Establishment and mitotic stability of an extra-chromosomal mammalian replicon

Stehle, Isa M.; Postberg, Jan; Rupprecht, Sina; Cremer, Thomas; Jackson, Dean A.; Lipps, Hans J.

doi:10.1186/1471-2121-8-33

Cited by 65 publications

(116 citation statements)

References 29 publications

Supporting

Mentioning

108

Contrasting

Order By: Relevance

“…This stability is fairly comparable to that of other large mammalian artificial chromosomes. Similar observations were obtained previously with other cell lines (18,22).…”

Section: Analysis Of the Supernatantsupporting

confidence: 81%

See 1 more Smart Citation

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Jenke

Wilhelm

Orth

et al. 2008

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

show abstract

“…This stability is fairly comparable to that of other large mammalian artificial chromosomes. Similar observations were obtained previously with other cell lines (18,22).…”

Section: Analysis Of the Supernatantsupporting

confidence: 81%

“…2, the hybridization pattern clearly shows that the vector was retained as an episome in the multiclonal cell population for at least 8 months. From the intensity of the signal obtained, we estimated that the vector was maintained at a copy number below 10 per cell, as observed for other cell lines (1,14,22).…”

Section: Resultsmentioning

confidence: 99%

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Jenke

Wilhelm

Orth

et al. 2008

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…The findings that EBV genomes localize in perichromatic regions and in open epigenetic domains are similar to a different extrachromosomal replicon model, pEPI (50). As such, oriP-based replicons might function as a model system regulated by higher order nuclear structures.…”

Section: Discussionmentioning

confidence: 62%

The Latent Origin of Replication of Epstein-Barr Virus Directs Viral Genomes to Active Regions of the Nucleus

Deutsch

Ott

Papior

et al. 2010

J Virol

View full text Add to dashboard Cite

The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions.To maintain genetic stability, proliferating cells must pass on their genomes as exact copies to their daughter cells. Viruses that establish latent infections have developed various strategies to ensure genetic stability as they integrate their genomes into the host's chromosomes or infect nonproliferating cells. Only a limited number of viruses with extrachromosomally maintained genomes establish a latent infection in proliferating cells. The gammaherpesvirus Epstein-Barr virus (EBV) is a 170-kbp circular double-stranded DNA molecule that resides as a nucleosome-packaged episome in latently infected B cells. The cis-acting origin of plasmid replication (oriP) is essential for DNA replication, nuclear retention, and viral gene regulation (30, 56). These functions are dependent on the EBVencoded nuclear antigen EBNA1 (33, 58). EBNA1 has a modular structure: its C-terminal DNA binding and dimerization domain recognizes consensus motifs located within oriP. A mutational analysis of the N terminus led to the identification of two regions (amino acids [aa] 40 to 89 and aa 329 to 379) capable of linking DNA elements, which have therefore been termed linking regions (37). Later studies demonstrated that these domains tether EBV genomes to me...

show abstract

“…30 It has been suggested that, eventually, only properly established vector molecules, influenced by higher nuclear architecture in the hepatocyte nucleus, will be able to express the luciferase transgene for extended periods of time. 31 Without …”

Section: S/mar Plasmids Do Not Become Established As Actively Replicamentioning

confidence: 99%

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

et al. 2008

View full text Add to dashboard Cite

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo

show abstract

Establishment and mitotic stability of an extra-chromosomal mammalian replicon

Cited by 65 publications

References 29 publications

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

Long-Term Suppression of Hepatitis B Virus Replication by Short Hairpin RNA Expression Using the Scaffold/Matrix Attachment Region-Based Replicating Vector System pEPI-1

The Latent Origin of Replication of Epstein-Barr Virus Directs Viral Genomes to Active Regions of the Nucleus

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

Contact Info

Product

Resources

About