Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

Alhaji, Suleiman Yusuf; Ngai, Siew Ching; Abdullah, Saifollah

doi:10.1080/02648725.2018.1551594

Cited by 28 publications

(26 citation statements)

References 120 publications

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“…Potential mechanisms of action include progressive transcriptional silencing via DNA methylation or histone modification, including de novo DNA methylation triggered by transgene recognition (Kohli et al, 2010); and posttranscriptional silencing known as RNA interference (Meyer, 1995;León-Bañares et al, 2004;Cerutti et al, 2011;Doron et al, 2016). Epigenetic silencing of nuclear-integrated exogenous DNA have been attributed to defense mechanisms against viruses and transposable elements in plants (Rajeevkumar et al, 2015) and mammalian cells (Alhaji et al, 2019) alike. Silencing mechanisms, and indeed transgene regulation mechanisms yet to be identified, can influence daughter cells from the same original clonal population differently (Kaufman et al, 2008).…”

Section: Rice Cell Lines Exhibit Dramatically Varied Stability That Dmentioning

confidence: 99%

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

George

Kahlke

Abbriano

et al. 2020

Front. Bioeng. Biotechnol.

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Section: Rice Cell Lines Exhibit Dramatically Varied Stability That Dmentioning

confidence: 99%

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

George

Kahlke

Abbriano

et al. 2020

Front. Bioeng. Biotechnol.

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“…We next examined whether Candidate 6 functioned on an epigenetic level to resist transgene silencing. It is well understood that transgene silencing is mediated by the loss of histone acetylation at the locus and addition of DNA methylation (4). Two small molecule drugs have been widely used to probe this effect, trichostatin A (TSA), which is a specific inhibitor of histone deacetylase, and 5azacytidine (5-aza), a cytidine analog that inhibits methylation upon its incorporation into DNA.…”

Section: Effective Ucoes Confer Resistance To Dna Cpg Methylation Andmentioning

confidence: 99%

“…Similarly, most gene and cell therapies rely on the stable incorporation and expression of therapeutic genetic material, but are plagued by transgene expression variability and silencing, reducing the treatment efficacy (3). This loss of expression has been attributed to epigenetic silencing mechanisms, including DNA methylation, histone deacetylation, and chromatin condensation at the integration site (3,4). Random integration methods, such as retroviral vectors, amplify this effect due to the transgene's interaction with its immediate genetic neighborhood, which may be heterochromatic (5).…”

Section: Introductionmentioning

confidence: 99%

A Novel Chromatin-Opening Element for Stable Long-term Transgene Expression

Rudina¹,

Smolke²

2019

Preprint

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Long-term stable expression of transgenes in mammalian cells is a challenge in gene therapy, recombinant protein production, and mammalian synthetic biology due to epigenetic silencing and position effect variegation. While multiple classes of regulatory elements have been discovered and proposed to help stabilize expression, the most efficacious has been the Ubiquitous Chromatin Opening Element (UCOE), and in particular, the prototypical A2UCOE from the HNRPA2B1-CBX3 locus. We developed a feature-driven bioinformatics algorithm to discover other putative UCOEs from the human genome , and identified a novel UCOE (SRF-UCOE) that can resist transgene silencing in the methylation-prone P19 cell line. We demonstrate that a 767 bp core sequence of SRF-UCOE is modular to four common mammalian promoters. Notably, SRF-UCOE stabilizes gene expression in transduced P19 cells up to 2.4-fold better over 26 days than the existing A2UCOE by resisting constructs' susceptibility to DNA methylation and histone deacetylation. Unlike existing UCOEs, SRF-UCOE lacks inherent transcriptional initiation activity, which can bolster its safe and predictable use in gene therapy constructs. We expect that expanding the set of UCOEs available will expand their utility to novel applications in gene therapy, synthetic biology, and biomanufacturing, as well as contribute to understanding their molecular mechanism.

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“…42 These promoters may also be subjected to progressive silencing in certain cell types. 43,44 In addition, uncontrolled expression may be of particular importance in the case of FRDA and other diseases where overexpression of the gene may lead to toxic side effects, potentially reducing the therapeutic benefit of the therapy. [26][27][28]30 Prior studies in Drosophila as well as more recent work in human cell models demonstrated that overexpression of frataxin affects cellular metabolism, increases oxidative stress and labile iron pool levels.…”

Section: Discussionmentioning

confidence: 99%

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Wang

et al. 2020

Human Gene Therapy

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Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

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Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

Cited by 28 publications

References 120 publications

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

A Novel Chromatin-Opening Element for Stable Long-term Transgene Expression

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

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