Team:Greece
Greece
Project Description
Early diagnosis and successful treatment of colorectal cancer (CRC), the second most frequent type of cancer, constitute a modern medical challenge. We aim to design a toolbox that integrates miRNA profile data and the natural propensity of bacteria to colonize cancer tissue, in order to engineer a novel anti-cancer programmable agent. Our goal is to engineer an E. coli strain capable of adhering exclusively to CRC cells and facilitating the transference of a synthetic RNAi-based logic circuit, which can differentiate between healthy and tumor cells due to the different miRNA profile the two cell types exhibit and induce apoptosis only in the latter. Our project can be broken down into two major devices, one in charge of accomplishing selective adhesion to CRC cells and successful internalization of our bacteria carrying the second device, the aforementioned RNAi-based logic circuit which will code for an apoptotic protein under the control of the inhibitory and disinhibitory action of the miRNAs we have identified as necessary and sufficient to discriminate between the two populations.
Device 1: To achieve selective adhesion to CRC cells we aim to modify type I pili to disrupt their natural ability to bind to a-D-mannose and introduce a mechanism to facilitate adhesion to CRC cells by a mannose-independent mechanism. We intend to employ part BBa_K1850011 that was submitted by iGEM Harvard 2015 in a fimH KO strain. Moving on to the second half of our device (internalization and transference of genetic material), we hope to modify our bacteria so that they will express two key proteins, invasin and listeriolysin O, which will allow them to enter epithelial and other non phagocytic cells and free themselves of the vesicle that was used for their phagocytosis (parts BBa_K299812, BBa_K177010 and BBa_K177026 by iGEM Warsaw 2009). With the simultaneous utilization of quorum sensing for their propagation in the tumor microenvironment (by placing invasin and listeriolysin O under quorum control through the use of the lux genetic circuit of Vibrio fischeri-part BBa_K546000 by iGEM Wageningen 2011), cell-density dependent invasion can be achieved. The construction of Device 1 leads to the development of a target-specific bacterial delivery agent that has multiple safety levels in order to ensure effective targeting and minimal off-site effects.
Device 2: Sophisticated multi-input sensors of molecular markers, specifically miRNAs, that are able to ascertain if a set of endogenous miRNAs matches a neoplasmatic or healthy cell profile, have been previously described. Our vision is to design a novel transcriptional/posttranscriptional synthetic RNAi-based logic circuit as a cell "classifier" of colorectal cancer cells, which has as inputs multiple up- or down-regulated miRNAs and as an output a fluorescent or apoptotic protein. By predetermining a reference miRNA profile of our working colorectal cancer cell line (Caco-2), a Boolean expression can be created that describes the miRNA profile of a cancer cell and the circuit can be constructed either by relying heavily on DNA synthesis services or by combining parts submitted by previous iGEM teams, such as iGEM BIT 2016, iGEM MIT 2014 and iGEM SYSU-China 2013 (e.g. part BBa_K2041000 and part BBa_K2041003). After carrying out a series of transfection experiments and elucidating the components of the circuit, the final step is the transfer of the "classifier" into cancer cells (bactofection) through the use of the genetically engineered bacterial strains which have already been transformed with Device 1.
Since bacteria have a natural propensity to colonize cancer tissue, we wanted to increase this specificity for the cancer microenvironment by adding a modified adhesion system and quorum sensing mechanics in Device 1. Conceptually, this feature would be applicable to any cancer site. Moreover, with Device 2, we hope to follow a personalized-medicine approach, where given sufficient information about the differential expression of miRNAs in a cancer tissue obtained by biopsy, a highly-potent anti-cancer agent can be delivered into cancer cells without affecting healthy cells, even if it is accidentally transferred into them by bactofection. As a result, by utilizing a bottom-up approach and adding multiple levels to ensure safety and prevent adverse systemic effects, we hope that our toolbox of genetically engineered bacterial strains can be applied to a wide spectrum of cancer types and simultaneously tackle the polyclonal origin of many neoplasms, advancing cancer therapeutics throughout the world.
