Evaluation of the allelic variant (L162V) of the human PPARalfa gene in the establishment of Leishmania infantum infection: use of CRISPR-Cas9 technology for knock-in in THP-1 cell line

• Grant number: 17/25854-3
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2018
• End date: March 31, 2021
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Paulo Eduardo Martins Ribolla
• Grantee: Isabela Gaseta Ferraz Paiva
• Host Institution: Instituto de Biotecnologia (IBTEC). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated scholarship(s): 18/23089-0 - Evaluation of CRISPR-Cas9 delivery methods in THP-1 cell lines for genomic editing targeting PPAR± gene, BE.EP.MS

Abstract

Activated peroxisome proliferation receptors (PPARs) make up a subfamily ofnuclear receptors. Three isoforms, encoded by separate genes, were identified sofar: PPARa, PPARb and PPARg. These receptors, which act as transcription factors,exhibit different functions and distributions in tissues. To date, it has been knownthat the genes regulated by PPARs participate mainly in the regulation of keyproteins, involved in the intra- and extracellular metabolism of lipids, oxidation offatty acids and inflammatory processes. PPARa, in particular, is expressed in anumber of metabolically active tissues, including liver, kidney, heart, skeletal muscle,adipose tissue, and macrophages that play an important role in lipid metabolism aswell as being part of the immune system. Due to its influence on lipid regulation andthe fact that its activity is easily modulated by synthetic drugs, PPARa is considered avery important target for the effective treatment of dyslipidemias. Lipid disordershave been reported in human patients and even in dogs with active VisceralLeishmaniasis (LV). LV in Brazil is a parasitic disease caused by infection of hostmacrophages by the protozoan Leishmania infantum and transmitted by the sandflyLutzomyia longipalpis, with wild canids and domestic dogs as its main reservoirs. Inthis context, PPARa might be a possible candidate gene to contribute to thegenetically determined risk of VL. In a case-control study conducted by our group inthe endemic area of Teresina-PI genotypes containing the mutated 162V allele weresignificantly more frequent among individuals with VL than among all uninfectedindividuals (p = 0.007). In addition healthy individuals having at least one mutated162V allele are nearly four times more likely to contract the disease than individualswho do not have the mutation (odds ratio 3.91 and confidence interval 1.38-11.07)The present project aims to use CRISPR-Cas9 technology for the generation ofgenetically modified macrophages to contain the human PPARa gene L162Vmutation for in vitro infection assays with Leishmania infantum as well as an overallanalysis of the gene expression profile of the modified cells with the purpose ofevaluating the role of the L162V allelic variant of the PPARa gene in theestablishment and development of Visceral LeishmaniasisThe present project aims to use CRISPR-Cas9 technology for the generation ofgenetically modified macrophages to contain the L162V mutation of the humanPPAR± gene to perform in vitro infection trials with Leishmania infantum in order toevaluate the role of the allelic variant L162V of the PPAR± gene in the establishmentand development of Visceral Leishmaniasis. (AU)