Construção e avaliação funcional de um plasmídeo terapêutico para expressão da Interleucina 10 (IL-10) de Mus musculus em células mamíferas, utilizando como veículo carreador linhagens de Lactococcus lactis invasivas: desenvolvimento de uma estratégia alternativa para o tratamento de doenças inflamatórias intestinais

Abstract

Mucosal surfaces are the principal sites of interaction between the body and the external environment, constituting one of the first lines of defense of an organism, and as such, are considered to be an attractive target for the delivery of biological drugs. In this regard, special attention has been given, in the last decades, to the development of more efficient strategies for delivery of vaccine and/or therapeutic plasmids to the mucosa. Theuse of bacteria carrying these plasmids, by oral route, constitutes a promising strategy; hereupon, the use of non pathogenic bacteria, such as Lactic Acid Bacteria (LAB), constitutes a safe choice for such purposes. Lactococcus lactis is the LAB model and has been extensively used for the production and delivery of antigens and cytokines to themucosal level. In this context, with the goal of optimizing plasmid delivery to human epithelial cells, invasive strains of L. lactis as well as a replicative plasmid in L. lactis, containing a eukaryotic expression cassette called pValac were constructed. Thus, the use of the invasiveL. lactis strain capable of delivering the pValac vector coding for Interleukin 10 (IL-10) of Mus musculus, a powerful anti-inflammatory cytokine, could represent a new strategy for the treatment of inflammatory diseases such as inflammatory bowel diseases (IBDs). So, thegoal of this project is to construct a therapeutic plasmid, pValac::IL-10, and verify its functionality in vitro, as well as its cloning into the invasive L. lactis strain, aiming at developing an alternative and more efficient therapeutic treatment against IBDs. The coding sequence of IL-10 was obtained from a synthetic plasmid containing the ORF of IL-10 fromM. musculus, which was first cloned into the Zero Blunt® TOPO® and subsequently into the pValac vector. To evaluate the functionality of the pValac::IL-10 plasmid, cells of the CHO (Chinese Hamster Ovary) cell line were transfected with the constructed plasmid and expression of IL-10 by these cells was evaluated by ELISA, Western Blot, Confocal Microscopy and FACS. The pValac::IL-10 plasmid was finally transformed into the invasive L. lactis strain and the same was then used in an in vivo pilot trial to evaluate its therapeutic and immunomodulatory capacity in mice with experimental IBD. Ultimately, this work constitutes a first step towards the development of a new strategy to control inflammatory diseases, besides validating the efficiency and effectiveness of the invasive strain of L. lactisas vehicle for new DNA therapies and vaccines.