Pluristyx, Inc. Seattle, Washington, United States
Abstract: Engineered iPSC lines are critical for advancing allogeneic cell therapies by addressing immune rejection and ensuring that transplanted therapies can be safely removed from the patient if needed. However, current workflows for generating iPSCs harboring multiple edits are costly, time-intensive, and compromise genetic and epigenetic diversity. There is a pressing need for innovative approaches to reduce timelines and manufacturing costs while improving process efficiency and scalability. We leveraged our highly efficient, mRNA-based reprogramming technology to generate early-passage, genetically stable, polyclonal iPSC Seed Banks. These polyclonal banks serve as a robust starting material for engineering iPSC lines with enhanced safety and immune tolerance. To achieve this, we integrated a Ganciclovir-inducible suicide gene (HSV-TK) into the CDK1 locus, enabling selective elimination of dividing cells (FailSafe®+). Additionally, we performed targeted knockouts of the B2M and CIITA genes to suppress Human Leukocyte Antigen I and II expression (B2M/CIITA-/-), creating immune-evading iPSC lines. Using CRISPR-Cas9 and MAD7 nuclease, we conducted a multi-parameter process optimization to improve knock-in and multiplex knock-out efficiencies. These enhancements allowed us to maintain polyclonal populations through sequential edits, deferring clonal selection to the final process stages. This polyclonal editing strategy significantly reduced processing time and stressful passaging events, and enabled the cryopreservation of intermediate backups at critical stages to ensure process flexibility. Polyclonal editing allowed us to derive clinically relevant FailSafe®+, B2M/CIITA-/- iPSC lines while preserving genetic and epigenetic diversity. The optimized workflow is fully compatible with GMP manufacturing and significantly reduces timelines and costs for generating engineered iPSCs. By maintaining polyclonal populations throughout, our approach offers a scalable, cost-efficient solution for producing safe and immune-tolerant iPSC lines for allogeneic cell therapies, accelerating their translation to clinical applications.
