Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder caused by mutations in the DMD gene, which encodes dystrophin. Although gene therapy using viral vectors has shown promise for the treatment of DMD, the clinical application of viral gene therapies is limited by vector toxicity, immunogenicity and the inability to package full-length dystrophin. Recent advances in messenger RNA (mRNA) technology offer a non-integrating, transient approach to restoring protein expression. Here we report the systemic delivery of skeletal-muscle-targeted full-length DMD mRNA in a murine model of DMD using allogenically engineered targeting extracellular vesicles (DMD t-EVs). This approach restores the endogenous translation of wild-type dystrophin and substantially improves muscle function. We further demonstrate the safety and biocompatibility of DMD t-EVs in non-human primates, supporting their translational potential. These findings highlight the promise of mRNA-loaded extracellular vesicles as a therapeutic platform for treating genetic disorders involving large, difficult-to-package genes.

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-NO2) into CD86-positive macrophages. Upon irradiation with low-intensity focused ultrasound, FA-PNBs release siSTAT1 and OA-NO2 at the ischemic injury zone. The results demonstrated the system's precise targeting and efficient delivery capabilities. The combined modulation of OA-NO2 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.

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 preclinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischemic 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 ischemic 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 ELISA. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischemic 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 AAV and LNP 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 ischemic vascular disease.

Adeno-associated virus (AAV)-mediated gene therapies face critical clinical limitations, including immune-mediated neutralization by pre-existing antibodies and dose-dependent hepatotoxicity. Extracellular vesicle-encapsulated AAVs (EV-AAVs) offer a promising solution by shielding AAVs from antibody recognition, yet existing production methods remain inefficient and impractical for clinical application. Here, we developed a cellular nanoporation (CNP) platform that enables scalable, high-yield generation of EV-AAVs, achieving an approximately 11-fold increase in production efficiency compared with conventional methods. In LDLR-deficient murine models with pre-existing neutralizing antibodies (1:200), EV-AAV-LDLR at half the standard AAV dose robustly restored hepatic LDL receptor expression and attenuated atherosclerosis progression. Notably, EV-AAV exhibited superior immune evasion capabilities, maintaining 2.3-fold higher hepatic transduction efficiency than conventional AAV upon secondary dosing due to antibody shielding. Importantly, EV-AAV therapy markedly reduced hepatotoxicity, with serum AST/ALT levels comparable to saline-treated controls, thereby overcoming a critical safety barrier of high-dose AAV treatment. These results demonstrate CNP as a clinically translatable platform for scalable EV-AAV manufacturing, enabling effective multi-dose regimens while overcoming key immunological and toxicity barriers in liver-directed gene therapy for familial hypercholesterolaemia.

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.

Rationale CRISPR/Cas9 has been extensively used to knock out genes, allowing the study of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, the current use of the Cas9-sgRNA plasmid or iCas9 system for gene editing in hPSCs has resulted in limited and inconsistent editing efficiency, as well as labor-intensive work. Additionally, identifying single-guide RNAs (sgRNAs) with high cleavage efficiency and distinguishing them from ineffective ones, which efficiently induce frameshift INDELs (Indels and Deletions) but fail to eliminate target proteins expression, are major challenges in gene knockout experiments. Methods This study addresses above issues using an optimized doxycycline-induced spCas9-expressing hPSCs (hPSCs-iCas9) system. We initially developed this system by optimizing a number of parameters to maximize INDELs introducing efficiency in hPSCs-iCas9 cells. The INDELs determined by this system were then compared to predicted scores from three cleavage efficiency scoring algorithms to validate the algorithms’ accuracy and consistency. Furthermore, we conducted gene knockout using a set of sgRNAs targeting different exons of the ACE2 gene to achieve approximately 80% INDELs for each targeting locus. Western blotting was then performed to detect ACE2 protein expression levels, enabling the identification of potentially ineffective sgRNAs. Results Several critical factors, including cell tolerance to nucleofection stress, sgRNA stability, nucleofection frequency, and the cell-to-sgRNA ratio, were found to have significant impact on editing efficiency in hPSCs-iCas9. Fine-tuning these parameters markedly improved this efficiency, resulting in up to 93% INDELs for single gene knockout. The three scoring algorithms exhibited significant differences or even conflicts in scoring cleavage efficiency. Through comparing experimental observations to predicted scores, we discovered that the Benchling algorithm outperformed the other two in terms of accuracy and consistency. Furthermore, a sgRNA targeting exon 2 of ACE2 gene was quickly identified as ineffective, as evidenced by the edited cells pool containing 80% INDELs while ACE2 protein expression retained unchanged detected by Western blot. Conclusion The findings of this study offer valuable insights into the optimal design of gene knockout experiments in hPSCs and provide practical solutions to sgRNA selection challenges for gene editing. ### Competing Interest Statement The authors have declared no competing interest.

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.