Team:UFMG Brazil/backup2

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LeishMANIA: from pathogens to drug delivery systems

Rheumatoid arthritis (RA) is an autoimmune disease that culminates in a chronic inflammatory condition affecting the joint lining. Inflammatory macrophages are central cells to the pathophysiology of this disease due to their prominent numbers in the inflamed synovial membrane and at cartilage-pannus junction, their clear activation status and their response to successful anti-rheumatic treatment. There is no cure for RA and most of the used drugs are anti-inflammatory compounds with oral and/or parenteral administration. Because of this, the typically prescribed medications have potentially serious systemic side effects. Besides, biological characteristics of the joints difficult drug delivery to the target site, compromising the efficacy of treatment. Therefore, we propose the use of an optimized visceral Leishmania strain to direct drugs to specific macrophages associated to RA. Leishmania was selected because it is able to infect macrophages and it produce recombinant proteins with post-translate modifications similar to humans. This last characteristic is important because the drug of choice is interferon beta (INF-β) that has N-glycosylation sites that increase stability and activity of molecule and N-glycosylation process in Leishmania adds a glycidic chain very similar to humans unlike bacteria, yeast and other fungus. INF-β is known to induce clinical improvement due to decreased synovial inflammation and protection against joint destruction during RA, but efficient treatment of RA patients has been unsuccessful due to pharmacokinetic issues and novel approaches leading to constitutive INF-β production at the site of inflammation may be required to induce clinical efficacy.

The project will start with a visceral Leishmania with previous deleted virulence factor. This modified strain of the parasite Leishmania donovani has the virulence factor centrin-1 deleted. This parasite is a good new chassi parasite because it has ability to express proteins with proper post-translational modifications, such as glycosilations and dissulfide bonds and deliver these proteins inside macrophages. This strain was already proved to have very low risk of contamination and the virulence reduction of this Leishmania strain has been evaluated and confirmed with different animal models, such as mice (Selvapandiyan et al., 2006), hamsters (Selvapandiyan et al., 2009) and dogs (Fiuza et al., 2014 ). Besides, human macrophage infection by this strain has also been proved to be reduced in vitro (Selvapandiyan et al., 2004).

First, the parasite will be transfected with genes that compose the machinery to control to produce of INF-β in amastigotes specifically localized in inflammatory macrophages. This molecular switch will be controlled by A2 protein 5`UTR and 3`UTR, a amastigote specific regulatory region and it will respond to three biomarkers present in high concentration in inflammatory macrophages from joints during RA. After, the resulting parasites will also be handled by incorporation of set of vectors containing kill switch machinery previously developed by our group to prevent replication of mutant parasites in insect hosts during accidental infection of these vectors. . Genetic manipulation of Leishmania will be performed by CRISPR/Cas system that allows insertion or deletion of multiples genes in one step. Mutant parasites will be tested firstly using in vitro inflammatory macrophage models and RA mouse models. The engineered parasite will be labeled with technetium-99m and intravital imaging of parasite distribution and concentration in inflamed joints from RA mouse models will be captured by gamma counter equipment.

In addition to the LeishMANIA project, the UFMG_Brazil iGEM team also focused on the scientific divulgation problem. As the project depends on public acceptance, since the team have the objective to use a genetically modified organism to treat a disease, the group are aware that it will face some resistance especially from people who are not close to science, because of the various prejudices involved. Thereby, we believe that is our responsibility also promote a way to bring people closer to the scientific development, whereas people become more open when they have more knowledge. In that way, the team created the project EMBRYO, aiming to increase the exchange of information between scientific and general public, through a communication channel in which society can pose their questions and demands to the scientific community.

References

Selvapandiyan A, Debrabant A, Duncan R, Muller J, Salotra P, Sreenivas G, Salisbury JL, Nakhasi HL. Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania. J Biol Chem. 2004 Jun 11;279(24):25703-10. Epub 2004 Apr 14. PubMed PMID: 15084606.

Selvapandiyan A, Duncan R, Debrabant A, Lee N, Sreenivas G, Salotra P, Nakhasi HL.Genetically modified live attenuated parasites as vaccines for leishmaniasis.Indian J Med Res. 2006 Mar;123(3):455-66. Review. PubMed PMID: 16778323.

Selvapandiyan A, Dey R, Nylen S, Duncan R, Sacks D, Nakhasi HL. Intracellular replication-deficient Leishmania donovani induces long lasting protective immunity against visceral leishmaniasis. J Immunol. 2009 Aug 1;183(3):1813-20. doi: 10.4049/jimmunol.0900276. Epub 2009 Jul 10. PubMed PMID: 19592661.

Fiuza JA, Gannavaram S, Santiago Hda C, Selvapandiyan A, Souza DM, Passos LS, de Mendonça LZ, Lemos-Giunchetti Dda S, Ricci ND, Bartholomeu DC, Giunchetti RC, Bueno LL, Correa-Oliveira R, Nakhasi HL, Fujiwara RT.Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum. Vaccine. 2015 Jan 3;33(2):280-8. doi:10.1016/j.vaccine.2014.11.039. Epub 2014 Dec 1. PubMed PMID: 25475955.