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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) vectors are a promising platform for in vivo gene therapies. However, cost‐effective, well‐characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two‐dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid‐high setpoints, and PEI:DNA ratio and total DNA/cell were at low‐mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin‐doped indium oxide nanocrystals. Tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonicShewanella oneidensiscultures. Using this method, we differentiated between starved and actively respiring cells, cells of varying genotype, and cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in aqueous environments and contribute to a fundamental understanding of planktonicS. oneidensiselectrophysiology using simplein situspectroscopy.