Engineering and optimization of the mir-106b-cluster for ectopic expression of multiplexed anti-HIV RNAs

Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 siRNAs processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron embedded, tri-cistronic mir106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the mir106b cluster demonstrated a strict requirement for maintenance of the native flanking pri-miRNA sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of HIV-1. This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted ina substantial enhancement of HIV suppression in long term acute infectious HIV-1 challenges.