Evelina e le fate (Italiana) (Italian Edition)

Because of their tunable features, nanoparticles are being investigated in an attempt to overcome the safety and delivery issues associated with siRNA-based drugs. Given the exceptional promise of these combined technologies, this review aims to address fully the main strengths and weaknesses of their applications, highlighting the potential problems and solutions that one day could transform RNAi into a conventional treatment for human malignancy. Second, we discuss the possible use of siRNA in the clinic, highlighting several challenges that could limit their applications, with particular emphasis on siRNA delivery and biological barriers.

Third, we review the wide range of strategies proposed for siRNA delivery, with attention paid to the advantages and current limitations of the nanoparticle-based approach. Further, we discuss strategies which have shown in vivo success in humans, reporting the recent promising results from clinical trials in cancer and other diseases. Finally, we provide a critical evaluation of future prospects and challenges for siRNA-based therapy in the treatment of cancer. RNAi is a fundamental regulatory pathway for most eukaryotic cells. It consists of complex enzymatic machinery able to control post-transcriptional gene expression through homology-dependent degradation of target mRNA by siRNA, ie, nontranslated dsRNA molecules.

Once cleaved, siRNAs interact withArgonaute-2, a multifunctional protein, and become incorporated into RNA-induced silencing complexes. This activated complex can propagate gene silencing further, destroying additional mRNA targets. This effect may last for 3—7 days in rapidly dividing cells and for many weeks in nondividing cells. Pre-miRNA hairpins are exported from the nucleus in a process involving the nucleo-cytoplasmic shuttle Exportin-5 Exp. See text for details. In certain cases, only partial complementarity can occur between siRNA and its target mRNA, leading to suppression of translation or destabilization of transcripts.

This phenomenon usually causes the so-called off-target effects of siRNA see later and mimics interactions with target sites of another class of regulatory small RNA, ie, miRNA. These molecules are thought to be the endogenous substrate for the RNAi machinery, because endogenous siRNA has not been found in mammalian systems. They are then exported to the cytoplasm by Exportin-5, where they are further processed by the Dicer enzyme which removes the loop, and one of the two strands is loaded into the RNA-induced silencing complex. Therefore, the primary mechanism of action is translational repression, although miRNA molecules with full sequence complementarity can degrade mRNA, so it could be said that while siRNAs are able to control single gene expression fully, microRNAs can moderate the expression of gene networks.

The need for the short hairpin RNA gene to enter the nucleus adds complexity to the delivery and reduces the potency of siRNA gene silencing. On the other hand, the potential advantage of DNA-based RNAi is its stable introduction into cells as gene therapy, bypassing transient effects, so avoiding repeated or continuous administration in the clinical setting. Another variation of siRNA-based therapy is administration of longer precursors processed by Dicer endonuclease. Recent reports indicate that these precursors are several nucleotides longer and are much more powerful than the siRNAs widely used for inducing target gene silencing.

Cancer is a disease that is often characterized by mutations in multiple signal transduction pathways, leading to uncontrolled cell proliferation.

It is usually difficult to identify the key genes governing cell proliferation or survival which should be targeted to induce cell death. RNAi technology can be useful for cancer therapy because of its high efficacy and specificity in downregulating gene expression, and it is clearly important to understand the role of altered genes in the development of cancer. It must be stressed that blockage of a single gene is not sufficient to cure or control most cancers.

One of the major likely benefits of the RNAi technique in the treatment of cancer is the feasibility of developing combination therapeutic RNAi approaches to inhibit multiple oncogenes or genes of proteins involved in tumorigenesis. Many in vitro and in vivo studies have evaluated the silencing of genes involved in pathways that drive cancer, such as oncogenesis, apoptosis, cell cycle regulation, cell senescence, tumor-host interaction, and resistance to conventional therapies.

Notably, miRNAs can be detected in body fluids, such as blood and cerebrospinal fluid, enabling noninvasive and early detection of disease. A further example of the potential applications of miRNA is expression profiling in order to distinguish cancer from normal tissue and to classify, eg, cancer subtypes or the origin of metastatic cancer tissue. As discussed, siRNAs hold promise as therapeutics in view of their ability to silence any gene with a known sequence. However, effective and well controlled in vivo delivery is challenging, and presently limits the use of RNAi in the clinic 27 see Table 1.

