| Adeno-associated virus is nonpathogenic to the human body, and AAV vectors are even safer than their wild-type precursor because AAV vectors are devoid of all AAV genes, except for the two Inverted Terminal Repeats. Furthermore, AAV-mediated gene transfer can lead to long-term expression. For these characteristic features, AAV vectors have been being given more and more attention. However, the major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. Exploring an efficient, safe and scalable production method to get a high quantity of AAV vectors is necessary to meet the clinical requirement.Apart from Adenovirus and Herpes simplex virus, Baculovirus, which will not elicit toxicity to the human body, is also a helper virus which can provide helper functions for the replication and package of AAV. So it will be an ideal production method if we use baculovirus to carry the cis and tran elements necessary for AAV package to produce rAAV in insect cells.The rAAV production system based on baculovirus includes two tran elements: two AAV replication proteins (Rep78,Rep52) and three AAV capsid proteins( Cap1,Cap2,Cap3). The two tran elements can be carried by two separate baculoviruses or be incorporated into a single baculovirus. Besides, another baculovirus carrying the transgene(EGFP) flanked by two AAV ITRs is the cis element which can be excised, replicated and packaged into rAAV particles with the helper functions of the trans elements.According to the sequences of Rep and Cap genes, we designed specific primers to amplify the Rep78,Rep52,Cap1 and Cap2+3 genes. Then the Rep78 and Cap2+3 genes were together constructed into the pFastBacDual plasmid to get the vector pFBD-AAV2Rep78Cap2+3, as well as Rep52 and Cap1 in the same way to get the vector pFBD-AAV2Rep52Cap1. Furthermore, An artificial intron containing the insect cell polyhedron promoter was constructed and inserted into the Rep and Cap coding sequences, which then were together constructed into the pFastBacDual plasmid. With the intron's dual functions in insect cells, the Rep coding sequence can express Rep78 and Rep52 simultaneously, while the Cap coding sequence can express both Cap1 and Cap2+3. Transform the above pFastBacDual constructs into MAX Efficiency? DH10BacTM competent E. coli, and site-specific transposition of the expression cassette into a baculovirus shuttle vector (bacmid) occured to form recombinant Bacmids, which in turn were transfected into the Sf9 cells to generate recombinant baculoviruses: Bac-AAV2Rep78Cap2+3, Bac-AAV2Rep52Cap1 and Bac-AAV2RepiCapi. Similar method was used to generate recombinant baculovirus Bac-AAV2EGFP. We used two strategies to produce rAAV: the first is to use three baculoviruses Bac-AAV2Rep78Cap2+3, Bac-AAV2Rep52Cap1 and Bac-AAV2EGFP, and the second is to use two baculoviruses Bac-AAV2RepiCapi and Bac-AAV2EGFP.Primary research proved that production systems based on three baculoviruses and two baculoviruses can both produce rAAV2-EGFP, with production capability of 4.85×103VG/cell and 6.12×104VG/cell separately. Further results detected by Flow Cytometry showed that the two systems can produce 3 transduction units and 30 transduction units per Sf9 cell separately. When the rAAV-EGFP produced from the above two systems were used to infect HEK293 cells, strong Green fluorescence can be seen under a fluorescence microscope, which implies that the activity of the rAAV2-EGFP is good enough to transduce gene of interest into targeted cells. According to the above results, the system based on two baculoviruses has a more powerful capability in producing rAAV2-EGFP. It is assumed that if we scale up the system to large-scale suspension infection, the quantity of rAAV and production efficiency will be elevated greatly. Hence, the efficient and scalable production method simplifies the production process, reduces the cost and can meet the need of gene therapy and clinical applications. |
