Tsutsui Makusu, Tsunekawa Yuji, Wada Mikako, Arima Akihide, Onodera Azusa, Nishina Masumi, Nagoya Miho, Baba Yoshinobu, Kawai Tomoji, Okada Takashi
The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka, 567-0047, Japan.
Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.
Small Methods. 2025 Jul;9(7):e2401321. doi: 10.1002/smtd.202401321. Epub 2025 Jan 2.
Achieving safe and efficient gene therapy hinges upon the inspection of genomes enclosed within individual nano-carriers to mitigate potential health risks associated with empty or fragment-filled vectors. Here solid-state nanopore sensing is reported for identifications of intermediate adeno-associated virus (AAV) vectors in liquid. The method exploits the phenomenon of translocation slowdown induced by the viscosity of salt water-organic mixtures. This enables real-time ionic current measurements allowing precise tracking of the electroosmotic flow-driven motions of recombinant AAV vectors in a nanopore. The resulting ionic signals facilitate discrimination between replicative intermediates carrying ssDNA fragments and its full vector counterparts based on genome length-derived subtle nanometer differences in the viral diameters. This rapid and non-destructive means of genome analysis within virus capsids provides a promising avenue toward a robust methodology for ensuring the integrity of AAV vectors before administration.
实现安全有效的基因治疗取决于对单个纳米载体中封装的基因组进行检测，以降低与空载体或片段填充载体相关的潜在健康风险。本文报道了利用固态纳米孔传感技术在液体中鉴定中间腺相关病毒（AAV）载体。该方法利用了盐水 - 有机混合物粘度引起的转位减速现象。这使得能够进行实时离子电流测量，从而精确跟踪纳米孔中电渗流驱动的重组AAV载体的运动。由此产生的离子信号有助于根据病毒直径中基于基因组长度的细微纳米差异，区分携带单链DNA片段的复制中间体及其完整载体对应物。这种在病毒衣壳内进行基因组分析的快速且无损的方法，为在给药前确保AAV载体完整性的强大方法提供了一条有前景的途径。
