Overview

Rare, or “orphan”, diseases are frequently ignored by the pharmaceutical industry. They encompass a huge range of disorders, from ALS to Tourette’s Syndrome, but individually have a relatively low number of patients. The low patient numbers mean that there is very little impetus for the pharmaceutical industry to research and produce novel, innovative therapeutics. This means that patients are often left with unsatisfactory treatments. Our goal is to produce a probiotic therapeutic to treat one such disorder: Wilson’s Disease.

Wilson's Disease

Wilson’s Disease is a genetic disorder characterised by an inability of the body to fully metabolise copper. Normally, when copper is ingested, it is taken up from the small intestine into the liver and subsequently transported into the blood or excreted into the bile. In Wilson’s Disease, there is a mutation in the gene: ATP7B. ATP7B encodes a copper-transporting protein that is responsible for loading copper onto ceruloplasmin for transport in the blood, and into the bile for removal from the body. In the absence of a functional form of this protein, copper is unable to be removed from the liver after absorption. This results in toxic accumulation, as cuprous ions react with hydrogen peroxide to produce dangerous free radicals that damage the tissue. This allows copper ions to leak into the blood and eventually accumulate in, and damage, other tissues, such as the kidneys and brain. You can read more about Wilson's disease here.

Current treatments are regarded by patients as unsatisfactory. From discussions with these individuals, we have surmised that there are three main problems with current treatments:

Our CuRE aims to address these limitations.

Probiotics

A probiotic constitutes a microorganism that is introduced into the body for its beneficial properties. The concept of a probiotic, meaning “for life”, was introduced by Elie Metchnikoff in 1907, when he hypothesised that replacing or diminishing the populations of ‘putrefactive’ bacteria in the gut with lactic acid bacteria could positively affect bowel health.

Products that are commonly sold as probiotics include food stuffs, such as yoghurts and cheeses. However, recently there has been an increase in the amount of research going into the use of probiotics as therapeutics, with the genetic engineering of organisms to produce useful substances. Currently there is limited legislation regarding probiotics, as probiotics sold as dietary supplements do not require FDA approval. A genetically-engineered probiotic therapeutic would require more stringent legislation and FDA approval to ensure, through clinical trials, that it works as expected.

Although advancing rapidly, the field of probiotics still requires significant research particularly in areas such as safety. Although regarded as safe for relatively healthy humans to consume, there have been some reports of probiotic-related side effects in people with serious underlying medicals conditions. We carried out a comprehensive safety review when completing our project.

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Parts

In order to detect and chelate dietary copper we investigated the copper sensing systems of E. coli and attempted to redesign them to fit our requirements. We also looked for copper chelating proteins. You can read about how we chose our parts here.

Delivery

From discussion with patients and the public, and the work carried out by previous Oxford iGEM teams, we decided to investigate the use of a bead to deliver our bacteria to the small intestine. Our bacteria will initially be encapsulated in an alginate matrix, and then be alternately coated in layers of alginate and chitosan. The goal of the multiple polymer-coatings is to protect the bacteria from the harsh conditions of the stomach, whilst having the ability to degrade in the more alkaline pH of the small intestine. This degradation releases our bacteria into the favourable conditions of the small intestine, where they can colonise the area and chelate dietary copper.

Results

Through our experimental work we have been able to obtain data suggesting the validity of these points:

• Different arrangements of both our CueR-linked and CusS/CusR-linked promoter systems are sensitive over a range of copper concentrations, including at the lower concentrations mimicking the gut.
• MymT is able to chelate a measurable amount of copper in vivo.
• Alternately layered chitosan-alginate beads release material in the small intestine, following passage through the stomach.

Please see our experiments and results pages for more discussion of our methods and the outcomes.

Improving Registry Parts

As part of our project, we have improved the function/characterisation of three poorly document parts.

Please visit our parts and results pages for further information.

BBa_I760005:

The copy of pCusC located in the registry had been previously used 11 times, but had no associated useful characterisation data. From discussions with a mentor, Tom Folliard, we came to the conclusion that this may have been because the promoter region was too short, and thus, missing key binding sites. We decided to deposit an elongated form of this promoter, for which we have achieved thorough characterisation.

BBa_K190020:

MymT can be found in the registry with no associated characterisation data. We codon-optimised the sequence for expression in E. coli and added a hexahistidine tag for purification. We also produced a form with a C-terminal sfGFP tag, also his-tagged, to aid purification and allow analysis by FLIM. This has provided us with preliminary characterisation data for the protein, suggesting copper-binding activity in vivo.

BBa_K1758324:

This is a composite part comprised of 2 subparts. One is CueR expressed from a constitutive promoter, the second is sfGFP expressed from pCopA. When the part was assembled in the registry, these two subparts were incorrectly joined so that the constitutive promoters was on the same strand as the sfGFP, in the same direction, but labelled as if it were correct. In the construction of our part, we corrected this, codon-optimised to E. coli and removed the 5’ UTR to allow synthesis by IDT. Though unable to then obtain this part due to a likely synthesis issue we obtained two other versions containing our sfGFP-tagged chelators.

Conclusion

Over the course of the summer we have successfully created and submitted 13 sequence-confirmed BioBrick parts, 8 of which have been characterised. Through our experimentation we have been able to examine the copper-sensitivity of a variety of promoters integral to our CuRE. In addition, we have tested the chelation ability of our copper chelators with a variety of different assays. Although these have not all been successful, through a collaboration with Cardiff we have been able to obtain preliminary data suggesting that MymT may be able to successfully lower the intracellular copper concentration. We have also obtained promising preliminary data relating to the delivery of substances to the small intestine via alginate-chitosan beads.

In conclusion, although we may have not achieved our final goal of producing a working system capable of reducing the extracellular copper concentration. We have contributed to our understanding of these systems, in addition to investigating a treatment that addresses the needs and concerns of the patients themselves.

Future

To further develop our project, we would hope to carry out a number of further experiments to allow us to generate something more than a proof-of-concept model. Primarily, we would hope to properly characterise the copper-chelating ability of Csp1 and confirm its localisation in the periplasm probably involving redesigning the TAT sequence. Additionally, we would carry out further experiments to refine our delivery system.

Once the system was able to successfully operate in E. coli DH5-α, we would hope to express it in a strain that more closely mimics the bacteria of the gut. More information on a potential future chassis choice can be found on our safety page.

Finally, the design of our system went through many iterations over the summer. One of our most ambitious designs was based on logic gates that would provide an additional safety mechanism, with more time we would love to test this system. We discuss this design further on our safety page.