Cell cycle dependent histone dynamics of an episomal non-viral vector

Rupprecht, Sina; Lipps, Hans J.

doi:10.1016/j.gene.2009.03.010

Cited by 20 publications

(30 citation statements)

References 35 publications

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“…Thus, in both normal and transformed cells, only certain cis-acting sequences, containing a version of the 20bp consensus sequence, were bound by the required trans-acting factors for episomal replication to occur, due to the presence of a more favorable chromatin environment (necessary for replication and other cellular processes to occur), which has been recently observed in other episomes. [53][54][55] The chromatin context encompassing chromosomal replication origins regulates the association of pre-RC proteins with them, which in turn correlates with their activity in both transformed and normal cells. The 20mer1, 20mer2, c-myc, and lamin B2 chromosomal loci each contain replication origins in both transformed and normal cells, as indicated by the abundance of nascent DNA at those regions, but the 20mer1, 20mer2, and c-myc origins displayed 2-to 3-fold more activity in the transformed cells (Fig.…”

Section: Discussionmentioning

confidence: 99%

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

et al. 2012

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This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells. Quantitative comparative analyses of episomal and in situ chromosomal replication origin activity as well as chromatin immunoprecipitation (ChIP) assays, using specific antibodies targeting members of the pre-replication complex (pre-RC) as well as open/closed chromatin markers encompassing both episomal and chromosomal origins, revealed that episomal origins had similar levels of in vivo activity, nascent DNA abundance, pre-RC protein association, and elevated open chromatin structure at the origin in both cell types. In contrast, the chromosomal origins corresponding to 20mer1, 20mer2, and c-myc displayed a 2- to 3-fold higher activity and pre-RC protein abundance as well as higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited similar levels of activity, pre-RC protein abundance, and higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in both cell types. Nucleosomal positioning analysis, using an MNase-Southern blot assay, showed that all the origin regions examined were situated within regions of inconsistently positioned nucleosomes, with the nucleosomes being spaced farther apart from each other prior to the onset of S phase in both cell types. Overall, the results indicate that cellular transformation is associated with differential epigenetic regulation, whereby chromatin structure is more open, rendering replication origins more accessible to initiator proteins, thus allowing increased origin activity.

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Section: Discussionmentioning

confidence: 99%

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

et al. 2012

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“…In nontransduced negative control cells (a-c), rare background spots positive for only one probe color were observed. In the clone transduced with integrating lentivector Vector genome visualization using FISH FISH is commonly used to scan for relatively large chromosomal abnormalities, and successes have also been reported with the detection of smaller single-copy targets (Rupprecht and Lipps, 2009). To examine the cytogenetic distribution of the vector genomes in the CHO cell clones stably transduced with IDLVs, FISH analyses were performed on 15 cell populations: IDLV-SG-transduced polyclonal population and clones 4, 7, and 10; IDLV-SGmtransduced polyclonal population and clones 5, 8, and 11; two control polyclonal populations transduced with IDLV-SG and IPLV-SG and harvested 24 hr after transduction; a control polyclonal population transduced with IPLV-SG and three derived clones; and an untransduced control population.…”

Section: Integration Analysis By Southern Blottingmentioning

confidence: 99%

Long-Term Episomal Transgene Expression from Mitotically Stable Integration-Deficient Lentiviral Vectors

et al. 2014

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Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a scaffold/matrix attachment region did not result in improved episomal retention in this system, and episomes did not form after transduction with adeno-associated viral or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (1) linear amplification-mediated polymerase chain reaction followed by deep sequencing of vector-genome junctions, (2) Southern blotting, and (3) fluorescent in situ hybridization strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.

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“…It has been previously reported that established cell clones containing S/MAR plasmids are found exclusively in euchromatin nuclear compartments of active transcription 18 and are associated with histone markers of active transcription (such as H3K4m3, H3K4m1 and H3K36m3) during most phases of the cell cycle. 19 During mitosis, histone modifications on the S/MAR plasmid decreases, allowing the plasmid to adopt an open permissive conformational change, similar to endogenous active genes in the S-phase. 19 The initial selection step, however, is critical to enrich the mitotically stable episomal plasmids in vitro, and lack of selective pressure results in less than 1% of replicating cells retaining the vector after 1 month.…”

Section: Discussionmentioning

confidence: 99%

“…19 During mitosis, histone modifications on the S/MAR plasmid decreases, allowing the plasmid to adopt an open permissive conformational change, similar to endogenous active genes in the S-phase. 19 The initial selection step, however, is critical to enrich the mitotically stable episomal plasmids in vitro, and lack of selective pressure results in less than 1% of replicating cells retaining the vector after 1 month. 7,20 Both in vitro and in vivo the stable attachment of S/MAR plasmid to the chromatin and its subsequent replication is a very rare event.…”

Section: Discussionmentioning

confidence: 99%

Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage

et al. 2010

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Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage SP Wong, O Argyros, C Coutelle and RP HarbottleThe ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.

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Cell cycle dependent histone dynamics of an episomal non-viral vector

Cited by 20 publications

References 35 publications

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

Long-Term Episomal Transgene Expression from Mitotically Stable Integration-Deficient Lentiviral Vectors

Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage

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