All AAVs serotypes were produced in 293T cells cultured adherently using FectoVIR®-AAV complexed with RepCap plasmid, GOI plasmid and Sartorius Polyplus proprietary pPLUS® AAV-Helper for the triple transfection. The three plasmids were co-transfected using FectoVIR®-AAV at an ratio of 1:1:1 in a total of 1.E+4 239T cells. The cells were harvested, and the virus was purified by preformed gradient of 15%, 25%, 40%, 60% iodixanol gradient centrifugation 27h after transfection, The titers (copies/ml) was determined by qPCR after alkaline lysis of the AAVs to release the packaged VG.

Introduction: Adeno-associated virus (AAV) vectors are promising tools for cancer gene therapy, yet their clinical application in head and neck squamous cell carcinoma (HNSCC) is hindered by suboptimal transduction efficiency and off-target risks. Bioengineered AAV capsids require optimization to enhance tumor-specific targeting while minimizing systemic toxicity. Methods: We employed a directed evolution strategy combining DNA shuffling and site-directed mutagenesis to generate AAV variants. Five rounds of in vitro selection were performed using HNSCC cell lines (SCC-090, SCC-152, FaDu), followed by validation through in vitro transduction assays and in vivo studies in HNSCC xenograft mouse models. AAVzy9-3, a lead capsid variant, was further tested for α2δ1-targeted gene silencing efficacy. Results: 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 validated through both in vitro and in vivo methods, employing a transplanted tumor mouse model. The results showed that AAVzy9-3 was more effective at infecting HNSCC cells than the wild type, while demonstrating reduced infectious potential toward other cells and organs. Additionally, the study used AAVzy9-3 to knockdown α2δ1 expression in a mouse model of HNSCC transplanted tumors, resulting in reduced tumor size. Discussion: The development of AAVzy9-3, a novel AAV variant with HNSCC-specific tumor tropism, addresses critical limitations of conventional AAVs. The in vivo antitumor activity validates its therapeutic potential for HNSCC.