Rational design and construction of multi-copy biomanufacturing islands in mammalian cells
| العنوان: | Rational design and construction of multi-copy biomanufacturing islands in mammalian cells |
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| المؤلفون: | Raffaele Altamura, Jiten Doshi, Yaakov Benenson |
| المصدر: | Nucleic Acids Research, 50 (1) Nucleic Acids Research |
| بيانات النشر: | ETH Zurich, 2022. |
| سنة النشر: | 2022 |
| مصطلحات موضوعية: | Mice, Cricetulus, AcademicSubjects/SCI00010, Narese/1, Gene Targeting, Genetic Vectors, Genetics, Animals, Humans, CHO Cells, Transgenes, Synthetic Biology and Bioengineering, Recombinant Proteins |
| الوصف: | Cell line development is a critical step in the establishment of a biopharmaceutical manufacturing process. Current protocols rely on random transgene integration and amplification. Due to considerable variability in transgene integration profiles, this workflow results in laborious screening campaigns before stable producers can be identified. Alternative approaches for transgene dosage increase and integration are therefore highly desirable. In this study, we present a novel strategy for the rapid design, construction, and genomic integration of engineered multiple-copy gene constructs consisting of up to 10 gene expression cassettes. Key to this strategy is the diversification, at the sequence level, of the individual gene cassettes without altering their protein products. We show a computational workflow for coding and regulatory sequence diversification and optimization followed by experimental assembly of up to nine gene copies and a sentinel reporter on a contiguous scaffold. Transient transfections in CHO cells indicates that protein expression increases with the gene copy number on the scaffold. Further, we stably integrate these cassettes into a pre-validated genomic locus. Altogether, our findings point to the feasibility of engineering a fully mapped multi-copy recombinant protein ‘production island’ in a mammalian cell line with greatly reduced screening effort, improved stability, and predictable product titers. Nucleic Acids Research, 50 (1) ISSN:1362-4962 ISSN:0301-5610 |
| وصف الملف: | application/application/pdf |
| اللغة: | English |
| تدمد: | 1362-4962 0301-5610 |
| DOI: | 10.3929/ethz-b-000528625 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b2dc1898de35e3757050b2542e3c146eTest |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....b2dc1898de35e3757050b2542e3c146e |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 13624962 03015610 |
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| DOI: | 10.3929/ethz-b-000528625 |