Team:Oxford/Our solution
Our Solution
Overview
We decided to develop a probiotic strain of E. coli as a novel, self-tuning therapeutic for IBD. Our core design consists of the following:
- NO-dependent IL-10 secretion system
- Membrane-anchored nucleoside hydrolase
- Riboswitch-ribozyme-sRNA construct
- Inducible kill switch
This provides a framework that can be used in a huge range of new projects by future iGEM teams, including biosensors and therapeutics.
Due to the size and impermeability of regulatory interleukin proteins to the bacterial cell membrane, we used adenosine and nitric oxide (NO) as metabolic markers of Treg and Th-17 function respectively. An imbalance in the levels correlates with the autoimmune response. We aim to restore these to healthy proportions by secreting Interleukin 10 (IL-10) - a signal protein which stimulates cell differentiation into T-reg cells. The localised action of IL-10 secreted from the engineered bacteria makes our therapeutic best suited to gastrointestinal-based autoimmune diseases. Thus, we decided to focus specifically on IBD.
Our bacteria produce personalised and easily administered doses of IL-10. This avoids impracticalities of current, injection based techniques. The gut has greater ease of introduction to the body over other sites of T-cell populations and no biological barriers need to be crossed for the probiotic bacteria to reach their target. Our focus is on autoimmune diseases in developing countries triggered by ingested pathogenic bacteria. In these cases the disease starts in the gut making it the obvious place for us to target. We propose the cultures could be ingested in tablet form; this avoids the training needed for injections and minimises the risk of infection.
NO-dependent IL-10 secretion system
As well as being a generalised marker of inflammation, NO is thought to be produced by Th cells as a signal to induce differentiation of precursors into Th cells, thereby influencing the relative populations of Th and Treg cells. Continuous production of NO makes it a suitable marker of the size of the population and severity of the autoimmune disease.
Membrane-anchored nucleoside hydrolase
A range of purines are used by Treg cells as immune signals. ATP is released from cells during stress or injury. To prevent it from causing excessive inflammation, ATP is rapidly metabolised into AMP then into Adenosine (Ado) by Treg cell surface enzymes. Ado is detected by GPCRs of Th cells, triggering increased cAMP in the cells. Inflammatory signals are inhibited and IL-10 production is enhanced in mature dendritic cells. Ado also induces semi-maturation of immature dendritic cells. Adenosine promotes Treg populations, adenosine generation and increases immunoregulatory activity. Production by Treg cells and its role in regulating T cell populations and IL-10 production make it a suitable marker of Treg function and degree of immunodeficiency.
Riboswitch-ribozyme-sRNA construct
In our circuit, IL-10 is secreted in the presence of NO and absence of elevated adenosine levels. IL-10 expression is stimulated by the endogenous E. coli SoxR transcription factor, activated by free radicals and oxidative stress, while expression is inhibited in response to adenosine by means of an adenine riboswitch linked to sRNA synthesis which will selectively inhibit translation of IL-10 mRNA. We have implemented a membrane-anchored adenosine hydrolase to generate adenine which can then diffuse into the cytoplasm and control the negative feedback loop.
We decided to use NO partly due to the work of the Stanford 2009 iGEM team, who used NO detection to activate synthesis of retinoic acid. We used the same SoxR/SoxS promoter system to detect NO but instead, are using it to stimulate IL-10 production.
A single stimulus can result in false positives and excessive suppression, leading to immunodeficiency. Incorporation of a negative feedback loop signaling high Treg populations avoids oversuppression of the immune system. The defining aspect of our design is the integration of two signals in order to increase the specificity and accuracy of our system in equilibrating the populations of Treg and Th-17 cells.
Inducible kill switch
Another important consideration was to develop a system to enhance the safety of our probiotic. Therefore, we decided to design a kill switch which would be activated by an external supplement in order to account for the possibility of adverse reactions in patients. We linked the antimicrobial artilysin Art-175 with a DsbA periplasmic secretion tag, under the control of an inducible pTet promoter. Therefore, upon induction by a synthetic TetR inducer, expression of the artilysin composite would promote host cell lysis. Further biosafety considerations are explained on the ‘Safety’ page.