Controlled removal of a nonviral episomal vector from transfected cells

Rupprecht, Sina; Hagedorn, Claudia; Seruggia, Davide; Magnusson, Terese; Wagner, Ernst; Ogris, Manfred; Lipps, Hans J.

doi:10.1016/j.gene.2010.07.001

Cited by 22 publications

(23 citation statements)

References 27 publications

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“…A co-localization with active histone modifications, early replicating foci, and splicing speckles in the absence of repressive chromatin markers has been shown before (24) and could now be globally confirmed. Since we have described before that an active transcription running into or over the S/MAR is essential for episomal replication and maintenance this observation supports our previous data (50). At this point, we can only speculate about cell-type specificity of detected contact pattern.…”

Section: Discussionsupporting

confidence: 92%

Genome-wide profiling of S/MAR-based replicon contact sites

Hagedorn

Gogol-Döring

Schreiber

et al. 2017

Nucleic Acids Research

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Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.

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Section: Discussionsupporting

confidence: 92%

Genome-wide profiling of S/MAR-based replicon contact sites

Hagedorn

Gogol-Döring

Schreiber

et al. 2017

Nucleic Acids Research

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“…The minimal expression levels of the SM22 promoter in the HEK293 cells (Figure 4A) did not achieve episomal retention of this plasmid. It is known, that transcription in the SMAR is a prerequisite for episomal establishment [15]. We confirmed the establishment as an episome by rescue experiments (Table 1).…”

Section: Resultssupporting

confidence: 77%

“…The SM22 only developed stable clones in TE-671 cells at lower efficiency when compared to the EF1α driven pEPito. As the transcription into the SMAR is crucial for the establishment of episomes [15], the strength of constitutive promoters influences the extent of the expression from the established clones [17]. These facts are also known for minicircles (J.…”

Section: Discussionmentioning

confidence: 99%

“…Our approach here was to modify this vector system by exchanging the constitutive promoter with tissue- or tumor-specific ones to generate a vector, which will episomally express the transgene in the target cell type only, but being lost in all other cell types. Recently, similar effects with pEPI vectors utilizing chemically inducible promoters could be shown [15]. …”

Section: Introductionmentioning

confidence: 95%

See 1 more Smart Citation

Generation of a tumor- and tissue-specific episomal non-viral vector system

Haase¹,

Magnusson²,

Su³

et al. 2013

BMC Biotechnol

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BackgroundA key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR).ResultsHere, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer.ConclusionsIn this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

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“…This provides an exciting way to reversibly modify cells with RGs after a prolonged imaging window (months), and is even safer and may have many more specific applications in the clinic compared to a system that modifies cells indefinitely. Future work will focus on development of MC constructs that utilize mammalian promoters, such as the Ubiquitin C promoter [35], that do not easily get silenced to cover applications that require longer cell imaging windows and, as has been recently explored [40], the use of inducible promoters that can be turned off once the appropriate imaging window has been achieved.…”

Section: Discussionmentioning

confidence: 99%

Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

et al. 2013

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Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×106 cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

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Controlled removal of a nonviral episomal vector from transfected cells

Cited by 22 publications

References 27 publications

Genome-wide profiling of S/MAR-based replicon contact sites

Genome-wide profiling of S/MAR-based replicon contact sites

Generation of a tumor- and tissue-specific episomal non-viral vector system

Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies

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