Abstract 469: Bladder cancer gene therapy via Nectin-4-targeted PLGA nanoparticles delivering CRISPR/Cas9 for double-strand break induction

Abstract Nectin-4-targeted antibody-drug conjugates (ADCs) linked with monomethyl auristatin E (MMAE) have revolutionized the treatment paradigm for metastatic bladder cancer. In combination with pembrolizumab, this therapy has doubled overall survival compared to conventional platinum-based chemotherapy. However, challenges such as drug toxicity and the accumulation of resistance to MMAE limit its therapeutic potential. In this study, we introduce a novel Nectin-4-targeted gene therapy strategy employing the CRISPR/Cas9 system. This advanced gene-editing tool, known for its precise induction of double-strand DNA breaks, was adapted to create over 50 random double-strand breaks (referred to as MT50) in target cells, leading to apoptotic cell death. To address the critical challenge of delivering gene-editing tools efficiently and specifically to cancer cells, we developed a highly efficient nucleic acid encapsulation system with poly(lactic-co-glycolic acid) (PLGA) nanoparticle. This platform demonstrated high nucleic acid encapsulation efficiency, exceeding 60% for plasmids (∼1 kb) and 80% for shorter RNAs, such as siRNA. The system was further optimized for encapsulating CRISPR/Cas9 components, including guide RNA (gRNA) and Cas9 mRNA. The T24 bladder cancer cell line, characterized by high Nectin-4 expression, was used as the experimental model. In vitro experiments demonstrated that MT50 delivery via Nectin-4-conjugated PLGA nanoparticles inhibited cellular proliferation by over 70%. In vivo, the nanoparticles were administered systemically through intraperitoneal injection into a murine model bearing T24 bladder cancer tumors. Tumor volume decreased by 70% following MT50 treatment, with significant downregulation of Nectin-4 expression observed in treated tumors compared to controls. While this experiment was conducted in immunocompromised mice, precluding the assessment of immunogenic cell death or synergistic effects with immune checkpoint inhibitors, we anticipate enhanced anti-tumor efficacy in immunocompetent models. Specifically, combining MT50 treatment with immune checkpoint inhibitors is expected to amplify therapeutic outcomes. This study highlights the successful development of a Nectin-4-targeted PLGA nanoparticle system capable of efficiently encapsulating and delivering CRISPR/Cas9 components to bladder cancer cells. The MT50 delivery system demonstrated significant anti-tumor effects both in vitro and in vivo, offering a promising alternative to MMAE-linked ADCs. By circumventing cumulative resistance and leveraging the precision of gene editing, this approach has the potential to redefine bladder cancer therapeutics. Citation Format: Seung-hwan Jeong, Seokbong Hong, Hyeong Dong Yuk, Cheol Kwak. Bladder cancer gene therapy via Nectin-4-targeted PLGA nanoparticles delivering CRISPR/Cas9 for double-strand break induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 469.