Team:UFMG Brazil/Devices

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kill switch

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Devices and Kill Switch

IFN-β generator

In order to enable Leishmania to act as an in situ drug delivery system to treat articulatory inflammatory diseases, we decided to make it constitutively expressing IFN-β, an important inflammation modulator. However, even if the production of this substance occurs directly within the target cells - macrophages that phagocyte our expression chassis - systemic side effects could still occur, once IFN-β reached circulation. To minimize that, we decided to alter the N-terminus of the protein, in order to add the sequence for a latency peptide (“LAP”), obtained from TGF-β. As long as LAP is present on the N-terminus, IFN-β remains in an inactive state. To separate LAP from the main protein and to allow its cleavage when needed, we added a spacer containing the amino acid sequence recognized by Aggrecanase, a protease specifically expressed on articulations. Thus, after being synthesised and falling onto the bloodstream, IFN-β will only have its LAP removed by Aggrecanase at articulatory regions, where it will be allowed to mediate local inflammation.

Before that, though, IFN-β produced by the Leishmania chassis must be firstly secreted into the macrophage cytoplasm, and then once again (this time, by the macrophage machinery) to reach the target organism’s system. The protein N-terminus was then further modified to include a generic secretion peptide signal that can be recognized by both Leishmania and macrophage cells.

It should be noted, however, that transformation of this system into our chassis is not as trivial as it would be on bacteria or yeast, because transcriptional regulation in Leishmania (and in other trypanosomatids) does not follow the usual model of promoter-based control. There are very few natural promoters on the entire genome, in such a way that pretty much every gene is constitutively expressed within large polycistronic messages. Later, specific sequences on the 5’- and 3’-untranslated regions of the mRNA are used by the cell machinery to determine whether it should remain on the cytoplasm at that specific moment or if it should be degraded. Therefore, to create a constitutive IFN-β generator, we had to transform into the genome a codon-optimized ORF for the secretion peptide/LAP/Aggrecanase linker/IFN-β construct, flanked by 5’- and 3’-UTR of highly, constitutively expressed Leishmania genes, instead of with the typical promoter, RBS and terminator parts.