A status report on RNAi therapeutics

Abstract

Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.

Figure 1

Small interfering (si)RNA Lead selection. A large panel of siRNAs identified by a bioinformatic screen were synthesized and tested in vitro for activity against the transthyretin transcript as measured by quantitative PCR. The upper part of the panel shows the entire panel tested in parallel at a given nanomolar siRNA concentration, and data are shown in rank order of potency, with each vertical line representing an individual siRNA. The bottom panel illustrates in vitro dose response curves for a potent versus a less potent molecule.

Figure 2

In vivo pharmacokinetic profile of a cholesterol-conjugated (squares) and unconjugated (circles) radio-labeled small interfering (si)RNA against ApoB in the mouse [16]. The half-life and clearance were calculated to be 95 minutes and 0.5 mL/minute and 6 minutes and 17.6 mL/minute, for the conjugated and unconjugated molecules, respectively.

Figure 3

Comparison of on- and off-target effects. A putative lead molecule was tested in vitro to evaluate potency against the intended target, transthyretin and four sequence-related off targets defined by the bioinformatic screen. The percentage reduction in transythyretin levels was measured by quantitative PCR.

Figure 4

Profiling immunostimulatory small interfering (si)RNAs. (a) A panel of siRNAs including negative and positive controls were evaluated in an in vitro human peripheral blood mononuclear cell (PBMC) assay as described previously [10] with supernatants examined for tumor necrosis factor (TNF) (left panel) and interferon-α (right panel) levels. The right-hand side of each panel has a chemically unmodified siRNA, which is compared with the same sequence after incorporation of a combination of phosphorothioate and 2'-O-methyl chemical modifications. (b) The left-hand panels show a series of immunostimulatory (A-D) and non- immunostimulatory (X-Z) siRNAs evaluated in an in vitro PBMC assay. The right-hand panel shows plasma cytokine profiles in mice injected intravenously with the same siRNAs formulated in LNP01 [26]. Common immunostimulatory siRNAs are identified by the in vitro and in vivo assays.