Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

Abstract

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 largescale 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.

Solutions against emerging infectious and noninfectious human diseases through the application of baculovirus technologies

“…In the first report where BEVS were employed for this purpose, three rec-baculoviruses were used (rep and cap genes in two independent BVs and the ITR-GOI-ITR construct in the third: the socalled ThreeBac system) along with Sf9 cells (Urabe et al 2002). Subsequently, different optimizations were performed including the use of only two rec-baculoviruses-i.e., the TwoBac system; one combing both the rep and the cap genes and the other carrying the ITR-GOI-ITR (Wu et al 2019) plus the later generation of a OneBac system consisting of packaging-Sf9 cells that expressed rep and cap genes when infected with a rec-baculovirus that carried the therapeutic nucleic acid (Mietzsch et al 2015). Subsequent optimizations of the OneBac platform enabled quite good yields of whole particles with little contaminating DNA (Mietzsch et al 2015;Joshi et al 2019).…”

Viral vector platforms within the gene therapy landscape

“…Since the baculovirus provides helper function, 208 a single BEV system in conjunction with Sf9 cell lines that stably express rep , can be employed for flexible and high-titer large-scale vector production. 209 Interestingly, a report that vectors produced from Sf9 cells are differentially posttranslationally modified, as compared to those generated from human- or mammalian-derived cell lines raised question as to whether differences in production schemes may impact tropism and transduction efficacies, 210 although further work in this area is necessary to cross-validate these findings. Nonetheless, BEV/Sf9 systems exhibit reduced encapsidation of contaminating DNAs 205 and therefore remain an attractive production strategy for large-scale, clinical-grade vectors.…”

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

“…[48,87] Similarly, a modified version of One-Bac consisting of only the rep-expressing Sf9 cell line and Bac-CapX-GOI for AAVX (X = serotype) production has also been recently reported. [88] A study by Joshi et al [43] reported AAV5 production in high cell density fed-batch cultures using One-Bac3.0, with a final volumetric yield exceeding 2 × 10 14 VG per L of cell culture. In contrast to the previously reported fed-batch process, [68] the culture at a 10 million cells per mL cell density was infected at a higher optimal MOI of 3, which resulted in the transition of the cell culture to the AAV-production phase from the growth phase within 24 hpi.…”

“…[ 48,87 ] Similarly, a modified version of One‐Bac consisting of only the rep‐ expressing Sf9 cell line and Bac‐CapX‐GOI for AAVX (X = serotype) production has also been recently reported. [ 88 ]…”