开发间充质干细胞膜包纳米囊泡以增强基因传递
2026年2月17日
本方案旨在引入一种生产和纯化间充质干细胞（MSC）来源的细胞外囊泡仿生物的方法，用于基因治疗。纳米囊泡被MSC来源的脂质双层包裹，并包裹携带目标基因的重组AAVs。该纳米囊泡为 体内 基因递送提供了增强载体。
在本方案中，我们介绍了一种制备间充质干细胞来源纳米囊泡的方法，这些囊泡类似于胞外囊泡，并包裹AAVs以提升基因递送效率。我们称含AAV的纳米囊泡为CME-AAVs。与新兴的基因递送载体EV-AAV相比，CME-AAV在细胞来源上提供了更大的灵活性和更高的产率。
首先，从含有人类间充质干细胞的培养皿中取出培养基，浓度为70%至90%。使用细胞刮刀将细胞从培养皿中取出，转移到含有PBS的锥形管中。将收集的细胞以300克离心机，在四摄氏度下离心五分钟，以沉淀细胞。
然后用PBS清洗细胞沉淀两次，再用离心机清洗。将最终细胞沉淀剂重新置于PBS中，并补充蛋白酶抑制剂混合物。然后，对细胞悬浮液进行三次冻融循环，破坏质膜并生成粗糙的裂解液。
使用移液管彻底均质裂解液。然后，以1000克离心机，温度4摄氏度20分钟，去除细胞残渣。把上清液转移到新的管子里。
在18,000克离心机温度下，四摄氏度，离心一小时，以沉淀膜分馏。丢弃上清液，将膜沉淀重新悬浮于PBS中，获得粗糙的间充质干细胞膜分馏。现在，通过依次通过装有20到27号针头的注射器来均匀化膜悬浮液。
利用双戒戊酸测定均质膜悬浮液的蛋白质浓度。将悬挂液分开，储存在零下80摄氏度，直到需要时再使用。每次解冻后用27号注射器重新均质悬浮液。
将预备好的膜悬浮液与腺相关病毒（AAVs）混合，比例为150微克蛋白质每1乘以10倍基因组拷贝的幂次方。将混合物在37摄氏度下孵育30分钟，轻轻摇晃。将膜和病毒混合物装入装有400纳米聚碳酸酯膜的迷你挤出机中。
将混合物通过挤出机20次以促进封装。将400纳米的膜替换为200纳米的膜，重复挤出。收集含有包裹病毒的工艺悬浮液，以及未包膜病毒和空囊泡。
如有需要，将多批分组合并，并立即进行净化。将原装碘沙醇溶液用10倍PBS和无菌去离子水稀释，制备40%和25%溶液。用移液管在13.2毫升开顶薄壁超透明管底部加入一毫升40%碘沙醇。
然后在40%层上叠加3毫升25%碘沙醇，形成不连续梯度。轻轻地将7毫升处理过的悬浮液覆盖在梯度顶部。平衡管子，然后装入摆动桶超速离心机转子。
离心机在150,000克下，温度四摄氏度，持续三小时，采用快速加速和慢减速。然后，小心地取出管子，同时不扰动梯度。从上到下收集一毫升分馏液，放入标记为1.5毫升的微型离心管中。
把所有分数都冷藏起来。现在，识别并将含有纯化CME-AAV的第七和第八组分，汇入一个新的开顶超离心管中。用PBS把体积调节到11毫升。
然后，用一毫升的移液器轻轻混合。利用快速加速和减速，将聚集的组分以150,000克、四摄氏度离心三小时。小心地丢弃上清液。
根据所需浓度，将沉淀重新悬浮于50至200微升的无菌PBS中。把纯净的制剂分开。然后在80摄氏度下储存，等待进一步使用。
在无菌PBS中制备包装病毒和控制病毒的工作溶液。将包膜病毒或对照病毒的基因组副本直接添加到每个孔的培养基中。包括含有等量PBS或空病毒颗粒的负对照井。
轻轻旋转平板以确保均匀分布，并根据实验设计培养24至72小时。接着，轻轻吸入培养基。用PBS清洗细胞两遍，但不要让它们脱落。
按照制造商说明用Hoechst 33342染色核。使用倒置荧光显微镜配合相应滤光片进行活细胞荧光成像，以增强绿色荧光蛋白和霍赫斯特。如有需要，选择10倍或20倍物镜进行定量，选择63倍物镜进行高分辨率成像。
每口井至少获得五个视场，使用相同的成像参数。将所有图像保存为原始和灰度格式以便定量分析。透射电子显微镜显示，CME-AAVs的尺寸尺寸和膜结合结构类似于细胞外囊泡。
间充质干细胞膜标记CD73和CD90通过西方印迹分析在CME-AAV中检测到。三项主要腺相关病毒衣壳蛋白VP1、VP2和VP3通过SDS-PAGE在CME-AAV中被鉴定。NanoSight分析显示，CME-AAV9。
EGFP的平均直径为128.6纳米，总颗粒浓度为1.32乘以10的幂次方/毫升。荧光显微镜显示CME-AAV9。EGFP在HEK293T细胞中显著提高了增强的绿色荧光蛋白表达水平。
膜包膜病毒的基因表达提升了约2.5倍，表明细胞吸收增强，细胞内运输能力可能有所改善。体内生物发光成像显示，CME-AAV给药后肝脏表现出最强的发光信号。对肝脏区域的定量分析显示，CME-AAV给药导致基因表达水平显著提高。
这些方案描述了通过挤出和密度梯度超离心获得纯化细胞膜包覆AAVs（CME-AAV）的步骤。需要仔细考虑两个关键因素：对细胞片段进行预处理以进行挤出，以及优化膜片段与AAV混合比以控制CME-AAV颗粒与基因组比。未来研究将进一步探索细胞膜包覆，以增强免疫保护和功能表面特性，优化CME-AAV作为AAV递送的多功能平台。
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Overview
Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are emerging as promising tools for therapeutic applications and regenerative medicine. This article introduces a novel platform utilizing MSC membrane-enveloped nanovesicles, generated via a size-defined extrusion method, to efficiently deliver gene therapy vectors. These engineered vesicles encapsulate recombinant adeno-associated virus (AAV) vectors, offering improved gene delivery efficiency and production scalability compared to conventional approaches.
