Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

Skipper, Kristian Alsbjerg; Hollensen, Anne Kruse; Antoniou, Michael; Mikkelsen, Jacob Giehm

doi:10.1186/s12896-019-0570-2

Cited by 13 publications

(11 citation statements)

References 53 publications

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“…Therefore, the proper selection of these elements can increase recombinant protein expression levels and stability ( Lee JS et al, 2018 ; Lee CP et al, 2018 ; Yeo et al, 2017 ). However, most studies have focused on the role of the enhancer, internal ribosome entry site (IRES), intron, promoters, and combinations of regulatory elements with promoters ( Hunter et al, 2019 ; Skipper et al, 2019 ; Yeo et al, 2017 ; Chai et al, 2018 ), few studies have been performed on the effects of poly A elements on transgene expression in CHO cells.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Wang

Zhang

et al. 2022

Front. Bioeng. Biotechnol.

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The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. Expression vector optimization is an effective strategy to increase transgene expression levels and stability, and the choice of suitable poly A element is crucial for the expression of recombinant protein. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. Five poly A elements, including bovine growth hormone (BGH), mutant BGH, herpes simplex virus type 1 thymidine kinase (HSV-TK), SV40, and a synthetic (Synt) poly A, were cloned into the expression vector and transfected into CHO cells. The results indicated the SV40 and Synt poly A sequences can significant improve eGFP transgene expression in stable transfected CHO cells and maintain long-term expression. However, qPCR results showed that the eGFP expression at protein level was not related to the gene copy number and mRNA level. Importantly, the SV40 and Synt poly A elements decreased the variation of eGFP transgene expression. Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. In conclusion, SV40 poly A and Synt poly A are stronger elements that increase stable transgene expression and decrease the variation of expression, and the choice of suitable poly A element is helpful to improve the expression of recombinant protein.

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Section: Introductionmentioning

confidence: 99%

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Wang

Zhang

et al. 2022

Front. Bioeng. Biotechnol.

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“…However, due to safety concerns, considerable efforts have been made to substitute viral enhancer sequences with cellular elements that avoid the spread of heterochromatin and hypermethylation. Several different of such protective elements, such as the 5′HS chicken β-globin (cHS4) insulator [ 79 , 80 , 81 , 82 ], the D4Z4 insulator [ 82 ], matrix attachment regions (MAR) [ 83 , 84 ], and Ubiquitous Chromatin Opening Elements (UCOE) [ 81 , 82 ], have already been exploited for transgene protection in both, viral and non-viral vector systems. The UCOE elements have been identified within the TBP-PSMB1 and HNRPA2B1-CBX3 gene loci.…”

Section: Transgene Expressionmentioning

confidence: 99%

“…Moreover, a core fragment within the 1.5 kb-UCOE was identified using bioinformatic approaches. This core fragment, only 0.86 kb in size, offered effective protection from transcriptional silencing [ 82 ].…”

Section: Transgene Expressionmentioning

confidence: 99%

Episomes and Transposases—Utilities to Maintain Transgene Expression from Nonviral Vectors

Kreppel

Hagedorn

2022

Genes

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The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology

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“…Epigenetic regulatory elements have been described as DNA sequences that are able to modulate the chromatin state, thereby affecting gene expression. The major representatives of this category of such regulatory elements are locus control regions (LCRs), matrix attachment regions (MARs), insulators, ubiquitously acting chromatin opening elements (UCOEs), and stabilizing and anti‐repressor elements (STARs) 11,13–18 . Out of them, the MARs have been extensively studied and are often integrated as cis‐acting elements into expression vectors 19 .…”

Section: Introductionmentioning

confidence: 99%

“…The major representatives of this category of such regulatory elements are locus control regions (LCRs), matrix attachment regions (MARs), insulators, ubiquitously acting chromatin opening elements (UCOEs), and stabilizing and anti-repressor elements (STARs). 11,[13][14][15][16][17][18] Out of them, the MARs have been extensively studied and are often integrated as cis-acting elements into expression vectors. 19 MAR sequences are DNA regions that can link the nuclear matrix and are characterized by an AT-rich core surrounded by two flanking regions with binding sites for various factors (insulators, transcription factors, nuclear matrix protein, etc.).…”

mentioning

confidence: 99%

Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

Oliviero

Hinz

Bogen

et al. 2022

Biotechnology Progress

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In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

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Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

Cited by 13 publications

References 53 publications

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

Episomes and Transposases—Utilities to Maintain Transgene Expression from Nonviral Vectors

Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

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