To be efficacious, they need to reach the cytoplasm of the cell in sufficient amounts to enable sustained target inhibition. Unmodified siRNA molecules also have potential toxicities, 52 , 53 via three main mechanisms, ie, saturation of RNAi machinery and competition within the miRNA pathway, stimulation of the immune response, and off-target effects.

RNAi is an important regulatory mechanism in the cell which could be perturbed by exogenous introduction of dsRNA. The components of the cellular machinery involved in gene silencing could be less accessible to miRNAs entering the natural cellular pathway. Some of the sequence motifs in siRNA molecules could have the ability to induce the innate immune response. The innate immune response is mediated principally by type I interferon and proinflammatory cytokines, 56 — 58 and can be triggered in different ways, ie, mediated or not mediated by the Toll-like receptor TLR.

Briefly, when nucleic acids enter endosomal and lysosomal compartments, TLR-3, 7, and 8 are primed and activate interferon-alpha and inflammatory cytokines via nuclear translocation of the nuclear factor k-light chain enhancer of activated B cells. This mechanism can lead to activation of interferon-beta and other inflammatory mediators. However, early genome-wide monitoring of gene activity in siRNA-treated cells disproved its almost ideal specificity. Specific research efforts have been made to understand better and control these undesirable off-target effects and their long-term implications, and even more so now, considering the possible clinical reality of siRNA.

In these contexts, siRNA therapy has higher bioavailability and may have fewer of the adverse effects associated with nonspecific siRNA delivery. The ideal route and obstacles which can be encountered by molecules introduced into the human body through systemic delivery are still a matter of debate. Much research effort using in vivo tracking and fluorescent methods is presently focused on these issues. It is generally accepted that, after intravenous injection, the molecules travel throughout the whole body, navigate the circulatory system, in which they need to avoid renal filtration, are taken up by phagocytes both in the bloodstream and in the extracellular matrix tissue and aggregate with serum proteins Figure 2.

Specifically, it has been reported that molecules larger than 5 nm in diameter do not readily cross the capillary endothelium, and therefore remain in the body if they are not cleared. However, larger molecules up to nm in diameter are able to enter some tissues, including the spleen, liver, and several types of tumor, due to their different vasculature.

The molecules must also diffuse through the extracellular matrix, which consists of a dense network of polysaccharides and fibrous proteins. The extracellular matrix is also rich in macrophages, which can obstruct transport of macromolecules and nanoparticles. The siRNA complex must then be taken up by target cells, at which point they have to evade the endosome system to reach the cytoplasm. Finally, to be effective, siRNA must be released from its carrier to reach the cellular machinery.

Encapsulation technologies for siRNA delivery in vivo: Readapted from Shim et al. Generally speaking, these protective strategies can be divided in three categories, ie, mechanical, chemical, and biological.

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Mechanical approaches seek to enhance permeability of biological barriers, and in this way allow gene material to reach the cellular environment. The main mechanical permeabilization techniques are sonoporation and electroporation. In this context, viral vectors provide the most efficient delivery, but their use is limited due to the host immune response and the inner complexities of this method. State-of-the art chemical approaches are the most promising, as a result of ever-increasing insight into techniques of synthesis and the biological behavior of nonbiological structures.

However, bioavailability remains an unresolved issue because these vectors are unable to stabilize and protect siRNA in the biological environment. Nanotechnology has been a most promising spinoff in molecular medicine. In this field, technical advances have suddenly accelerated towards more refined fabrication techniques and new materials with unique and innovative features. The impact of nanostructured therapeutics in terms of efficacy and safety is promising to be revolutionary.

The dawn of nanotechnology, seen as the manipulation of matter on a nanometric scale, allows the design and fabrication of novel devices with uniquely tailored physicochemical properties and enormous therapeutic and diagnostic potential. Over the past two decades, there has been a steady rise in the number of commercially available nanoparticle therapeutics.

Liposomes became used as drug delivery systems, because of their circumscribed inner volume which can be used to contain and transport a drug to its target site of action. Chemical surface modifications, such as poly ethylene glycol PEG , have been developed to increase bioavailability. Limiting factors to their successful use as carriers remain, especially concerning their stability in biological environments and their ability to transport drugs across biological barriers.

Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

In the following sections, we briefly highlight the advantages of nanoparticles which make them suitable for integration into siRNA technology. A broad overview is also provided of almost all delivery strategies that have been attempted to date, underlining the eventual limitations and pitfalls. Nanoparticles are physical entities with a characteristic length of 1— nm. They can be considered as a unit in term of physicochemical properties. Nanoparticles can be categorized as organic or inorganic according to the bulk constituent materials in their structure.

Classifications based on size, shape, surface, structure, and chemical behavior can be devised. Overall, what is particularly attractive about nanostructures is their surface to volume ratio. Nanosized entities enable a very limited volume to provide an enormous surface area for transport, chemical reactions, and interaction with biological systems. These features, combined with enhanced permeability to biological barriers, make nanoparticles the basis of nanomedicine and a powerful and promising tool in the design of new diagnostic and therapeutic devices.

Interest in nanoparticles has consistently grown in every branch of medicine. It has been driven by the potential of nanoparticles to achieve a high therapeutic index and corresponding clinical success with improved patient compliance and reduced side effects. They may be capable of improving bioavailability and reducing the frequency and dosage needed for currently used drugs.

Residence time in the circulation, maximal tolerated dose, and selectivity are the most important factors to consider when determining the optimal dosage. It is important that the nanomaterial carrier is able to be eliminated harmlessly from the body in a reasonable period of time after releasing its cargo, and, in case of theranostic nanoparticles, accomplishing a desired diagnostic function.

In the body, the drug loaded in nanoparticles is usually released from the matrix by diffusion, swelling, erosion, or degradation. Two kinds of targeting strategies are possible, ie, passive targeting and active targeting. Passive targeting does not require surface modification of the nanoparticles, and is achieved using the unique pathophysiology of diseased cells and controlling particle mobility through human tissues, according to particle size and route of administration. On the other hand, active targeting can be achieved by surface modification of nanoparticles and their functionalization with ligands or antibodies that can recognize and bind to complementary molecules or receptors found on the surface of specific cells.

A further advance in drug delivery is represented by the multistage delivery system, 80 which provides more accurate spatiotemporal targeting with efficient cargo release. Nanoparticles suitable for multistage drug delivery systems are hybrid nanostructures for instance, with a core-shell structure designed to reach injured tissues in a selective manner. These features, combined with conventional targeting against upregulated cell surface antigens, allow precise spatiotemporal drug delivery in selected areas of the host system.

In this sense, nanoparticles are designed to be part of a delivery system and it is necessary to consider the overall behavior of such systems, ie, carrier, payload, and host system properties, as well as mutual interactions. In a gene delivery context, a key feature of the carrier is the ability to protect the gene payload until it reaches the target site, with no modification in molecular structure of the gene or its biochemical activity. This review explores strategies for siRNA delivery by nanoparticles.

The pursuit of gene delivery has been undertaken using viral-based 84 and nonviral-based 85 , 86 vectors. These topics have already been extensively reviewed, 87 , 88 so are not discussed in depth in this paper. Nanoparticle features in drug delivery are likely to overcome a number of issues concerning therapeutic applications of siRNA. The challenge of effective and nontoxic delivery is a crucial point and remains the most significant barrier to therapeutic application of siRNA technology.

As discussed above, slow, sustained, and controlled release could be helpful for decreasing the frequency of treatment and lead to more effective therapies, especially in siRNA therapeutics. In order to achieve effective siRNA delivery, several strategies have been studied. Leaving apart the viral vector delivery setup, we consider here the chemical modifications of siRNA, ligand-based targeted or conjugated siRNA, polymers, cationic and neutral liposomes, sensu stricto nanoparticles, and combined approaches.

A short description and a few examples of each of these strategies is presented see Table 2 , and a more detailed revision can be found elsewhere. Various chemical modifications have been introduced to increase the in vivo metabolic stability of siRNA molecules less than 10 nm in size. Examples of such modifications and their advantages without affecting the efficiency of RNAi are listed below and have been reviewed extensively elsewhere: These chemical modifications, added to the sugars, backbone, or bases of dsRNA, improve intravascular stabilization and are able to reduce activation of the innate immune response, without significant loss of RNAi activity.

These can be useful for improving resistance to degradation, and also for introducing targeting or conjugating ligands less than 10 nm in size, such as peptides and aptamers. Indeed, the smallest siRNA nanoparticles derive from direct conjugation of small molecules, peptides, or polymers to the sense strand of siRNA. Based on the additional features attributed to siRNA molecules, these further modifications can be classified as ligand-targeted affecting target specificity or ligand-conjugated mostly affecting stability.

