Small-molecule pharmaceutical drugs, almost without exception, meet the 'Lipinski Rules', criteria including high lipophilicity and molecular weight of not more than In sharp contrast, siRNAs naturally lack these drug-like properties owing to their large size two turns of a nucleic acid double helix , nearly 40 anionic charges due to the phosphodiester backbone, and high molecular weight over 13 kDa.
In aqueous solution, with their sugar-phosphate backbone exposed to water, siRNAs are extremely hydrophilic and heavily hydrated. Furthermore, siRNAs are unstable in serum as a result of degradation by serum nucleases, contributing to their short half-lives in vivo 7. Although the molecular weight of siRNAs cannot be reduced, these molecules can be made more 'drug-like' through judicious use of chemical modification to the sugars, backbone or bases of the oligoribonucleotides.
Chemically modified siRNA duplexes have been evaluated in cell-based assays and in animal models. The modifications discussed are shown in Figure 2. The activity depends on the position of the modification in the guide-strand sequence. These optimized siRNAs are generally equipotent with or superior to native siRNAs and show increased thermal and plasma stability. In the boranophosphate linkage, a nonbridging phosphodiester oxygen is replaced by an isoelectronic borane BH 3 - moiety.
Boranophosphate siRNAs have been synthesized by enzymatic routes using T7 RNA polymerase and a boranophosphate ribonucleoside triphosphate in the transcription reaction. Boranophosphate siRNAs are more active than native siRNAs if the center of the guide strand is not modified, and they may be at least ten times more nuclease resistant than unmodified siRNAs 17 , Thermal melting analysis showed that the base pair between rF and adenosine is destabilizing relative to a uridine-adenosine pair, although it is slightly less destabilizing than other mismatches.
The crystal structure of a duplex containing rF-adenosine pairs shows local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, a well-ordered water structure is not seen around the rF residues in both grooves. Certain terminal conjugates have been reported to improve or direct cellular uptake. For example, siRNAs conjugated with cholesterol improve in vitro and in vivo cell permeation in liver cells 6.
As described below, cholesterol and an RNA aptamer conjugation show promise in animal models. Critical design concerns in the selection of siRNA duplexes for therapeutic use are potency and specificity. There are two major considerations with regard to siRNA specificity: 'off-targeting' due to silencing of genes sharing partial homology with the siRNA, and 'immune stimulation' due to the engagement of components of the innate immune system by the siRNA duplex.
A combination of bioinformatics methods, chemical modification strategies and empirical testing is required to address these issues. Concomitant with the first description of the structure of active siRNAs, a set of 'rules' was proposed for selecting potent siRNA duplex sequences 21 , Several groups have subsequently developed more sophisticated extensions of these largely empirical criteria, leading to the development of algorithms for siRNA design 23 , Recent biochemical studies of the molecular mechanism of RNA interference have highlighted some key features of potent siRNA duplexes Fig.
Most notably, it has been found that the efficiency with which the guide strand is incorporated into the RISC complex is perhaps the most important factor determining siRNA potency. Because siRNA duplexes are symmetric, the question arose of how the RISC machinery is able to determine which strand to use for target silencing. Examination of the sequences of a large number of vertebrate and invertebrate miRNA precursor sequences showed that the predicted thermodynamic stabilities of the two ends of the duplex are unequal 25 , In short, miRNA precursors show thermodynamic asymmetry.
It was hypothesized that components of the RISC machinery select the guide strand based on this asymmetry. Nucleotides that are important for potency, mRNA recognition, mRNA cleavage and cleavage specificity, including minimization of off-targeting, are shown. Experimental evidence supporting the asymmetry hypothesis has been derived from studies using chemically synthesized siRNAs in transfection experiments.
Through an elegant assay in which each strand of the siRNA targets a different reporter gene, Schwarz et al. In fact, strand selection could be switched by making a single nucleotide substitution at the end of the duplex to alter relative binding of the ends.
A similar conclusion was reached by another group based on in vitro screening of a large collection of siRNAs with varying potency 26 , The issue of off-target silencing has been the subject of intensive study in a number of different laboratories over the past several years.
Transcriptional profiling studies have confirmed that siRNA duplexes can potentially silence multiple genes in addition to the intended target. As expected, genes in these so called off-target 'signatures' contain regions that are complementary to one of the two strands in the siRNA duplex 28 , 29 , Evidence in support of this concept came from a closer look at the determinants of siRNA off-targeting.
