Interfering with disease: a progress report on siRNA-based therapeutics

Abstract

RNA interference (RNAi) quietly crept into biological research in the 1990s when unexpected gene-silencing phenomena in plants and flatworms first perplexed scientists. Following the demonstration of RNAi in mammalian cells in 2001, it was quickly realized that this highly specific mechanism of sequence-specific gene silencing might be harnessed to develop a new class of drugs that interfere with disease-causing or disease-promoting genes. Here we discuss the considerations that go into developing RNAi-based therapeutics starting from in vitro lead design and identification, to in vivo pre-clinical drug delivery and testing. We conclude by reviewing the latest clinical experience with RNAi therapeutics.

Figure 1. Mechanism of RNA interference in mammalian cells.

RNA interference (RNAi) pathways are guided by small RNAs that include small interfering RNA (siRNA) and microRNAs (miRNAs). The siRNA pathway begins with cleavage of long double-stranded RNA (dsRNA) by the Dicer enzyme complex into siRNA. These siRNAs are incorporated into Argonaute 2 (AGO2) and the RNAi-induced silencing complex (RISC). If the RNA duplex loaded onto RISC has perfect sequence complementarity, AGO2 cleaves the passenger (sense) strand so that active RISC containing the guide (antisense) strand is produced. The siRNA guide strand recognizes target sites to direct mRNA cleavage (carried out by the catalytic domain of AGO2). RNAi therapeutics developed to harness the siRNA pathway typically involve the delivery of synthetic siRNA into the cell cytoplasm. The microRNA pathway begins with endogenously encoded primary microRNA transcripts (pri-miRNAs) that are transcribed by RNA polymerase II (Pol II) and are processed by the Drosha enzyme complex to yield precursor miRNAs (pre-miRNAs). These precursors are then exported to the cytoplasm by exportin 5 and subsequently bind to the Dicer enzyme complex, which processes the pre-miRNA for loading onto the AGO2–RISC complex. When the RNA duplex loaded onto RISC has imperfect sequence complementarity, the passenger (sense) strand is unwound leaving a mature miRNA bound to active RISC. The mature miRNA recognizes target sites (typically in the 3′-UTR) in the mRNA, leading to direct translational inhibition. Binding of miRNA to target mRNA may also lead to mRNA target degradation in processing (P)-bodies. Modified with permission from Nature Rev. Genet. Ref © (2007) Macmillan Publishers Ltd.

Figure 2. Turning siRNA into drugs.

This three-step process begins with in silico design and in vitro screening of target siRNAs, is followed by incorporating stabilizing chemical modifications on lead siRNAs as required, and ends with the selection and in vivo evaluation of delivery technologies that are appropriate for the target cell type/organ and the disease setting. A schematic illustration of the important features of siRNA structure (two base pair overhangs, seed region and mRNA cleavage site) is shown. An example of an optimized siRNA molecule that incorporates chemical modifications to increase nuclease stability and to minimize off-targeting is shown below. The phosphorothioate (P=S) and 2′-base sugar modifications (green circles) are illustrative of exonuclease and endonuclease stabilizing chemistries, respectively; other chemistries that confer similar properties on an siRNA duplex also exist. The passenger strand and the guide strand are represented in blue and orange, respectively.

Figure 3. Delivery of small interfering RNAs.

Different strategies have been used to deliver and achieve RNAi-mediated silencing in vivo. a | Direct injection of naked siRNA (unmodified or chemically modified) has proven efficacious in multiple contexts of ocular, respiratory and central nervous system disease^{,,,,,,,,,,,,,,,,,}. b | Direct conjugation of siRNA to a natural ligand such as cholesterol has demonstrated in vivo silencing in hepatocytes following systemic administration. c | Aptamer–siRNA conjugates silence target genes in tumour cells following local injection in a tumour xenograft model. d | Liposome-formulated delivery of siRNA has been used to silence multiple targets following systemic administration^,,,. The composition of a stable nucleic acid–lipid particle (SNALP) is shown. Cationic lipids aid formulation, cellular uptake and endosomal release. Fusogenic lipids also function in endosomal release. Polyethylene glycosylated (PEG) lipids stabilize the formulation, regulate fusogenicity and shield surface charges. Cholesterol also helps to stabilize the formulation. Other cationic liposome formulations exist which may or may not contain a fusogenic or PEG lipid. The ratios of different components can vary between formulations. e | Antibody–protamine fusion proteins have been used to non-covalently bind siRNAs through charge interactions and to deliver siRNA specifically to cells that express the surface receptor that is recognized by the antibody^,. The passenger strand and the guide strand are represented in blue and orange, respectively.