Enabling Commercial AAV Manufacturing By Xcite® AAV Stable Producer Cell Lines

By Gang Li, Chien-Ting Li, Khanh Ho, Friedrich Michael Haller, Leila Aneke, Jiasong Jiang, Peng Wang, Tam Duong, Senthil Ramaswamy, and Bingnan Gu

AAV's superior safety profile and transduction efficiency make it an exceptional viral vector for human gene therapy. However, current AAV manufacturing processes are hindered by low productivity, scalability challenges, high raw material costs, supply chain complexities, and significant batch-to-batch variation. To address these limitations and industrialize AAV production, we have developed a high-performance, helper virus-free Xcite® AAV stable producer cell line (PCL). This cell line is derived from our proprietary high-producer HEK293 cells used in our AAV transient transfection platform and is cultivated in a chemically defined, serum-free, ADCF medium in suspension. The DNA expression vectors are meticulously designed to tightly regulate viral gene expression through engineered inducible promoters and are stably integrated into the cell genome in a single step using our VS piggyBac™ transposon system, substantially enhancing cell line construction efficiency. Upon low-dose doxycycline induction at standard cell density, our selected PCL clones outperform the transient transfection method, achieving even higher AAV titers (exceeding 1E12 vg/mL) and comparable AAV genome packaging (more than 30% full capsids) at harvest. Furthermore, these cells maintain stable AAV productivity and packaging over 30 cell passages, corresponding to a scale far exceeding 2000 liters. Our Xcite® AAV stable PCL represents a cutting-edge platform capable of enabling large-scale, high-quality commercial AAV manufacturing with significantly reduced costs and process complexity.