What is rnai therapy

Open in a separate window. Table 2 Pros and cons of delivery approaches. Figure 2. Delivery strategies for RNAi The cell grey ellipse contains a nucleus dark circle and a cell membrane dark ellipse.

Tissue delivery Some of the delivery strategies that have been successfully utilized in animal models during pre-clinical studies have not been tried yet in a patient setting.

Adenoviruses Non-enveloped icosahedral viruses containing double stranded DNA genome that are commonly used as vectors for gene therapy.

Antagomirs Backbone modified antisense oligomers complementary to microRNAs, blocking their function. ApoB Apolipoprotein B is the main apolipoprotein of low density lipoproteins that transport cholesterol and triglycerides through the blood stream.

Blood—brain barrier A selectively permeable barrier between the capillaries and the brain that acts as a filter preventing many substances from entering the central nervous system. Catalytic RNAs Ribonucleic acids with enzymatic activity. Endosome A membrane-bound organelle that sorts endocytosed material and recycles it back to the cell surface or delivers it to other compartments as the lysosome where such cargo can be degraded. Lentiviral vectors Gene therapy vectors engineered from HIV or other members of the lentivirus family a class of retrovirus.

Locked nucleic acid technology Backbone modification of nucleic acids that locks the sugars into a single constrained conformation, making the oligomers they are associated with nuclease-resistant and forming thermodynamically stable hybrids with ribonucleic acids. Polycation polymers Positively charged polymers that interact with negatively charged nucleic acids and can be used as delivery vehicle for gene therapy.

RNA-aptamer In vitro evolved, 2' Fl backbone modified RNA molecules that take on three-dimensional shapes allowing them to bind with high affinities to targeted proteins. RNA interference A cellular process in which small double stranded RNAs are used as precursors for selection of a guide strand that directs sequence specific base pairing to complementary sequences. SNALPs Stable nucleic acid lipid particles that can be modified with targeting ligands and are used as delivery vehicle for gene therapy.

Toll-like receptors Proteins that recognize pathogen molecules and activate immune cell responses. Vector Carrier and delivery vehicle for gene therapy. Targeted cellular delivery Targeted delivery is another important goal for siRNA therapeutics. The endosome Once delivered to the right tissue or the right cell, another obstacle that has to be overcome with siRNA delivery systems is the endosome.

RNAi in the clinic There are currently only two clinical trials using ex vivo delivery, whereas most of the trials employ systemic delivery including injections directly into the target tissues such as the eyes for treatment of age-related macular degeneration AMD or directly into tumours, inhalation or infusion with targeted delivery vehicles with incorporated siRNAs Fig 1.

Figure 1. Delivery of therapeutics to patients Systemic delivery: a double stranded siRNA is packaged into delivery vehicle targeted nanoparticles, polymers, liposomes, etc. Unexpected problems and possible solutions for RNAi based therapies Given that RNAi was first described slightly over a decade ago and the mechanisms of this phenomenon are still being unraveled, it is quite amazing that there is such a collection of ongoing clinical trials.

Bridge the Gap The Gap RNAi-based therapeutics are currently being tested in various phase I and II clinical trials, however there are still several problems to overcome before their clinical application becomes widespread. The Bridge This review describes the current status of RNAi-based therapeutics and summarizes the many approaches the field is taking to tackle the issue of the RNA delivery. Looking to the future The promise of RNAi as a powerful new approach for therapeutic treatment of diseases has propelled early stage clinical testing of siRNAs for a variety of diseases.

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J Cell Biol. Systemic RNAi in C. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Durable protection from Herpes Simplex Virus-2 transmission following intravaginal application of siRNAs targeting both a viral and host gene. This malignancy often develops in patients with primary sclerosing cholangitis, a condition that starts with bile duct inflammation and subsequent fibrosis.

Similarly, the Sirnaomics antifibrosis pipeline contains local and systemic therapeutics. The local administration product targets keloid, a fibrous nodule that forms during scar healing, and the systemic product targets primary sclerosing cholangitis.

