The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Recombinant adeno-associated virus (rAAV) is one of the prominent gene delivery vehicles that has opened promising opportunities for novel gene therapeutic approaches. However, the current major viral vector production platform, triple transfection in mammalian cells, may not meet the increasing demand. Thus, it is highly required to understand production bottlenecks from the host cell perspective and engineer the cells to be more favorable and tolerant to viral vector production, thereby effectively enhancing rAAV manufacturing. In this review, we provided a comprehensive exploration of the intricate cellular process involved in rAAV production, encompassing various stages such as plasmid entry to the cytoplasm, plasmid trafficking and nuclear delivery, rAAV structural/non-structural protein expression, viral capsid assembly, genome replication, genome packaging, and rAAV release/secretion. The knowledge in the fundamental biology of host cells supporting viral replication as manufacturing factories or exhibiting defending behaviors against viral production is summarized for each stage. The control strategies from the perspectives of host cell and materials (e.g., AAV plasmids) are proposed as our insights based on the characterization of molecular features and our existing knowledge of the AAV viral life cycle, rAAV and other viral vector production in the Human embryonic kidney (HEK) cells.

The development of gene therapies based on recombinant adeno-associated viruses (rAAVs) has grown exponentially, so the current rAAV manufacturing platform needs to be more efficient to satisfy rising demands. Viral production exerts great demand on cellular substrates, energy, and machinery; therefore, viral production relies heavily on the physiology of the host cell. Transcriptomics, as a mechanism-driven tool, was applied to identify significantly regulated pathways and to study cellular features of the host cell for supporting rAAV production. This study investigated the transcriptomic features of two cell lines cultured in their respective media by comparing viral-producing cultures with non-producing cultures over time in parental human embryonic kidney cells (HEK293). The results demonstrate that the innate immune response signaling pathways of host cells (e.g., RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytosolic DNA sensing pathway, JAK-STAT signaling pathway) were significantly enriched and upregulated. This was accompanied by the host cellular stress responses, including endoplasmic reticulum stress, autophagy, and apoptosis in viral production. In contrast, fatty acid metabolism and neutral amino acid transport were downregulated in the late phase of viral production. Our transcriptomics analysis reveals the cell-line independent signatures for rAAV production and serves as a significant reference for further studies targeting the productivity improvement in the future.

Recombinant adeno-associated virus (rAAV) is the most effective viral vector technology for directly translating the genomic revolution into medicinal therapies. However, the manufacturing of rAAV viral vectors remains challenging in the upstream processing with low rAAV yield in large-scale production and high cost, limiting the generalization of rAAV-based treatments. This situation can be improved by real-time monitoring of critical process parameters (CPP) that affect critical quality attributes (CQA). To achieve this aim, soft sensing combined with predictive modeling is an important strategy that can be used for optimizing the upstream process of rAAV production by monitoring critical process variables in real time. However, the development of soft sensors for rAAV production as a fast and low-cost monitoring approach is not an easy task. This review article describes four challenges and critically discusses the possible solutions that can enable the application of soft sensors for rAAV production monitoring. The challenges from a data scientist\'s perspective are (i) a predictor variable (soft-sensor inputs) set without AAV viral titer, (ii) multi-step forecasting, (iii) multiple process phases, and (iv) soft-sensor development composed of the mechanistic model.

Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.

Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae.