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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.
Environmental Kill Switch
Although our modified Leishmania strain is expected to be harmless to the human host, it could still pose an environmental threat if a sandfly be infected by this genetically modified strain from a bloodstream of a undergoing treatment patient. This could be a huge problem, since the Leishmania would revert to the promastigote form inside the insect’s gut, being able to replicate once again, and releasing a viable GMO into the wilderness.
To prevent that, we designed a kill switch that takes advantage of one of Leishmania's metabolic deficiency: its inability to synthesize purines. This protozoa needs to import such nucleotides from the environment - be it the insect’s gut or the vertebrate host cells; however, a different uptake mechanism is used on each life form, with the purine capture in promastigotes being performed by a membrane 3’-nucleotidase/nuclease. Thus, by restraining this enzyme to only work under external activation, it is possible to make sure that promastigotes only grow in media containing the activator molecule, which must be provided when cultivating the cells on the lab; after injection on the patient, the Leishmania will change to the amastigote life form, which does not require 3’-nucleotidase/nuclease for the purine uptake; and if one of these cells happens to be ingested by a sandfly feeding on the patient, it will not be able to survive due to lack of nucleotides after it changes to the promastigote form.
To do that, we designed two constructs to make sure that both alleles for the 3’-nucleotidase/nuclease would be modified. Each construct contains either the T7 RNA polymerase ORF or the repressor protein TetR flanked by the 5’- and 3’-UTR promastigote constitutively and highly expressed genes; a drug resistance marker (puromycin or nourseothricin), were also flanked by the same UTR; and a T7 RNA polymerase promoter with a double operator for the TetR repressor protein. Both of the constructions were entirely flanked by genomic sequences preceding the native 3’-nucleotidase/nuclease gene to allow recombination to occur. The cell resistant selection for both puromycin and nourseothricin ensures that both alleles have undergone recombination.
