Chemically Defined, High-Density Insect Cell-Based Expression System for Scalable AAV Vector Production

Abstract

The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 10¹¹ genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.

Keywords: AAV; baculovirus; insect cell.

Graphical abstract

Figure 1

Design of Constructs and Confirmation of Infectivity (A) The packaging construct (top) containing both the AAV2 Rep and Cap genes from AAV2, AAV8, or AAV9 were cloned into a pFastBac Dual vector. The Cap cassette is driven by the p10 promoter and contains the herpes simplex virus (HSV) thymidine kinase (tk) polyadenylation signal (pA). The Rep cassette is driven by the polyhedrin promoter (pH) and contains the simian virus 40 (SV40) pA. The transgene construct (bottom) contains either the ZsGreen or luciferase gene, which is driven by the cytomegalovirus (CMV) promoter. It also contains a beta-globin (B-globin) intron and a human growth hormone (hGH) pA signal. Lastly, the transgene cassette is flanked by the AAV2 inverted terminal repeat (ITR). (B) Purified AAV2 containing the ZsGreen transgene cassette was used to infect 293T cells at an MOI of 200. The cells were then imaged for ZsGreen expression 48 h postinfection. (C) Purified baculovirus containing the ZsGreen transgene cassette only was used to infect 293T cells at an MOI of 200. The cells were then imaged for ZsGreen expression 48 h postinfection.

Figure 2

Optimization of AAV Production in ExpiSf Expression System (A) Infection with varying cell densities (cells/mL) at the time of infection was tested using an MOI of 1 per baculovirus (left panel). qPCR was used to measure the viral genomes per milliliter (vg/mL) of each sample. Percentage of viable cells (% viability) was monitored during the optimization of the cell density (right panel). (B) Infection with varying MOIs of the baculoviruses was tested using a cell density of 5 × 10⁶ cells/mL (left panel). qPCR was used to measure the titers (vg/mL). Percentage of viable cells (% viability) was monitored during the optimization of the MOI (right panel). (C) Infection with varying MOIs of the baculoviruses was tested using a cell density of 5 × 10⁶ cells/mL (left panel). Samples were taken at 48 h, 72 h, and 96 h postinfection. qPCR was used to measure the titers (vg/mL). Percentage of viable cells (% viability) was monitored during the optimization of the MOI and harvest time (right panel). For statistical analysis, each condition was compared to the 72-h MOI of 1 condition. (D) Crude cell lysates of AAV2 produced with varying baculoviral MOIs were harvested at 48 h, 72 h, and 96 h postinfection. The infectivities were assessed by transducing Ad293 cells with a MOI of 100. The percentage of ZsGreen-positive cells was measured by flow cytometry. Statistical analysis was performed within each MOI group and was compared to its 72-h time point. ∗p < 0.05, ∗∗p < 0.005.

Figure 3

Viral Genome Titers and Infectivity of Purified AAV Preparations (A) Purified AAV preparations of various serotypes were produced using either the Bac-to-Bac System in Sf9 cells or the ExpiSf expression system in ExpiSf9 cells, and total viral genomes obtained from 300 mL of culture were measured using qPCR. (B) The infectivity of the purified AAV preparations produced in either Sf9 or ExpiSf9 cells was assessed by measuring the percentage of ZsGreen-positive cells by flow cytometry. ∗p < 0.05. (C) The infectivity of the purified AAV preparations produced in Sf9, ExpiSf9, or Ad293 cells was assessed by measuring the percentage of ZsGreen-positive cells by flow cytometry. ∗p < 0.05. (D) AAV9 yields from the three cell lines tested are shown in terms of viral genomes per cell (vg/c) along with the standard deviation (SD).

Figure 4

Sodium Dodecyl Sulfate (SDS)-PAGE and Capsid Ratio Analysis of AAV9 Produced in Sf9 and ExpiSf9 Cells Using Capillary Electrophoresis SDS (A) Purified AAV8 and AAV9 from both systems were assessed by SDS-PAGE gel stained with SYPRO Red. Lane 1, AAV8 produced in Sf9 cells (~2.6 × 10¹¹ vg); lane 2, AAV8 produced in ExpiSf9 cells (~2.6 × 10¹¹ vg); lane 3, AAV9 produced in Sf9 cells (~1.0 × 10¹¹ vg); lane 4, AAV9 produced in ExpiSf9 cells (~1.0 × 10¹¹ vg). (B) Chromatogram showing the overlay of the AAV9 samples produced in either Sf9 (red) or ExpiSf9 (blue) cells. Bottom table shows the number of VP1, VP2, and VP3 proteins per AAV9 particle produced in either Sf9 or ExpiSf9 cells.

Figure 5

Analytical Ultracentrifugation-Sedimentation Velocity Profiles of AAV9 Produced in ExpiSf9 Cells and Alkaline Gel Electrophoresis of Insect Cell-Derived AAV9 Genomes (A) Empty AAV9 capsid was analyzed by AUC in order to identify the lower boundary. (B) Once the lower empty capsid boundary was set, AAV9 containing the transgene cassette was run. Each peak was then integrated using the SEDFIT software to determine the percentage of full viral particles. The sedimentation coefficient on the x axis is in Svedberg units (S). The y axis represents the concentration (C) as a function of S. (C) Diagram showing the sequencing coverage of the recombinant AAV9 genome. Bottom table shows the percentages of the AAV genome in the recombinant AAV9 preparations. (D) Sf9- and ExpiSf9-derived AAV9 ZsGreen genomes were isolated and run on an alkaline gel. Lane 1, DNA ladder; lane 2, Sf9-derived AAV9 ZsGreen genome; lane 3, ExpiSf9-derived AAV9 ZsGreen genome. 500 ng of DNA was loaded on the left gel, and 7,500 ng of DNA was loaded on the right gel. The predicted genome size is ~2.6 kilobase (kb) pairs.

Figure 6

Luciferase Expression and Biodistribution in Mice Mice were tail-vein injected with AAV9 containing luciferase produced from Sf9, ExpiSf9, or Ad293 cells. (A) The mice were imaged by the In Vivo Imaging System (IVIS) at day 14, with the Sf9-derived AAV9 group (top), ExpiSf9-derived AAV9 group (middle), and Ad293-derived group (bottom). (B) In vivo luciferase signals were quantitated, and the average total flux (photons per second [p/s]) for each group of five mice is shown. ∗∗p < 0.005 and ∗∗∗p < 0.0005. (C) Ex vivo images of the mice organs were also taken at day 21, with the Sf9-derived AAV9 group (top), ExpiSf9-derived AAV9 group (middle), and Ad293-derived AAV9 group (bottom). (D) Ex vivo luciferase signals were quantitated, and the average total flux (p/s) for each group of two mice is shown (upper panel). qPCR was performed on DNA extracted from the collected mice tissues to quantitate how many copies of AAV viral genomes were present (lower panel). Sf9-derived AAV9 (green), ExpiSf9-derived AAV9 (yellow), and Ad293-derived AAV9 (blue). ∗p < 0.05, comparing Sf9-derived AAV9 to ExpiSf9-derived AAV9; &p < 0.05, comparing ExpiSf9-derived AAV9 to Ad293-derived AAV9; #p < 0.05, comparing Sf9-derived AAV9 to Ad293-derived AAV9.