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Evaluating the gene delivery potential of E1L4-deficient adenovirus vectors

评估 E1L4 缺陷腺病毒载体的基因传递潜力

基本信息

批准号：
BB/E014550/1
负责人：
Keith Leppard
金额：
$ 45.25万
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE014550%2F1
关键词：
Evaluating gene delivery potential E1L4

项目摘要

The aim of this project is to evaluate the performance of a new version of the adenovirus gene delivery system that has been widely considered as a way of achieving gene therapy in people. Adenoviruses, which naturally infect people, can be made incapable of growing and causing disease by removing parts of their genome DNA, and then foreign gene sequences can be added in their place to create a gene delivery vector. When the vector infects a cell, the foreign gene is efficiently taken into the cell but, ideally, no further events of the infectious cycle occur. Instead, the foreign gene becomes operative in the cell and has a beneficial effect. However, many earlier versions of adenovirus vectors suffered from two related problems: the gene delivery effect did not last very long and powerful immune responses were generated to viral proteins made from the so-called 'late' genes that had not been removed from the genomes of these vectors. Adenovirus late genes provide proteins that are essential for the vector to be grown in the laboratory. If you take the late genes out of the vector, you cannot grow the vector particles that you need to use in therapy unless you provide the proteins in a different way. The two options are to mix the vector with a second virus that still has these late genes, so it can help the vector to grow, or to make special cells that contain the missing genes and to use these to grow the vector. In the first case you end up with a mixture from which you have to separate out the vector particles that you want, which is difficult on a large scale, while in the second case, persuading cells to make all these proteins in the large amounts needed has so far been impossible. We have been working on a new way to prevent the production of these late viral proteins when a vector is used, that works without actually removing all the genes. This means that the genes can still be used to provide the necessary proteins when growing the vector in the laboratory. The trick is to remove the genes for just two late proteins, which we have discovered turn on the production of all the others. When these two proteins are provided the vector grows well but otherwise, none of the late genes it carries will work. We have made cells that will make these two key proteins 'on demand' / the cells cannot be asked to make them all the time because they harm the cells / and have used them to grow a virus that has the two genes removed. Now we want to find out how this deleted virus behaves when used for gene delivery. First of all, we need to study its basic properties, such as how easy it is to grow and how stable its particles are. Then, we are planning to use it to study the delivery of a test gene into cells and tissues, to find out how long the gene stays around and keeps working.

该项目的目的是评估新版本腺病毒基因传递系统的性能，该系统已被广泛认为是实现人类基因治疗的一种方式。自然感染人类的腺病毒可以通过去除部分基因组 DNA 使其无法生长和引起疾病，然后可以在其位置添加外源基因序列以创建基因传递载体。当载体感染细胞时，外源基因被有效地带入细胞，但理想情况下，不会发生感染周期的进一步事件。相反，外源基因在细胞中开始发挥作用并产生有益的作用。然而，许多早期版本的腺病毒载体都存在两个相关问题：基因传递效应不能持续很长时间，并且对由尚未从这些载体的基因组中删除的所谓“晚期”基因制成的病毒蛋白产生强大的免疫反应。腺病毒晚期基因提供了载体在实验室中生长所必需的蛋白质。如果您从载体中取出晚期基因，则无法生长治疗中需要使用的载体颗粒，除非您以不同的方式提供蛋白质。两种选择是将载体与仍具有这些晚期基因的第二种病毒混合，因此它可以帮助载体生长，或者制造包含缺失基因的特殊细胞并使用它们来生长载体。在第一种情况下，你最终会得到一种混合物，你必须从中分离出你想要的载体粒子，这在大规模上是很困难的，而在第二种情况下，说服细胞大量产生所需的所有这些蛋白质迄今为止是不可能的。我们一直在研究一种新方法，以防止使用载体时产生这些晚期病毒蛋白，该方法无需实际去除所有基因即可发挥作用。这意味着在实验室中培养载体时，这些基因仍然可以用来提供必需的蛋白质。诀窍是仅删除两种晚期蛋白质的基因，我们发现这两种蛋白质会启动所有其他蛋白质的产生。当提供这两种蛋白质时，载体生长良好，但否则，它携带的晚期基因都不会发挥作用。我们已经制造了能够“按需”制造这两种关键蛋白质的细胞/不能要求细胞一直制造它们，因为它们会伤害细胞/并用它们来培养去除了这两种基因的病毒。现在我们想了解这种被删除的病毒在用于基因传递时的表现如何。首先，我们需要研究它的基本特性，比如它的生长难易程度、颗粒的稳定性如何。然后，我们计划用它来研究测试基因向细胞和组织的传递，以找出该基因保留并继续发挥作用的时间。