Polo-like kinase inhibitors increase AAV production by halting cell cycle progression

Recombinant Adeno-associated viruses (rAAV) are commonly used in gene therapy for preclinical research and therapeutic applications. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. In this study, we aimed to identify compounds that increase the capacity of cells to produce AAV9 with a high-throughput small-molecule screening strategy. With the Arrayed Targeted Library for AAV Screening platform, we screened a library of 3,300 small molecules and identified several targets, including cell-cycle modulators, G protein-coupled receptor modulators, histone deacetylate inhibitors, Janus kinase inhibitors, and metabolic modulators. Most notably, we identified Polo-like kinase isoform 1 (PLK1) inhibitors as enhancers of AAV production. PLK1 inhibition using HMN-214 increased AAV production, and was largely consistent across HEK293 cell lines, vector payloads and capsid serotypes. Using cell cycle and RNA-sequencing analysis, we show that PLK1 inhibition halts cells in the G2/M phase and blocks their exit from the M to G1 phase. These findings can be used to develop more cost-effective methods to increase AAV yield during production in the manufacturing process. Overall design: We performed a small molecule screen for enhancers of AAV production in HEK293 cells. We used a triple plasmid transfection for AAV production. After identifying HMN-214 as a top hit, we performed RNA-seq in the presence and absence of this compound, as well as with and without AAV production. We collected total RNA for transcriptome analysis at 3 time points: before treatment (0 hr), 24 hours, and 72 hours after treatment. We carried out differential expression analysis between the HMN-214 treated group at 24 and 72 hours post treatment, and in the presence and absence of AAV production to identify gene products which may support enhanced AAV production when PLK1 is inhibited.