Introduction/Contextualization

The ability to genetically manipulate a single cell type within a complex mixture of cells, as those found in the human body, carries enormous therapeutic and diagnostic potential. However, the generation of devices for the delivery of genetically encoded messages to specific cell types is not possible. One major limitation is the paucity of truly cell‐type specific regulatory elements with gene-driving capacity (e.g., enhancers). Tests using a human regulatory element activity atlas have demonstrated that most cells do not possess reliable cell-type specific enhancers. Intersectional genetics approaches, employing regulatory elements, can increase both the breadth and robustness of cells amenable to specific genetic targeting. This observation led to the concept of Versatile Entry Codes (VEnCodes), which are the smallest intersections of co-activated regulatory elements capable of distinguishing the target cell from non-target cells within a complex mixture.

Objectives

This study aimed to generate a genetic biosensor for human induced Pluripotent Stem Cells (hiPSCs) by developing a split-protein strategy as an intersectional method and coupling it to hiPSC VEnCodes obtained via different methodologies.

Methodology

We generated a functional split version of the reverse tetracycline-dependent transcriptional activator (rtTAv) using a combination of 3 orthogonal split inteins (NpuDnaE, gp41-1, and NrdJ-1). Two hiPSC lines (IMR90-4 and Nelson) were used to validate the activity of known pluripotent stem cell enhancers (OCT4, SOX2, and Nanog), as well as an enhancer identified from publicly available epigenomics data—the pSCE2. These intersections were assessed with our custom 2× and 3× split-rtTAv systems, and specificity was confirmed by testing in a non-hiPSC line (HeLa).

Results and Conclusions

Several functional intersections were identified, demonstrating robust activity in hiPSCs and negligible activation in HeLa cells. Moreover, by leveraging VEnCode algorithms developed in our laboratory on curated public CAGE-seq (FANTOM5) data, an additional 12 hiPSC enhancers were validated. These findings indicate that VEnCodes, when integrated with intersectional genetic methods, hold promise for overcoming the limitations of current gene therapies and genetic modification techniques through enhanced specificity and sensitivity in genetic delivery.