Monobac System-A Single Baculovirus for the Production of rAAV

Abstract

Large-scale manufacturing of rAAV is a bottleneck for the development of genetic disease treatments. The baculovirus/Sf9 cell system underpins the first rAAV treatment approved by EMA and remains one of the most advanced platforms for rAAV manufacturing. Despite early successes, rAAV is still a complex biomaterial to produce. Efficient production of the recombinant viral vector requires that AAV replicase and capsid genes be co-located with the recombinant AAV genome. Here, we present the Monobac system, a singular, modified baculovirus genome that contains all of these functions. To assess the relative yields between the dual baculovirus and Monobac systems, we prepared each system with a transgene encoding γSGC and evaluated vectors' potency in vivo. Our results show that rAAV production using the Monobac system not only yields higher titers of rAAV vector but also a lower amount of DNA contamination from baculovirus.

Keywords: baculovirus; gene therapy; rAAV.

Figure 1

(a) Kinetics of production of rAAV8-γSGC and of baculovirus amplification, a comparison of dual baculovirus production system with and without reporter genes. Production of rAAV was performed by the co-infection of Sf9 cells with baculoviruses bac-rAAV-γSGC-GFP and bac-R2C8-YFP or with bac-rAAV-γSGC and bac-R2C8. Infections were performed at a MOI of 0.05 per baculovirus. rAAV titers, based on ITR amplicon qPCR, and baculovirus titers, based on DNApol amplicon qPCR, were determined at time points 0 h, 6 h, 24 h, 30 h, 48 h, 54 h, 72 h, 78 h, and 96 h post-infection. Titers of bulk samples were presented on a logarithmic scale and expressed as vg mL⁻¹. “Dual-baculovirus-rAAV vg mL⁻¹” represents the rAAV8- γSGC titers obtained from the co-infection performed with bac-rAAV-γSGC and bac-R2C8. “Fluorescent dual-baculovirus-rAAV vg mL⁻¹” represents the rAAV titers obtained from the co-infection performed with bac-rAAV-γSGC-GFP and bBac-R2C8-YFP. “Dual-baculovirus-baculovirus vg mL⁻¹” titer represents the baculovirus titers obtained from the co-infection performed with baculoviruses bac-rAAV-γSGC and bac-R2C8. “Fluorescent dual-baculovirus-baculovirus vg mL⁻¹” represents the baculovirus titers obtained from the co-infection performed with baculoviruses bac-rAAV-γSGC-GFP and bac-R2C8-YFP. (b) Kinetics of baculovirus co-infection during rAAV production. Percentage of infection of Sf9 cells determined by FACS, during rAAV8-γSGC production, with baculovirus bac-R2C8-YFP, encoding the rep2-cap8 expression cassette and the YFP reporter gene, and baculovirus bac-rAAV-γSGC-GFP. FACS determination of the percentage of cells co-expressing GFP and YFP reporter genes, cells expressing only one of the fluorescent proteins representing single infection only with bac-rAAV-γSGC-GFP or bac-R2C8-YFP, and cells negative for both fluorescent markers. Time points were 0 h, 6 h, 24 h, 30 h, 48 h, 54 h, 72 h, 78 h, and 96 h post-infection. (c) Kinetics of single infection with baculovirus encoding the rAAV-γSGC and baculovirus encoding rep2-cap8 expression cassette. Percentage of infection of Sf9 cells determined by FACS during single infection performed with bac-rAAV-γSGC-GFP and bac-R2C8.

Figure 2

(a) Time course of replication of baculovirus DNA during rAAV8-γSGC production in the dual baculovirus system with one baculovirus-encoding recombinant rAAV8-γSGC transgene inserted at the Tn7 site and the second baculovirus encoding cap8 gene at the egt locus and rep2 gene inserted at the Tn7 site. Total MOI was 0.1 (n = 2). Follow-up was performed for 96 h. Baculovirus DNA copy number, based on DNA Polymerase qPCR amplicon measurement, is expressed in number of copies per mL of cell culture. (b) Ratio of rAAV8-γSGC gene copy number to baculovirus DNA copy number, measured by qPCR of the baculovirus DNA polymerase gene amplicon, during rAAV8-γSGC production.

Figure 3

Schematic representation of the Monobac system. Homologous recombination is performed at the egt locus using plasmid pKD46 encoding the red recombination genes [28] in DH10 bac cells. The cat gene used to select positive clones can then be removed using a Cre-encoding plasmid (this study) leading to a Cre-insensitive lox72 DNA scare [24]. With this method, additional genes can be inserted or removed from the AcMNPV bacmid genome, like the deletion of AcMNPV cathepsin-chitinase genes [35]. pMON7124 [26] is then used to promote recombination at the Tn7 site of the bacmid, using a donor plasmid encoding the rAAV transgene and flanked by the Tn7L and R sequences.