In particular, it increases binding to serum albumin, with consequent improved biodistribution in certain target tissues, eg, the liver. The improvement in cellular uptake is mediated by in vivo interaction and incorporation into low-density and high-density lipoproteins. Cholesterol-modified siRNA are capable of silencing apolipoprotein B targets in the mouse liver and jejunum, and of ultimately reducing total cholesterol levels.

These interact with high-density and low-density lipoprotein receptors, enhancing delivery into the liver and gene silencing in vivo. Moreover, albumin serves as a primary carrier for siRNA conjugated to medium-chain fatty acids. Many promising Phase II trials have been reported in this regard, with encouraging safety and efficacy results.

The folate receptor is a membrane glycoprotein over-expressed in several human tumors, including ovarian, colorectal, and breast cancer, but is minimally present in normal tissues. This feature makes it an attractive target for drug delivery. Folate-mediated targeting has several advantages, including the small size of folic acid, its lack of immunogenicity, convenient availability, and easy chemical conjugation. Like the folate receptor, the transferrin receptor is a glycoprotein frequently overexpressed by tumor cells. The intracellular routing of transferrin receptor-mediated endocytosis has been well characterized, and this molecule is easily available.

For this reasons, transferrin receptor-mediated delivery has been extensively explored in a variety of targets, including tumors, endothelial cells, and the brain. Antibodies have also been used extensively in the treatment of cancer. Because of their high specificity and affinity for cancer cell antigens, they have the capacity to induce an immune response against target cancer cells. Several studies have demonstrated antibody-targeted delivery of drugs and nucleic acids. Antibodies or their fragments can be used as targeting agents for nanoparticles. Among the successful in vivo demonstrations, are antibody-protamine fusions that bind siRNA.

For instance, prostate-specific membrane antigen is a cell surface receptor usually detected at abnormal levels in prostate cancer cells and in tumor vascular endothelium, and has been proposed for targeting siRNA to these sites. Delivery of siRNA mediated by aptamers is promising, albeit limited by some challenges that need to be investigated further, in particular, the pharmacokinetics and biodistribution of these molecules.

To increase in vivo transfer, siRNA can be conjugated with other molecules, eg, peptides or PEG, which are able to overcome steric hindrance, or linked with drugs or nucleic acids and nanoparticles. Cell-penetrating peptides, protein transduction domains, and membrane translocation sequences are counted among the peptides.

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Overall, these mechanisms result in increased target gene knockdown in vitro and in vivo. Polymers are organic macromolecules that protect RNA from degradation and facilitate its sustained delivery to tissues. Many different types of polymers, varying in size, chemistry, and pharmacological properties, can be considered. Cationic polymers 62 , 87 , 88 , — can be useful as transfection agents, given that they can bind one or more large nucleic acids, package them reversibly into stabilized nanoparticles, and protect them against a degrading bioenvironment.

These vectors avoid enzymatic degradation by nucleic acids by forming condensed complexes along with targeted delivery to cells and tissue. Cationic polymers are generally divided into synthetic and natural polymers, with a linear or branched structure. Synthetic polymers include branched or linear polyethylenimine, poly-L-lysine, and cyclodextrin-based polycations. On the other side, natural cationic polymers include chitosan, atelocollagen, and cationic polypeptides. Another advantage of cationic polymer-based delivery is the ease with which siRNA and polymer complexes can be formulated due to their opposite charges.

In fact, cationic polymers usually form a complex with negatively charged siRNA upon simple mixing. Despite these advantages, appreciable cytotoxicity both necrosis and apoptosis is a major problem with the use of these compounds. These molecules can be defined by the morphology and polymer composition in their core and corona 73 , Figure 2. The therapeutic load in this case siRNA is usually conjugated to the surface of the nanoparticle or encapsulated and protected inside the core. Delivery systems composed of these compounds can be engineered to enable controlled or triggered release of therapeutic molecules.

For example, PEG allows steric stabilization or prevention of protein absorption. The final result is avoidance of opsonization and rapid clearance from bloodstream due to phagocytosis in the liver or spleen. As described, this delivery strategy combines the protective effect and loading capacity of the polymer with the specificity of an active targeting strategy through chemical molecular conjugation. During the past few years, several papers have reported delivery of nucleic acids mediated by cationic polymer carriers , and described the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport.

