Metabolic reprogramming as a novel therapeutic target for Coxsackievirus B3

국명욱,가윤지,김예진,박지윤,송은선,이하늘,이윤행,고가현,김재광,예정용,권형욱,변영주,박준태 한국통합생물학회 2022 Animal cells and systems Vol.26 No.6

Coxsackievirus B3 (CVB3) is a single-stranded RNA virus that belongs to the Enterovirus genus. CVB3 is a human pathogen associated with serious conditions such as myocarditis, dilated cardiomyopathy, and pancreatitis. However, there are no therapeutic interventions to treat CVB3 infections. In this study, we found that CVB3 induced metabolic alteration in host cells through increasing glycolysis level, as indicated by an increase in the extracellular acidification rate (ECAR). CVB3-mediated metabolic alteration was confirmed by metabolite change analysis using gas chromatography-mass spectrometry (GC-MS). Based on findings, a strategy to inhibit glycolysis has been proposed to treat CVB3 infection. Indeed, glycolysis inhibitors (2-Deoxy-Dglucose, sodium oxide) significantly reduced CVB3 titers after CVB3 infection, indicating that glycolysis inhibitors can be used as effective antiviral agents. Taken together, our results reveal a novel mechanism by which CVB3 infection is controlled by regulation of host cell metabolism.

Bacterial Artificial Chromosome-based Protein Expression Platform Using the Tol2 Transposon System

국명욱,박지윤,송은선,이하늘,이윤행,주정현,권형욱,박준태 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.3

Conventional vector systems, including plasmidbased vectors, are mainly used in mammalian cells for the production of biopharmaceuticals. Plasmid-based vectors express transgene by the random integration of recombinant transgenes into the genome. Transgene expression is greatly influenced by the surrounding chromatin, and in most cases, expression is weak and tends to be suppressed over time. Therefore, a novel strategy is required to create clones that maintain increased transgene expression. In this study, we used a bacterial artificial chromosome (BAC) containing the Rosa26 locus, which allows constitutive and ubiquitous gene expression. Moreover, we improved the Rosa26 BAC-mediated expression system by incorporating the Tol2 transposon system with helper vector, resulting in improved efficiency of protein production and maintained productivity even in single-cell clones. Furthermore, the recombinant Rosa26 BAC was improved in terms of protein productivity by using helper mRNA instead of helper vector. Finally, establishment of an optimal molar ratio between the recombinant Rosa26 BAC and helper mRNA helped to achieve maximal protein productivity. Taken together, our results provide an effective strategy for improving BAC-based expression systems for biopharmaceutical production.

Alleviation of Senescence via ATM Inhibition in Accelerated Aging Models

국명욱,김재원,이영삼,조경아,박준태,박상철 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.3

The maintenance of mitochondrial function is closely linked to the control of senescence. In our previous study, we uncovered a novel mechanism in which senescence amelioration in normal aging cells is mediated by the recovered mitochondrial function upon Ataxia telangiectasia mutated (ATM) inhibition. However, it remains elusive whether this mechanism is also applicable to senescence amelioration in accelerated aging cells. In this study, we examined the role of ATM inhibition on mitochondrial function in Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) cells. We found that ATM inhibition induced mitochondrial functional recovery accompanied by metabolic reprogramming, which has been known to be a prerequisite for senescence alleviation in normal aging cells. Indeed, the induced mitochondrial metabolic reprogramming was coupled with senescence amelioration in accelerated aging cells. Furthermore, the therapeutic effect via ATM inhibition was observed in HGPS as evidenced by reduced progerin accumulation with concomitant decrease of abnormal nuclear morphology. Taken together, our data indicate that the mitochondrial functional recovery by ATM inhibition might represent a promising strategy to ameliorate the accelerated aging phenotypes and to treat agerelated disease.

Cre/Lox-based RMCE for Site-specific Integration in CHO Cells

김재원,이윤행,국명욱,황수영,권형욱,박준태 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.5

Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/ Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.

Improvement of Tol2 Transposon System Enabling Efficient Protein Production in CHO Cells

황수영,이윤행,국명욱,김재원,오세경,박준태 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.5

Establishment of mammalian cell lines with high protein productivity is an important object in the field of biopharmaceutics. Toward this end, Tol2 transposonbased expression systems have been developed as effective means to facilitate protein productivity. Here, we proposed novel strategies to improve conventional Tol2 transposon systems. The use of Tol2 transposase mRNA as a helper vector improved the efficiency of transgene integration and protein production. Moreover, the use of the Tol2 transposon vector containing the minimum cis-sequences essential for transposition (mini-TP) also served as one of the efficient means to generate recombinant cells that enable higher protein production. Furthermore, mini-TP showed a more beneficial response to DNA methylation inhibitors, suggesting that the use of mini-TP with DNA methylation inhibitors could be used as a means of commercial production. Taken together, our results provide effective strategies to improve the Tol2 transposon-based expression system. These strategies will be applicable to the production of therapeutic proteins and open new avenues in biopharmaceutical research.

Improvement of Sleeping Beauty Transposon System Enabling Efficient and Stable Protein Production

이윤행,박지윤,송은선,이하늘,국명욱,주정현,노형민,박준태 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.3

The most essential goal in the field of biopharmaceuticals is to develop cell lines with higher protein yields. To this goal, the Sleeping Beauty (SB) transposonbased expression system has been developed as a powerful tool for increasing protein productivity. However, SB transposon system has fallen short of expectation in terms of the efficiency and stability of protein production, limiting its applicability to large-scale production of recombinant proteins. Here, we propose a novel strategy to increase the efficiency and stability of protein production, through modification of the traditional SB transposon vector. Adding a pair of inverted terminal repeats (ITRs) next to existing ITRs (i.e., double-ITRs) significantly increased the efficiency of transgene integration, resulting in high-yield and sustained protein production. Furthermore, double- ITRs responded more favorably to DNA methylation inhibitors in terms of protein yield, implying that using double-ITRs with DNA methylation inhibitors may be effective in increasing protein productivity. Taken together, our study introduces a new vector platform that is applicable to high-yield and sustained protein production, and will open new avenues in the field of biopharmaceuticals.

Improvement of Tol2 Transposon System by Modification of Tol2 Transposase

박지윤,이하늘,송은선,이윤행,국명욱,고가현,변영주,권형욱,박준태 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.6

Tol2 transposon-based vector platform has been developed as a useful strategy to improve protein yield. Tol2 transposase identifies inverted terminal repeats of transposons and enhances transgene integration efficiency in the genome. However, Tol2 transposon system does not reach a level of efficiency for use in commercial production, limiting its usefulness for mass production of recombinant proteins. Here, we proposed a novel strategy to improve the existing Tol2 transposase by adding a nuclear localization signal of histone H2B next to the Tol2 transposase (i.e., NLS-H2B-Tol2 transposase). Comparing the NLS-H2BTol2 transposase with the traditional Tol2 transposase, a significant increase in protein production was measured. Concurrently, a significant increase in nuclear transport of NLS-H2B-Tol2 transposase was observed, suggesting a causal relationship between increased nuclear transport and increased protein production. Furthermore, importin beta maximized NLS-H2B-Tol2-mediated protein productivity. Taken together, our findings offer useful ways for improving conventional Tol2 transposon-based vector platform. This new platform will be a breakthrough in the field of biopharmaceuticals to produce therapeutic proteins.