Efficient episomal gene transfer to human hepatic cells using the pFAR4–S/MAR vector

Giannakopoulos, Aristeidis; Quiviger, Michael; Stavrou, Eleana F.; Verras, Meletios; Marie, Corinne; Scherman, Daniel; Athanassiadou, Aglaia

doi:10.1007/s11033-019-04777-9

Cited by 9 publications

(16 citation statements)

References 30 publications

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“…Nonviable cells were excluded by gating based on propidium iodide (PI) (Pharmingen, BD). Transfected cells were sorted with a FACS Vantage (BD, Erembodegen, Belgium) as previously described 8,64 . Times and conditions for the estimation of eGFP and γ globin expression for each cell type is provided in the respective Results Section.…”

Section: Methodsmentioning

confidence: 99%

“…Probe labelling was performed by Nick Translation Mix (Roche) following the manufacturer’s instructions. For internal control, DLEU (13q14) hybridization probe was used, labelled with Fluorescein-12-2′-deoxy-uridine-5′-triphosphate (Roche) as well as Tetramethyl-Rhodamine-5-dUTP (Roche) that detects the endogenous 13q14 locus giving a double -green and red–signal respectively as reported previously 64 . A total of 50 metaphase and interphase spreads were analysed.…”

Section: Methodsmentioning

confidence: 99%

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Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

Stavrou¹,

Simantirakis²,

Verras³

et al. 2019

Sci Rep

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We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

Stavrou¹,

Simantirakis²,

Verras³

et al. 2019

Sci Rep

Self Cite

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“…19 One of the primary reasons for using AAV is that AAV has a long-term and efficient transgene expression in various cell types in many tissues such as liver, muscle, retina, and the central nervous system (CNS). [43][44][45] However, the application of AAV has some associated disadvantages. The preexisting immunity to human AAV vectors is comparable to the AV, and the integration into the host genome (if it occurs at all) is random, which can lead to accidental activation or inhibition of endogenous gene expression.…”

Section: Adeno-associated Virus Vectorsmentioning

confidence: 99%

Viral Vector Systems for Gene Therapy: A Comprehensive Literature Review of Progress and Biosafety Challenges

Ghosh

Brown

Jenkins³

et al. 2020

Applied Biosafety

134

113

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Introduction: National Institutes of Health (NIH) defines gene therapy as an experimental technique that uses genes to treat or prevent disease. Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be effective and safe. Methods: Applications of viral vectors and nonviral gene delivery systems have found an encouraging new beginning in gene therapy in recent years. Although several viral vectors and nonviral gene delivery systems have been developed in the past 3 decades, no one delivery system can be applied in gene therapy to all cell types in vitro and in vivo. Furthermore, the use of viral vector systems (both in vitro and in vivo) present unique occupational health and safety challenges. In this review article, we discuss the biosafety challenges and the current framework of risk assessment for working with the viral vector systems. Discussion: The recent advances in the field of gene therapy is exciting, but it is important for scientists, institutional biosafety committees, and biosafety officers to safeguard public trust in the use of this technology in clinical trials and make conscious efforts to engage the public through ongoing forums and discussions.

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“…However, despite the initial success of this strategy to achieve sustained J o u r n a l P r e -p r o o f transgene expression in the liver of mice and pigs, 14,26 as of yet S/MAR has not reached clinical trials.…”

Section: Administration Of Naked Genetic Materialsmentioning

confidence: 99%

“… 25 DNA vectors containing a S/MAR sequence can provide persistent mitotic stability in dividing cells and avoid epigenetic silencing allowing for sustained transgene expression. However, despite the initial success of this strategy to achieve sustained transgene expression in the liver of mice and pigs, 14 , 26 S/MAR has not yet reached clinical trials.…”

Section: Non-viral Vectorsmentioning

confidence: 99%

Novel vectors and approaches for gene therapy in liver diseases

Maestro

Weber²,

Zabaleta

et al. 2021

JHEP Reports

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Efficient episomal gene transfer to human hepatic cells using the pFAR4–S/MAR vector

Cited by 9 publications

References 30 publications

Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

Viral Vector Systems for Gene Therapy: A Comprehensive Literature Review of Progress and Biosafety Challenges

Novel vectors and approaches for gene therapy in liver diseases

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