<i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System
Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applic...
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MDPI AG
2021-04-01
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| Online Access: | https://www.mdpi.com/1999-4915/13/4/704 |
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| author | Qian Yu Pengfei Chang Xiaoxuan Liu Peng Lü Qi Tang Zhongjian Guo Jianming Qiu Keping Chen Qin Yao |
| author_facet | Qian Yu Pengfei Chang Xiaoxuan Liu Peng Lü Qi Tang Zhongjian Guo Jianming Qiu Keping Chen Qin Yao |
| author_sort | Qian Yu |
| collection | DOAJ |
| description | Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, <i>Bombyx mori</i> pupae were used as a new platform and infected with recombinant <i>Bombyx mori</i> nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 10<sup>4</sup> vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 10<sup>12</sup> VG/larva and 4.6 × 10<sup>12</sup> VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors. |
| first_indexed | 2024-03-10T12:12:54Z |
| format | Article |
| id | doaj.art-c9f62be6d05b4dd1b98fbcb308f2c77c |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T12:12:54Z |
| publishDate | 2021-04-01 |
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| series | Viruses |
| spelling | doaj.art-c9f62be6d05b4dd1b98fbcb308f2c77c2023-11-21T16:06:31ZengMDPI AGViruses1999-49152021-04-0113470410.3390/v13040704<i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression SystemQian Yu0Pengfei Chang1Xiaoxuan Liu2Peng Lü3Qi Tang4Zhongjian Guo5Jianming Qiu6Keping Chen7Qin Yao8School of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaDepartment of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USASchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaSchool of Life Sciences, Jiangsu University, Zhenjiang 212013, ChinaRecombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, <i>Bombyx mori</i> pupae were used as a new platform and infected with recombinant <i>Bombyx mori</i> nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 10<sup>4</sup> vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 10<sup>12</sup> VG/larva and 4.6 × 10<sup>12</sup> VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.https://www.mdpi.com/1999-4915/13/4/704rBmNPV/AAV2Rep-HBoV1CaprBmNPV/AAV2ITR-eGFPsilkworm pupaeBmN cellsrAAV2/HBoV1 |
| spellingShingle | Qian Yu Pengfei Chang Xiaoxuan Liu Peng Lü Qi Tang Zhongjian Guo Jianming Qiu Keping Chen Qin Yao <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System Viruses rBmNPV/AAV2Rep-HBoV1Cap rBmNPV/AAV2ITR-eGFP silkworm pupae BmN cells rAAV2/HBoV1 |
| title | <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System |
| title_full | <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System |
| title_fullStr | <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System |
| title_full_unstemmed | <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System |
| title_short | <i>Bombyx mori</i> Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a <i>Bombyx mori</i> Nuclear Polyhedrosis Virus Expression System |
| title_sort | i bombyx mori i pupae efficiently produce recombinant aav2 hbov1 vectors with a i bombyx mori i nuclear polyhedrosis virus expression system |
| topic | rBmNPV/AAV2Rep-HBoV1Cap rBmNPV/AAV2ITR-eGFP silkworm pupae BmN cells rAAV2/HBoV1 |
| url | https://www.mdpi.com/1999-4915/13/4/704 |
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