Adeno-associated virus vector modification based on directed evolution technology for gene therapy targeting head and neck squamous cell carcinoma

Yiyuan Zhu^1*Wei Ji²Qi Zhang³Yanbo Dong¹Liangfa Liu^1*

• ¹Beijing Friendship Hospital, Capital Medical University, Beijing, China
• ²Qilu Hospital, Shandong University, Jinan, Shandong Province, China
• ³Beijing Institute of Biotechnology, Beijing, Beijing, China

Adeno-associated virus (AAV) gene therapy has been explored as a potential strategy in cancer treatment. However, in the treatment of head and neck squamous cell carcinoma (HNSCC), there are concerns about low transduction efficacy. Additionally, off-target expression remains an issue that requires attention. Bioengineering of viral vectors for therapeutic gene delivery is a key strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. In this study, we engineered AAV vectors using a directed evolution approach that combines DNA shuffling and point mutation. Following five rounds of in vitro selection, a chimeric AAV capsid, designated as AAVzy9-3, was obtained. This capsid demonstrated superior transduction efficiency in SCC-090, SCC-152 and FaDu cells when compared to the most efficient parental capsid. The validation of AAVzy9-3 targeting of HNSCC cells was conducted through both in vitro and in vivo methods, employing a transplanted tumor mouse model. The results demonstrated that AAVzy9-3 exhibited superior efficacy in HNSCC infection compared to the wild type, while demonstrating reduced infectious potential towards other cells and organs. In addition, the study employed AAVzy9-3 to knock down α2δ1 expression in a mouse model of HNSCC transplanted tumors, which resulted in reduced tumor size. These findings underscore the potential of AAVzy9-3 for future utilization in clinical settings.