RNA interference-based therapeutics and diagnostics.

Abstract

The 2006 Nobel Prize in Medicine and Physiology was awarded to Andrew Fire and Craig Mello only 8 years after their Nature publication showing small double-stranded RNA (siRNA)-mediated gene silencing by the process of RNA interference (RNAi). This remarkable recognition highlights the importance and immense implications of understanding and controlling cellular gene expression as a functional genomic study tool and its potential exploitation as a therapeutic modality. The discovery in 2001 by Thomas Tuschl's group that RNAi is found in mammals and exogenous synthetic siRNA introduced into the cell could silence genes fuelled initial excitement that small RNAs were a paradigm shift in therapeutics. Expectations were high because of the potential of these molecules to “silence” or “knock-down” any gene of interest to treat almost any disease by targeting otherwise “undruggable” targets such as molecules without ligand-binding domains or enzymatic function. A greater understanding of the RNAi mechanisms and rapid expansion of the field has resulted in the identification of a wide repertoire of RNAi triggers that engage at various levels of the RNAi cascade, collectively termed small interfering nucleic acids (siNA) that could be exploited as molecular medicines. MicroRNAs (miRNA), for example, are endogenous small non-coding RNA that control cellular gene expression whose deregulation can be associated with disease states, and as a consequence, have gained considerable attention as disease biomarkers and therapeutic drugs or targets. Despite the promise, the clinical translation of siNA therapeutics has proven challenging. The main obstacle is delivery of these molecules to target sites and into cells at therapeutically relevant levels without toxicity. The expanding classes of siNA include mimics of endogenous microRNAs to suppress the expression of many genes, but with less efficient suppression of each one. The delivery requirements needed for conventional siRNA and imperfectly paired microRNA mimics are essentially the same although antagonizing endogenous microRNAs using single-stranded antisense oligonucleotides may be somewhat easier. When injected intravenously, siNA are rapidly cleared by renal filtration and are susceptible to degradation by extracellular RNases. The siNA circulatory half life can be increased—even to days—by chemical modifications to eliminate susceptibility to endogenous exonucleases and endonucleases and by incorporating the siNA into a larger moiety, above the molecular weight cutoff for kidney filtration. Intracellular entry is, however, a major hurdle due to the polyanionic nature and high molecular weight of siRNA. Although cells can endocytose many types of modified nucleic acids or nucleic acids-containing particles, another important bottleneck is getting these molecules efficiently out of the endosome into the cytosol where the RNAi machinery resides or into the nucleus for precursor miRNA or expression vectors for siNA. Nucleic acid therapeutics has been extensively studied both in academia and in the pharmaceutical industry with high D. Peer Laboratory of NanoMedicine, Department of Cell Research and Immunology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv 69978, Israel