Optimisation of Tet-On inducible systems for Sleeping Beauty-based chimeric antigen receptor (CAR) applications

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...

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Bibliographic Details
Published in:Scientific reports 2020-08, Vol.10 (1), p.13125, Article 13125
Main Authors: Ali Hosseini Rad, S. M., Poudel, Aarati, Tan, Grace Min Yi, McLellan, Alexander D.
Format: Article
Language:English
Subjects:
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Amino Acid Sequence
Antigens
Cell therapy
Chimeric antigen receptors
DNA Transposable Elements - genetics
Gene expression
Gene therapy
HEK293 Cells
Humanities and Social Sciences
Humans
Lymphocytes T
Models, Molecular
multidisciplinary
Mutation
Protein Conformation
Receptors, Chimeric Antigen - chemistry
Receptors, Chimeric Antigen - genetics
Response Elements - drug effects
Ribonucleic acid
RNA
Science
Science (multidisciplinary)
Tata box
Tetracycline - pharmacology
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-70022-0