A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells
Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an ar...
| Main Authors: | , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2023
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| Online Access: | https://hdl.handle.net/10356/168601 |
| _version_ | 1811693400639930368 |
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| author | Siddiqui, Asim Azhar Peter, Sabrina Ngoh, Eve Zi Xian Wang, Cheng-I Ng, Shirelle Dangerfield, John A. Gunzburg, Walter H. Dröge, Peter Makhija, Harshyaa |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Siddiqui, Asim Azhar Peter, Sabrina Ngoh, Eve Zi Xian Wang, Cheng-I Ng, Shirelle Dangerfield, John A. Gunzburg, Walter H. Dröge, Peter Makhija, Harshyaa |
| author_sort | Siddiqui, Asim Azhar |
| collection | NTU |
| description | Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based 32 applications for more effective and affordable therapies. |
| first_indexed | 2024-10-01T06:51:05Z |
| format | Journal Article |
| id | ntu-10356/168601 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T06:51:05Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | ntu-10356/1686012023-06-26T15:32:10Z A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells Siddiqui, Asim Azhar Peter, Sabrina Ngoh, Eve Zi Xian Wang, Cheng-I Ng, Shirelle Dangerfield, John A. Gunzburg, Walter H. Dröge, Peter Makhija, Harshyaa School of Biological Sciences Singapore Immunology Network, A*STAR Science Human Cell Line Engineering λ Integrase Expi293F Cells Biotherapeutics Site-Specific Transgenesis Microencapsulation Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based 32 applications for more effective and affordable therapies. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This work was supported through a grant from the National Research Foundation-Competitive Research Programme, Singapore to P.D. (NRF-CRP21-2018-0002), and the Core Fund from the Singapore Immunology Network, Agency for Science, Technology and Research, Singapore to C-I.W. 2023-06-23T02:33:58Z 2023-06-23T02:33:58Z 2023 Journal Article Siddiqui, A. A., Peter, S., Ngoh, E. Z. X., Wang, C., Ng, S., Dangerfield, J. A., Gunzburg, W. H., Dröge, P. & Makhija, H. (2023). A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells. Frontiers in Bioengineering and Biotechnology, 11, 1198465-. https://dx.doi.org/10.3389/fbioe.2023.1198465 2296-4185 https://hdl.handle.net/10356/168601 10.3389/fbioe.2023.1198465 11 1198465 en NRF-CRP21-2018-0002 Frontiers in Bioengineering and Biotechnology © 2023 Siddiqui, Peter, Ngoh, Wang, Ng, Dangerfield, Gunzburg, Dröge and Makhija. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. application/pdf |
| spellingShingle | Science Human Cell Line Engineering λ Integrase Expi293F Cells Biotherapeutics Site-Specific Transgenesis Microencapsulation Siddiqui, Asim Azhar Peter, Sabrina Ngoh, Eve Zi Xian Wang, Cheng-I Ng, Shirelle Dangerfield, John A. Gunzburg, Walter H. Dröge, Peter Makhija, Harshyaa A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title | A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title_full | A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title_fullStr | A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title_full_unstemmed | A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title_short | A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells |
| title_sort | versatile genomic transgenesis platform with enhanced λ integrase for human expi293f cells |
| topic | Science Human Cell Line Engineering λ Integrase Expi293F Cells Biotherapeutics Site-Specific Transgenesis Microencapsulation |
| url | https://hdl.handle.net/10356/168601 |
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