Also important in the field of delivery systems based on organic molecule carriers are lipid vectors, such as liposomes and cationic lipids. Liposomes are compounds characterized by a phospholipid bilayer and an aqueous core. They can be created from single or multiple types of lipid. Unilamellar and multilamellar liposomes are commonly used as vehicles for delivery of pharmaceuticals. Liposomes interact with siRNAs to form complexes stabilized by electrostatic interactions. Liposomes tend to accumulate in the reticuloendothelial system, leading to rapid clearance by the liver and a short half-life in serum, so require either continuous infusion or frequent administration.

Liposomes also lack target tissue specificity and have reduced access to other tissues. A possible approach to circumvent this problem is to develop sustained-release polymer formulations. It is also possible to construct amorphous structures with liposomes, in which lipids and nucleic acids alternate with each other. The additional flexibility of these molecules can optimize the physical and chemical properties of the nanoparticle. To avoid the potential toxicity associated with other delivery systems, neutral liposomes 30—40 nm in diameter which encapsulate siRNA are commonly being used.

Unilamellar liposomes, such as dioleoyl phosphatidylcholine, , with a hydrophilic core and a hydrophobic surface are able to protect siRNA from degradation by surrounding endonucleases and can enhance internalization through membrane fusion or receptor-mediated endocytosis.

Cationic lipids — nm in size are able to protect siRNA from degradation by nucleases and increase the circulating half-life and uptake by cells. Several cationic lipids have been synthesized for formulation with siRNA, and have been evaluated in preclinical studies or animal models. However, they show significant cell toxicity and elicit hypersensitivity reactions in vivo.

In particular, liposomal nanoparticles comprise electrostatic complexes of nucleic acids and cationic lipids, such as dioleoyl trimethylammonium propane and N,N-dimethylaminopropane carbamoyl cholesterol. A modern class of biodegradable solid lipid nanoparticles has also been developed.

These nanoparticles are used to incorporate various drugs or imaging agents, with the benefit of using physiological and nontoxic lipids that remain solid at body temperature. This strategy was used successfully to protect nonhuman primates from lethal Ebola virus infection.

Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

Finally, in very recent work by Lobovkina et al solid lipid nanoparticles were developed for sustained in vivo siRNA delivery in a mouse model. Even in this case, preparing nanoparticles from physiological lipids resulted in excellent biocompatibility, minimal toxicity, and less cost when compared with polymeric carriers. Nanoparticles and microspheres have also been developed as gene delivery vehicles.

These are promising strategies because they afford improved siRNA delivery and stability, with minimal toxicity in animal models. On the other hand, direct injection of synthetic oligonucleotides into solid tumors can be done for some neoplasms, such as mesothelioma by intrapleural injection , ocular tumors, brain tumors, and sarcomas, and could reduce or eliminate off-target effects. Using this approach, oligonucleotides are sequestered in various kinds of nanoparticles to protect them from degradation and to direct them to appropriate tissues.

The idea behind such an approach is that the nanoparticle is seen merely as a carrier, and thus the functions of the vector are to protect, stabilize, and transport the gene material, with the minimum of interaction. Here we give an overview of the main nanoparticles proposed for siRNA delivery and investigation, starting from inorganic crystals, noble metal nanoparticles, and other nanostructures see Table 3. Quantum dots are semiconducting inorganic crystals with superior photostability and tunable optical properties for an extensive selection of nonoverlapping colors.

These features make quantum dots basically useless in designing delivery systems, but they are a powerful tool in cancer targeting, imaging in living animals, and investigation of pathophysiology in tumor tissue. Incorporation of siRNA into gold nanoparticles was first accomplished by Oishi et al. To overcome such problems, new delivery strategies based on porous silicon 80 have been developed.

Multistage delivery systems show promise for meeting the challenges of targeting and overcoming biological barriers. Numerous, chemically different, core-shell nanoparticles with cationic cores and variable shells have been synthesized and tested for intracellular siRNA delivery. In general, this strategy enables tools for nanoparticle delivery to be equipped with the necessary targeting moieties, such as antibodies, aptamers, and small peptides, for direct delivery and improved specificity. These nanoparticles have been engineered to act in a depot manner, resulting in slow, sustained, and targeted release of siRNA.

