Team:Warwick/Project/Motivation

MOTIVATION
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 an issue 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

These graphs clearly demonstrate the growing problem posed by diabetes. People of all races, in all corners of the globe are requiring constant monitoring and treatment for this disorder. The treatment requires frequent hospital or doctor's appointments and daily injections and/or tablets to relieve the symptoms and damaging effects of diabetes. This costs the healthcare systems in many countries billions of dollars already and could double if those currently living unaware of their condition were to be diagnosed. Almost every individual in America and Europe will have a friend or family member affected with this disease which will cause further heartache to thousands more following death and pain 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 costing $900 for 90 tablets, 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 glucagon-like peptides which is accelerated by DPP-IV. Incretins act to increase the duration of insulin release and hence to increase the probability of response from the insulin resistant cells. In this way the insulin resistance can in part be masked.

Gene therapy has been long sought after in biology and has already been implemented as a treatment for many disorders, such as functional CFTR gene therapy in cystic fibrosis sufferers. Its applications are extremely wide ranging, since by manipulating the genome of a cell, you can in theory make it carry out the desired function or restore functionality where before there was disfunction. The key difference in our project to traditional gene therapy which focuses on modifying the genome of cells, is that we are attempting to produce the same effect across many cells rather than individual cells on their own. There are many problems with current gene therapy, including it being dangerous and being extremely difficult to perform, due to the possibility of modifying the DNA of cells in ways that cannot be predicted and causing further issues by altering a necessary gene. Our project is to try and solve some of these problems by bypassing DNA and using a self replicating RNA strand we termed a 'replicon'.

What is a replicon?
A replicon is a 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 to which it associates forming a double stranded RNA fragment. Double stranded RNA is not endogenous to the cell and hence is recognised as foreign by Dicer and RISC, in the process detailed on How it works. The double stranded RNA is destroyed and RISC retains a form of memory for this sequence and will digest it spontaneously in subsequent exposures. We exploited this protection system to target our unwanted mRNA.

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RNA: Meet iGEM

A self-replicating RNA system to produce an siRNA to target mRNA which would otherwise give rise to a deregulated protein is just one of the many applications of our system. We purposely as a team created a set of core parts and RNA promoters, essentially creating a tool kit which allows any future iGEM team or researcher to take and use in their own system. This plug and play modularity is at the heart and soul of our motivation to give something to the synthetic biology community. We have the RNA dependent RNA polymerase, which propagates RNA templates. We have the aptazyme kill switch, which ensures regulation of our system or any system, the RNA promoters, the RNA terminators, the testing modules - you name it, we've most likely tested it.

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 being modified in a way that is unwanted. This of course, prevents the problem of insertional mutagenesis with viral vectors that routinely integrate into the genome. 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 does exist but currently is limited in respect to specificity to particular cell types, avoiding recognition by the immune response and efficiency of uptake of the viral vector. In the future, we hope that someone may stumble across our idea and build upon it, so that the full potential of our system can be realized.