Evaluating the gene delivery potential of E1L4-deficient adenovirus vectors

该项目的目的是评估一种新版本的腺病毒基因递送系统的性能，该系统已被广泛认为是实现人类基因治疗的一种方法。腺病毒，自然感染人，可以通过去除其基因组DNA的一部分，使其不能生长和引起疾病，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，外源基因被有效地吸收到细胞中，但理想情况下，不会发生感染周期的进一步事件。相反，外源基因在细胞中起作用，并产生有益的影响。然而，许多早期版本的腺病毒载体遭受两个相关的问题：基因递送效果持续时间不长，并且对由所谓的“晚期”基因制成的病毒蛋白产生强大的免疫反应，这些基因尚未从这些载体的基因组中去除。腺病毒晚期基因提供载体在实验室中生长所必需的蛋白质。如果你把晚期基因从载体中取出，你就不能生长出你需要在治疗中使用的载体颗粒，除非你以不同的方式提供蛋白质。两种选择是将载体与仍然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，或者制造含有缺失基因的特殊细胞并使用这些细胞来生长载体。在第一种情况下，你最终得到的是一种混合物，你必须从中分离出你想要的载体颗粒，这在大规模上是困难的，而在第二种情况下，说服细胞大量制造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的方法，当使用载体时，可以防止这些晚期病毒蛋白的产生，这种方法实际上不需要删除所有的基因。这意味着当在实验室中培养载体时，这些基因仍然可以用来提供必要的蛋白质。诀窍是只去除两种晚期蛋白质的基因，我们已经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起作用。我们已经制造出了能够“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，我们还用它们来培养一种去除了这两种基因的病毒。现在我们想知道这种被删除的病毒在用于基因传递时的行为。首先，我们需要研究它的基本性质，比如它有多容易生长，它的粒子有多稳定。然后，我们计划用它来研究测试基因进入细胞和组织的过程，以了解基因在细胞和组织中停留和工作的时间。

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.