The Immunosuppressive TTAGGG Motif Improves Homology-Directed Insertion of DNASequences in Human Primary and Induced Pluripotent Stem (iPS) Cells

Abigail Blatchford1, Christopher B. Rohde1, I. Caglar Tanrikulu1, Taeyun Kim1, Matthew Angel1

1Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 30, No 4S1, 2022

Synthetic oligodeoxynucleotides (ODNs) have been used as repair templates in gene-editing applications to insert transgenic sequences into defined genomic loci, albeit with low efficiency. Cells engineered in this way are of interest for many therapeutic applications, including allogeneic NK and T cells engineered to express stealthing proteins, cytokines, and chimeric antigen receptors (CARs) for the treatment of a variety of cancers. To increase the efficiency of integration, gene-editing proteins can be co-expressed to create a double-strand break at the target locus. However, recognition of dsODNs by pattern recognition receptors activates signaling cascades resulting in the production of cytokines, including type I interferons such as IFIT1-3 and IFN-β. This immune response can lead to cell cycle arrest, differentiation, and apoptosis and may contribute to low insertion efficiency observed in primary and iPS cells. It has been shown in human immune cells that co-delivery of a short ODN comprising the immunosuppressive motif, TTAGGG, which is found in mammalian telomeric DNA, inhibits the activation of the damage-associate molecular pattern (DAMP) pathway in response to cytosolic DNA. This ODN competitively binds to inflammasomes, and reduces the secretion of proinflammatory cytokines. We hypothesized that the presence of the TTAGGG motif would decrease dsODN-related activation of a pro-inflammatory response in human cells, leading to higher transgene insertion efficiency. We incorporated the TTAGGG motif either at the 5’ end of dsODNs, or delivered it separately on a short single-stranded ODN (A151). Human primary fibroblasts, iMSCs and iPSCs were electroporated with a dsODN encoding a GFP reporter and containing an SfoI restriction site. Upregulation of pro-inflammatory markers including IFIT1-3, was measured by RT-PCR. We observed 29-fold higher expression of IFIT1 and IFIT3 in cells electroporated with dsODNs than in untreated controls. Interestingly, including TTAGGG motifs at the 5’-ends of the dsODNs limited the upregulation of IFIT1 and IFIT3 to 10- and 15-fold, respectively, while co-delivery of the TTAGGG motif prevented their upregulation altogether. We then used a gene-editing endonuclease targeting the AAVS1 safe-harbor locus on chromosome 19 to investigate the impact of the TTAGGG motif on the insertion of transgenes at this site. The TTAGGG motif (whether incorporated in the dsODN or co-transfected in the form of the A151 ODN) resulted in approximately 50% higher viability and approximately 50% more GFP-positive cells than when the motif was not present. We show that immunosuppressive sequences can increase ODN insertion efficiency and improve cell viability, and may therefore be a powerful tool for therapeutic knock-in applications, including the generation of knock-in iPS cell lines.