A shared feature of the products in the Sirnaomics pipeline is the use of a two-pronged target approach. To facilitate the design of RNAi therapeutics, the company has developed proprietary algorithms that trigger the RNAi mechanism and instigate rapid, deep, and durable knockdown of target genes.

RNAi therapeutics are ideally suited for targeting conditions that cannot be targeted with small molecules and biologics. The company asserts that its technology allows lead products to be translated into the clinic in less than two years. Besides emphasizing speed, the company is working to expand the number of tissues that RNAi therapeutics can target.

At present, the usual target is the liver, which is certainly an important target. But targeting other tissues would help address many unmet medical needs. Different tissues require different silencing molecules. To design these molecules, Arrowhead draws on its portfolio of RNA trigger structures and chemistries. Combining these molecular elements in different ways allows the company to optimize each drug candidate on a target-by-target basis and obtain the most potent RNAi trigger.

Because the molecules used for RNAi are large and highly charged, they may have difficulty crossing membranes. Advances in the RNAi space have been facilitated to a great extent by several layers of specificity for RNAi delivery, both in terms of the genes that are targeted and with respect to the cell type and cell population that the molecules are designed to enter. Many of these solutions need to be very specific to individual cell types. He notes, however, that if Arrowhead figures out what modifications a molecule needs to target a given cell population, the exercise opens the possibility of manipulating any genes in that cell population.

In the RNAi space, Arrowhead has pioneered potential treatments for hypertriglyceridemia. 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.

In these cases, local delivery with more focused exposure might circumvent undesired side effects resulting from systemic delivery. Liposomes, and lipid complexes or conjugates with small molecules, polymers, proteins and antibodies, have all been used to facilitate delivery of siRNAs to target cells. With these delivery partners, more robust efficacy can be achieved with doses of siRNA that are substantially lower, less frequent or both.

For the additional non-siRNA components, however, there are associated biological and large-scale manufacturing considerations. Lipids and polymers can have cytotoxic effects that might limit their use in siRNA delivery for particular disease indications and dosing paradigms. However, it seems to be possible to identify lipid-based formulations and dosing regimens for which cytotoxicity is minimal and the risk of histopathology is reduced 51 , 52 , Small molecules, proteins and antibodies used as conjugates also need to be considered from the standpoint of biological activity.

If the endogenous molecule for example, receptor with which they interact has an important role in normal physiology, then using this endogenous molecule to potentiate delivery may alter its normal function and produce undesired side effects. In all of these cases, the additional non-siRNA molecule or molecules increase the complexity of manufacturing, particularly at large scale. With scientific and technical advances, these approaches may provide marked enhancements to siRNA delivery with acceptable biological and manufacturing considerations.

In both these cases, highly validated disease targets are being inhibited with siRNAs. Furthermore, direct administration of siRNAs to the eye and lung for AMD and RSV infection, respectively, maximizes the chances of delivering sufficient and therapeutically relevant concentrations of drug to the tissue of interest.

As these and other trials advance through the clinic in the near future, the exciting potential of siRNAs may be demonstrated. As a therapeutic approach, RNAi provides solutions to the major drawbacks of traditional pharmaceutical drugs Table 1.

The principal advantages of RNAi over small-molecule and protein therapeutics are that all targets, including 'non-druggable' targets, can be inhibited with RNAi and that lead compounds can be rapidly identified and optimized. The primary challenge associated with small-molecule drugs is the identification of highly selective and potent compounds—a difficult and time-consuming process that, for some targets, can be unsuccessful.

With RNAi, the identification of highly selective and potent sequences is rapid and has been demonstrated with numerous molecular targets across all molecular classes.

With protein and antibody drugs, the main technical challenge is production. For proteins, acceptable cellular production levels are often difficult to achieve. For biologics as a therapeutic class, aggregation continues to be a major issue. In contrast, siRNAs are synthetic and easy to produce from a chemistry standpoint. Of course, with RNAi, by definition, only antagonism of the specific molecular target is possible, whereas small molecules, proteins and antibodies provide an opportunity for agonism of a molecular target.