Abstract

Despite the clinical success of siRNA therapeutics, limitations including rapid degradation, off-target effects, and high manufacturing costs hinder broader application. Circular siRNAs, particularly dumbbell-shaped variants (db-siRNAs), offer enhanced stability and prolonged activity but lack scalable production methods. Here, we developed a streamlined platform for db-siRNA biosynthesis using a minimized T4 phage thymidylate synthase ( Td ) group I intron permuted intron-exon (PIE) system. By embedding essential splicing motifs into one terminal loop, we achieved ∼85% cyclization efficiency both in vitro and in vivo . Using engineered Escherichia coli Nissle 1917 (EcN), we established a low-immunogenicity production system, which could yield functional db-siRNAs at 60 mg/L in a fed-batch fermentation with a 3 L bioreactor. Db-siRNAs with 23-bp stems demonstrated optimal gene silencing for over 10 days in vitro and effectively reduced target expression in vivo when formulated with lipid nanoparticles. Our platform enables rapid, cost-effective production of stable, potent db-siRNAs for preclinical development, addressing key manufacturing challenges in RNAi therapeutics.