Fig. 3

Principles and criteria for choosing appropriate safe harbors for CAR transgene integration into the human genome. A Three-dimensional nuclear organization. The DNA strands inside the cell nucleus are in the open form (Euchromatin) or compressed form (Heterochromatin). Inserting the CAR construct into the Heterochromatin regions will imping on its consequential expression. B Epigenetic markers and regulatory DNA. Another level of regulation of gene expression in the genome is through epigenetics. In this case, according to histone codes and DNA methylations, the structure of the nucleosomes is changed (chromatin remodeling) and the expression of a gene is allowed. If a CAR transgene is integrated into an active transcription region, it is more likely for the transgene to be expressed correctly. C Activation of adjacent genes and essential gene disruption. DNA strands inside the nucleus are organized into TAD regions. These areas are separated by insulator elements. If a CAR construct is positioned within a TAD region, it is possible that it might interact with the neighboring genes in the same region, but not with other genes outside that given TAD. D CAR expression level and tonic signaling. If the CAR transgene is expressed permanently without proper regulation, the CAR protein accumulates on the surface of the engineered cell as they also bind to each other, causing tonic signaling. Conversely, if the CAR transgene is adjusted by appropriate regulatory factors such as promoter, enhancer, and silencers, the expression is carried out dynamically when needed, the frequency of CAR protein on the cell surface is appropriate and does not cause tonic signaling. Tonic signaling leads to more T cell differentiation and exhaustion