Abstract
Regulated expression of genetic elements that either encode polypeptides or various types of functional RNA is a fundamental goal for gene therapy. Inducible expression may be preferred over constitutive promoters to allow clinician-based control of gene expression. Existing Tet-On systems represent one of the tightest rheostats for control of gene expression in mammals. However, basal expression in absence of tetracycline compromises the widespread application of Tet-controlled systems in gene therapy. We demonstrate that the order of P2A-linked genes of interest was critical for maximal response and tightness of a chimeric antigen receptor (CAR)-based construct. The introduction of G72V mutation in the activation region of the TetR component of the rtTA further improved the fold response. Although the G72V mutation resulted in a removal of a cryptic splice site within rtTA, additional removal of this splice site led to only a modest improvement in the fold-response. Selective removal of key promoter elements (namely the BRE, TATA box, DPE and the four predicted Inr) confirmed the suitability of the minimal CMV promoter and its downstream sequences for supporting inducible expression. The results demonstrate marked improvement of the rtTA based Tet-On system in Sleeping Beauty for applications such as CAR T cell therapy.