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Processes
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Transient Gene Expression for Rapid Protein and Virus-Vector Supply
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Transient Gene Expression for Rapid Protein and Virus-Vector Supply

A special issue of Processes (ISSN 2227-9717). This special issue belongs to the section "Biological Processes and Systems".

Deadline for manuscript submissions: closed (30 April 2019) | Viewed by 19343

Special Issue Editors

Prof. Dr. Florian M. Wurm

E-Mail Website
Guest Editor
Swiss Federal Institute of Technology Lausanne, Route Cantonale, 1015 Lausanne, Switzerland
Interests: gene transfer; protein production/manufacturing from cultivated mammalian cells, including regulatory issues, process development; innovative bioreactors for mammalian cells; suspension culture; CHO cells; large scale manufacturing - CHO; orbital shaking; optimizing mammalian cell culture processes; transient gene expression
Dr. Martin Jordan

E-Mail Website
Guest Editor
Merck Biopharma, Biotech Process Sciences, Corsier-sur-Vevey, Switzerland
Interests: media development; process optimization; transfection; transient expression; cell line generation; glycosylation of recombinant proteins; high throughput culture systems; process intensification

Special Issue Information

Dear Colleagues,

Transient Gene Expression (TGE) is a most important technique for investigating fundamental processes in the life sciences. Google give 34 million hits, when using these three words.

This Special Issue of “Transient Gene Expression for Rapid Protein and Virus-Vector supply” focuses on protein production and on the generation of innovative virus vectors for the widest range of applications in R&D and for potential use in the clinic. Different from stable expression, transient expression allows the delivery of the desired DNA of interest into a culture of animal cells and the protein expression begins literally within minutes when the DNA arrives at the nucleus of cells. Days after such a delivery of DNA, protein can be obtained from such cultures. This allows the study of structure and function of the desired protein, preferable in a purified form, but also frequently just separates from the cells that produced the protein, in sufficient quantities, to execute a large battery of experiments with it, including in vitro or in vivo studies.

We encourage submissions of papers that present various technologies, used with CHO, HEK-293, HeLa and other mammalian or non-mammalian derived cell lines providing access to rapid protein synthesis and to virus vectors. The papers should emphasize readily applicable methods, both for “standard” laboratories, but also for more specialized use at scales of operation that exceed the typical research laboratory (“large scale transient gene expression”). In spite of the fact that virus-mediated transfer of genetic information can be considered transient, such as with the help of Baculovirus vectors, the editors of this Special Issue wish to restrict the issue to submissions of “naked” DNA or RNA as carriers the genes of interests into the host cell system.

Observation, discussion points or concerns that could lead to the use of TGE for clinical manufacture and use of products derived thereof for therapy are encouraged as well.

Prof. Dr. Florian M. Wurm
Dr. Martin Jordan
Guest Editors

Review related to the Special Issue：
Wurm, F.M. CHO Quasispecies—Implications for Manufacturing Processes. Processes 2013, 1, 296-311.
Wurm, F.M.; Wurm, M.J. Cloning of CHO Cells, Productivity and Genetic Stability—A Discussion. Processes 2017, 5, 20.

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Processes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2400 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Transfection, transient gene expression, mammalian cells, insect cells, large-scale, protein production, disposable bioreactors, protein variant analysis, protein structure, protein function, chimaeric proteins, protein design, DNA transfection reagents

Published Papers (3 papers)

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Research

9 pages, 1728 KiB  
Article
Enhanced Production of Anti-PD1 Antibody in CHO Cells through Transient Co-Transfection with Anti-Apoptotic Gene Bcl-xL Combined with Rapamycin
by Yunxia Li, Xinyu Zhang, Lei Wang, Huifang Zong, Yuan Yuan, Lei Han, Xi Li, Chenxiao Xu, Jingyi Zhang, Jianwei Zhu and Baohong Zhang
Processes 2019, 7(6), 329; https://doi.org/10.3390/pr7060329 - 01 Jun 2019
Viewed by 5667
Abstract
CHO cells are often used to produce monoclonal antibodies in mammalian cell expression systems. In the process of large-scale cell culture, apoptosis is related to cell survival and product quality. Over-expressing an anti-apoptotic gene to delay apoptosis and improve cell growth is one [...] Read more.
CHO cells are often used to produce monoclonal antibodies in mammalian cell expression systems. In the process of large-scale cell culture, apoptosis is related to cell survival and product quality. Over-expressing an anti-apoptotic gene to delay apoptosis and improve cell growth is one of the strategies for improving productivity of monoclonal antibodies. Autophagy inducer rapamycin can extend the culture duration of CHO cells and affect the yield of antibodies. A method was developed for transient co-transfection of anti-apoptotic genes and genes of interest combined with rapamycin to increase the transient expression of the anti-PD1 antibody. Under the optimal transfection conditions, the combination of Bcl-xL and rapamycin can significantly delay cell apoptosis, inhibit cell proliferation, and prolong cell life-time. As a result, anti-PD1 monoclonal antibody expression levels are increased by more than 2 times. Full article
(This article belongs to the Special Issue Transient Gene Expression for Rapid Protein and Virus-Vector Supply)
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17 pages, 3163 KiB  
Article
A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells
by Chih-Yu Wu, Chao-Wei Huang, Yu-Shin Nai, Pei-Yu Chu, Chung-Hsiung Wang and Shih-Torng Ding
Processes 2019, 7(5), 291; https://doi.org/10.3390/pr7050291 - 15 May 2019
Cited by 3 | Viewed by 5426
Abstract
Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable [...] Read more.
Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications. Full article
(This article belongs to the Special Issue Transient Gene Expression for Rapid Protein and Virus-Vector Supply)
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17 pages, 3255 KiB  
Article
Non-Viral Transfection of Human T Lymphocytes
by Simon A. B. Riedl, Patrick Kaiser, Alexander Raup, Christopher V. Synatschke, Valérie Jérôme and Ruth Freitag
Processes 2018, 6(10), 188; https://doi.org/10.3390/pr6100188 - 11 Oct 2018
Cited by 19 | Viewed by 7066
Abstract
The genetic modification of human T lymphocytes with established non-viral methods is inefficient. Linear polyethylenimine (l-PEI), one of the most popular non-viral transfection agents for mammalian cells in general, only achieves transfection rates in the single digit percentage range for these cells. Here, [...] Read more.
The genetic modification of human T lymphocytes with established non-viral methods is inefficient. Linear polyethylenimine (l-PEI), one of the most popular non-viral transfection agents for mammalian cells in general, only achieves transfection rates in the single digit percentage range for these cells. Here, a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA) nanostar (number average of the molecular weight: 755 kDa, polydispersity: <1.21) synthesized via atom transfer radical polymerization (ATRP) from a silsesquioxane initiator core is proposed as alternative. The agent is used to prepare polyplexes with plasmid DNA (pDNA). Under optimal conditions these polyplexes reproducibly transfect >80% of the cells from a human T-cell leukemia cell line (Jurkat cells) at viabilities close to 90%. The agent also promotes pDNA uptake when simply added to a mixture of cells and pDNA. This constitutes a particular promising approach for efficient transient transfection at large scale. Finally, preliminary experiments were carried out with primary T cells from two different donors. Results were again significantly better than for l-PEI, although further research into the response of individual T cells to the transfection agent will be necessary, before either method can be used to routinely transfect primary T lymphocytes. Full article
(This article belongs to the Special Issue Transient Gene Expression for Rapid Protein and Virus-Vector Supply)
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