Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors

Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3′ position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi.