efficient amplification of chimeric adenovirus 540s vectors carrying the short fiber protein of ad40 in suspension cell cultures有效放大的嵌合540年代腺病毒载体携带的短纤维蛋白ad40悬浮细胞培养.pdfVIP

Efficient Amplification of Chimeric Adenovirus 5/40S Vectors Carrying the Short Fiber Protein of Ad40 in Suspension Cell Cultures 1 ´ 1 1 ´ 1 1,2 Marta Miralles , Marıa Mercedes Segura , Meritxell Puig , Assumpcio Bosch , Miguel Chillon * ` 1 Center of Animal Biotechnology and Gene Therapy (CBATEG), and Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona, Barcelona, ` Spain, 2 Institut Catala de Recerca i Estudis Avanc¸ats (ICREA), Barcelona, Spain Abstract The human adenovirus 40 (Ad40) is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber) during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors. Citation: Miralles M, S