Vascular-targeted gene therapy to block proliferation of smooth muscle cells using a novel adenovirus vector

为了改善急性损伤后血管壁的功能，已出现了许多策略，包括靶向内皮细胞功能、血管平滑肌细胞的增殖和迁移以及外膜祖细胞。MicroRNA是一种小的非编码RNA，它能够通过靶向3‘非编码区中的种子序列来靶向数百个基因。我们已经表明miRNA在血管平滑肌细胞功能和血管重塑的发展中是必不可少的(Dun2015/MacDonald2013)。我们已经鉴定出具有阻断血管平滑肌细胞增殖能力的新型miRNA。向前迈出的重要一步将是开发新的方法，通过基于病毒的递送方法将治疗性miRNA运送到血管壁中。与Havenga博士多年来的合作促进了新腺病毒载体的开发，用于基因治疗(Waddington Cell，2008)。最近，我们鉴定、纯化和测试了一种新型的人腺病毒载体Ad20/42/42，它可以有效地将基因输送到血管内皮细胞，但对肝脏的输送非常有限-腺病毒载体聚集的主要部位。此外，这种病毒在普通人群中显示出非常低的预先存在的中和水平(人腺病毒5型约为10%对60%)，这使其成为一种非常有吸引力的应用于人类基因治疗的病毒。因此，在这个项目中，我们将在血管系统损伤后的血管基因治疗环境中测试该载体(以及在项目过程中可用的其他新的有希望的载体)。因此，该项目将在以下方面具有突出的培训潜力：病毒学、载体构建和繁殖急性血管损伤的活体模型血管细胞的体外培养和病毒的传递活体评价治疗性miRNA。此外，该项目还受益于与意大利的里雅斯特的Giacca实验室合作开发治疗性miRNA。

Many strategies have emerged to improve the function of the blood vessel wall following acute injury, including targeting endothelial cell function, vascular smooth muscle cell proliferation and migration and adventitial progenitor cells. microRNA are small non-coding RNA that have the capacity to target many hundreds of genes through targeting seed sequences in the 3'UTR. We have shown the miRNA are essential in vascular smooth muscle cell function and development of vascular remodelling (Deng 2015/MacDonald2013). We have identified novel miRNA that have the capacity to block vascular smooth muscle cell proliferation. A major step forward would be the development of novel approaches to deliver therapeutic miRNA into the vessel wall by virus-based delivery approaches. Working with the Dr Havenga over many years has facilitated the development of novel adenovirus-based vectors for gene therapy (Waddington Cell, 2008). Recently, we have identified, purified and tested a novel human adenovirus vector called Ad20/42/42 that displays effective delivery of genes to endothelial cells but very restricted delivery to the liver - the main site of adenovirus vector accumulation. Further, this virus shows very low levels of pre-existing neutralisation in the general population (approximately 10% vs 60% for human adenovirus 5) making this a very attractive virus for application to human gene therapy. In this project we will therefore test this vector (and other novel promising vectors that become available during the course of the project) in the setting of vascular gene therapy post injury of the vasculature. The project will therefore have outstanding training potential in:Virology, vector construction and propagationIn vitro cell culture of vascular cells In vivo models of acute vascular injury and delivery of virusEvaluation of therapeutic miRNA in vivo.The project additionally benefits from a collaboration on the development of therapeutic miRNA with the Giacca lab in Trieste, Italy.