Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System
The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a ma...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-09-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/15/10/1983 |
| _version_ | 1797572087430250496 |
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| author | Oleksandr Kondratov Sergei Zolotukhin |
| author_facet | Oleksandr Kondratov Sergei Zolotukhin |
| author_sort | Oleksandr Kondratov |
| collection | DOAJ |
| description | The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological “fitness” of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre–recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding “fit” AAV capsids characterized by a high production yield and superior transduction efficiencies. |
| first_indexed | 2024-03-10T20:49:46Z |
| format | Article |
| id | doaj.art-458938bd46814838b3b01f744179ad89 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T20:49:46Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-458938bd46814838b3b01f744179ad892023-11-19T18:26:25ZengMDPI AGViruses1999-49152023-09-011510198310.3390/v15101983Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 SystemOleksandr Kondratov0Sergei Zolotukhin1Division of Cellular and Molecular Therapy, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610, USADivision of Cellular and Molecular Therapy, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610, USAThe widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological “fitness” of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre–recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding “fit” AAV capsids characterized by a high production yield and superior transduction efficiencies.https://www.mdpi.com/1999-4915/15/10/1983rAAVKozakSf9packagingtransductiondirected evolution |
| spellingShingle | Oleksandr Kondratov Sergei Zolotukhin Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System Viruses rAAV Kozak Sf9 packaging transduction directed evolution |
| title | Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System |
| title_full | Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System |
| title_fullStr | Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System |
| title_full_unstemmed | Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System |
| title_short | Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System |
| title_sort | exploring the comprehensive kozak sequence landscape for aav production in sf9 system |
| topic | rAAV Kozak Sf9 packaging transduction directed evolution |
| url | https://www.mdpi.com/1999-4915/15/10/1983 |
| work_keys_str_mv | AT oleksandrkondratov exploringthecomprehensivekozaksequencelandscapeforaavproductioninsf9system AT sergeizolotukhin exploringthecomprehensivekozaksequencelandscapeforaavproductioninsf9system |