Single-Stranded Hairpin\nLoop RNAs (loopmeRNAs) Potently\nInduce Gene Silencing through the RNA Interference Pathway

Synthetic small interfering RNAs conjugated to trivalent <i>N</i>-acetylgalactosamine (GalNAc) are clinically validated\n drugs for treatment of liver diseases. Incorporation of phosphorothioate\nlinkages and ribose modifications are necessary for stability, potency,\nand duration of pharmacology. Although multiple alternative siRNA\ndesigns such as Dicer-substrate RNA, shRNA, and circular RNA have\nbeen evaluated in vitro and in preclinical studies with some success,\nclinical applications of these designs are limited as it is difficult\nto incorporate chemical modifications in these designs. An alternative\nsiRNA design that can incorporate chemical modifications through straightforward\nsynthesis without compromising potency will significantly advance\nthe field. Here, we report a facile synthesis of GalNAc ligand-containing\nsingle-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant\nchemical modifications. We evaluated the efficiency of novel loopmeRNA\ndesigns in vivo and correlated their structure–activity relationship\nwith the support of in vitro metabolism data. Sequences and chemical\nmodifications in the loop region of the loopmeRNA design were optimized\nfor maximal potency. Our studies demonstrate that loopmeRNAs can efficiently\nsilence expression of target genes with comparable efficacy to conventional\ndouble-stranded siRNAs but reduced environmental and regulatory burdens.