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Nucleic acid-based gene therapies have huge potential for precise treatment of pancreatic ductal adenocarcinoma (PDAC), but their clinical translation still faces many challenges, such as the lack of an efficient delivery platform that can simultaneously minimize payload leakage, inherent toxicity, and immunogenicity. This paper reports an acid-activated nucleic acid nanoprodrug based on rolling-circle replication that can directly integrate large amounts of amplified Cas12/crRNA ribonucleoproteins (RNPs) and antisense oligonucleotides (ASOs), using only a small amount of excipient, achieving tumor-specific activation for PDAC combination gene therapy. The nanoprodrug is assembled from a polymer-crRNA strand conjugated with Cas12a (Pro-RNPs) and a polymer-ASO strand (Pro-ASOs). The latter includes an AS1411 aptamer for targeting cancer cells and an HhaI restriction site to trigger responsive release, while its surface is loaded with an acid-degradable polymer-coated HhaI enzyme. Once taken up by PDAC cells, the nanoprodrug is activated in the acidic lysosome through the degradation of the polymer-coated HhaI enzyme. The HhaI enzyme cuts specific recognition sites, and the overexpressed RNase H hydrolyzes the RNA strand in the DNA-RNA hybrid duplex. These combined cutting events break down the nanoprodrug, efficiently co-releasing ASO and Cas12a/crRNA RNP, enabling precise gene silencing and editing. Studies in PDAC-bearing mice show that this drug has strong anti-tumor activity, with minimal systemic toxicity or immunogenicity, offering a promising strategy for precise gene therapy in pancreatic cancer.
Summary
Gene therapy for pancreatic cancer is limited by payload leakage and immunogenicity of delivery platforms. We developed acid-activated nucleic acid nano-prodrugs based on rolling-circle replication, integrating Cas12a/crRNA ribonucleoproteins with antisense oligonucleotides (ASOs) in a single assembly. The prodrug surface is coated with an acid-degradable polymer. Once inside tumor cells, it triggers HhaI enzyme release in acidic lysosomes to cut recognition sites, while RNase H digests the RNA strands. This dual-cutting destroys the assembly, efficiently co-releasing ASOs and Cas12a/crRNA, enabling precise gene silencing and editing. In pancreatic cancer mice, it showed strong antitumor activity with minimal systemic toxicity and immunogenicity.
This work uses nucleic acid self-assembly for high-density loading and acid-responsive activation, reducing excipient use and off-target release risk. In the future, crRNA sequences or ASO targets can be swapped to fit different pancreatic cancer subtypes driven by various genes. The AS1411 aptamer can be replaced with other tumor-targeting ligands. The rolling-circle replication feature makes this platform scalable, though in vivo stability of nucleases and immunogenicity after repeated dosing need further study. Its dual mechanism of gene silencing and editing also provides a useful design framework for nucleic acid therapies against other hard-to-treat solid tumors.
Reference:
DOI: 10.1002/adma.75257
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