Donor Sequence Optimization Enables Targeted Insertion of Complex Stealthing Constructs in mRNA-Reprogrammed Induced Pluripotent Stem Cells

Elizabeth Belcher1, Raven Dance Hinkel1, Christopher B. Rohde1, Matthew Angel1, Kyle M. Garland1 1Factor Bioscience Inc., Cambridge, MA

 

2024 ISSCR Annual Meeting

Cellular therapies are an emerging field of medicine that involves the administration of cells as living agents to repair or replace damaged cells and tissues. While traditional cell therapies, such as autologous and donor-derived allogeneic therapies, hold great potential in combating intractable diseases, their clinical utility has been limited by several factors, including manufacturing complexities, high costs, safety considerations, scalability, and batch-to-batch consistency. Next-generation allogeneic cell therapies derived from mRNA-reprogrammed induced pluripotent stem cells (iPSCs) can directly address many of these challenges. However, iPSC-derived cells can still be subject to elimination by host immune cells, which can greatly reduce their efficacy. To address this challenge, we generated mRNA-reprogrammed iPSCs engineered to avoid elimination by host T cells and NK cells, primary drivers of immune-mediated rejection to allogeneic cell therapies. The engineered iPSCs do not express B2M but instead express a B2M-HLA-E fusion protein. UltraSliceā„¢ gene-editing mRNA was used to insert donor DNA containing a (G4S)4 linker and HLA-E gene sequence upstream of the endogenous B2M stop codon. Incomplete insertion of a linker sequence with high GC content (83.3%) was observed in edited cells, with one edited iPSC line missing 68 nucleotides from the transgene. We hypothesized that the high GC content of the linker sequence may promote secondary structure formation and lead to aberrant splicing during genomic insertion. Interestingly, a template containing a sequence-optimized linker with lower GC content (61.7%) resulted in full-length biallelic insertion in 2 of 16 colonies screened and exhibited detectable HLA-E expression via flow cytometry, while templates containing linkers with higher GC content (71.7% and 83.3%) did not (0 of 16 colonies in each case). We show that sequence optimization of donor DNA enabled the generation of mRNA-reprogrammed iPSCs with stealthing features. These cells may prove useful in the development of a broad range of engineered cell therapies.