Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem, Israel.
Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Nat Protoc. 2019 Apr;14(4):1015-1026. doi: 10.1038/s41596-019-0125-y. Epub 2019 Feb 25.
Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.
超声介导的基因传递（声孔作用）是一种微创、非病毒且可临床转化的基因治疗方法。与病毒载体相比，该方法具有更好的安全性，并且比其他物理基因传递方法（如电穿孔）的侵入性更小。我们之前曾使用声孔作用在不同的骨骼组织中过表达转基因，以诱导组织再生。在这里，我们提供了一个易于适应的方案，可以解决组织再生或其他应用的各种其他目标，例如癌症和神经退行性疾病。本方案描述了如何在小鼠和猪动物模型中制备、进行和优化超声介导的基因传递。该方案包括微泡-DNA 混合物的制备和超声成像下的体内声孔作用。超声介导的基因传递可以在 10 分钟内完成。在 DNA 传递后，可以对动物进行跟踪，以监测基因表达、蛋白质分泌和其他转基因特异性结果，包括组织再生。具有超声成像或体内操作经验的有能力的研究生或技术人员可以完成此过程。
