Autologous MPO Knock-out Hematopoietic Stem and Progenitor Cells for Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive condition for which there is no cure. Even with substantial pharmacologic advances in the modern treatment era, existing treatments provide only symptomatic relief and survival remains unacceptably poor. Patients who fail to respond to medications usually have the worst prognosis and patients with persistently elevated pulmonary pressure and right heart failure usually die within 5 years. PAH is a severe complication of Systemic sclerosis (SSc) affecting approximately 8%-12% of patients. SSc affects 13.5-44.3 per 100,000 individuals and is complex and diverse, with limited treatment options. SSc-associated PAH is the second largest group of patients with PAH. Once diagnosed, it becomes the leading cause of mortality among this patient population, with a 50% mortality rate within 3 years of PAH diagnosis. Inflammation and endothelial injury being the main components of the syndrome, investigation regarding the underlying mechanisms of this immune response may lead to the development of a new efficient therapy. There is extensive evidence for a pathogenic role for neutrophil myeloperoxidase (MPO) in PAH disease. PAH patients with higher levels of MPO have a poorer outcome. Studies in murine models of PAH demonstrate significant disease amelioration by elimination of neutrophil MPO by hematopoietic stem cell (HSC) transplantation with HSC lacking MPO from germline MPO gene knock-out. We developed a gene therapy approach to PAH in which autologous HSCs will undergo gene editing using CRISPR/Cas9 to achieve bi-allelic disruption of the MPO gene. These MPO-knock-out HSCs will then be autologous transplanted. That autologous transplantation of HSPC in which the MPO gene is disrupted is expected to prevent progression of PAH due to the essential role of neutrophil MPO activity on disease development. We identified a sgRNA that leads to high frequency disruption of MPO in primary human CD34+ hematopoietic stem and progenitor cells (HSPC), leading to efficient elimination of MPO protein in the resultant neutrophils. That sequence was assessed for genome-wide off-target and showed no detectable locus, validating it as a lead candidate for safe gene editing strategy. We validated in an in vitro neutrophil differentiation model the functional efficiency of the knock-out. MPO KO differentiated cells exhibit reduced neutrophil extracellular traps formation (NETosis) and absence of MPO protein. An optimized gene editing protocol for HSC was optimized and showed consistent allelic disruption over 80%. The edited cells retain hematopoietic potential and showed no skewing in the generated lineages in an in vitro colony forming assay. We developed a surrogate gene editing protocol for murine cells and optimized electroporation to achieve high allelic disruption of the murine MPO gene in lineage negative murine hematopoietic cells. The protocol will be tested in a hypoxia-induced PAH murine model. The presented work can represent a major improvement for PAH patient. The limited therapeutic options and severity of the disease urges to develop new approaches. The gene therapy protocol developed showed promising result in vitro and rely on clinical evidences for the implication of MPO in the disease. Results will be tested in a pre-clinical murine model