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

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 selection were performed using HNSCC cell lines (SCC-090, SCC-152, FaDu), followed by validation through transduction assays and 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 and 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 antitumor activity validates its therapeutic potential for HNSCC.