Intracellular gene delivery mediated by nanoparticles forms the foundation of many important nanomedicines, such as the COVID-19 mRNA vaccines, which have prevented deaths globally. However, a critical and longstanding challenge in gene delivery has been how to effectively deliver therapeutic genes into the cytosol and ensure that they function properly. A research team from The Chinese University of Hong Kong (CUHK), has developed a novel worm-like nucleic acid nanostructure, or nanoworm, that potentially offers a safe and effective approach to gene therapy. The findings have been published in the international journal Science Advances. Limitations of existing approachesThe efficacy of gene therapy hinges on the successful delivery of therapeutic nucleic acids into target cells using nanoparticles. A major bottleneck occurs once inside the cell, most nanoparticles become trapped in the vesicles known as endosomes, which eventually fuse with lysosomes, leading to enzymatic degradation of the gene cargo. Fewer than 0.04% of the published studies since 2019 showed efficient escape into endosomes, making endosomal entrapment a significant barrier that hampers therapeutic outcome of gene therapy. The two mainstream types of gene carriers for overcoming this barrier are cationic nanoparticles and lipid-based nanoparticles. Both have significant drawbacks: cationic nanoparticles can induce cytotoxicity due to their positive charge, while lipid-based nanoparticles present screening and engineering challenges and carry a risk of triggering inflammatory responses. Overcoming a longstanding bottleneck in gene therapyTo address this bottleneck, the team led by Professor Jonathan Choi Chung-hang, Professor in the Department of Biomedical Engineering, Faculty of Engineering, CUHK, has developed a novel worm-like nucleic acid nanostructure specifically engineered to enhance endosomal escape. This nanoworm consists of four or five gold nanoparticle cores, each 40 nm in diameter, surrounded by a polydopamine shell of 20 nm thick. Each nanoworm can carry 900-1,000 therapeutic oligonucleotides or about 30 therapeutic mRNA strands. Research has shown that this nanoworm enables delivery of diverse gene cargoes [DNA, small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA)] to multiple cell types in cultured cells and in animals. It can naturally enter various cells such as cancer cells, brain endothelial cells, primary macrophages and mesenchymal stem cells, without requiring cationic transfection agents. The researchers further discovered that the nanoworm has the remarkable ability to naturally activate the chloride voltage-gated channel 3 (ClC3) ion exchanger to drive endosomal escape; this is an unreported mechanism for nanoparticle-based gene carriers. This nanostructure has shown promising therapeutic applications, enabling miRNA-enabled macrophage polarisation, siRNA-enabled mesenchymal stromal cell differentiation, mRNA-enabled cell-based therapy for the reduction of kidney fibrosis and mRNA delivery to hepatocytes for the treatment of liver injury. In these applications, it outperformed the commercial transfection agent in endosomal escape and efficacy. Intravenous injection of the nucleic acid nanoworm into mice did not induce noticeable toxicity in vivo, realizing safe and effective gene delivery. Dr Reese Xiao Yu, the first author of the study and a PhD graduate from CUHK’s Department of Biomedical Engineering, explained: “The unique worm-like shape of the nucleic acid nanostructure significantly enhances its ability to escape endosomes by regulating the ClC3 ion exchanger, with minimal overlap between the nanostructure and endosomes, ranking it within the top 1% of nanoparticle-based gene carriers in the field.” Opening new directions for gene therapyProfessor Choi added: “This study not only propels the field of nucleic acid nanotechnology forward via a new worm-like nanoarchitecture that can achieve efficient endosomal escape but also offers a notable vista for designing safe and effective gene therapies based on non-cationic nanoparticle-based gene carriers.” This research was conducted in collaboration with Professor Xiaoqiang Yao from the School of Biomedical Sciences, Faculty of Medicine, CUHK. The team plans to expand the technology’s applications to other fields, such as gene editing and tissue engineering, and to work with the Faculty of Medicine on validating the safety and efficacy of this nanostructure in various animal models. This project was supported by the Research Matching Grant Scheme, the General Research Fund and Research Fellowship Scheme from the Research Grants Council of the Hong Kong Special Administrative Region of the People’s Republic of China , the InnoHK initiative of the Innovation and Technology Commission of the Government of the Hong Kong Special Administrative Region of the People’s Republic of China, the Chow Yuk Ho Technology Centre for Innovative Medicine and a CUHK Vice Chancellor Discretionary Fund. | 
(From left) Professor Yao Xiaoqiang, Dr Reese Xiao Yu and Professor Jonathan Choi Chung-hang. | 
Schematic illustration of the nucleic acid nanoworm. | 
Left: Proof of intracellular nucleic acid nanoworms (orange arrow) escaping from the ruptured membrane of endosomes to the cytosol by transmission electron microscopy. Dotted line denotes the intact portion of the endosomal membrane after rupture. Right: Mechanism of endosomal escape via the ClC3 ion exchanger. | 
Demonstration of endosomal escape of nucleic acid nanoworm in multiple types of cells by confocal microscopy (upper row) and transmission electron microscopy (lower row). The white number indicates the overlap between the endosomes (green) and nucleic acids (red). Purple arrow indicates cytosolic distribution of the nucleic acid nanoworms. |
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