Article

Baiker, A, Maercker, C, Piechaczek, C, Schmidt, SB, Bode, J, Benham, C et al.. Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix. Nat Cell Biol 2: 182-184

Institut für Zellbiologie, Universität Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany.
Nature Cell Biology (Impact Factor: 19.68). 04/2000; 2(3):182-4. DOI: 10.1038/35004061
Source: PubMed

ABSTRACT

DNA replication occurs in tight association with the nuclear matrix, where binding of the replication origin to the nuclear matrix must precede the onset of S phase. We have shown previously that the origin of replication of the simian virus 40 (SV40) genome linked to a human scaffold/matrix-attached region (S/MAR) allows sustained episomal replication (where an episome is autonomous, self-replicating DNA) that is independent of the expression of the virally encoded large T-antigen. A vector with this combination of SV40 origin and potential for matrix association is maintained in cultured cells for at least 100 cell generations, in the absence of selection. Here we show, by in situ hybridization
and nuclear-fractionation procedures, that there is a specific interaction of this vector with the nuclear matrix and the chromosome scaffold, presumably through proteins that both structures have in common. This interaction correlates with replication of the vector as an episome. These observations allow a mechanistic explanation for the episomal replication and mitotic stability of this new type of vector.

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Available from: Christoph Piechaczek
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    • "However, pEPI-HS4opp was found to establish with an increased efficiency of 25.3 ± 13.8% (P = 0.028). It has been shown that pEPI plasmid vectors interact with the nuclear matrix via an S/MAR–SAF-A interaction and thereby are stably maintained.28 Furthermore, it is known that the cHS4 insulator element associates with the nuclear matrix through its interaction with CTCF.31 "
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    ABSTRACT: The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.Molecular Therapy-Nucleic Acids (2013) 2, e118; doi:10.1038/mtna.2013.47; published online 3 September 2013.
    Full-text · Article · Sep 2013 · Molecular Therapy - Nucleic Acids
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    • "The first S/MAR vector to be described, called pEPI-1, was a plasmid containing the hIFN-ß S/MAR and the SV40 origin of replication (ORI) [13]. This vector was shown to maintain mitotic stability by binding to matrix proteins such as SAF-A (also known as hnRNP-U)[31] and replicate episomally once-per-cell-cycle in a semiconservative fashion [13], [32]. Later it was shown that constructs containing a transcription unit linked to a downstream S/MAR (i.e. "
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    ABSTRACT: 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, 1.5×10(6) 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.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "Since transformation of recipient cells is an inevitable consequence of these factors the development of a new vector generation is desirable in which their function is replaced by components of the endogenous cellular replication machinery. Such a vector, pEPI-1, which is based on the SV40 origin of replication but is independent on the virally encoded large T-antigen for replication has recently been introduced (Piechaczek et al., 1999, Baiker et al., 2000). pEPI-1 contains a well characterized scaffold/matrix attached region (S/MAR) from the human interferon-ß gene (Element I in Mielke et al, 1990), it replicates episomally in a variety of mammalian cell types and is stably maintained and segregated over several hundred generations even in the absence of selection. "
    Dataset: BodeGTMB01
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    ABSTRACT: DNA replication occurs in tight association with the nuclear matrix, where binding of the replication origin to the nuclear matrix must precede the onset of S phase1–4. We have shown previously that the origin of replication of the simian virus 40 (SV40) genome linked to a human scaffold/matrix-attached region (S/MAR) allows sustained episomal replication (where an episome is autonomous, self-replicating DNA) that is independent of the expression of the virally encoded large T-antigen5. A vector with this combination of SV40 origin and potential for matrix association is maintained in cultured cells for at least 100 cell generations, in the absence of selection5. Here we show, by in situ hybridization and nuclear-fractionation procedures, that there is a specific interaction of this vector with the nuclear matrix and the chromosome scaffold, presumably through proteins that both structures have in common. This interaction correlates with replication of the vector as an episome. These observations allow a mechanistic explanation for the episomal replication and mitotic stability of this new type of vector.
    Full-text · Dataset · Jun 2013
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