Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors

Monjezi, Razieh; Miskey, Csaba; Gogishvili, Tea; Schleef, Martin; Schmeer, Marco; Einsele, Hermann; Ivics, Zoltán; Hudecek, Michael

doi:10.1038/leu.2016.180

Cited by 207 publications

(228 citation statements)

References 51 publications

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“…The discovery that the PB closer end vectors could significantly increase transposition efficiency in both DNA-PK deficient (SCID) and normal cells (BALB/c) supports the importance of PEC formation. Consistent with previous discoveries in different vector systems (25,26), these data indicate that shorter external spacers increase transposition efficiency and provide a simple practical principle for the design of transposon vectors (31). Given that the DNA-PK complex is also involved in the transposition of other DNA transposons (27)(28)(29), the approach using closer end vectors could be applied to other mobile elements for designing efficient vector systems.…”

Section: Discussionsupporting

confidence: 69%

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Jin

Chen

Zhao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.piggyBac | DNA-PK | paired-end complex formation | transposition efficiency | CE transposon vectors T ransposition allows DNA transposons to serve as powerful genetic manipulation tools. During transposition, DNA transposons follow a "cut-and-paste" manner. A paired-end complex (PEC) is first formed by aligning both ends of the transposon, which is then excised and inserted into new loci (1, 2). Although the transposon-encoded transposase is suggested to be sufficient for PEC formation, it is unlikely to be the only protein involved in transposition. In fact, many DNA transposons are nonfunctional outside their natural hosts, suggesting the effects of host factors (3).piggyBac (PB) is a DNA transposon originally identified from the cabbage looper moth (4). Modified PB is highly active in mouse and human cells (5). Because of its potent transposition efficiency and ability to carry large insertions, PB provides unique opportunities for mutagenesis and transgenesis in mammalian systems (6-10). Although the PB transposon has a wide host range, including mammals, its transposition efficiency varies in cultured cells from different mammalian species (7,9). Thus, host factors may still be involved in the proces...

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Section: Discussionsupporting

confidence: 69%

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Jin

Chen

Zhao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Noteworthy, the ratio of integrations of the PEDF gene into exons, representing 4% of the total integration events, was lower than the ratio expected by random distribution. In human T cells, SB transposon insertions were shown to have the lowest deviation from a random genome-wide distribution as well as the highest theoretical chance of targeting a safe site of the genome 35, 55. Although site-specific gene insertion into safe harbor sites by designer nucleases, such as zinc finger nucleases, TALENs, and the CRISPR/Cas system, is appealing from a safety point of view, the overall efficiency of transgene integration in polyclonal RPE cell populations by these technologies is expected to be far lower than with our transposon-based approach.…”

Section: Discussionmentioning

confidence: 99%

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Thumann

Harmening

Prat-Souteyrand

et al. 2017

Molecular Therapy - Nucleic Acids

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Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

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“…Reducing the size of the DNA, and using supercoil DNA proved to be advantageous modifications in the delivery protocol [76,77]. Furthermore, the use of conventional plasmids as vectors that are propagated and isolated from bacteria raises a safety concern and a roadblock for broad clinical applications.…”

Section: Eliminating Bacterial Sequences From the Transposon Vectormentioning

confidence: 99%

“…They are produced by the inclusion of site-specific intramolecular recombination motifs between the GOI and bacterial backbone in the parental plasmid. SB transposon and transposase minicircle constructs have been examined and optimized for safety and efficacy in various cell types [77], including primary T cells [76]. A minicircles-based SB system has been also used for efficient germline transgenesis [81].…”

Section: Eliminating Bacterial Sequences From the Transposon Vectormentioning

confidence: 99%

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

Narayanavari¹,

Izsvák²

2017

Cell Gene Therapy Insights

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Transposable elements are natural, non-viral gene delivery vehicles capable of mediating stable genomic integration. The Sleeping Beauty (SB) transposon has the ability to cut-and-paste the 'gene of interest' into the genome, providing the basis for long-term, permanent transgene expression in transgenic cells and organisms. The SB transposon system is relatively well characterized, and has been extensively engineered for efficient gene delivery and gene discovery purposes in a wide range of vertebrates, including humans. The SB system is a safe and simple-to-use vector that enables cost-effective, rapid preparation of therapeutic doses of cell products. Recently, there has been a growing interest in using the SB system for therapy as evidenced by the large number of pre-clinical studies. SB moved swiftly from pre-clinical to clinical trials in almost a decade. In this article, we highlight the advancements and challenges associated with the SB system in various therapeutic applications. We also provide an overview that has been exploited by spin-off companies based on the SB system.

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Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors

Cited by 207 publications

References 51 publications

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

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