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: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-06-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1198465/full |
| _version_ | 1827919092765425664 |
|---|---|
| author | Asim Azhar Siddiqui Sabrina Peter Eve Zi Xian Ngoh Cheng-I. Wang Shirelle Ng John A. Dangerfield Walter H. Gunzburg Walter H. Gunzburg Peter Dröge Harshyaa Makhija |
| author_facet | Asim Azhar Siddiqui Sabrina Peter Eve Zi Xian Ngoh Cheng-I. Wang Shirelle Ng John A. Dangerfield Walter H. Gunzburg Walter H. Gunzburg Peter Dröge Harshyaa Makhija |
| author_sort | Asim Azhar Siddiqui |
| collection | DOAJ |
| 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 applications for more effective and affordable therapies. |
| first_indexed | 2024-03-13T03:52:55Z |
| format | Article |
| id | doaj.art-7d08271bf26747a0830e808c29952bae |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-13T03:52:55Z |
| publishDate | 2023-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-7d08271bf26747a0830e808c29952bae2023-06-22T09:39:33ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-06-011110.3389/fbioe.2023.11984651198465A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cellsAsim Azhar Siddiqui0Sabrina Peter1Eve Zi Xian Ngoh2Cheng-I. Wang3Shirelle Ng4John A. Dangerfield5Walter H. Gunzburg6Walter H. Gunzburg7Peter Dröge8Harshyaa Makhija9School of Biological Sciences, Nanyang Technological University, Singapore, SingaporeSchool of Biological Sciences, Nanyang Technological University, Singapore, SingaporeSingapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, SingaporeSingapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore, SingaporeAustrianova Singapore Pte. Ltd., Singapore, SingaporeAustrianova Singapore Pte. Ltd., Singapore, SingaporeAustrianova Singapore Pte. Ltd., Singapore, SingaporeDepartment of Pathobiology, Institute of Virology, University of Veterinary Medicine, Vienna, AustriaSchool of Biological Sciences, Nanyang Technological University, Singapore, SingaporeLambdaGen Pte. Ltd., Singapore, SingaporeReliable 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 applications for more effective and affordable therapies.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1198465/fullhuman cell line engineeringλ integraseExpi293F cellsbiotherapeuticssite-specific transgenesismicroencapsulation |
| spellingShingle | Asim Azhar Siddiqui Sabrina Peter Eve Zi Xian Ngoh Cheng-I. Wang Shirelle Ng John A. Dangerfield Walter H. Gunzburg Walter H. Gunzburg Peter Dröge Harshyaa Makhija A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells Frontiers in Bioengineering and Biotechnology human cell line engineering λ integrase Expi293F cells biotherapeutics site-specific transgenesis microencapsulation |
| 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 | human cell line engineering λ integrase Expi293F cells biotherapeutics site-specific transgenesis microencapsulation |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1198465/full |
| work_keys_str_mv | AT asimazharsiddiqui aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT sabrinapeter aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT evezixianngoh aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT chengiwang aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT shirelleng aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT johnadangerfield aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT walterhgunzburg aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT walterhgunzburg aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT peterdroge aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT harshyaamakhija aversatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT asimazharsiddiqui versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT sabrinapeter versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT evezixianngoh versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT chengiwang versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT shirelleng versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT johnadangerfield versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT walterhgunzburg versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT walterhgunzburg versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT peterdroge versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells AT harshyaamakhija versatilegenomictransgenesisplatformwithenhancedlintegraseforhumanexpi293fcells |