Genetic engineering of macrophages to acquire Phd2 +/- phenotype from induced pluripotent stem cells and its use for treatment of Limb Ischemia

Abstract

The main targets of thematic project are the modulation of macrophages by CSFs to promote the formation of M2 macrophages. These cells produce a series of growth factors, which promote vessel formation and remodeling, and myogenesis in ischemic and inflamed skeletal muscle. The main oxygen sensor in cells is the proline hydroxylase of HIF (Hypoxia Inducible Factor), which is called the PHD; PHD2 is believed to be the major isoform in this activity. PHD2 is a Fe(II)/2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of specific proline residues of HIF-5alfa, leading to degradation by ubiquitin pathway. Haplodeficient macrophages of the PHD2 (Phd2 +/-) gene have been reported to possess a M2-like phenotype which are capable of promoting arteriogenesis in ischemic animals. Therefore, these macrophages can be used in cell therapy for the treatment of Limb Ischemia; however, this type of cell (Phd2 +/- macrophages) does not exist naturally, being necessary to create and produce from iPS.In the recently published two papers with the support of this thematic project, we demonstrated that the alginate biomaterial is a great material to carry lentiviral vector and to release slowly this vector at the injection site. In addition, this delivery system allowed retention of this vector at the injection site, which is a very relevant issue in terms of biosafety. The injection of the macrophage suspension into the muscle can lead to the loss of these cells, which will decrease efficacy and time of therapeutic activity. An alternative to overcome this point could be the use of alginate microgels to deliver these cells.The objectives of this project are to create macrophages with the Phd2 +/- phenotype using induced pluripotent stem cells modified by Crispr-Cas9 and to evaluate the therapeutic effect of these macrophages in the free form or in the alginate gel in the murine Ischemic Limbs. (AU)