Although substantial progress has been achieved in the field of immuno-oncology—including the development of immune checkpoint inhibitors, adoptive cell therapies, and therapeutic antibodies—numerous clinical challenges remain unresolved. In particular, triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), exhibits limited responsiveness to existing immunotherapeutic strategies. This underscores the pressing need for alternative therapeutic platforms with enhanced tumor specificity. Among available delivery vectors, adeno-associated virus (AAV) has emerged as a promising candidate for gene therapy, owing to its low immunogenicity, established safety profile, and capacity for tissue-specific tropism. These properties render AAV particularly well-suited for the development of targeted therapeutic systems engineered to maximize tumor-specific gene delivery efficiency. In this study, we sought to engineer an AAV vector with enhanced specificity for TNBC by employing a directed evolution strategy. A highly diverse peptide library was incorporated into the AAV capsid, generating a large pool of capsid variants. These were subjected to positive selection using TNBC cell models to isolate variants exhibiting preferential tumor tropism. To further eliminate off-target delivery, we implemented an additional negative screening step based on the hepatic system, allowing for the exclusion of liver-tropic variants. Through this dual-screening process, we identified engineered AAV variants with significantly improved TNBC-targeting efficiency compared to wild-type AAV, as confirmed via both in vitro and in vivo transduction assays. These findings support the potential application of the selected vectors in precise and tumor-selective gene therapies, including gene knockout or correction, and lay a foundation for the advancement of next-generation targeted delivery systems in the field of precision oncology.
