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

Recombinant adeno-associated viruses (rAAVs) are commonly used in gene therapy for preclinical research and therapeutic applications. Despite the clinical efficacy of rAAVs, their manufacturing involves 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 adeno-associated virus (AAV) production. Inhibiting PLK1 with HMN-214 increased AAV production, which was largely consistent across HEK293 cell lines, vector payloads, and capsid serotypes. Using cell cycle and RNA-sequencing analysis, we showed that PLK1 inhibition halts cells in the G2/M phase and blocks their exit from the M to G1 phase. These findings support that inhibiting PLK1 may enhance AAV production and could be used to develop more cost-effective methods to manufacture AAV for gene therapies.