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)