Yi You

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Following myocardial infarction (MI), the accumulation of CD86-positive macrophages in the ischemic injury zone leads to secondary myocardial damage. Precise pharmacological intervention targeting this process remains challenging. This study engineered a nanotherapeutic delivery system with CD86-positive macrophage-specific targeting and ultrasound-responsive release capabilities. A folic acid (FA)–modified ultrasound-responsive gene/drug delivery system, assembled from DOTAP, DSPE-PEG2000-FA, cholesterol, and perfluorohexane (PFH)—termed FA-PNBs—was developed to codeliver small interfering RNA of STAT1 (siSTAT1) and the small-molecule nitro-oleic acid (OA-NO 2 ) into CD86-positive macrophages. Upon irradiation with low-intensity focused ultrasound, FA-PNBs release siSTAT1 and OA-NO 2 at the ischemic injury zone. The results demonstrated the system’s precise targeting and efficient delivery capabilities. The combined modulation of OA-NO 2 and siSTAT1 optimizes the immune microenvironment in the infarcted region, alleviates ventricular remodeling, preserves cardiac function, and holds promise for clinical intervention strategies after MI.

Bone regeneration remains a clinical challenge due to the inherent limitations of conventional grafts and synthetic materials. While mRNA-based strategies offer promising therapeutic potential, their clinical application is hindered by systemic delivery barriers and rapid degradation. Here, we present a hybrid extracellular vesicle (EV)-GelMA hydrogel system for the localized delivery of VEGFA mRNA to enhance both angiogenesis and osteogenesis through precise spatiotemporal control. In vitro, the VEGFA-EVs-GelMA system significantly enhanced migration and osteogenic differentiation of pre-osteoblastic cells, as evidenced by upregulation of ALP, RUNX2, and OPN expression compared to GelMA controls. In a rat cranial defect model, the VEGFA-EVs-GelMA group showed a marked increase in bone volume/total volume (BV/TV), supported by microcomputed tomography (Micro-CT), at both 4 and 8 weeks post-implantation . Mechanistically, this dual-component platform leverages the biomolecular protection and targeted delivery afforded by EVs, together with the tunable release properties of GelMA hydrogels, to overcome key mRNA delivery barriers. Our results highlight the potential of this system as a clinically translatable strategy for promoting vascularized bone regeneration and provide new insights for the treatment of complex bone defects.

Abstract Introduction: Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs) which hold promise in therapy for SCI by transplantation. In our study, we aimed to establish a chemically defined culture system by using serum-free medium and ascorbic acid (AA) to generation and expansion of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods: We induce hESC/iPSC to neurospheres by using a newly established induction system in vitro in our study. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction systems, including conventional N2 medium with gelatin-coated (coated) and N2+AA medium without pre-coated (AA) were characterized by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons and the microelectrode array (MEA) recording system was used to evaluate the functionality of neurons differentiated from lt-NES cells. Moreover, the mechanism of AA-induced lt-NES cells was explored through RNA-seq and the use of inhibitors. Results: HESCs/iPSCs were efficiently induced to neurospheres by using a newly established induction system in vitro. And lt-NES cells derived from hESCs/iPSCs-neurospheres using two induction system (coated vs AA) both expressed neural pluripotency-associated genes PAX6, NESTIN, SOX1, SOX2. After long-term cultivation, we found that they both can maintain the long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retain the ability to differentiate into general functional neurons that highly express β-tubulin. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathways. Conclusions: Taken together, this new chemically defined culture system is stable and effective to generate and culture the lt-NES cells induced by hESCs/iPSCs using serum-free medium combined with ascorbic acid (AA). The lt-NES cells under this culture system can maintain the long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Keywords: Spinal cord injury, Neurospheres, Ascorbic acid, lt-NES cells, Human pluripotent stem cells.