Transcriptomic features reveal molecular signatures associated with recombinant adeno‐associated virus production in HEK293 cells

Abstract

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.

Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

“…Recent transcriptional studies have implicated many of the same pathways and processes we identified in AAV production, despite variations in study design including plasmid and cell sources, transfection conditions, and growth media (Supplementary file 3). Wang et al (2023) identified several upregulated stress response and homeostatic proteins which align with our data, including HBB, HSPA6, and CCL5, as well as microtubule-interacting proteins including STMN4 and DNAH17 (Figure 3 -figure supplement 1A-C).…”

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

“…Given that viral proteins and viral nucleic acids are perceived as exogenous entities within the host cell milieu, their presence has the potential to trigger a series of host cell stress reactions, including but not limited to autophagy, endoplasmic reticulum (ER) stress, and programmed cell death (apoptosis). Our investigation unveiled an increased activation of these stress responses during phases associated with viral production 4,7,26,37–41 (Fig. 5-7).…”

“…Further, the host cell's innate immune response typically serves as the initial barrier against viral dissemination. It has been documented that, within the conventional transfection parameters applied to the HEK293 cell line, the host cell's innate immune system tends to exhibit an upregulation 8,[26][27][28][29][30][31][32] . However, in our case, when comparing our optimal transfection conditions to the standard ones, we observed a notable downregulation or reduced expression of proteins associated with immune response (such as ASAH1, CREB1, CD81, POLR3F, STOM, ATP11B, ATP6V1C1, NFKB2) across the time points (Supplementary Data GO Analysis).…”

Comparative transcriptomic and proteomic kinetic analysis of adeno-associated virus production systems

“…In a proteomic investigation of rAAV5 production by plasmid transfection of HEK293 cells, it was reported that the defense response was significantly enriched compared to untransfected cells (Strasser et al 2021 ). Another transcriptomic study of rAAV2 production by plasmid transfection of HEK293 cells reported significant upregulation of the signaling pathways of pattern recognition receptors (PRR), including Toll-like receptor (TLR), retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR), nucleotide oligomerization domain (NOD)-like receptor, and cytosolic DNA-sensing pathways (Wang et al 2023 ).…”