Figure 4

Comparison of rAAV8-γSGC productivity between the dual baculovirus and the Monobac systems in spinner and bioreactor cultures. (a) Purified rAAV8-γSGC produced in Spinner (in red) (n = 3) and bioreactor vessels (in blue) (n = 2); comparison between the dual baculovirus (●, ▲) and Monobac (■, ▼) systems. rAAV8-γSGC titers are expressed in vg mL⁻¹. Each data label represents an individual production run. Horizontal black lines represent the average titers obtained. Error bars represent standard deviation. (b) Protein profiles of each rAAV8-γSGC run after vector purification are visible on Western blots using B1 progen antibody for the detection of AAV VP proteins. 5 × 10⁹ vg of purified vector have been deposed per lane. The SDS-PAGE gel (top) represents rAAV8-γSGC produced using the dual baculovirus production system. The SDS-PAGE gel (bottom) represents rAAV8-γSGC produced using the Monobac production system.

Figure 5

Impact of rep2-cap8 cloning position in the baculovirus genome on rAAV production. rAAV8-γSGC titers in bulk are expressed in vg mL⁻¹ of cell culture and represent the mean of 3 production runs. Error bars represent standard deviation. “Dual system” represents production performed using the dual-baculovirus rAAV production system [20]. “Monobac dual” represents productions performed with one baculovirus encoding rAAV8-γSGC transgene at Tn7 site while the second baculovirus encodes rep2-cap8 expression cassette from the egt locus. “Monobac” represents productions performed using the single baculovirus with rAAV8-γSGC genome inserted at the Tn7 site and rep2-cap8 expression cassette cloned at the egt locus.

Figure 6

Comparison of rAAV8-γSGC production performed using the dual baculovirus and Monobac systems. Comparison of rAAV8-γSGC production performed with dual baculovirus (Dual WT) and Monobac systems (MB WT) analyzed by (a) analytical ultracentrifugation sedimentation coefficient results for purified rAAV8-γSGC produced with dual baculovirus and Monobac production systems (shown are the data obtained by interference monitoring). Values have been normalized to c(s). Empty rAAV particles sediment between 64 and 66 S, full rAAV8 particles have sedimentation values comprised between 99 and 103 S. Values between 76 and 90 S correspond to capsids partially filled with a genome size between 1 and 3 kb. The values < 60 S correspond to impurities smaller than rAAV particles that could be free proteins or DNA. (b) qPCR ratio between baculovirus DNA polymerase amplicon per purified rAAV8-γSGC vg titers (t-test, p = 0.002, ***).

Figure 7

Genetic stability of Monobac- γSGC over 10 passages in culture flasks. We studied the genetic stability of the Monobac with and without the rAAV- γSGC transgene and the rep2-cap8 baculovirus from the dual rAAV production system. The x-axis represents the number of passages. Results were obtained by qPCR. The ITR sequence allows the follow-up of the rAAV-γSGC transgene cloned in the Monobac genome and, at the same time, the rAAV-γSGC produced during the infection. The BAC sequence quantifies the baculovirus DNA polymerase gene. The Rep52 and cap8 sequences are used to quantify the rep2-cap8 expression cassette inserted in the egt gene in the Monobac and in the Tn7 site of the baculovirus in the dual-rAAV production system. Results are expressed either as copies per mL or as ratio of either ITR, cap8, and rep52 sequences in comparison to the BAC sequence. Individual clones are represented with the horizontal line indicating the mean or data are shown as histogram bars with the SD. The x-axis indicates the number of passages. P2 represents the baculovirus seed stocks used in this experiment, while P4, P7, and P10 represent the baculovirus stocks at passages 4, 7, and 10. (a) Monobac-γSGC, stability of the rep2-cap8 expression cassette. We followed the ratio between the rep2 gene and the cap8 gene over the baculovirus DNA polymerase gene. (b) Monobac-γSGC, rAAV versus baculovirus productivity. We followed the number of rAAV particles produced per baculovirus through the ratio between ITR-based qPCR and baculovirus DNApol qPCR. (c) Monobac-γSGC, copies per mL of ITR BAC, rep and cap sequences. Copies per mL of the ITR, cap8, rep52 and BAC sequences. (d) Monobac without any rAAV transgene. Copies per mL of cap8, rep52, and BAC sequences. (e) The rep2-cap8 containing baculovirus of the dual rAAV production system. Copies per mL of cap8, rep52, and BAC sequences. (f) The rep2-cap8-containing baculovirus of the dual rAAV production system; stability of the rep2-cap8 expression cassette. We followed the ratio between the rep2 gene and the cap8 gene over the baculovirus DNA polymerase gene.

Figure 8

In vivo evaluation of rAAV8-γSGC vector potency. Quantification of muscle fibers positive for γSGC protein expression presented as percentage of muscle fibers (+/-SD) positive for γSGC protein expression 28 days after transduction with rAAV8-γSGC produced using the Monobac (red dots) or the dual baculovirus production (blue dots) system at a dose 5 × 10¹² vg kg⁻¹ of vector were injected in the tail vein of the γSGC mouse model following delivery of AAV produced with dual baculovirus and Monobac systems. The muscles studied were deltoid, diaphragm, anterior loge, psoas, quadriceps, and heart.