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.