Fig. 1

A Lentiviral components of RB‐312. RB-312 includes two lentiviral (LV) constructs. LV#1 (HER2‐TEV) encodes an anti‐HER2 (4D5 clone) scFv combined to the CD28 and CD3ζ co‐stimulatory domains, the TEV protease and IL12Asg and IL12Bsg targeting the TSS of the endogenous IL12A (encoding p35 subunit) and IL12B (encoding p40 subunit) genes, which are driven by human U6 (hU6) and H1 promoters (named “RB‐312_hh”) or hU6 and mouse U6 (mU6) promoters (named “RB‐312_hm”). LV#2 (LdCV) encodes LAT, fused to dCas9‐VPR via a TEV‐cleavable sequence (TCS). Bicistronic expression of tNGFR and Q8 was used for detection of HER2-TEV and LdCV, respectively. B Mechanism of activation of HER2‐TEV/LdCV CRISPRa platform—Activation of HER2 CAR brings TEV in proximity of LdCV releasing dCas9‐VPR for nuclear translocation to the IL12A and IL12B TSS and conditionally and reversibly upregulated IL-12/p70 heterodimer expression. The figure represents the CRISPRa logic at steady‐state levels of LdCV expression. However, LdCV expression varies according to the physio‐metabolic status of individual cells resulting in variable degree of transcriptional activity and consequently different substrate availability for TEV cleavage