Name: Development of Mesenchymal Stem Cell Membrane-Enveloped Nanovesicles for Enhanced Gene Delivery (Video) | JoVE Journal
Uploaded: 2026-02-17T14:40:00.000+00:00
Duration: 10 min
Description: Learn to prepare MSC membrane-enveloped nanovesicles using extrusion for enhanced AAV gene delivery. This method improves yield and efficiency over conventional approaches for therapeutic applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The work was supported by Guangdong S&#38;T Program (2023B0303010002) to DM, and Guangdong Basic and Applied Basic Research Foundation (2025A1515011134) and BNU Faculty of Arts and Sciences Cross-disciplinary Research Project (12900-311324240581) to YL. This work was further supported by the instrumentation and technical expertise provided by the Instrumentation and Service Center for Science and Technology at Beijing Normal University.

<ol><li>Wang, D., Tai, P. W. L., Gao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adeno-associated+virus+vector+as+a+platform+for+gene+therapy+delivery%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nat Rev Drug Discov. 18 (5), 358-378 (2019).</li>
<li>Li, C., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+adeno-associated+virus+vectors+for+gene+therapy%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Engineering adeno-associated virus vectors for gene therapy.</a> Nat Rev Genet. 21 (4), 255-272 (2020).</li>
<li>Mendell, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+clinical+applications+of+in+vivo+gene+therapy+with+AAVs%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Current clinical applications of in vivo gene therapy with AAVs.</a> Mol Ther. 29 (2), 464-488 (2021).</li>
<li>Crunkhorn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profiling+aav+tropism%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Profiling aav tropism.</a> Nat Rev Drug Discov. 22 (3), 183(2023).</li>
<li>Kosaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+loading+capacity+and+patterns+of+packaged+DNA+in+AAV+genomes+of+different+sizes+using+long-read+sequencing%5BTitle%5D)+AND+%22Mol+Ther+Methods+Clin+Dev%22%5BJournal%5D)">Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing.</a> Mol Ther Methods Clin Dev. 33 (2), 101474(2025).</li>
<li>McCarty, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-complementary+AAV+vectors%3B+advances+and+applications%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Self-complementary AAV vectors; advances and applications.</a> Mol Ther. 16 (10), 1648-1656 (2008).</li>
<li>Lopez-Gordo, E., Chamberlain, K., Riyad, J. M., Kohlbrenner, E., Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+adeno-associated+virus+serotypes+and+engineered+adeno-associated+virus+capsid+variants%3A+Tropism+differences+and+mechanistic+insights%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Natural adeno-associated virus serotypes and engineered adeno-associated virus capsid variants: Tropism differences and mechanistic insights.</a> Viruses. 16 (3), 442(2024).</li>
<li>Louis Jeune, V., Joergensen, J. A., Hajjar, R. J., Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pre-existing+anti-adeno-associated+virus+antibodies+as+a+challenge+in+AAV+gene+therapy%5BTitle%5D)+AND+%22Hum+Gene+Ther+Methods%22%5BJournal%5D)">Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy.</a> Hum Gene Ther Methods. 24 (2), 59-67 (2013).</li>
<li>Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+adeno-associated+virus-9-neutralizing+antibody+in+Chinese+patients+with+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Prevalence of adeno-associated virus-9-neutralizing antibody in Chinese patients with Duchenne muscular dystrophy.</a> Hum Gene Ther. 35 (1-2), 26-35 (2024).</li>
<li>Directed evolution of AAV mutants for enhanced gene delivery. Schaffer, D. V., Maheshri, N. Conf Proc IEEE Eng Med Biol Soc, 2004, 3520-3523 (2004).</li>
<li>Zhao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+sialylated+N-glycans+on+adeno-associated+virus+capsids+for+targeted+gene+delivery+and+therapeutic+applications%5BTitle%5D)+AND+%22J+Control+Release%22%5BJournal%5D)">Engineering sialylated N-glycans on adeno-associated virus capsids for targeted gene delivery and therapeutic applications.</a> J Control Release. 380, 563-578 (2025).</li>
