5 Conclusion and Future Perspectives
From a molecular design standpoint, the Three-Bac system offers the
greatest flexibility by providing many possible combinations of
expression of VP proteins, replicase protein, and the gene of interest
independently. It is also a modular tool for pseudotyping various AAV
capsids[45],[32] allowing the production of mosaic AAV capsids
with different combinations of one or more VP proteins from other
serotypes. The Two-Bac system somewhat restricts this flexibility,
especially in a case where rep and cap sequences are
combined and inserted in a single baculovirus construct. The One-Bac
system, which involves Rep2CapX packaging cell line and a Bac-GOI,
offers restricted flexibility. The generation of the stable packaging
cell line represents a strategy, though effective and simple from a
processing standpoint, often complex and resource intensive. An
alternative and more flexible variant to existing One-Bac system
reported is the combination of RepX (X=Serotype dependent Rep) insect
cell line and Bac-CapY-ITR-GOI BV (Y=Serotype of interest) for AAV
serotypes production[70]. Overall, the modular Three-Bac system
provides the necessary flexibility at the early stage of development
that involves screening of multiple serotypes. However, at larger
production scales, it is the less attractive system due to inherent
manufacturing challenges, as discussed in previous sections. On the
other hand, One-Bac, though less flexible and potentially difficult to
implement at an early stage of development, offers a robust yet simple
manufacturing process for well-established clinically relevant serotypes
such as AAV5 or AAV9.
A recently added tool offers a number of advantages, the
Mono-Bac[71] or Bac-RepX/CapY/ITR (Figure 2A) combines all the AAV
elements in a single baculovirus construct. Upon infection with this
single rBV vector, the insect cells produce AAV. Yet the Mono-Bac system
has not been extensively used.
Although insect cells are being used for AAV production for more than
fifteen years, the first major question, yet unanswered, relates to the
uncertainty whether the maximum cellular protein production/processing
capacity has been reached. The second question is the efficiency of
vector genome packaging in insect cells. Recent reports suggested that
AAV serotypes produced in insect cells show more empty particles and
consequently less viral genome containing particles when compared
side-by-side to mammalian cell productions [72] -submitted]. This
necessitates active efforts towards engineering insect cells to produce
more genomic and functional particles. The production of wild type AAV
serotypes may provide a model for such study since nature has perfected
the process of generating 100% genomic and functional particles in the
presence of helper virus function in mammalian cells, the natural host
of AAV[73]. Moreover, another important factor, requiring
significant consideration is the selection of appropriate combination of
ITR and Rep proteins for their optimal functionality in genome
replication and its packaging into pre-formed capsids. Typical
production protocols of different AAV serotypes production in insect
cells involves rep and ITR sequences from AAV2 and the cap sequence of
choice. The selection of appropriate combinations of serotype-specific
rep/ITR remains an open area of investigation to further support the
findings of a single report studying its effect on genome packaging
efficiency and genomic particle yield of AAV5[42]. In addition to
molecular and cellular engineering, the improvement in high cell density
production can also contribute to address the challenges of high-yield
productions taking advantage of advancements in the field of biologics
manufacturing. This involves and not limited to, optimization of medium
formulation, design and optimization of nutrient feed for insect cells,
and detailed understanding of the critical process parameters guided by
the critical quality attributes of AAV serotype therapeutic products.
However, increasing the cell density in a production process beyond a
point may turn counterproductive at a downstream process stage. Because
AAV is an inherently intracellular product, the primary recovery step
involves the cell lysis and release of AAV in lysis buffer. In the high
cell density culture process, the cellular components (host cells genome
and proteins) are co-extracted with AAV in the lysate during the harvest
and clarification process. Their removal exerts a significant burden on
downstream processing. It would be appealing to study if the AAV capsid
sequences can be modified, without altering the functionality, to direct
its secretion in the extracellular environment as is the case with
secretory proteins such as monoclonal antibodies. This way, a high cell
density-high volumetric yield process can be derived devoid of the
presence of major interfering cellular components. Such modification can
also be adopted for a perfusion production process with an ultra-high
cell density culture, which will ultimately lead to a very high
volumetric yield of AAV and reduce the production scale. In conclusion,
comforted by the increased regulatory acceptance, ease of scale-up, and
recent advancements in production technologies, insect cell baculovirus
system is being more broadly adopted for the production of multiple AAV
serotypes (Table 1). As a result, more insect cell produced AAV vectors
can be expected in clinical trials in the future.