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After much deliberation over many project ideas, either expanding on previous projects or alternative substrates for existing parts, we decided we wanted to open up a whole new world of opportunities for Synthetic Biology. Developing the basics for a new realm in the field of RNA. Using a combination of experiments in Escherichia coli (E.coli) and human cells, both HeLa and Huh7.5 we attempted to turn on the lights to RNA world experimentation.

Until now RNA has been used sparingly in iGEM with teams tiptoeing around the idea with little advancement, we wanted to set the groundwork for future teams to have the option of classical Synthetic Biology i.e. DNA projects or new projects in RNA world. We feel this is a hugely exciting new area for research to begin as we were initially struggling with originality of our project due to the exponential increase in iGEM teams and projects done previously and underway. RNA is a fascinating alternative for projects. We decided the fundamentals were: an RNA repressor, promoter, ribosome binding site (RBS), kill switch, a replication system and demonstrating the potentials with our own part. These were combined into a self-replicating RNA strand or “Replicon”. These demonstrate a use of all the elements together however the potential permutations and adaptations of these parts are endless.

Deciding on how to utilise our system we had a huge number of potential experiments but decided we would focus our efforts on current and important world health problems. These were narrowed down to; type II diabetes mellitus and, on further research into current events, Ebola. Type II diabetes is a pandemic of epic proportions, on the rise in all corners of the globe of all race and age, in part due to the increase trend in obesity and glucose consumption.

America	England
Comparison by race	Worldwide

This costs the healthcare systems in all countries billions of dollars already and many are living undiagnosed that could double this figure. Almost every individual in America and Europe will have a friend or family member affected with this disease and causes heartache to thousands more following deaths of sufferers.

Treatments for Type II diabetes range from the simple; lose weight and consume less sugar to the expensive; gene therapy and dipeptidylpeptidase IV (DPP-IV) inhibitors, and the painful; amputations. DPP-IV, due to its cellular, genomic origin seemed like an appropriate target to tackle using our system. DPP-IV inhibitors are administered in stage two of treatment directly after lifestyle changes and are used to slow the degradation of incretins such as glucose like peptides which is accelerated by DPP-IV. Incretins act to increase the duration of

Gene therapy has been long sought after in biology. Its applications are extremely wide ranging, as by manipulating the genome a cell, you can in theory completely manipulate a cell to make it do whatever you want. This is essentially the same goal as synthetic biology, however the key difference is that gene therapy focuses on modifying the genes of cells in animals that have already grown beyond embryonic stage, rather than individual cells on their own. There are many problems with current gene therapy, including it being dangerous, being extremely difficult to perform, its possibility of modifying the DNA of cells in ways that can't be predicted, or the gene therapy not being permanent. Our project is to try and solve some of these problems by using a type of RNA called a 'replicon'.

What is a replicon?

A replicon is RNA that acts to replicate itself on its own using only the ribozymes of the cell. RNA usually degrades very quickly in cells, but a replicon should last permanently, because it should replicate faster than it can be degraded. Several viruses use replicons as their method of manipulating cells. Our idea is to take parts from the genome of hepatitis C (HCV) and modify it so that instead of doing damage to the body, it 'silences' harmful genes. To do this, we want to add an siRNA sequence to a replicon sequence. siRNA (small interfering RNA) is RNA that contains part of a complimentary nucleotide sequence to a particular RNA sequence. The human cell breaks this sequence down, and then continues to break down complimentary RNA sequences to this, including the sequence we want to target.

What are the advantages of this over conventional gene therapy?

Nowhere in this process is the actual DNA of the cell modified, so this removes the danger of DNA of the cell being modified in a way that is unwanted. This also improves upon conventional gene silencing, which involves siRNA only, as the replicon represents a permanent source of siRNA, greatly increasing the efficiency of the gene silencing. Our proposed method of delivery is to use a viral vector, technology which unfortunately doesn't exist yet, but could be developed within the next few decades. This method would allow for gene silencing in multicellular organisms, the holy grail of gene therapy.

Alternative Applications of our strand

Due to the way in which the RNA strand was engineered, the construct can easily be altered to act as a chassey for any number of potential uses, both in siRNA and other genes. This is due to the prescence of restriction sites abundant in our construct. One particularly current application is for the treatment of Ebola. Ebola has come to the forefront of public knowledge and fears due to the rampant outbreak in Africa which has resulted in a death toll of 9000 recorded deaths.

Ebola

Ebola is a viral haemorrhagic fever which attacks the blood vessels throughout the body causing internal and external bleeding and multiple organ failure leading to death in 50% of cases. The most recent outbreak of Zaire ebolavirus originated in Guinea and has now spread to many other countries in Africa including; Sierra Leone, Nigeria, Liberia and further afield with the first death in the U.S recorded in mid-October. The most effective treatment to date was ZMAPP, a combination of three humanised monoclonal antibodies used in tandem with rehydration to replenish water loss from hemorrhage reducing the mortality rate from 90% to 50%. There has recently been a worldwide exhaustion of reserves of ZMAPP which is complex and lengthy to prepare, therefore driving endeavors to find alternative therapies. The most promising of these is siRNA therapies targetting the Ebolavirus genome.

The siRNA is transfected into cells using lipid nanoparticle technology, as used in our transfection in vitro, and targets the L, VP24 and VP30 genes. This has been tested in chimpanzees and has shown reduction in Ebolavirus found in the blood when administered both pre and post infection, at the time when symptoms would present. If these siRNAs were inserted into our replicon it could be used as a prophylactic or post-infection treatment of those at risk or infected with, respectively, Ebola. This treatment has been fast tracked to Stage 3 clinical trials due to the severity of the disease.

Alzheimer's Disease

Another potential application for our replicon in an alternative location is Alzheimer's Disease. This is a neurodegenerative disorder usually affecting those in old age and causing, memory loss, inflammation and neuronal cell death. These symptoms arise from the build-up of β-amyloid peptides causing neurofibrillary tangles in the brain. The disease shares a number of physiological markers with type II diabetes mellitus and shows an improvement in symptoms following administration of incretins. This is due to cAMP coupled incretin receptors present in the brain, suggesting a central role of these hormones in the regulation of neuronal function and plasticity as well as neuronal survival which is severely reduced in those with Alzheimer's, as seen in the figure below.

The effect of long-term suppression of DPP-IV on the alzheimer's symptoms was investigated in D'Amico et al. 2009. The reduction in DPP-IV increases the life span of incretins such as GLP-1. Transgenic mice prone to the development of Alzheimer's, when administered with DPP-IV inhibitors showed a significantly increased level of GLP-1 in a tissue assay and subsequently a reduction in nitrosative stress, inflammation and diminished build-up of β-amyloid peptides. Suggesting that if the replicon was administered to neuronal cells there may be an extension of neuronal cell life an aleviated symptoms of Alzheimer's, increasing the quality of life in old age.