<li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aav-containing+exosomes+as+a+novel+vector+for+improved+gene+delivery+to+lung+cancer+cells%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Aav-containing exosomes as a novel vector for improved gene delivery to lung cancer cells.</a> Front Cell Dev Biol. 9, 707607(2021).</li>
<li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicle-encapsulated+adeno-associated+viruses+for+therapeutic+gene+delivery+to+the+heart%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Extracellular vesicle-encapsulated adeno-associated viruses for therapeutic gene delivery to the heart.</a> Circulation. 148 (5), 405-425 (2023).</li>
<li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravitreal+injection+of+an+exosome-associated+adeno-associated+viral+vector+enhances+retinoschisin+1+gene+transduction+in+the+mouse+retina%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Intravitreal injection of an exosome-associated adeno-associated viral vector enhances retinoschisin 1 gene transduction in the mouse retina.</a> Hum Gene Ther. 32 (13-14), 707-716 (2021).</li>
<li>Breuer, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+engineering+of+lymphocytes+after+systemic+exosome-associated+AAV+delivery%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery.</a> Sci Rep. 10 (1), 4544(2020).</li>
<li>Cheng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+aspects+of+extracellular+vesicle-associated+AAV+allows+increased+vector+yield+and+insight+into+its+transduction+and+immune-evasive+properties%5BTitle%5D)+AND+%22Mol+Ther+Methods+Clin+Dev%22%5BJournal%5D)">Probing aspects of extracellular vesicle-associated AAV allows increased vector yield and insight into its transduction and immune-evasive properties.</a> Mol Ther Methods Clin Dev. 33 (1), 101407(2025).</li>
<li>Elmore, Z. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+membrane-associated+accessory+protein+is+an+adeno-associated+viral+egress+factor%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">The membrane-associated accessory protein is an adeno-associated viral egress factor.</a> Nat Commun. 12 (1), 6239(2021).</li>
<li>Deshetty, U. M., Sil, S., Buch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+therapy+for+the+heart%3A+Encapsulated+viruses+to+the+rescue%5BTitle%5D)+AND+%22Extracell+Vesicles+Circ+Nucl+Acids%22%5BJournal%5D)">Gene therapy for the heart: Encapsulated viruses to the rescue.</a> Extracell Vesicles Circ Nucl Acids. 5 (1), 114-118 (2024).</li>
<li>Fan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+repair+of+spinal+cord+injury+based+on+miR-124-3p-loaded+mesoporous+silica+camouflaged+by+stem+cell+membrane+modified+with+rabies+virus+glycoprotein%5BTitle%5D)+AND+%22Adv+Sci+%28Weinh%29%22%5BJournal%5D)">Targeted repair of spinal cord injury based on miR-124-3p-loaded mesoporous silica camouflaged by stem cell membrane modified with rabies virus glycoprotein.</a> Adv Sci (Weinh). 11 (21), e2309305(2024).</li>
<li>Yao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembly+of+stem+cell+membrane-camouflaged+nanocomplex+for+microRNA-mediated+repair+of+myocardial+infarction+injury%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Self-assembly of stem cell membrane-camouflaged nanocomplex for microRNA-mediated repair of myocardial infarction injury.</a> Biomaterials. , 257-120256 (2020).</li>
<li>Tian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomimetic+mesenchymal+stem+cell+membrane-coated+nanoparticle+delivery+of+mkp5+inhibits+hepatic+fibrosis+through+the+Ire%2Fxbp1+pathway%5BTitle%5D)+AND+%22J+Nanobiotechnology%22%5BJournal%5D)">Biomimetic mesenchymal stem cell membrane-coated nanoparticle delivery of mkp5 inhibits hepatic fibrosis through the Ire/xbp1 pathway.</a> J Nanobiotechnology. 22 (1), 741(2024).</li>
<li>Kang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exosomes+secreted+from+CXCR4+overexpressing+mesenchymal+stem+cells+promote+cardioprotection+via+akt+signaling+pathway+following+myocardial+infarction%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Exosomes secreted from CXCR4 overexpressing mesenchymal stem cells promote cardioprotection via akt signaling pathway following myocardial infarction.</a> Stem Cells Int. 2015, 659890(2015).</li>
