Mitochondrial Augmentation of Hematopoietic Stem and Progenitor Cells As a New Therapeutic Approach for Myelodysplasia

Currently the only curative treatment option for patients with myelodysplastic syndromes (MDS) is allogeneic bone marrow transplant. However, allogeneic transplant is not feasible for the majority of MDS patients. Several studies have identified that mitochondrial dysfunction may underlie hematopoietic defects of multiple MDS subtypes. Mitochondrial augmentation technology has been recently developed whereby healthy, functional mitochondria are introduced into diseased hematopoietic stem/progenitor cells (HSPCs) characterized by mitochondrial dysfunction. The “augmented” autologous cells are then systemically infused to the recipient patient. This technology was initially tested in primary mitochondrial diseases using syngeneic, maternal blood derived mitochondria (MNV-101). Recently, an allogeneic placental mitochondria source was developed (MNV-201) and is undergoing a phase I trial to treat Pearson Syndrome, a pediatric disease caused by single mitochondrial DNA deletions, resulting in sideroblastic anemia and high frequency of MDS. In 15 primary mitochondrial disease patients treated to date with MNV-101 or MNV-201, no conditioning was applied, and preliminary safety and efficacy were observed. We therefore sought here to evaluate a similar mitochondrial augmentation cell therapy approach in spontaneous adult MDS. We hypothesized that enrichment of MDS patient-derived cells with healthy, functional mitochondria may improve HSPC functionality and rescue ineffective hematopoiesis. We subjected bone marrow cells from low-risk (LR) MDS patients to augmentation with exogenous allogeneic mitochondria from healthy term placenta or a negative control. Mitochondrial augmentation increased erythroid differentiation capacity in all five LR MDS patient samples based on culturing HSPCs in Stemspan medium with erythroid promoting cytokines (a 2-fold mean increase in cell number ± 0.8 with mitochondrial augmentation was seen, p=0.02, one sample Wilcoxon test). Immunophenotypic assessment confirmed increased absolute numbers of mature erythroid cells (CD71+/Ter119+) in the augmented group. Xenotransplantation of patient MDS cells into immunocompromised mice has been a historical challenge. Out of six bone marrow derived LR MDS patient samples, five showed engraftment (defined as >1% human CD45+ cells) ~17 weeks post-transplantation into unirradiated NBSGW mice. Interestingly, intravenous infusion of mitochondrially augmented bone marrow derived cells from LR MDS patients resulted in greater (1.6-5.3-fold increase) engraftment in bone marrow relative to non-augmented samples with multilineage (myeloid and B- and T- lymphoid) reconstitution in two of five patients. Studies to determine the impact of mitochondrial augmentation on clonality based on MDS-associated somatic mutations did not demonstrate alterations in variant allele frequency after in vitro or in vivo studies, and no increase in AML-related mutations was observed. To further assess the impact of mitochondrial augmentation on MDS therapeutic differentiation and transformation potential in vivo, we utilized lineage negative HSPCs from the NUP98-HOXD13 (NHD13) mouse model of MDS. Irradiated C57BL mice received an infusion of 200,000 HSPCs from aged (6-month-old) wild-type C57BL mice or NHD13 mice that were either unprocessed or mitochondrially augmented with placenta-derived mitochondria from healthy mice of a distinct strain (NZB mice). Within 35 days, all ten animals which received non-augmented NHD13 cells were dead or moribund due to AML development, whereas all animals which received augmented NHD13 HSPCs survived with a median of 114 days (p<.0001, log-rank test). Importantly, blood counts were not affected by mitochondrial augmentation in wild-type control bone marrow. Taken together, the accumulated studies with mouse and human MDS models suggest the potential for mitochondrial augmentation of HSPCs to improve the differentiation defect of a subset of human MDS, enhance in vivo reconstitution potential, and postpone transformation to acute leukemia. These preclinical results support the potential of mitochondrial augmentation of HSPCs as a novel approach to treat MDS patients utilizing autologous cell therapy.