Two strategies for avoiding seed region—mediated off-targeting can be envisioned. The first is simply to ensure that nucleotides complementary to positions 2—8 of the guide strand are unique to the intended target. Though theoretically possible, this approach may prove impractical, as the universe of possible seed-region heptamers is only 16, distinct sequences.
As one alternative, recent published work has reported that off-targeting can be substantially reduced by chemical modification of nucleotides within the seed region In fact, introduction of the modification at a single nucleotide position position 2, Fig. The mechanism, anticipated by recently published crystal structure data, appears to involve perturbation of RISC interaction with the modified nucleotide.
Interactions outside of the seed region can also substantially affect siRNA specificity. In a recent study, Schwarz et al. The authors hypothesized that mismatches at these positions are particularly disruptive to the helical structure of the siRNA—mRNA complex required for target cleavage.
A second mechanism whereby siRNA duplexes can induce unintended effects is through stimulation of the innate immune system in certain specialized immune cell types. It has been demonstrated that siRNA duplexes harboring distinct sequence motifs can engage Toll-like receptors TLRs in plasmacytoid dendritic cells, resulting in increased production of interferon Such immune stimulation could pose a significant problem in a therapeutic setting.
This phenomenon is reminiscent of the results of earlier studies with DNA antisense oligonucleotides in which distinct sequences so-called CpG motifs were shown to be immunostimulatory Subsequent studies established that TLR-9, the receptor for unmethylated CpG-containing pathogen DNA, is the innate immune regulator engaged by antisense oligonucleotides Several possible strategies exist for avoiding immune stimulation by siRNA duplexes, including avoidance of the offending sequences during siRNA design and chemical modification to inactivate the motifs.
The former approach is not feasible at present because the full spectrum of stimulatory motifs has not been identified. Another possibility would be to use siRNA delivery strategies that avoid the cell types responsible for immune stimulation. Prediction of the nucleotide sequence and chemical modifications required to yield an ideal siRNA duplex remains a work in progress.
Still, the recent advances described above have allowed the development of design algorithms that greatly increase the likelihood of success. It is nonetheless important to note that the relevance of in vitro measurements of potency and specificity to in vivo activity in a therapeutic setting has yet to be established. For example, the spectrum of off-target genes identified in tissue culture studies can differ depending on the method by which siRNAs are introduced into cells Also, the induction of an innate immune response by certain siRNA sequences is cell type dependent At present, the most prudent and robust strategy is to synthesize and screen a substantial library of siRNA duplexes for each target of interest perhaps even 'tiling' the entire messenger RNA to identify the most promising candidates.
During the past several years, numerous studies have been published demonstrating efficacious silencing of disease genes by local administration of siRNAs or shRNAs in animal models of human disease. Both exogenous and endogenous genes have been silenced, and promising in vivo results have been obtained across multiple organs and tissues. Efficacy has been demonstrated for viral infection respiratory and vaginal , ocular disease, disorders of the nervous system, cancer and inflammatory bowel disease Fig.
An important aspect of these proof-of-concept studies is that they have supported the expected high specificity of RNAi. Direct RNAi represents local delivery of RNAi, and has been carried out successfully to specific tissues or organs, including lung, eye, the nervous system, tumors, the digestive system and vagina.
Systemic RNAi represents intravenous delivery of RNAi and has been carried out successfully to lung, tumors, liver and joint. Specific disease models are indicated where efficacy was achieved. Local RNAi can protect against both respiratory 42 , 9 and vaginal 43 viral infections. Two reports illustrate efficacious direct delivery of siRNA to the lung in rodent and monkey models of RSV, influenza and severe acute respiratory syndrome SARS infection with and without lipid formulation.
In addition, siRNA targeting RSV reduced pulmonary pathology, as assessed by respiratory rate, leukotriene induction and inflammation. Another system for which there have been multiple examples of efficacious local delivery of siRNA is the eye, where proof of concept has been successfully achieved in animal models of ocular neovascularization and scarring using saline and lipid formulations 44 , 45 , Intravitreal injection of siRNA targeting vascular endothelial growth factor VEGF receptor-1, formulated in phosphate-buffered saline, was effective in reducing the area of ocular neovascularization by one-third to two-thirds in two mouse models As with the lung, multiple siRNA formulations were effective in the eye.
