Regenerative Medicine & Cellular Therapies (RMCT), Biodiscovery Institute (BDI), School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
Scancell Ltd, University of Nottingham Biodiscovery Institute, University Park, Nottingham NG7 2RD, UK.
J Control Release. 2024 Mar;367:209-222. doi: 10.1016/j.jconrel.2024.01.031. Epub 2024 Jan 26.
Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.
基于物理的基因传递方法（如 Helios 基因枪）涉及将核酸沉淀到微粒上，并通过高速加速直接转染暴露组织（如皮肤）的细胞膜。糖胺聚糖（GAG）结合增强转导（GET）系统利用新型融合肽，其包含细胞结合、核酸浓缩和细胞穿透结构域，可增强多种细胞类型的转染效率。在这项研究中，我们将化学（GET）和物理（基因枪）DNA 传递系统相结合，并假设这种组合将在最初未通过生物穿透进行转染的细胞中产生增强的分布和有效摄取。在细胞单层和工程组织中的体外物理化学特性表征、子弹内容物的优化和转染实验表明，这些制剂能够高效转染，包括 DC2.4 树突状细胞。我们将这些制剂纳入基因枪的生物弹道学格式中，通过冻干（冷冻干燥）形成可发射的干子弹。与传统方法相比，这种系统更简单，并且具有增强的可扩展性，可用于生成子弹。冲洗后的 GET 子弹内容物保持介导转染的能力（在不存在和存在血清的情况下，与标准亚精胺子弹相比，报告基因表达分别增加了 17 倍和 13 倍）。体外（细胞和胶原凝胶）和体内（小鼠）的发射 GET 子弹显示与未处理对照相比，报告基因转染增加，而体外保持细胞活力，体内无明显毒性。最后，使用 GET 子弹通过基因枪递送含有刺突（S）蛋白受体结合域（S-RBD）的 SARS-CoV-2 质粒 DNA 疫苗。与传统系统相比，产生了具有可比性的特异性 T 细胞和抗体反应。使用基因枪的 GET-金-DNA 载体的非物理和物理组合显示出作为一种替代 DNA 传递方法的潜力，该方法可扩展用于大规模可部署疫苗接种和皮内基因传递。
