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.

Methods. Novel, small molecules, SR1, HDAC inhibitors, and UM171 were evaluated in the presence of cytokines to expand primary human CD34+ cells ex vivo . Cell number and immunophenotype were assessed by flow cytometry, and HSC function was evaluated by cell and molecular assays in vitro . The expanded cells were transplanted into sub-lethally irradiated NSG mice to evaluate engraftment potential in vivo . For editing studies, mPB and BM CD34+ cells were electroporated with CRISPR/Cas9 RNPs targeting the beta-2 microglobulin (B2M) cell surface protein. Editing rates were evaluated by flow cytometry based on loss of protein expression and TIDE analysis. Edited cells were expanded in the presence of AHR antagonist or vehicle and transplanted into NSG mice. Engraftment and editing rates were evaluated by flow cytometry of the peripheral blood and bone marrow.

Results. Based on our results, cultures expanded with an AHR antagonist showed the largest improvement in NSG engraftment levels compared to unmanipulated cells. Culture of CD34+ cells with SR1 or a novel AHR antagonist led to a 6-fold increase in CD34+ number and a significant increase in engraftment in NSG mice relative to vehicle-cultured CB derived CD34+ cells. Our proprietary compound displayed complete AHR antagonism in the dioxin response element luciferase reporter assay and was a more potent antagonist compared to SR1 (a 12-fold increase in potency). To evaluate the ability of the AHR antagonist compound to effectively expand gene edited cells, mPB and BM derived CD34+ cells were treated with either vehicle or AHR antagonist and the following day edited with CRISPR/Cas9 RNPs targeting B2M. Following 7 days of expansion, the vehicle or AHR antagonist-treated cells showed 87% and 84% loss of the target protein, respectively. The expanded culture contained 3.4-fold more CD34+CD90+ cells than the vehicle-treated cells. Upon transplant, mice receiving the expanded cells showed greater than 2-fold increase in engraftment compared to those receiving vehicle-treated cells. Importantly, the editing rates of the expanded cells are maintained in vivo with an average of >75% of the human cells in the periphery of the mice showing loss of target protein.

Conclusions. We have demonstrated that AHR antagonism is an effective strategy to expand functional HSCs and that novel small molecules inhibiting AHR can expand gene modified HSCs from mPB and BM. Studies are ongoing to confirm expansion with our proprietary compound of HSCs transduced with lentiviral vectors also leads to improved engraftment. This approach is a promising strategy to improve autologous gene therapy and gene editing via ex vivo HSC expansion.