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. 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.

重组腺相关病毒（rAAV）是基因治疗领域中用于临床前研究与治疗应用的常用载体。尽管其临床疗效已获验证，但rAAV的生产仍面临生产力与质量控制层面的双重挑战，导致其供应受限。本研究旨在通过高通量小分子筛选策略，筛选可提升细胞生产AAV9能力的活性化合物。我们借助AAV筛选阵列化靶向文库平台，对包含3300种小分子的文库进行了筛选，最终鉴定出多个靶点类别，包括细胞周期调节剂、G蛋白偶联受体调节剂、组蛋白去乙酰化酶抑制剂、Janus激酶抑制剂以及代谢调节剂。尤为关键的是，我们发现polo样激酶1（PLK1）抑制剂可作为AAV生产的增强剂。使用HMN-214抑制PLK1能够提升AAV的生产效率，且该效果在HEK293细胞系、载体载荷与衣壳血清型中均保持高度一致性。通过细胞周期分析与RNA测序（RNA-seq），我们证实PLK1抑制可将细胞阻滞于G2/M期，并阻断细胞从M期向G1期的过渡。上述研究结果可用于开发更具成本效益的工艺方法，以提升工业化生产过程中AAV的产量。我们在HEK293细胞中开展了针对AAV生产增强剂的小分子筛选实验。AAV的生产采用三质粒转染方案。在将HMN-214鉴定为最优候选化合物后，我们分别在添加与不添加该化合物、存在与不存在AAV生产的条件下开展了RNA测序实验。我们于三个时间点收集总RNA用于转录组分析：处理前（0小时）、处理后24小时以及处理后72小时。我们针对处理后24小时与72小时的HMN-214处理组，以及存在与不存在AAV生产的各组样本开展了差异表达分析，以鉴定出在PLK1受抑制时可辅助提升AAV生产的基因产物。