Engineering redirected NF-κB/OIP5 expression programs to enhance tumor responses to chemotherapy in bladder cancer

Abstract

Nuclear factor kappa-B (NF-κB), a pivotal transcriptional regulator, plays a crucial role in modulating downstream genes implicated in tumor drug resistance. We establish a programmable system within bladder cancer cells to tailor drug responses by employing a synthetic clustered regularly interspaced short palindromic repeats (CRISPR)-based expression strategy that emulates natural transcriptional regulators. Our investigation uncovers the functional significance of Opa-interacting protein 5 (OIP5), upregulated upon NF-κB activation, as a key regulator governing drug-resistance to vincristine (VCR) treatment in bladder cancer. Through engineered guide RNAs (sgRNAs) targeting OIP5 to integrate NF-κB aptamers, we construct a modular scaffold RNA that encodes both the target locus and regulatory functionality. This engineered CRISPR scaffold RNA effectively responds to VCR stimulus by binding with activated NF-κB. Intriguingly, it redirects NF-κB to attenuate OIP5 expression-a reversal of its original role-while concurrently obstructing multiple NF-κB-mediated drug resistance pathways. This dual action thwarts drug resistance development. Further enhancing therapeutic potential, we develop a versatile nanoparticle system capable of co-delivering CRISPR scaffold RNAs and VCR. This synergistic approach demonstrates potent anti-tumor effects in both in vitro and in vivo settings. Our nanoparticle-mediated combination presents a compelling proof-of-concept, showcasing the utility of engineered CRISPR-based synthetic expression programs to reconfigure cellular drug responses and heighten tumor cell susceptibility to chemotherapy.

Keywords: Drug resistance; Gene circuit; NF-κB; OIP5; RNA engineering; Vincristine.