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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.

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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.

Our motivation

Diabetes The majority of our siRNA testing will revolve around inhibiting Dipeptidyl peptidase IV (DPP-IV). DPP-IV causes increased speed of degradation of incretins, which when undegraded increase the duration of production of insulin. If our system works, it could act as a long-term treatment for type II diabetes as the treatment. Current DPP-IV inhibitors are very expensive at $885 for 90 tabs, four of which must be taken each day These act non-specifically causing side effects such as headaches, nausea and gastrointestinal issues. DPP-IV also acts as a tumour suppressor so administering these non specifically means tumour risk is increased throughout the body. Our system enables administration to specific cell types using a programmed viral vector and can be destroyed using an exogenous compound in the event of tumour formation. The administration of this would be very infrequent due to the self-replicating characteristic of the RNA strand and could improve patient compliance and quality of life.

Ebola Ebola has recently come to the headlines due to the epidemic currently occurring in Central Africa. Due to its initial appearance in a township the infectivity has been revealed to be much higher than initially suspected and is spreading devastatingly through multiple countries. The most effective treatment to date is re-hydration, reducing the mortality rate to 50% and ZMAPP, a combination of three humanised, monoclonal antibodies produced by genetic engineering targeting a specific gene of the Zaire ebolavirus aiding the immune system in combating the disease. Unfortunately the world reserves were exhausted at the end of September and research into other treatments has been undertaken. There looks to be significant promise in the use of siRNAs, such as TKM-Ebola, this interferes with the replication of the Ebola virus at an RNA level and has been shown to give 100% protection in macaques from lethal doses of Zaire ebolavirus. Our system could be adapted to deliver the siRNA against Ebola and could act as a prophylactic or poat infection treatment during an outbreak.

Alzheimer's

liver cancer

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