Development of efficient disease-regulated expression cassettes for gene therapy using microRNA targeting sequences

基因疗法，即使用核酸材料（例如DNA、RNA），正在被开发用于治疗目前难以用常规药物治疗的许多疾病。尽管有大量的候选治疗基因，但主要问题是将基因安全有效地递送到靶细胞和组织中，以便在足够长的时间内达到适当的基因水平以治疗疾病。一旦确定了治疗基因，第一步就是开发“表达盒”。表达盒是连接到DNA序列的基因，该DNA序列被称为“转录”，是“启动”基因所需的。实现成功的基因治疗的限制之一是基因在细胞内表达的程度。大多数基因在所有细胞类型中都以高水平表达，有时这会导致基因转录被关闭，因为细胞将表达盒识别为“外源”，或者转录太高并且对细胞有毒，因为它一直表达，而不仅仅是在疾病活跃时。为了使细胞正常发挥功能，它们使用几种方法来打开和关闭自己的基因，这使得不同组织/器官中的不同细胞能够执行其正常功能，并对使它们功能异常的变化（例如疾病）做出反应。细胞使用的方法包括称为“启动子”的DNA开/关开关，其仅在一种细胞类型（例如肝细胞、心脏细胞或血细胞）中发现。此外，最近发现了一种新的基因表达控制途径，称为“RNA干扰”，它“整理”基因表达，以确保它在不同细胞类型之间更具特异性。重要的是，最近的论文表明，这一途径在细胞生长和疾病期间控制细胞自身基因表达方面很重要。在这个提议中，我试图通过使用启动子和RNA干扰基因“开/关”系统来改进基因治疗表达盒，因此只有当疾病在细胞中活跃时，基因才被打开。RNA干扰最近已被证明是重要的基因调控过程中的疾病称为心脏肥大（心脏扩大）。因此，作为使用这些鉴定的RNA干扰“开关”的概念证明，我将使基因递送表达盒仅在经历肥大的心脏细胞中表达基因。重要的是，我将使用一种病毒载体，它可以有效地将基因传递到体内的这些心脏细胞中，并在心脏扩大发生的动物模型中测试这个概念。这利用了我们最近对RNA干扰及其在肥大中的功能的了解，理想的基因递送载体的可用性，以在理想的模型中将基因递送到正确的细胞类型（心脏细胞）。这项工作将为在特定疾病情况下使用RNA干扰来开启转基因表达提供概念证明，然而，这些概念将适用于未来针对许多不同疾病中许多不同细胞类型的基因疗法的开发。

Gene therapy, the use of nucleic acid material (e.g. DNA, RNA), is being developed to treat a number of diseases that are currently difficult to treat with conventional drugs. Although there are a large number of candidate therapeutic genes a major problem has been the safe and efficient delivery of the gene into target cells and tissues in order to achieve appropriate levels of the gene for long enough to treat the disease. Once a therapeutic gene has been identified, the first step is to develop an 'expression cassette'. The expression cassette is the gene linked to DNA sequences required to 'switch on' the gene, termed 'transcription'. One of the limitations to achieving successful gene therapy has been how well the gene is expressed once inside a cell. Most genes are expressed at high levels, in all cell types and sometimes this results in gene transcription being switched off because the cell identifies the expression cassette as 'foreign', or transcription is too high and is toxic to the cell because it is expressed all the time, not just when the disease is active. For cells to function normally they use several methods to switch their own genes on and off and this allows different cells in different tissues/ organs to perform their normal functions and respond to changes which make them function abnormally (e.g. disease). The methods used by cells include DNA on/ off switches called 'promoters' which are only found in one cell type (e.g. liver cells, heart cells, or blood cells). Also, recently a new gene expression controlling pathway has been found termed 'RNA interference' which 'tidies up' gene expression to ensure it is even more specific between different cell types. Importantly recent papers have indicated that this pathway is important in controlling cell's own gene expression while they are growing and also during disease. In this proposal I seek to improve gene therapy expression cassettes by using promoters and the RNA interference gene 'on/ off' system, so the gene is only switched on when disease is active in the cells. RNA interference has very recently been shown to be important in the regulation of genes during a disease termed cardiac hypertrophy (heart enlargement). Therefore, as proof of concept for using these identified RNA interference 'switches' I will make gene delivery expression cassettes to express genes only in heart cells undergoing hypertrophy. Importantly, I will use a virus vector which can efficiently deliver genes to these heart cells in vivo and test the concept in an animal model of in which heart enlargement take place. This takes advantage of our recent knowledge of RNA interference and its function in hypertrophy, the availability of an ideal gene delivery vector to deliver the gene to the correct cell type (heart cells) in an ideal model. This work will provide proof of concept for the use of RNA interference to switch on transgene expression in a specific disease situation, however the concepts will be applicable to the development of gene therapies aimed at many different cell types in many different diseases in the future.