Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix.
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.
Full-textDOI: · Available from: Christoph Piechaczek, May 30, 2015
SourceAvailable from: Lorena Cussó[Show abstract] [Hide abstract]
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.PLoS ONE 08/2013; 8(8):e73138. DOI:10.1371/journal.pone.0073138 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.Nucleic Acids Research 01/2014; 42(7). DOI:10.1093/nar/gku082 · 8.81 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: In der modernen molekularen Medizin ist ein stabiler Gentransfer vor allem bei der lebenslangen Behandlung von Erbkrankheiten und bei der Therapie von chronisch-infektiösen Erkrankungen erforderlich. In diesem Artikel haben wir die Möglichkeiten des stabilen Gentransfers dargestellt und diskutiert. Prinzipiell unterscheidet man hierbei Ansätze, die zur somatischen Integration in das Wirtsgenom führen, oder Ansätze die auf nichtintegrierenden und somit episomalen Systemen basieren. Obwohl die Entwicklung der integrierenden Systeme auch im Hinblick auf die präklinische Testung weiter fortgeschritten ist, werden verbesserte episomale Systeme in der Zukunft weiterhin eine wichtige Rolle spielen, da diese das Risiko der Genotoxität, also die Veränderungen des Expressionsmusters der transduzierten Zelle, signifikant verringern würden. Aufgrund der größeren Erfahrung mit integrierenden Systemen sind diese bereits heute in der klinischen Testung Realität. Es ist aber anzunehmen, dass in der Zukunft die episomalen Systeme mit weiterer Entwicklung eine zunehmend wichtigere Rolle spielen werden.Pharmazie in unserer Zeit 05/2011; 40(3). DOI:10.1002/pauz.201100419