You Yi, Tian Yu, Guo Rui, Shi Junfeng, Kwak Kwang Joo, Tong Yuhao, Estania Andreanne Poppy, Hsu Wei-Hsiang, Liu Yutong, Hu Shijun, Cao Jianhong, Yang Liqun, Bai Rui, Huang Pufeng, Lee Ly James, Jiang Wen, Kim Betty Y S, Ma Shuhong, Liu Xujie, Shen Zhenya, Lan Feng, Phuong Nguyen Patricia Kim, Lee Andrew S
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, 2199 Lishui Rd, Nanshan, Shenzhen, Guangdong Province 518055, China.
Institute for Cancer Research, Shenzhen Bay Laboratory, Guangqiao Road, Guangming District, Shenzhen 518055, China.
Eur Heart J. 2025 May 2;46(17):1662-1676. doi: 10.1093/eurheartj/ehae883.
Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the pre-clinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischaemic vascular disease.
After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models of ischaemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays.
VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by western blot, microscopy, and enzyme-linked immunosorbent assay. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischaemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with adeno-associated viral and lipid nanoparticle VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically.
This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischaemic vascular disease.
近期完成的通过病毒载体递送VEGF-A的基因治疗临床试验结果不佳，这使得开发替代递送策略的需求更为迫切。本研究旨在证明装载VEGF-A mRNA的细胞外囊泡（EVs）在治疗缺血性血管疾病方面的临床前疗效和安全性。
通过细胞纳米穿孔（CNP）将全长VEGF-A mRNA封装到成纤维细胞衍生的EVs中后，将收集到的VEGF-A EVs递送至缺血性损伤小鼠模型。通过蛋白质和基因表达的原位分析验证靶组织递送情况。通过体内成像和组织学确认功能恢复情况。使用基于免疫的检测方法证明单次和连续递送的安全性。
使用CNP方法生成了具有高mRNA含量的VEGF-A EVs。VEGF-A EV给药通过原位RNA杂交显示外源性VEGF-A mRNA的表达，并通过蛋白质印迹、显微镜检查和酶联免疫吸附测定显示蛋白质表达升高。在股动脉或冠状动脉结扎后用人VEGF-A EV治疗的小鼠，缺血组织中的新生血管形成增加，动脉灌注增加，左心室功能分别得到改善。在受伤皮肤中连续递送VEGF-EVs显示重复给药后伤口愈合得到改善。重要的是，与腺相关病毒和脂质纳米颗粒VEGF-A基因治疗方式相比，小鼠VEGF-A EV递送在注射部位或全身均未触发先天性或适应性免疫反应。
本研究表明，VEGF-A EV疗法可高效、剂量依赖性地形成VEGF-A蛋白，免疫原性低，在缺血性血管疾病小鼠模型中可促进新血管形成。
