ST-101: Manufacturing GMP-compliant HLA-compatible definitive human hematopoietic stem and progenitors cells from iPSCs using Piezo1 agonists for clinical transplant

Abstract Making transplantable human hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has been a longstanding objective of the field. Such a product would potentially provide the ability to produce unlimited numbers of HSC in vitro andeliminate the need to find HLA compatible donors for every patient requiring a transplant. However, an incomplete understanding of the signals driving the emergence of definitive HSC during development has limited this work. Recent studies demonstrated that the first heartbeat in the developing embryo induces activation of the mechano-sensitive Ca2+ channel Piezo1 in the ventral aorta-gonad-mesonephros (AGM) and promotes the formation of definitive HSC from bipotential hemogenic endothelial (HE) cells. Additional studies identified that the Piezo1 agonist Yoda1, can be used during in vitro culture of iPSC to generate transplantable hematopoietic stem and progenitor cells (HSPCs). These cells show high level long-term multilineage engraftment following primary and secondary transplant in immunodeficient mice with activity that is comparable to CD34+ cells isolated from umbilical cord blood (UCB)1. To manufacture HPSC for use in clinical transplant, a GMP compliant process was established that includes iPSC expansion, embryoid body (EB) culture to induce CD34+ mesoderm and a four-day endothelial to hematopoietic transition (EHT) culture. The final product, which we call PRIME HSPCTM contains 95% CD34+38- cells with 12% of these expressing markers of long-term HSPCs (CD34+90+45-) and ~ 88% expressing markers of multilineage progenitors (CD34+90-45+). The CD34 negative cells have a phenotype consistent with multipotent progenitors (CD34-90-45+). The product has no T cells, eliminating the potential risk of GvHD. To minimize the risk of graft rejection while preserving immune function, we established a process to create HLA-compatible banks of Stratus Prime HSPCs. High frequency HLA haplotypes were identified using NMDP and IPD-AFND databases along with an HLA editing strategy to maximize HLA matching for a single bank. The HLA editing approach consists of CRISPR disruption of HLA-A, HLA-DPB1, and HLA-DQB1. Using this approach, 7 banks made from high frequency donor haplotypes will cover 43% of the US population and 36% of the European population. To establish this process the most common haplotype covering 14.5% U.S., 12.9% EU population was used to produce the drug product ST-101. The Stratus Prime™ platform was employed to reprogram G-CSF mobilized peripheral blood (mPB) CD34+ cells from a healthy donor homozygous for the most common haplotype (HLA- B08:01, C07:01 and DRB1 03:01) into iPSCs to make the initial iPSC lines. Next, CRISPR editing was used to make HLA-edited iPSCs and establish clonal cell lines. These clonal lines were molecularly characterized to confirm on-target editing and examine potential off target sites. Genome integrity was confirmed by whole genome sequencing, optical genome mapping and targeted examination of potential oncogenes. The edited iPSCs retained stemness, pluripotency, and tri-lineage potential. Individual HLA-edited iPSC clonal lines passing these criteria were tested in the Stratus Prime Process for the ability to make HSPCs. Master and Working Cell Banks were established for the selected clone and the GMP process was employed to produce the ST-101 drug product. Upon transplant, ST-101 showed high level multilineage engraftment in primary and secondary mice comparable to the unedited line product and CD34+ cells from UCB. These results demonstrate high potency and safety of the ST-101 product. The Stratus PrimeTM Process demonstrates the ability to make HLA-compatible HSPCs in vitro to overcome donor dependency for HSCT. The ST-101 product has a high number of HPSCs which should facilitate rapid engraftment, does not contain T cells eliminating the risk of GvHD and is matched to HLA-compatible patients minimizing the possibility of graft rejection and enhancing immune function. Detailed molecular characterization of the ST-101 product is ongoing as are additional limit dilution transplant experiments in immune deficient mice to fully define the potency of the product in relation to current sources of human HSC. The strong pre-clinical safety and efficacy data provides support for advancing ST-101 into human clinical trials. 1 Scapin, et al. American Journal of Hematology 100.6 (2025): 963-979.