Acid‐Activatable Rolling Circle Replication‐Based Nucleic Acid Nano‐Prodrug for Combined Gene Therapy of Pancreatic Cancer

ABSTRACT

Nucleic acid‐based gene therapeutics hold great potential for precisely treating pancreatic ductal adenocarcinoma (PDAC), yet their clinical translation remains challenged by the lack of efficient delivery platforms capable of simultaneously minimizing payload leakage, intrinsic toxicity, and immunogenicity. Herein, we report an acid‐activatable nucleic acid nano‐prodrug based on rolling circle replication, which directly integrates a large number of amplified Cas12/crRNA ribonucleoproteins (RNPs) and antisense oligonucleotides (ASOs) with minimal excipients, achieving tumor‐specific activation for PDAC combined gene therapy. The nano‐prodrug is assembled from a polymeric crRNA chain conjugated with Cas12a (Pro‐RNPs) and a polymeric ASOs chain (Pro‐ASOs)—the latter incorporating AS1411 aptamers for cancer targeting and HhaI cleavage sites for stimuli‐responsive release—while being surface‐loaded with acid‐degradable polymer‐coated HhaI enzymes. Upon PDAC uptake, the nano‐prodrug is activated within acidic lysosomes through degradation of HhaI enzymes’ polymer coating. HhaI cleaves specific recognition sites while overexpressed ribonuclease H hydrolyzes the RNA strand in DNA‐RNA heteroduplexes. These combined cleavage events disrupt the nano‐prodrug, enabling efficient co‐release of ASOs and Cas12a/crRNA RNPs for precise gene silencing and editing. In vivo studies in PDAC‐bearing mice demonstrate potent antitumor activity with minimal systemic toxicity or immunogenicity, presenting a promising strategy for precision gene therapy in pancreatic cancer.