LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors

Chow, Cheok-man; Athanassiadou, Aglaia; Raguz, Selina; Psiouri, Lambrini; Harland, Lee; Malik, MM; Aitken, M. Alexandra; Grosveld, Frank; Antoniou, Michael

doi:10.1038/sj.gt.3301654

Cited by 26 publications

(23 citation statements)

References 45 publications

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“…16,36,37 The average copy number of the vector in K562 cells was estimated to be low, similar to previously published reports of pEPI copy number in HeLa and CHO cells, 16,18,36 as well as of that of EBV-based episomes in K562 cells. 24 This is in accordance with a tightly regulated once-per cell cycle replication of pEPI vector. 36 Thus, pEPI-eGFP can be a valuable tool in basic research as an expression vector conferring sustained and regulated expression of transgenes, being easy and simple to use as a nonviral gene transfer system.…”

Section: Discussionsupporting

confidence: 76%

“…BamHI-digested total DNA from stably transfected K562 cells was Southern blotted and probed simultaneously with a pEPI-eGFP-specific probe, as above, and a probe specific for the endogenous b-globin gene. By comparing the intensities of the bands corresponding to the two probes and considering b-globin band intensity as a three-copy gene reference (since K562 cells are trisomic for chromosome 11 and therefore possess three copies of the b-globin gene), 21,24 we estimated the average copy number of the vector in K562 cells to be 2.5 ( Figure 1e). The plasmid band generated on Southern blots of MEL cells at various time points did not exhibit a decrease in intensity (Figure 1b), suggesting that plasmid loss could not account for the decline in eGFP expression.…”

Section: Pepi-egfp Functions As a Stable Episome In Hematopoietic Promentioning

confidence: 99%

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Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

et al. 2005

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Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/ matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34 + cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

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Section: Discussionsupporting

confidence: 76%

Section: Pepi-egfp Functions As a Stable Episome In Hematopoietic Promentioning

confidence: 99%

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

et al. 2005

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“…LCR elements have been used in a number of studies with the aim of enhancing and prolonging transgene expression. A combination of HS 2, 3 and 4 from the globin LCR was shown to endow an episomal plasmid vector with the ability to maintain stable transgene expression in the absence of drug selection for 60 days (more than 60 cell replications) [Chow et al, 2002]. Miao et al [Miao et al, 2000;Miao et al, 2001] also incorporated the apolipoprotein E LCR into episomal plasmid vectors and showed significant improvement over constructs lacking the LCR.…”

Section: Locus Control Regions (Lcrs)mentioning

confidence: 99%

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

Papadakis

Nicklin²,

Baker³

et al. 2004

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It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailormade' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

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“…The inclusion of bLCR minilocus in the cosmid construct prevented transgene silencing over time. 126 Satoh et al 81 reported successful transfer and expression of human ADA gene in human hematopoietic progenitor cells with colony-forming capacity within an EBV-based vector.…”

Section: 78mentioning

confidence: 99%

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

2005

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LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors

Abstract: Locus control regions (LCRs)

Cited by 26 publications

References 45 publications

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

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