<li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+in+treating+human+diseases%3A+Molecular+mechanisms+and+clinical+studies%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Mesenchymal stem cells in treating human diseases: Molecular mechanisms and clinical studies.</a> Signal Transduct Target Ther. 10 (1), 262(2025).</li>
<li>Ong, S. G., Chitneni, M., Lee, K. S., Ming, L. C., Yuen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+extrusion+technique+for+nanosizing+liposomes%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Evaluation of extrusion technique for nanosizing liposomes.</a> Pharmaceutics. 8 (4), 36(2016).</li>
<li>Tackenberg, M. W., Kleinebud, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Encapsulation+of+liquids+via+extrusion--A+review%5BTitle%5D)+AND+%22Curr+Pharm+Des%22%5BJournal%5D)">Encapsulation of liquids via extrusion--A review.</a> Curr Pharm Des. 21 (40), 5815-5828 (2015).</li>
<li>Bai, J. -J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dean-flow-coupled+elasto-inertial+focusing+accelerates+exosome+purification+to+facilitate+single+vesicle+profiling%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Dean-flow-coupled elasto-inertial focusing accelerates exosome purification to facilitate single vesicle profiling.</a> Anal Chem. 95 (4), 2523-2531 (2023).</li>
<li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomimetic+gene+delivery+system+coupled+with+extracellular+vesicle-encapsulated+AAV+for+improving+diabetic+wound+through+promoting+vascularization+and+remodeling+of+inflammatory+microenvironment%5BTitle%5D)+AND+%22J+Nanobiotechnology%22%5BJournal%5D)">Biomimetic gene delivery system coupled with extracellular vesicle-encapsulated AAV for improving diabetic wound through promoting vascularization and remodeling of inflammatory microenvironment.</a> J Nanobiotechnology. 23 (1), 242(2025).</li>
<li>Smith, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+equivalence+of+stem+cell+and+stem+cell-derived+extracellular+vesicle+transplantation+to+repair+the+irradiated+brain%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">Functional equivalence of stem cell and stem cell-derived extracellular vesicle transplantation to repair the irradiated brain.</a> Stem Cells Transl Med. 9 (1), 93-105 (2020).</li>
<li>Liang, Y., Lehrich, B. M., Zheng, S., Lu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+methods+in+biomarker+identification+for+extracellular+vesicle-based+liquid+biopsy%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Emerging methods in biomarker identification for extracellular vesicle-based liquid biopsy.</a> J Extracell Vesicles. 10 (7), e12090(2021).</li>
<li>Verma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seroprevalence+of+adeno-associated+virus+neutralizing+antibodies+in+males+with+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Seroprevalence of adeno-associated virus neutralizing antibodies in males with Duchenne muscular dystrophy.</a> Hum Gene Ther. 34 (9-10), 430-438 (2023).</li>
<li>Yan, B., Liang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+therapeutics+for+extracellular+vesicles%3A+Delivering+CRISPR+for+cancer+treatment%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">New therapeutics for extracellular vesicles: Delivering CRISPR for cancer treatment.</a> Int J Mol Sci. 23 (24), 15758(2022).</li>
<li>Hu, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythrocyte+membrane-camouflaged+polymeric+nanoparticles+as+a+biomimetic+delivery+platform%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform.</a> Proc Natl Acad Sci U S A. 108 (27), 10980-10985 (2011).</li>
<li>Aryal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythrocyte+membrane-cloaked+polymeric+nanoparticles+for+controlled+drug+loading+and+release%5BTitle%5D)+AND+%22Nanomedicine+%28Lond%29%22%5BJournal%5D)">Erythrocyte membrane-cloaked polymeric nanoparticles for controlled drug loading and release.</a> Nanomedicine (Lond). 8 (8), 1271-1280 (2013).</li>
<li>Zhang, W., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell+membrane-camouflaged+targeted+delivery+system+in+tumor%5BTitle%5D)+AND+%22Mater+Today+Bio%22%5BJournal%5D)">Stem cell membrane-camouflaged targeted delivery system in tumor.</a> Mater Today Bio. 16, 100377(2022).</li>
</ol>

