Journal
HUMAN GENE THERAPY METHODS
Volume 30, Issue 5, Pages 172-183Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/hgtb.2019.123
Keywords
rAAV production; OneBac; recombinant baculovirus; Sf9 packaging cell line; gene therapy
Categories
Funding
- National Natural Science Foundation of China [31500868, 31771156]
- National Basic Research Program (973 Program) of China [2015CB755600]
- State Key Program of National Natural Science Foundation of China [31830035]
- Strategic Priority Research Program (B) [XDB0205005]
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Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 10(5) vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.
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