In the nervous system, RNAi has been particularly useful for validating disease targets in vivo. Again, several formulations, including saline, polymer complexation and lipid or liposomal formulations, have been efficacious for delivering siRNAs locally to the nervous system in numerous disease models.
The simplest mode of delivery is intracerebroventricular, intrathecal or intraparenchymal infusion of naked siRNA formulated in buffered isotonic saline, which results in silencing of specific neuronal molecular mRNA targets in multiple regions of the central and peripheral nervous systems 47 , 48 , 49 , With naked siRNA formulated in buffered isotonic saline, doses of 0. Local viral delivery of shRNA to the nervous system has been reported in vivo with adenoviral, adeno-associated viral AAV and lentiviral delivery in normal mice 55 as well as in animal models of spinocerebellar ataxia 56 , Huntington disease 57 , 58 , amyotrophic lateral sclerosis ALS 59 , 60 and Alzheimer disease 61 , where abnormal, disease phenotypes including behavior and neuropathology were normalized.
Notably, all of the in vivo studies to date have targeted genes expressed in neurons; it remains to be seen whether silencing in vivo can be achieved in other nervous-system cell types such as oligodendrocytes and astrocytes. For application to oncology, direct delivery of siRNAs and viral delivery of shRNAs to tumors have been successful in inhibiting xenograft growth in several mouse models.
A number of approaches—including lipid-based formulation TransMessenger 62 and complexation with PEI 63 , cholesterol-oligoarginine 64 , a protamine-Fab fusion protein 65 and atelocollagen 66 , 67 —have been shown to facilitate delivery into tumor cells. Notably, these siRNA delivery approaches are effective with several or even a single intratumor injection of siRNA, at microgram doses. Very recently, aptamer-siRNA chimeric RNAs have also been used successfully to facilitate siRNA delivery in vivo , resulting in tumor regression in a xenograft model of prostate cancer Viral and vector-based delivery of shRNAs directly to the tumor site 69 , 70 has also been used effectively in mouse models of adenocarcinoma, Ewing sarcoma and prostate cancer.
Of the multiple delivery strategies that have been effective in mouse tumor models, the aptamer approach has the potential of substantially simplifying delivery, if an aptamer is available for a tumor-specific receptor such as prostate-specific membrane antigen PMSA and the large-scale synthesis of such a construct is feasible.
This report, together with a study of siRNA targeting herpes simplex virus-2 ref. Over the past several years, a number of studies have been published demonstrating the silencing of disease genes by systemic administration of siRNAs Fig. In some of these studies, silencing of endogenously expressed genes has shown promising in vivo results in different disease contexts.
For example, efficacy has been demonstrated in mouse models of hypercholesterolemia and rheumatoid arthritis. In other work, systemic RNAi targeting exogenous genes has shown promise in models of viral infection hepatitis B virus HBV , influenza virus, Ebola virus and in tumor xenografts.
Critical to the success of most of these studies has been the use of chemical modifications or delivery formulations that impart desirable pharmacokinetic properties to the siRNA duplex and that also promote cellular uptake in tissues. In , Soutschek et al. These therapeutically relevant findings were completely consistent with the known function of apoB in lipid metabolism.
Cholesterol conjugation imparted critical pharmacokinetic and cellular uptake properties to the siRNA duplex. Further advances in systemic RNAi with optimized delivery have recently been reported.
Recently, Zimmermann et al. More importantly, therapeutic silencing of apoB was also demonstrated in nonhuman primates. A single dose of 2. Furthermore, silencing was shown to last for at least 11 d after a single dose. In addition, the treatment seemed to be well tolerated, with transient increases in liver enzymes as the only reported evidence of toxicity.
This primate study represented an important step forward in the development of systemic RNAi for therapeutic applications. In mouse tumor xenograft models, the efficacy of systemic RNAi has been demonstrated using a variety of delivery strategies reviewed in refs.
Systemically delivered cationic cardiolipin liposomes containing siRNA specific for Raf-1 inhibit tumor growth in a xenograft model of human prostate cancer Intravenous administration of these complexes into tumor-bearing mice inhibits both tumor angiogenesis and growth rate Simpler PEI formulations have also shown efficacy in xenograft tumor models 79 , as have complexes of siRNA duplexes with atelocollagen.
Systemic administration of atelocollagen—siRNA complexes has marked effects on subcutaneous tumor xenografts 66 as well as bone metastases Another recently described delivery strategy made use of a recombinant antibody fusion protein to achieve cell type—specific delivery.
