The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

Tsukahara, Tomonori; N, Iwase; Kawakami, Koichi; Iwasaki, Miki; Yamamoto, Chiyo; Ohmine, Ken; Uchibori, Ryosuke; Teruya, Takeshi; Ido, Hiroyuki; Saga, Yasushi; Urabe, Masashi; Mizukami, Hiroaki; Kume, Akihiro; Nakamura, Masataka; Brentjens, Renier J.; Ozawa, Keiya

doi:10.1038/gt.2014.104

Cited by 30 publications

(30 citation statements)

References 38 publications

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“…Also, Tol2 vectors have been used for transfection of foreign DNAs into mammalian cells including ESCs, iPSCs and human lymphocytes [86, 93, 94]. In addition, Tol2 vectors with a fluorescent marker or a transcription activator such as rtTA have been introduced to chick and mouse embryos by electroporation to label and analyze specific cell types in vivo [95, 96].…”

Section: Transposons and Functional Genomicsmentioning

confidence: 99%

Transposons As Tools for Functional Genomics in Vertebrate Models

2017

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Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice and rats.

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Section: Transposons and Functional Genomicsmentioning

confidence: 99%

Transposons As Tools for Functional Genomics in Vertebrate Models

2017

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“…136 Tol2-mediated CD19specific CAR expression in T-lymphocytes was recently demonstrated to suppress B cell lymphoma progression both in vitro and in vivo. 73 Comparative analysis indicated that PB was more robust than SB11 and Tol2 systems. 137 In vivo transposon delivery Transposon-mediated gene transfer has also been validated for in vivo transfection.…”

Section: Hscmentioning

confidence: 99%

“…70 Tol2 transposon is able to deliver a genetic cargo up to 10-11 kb in several mammalian cells without compromising transposition efficiency. [71][72][73][74] Moreover, the Tol2two-component system has been used successfully to generate transgenic animals such as mouse, chicken, frog, [75][76][77][78][79] and particularly zebrafish models. 72,77,80,81 Like SB and PB, the Tol2 system enables sustained transgene expression after gene delivery, thereby providing the advantage of being suitable vector for gene therapy 72,73 (Fig.…”

Section: Tol2mentioning

confidence: 99%

Transposons: Moving Forward from Preclinical Studies to Clinical Trials

Tipanee¹,

VandenDriessche

Chuah

2017

Human Gene Therapy

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Transposons have emerged as promising vectors for gene therapy that can potentially overcome some of the limitations of commonly used viral vectors. Transposons stably integrate into the target cell genome, enabling persistent expression of therapeutic genes. Transposons have evolved from being used as basic tools in biomedical research to bona fide therapeutics. Currently, the most promising transposons for gene therapy applications are derived from Sleeping Beauty (SB) or piggyBac (PB). Stable transposition requires co-delivery of the transposon DNA with the corresponding transposase gene, mRNA, or protein. Stable transposition efficiency can be substantially increased by using "next-generation" transposon systems that combine codon-usage optimization with hyper-activating mutations in the SB or PB transposases. By virtue of their relatively large capacity, gene therapy applications with relatively large therapeutic transgenes, such as full-length dystrophin, can now be envisaged. The authors and others have shown that efficient and stable gene transfer can be achieved with these next-generation transposons in several clinically relevant primary cells, such as CD34 hematopoietic stem/progenitor cells, T cells, and mesenchymal and myogenic stem/progenitor cells that are amenable for ex vivo transfection. Alternatively, in vivo transposon gene delivery has been explored using non-viral vectors or nanoparticles or in combination with viral vectors. The therapeutic potential of these SB- and PB-based transposons has been demonstrated in preclinical models that mimic the cognate human diseases. However, there are still challenges impeding clinical translation of transposons pertaining mainly to the typical limiting efficiencies of most non-viral transfection methods and the intrinsic DNA toxicity. Nevertheless, it is particularly encouraging that transposons have now been used in gene therapy clinical trials. In particular, transposon-engineered T cells expressing chimeric antigen receptors are starting to yield promising results in patients with hematological malignancies.

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“…The potential for this risk arises from the random nature of integration into the host genome, although no instances of this occurring in clinical trials using CAR-T cells engineered through these methods have been reported. Alternatively, transposon-based systems are capable of delivering a larger payload (~100 kb), cost effective, scalable and potentially less immunogenic than viral vectors [220][221][222] . This approach requires delivery of plasmid DNA encoding the transgene and transposase components, which is most commonly achieved through electroporation 221 , a method that can be toxic to the cells and is not as efficient as viral-based delivery.…”

Section: Delivery Of Car Genesmentioning

confidence: 99%

Programming CAR-T cells to kill cancer

2018

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T cells engineered to express chimeric antigen receptors (CARs) that are specific for tumour antigens have led to high complete response rates in patients with haematologic malignancies. Despite this early success, major challenges to the broad application of CAR-T cells as cancer therapies remain, including treatment-associated toxicities and cancer relapse with antigen-negative tumours. Targeting solid tumours with CAR-T cells poses additional obstacles because of the paucity of tumourspecific antigens and the immunosuppressive effects of the tumour microenvironment. To overcome these challenges, T cells can be programmed with genetic modules that increase their therapeutic potency and specificity. In this Review Article, we survey major advances in the engineering of next-generation CAR-T therapies for haematologic cancers and solid cancers, with particular emphasis on strategies for the control of CAR specificity and activity and on approaches for improving CAR-T-cell persistence and overcoming immunosuppression. We also lay out a roadmap for the development of off-the-shelf CAR-T cells.

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The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

Cited by 30 publications

References 38 publications

Transposons As Tools for Functional Genomics in Vertebrate Models

Transposons As Tools for Functional Genomics in Vertebrate Models

Transposons: Moving Forward from Preclinical Studies to Clinical Trials

Programming CAR-T cells to kill cancer

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