Recombinant adeno-associated virus (rAAV) vector is one of the most promising viral vectors in gene therapy. Currently, three-plasmid transfection based on human embryonic kidney 293 (HEK293) cells is the most commonly used rAAV vector production system, but its low production efficiency has become one of the challenges faced by rAAV gene therapy drugs on the road to commercialization. In view of this challenge, researchers are working to develop improved methods to increase the production of rAAV in HEK293 cells.
Studies have shown that HEK293 cells exhibit a series of responses including endoplasmic reticulum (ER) stress when producing viral vectors such as rAAV. ER stress activates the downstream unfolded protein response (UPR) to reduce the stress of host cells, ensure cell survival and produce viral vectors. Based on transcriptomics data, researchers found that when endoplasmic reticulum stress was upregulated, the expression of the following three genes was significantly enhanced: GADD34/PPP1R15A, heat shock protein family member 6 (HSPA6) and X-box binding protein 1 (XBP1). These genes are believed to play a more important role in the process of virus generation. GADD34 plays a key role in the process of recovering protein synthesis after the unfolded protein response signal temporarily inhibits translation. HSPA6 is an ER chaperone protein regulated by UPR signaling through activating transcription factor 6 (ATF6), and its expression can promote the transport, folding, secretion and degradation of misfolded proteins in the ER. Activation of XBP1 can also promote the transport, folding and secretion of ER proteins. Based on this, the researchers hypothesized that overexpression of these protein processing genes can alleviate host cell stress caused by viral protein synthesis, reduce the accumulation of erroneous or unfolded proteins, and enhance viral production. In addition, studies have shown that overexpression of the anti-apoptotic gene BCL2 can increase the yield of lentiviral vectors in HEK293 cells by 53%. The above demonstrates the important role of ER protein processing and anti-apoptotic pathways in viral vector production.
Figure 1. The overall workflow of this overexpression study, including plasmid construction, stable integration, and assessment of rAAV productivity in overexpressing cell pools. (Fu Q, et al., 2024)
In order to improve the production of rAAV vectors, the researchers selected the above four candidate genes: XBP1, GADD34/PPP1R15A, HSPA6 and BCL2, and stably integrated these genes into the safe harbor site (i.e., AAVS1 site) of the HEK293 host cell line to achieve their overexpression. The traditional three-plasmid transient transfection method was used to evaluate the yield and quality of rAAV vectors.
Figure 2. Transient transfection for AAV2 production in HEK293T cells for both overexpressed stable pools and parental cells in the high-producing condition. (Fu Q, et al., 2024)
The results showed that under high-yield conditions of viral vectors, overexpression of XBP1, GADD34, HSPA6 and BCL2 in HEK293T cells increased rAAV2 vector genome titer and packaging efficiency. Among them, the rAAV production increased by up to 100%, and the rAAV vector full capsid ratio increased by up to 37%. Under low-yield conditions, overexpression of any of the above candidate genes did not improve rAAV vector yield and quality. The study speculated that under high-yield conditions, more protein synthesis would lead to more viral protein accumulation in the ER, thereby increasing ER stress.
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Overexpression of ER protein processing genes plays a more important role in the adaptive UPR pathway, helping to alleviate ER stress and thus improve protein folding for vector packaging. In addition, cell growth in the overexpression group was lower than that in the negative control group, but a significant increase in productivity (about one-fold) was observed. After stable integration of these ER protein processing genes, the continuous expression of the stable transgene consumes nutrients and energy, slowing overall cell growth. At the same time, the enhanced viral packaging process consumes additional nutrients and energy, which may be the reason for the poor cell growth performance.
The study also pointed out that overexpression of ER protein processing genes is of great significance for improving rAAV production. In the future, various overexpression combinations of ER processing genes may better improve viral vector productivity and overall product quality. The increase in viral vector genome titer observed in this study was due to improved intracellular protein production, improved viral vector capsid quality, and improved viral vector packaging efficiency. In addition, this increase in productivity has been verified in different HEK293 cell lines and various AAV serotypes (AAV2 and 8), although the degree of improvement is slightly different.
Reference
Fu Q, et al. Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells. Applied Microbiology and Biotechnology, 2024, 108(1): 459.