Geguchadze Ramaz, Wang Zhimin, Zourelias Lee, Perez-Riveros Paola, Edwards Paul C, Machen Laurie, Passineau Michael J
Gene Therapy Program, Allegheny Health Network, Pittsburgh, Pennsylvania, USA.
National Institute of Dental and Craniofacial Research, Bethesda, Maryland, USA.
Mol Ther Methods Clin Dev. 2014;1:14007. doi: 10.1038/mtm.2014.7.
In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of AAV. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis.
在本研究中，我们比较了在小鼠下颌下唾液腺中，超声辅助的非病毒基因转移与传统质粒和微型环状载体的基因转移效率及宿主反应。最初，我们使用等摩尔量的质粒和微型环状载体观察基因转移效率，证实了先前的一份报告，该报告表明在物理基因转移方法中微型环状载体更有效。然后，我们试图通过整体蛋白质组学分析来表征唾液腺对外源基因转移的生理反应。有点令人惊讶的是，我们发现单独的声孔效应（不存在基因转移载体时）对唾液腺蛋白质组几乎没有影响。然而，当使用质粒载体时，我们观察到唾液腺蛋白质组发生了深刻的扰动，其程度与先前一份关于高剂量腺相关病毒（AAV）后的报告中所见相当。最后，我们发现用微型环状载体进行基因转移仅诱导了轻微的蛋白质组学改变，类似于单独的声孔效应。我们使用质谱法为质粒和微型环状载体之间存在差异的218个凝胶斑点确定了蛋白质ID。对这些蛋白质的生物信息学分析表明，它们汇聚在68条已知的蛋白质相互作用途径上，最显著的是那些与先天免疫、细胞应激和形态发生相关的途径。