Dr. Daopin Wu is an employee of Guangdong Hengqin United Life Science Co., Ltd. The company did not provide funding for this study and had no role in study design, data collection, data analysis, manuscript preparation, or the decision to publish. The remaining authors declare no competing financial interests.

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 ...

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 cells1,2. To date, multiple AAV-based therapeutics have received regulatory approval for clinical use, including treatments for spinal muscular atrophy, retinal dystrophies, and hemophilia, marking important milestones in translational medicine3. The broad tropism and low immunogenicity of AAV vectors have also enabled their application across a variety of tissues, including the liver, muscle, central nervous system, and eye4. As a result, AAV has become a leading platform for in vivo gene delivery, supporting ongoing preclinical and clinical studies targeting metabolic, neurological, and cardiovascular disorders.
Despite its remarkable clinical success, AAV-based gene therapy faces several intrinsic challenges that continue to limit its broader application. One major constraint lies in the limited genome packaging capacity of the AAV capsid (~4.7 kb), which restricts its use for delivering large or multiple therapeutic genes5,6. In addition, the biodistribution and cellular tropism of AAV vectors are largely determined by their serotypes, each exhibiting distinct affinities for specific tissues. Although extensive capsid engineering and peptide display strategies have been employed to enhance tissue targeting and transduction efficiency, achieving precise and predictable tropism across diverse biological contexts remains a significant hurdle7. Another critical limitation is the prevalence of pre-existing neutralizing antibodies (NAbs) against natural AAV serotypes in the human population. Even low titers of NAbs can substantially reduce vector transduction efficiency, limiting patient eligibility and complicating repeated dosing8,9. Various strategies, such as rational capsid redesign, immunosuppressive regimens, and transient plasma exchange, have been explored to mitigate antibody-mediated neutralization10,11, yet these approaches offer only partial or transient relief. Collectively, these limitations reduce the overall therapeutic efficacy and reproducibility of AAV-mediated gene delivery in clinical settings.
In recent years, extracellular vesicle-enveloped AAV (EV-AAV) has been explored as an alternative vector with the potential to overcome several key limitations of conventional AAVs12,13,14,15. During vector packaging, a portion of AAV particles can be secreted through vesicular trafficking pathways and become enclosed within extracellular vesicles. This process is believed to involve AAV-encoded membrane-associated accessory protein (MAAP), which facilitates viral egress and promotes membrane association16,17. The resulting EV-AAV particles exhibit improved transduction efficiency and enhanced resistance to neutralizing antibodies compared with conventional AAV, primarily due to the vesicular membrane that promotes cellular uptake and provides partial shielding from antibody recognition18. However, despite these advantages, EV-AAV production remains inefficient. Typically, only a small fraction of vector genomes (approximately 0.5%-12%) are secreted into the culture medium as EV-associated AAVs, while the majority remain intracellular or exist as free AAVs12,13. Consequently, large-scale production is required to obtain sufficient vector yield. Furthermore, EV-AAV generation has been primarily demonstrated using HEK293T producer cells, whose EVs may exhibit rapid hepatic clearance, short circulation half-life, and potential biosafety concerns. These limitations highlight the need for a more efficient and customizable membrane-envelopment strategy, motivating the development of the cell membrane-enveloped AAV (CME-AAV) platform presented in this study.
Mesenchymal stem cells (MSCs) and their EVs have been extensively studied for their regenerative potential and immune-evasive properties. Beyond therapies based on cells or naturally secreted EVs, recent studies have demonstrated an alternative approach for enhanced drug delivery using nanoparticles cloaked with MSC membranes19,20,21. The MSC membrane coating confers active targeting capability, reduces immune clearance, and prolongs circulation time, of which effects are likely attributed to the intrinsic molecular composition of MSC-derived membranes22,23. Building on this concept, we utilized MSC-derived membrane material to construct a cell membrane-enveloped AAV (CME-AAV) nanovesicle for gene delivery. To achieve this, purified MSC membrane fragments were mixed with recombinant AAV particles and subjected to a controlled extrusion process, a technique originally established for liposome fabrication24,25. Through size-defined extrusion, the membrane components spontaneously assemble into nanoscale vesicles encapsulating AAV particles. The resulting CME-AAVs were further purified from free, unencapsulated AAVs using density gradient-based centrifugation. This strategy enables efficient and reproducible preparation of membrane-enveloped AAV vectors, which exhibit markedly enhanced transduction efficiency compared with conventional AAVs and maintain comparable resistance to neutralizing antibodies as EV-AAVs. By substituting different transgenes, the CME-AAV platform holds broad potential for the treatment of various genetic and acquired diseases.

