This dissertation shall discuss and consider the design of a suitable animal model to study a severe autosomal dominant disease known as Huntington's disease. It will also discuss why SiRNA strategies may provide a potential therapy for this incurable disease.
Huntington's disease (HD) is an autosomal dominant inheritable neurodegenerative disorder, in which repetitive sequences of DNA lead to the production of a faulty protein. The cause of Huntington's disease is thought to be caused by a mutant form of the huntingtin protein (mHtt), the gene that encodes huntingtin is found on chromosome 4. The mutant form of the protein is expressed during development through adulthood, causing neuronal dysfunction and neuronal cell death in the striatum. The disease is slow and progressive and symptoms of the disease usually worsen in HD patients around mid-adulthood. As adults, victims lose their cognitive abilities, suffer involuntary movements and after about ten years or more patients usually die from the disease. The precise function of the huntingtin protein is not known as yet, it appears to play a vital role in nerve cell function. However the protein is not only found in nerve cells, it can be found in all cells throughout the body. Huntington's does not kill all the cells in the body, it has only been found to selectively kill nerve cells. The cause of Huntington's disease is thought to be due to the expansion of a polyglutamine sequence, the presence of expanded polyglutamine produces the toxic gain of function in the huntingtin protein. The mutated huntingtin protein responsible for HD has been shown to contain more of the amino acid glutamine than the normal huntingtin protein. The abundance of glutamine is due to the repetitive copies of the CAG codon in the Huntington gene. These proteins have affinity for each other and form clumps or aggregations, often called Neuronal inclusions (NIs) in the cell's nucleus. Various experiments have revealed that the huntingtin protein interacts with two other proteins only found in the brain, known as Huntingtin's interactor protein (HIP-1) and Huntingtin's associated protein (HAP-1). The number of CAG repeats in the HD gene has been shown to have an effect on how the huntingtin protein interacts with (HIP-1) and (HAP-1). If there is an increase in the number of CAG repeats, huntingtin binds less to (HIP-1) and more to (HAP-1). Therefore if the protein is altered in any way, it can be disruptive to nerve cells. The prevalence of Huntington's disease is around 4-10 individuals per 100,000 in the general western population, but many more people are thought to be at risk from developing the disease.
What is RNAi? How does it work?
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Small interfering RNA (SiRNA) or silencing RNA are a class of double-stranded RNA molecules, 20-25 nucleotides in length, they occur naturally, as part of the cells defence system against viruses and to control gene activity. RNAi comes into action between transcription and translation steps, RNA is introduced into the cell and binds to and destroys its messenger RNA (mRNA) target. The RNA pieces that are introduced into the cell are exactly complementary to the specific strand of mRNA; this is introduced into the cell through an enzyme known as dicer, which cuts it into smaller fragments once it is inside the cell. When the smaller pieces of SiRNA match up with mRNA, they initiate a process that cuts the mRNA into tiny fragments. The cell then recognises these fragments as waste and degrades them, preventing that mRNA from being translated into the protein. The SiRNA shoots the messenger, in a manner of speaking, and in doing so the protein cannot be formed.
SiRNA have been of interest as a possible therapy for Huntington's disease since 2004. A leading researcher from Iowa University, Beverly Davidson and her team have been successful in showing how effective SiRNA is in reducing the disease-causing huntingtin gene by RNAi based experimental work in knockout mice. Human Huntingtin injected into mice
How can RNAi be used as a possible therapy for HD?
Huntington's disease symptoms may be able to be halted with the development of a therapy designed to knock out the production of the defective mutant protein (mHtt) which causes the condition. Using gene therapy to switch off genes instead of adding new ones, could possibly slow down or prevent fatal brain disorders like Huntington's disease. The method exploits a mechanism called RNA interference (RNAi), and it may also be useful as a possible therapeutic tool for treating a wide range of other genetic diseases. It has been shown that reducing the amount of protein aggregation in the cell may be benefit for patients with Huntington's disease.
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Possible animal models and challenges faced with RNAi
RNAi has been shown to have great success in reducing the amount of mutant huntingtin protein in animal models. The animal models needed for studying possible RNAi therapies for Huntington's disease, would be transgenic animal models. Transgenesis is a process which introduces an exogenous gene known as a transgene into another living organism. Transgenic animal models are able to take in DNA from another species and display the phenotype or disease symptoms of that gene. Transgenic mice allow researchers to study certain aspects of HD and other diseases which would not be possible to do with human subjects. The shorter lifespan of mice is also beneficial as testing can be carried out faster. However there are certain drawbacks with using mouse models in studying HD, such as not being able to display some of the brain changes and behavioural features observed in patients with HD. Although knockout mice are not a viable model for studying HD, the use of the knockout mouse model, to a certain extent, increased the understanding of Huntington's disease.
The essential role of wild type huntingtin protein in humans has not been proven as yet, although experiments on knockout mice have shown that if HD expression is halted it can have disastrous effects. The knockout of the HD gene has been found to cause death of the mice during early embryonic development. The design of specific SiRNA that only targets the disease-causing transcript without causing any harm to the normal allele is currently being researched.