Key Study Components
Area of Science
- Gene therapy
- Regenerative medicine
- Stem cell biology
- Nanotechnology
Background
- Extracellular vesicles (EVs) are nanoscale vesicles secreted by all cell types, carrying proteins, RNAs, and metabolites.
- MSC-derived EVs have therapeutic potential due to their ability to modulate recipient cell behavior.
- Efficient DNA delivery via EVs is challenging due to poor DNA sorting mechanisms.
- Previous studies showed that AAV-containing EVs can deliver genes, but production is limited by low yield and time-consuming processes.
Purpose of Study
- To develop a scalable and efficient method for producing MSC membrane-enveloped nanovesicles for gene delivery.
- To encapsulate recombinant AAV vectors within these vesicles for enhanced gene transfer.
- To compare the efficiency and yield of this platform with conventional AAV and naturally secreted EV-AAVs.
Methods Used
- Generation of MSC membrane-enveloped nanovesicles using a size-defined extrusion technique.
- Encapsulation of recombinant AAV vectors carrying therapeutic gene sequences within the nanovesicles.
- Characterization of vesicle size (approximately 200 nm diameter) and properties.
- Comparison of gene delivery efficiency and production yield with conventional AAV and EV-AAVs.
Main Results
- The engineered MSC nanovesicles mimic natural EVs and efficiently encapsulate AAV vectors.
- These vesicles achieve significantly higher gene delivery efficiency compared to conventional AAVs.
- Production yield is greatly improved, with reduced time and cost relative to naturally secreted EV-AAVs.
- The platform supports scalable production suitable for clinical translation.
Conclusions
- The MSC-based membrane nanovesicle platform enhances gene delivery efficiency and scalability.
- This approach combines the advantages of EV-mimicking structures with AAV-mediated gene transfer.
- The technology offers a promising strategy to advance gene therapy toward clinical applications.
Frequently Asked Questions
Executive Industry Relevance
Efficient and scalable gene delivery remains a critical challenge in gene therapy and regenerative medicine pipelines. The development of mesenchymal stem cell (MSC) membrane-enveloped nanovesicles encapsulating recombinant AAV vectors addresses key bottlenecks in delivery efficiency and production yield. This platform offers enhanced predictive confidence for translational gene therapy programs and supports risk-adjusted portfolio advancement.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of gene transfer mechanisms in disease-relevant cellular systems.
- Supports biological de-risking by mimicking natural extracellular vesicle properties.
- Facilitates functional validation of gene delivery targets with improved efficiency.
Screening & Assay Development
- Provides standardized nanovesicle preparations for reproducible gene delivery assays.
- Improves assay scalability and throughput by reducing production time and cost.
- Enables quantitative comparison of gene transfer efficiency across candidate vectors.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by supporting sustained gene expression in vitro and in vivo.
- Bridges discovery and preclinical validation with scalable, EV-mimicking delivery vehicles.
- Reduces risk in advancing gene therapy candidates toward clinical translation.
Pipeline & Workflow Integration
This nanovesicle platform integrates from early discovery through preclinical gene therapy development, supporting both mechanistic studies and translational research.
- Discovery Biology: Advances hypothesis testing for gene delivery and expression in MSC-derived systems.
- Screening: Delivers reproducible, quantitative gene transfer outputs for candidate evaluation.
- Analytics: Enables measurement of delivery efficiency and yield for cross-condition comparison.
- Translational Research: Supports continuity from in vitro validation to in vivo preclinical models.
- Enterprise Reuse: Establishes a scalable, reusable platform for diverse gene therapy programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in gene delivery and target validation.
- Operational Value: Standardizes production and reduces time-to-assay for gene transfer studies.
- Strategic Value: Improves go/no-go decision quality and capital allocation for gene therapy assets.
- Portfolio Impact: Enables risk-adjusted prioritization of gene delivery platforms for clinical advancement.
Implementation Considerations
- Requires expertise in MSC culture, nanovesicle engineering, and AAV vector handling.
- Needs access to extrusion instrumentation and analytical tools for vesicle characterization.
- Demands cross-team standardization for reproducible vesicle production and gene delivery assays.
- Adaptation may be needed for different gene payloads or disease models.
- Production scalability and vesicle characterization must be validated for each application.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:42
Preparation of Human Mesenchymal Stem Cell (hMSC) Membrane Fractions
2:41
Encapsulation of Adeno-Associated Virus (AAV) Within Mesenchymal Stem Cell Membrane Vesicles by Extrusion
3:43
Purification of Membrane-Encapsulated Adeno-Associated Virus by Iodixanol Density Gradient Ultracentrifugation
5:48
In Vitro Transduction and Fluorescence Imaging of Cells with Encapsulated and Control Adeno-Associated Virus
7:14
Results
9:02
Conclusion
