Leveraging Stage-Specific Promoters to Enhance Immune Cell Engineering in iPSC-Derived Cells for Cancer Immunotherapy
Authors: Perez Arina , Hall Matthew S , Lu Sumei , Peterka Brad , Naso Michael , Levitsky Hy , Gurung Buddha 
Published: December 2024

iPSC-derived immune effectors including iNK cells and iT cells hold tremendous promise as therapeutics for cancer and autoimmunity. A key advantage of the iPSC platform is the ability for unlimited genetic engineering thereby enabling the generation of bespoke therapies tailored to specific malignancies. This includes knocking out genes of interest (GOI) as well as site-specific knock-in of transgenes using CRISPR nuclease technology. Commonly used constitutive promotors, including CAG and EF1α, drive robust transgene expression in iPSCs that is maintained through the differentiation process. For a small subset of transgenes however, including homeostatic cytokines and Chimeric Antigen Receptors (CAR), that might interfere with the differentiation of iPSC to a particular immune effector cell, utilizing a constitutive promoter might not be an ideal strategy. Devising new methodologies of iPSC gene-editing that enable expression of such transgenes in a context-dependent fashion would be highly desirable for iPSC-based therapies. Here we describe a differentiation-stage-specific gene expression system that utilizes CRISPR nuclease technology to engineer transgenes in a site-specific manner in iPSCs ensuring expression exclusively upon differentiation into the desired effector cell. Comparative global transcriptomic analysis of cells throughout the differentiation process from iPSCs to gamma delta (γδ) iT cells was conducted to identify endogenous genes with differentiation-stage-specific expression. Longitudinal analysis revealed several dozen genes that are silent at both the iPSC and Hematopoietic Progenitor Cell (HPC) stage with meaningfully detectable transcript levels late during γδ iT cell differentiation process. Based on the kinetics and magnitude of transcript levels in terminal effector cells and an understanding of their inherent function in T cells, we selected five candidate genes for engineering using our differentiation-stage-specific expression system.

Transgenes of interest were knocked into the locus of the set of genes identified above by Homology Directed Repair (HDR) using a CRISPR nuclease and guide RNAs (gRNAs) targeting the COOH terminus. The donor template plasmid facilitated removal of the stop codon in the target gene locus, introduction of a ribosomal skip site (P2A) followed by the transgene of interest. Successful insertion of the transgene was confirmed by junction-PCR, and furthermore NGS sequencing of the amplicon ensured retention of the proper reading frame. Clonal iPSC lines engineered with the transgene were generated, characterized for transgene copy number, evaluated for mono- or bi-allelic insertion(s) and subsequently differentiated to γδ iT cells. The engineered iPSC clones were monitored for transgene expression at different stages of the differentiation process, and the trajectory of the differentiation process towards a desired effector cell was monitored throughout by lineage-specific biomarker expression. As expected, transgene expression was not detected at the iPSC and HPC stages. The emergence of transgene expression coincided with the emergence of early iT cell markers, peaking in mature γδ iT cells. Comparative analysis of lineage-specific biomarkers revealed a differentiation trajectory concomitant with un-engineered γδ iT cells, in stark contrast to cells derived from iPSCs where the transgene is constitutively expressed using a strong synthetic promoter. Assays to evaluate the impact on cell fitness and functionality in vitro and in vivo are currently underway. Preliminary observations indicate significant increases in cell viability, proliferation, and lineage-specific markers, suggesting promising improvement in functional outcomes of the cell therapy product.