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

Rad, Seyed Mohammad Ali Hosseini; Poudel, Aarati; Tan, Guan Huat; McLellan, Alexander D.

doi:10.1038/s41598-020-70022-0

Cited by 14 publications

(26 citation statements)

References 47 publications

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“…The target gene expression can be detected within 30 min after nontoxic Dox added and maximal expression levels can be higher than expression levels obtained from the CMV promoter 42 . Therefore, the Tet‐on system has been applied to develop a tissue‐specific delivery system on cell therapy and generate controllable transgene expression systems to produce animal models of disease 43–45 . Herein, we first generated inducible cell lines to quantify ZIKV C–C interaction based on the SLCA and Tet‐on system.…”

Section: Discussionmentioning

confidence: 99%

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Development and characterization of an inducible assay system to measure Zika virus capsid interactions

Huang

Wang

et al. 2022

Journal of Medical Virology

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The global spread of the mosquito‐borne Zika virus (ZIKV) infection and its complications including Guillain‐Barré syndrome and fetus microcephaly in 2015 have made ZIKV as a significant public health threat. The capsid protein plays crucial roles in ZIKV replication and thus represents an attractive therapeutic target. However, inhibitors of ZIKV capsid assembly have not been rigorously identified due to the lack of a target‐based screening system. In this study, we developed a novel ZIKV capsid interaction method based on a split‐luciferase complementation assay, which can be used to measure and quantify ZIKV capsid–capsid (C–C) interaction by the restored luciferase signal when capsid proteins interact with each other. Furthermore, a Tet‐on inducible stable cell line was generated to screen inhibitors of capsid dimerization. By using of this system, peptides (Pep.15‐24 in the N‐terminal region of ZIKV capsid protein and Pep.44‐58 in the α2 helix of ZIKV capsid protein) were identified to inhibit ZIKV C–C interaction. Overall, this study developed a novel inducible assay system to measure ZIKV capsid interaction and identify ZIKV capsid multimerization inhibitors, which will be applied for future discovery of ZIKV assembly inhibitors.

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Section: Discussionmentioning

confidence: 99%

“…By using of this system, synthetic peptides Pep.15-24 and Pep. [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] were identified to inhibit ZIKV C-C interaction. This well-established inducible assay system targeting ZIKV capsid interaction can be applied for future discovery of anti-ZIKV drugs.…”

mentioning

confidence: 99%

Development and characterization of an inducible assay system to measure Zika virus capsid interactions

Huang

Wang

et al. 2022

Journal of Medical Virology

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“…Recently, our group developed a Tet‐On system for CAR T cell applications. We showed that introducing a G72V mutation in rtTA (G72V‐rtTA) described previously for yeast, 105 significantly enhanced the Tet‐On system function in large gene cassettes containing a CAR 106 . Such a system might be beneficial when the expression of a miRNA needs to be regulated during T cell differentiation.…”

Section: Introductionmentioning

confidence: 94%

“…We showed that introducing a G72V mutation in rtTA (G72V-rtTA) described previously for yeast, 105 significantly enhanced the Tet-On system function in large gene cassettes containing a CAR. 106 Such a system might be beneficial when the expression of a miRNA needs to be regulated during T cell differentiation. For instance, let-7 has an increased expression within T N and T M cells whilst is downregulated in T EFF cells.…”

Section: Strategies To Overexpress Mirna In Car T Cellsmentioning

confidence: 99%

MicroRNA‐mediated metabolic reprogramming of chimeric antigen receptor T cells

et al. 2022

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Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti‐tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non‐immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre‐miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells.

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“…A solution to the basal activity of the Tet-on system has also been proposed in the CAR context, by introducing the G72V mutation in the TetR region. This resulted in a 40-fold reduction in Dox-independent activation [ 54 ]. An interesting study that proposed the possibility of additional control coupled the activation of the Tet system, not only to Tc but also to blue light similar to the Cre system [ 55 ].…”

Section: Receptor Switchesmentioning

confidence: 99%

Application and Design of Switches Used in CAR

Głowacki

Rieske

2022

Cells

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Among the many oncology therapies, few have generated as much excitement as CAR-T. The success of CAR therapy would not have been possible without the many discoveries that preceded it, most notably, the Nobel Prize-winning breakthroughs in cellular immunity. However, despite the fact that CAR-T already offers not only hope for development, but measurable results in the treatment of hematological malignancies, CAR-T still cannot be safely applied to solid tumors. The reason for this is, among other things, the lack of tumor-specific antigens which, in therapy, threatens to cause a lethal attack of lymphocytes on healthy cells. In the case of hematological malignancies, dangerous complications such as cytokine release syndrome may occur. Scientists have responded to these clinical challenges with molecular switches. They make it possible to remotely control CAR lymphocytes after they have already been administered to the patient. Moreover, they offer many additional capabilities. For example, they can be used to switch CAR antigenic specificity, create logic gates, or produce local activation under heat or light. They can also be coupled with costimulatory domains, used for the regulation of interleukin secretion, or to prevent CAR exhaustion. More complex modifications will probably require a combination of reprogramming (iPSc) technology with genome editing (CRISPR) and allogenic (off the shelf) CAR-T production.

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

Cited by 14 publications

References 47 publications

Development and characterization of an inducible assay system to measure Zika virus capsid interactions

Development and characterization of an inducible assay system to measure Zika virus capsid interactions

MicroRNA‐mediated metabolic reprogramming of chimeric antigen receptor T cells

Application and Design of Switches Used in CAR

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