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

Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. To address the growing demand, next-generation process development should be informed by a mechanistic understanding of the cellular response to rAAV. In this study, we performed transcriptomic analysis of 5 cell lines with variable capacities for rAAV production. Using an intersectional approach, we assessed the transcriptional response to rAAV production and compared transcriptional profiles between high and baseline producers to identify possible targets for enhancing production. Modulation of cell cycle and nucleosome components suggested a reduction of proliferative capacity and a shift toward DNA replication to support rAAV production. During rAAV production, we observed upregulation of several core functions including transcription, stress response, and Golgi and endoplasmic reticulum organization. Conversely, inhibitors of DNA-binding proteins and mitochondrial components were consistently downregulated during rAAV production. We next performed a drug connectivity analysis of these results and identified 5 classes of drugs predicted to enhance rAAV production. Validation studies confirmed the efficacy of HDAC and microtubule inhibitors. Our data uncover novel and previously identified pathways that may enhance rAAV productivity, potentially enabling a path to engineer improved processes and cell lines for higher yields and better quality rAAV production. Overall design: Using a triple plasmid transfection, we produced rAAV in 5 different HEK293 cell lines. We collected total RNA for transcriptome analysis at 3 time points: before transfection (0 hr), 24 hours, and 72 hours after transfection. We identified differentially expressed genes between time points to determine how AAV production affects gene expression. We also compared gene expression profiles between 2 high and 2 baseline AAV-producing cell lines to identify genes that are associated with enhanced AAV production.