Development of Mesenchymal Stem Cell Membrane-Enveloped Nanovesicles for Enhanced Gene Delivery

Summary

This protocol aims to introduce a method to produce and purify mesenchymal stem cell (MSC)-derived extracellular vesicle biomimetics for gene therapy. The nanovesicle is confined with MSC-derived lipid bilayers and encapsulates recombinant AAVs carrying the gene of interest in the lumen. This nanovesicle offers an enhanced vector for in vivo gene delivery.

Abstract

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) hold great promise for therapeutic applications and regenerative medicine. EVs are nanoscale vesicles secreted by all known cell types, carrying diverse cargos including membrane-anchored proteins, soluble factors, multiple RNA species, and metabolites that regulate the physiology and behavior of recipient cells. While MSC-derived or engineered EVs can deliver therapeutic proteins and RNAs, EV-mediated DNA delivery remains challenging due to the lack of efficient mechanisms for sorting DNA sequences into vesicles. Previous work from our group and others demonstrated that adeno-associated virus (AAV)-containing EVs enable targeted nuclear delivery and sustained gene expression in vitro and in vivo. However, their production and isolation have been limited by low yield and time-intensive procedures. Here, we report the development of MSC membrane-enveloped nanovesicles generated by a size-defined extrusion method for efficient gene delivery. These vesicles, approximately 200 nm in diameter, mimic the properties of natural EVs while encapsulating recombinant AAV vectors carrying therapeutic gene sequences. Compared with conventional AAVs, the engineered MSC vesicles improved gene delivery efficiency and achieved significantly higher yields with reduced time and cost relative to naturally secreted EV-AAVs. In summary, we present a novel MSC-based membrane nanovesicle platform that combines the advantages of EV-mimicking structures with AAV-mediated gene transfer. This approach enhances delivery efficiency and production scalability, offering a promising strategy to advance gene therapy toward clinical translation.

Introduction

Gene therapy represents a transformative approach for treating genetic and acquired diseases by introducing, replacing, or silencing specific genes to restore normal cellular function. Over the past two decades, advances in vector engineering and delivery strategies have significantly expanded the therapeutic landscape of gene therapy. Among the various vector systems developed, adeno-associated virus (AAV) has emerged as one of the most successful and widely adopted platforms due to its favorable safety profile, efficient transduction, and long-term gene expression in both dividing and non-dividing cells^1,2. ....

Protocol

All animal procedures were approved by the institutional animal care and use committee of Beijing Normal University at Zhuhai, China. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation and formation of CME-AAV via pore size-defined extrusion

1. Production of recombinant AAV vectors
   ​NOTE: The AAV production protocol follows previously published and widely used procedures. Detailed optimization steps and variations have been described in prior studies^12,26.
   1. Culture HEK293T cells in complete grow....

Results

MSCs used in this study were isolated from human umbilical cord tissue under protocols in accordance with relevant ethical guidelines. MSC membrane fractions were prepared according to the described protocol. AAV9.EGFP was produced in-house using a transient dual-plasmid transfection system in HEK293T cells, as previously reported^12,26. For CME-AAV fabrication, membrane preparations, and AAV9.EGFP was co-incubated at a ratio of 150 µg of membrane protein per 1 × .......

Discussion

Critical steps of the CME-AAV protocol

This protocol describes a method for generating MSC membrane-enveloped AAVs, and several steps are critical for successful CME-AAV formation. First, careful preprocessing of the cell membrane is essential. The goal of this step is to obtain membrane fragments that are suitable for extrusion while minimizing contamination from nuclear DNA, cytosolic proteins, and subcellular organelles. In addition, the size of membrane fragments is a key .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bioluminescence imaging system	PerkinElmer	IVIS Spectrum optical imaging system (Lumina III)
D-Luciferin potassium salt	Yeasen Biotechnology	40902ES03
Fetal bovine serum	Yeasen Biotechnology	40130ES76
Fluorescence microscope	Zeiss	Axio Observer 7
Formvar/carbon-coated copper grid 200 mesh	Millipore-Sigma	TEM-FCF400CU
Glutaraldehyde	Millipore-Sigma	354400
HBSS	Gibco	C14175500BT
Hieff Unicon qPCR TaqMan Probe Master Mix	Yeasen Biotechnology	11205ES08
High-glucose Dulbecco's modified Eagle's medium	Hyclone	SH30243
High-speed centrifuge	Beckman Coulter	Avanti J-E
Hoechst 33342	Beyotime	C1022
NTA instrument	Malvern Panalytical	NanoSight NS300
Open-top thinwall ultra-clear tube for ultracentrifuge	Beckman Coulter	344059
OptiPrep iodixanol	Millipore-Sigma	D1556
Paraformaldehyde 4%	Beyotime	P0099
PBS	Biosharp	BL302A
Penicillin-streptomycin	Millipore-Sigma	V900929
qPCR instrument	Applied Biosystems	QS6 real-time PCR system
Rotor for high-speed centrifuge	Beckman Coulter	JA-25.50
SW 41 Ti Swing-bucket rotor for ultracentrifuge	Beckman Coulter	331362
Transmission electron microscope	JEOL	JEM-2100F
Ultracentrifuge	Beckman Coulter	Optima XE-100
Uranyl acetate	Electron Microscopy Sciences	NC1375332
Whatman Nuclepore polycarbonate membranes filters (200 nm)	Cytiva	10417004
Whatman Nuclepore polycarbonate membranes filters (400 nm)	Cytiva	10417104