Figure legends
Figure 1. AAV Family Tree and wtAAV2 Genome Map and Expression Profile
(A) AAV family tree. The Dependovirus AAV mainly infects mammalian hosts and replicates in the presence of a helper virus. Though evolutionary distant yet related to Densovirinae subfamily, it shares common structural and functional features with viruses from this subfamily, which mainly infect invertebrates hosts such as insects. (B) wtAAV2 genome map and protein expression profile. The numbers represent the nucleotide position. The viral genome is flanked by two ITRs, one on each end. Two promoters in the Left ORF drive the expression four functional Rep proteins, whereas a single promoter in the right ORF drives the expression of three capsid protein subunits (VP1, VP2, and VP3) from a single mRNA transcript. The intron splicing generates four Rep proteins of different sizes from two mRNA transcripts, whereas the leaky scanning of weak translational codons results in three VP proteins production in a stochastic ratio of 1:1:10. The diamond shape end represents the N-terminal of the peptide. Two polyadenylation (PA) signal sequences are shown via orange line.
Figure 2. AAV Insect Cell Baculovirus Expression Systems for AAV Production and Mechanism of Inducible Expression in One-Bac.
(A) Four major systems for rAAV production using IC-BEVS. The initial Three-Bac system consisted of three rBV vectors, each carrying specific nucleotide sequence. Further study with this system identified critical shortcomings, related explicitly to expression stability of rBVs and mechanism behind it. The second-generation systems (Two-Bac) exhibited better expression stability of rBVs over the extended passage numbers and required only two rBV co-infection for AAV production. Third-generation systems such as One-Bac or Mono-Bac further simplified the manufacturing process requiring only single rBV co-infection. The One-Bac consisted of a Rep2CapX packaging cell line and a Bac-GOI, whereas in Mono-Bac, a single baculovirus carries all the necessary gene (Bac-ITR-GOI-Rep2-CapX) sequences. (B) The postulated mechanism of induction and amplification of rep/cap genes in One-Bac. Bac-ITR infection provides IE-1, which activates hr2-0.9 (1) and induces the Rep78/52 or Cap expression (2). The expressed Rep78 further forms a complex with RBE (3) and induces the second round of amplification of Cap or Rep52 expression (4), resulting in a lower Rep78: Rep52 ratio. (The artwork in the figure was created with Biorender.com)
Figure 3. An Overview of Process-Flow for rAAV Production Employing IC-BEVS Platform
The AAV production using the IC-BEVS platform consists of two stages, 1. Generation of recombinant baculovirus expression vectors (rBEVS) and additionally Rep2CapX Sf9 packaging cells in case of One-Bac system (Top section), and 2. AAV production at bioreactor scale in suspension culture of insect cells followed by purification and formulation (Bottom part). The first part requires molecular cloning, transient expression, plaque purification, and characterization of rBVs. Similarly, the Rep2CapX cells are generated via stable transfection of respective plasmid vector and selection followed by characterization and master cell bank preparation. During the production stage, the rBVES and insect cells are (Sf9 or Hi5) sequentially expanded in quantity required for stirred tank or Wave™ production scale bioreactor. At the production stage, the insect cells are infected with rBEVS at an appropriate multiplicity of infection (Low:<1 or High:1-10) and at optimal cell density. Generally, under the batch mode of cultivation, the cells are infected at low cell density around 2-4 million cells/mL, whereas under fedbatch or perfusion mode, the cells reach higher peak cell density before infection:7-12 million cells/mL. The cultivation temperature for insect cells is generally set at 27°C, although one study reported AAV production at higher and lower temperatures and their effect on yield and production kinetic. Post infection, at 72-96 hours, the culture is harvested, and cells are lysed to recover AAV. Next, the lysate is subjected to multistep purification process followed by formulation in appropriate buffer as a final step. (The artwork in the figure was created with Biorender.com)