Genetic engineering of mouse ESCs by CRISPR-Cas9 to produce macrophages-Phd2 +/-

• Grant number: 19/04597-8
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: August 01, 2019
• End date: March 20, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Sang Won Han
• Grantee: Patrícia Terra Alves
• Supervisor: Lesley M Forrester
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Institution abroad: University of Edinburgh, Scotland
• Associated to the scholarship: 18/09203-5 - Genetic engineering of macrophages to acquire Phd2 +/- phenotype from induced pluripotent stem cells and its use for treatment of Limb Ischemia, BP.PD

Abstract

Peripheral arterial disease (PAD) is a circulatory disorder caused by atherosclerosis, which affects millions of people every year. In the advanced stage of PAD, this disease evolves to critical limb ischemia (CLI) which severely affects the limb's blood circulation, causing pain at rest and nonhealing wounds. Therefore, the promotion of angiogenesis and control of inflammation should favor the improvement of CLI. The main oxygen sensor in cells is the proline hydroxylase of HIF (hypoxia inducible factor), which is called the PHD, and PHD2 is the major isoform. PHD2 is a Fe (II) / 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of specific proline residues of HIF1-±, leading to degradation via ubiquitin. According to Takeda et al. (Nature 2011;479(7371):122), macrophages that are haplodeficient at the PHD2 locus (PHD2+/-) present a M2-like phenotype and are capable of promoting arteriogenesis. These macrophages could be a good candidate for the treatment of limb ischemia, however, these cells are no available for test. This project aims to generate mouse embryonic stem cells (ESCs) that have been genetically modified to be haplodeficient by knocking out one allele PHD2 using CRISPR-Cas9. These ESCs will be differentiated into PHD2+/- macrophages and their therapeutic efficacy will be assessed in a mouse model of limb ischemia.