endosomal trafficking were observed, with disassembly in reductive environments without toxic byproducts.

Conclusions:

• HBpep-SS4 represents a minimalist, single-component peptide system with sequence-encoded environmental responsiveness for safe and efficient mRNA delivery.
• The system simplifies synthesis, enhances scalability, and shows significant potential for various RNA-based therapeutic applications.
• Primary sequence-encoded responsiveness offers a promising strategy for integrating structure, function, and controlled release in peptide-based delivery platforms.