<table><tbody><tr><td>Bioluminescence imaging system</td><td>PerkinElmer</td><td>IVIS Spectrum optical imaging system (Lumina III)&amp;nbsp;</td><td></td></tr><tr><td>D-Luciferin potassium salt</td><td>Yeasen Biotechnology</td><td>40902ES03</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Yeasen Biotechnology</td><td>40130ES76</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss&amp;nbsp;</td><td>Axio Observer 7</td><td></td></tr><tr><td>Formvar/carbon-coated copper grid 200 mesh</td><td>Millipore-Sigma</td><td>TEM-FCF400CU</td><td></td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>Millipore-Sigma</td><td>354400</td><td></td></tr><tr><td>HBSS&amp;nbsp;</td><td>Gibco</td><td>C14175500BT</td><td></td></tr><tr><td>Hieff Unicon qPCR TaqMan Probe Master Mix</td><td>Yeasen Biotechnology</td><td>11205ES08</td><td></td></tr><tr><td>High-glucose Dulbecco&#039;s modified Eagle&#039;s medium</td><td>Hyclone</td><td>SH30243</td><td></td></tr><tr><td>High-speed centrifuge</td><td>Beckman Coulter</td><td>Avanti J-E</td><td></td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Beyotime</td><td>C1022</td><td></td></tr><tr><td>NTA instrument</td><td>Malvern Panalytical</td><td>NanoSight NS300&amp;nbsp;</td><td></td></tr><tr><td>Open-top thinwall ultra-clear tube for ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>OptiPrep iodixanol&amp;nbsp;</td><td>Millipore-Sigma</td><td>D1556</td><td></td></tr><tr><td>Paraformaldehyde 4%</td><td>Beyotime</td><td>P0099</td><td></td></tr><tr><td>PBS</td><td>Biosharp</td><td>BL302A</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Millipore-Sigma</td><td>V900929</td><td></td></tr><tr><td>qPCR instrument</td><td>Applied Biosystems&amp;nbsp;</td><td>QS6 real-time PCR system&amp;nbsp;</td><td></td></tr><tr><td>Rotor for high-speed centrifuge</td><td>Beckman Coulter</td><td>JA-25.50&amp;nbsp;</td><td></td></tr><tr><td>SW 41 Ti Swing-bucket rotor for ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Transmission electron microscope&amp;nbsp;</td><td>JEOL</td><td>JEM-2100F&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima XE-100</td><td></td></tr><tr><td>Uranyl acetate&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>NC1375332</td><td></td></tr><tr><td>Whatman Nuclepore polycarbonate membranes filters (200 nm)</td><td>Cytiva</td><td>10417004</td><td></td></tr><tr><td>Whatman Nuclepore polycarbonate membranes filters (400 nm)</td><td>Cytiva</td><td>10417104</td><td></td></tr></tbody></table>

development of mesenchymal stem cell membrane-enveloped nanovesicles for enhanced gene delivery

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

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.

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 reported12,26. For CME-AAV fabrication, membrane preparations, and AAV9.EGFP was co-incubated at a ratio of 150 µg of membrane protein per 1 × ...

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.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) hold great promise for therapeutic applications and regenerative medicine. EVs are ...

