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

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.

Gene therapy is being developed as a therapeutic intervention in many monogenic and polygenic disorders. For gene therapy to succeed there is a requirement to refine gene delivery vectors and expression cassettes to regulate transgene expression in a cell/ tissue-specific and disease-regulated manner. Using viral promoters results in high level transgene expression in most cell types which may be undesirable, (e.g. cytotoxic or cytostatic gene therapies) and viral promoters are sensitive to methylation-mediated transcriptional silencing. There are many candidate cell-specific eukaryotic promoters, although some show low transcription compared to viral promoters or lose their cell specificity once engineered into gene transfer vectors. In this proposal I will take two approaches to develop efficient, cell-specific, disease regulated expression cassettes. First, I will take advantage of recent data which indicates that RNA interference (through the function of microRNAs) plays a fundamental role in the activation/ suppression of gene expression in response to disease stimulation. Second, I will use a cell-specific promoter (which will be improved by randomly combining it with transcriptional response elements (TREs) that respond to transcription factors which are activated in disease processes. MicroRNA target sequneces which regulate gene expression during hypertrophy will be combined with cardiac myocyte promoters and synthetic TREs linked to genes which are activated in response to cardiac hypertrophy. These novel expression cassettes will be tested in the situation of in vivo cardiac hypertrophy in a rat model using the cardiac tropic adeno-associated virus serotype 6. Importantly, this will provide proof of concept for using microRNA to regulate transgene expression in a disease situation using recent data and a relevant vector and model, however the principles will be applicable to many gene therapies in the futur