The mechanism of phiC31 integrase; a tool for gene therapy and genome manipulation

这是真的很难治愈的人与遗传疾病，如肌肉萎缩症，他们有错误的基因。最好的治疗方法是给他们正确的基因，一个工作良好的基因。理想情况下，这种治疗在他们的一生中只需要一次，因为基因，当它们是染色体的一部分时，会忠实地从一个细胞传递到下一个细胞，因此治疗将永久存在。虽然这听起来很简单，但实际上很难。这个项目涉及一种可能的方法，将正确的基因导入病人的染色体。一些病毒，主要是那些感染细菌的病毒，有一种方法可以将自己的基因植入宿主的染色体。这个过程涉及蛋白质，称为整合酶，因为它们将两段DNA整合成一段。大多数整合酶优先使用宿主染色体中的特定位点，病毒DNA进入该位点。在病毒DNA中也存在优选位点。为了将正确的基因导入人体以治疗疾病，我们需要改造整合酶，使其能够在该人的染色体中找到其首选位点。因此，这个项目是关于了解这些整合是如何工作的，以便我们可以合理地改变它们。例如，我们想知道整合酶的哪一部分负责识别其优选的整合位点？整合酶的另一个特征是在没有任何其他病毒蛋白的情况下它是不可逆的。这意味着一旦正确的基因被插入，它就永远存在，因此只需要一次治疗。我们认为整合酶可以在反应途径的早期检测到其优选位点的存在，这一阶段将两个优选位点结合在一起。在整合酶中有一种锁和钥匙的相互作用，它激活了路径的其余部分以完成整合。如果没有正确的锁和钥匙的相互作用，路径就会被阻塞。几乎所有的这个项目都将用我们通过突变以某种方式改变的整合酶来完成。通过研究整合酶的性质如何变化，我们可以了解蛋白质如何工作。一些工作将与能够确定蛋白质三维结构的科学家合作完成。利用它们，我们的目标是获得整合酶的3-D结构及其整合的首选位点。这项工作的第三部分涉及一个称为切除的过程，与整合相反，是病毒DNA从宿主染色体上切除的过程。虽然整合酶本身是不可逆的，并且只整合DNA，但编码它的病毒必须能够从其宿主染色体上切除其DNA。我们打算寻找一种蛋白质，与整合酶相互作用，改变其性质，做切除。这将有助于我们更多地了解整个整合/切除过程，并增加我们设计更好的方法将基因传递给病人的能力。

It is really difficult to cure people with genetic diseases, such as muscular dystrophy, where they have the wrong gene. The best cure would be to give them the right gene, one that works well. Ideally this treatment would need to be given only once in their lifetime because genes, when they are part of the chromosome, are passed faithfully from one cell to the next and so the cure would perpetuate. Although this sounds simple, in practice its very hard. This project concerns a possible way of getting the right gene into a sick person's chromosome. Some viruses, mostly those that infect bacteria, have a way of getting their own genes into the chromosome of their hosts. This process involves proteins, called integrases, because they integrate two pieces of DNA into one. Most integrases use a particular site in the host chromosome preferentially over all others and the virus DNA goes into that site. There is also a preferred site in the virus DNA. In order to introduce correct genes into people as a cure for disease, we need to engineer the integrase so that it can find its preferred site in that person's chromosome. Consequently this project is about understanding how these integrases work so that we can alter them rationally. We would like to know, for instance, which part of the integrase is responsible for recognising its preferred integration site? Another feature of integrase is that it is irreversible in the absence of any other virus proteins. This means that once the correct gene is inserted it is there forever, hence the need for only one treatment. We think that integrase can detect the presence of its preferred sites very early on in the reaction pathway, the stage that brings the two preferred sites together. There is a kind of lock and key interaction within integrase that activates the rest of the pathway to complete integration. Without the right lock and key interaction the pathway is blocked. Almost all of this project will be done with integrases that we have altered in some way by mutation. By studying how the properties of the integrases change we can understand how the proteins work. Some of the work will be done in collaboration with scientists who can determine the 3-dimensional (3-D) structure of proteins. With them we aim to obtain a 3-D structure of integrase with its preferred sites for integration. A third part of this work addresses a process called excision, the opposite of integration and is where virus DNA is excised from the host chromosome. Although integrase by itself is not reversible and only integrates DNA, the virus that encodes it must be able to excise its DNA from its host chromosome. We intend to search for a protein that interacts with integrase to change its properties to do excision. This will help us to understand more about the whole integration/excision process and add to our ability to design better ways to deliver genes to sick people.