Key result
Sf9 cells match rAAV2/HBoV1 yield but have ~6-fold lower transduction potency than HEK293 cells.
Why the study?
The production of rAAV2/HBoV1 vectors in insect cells using baculovirus expression vectors was not established and required optimization compared to production in HEK293 cells.
A baculovirus expression vector system can produce rAAV2/HBoV1 in insect cells at high yields, though further optimization is needed to improve capsid packaging efficiency and transduction potency.
NS-free production boosts rAAV2/HBoV1 yields >16-fold; extends manufacturing but leaves clinical translation open.
We have previously developed an rAAV2/HBoV1 vector in which a recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged into a human bocavirus 1 (HBoV1) capsid. Recently, the production of rAAV2/HBoV1 in human embryonic kidney (HEK) 293 cells has been greatly improved in the absence of any HBoV1 nonstructural proteins (NS). This NS-free production system yields over 16-fold more vectors than the original production system that necessitates NS expression. The production of rAAV with infection of baculovirus expression vector (BEV) in the suspension culture of Sf9 insect cells is highly efficient and scalable. Since the replication of the rAAV2 genome in the BEV system is well established, we aimed to develop a BEV system to produce the rAAV2/HBoV1 vector in Sf9 cells. We optimized the usage of translation initiation signals of the HBoV1 capsid proteins (Cap), and constructed a BEV Bac-AAV2Rep-HBoV1Cap, which expresses the AAV2 Rep78 and Rep52 as well as the HBoV1 VP1, VP2, and VP3 at the appropriate ratios. We found that it is sufficient as a trans helper to the production of rAAV2/HBoV1 in Sf9 cells that were co-infected with the transfer Bac-AAV2ITR-GFP-luc that carried a 5.4-kb oversized rAAV2 genome with dual reporters. Further study found that incorporation of an HBoV1 small NS, NP1, in the system maximized the viral DNA replication and thus the rAAV2/HBoV1 vector production at a level similar to that of the rAAV2 vector in Sf9 cells. However, the transduction potency of the rAAV2/HBoV1 vector produced from BEV-infected Sf9 cells was 5-7-fold lower in polarized human airway epithelia than that packaged in HEK293 cells. Transmission electron microscopy analysis found that the vector produced in Sf9 cells had a high percentage of empty capsids, suggesting the pseudopackage of the rAAV2 genome in HBoV1 capsid is not as efficient as in the capsids of AAV2. Nevertheless, our study demonstrated that the rAAV2/HBoV1 can be produced in insect cells with BEVs at a comparable yield to rAAV, and that the highly efficient expression of the HBoV1 capsid proteins warrants further optimization.
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Deng et al. (2020) studied this question. rAAV2/HBoV1 vector production in Sf9 cells using BEV system vs. rAAV2/HBoV1 vector production in HEK293 cells was evaluated on Vector yield and transduction efficiency. The rAAV2/HBoV1 vector can be produced in Sf9 insect cells at a yield comparable to rAAV2 when HBoV1 NP1 is co-expressed, though its transduction potency was 5-7-fold lower than vectors packaged in HEK293 cells.
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