Introduction

RNA interference (RNAi) is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes (1). Using small interfering RNA (siRNA) molecules, RNAi can selectively silence essentially any gene in the genome. Once in a cell, a short double-stranded RNA (dsRNA) molecule is cleaved by an RNase called Dicer (2) into 21- to 23-nucleotide guide RNA duplexes called siRNAs that become bound to the RNA-induced silencing complex (RISC) (3,4). Within the RISC, one of the two strands of the siRNA is chosen as the antisense strand via cleavage of the passenger strand (5,6,7), so that they can target complementary sequences in messenger RNAs (mRNAs) involved in a disease (8). After pairing with an siRNA strand, the targeted mRNA is cleaved and undergoes degradation thereby interrupting the synthesis of the disease-causing protein (9). The RISC complex is naturally stable within the cell, enabling siRNAs to cut multiple mRNA molecules consecutively and, therefore, suppressing protein synthesis in a potent and targeted way.

RNAi was described by the journal Science as the “Breakthrough of the Year” in 2002 having the potential to become a powerful therapeutic drug. RNAi-based therapeutics has potentially significant advantages over traditional approaches to treating diseases, including broad applicability, therapeutic precision, and selectivity avoiding side effects. This widespread applicability, coupled with relative ease of synthesis and low cost of production make siRNAs an attractive new class of small-molecule drugs. RNAi-based drugs are designed to destroy the target RNA and therefore stop the associated undesirable protein production required for disease progression.

Several recent studies using highly sensitive microarray analyses have shown that siRNAs can have off-target effects by silencing unintended genes (10,11). These off-target effects can be minimized by modifying the siRNAs to prevent incorporation of the sense strand into RISC and by choosing sequences with minimal complementarities to known genes in the database (12). Finding siRNAs that are active at low concentrations should help to abrogate some of these problems.

For RNA-based therapies, manufacture has been seen as a solvable problem, while delivery and stability have been the most significant obstacles. There are two strategies for delivering siRNAs in vivo (13). One is to stably express siRNA precursors, such as short hairpin RNAs (shRNAs), from viral vector using gene therapy; the other is to deliver synthetic siRNAs by complexing or covalently linking the duplex RNA with lipids and/or delivery proteins. To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Despite the questions and unsolved problems, several companies are moving ahead to develop RNAi therapy for many diseases including diabetes.

RNAi in Vivo Targeting Liver

Liver is the prime organ target for systemically delivered siRNA, which tends to concentrate in this organ whether delivered hydrodynamically or with cholesterol or lipid carriers (14,15,16,17,18,19,20). Initial studies showing the activity of RNAi in vivo involved delivery of siRNA in mouse liver by using the hydrodynamic method, this consists of a rapid injection of a large volume of aqueous solution into the mouse tail vein, which creates a high pressure in the vascular circulation, leading to an extensive delivery of siRNA into hepatocytes (14,15,16,17,18). However, the sudden volume load induces right-sided heart failure, and the resulting high venous pressures permit the siRNAs to enter into the cells (21). This procedure allows high efficiency of siRNA uptake and potent siRNA activity in hepatocytes, but is not clinically viable because of the potential damage of the liver and other organs and, therefore, is limited only to research on liver function and metabolism or liver infectious diseases such as hepatitis (15,16,17).

As one step toward the liver-targeting delivery, liver delivery of chemically modified oligonucleotides with cholesterol conjugates was tested, as described in recent publications (19,20). Soutschek et al. (19) developed chemically modified siRNAs to silence an endogenous gene encoding apolipoprotein B (apoB). apoB is a molecule involved in the metabolism of cholesterol, and the concentrations of this protein in human blood samples correlate with those of cholesterol, and higher levels of both compounds are associated with an increased risk of coronary heart disease (22,23,24). The siRNAs synthesized by this group contained selective stabilizing modifications and were joined to a cholesterol group that was chemically linked to the terminal hydroxyl group of the sense-strand RNA (19). Intravenous injections of the siRNA-cholesterol conjugates in mice resulted in uptake into several tissues, including the liver, jejunum, heart, kidneys, lungs, and fat tissue and efficiently reduced the levels of apoB mRNA by more than 50% in the liver and by 70% in the jejunum. This reduction resulted in a lowering of the levels of blood cholesterol comparable to that observed in mice in which the apoB gene had been deleted (23). These results demonstrated that siRNA can be delivered systemically targeting the liver and suggest that RNAi has the potential to become a new therapeutic for the treatment of metabolic diseases.