DISCUSSION
Our study compares two systems to generate recombinant baculovirus vectors in the context of rAAV production. Indeed, baculovirus backbone can be engineered by exploiting both homologous recombination (HR) or Tn7 transposition, the latter being the most widely used system. First, we show that the recombinant baculoviruses derived from the Tn7 system tends to loose the exogenous expression cassette after few rounds of viral amplification while HR system preserves baculoviral genome stability over multiple passages. This is an advantage for manufacturing at industrial scale, because multiple rounds of baculovirus amplifications could be needed to infect large volumes of culture. Secondly, we show that BEVs generated with the HR system preserve rAAV yields and infectivity comparable to the standard Tn7 system. Importantly, HR system is devoid of any bacterial DNA sequences in rAAV biotherapeutic final product which is an additional safety advantage to meet requirements from the regulatory agencies.
The genetic instability, and thus, substantial loss of the expression cassette, observed during recombinant baculovirus amplification after Tn7 transposition was specific to this system. The mechanism underlying this genetic instability is not fully understood, although the presence of residual bacterial elements could be detrimental for baculovirus genome replication. Our data show that HR system resulted in increased genetic stability of the inserted transgene, due to knock-out the BAC sequence from parental bacmid, when homologous recombination occurred. Nonetheless, some limitations of HR system have also been identified, i) the low frequency of natural HR events that can occur in Sf9 insect cell could lead to the generation of fewer number of clones of recombinant baculoviruses; ii) the risk to generate baculovirus defective interfering particles (DIs) due to cross-complementation of ORF1629 essential protein from viable recombinant baculovirus. Here we show that the use of circular donor plasmid induced very low HR frequency and lead to accumulation of DIs that are not carrying the cassette of interest. However, we solved this issue by using a linearized donor plasmid that maximizes the on-target cassette integration and finally leads to similar yield of baculoviral clones than Tn7 system. This observation suggests again the need for HR system to still proceed with one round of viral plaque isolation to definitively eliminate DIs and generate a pure baculoviral reagent before amplification for generating master viral seed (MVS). Whereas the standard Tn7 system seemed to be limited to a few numbers of passages, in this study we show that HR system may allow extended number of rounds (up to 10 passages) without cassette instability. This is a major advantage for industrial applications if large batches of biotherapeutic products require the use of high doses of baculovirus reagents.
Our study also shows that HR and Tn7-derived BEVs generated similar amounts of rAAV vectors, despite the fact that total capsid titer seemed to be slightly higher with HR. This observation was confirmed by SDS-PAGE analyses when running AAV proteins normalized by the vector genome copy number. Further investigations are necessary to determine if the higher proportion of empty AAV capsids in the HR system is due to the absence of chitinase and cathepsin in the BacMagic-2 system, as suggested by another group (Chen, 2017), or a direct consequence of the higher genetic stability of Cap expression along the rAAV production.
Importantly, we show that final rAAV products generated with HR-derived BEV are exempted of encapsidated bacterial sequences including antibiotic resistance genes, such as gentamycin and kanamycin. In contrary to the Tn7 system, the HR system knock-out the BAC sequence from parental bacmid. Gentamycin sequence is detectable in rAAV generated with Tn7-derived BEVs, likely due to the proximal location with the AAV ITRs. Nonetheless, none of the two methods can avoid this reverse packaging phenomenon of baculoviral sequences from the AAV ITRs (Penaud-Budloo et al., 2017).
In summary, we conducted a side-by-side comparison of different methods to generate recombinant baculovirus vectors and concluded that all methods are equally efficient to generate high yields of rAAV vectors. HR-derived BEVs showed higher genetic stability over passages and lower amounts of nucleic acid impurities (i.e. bacterial sequences) which are significant advantages for large scale manufacturing of rAAV biotherapeutics.