Aryl Hydrocarbon Receptor Antagonists Expand Adult Hematopoietic Stem Cells From Mobilized Peripheral Blood and Bone Marrow and Increase the Dose of CRISPR/Cas9 Gene-Edited NSG-Repopulating Cells

Background. Gene modified hematopoietic stem cell (HSC) transplant is emerging as a promising approach to treat a variety of inherited genetic diseases, but has been hampered by the inability to generate a sufficient dose of gene modified HSCs and the requirement for toxic myeloablative conditioning protocols to achieve engraftment. Ex vivo expansion of gene-modified HSCs is a promising approach to circumvent these challenges as high doses of gene modified cells have the potential to achieve robust engraftment and reduce the requirement for myeloablative conditioning. Several small molecule approaches (SR1, UM171, and HDAC inhibitors) have been reported to expand cord blood (CB) derived HSCs ex vivo but the ability of these agents to expand gene modified adult HSCs is not known. Experiments comparing these approaches for CB expansion has been reported (see Goncalves et al “Phenotype Does Not Always Equal Function: HDAC inhibitors and UM171, but not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-negative Human Cells”) and found that aryl hydrocarbon receptor (AHR) antagonism is the most effective at expanding NSG-engrafting cells. In a clinical trial of patients receiving MGTA-456, an AHR antagonist-expanded CB CD34+ cell product, engraftment occurred at a median of 14.5 days (r 7-23), which was significantly better than identically-treated historical controls (n=151, p<0.01) (Wagner et al, Cell Stem Cell, 2016). Because of the encouraging clinical results in CB, we identified novel AHR antagonists and evaluated the ability of SR1 and the proprietary AHR antagonists to increase the number of gene modified NSG engrafting cells manufactured from mobilized peripheral blood (mPB) and bone marrow (BM) CD34+ cells.