Design of Synthetic shRNAs for Targeting Hepatitis C: A New Approach to Antiviral Therapeutics

Small hairpin RNAs (shRNAs) are widely used as gene silencing tools and typically consist of a duplex stem of 19–29 bp, a loop, and often a dinucleotide overhang at the 3′ end. Like siRNAs, shRNAs show promise as potential therapeutic agents due to their high level of specificity and potency, although effective delivery to target tissues remains a challenge. Algorithms used to predict siRNA performance are frequently used to design shRNAs as well. However, the differences between these two kinds of RNAi mediators indicate that the factors affecting target gene silencing will not be the same for siRNAs and shRNAs. Stem and loop lengths, structures of the termini, the identity of nucleotides adjacent to and near the loop, and the position of the guide (antisense) strand all affect the efficacy of shRNAs. In addition, shRNAs with 19-bp or shorter stem lengths are processed and function differently than those with longer stems. In this review, we describe studies of targeting the hepatitis C virus that have provided guidelines for an optimal design for short (19 bp) shRNAs (sshRNAs) that are highly potent, stable in biological fluids, and have minimal immunostimulatory properties.