As described above, Song et al. After systemic administration, the Fab-protamine fusion was able to deliver an siRNA mixture to mouse melanoma cells engineered to express the envelope protein, leading to substantial inhibition of tumor growth in mice. Tumors derived from cells not expressing the envelope protein were unaffected.
In another example of ligand-directed delivery, Hu-Lieskovan et al. Removal of the targeting ligand or the use of a control siRNA sequence eliminated the antitumor effects. Effective delivery is perhaps the most challenging remaining consideration for successful translation of RNAi to the clinic and to broad use in patients.
In the animal studies reviewed above, nonviral and viral approaches, local and systemic administration, and multiple formulations saline, lipids, and complexes or conjugates with small molecules, polymers, proteins and antibodies have all been used to achieve efficacy. However, each of these approaches has distinct advantages and disadvantages for clinical translation, which require careful consideration. Although viral delivery provides the potential advantage that a single administration could lead to durable down-modulation of the targeted pathological protein, a major risk was highlighted recently Clearly, for all drugs, it is critical to be able to control the level of drug and the duration of drug action, such that the exposure is safe while still being efficacious.
In distinct contrast to nonviral delivery of siRNAs, a substantial liability of viral delivery is that it is impossible to fully predict drug exposure, with regard to both amount and timing. The principal considerations for selecting local versus systemic siRNA administration are the doses needed to achieve sufficient drug concentration in the target tissue and the possible effects of the exposure of nontargeted tissues to drug.
At one extreme, with certain tissues, efficacy has so far been demonstrated only with local delivery; current formulations may not provide sufficient drug concentration in the target tissue after systemic delivery. In general, and as with any pharmacologic approach, the doses of siRNA required for efficacy are substantially lower when siRNAs are injected into or near the target tissue than when they are administered systemically.
Given the high specificity of siRNAs for their intended molecular target, exposure of nontargeted tissues to drug is an issue only if the molecular target is expressed in nontargeted tissue and has an important role in normal cellular function within that tissue. Brummelkamp, T. A system for stable expression of short interfering RNAs in mammalian cells. Science , — Chiu, Y. RNA 9, — Cihlar, T. Current status and prospects of HIV treatment.
Cross, R. Post-exposure treatments for Ebola and Marburg virus infections. DeVincenzo, J. Antiviral Res. A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus. DiGiusto, D. Ding, S. RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res. Dominska, M. Breaking down the barriers: siRNA delivery and endosome escape. Cell Sci. Nucleic Acids Res.
Dunning, J. PLoS Med. Elbashir, S. Nature , — Enayati, S. FEMS Microbiol. Fabozzi, G. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Fire, A. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Flisiak, R. Expert Opin. Ge, Q. Geisbert, T. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study.
Lancet , — Gimenez-Barcons, M. Endoribonuclease-prepared short interfering RNAs induce effective and specific inhibition of human immunodeficiency virus type 1 replication. Gish, R. Gitlin, L. Short interfering RNA confers intracellular antiviral immunity in human cells. Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches.
Gottlieb, J. ALN-RSV01 for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients. Heart Lung Transplant. Hannus, M. Heninger, A. Production of endoribonuclease-prepared short interfering RNAs esiRNAs for specific and effective gene silencing in mammalian cells.
CSH Protoc. Huang, L. Production of highly potent recombinant siRNAs in Escherichia coli. Jacque, J. Kaczmarek, J. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Kapadia, S. Kim, D. Kok, K. Cell Host Microbe 9, — Kraft, C. The use of TKM and convalescent plasma in 2 patients with ebola virus disease in the United States. Kumar, P. Cell , — Macrae, I. Structural basis for double-stranded RNA processing by Dicer.
Maillard, P. EMBO J. Makeyev, E. Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6. Minks, M. Structural requirements of double-stranded RNA for the activation of 2',5'-oligo A polymerase and protein kinase of interferon-treated HeLa cells.
Morrissey, D. Musacchio, T. Nair, J. Naso, M. Adeno-associated virus AAV as a vector for gene therapy. BioDrugs 31, — Niehl, A. Synthetic biology approach for plant protection using dsRNA. Plant Biotechnol. Olejniczak, M. Probe Reagents for Functional Genomics. A simplified model for the RNAi pathway A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. The high degrees of efficiency and specificity are the main advantages of RNAi.
You are here: NCBI.
0コメント