Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system
Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-base...
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| Veröffentlicht in: | Gene therapy 2018-09, Vol.25 (6), p.415-424 |
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| creator | Bosma, Bas du Plessis, Francois Ehlert, Erich Nijmeijer, Bart de Haan, Martin Petry, Harald Lubelski, Jacek |
| description | Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry. |
| doi_str_mv | 10.1038/s41434-018-0034-7 |
| format | Article |
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High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.</description><identifier>ISSN: 0969-7128</identifier><identifier>EISSN: 1476-5462</identifier><identifier>DOI: 10.1038/s41434-018-0034-7</identifier><identifier>PMID: 30069004</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>42 ; 42/41 ; 42/44 ; 631/1647/1513 ; 631/1647/2300/1514 ; Baculoviridae - genetics ; Biomedical and Life Sciences ; Biomedicine ; Capsid Proteins - genetics ; Cell Biology ; Expression vectors ; Gene Expression ; Gene Therapy ; Genetic Therapy ; Genetic Vectors - genetics ; Genetic Vectors - therapeutic use ; Human Genetics ; Humans ; Nanotechnology ; Operon - genetics ; Parvovirinae - genetics ; Serogroup ; Stoichiometry ; Translation initiation ; Viral Proteins - genetics ; Viral Proteins - therapeutic use ; VP1 protein ; VP3 protein</subject><ispartof>Gene therapy, 2018-09, Vol.25 (6), p.415-424</ispartof><rights>The Author(s) 2018</rights><rights>2018. 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High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. 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Academic</collection><jtitle>Gene therapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bosma, Bas</au><au>du Plessis, Francois</au><au>Ehlert, Erich</au><au>Nijmeijer, Bart</au><au>de Haan, Martin</au><au>Petry, Harald</au><au>Lubelski, Jacek</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system</atitle><jtitle>Gene therapy</jtitle><stitle>Gene Ther</stitle><addtitle>Gene Ther</addtitle><date>2018-09-01</date><risdate>2018</risdate><volume>25</volume><issue>6</issue><spage>415</spage><epage>424</epage><pages>415-424</pages><issn>0969-7128</issn><eissn>1476-5462</eissn><abstract>Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. 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| ispartof | Gene therapy, 2018-09, Vol.25 (6), p.415-424 |
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| subjects | 42 42/41 42/44 631/1647/1513 631/1647/2300/1514 Baculoviridae - genetics Biomedical and Life Sciences Biomedicine Capsid Proteins - genetics Cell Biology Expression vectors Gene Expression Gene Therapy Genetic Therapy Genetic Vectors - genetics Genetic Vectors - therapeutic use Human Genetics Humans Nanotechnology Operon - genetics Parvovirinae - genetics Serogroup Stoichiometry Translation initiation Viral Proteins - genetics Viral Proteins - therapeutic use VP1 protein VP3 protein |
| title | Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system |
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