In this protocol, we introduce a method to prepare mesenchymal stem cell-derived nanovesicles, which resemble extracellular vesicles and encapsulate AAVs for improved gene delivery. We call this AAV-containing nanovesicles CME-AAVs. Compared with the emerging gene delivery vector EV-AAV, CME-AAVs offer greater flexibility in cell sources and higher yield.To begin, remove the culture medium from a culture dish containing human mesenchymal stem cells at 70 to 90%confluency. Using a cell scraper, detach the cells from the culture dish and transfer them into a conical tube containing PBS. Centrifuge the collected cells at 300g for five minutes at four degrees Celsius to pellet the cells.Then, wash the cell pellet twice with PBS and centrifuge again. Resuspend the final cell pellet in PBS supplemented with a protease inhibitor cocktail. Then, subject the cell suspension to three freeze-thaw cycles to disrupt the plasma membrane and generate a crude lysate.Using a pipette, homogenize the lysate thoroughly. Then, centrifuge at 1, 000g for 20 minutes at four degrees Celsius to remove cellular debris. Transfer the supernatant to a new tube.Centrifuge at 18, 000g for one hour at four degrees Celsius to pellet the membrane fraction. Discard the supernatant and resuspend the membrane pellet in PBS to obtain the crude mesenchymal stem cell membrane fraction. Now, homogenize the membrane suspension by sequentially passing it through syringes fitted with 20 to 27 gauge needles.Determine the protein concentration of the homogenized membrane suspension using a bicinchoninic acid assay. Aliquot the suspension and store at minus 80 degrees Celsius until needed. Re-homogenize the suspension using a 27 gauge syringe after each thaw cycle.Mix the prepared membrane suspension with adeno-associated virus, or AAVs, at a ratio of 150 micrograms protein per one times 10 to the power of 10 genome copies. Incubate the mixture at 37 degrees Celsius for 30 minutes with gentle shaking. Load the membrane and virus mixture into a mini extruder fitted with a 400 nanometer polycarbonate membrane.Pass the mixture through the extruder 20 times to promote encapsulation. Replace the 400 nanometer membrane with a 200 nanometer membrane and repeat extrusion. Collect the process suspension containing encapsulated virus, along with unencapsulated virus and empty vesicles.Pool multiple batches, if required, and proceed immediately to purification. Dilute the stock iodixanol solution with 10x PBS and sterile deionized water to prepare 40 and 25%solutions. Using a pipette, add one milliliter of 40%iodixanol to the bottom of a 13.2 milliliter open-top thin-wall ultra-clear tube.Then, overlay three milliliters of 25%iodixanol above the 40%layer to form a discontinuous gradient. Gently overlay seven milliliters of the processed suspension onto the top of the gradient. Balance the tubes and load them into a swing bucket ultracentrifuge rotor.Centrifuge at 150, 000g for three hours at four degrees Celsius, using fast acceleration and slow deceleration. Then, carefully remove the tubes without disturbing the gradient. Collect one milliliter fractions from top to bottom into labeled 1.5 milliliter microcentrifuge tubes.Keep all fractions on ice. Now, identify and pool fractions seven and eight containing purified CME-AAVs into a new open-top ultracentrifuge tube. Make up the volume to 11 milliliters with PBS.Then, gently mix with a one milliliter pipette. Using fast acceleration and deceleration, centrifuge the pooled fractions at 150, 000g for three hours at four degrees Celsius. Discard the supernatant carefully.Resuspend the pellet in 50 to 200 microliters of sterile PBS, depending on the desired concentration. Aliquot the purified preparation. Then, store at 80 degrees Celsius until further use.Prepare working solutions of encapsulated virus and control virus in sterile PBS. Add equal genome copies of encapsulated virus or control virus directly to the culture medium in each well. Include negative control wells with an equivalent volume of PBS or empty virus particles.Gently swirl the plate to ensure uniform distribution and incubate for 24 to 72 hours, depending on experimental design. Next, gently aspirate the culture medium. Wash the cells twice with PBS without dislodging them.Stain nuclei with Hoechst 33342 according to the manufacturer's instructions. Perform live-cell fluorescence imaging using an inverted fluorescence microscope with appropriate filter sets for enhanced green fluorescent protein and Hoechst. Select 10x or 20x objectives for quantification and 63x objective for high-resolution imaging, if needed.Acquire at least five fields of view per well, using identical imaging parameters. Save all images in raw and grayscale formats for quantitative analysis. Transmission electron microscopy revealed that CME-AAVs exhibited size dimensions and membrane-bound structures similar to extracellular vesicles.The mesenchymal stem cell membrane markers, CD73 and CD90, were detected in CME-AAV by Western blot analysis. The three major adeno-associated virus capsid proteins, VP1, VP2, and VP3, were identified in CME-AAV by SDS-PAGE. NanoSight analysis showed that CME-AAV9.EGFP had a mean diameter of 128.6 nanometers and a total particle concentration of 1.32 times 10 to the power of 10 particles per milliliter. Fluorescence microscopy demonstrated that CME-AAV9. EGFP produced a markedly higher level of enhanced green fluorescent protein expression in HEK293T cells.The membrane-enveloped virus achieved approximately 2.5-fold higher gene expression, suggesting enhanced cellular uptake and potentially improved intracellular trafficking. In vivo bioluminescence imaging showed that the liver exhibited the strongest luminescent signal following CME-AAV administration. Quantitative analysis of liver regions revealed that CME-AAV administration resulted in significantly higher gene expression levels.These protocols describe steps to obtain purified cell membrane-enveloped AAVs, or CME-AAV, via extrusion and density gradient ultracentrifugation. Two key factors require careful consideration:Preprocessing cell fragments for extrusion and optimizing membrane fragment-to-AAV mixing ratio to control CME-AAV particle-to-genome ratio. Future studies can further explore cell membrane enclosure to enhance immune protection and functional surface properties, optimizing CME-AAV as a versatile platform for AAV delivery.

development-mesenchymal-stem-cell-membrane-enveloped-nanovesicles-for

Research

JoVE Journal

Bioengineering

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

Here, we present a gene therapy approach that delivers ABE-coated chitosan directly to the bone marrow by intraosseous injection.

Enhanced Gene Delivery and Expression using Intraosseous Injection of Chitosan Nanoparticles Encapsulated Adenine Base Editor Plasmids

Learn to prepare MSC membrane-enveloped nanovesicles using extrusion for enhanced AAV gene delivery. This method improves yield and efficiency over conventional approaches for therapeutic applications.

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

Explore More Videos