The majority of biodegradable formulations of this kind have used polymeric materials in which siRNA is incorporated in a polymeric core. Researchers have recently constrained siRNA between a cationic core composed of DOTAP and an outer lipid bilayer of 1,2-distearoyl-sn-glycerophosphoethanolamine-N- polyethylene glycol and egg phosphatidylcholine obtaining a bloodstream circulation time of up to 20 hours after injection. A novel approach has been proposed by Tanaka et al involving a multistage delivery system composed of two biodegradable and biocompatible carriers.

The first-stage carriers are mesoporous, microscale, biodegradable silicon particles that enable loading and release of the second-stage nanocarriers, ie, dioleoyl phosphatidylcholine nanoliposomal siRNA. In vivo mouse models provided the first evidence that single administration of multistage siRNA-dioleoyl phosphatidylcholine delivery resulted in sustained in vivo gene silencing for 3 weeks, with a significant antitumor effect in two orthotopic mouse models of ovarian cancer, and no observable concurrent toxicity.

Nanoparticle-based drug delivery still has some limitations, and the barriers and obstacles encountered by nanoparticle-siRNA complexes are schematized in Figure 3 adapted from Whitehead First of all, these vehicles must reach and enter target cells. The biological barriers encountered are the mucosa and the cellular and humoral arms of the immune system. These molecules tend to be sequestered by negatively charged serum proteins in the bloodstream. Moreover, immune cells can collect them via opsonization.

Obstacles of Nanoparticles-based siRNA delivery in vivo. After administration into blood circulation the siRNA-nanoparticles A must avoid rapid degradation by plasma components eg, cellular and humoral arm of the immune system and sequester by negatively charged serum protein. C To reach the target cells they must overcome the capillary endothelium through an extravasation process and D overcome the extracellular matrix ECM: E Furthermore these particles must be taken up into the cells, usually bound to cellular receptors and transported into the cytoplasm through a receptor mediated endocytosis process.

Nanoparticles can be engineered in terms of size, surface functionalization, and core structures to overcome these barriers. Indeed, addition of PEG or other hydrophilic conjugates to the surface of a delivery vehicle can reduce protein sequestration, help in evading the immune system, provide steric stabilization, and protect against the effects of the surrounding microenviroment. These nanocomplexes generally undergo rapid clearance. Thus, the optimal diameter of nanoparticles for intratumoral delivery, avoidance of the reticuloendothelial system, and renal clearance should be in the range of 5— nm.

Some tumor characteristics can be exploited for drug delivery. The enhanced permeability and retention effect takes advantage of the abnormal neovascularization typical of many cancers.

Introduction

The blood vessels are either disrupted or not fully formed, allowing molecules of a certain size to be retained to a greater extent in abnormal tissues than in healthy ones. It has been shown that therapeutic nanoparticles can accumulate in specific target tissues as a result of this effect, potentially allowing for drug release over a longer period of time than for the same drug administered as a conventional preparation.

Conversely, limitations can arise from the intricate tumor microenvironment and interfere with achieving therapeutic concentrations of these molecules. The higher interstitial fluid pressure found in solid tumor tissue could prevent diffusion of nanoparticles. The molecules must also face the extracellular matrix, and often the desmoplastic process in cancer creates difficulties in this respect.

Once inside the target cells, the nanoparticles must enter the intracellular trafficking pathway. Many nanoparticles enter cells through endocytosis, and the endosomal vesicles containing RNAi must escape the late endosomal phase in the lysosome that degrades RNA. Fusogenic lipids, fusogenic peptides, photosensitive molecules, pH-sensitive lipoplexes, and pH-sensitive polyplexes are some of the mechanisms used to improve endosomal escape.

Nanoparticles have often been reported to have systemic toxicity, mostly in the liver. In the oncology field, given the heterogeneity of tumors, development of resistance seems inevitable. Furthermore, some cancers could have inherent resistance to some types of RNAi, owing to factors such as ethnicity, somatic mutations, altered RNAi processing machinery Dicer, Drosha, RNA-induced silencing complex , and germline single nucleotide polymorphisms. The clinical utility of RNAi is still a matter of debate.

There has been increasing interest in harnessing this versatile and multifaceted mechanism as a novel pharmacological approach to the treatment of human disease. As has already happened in other developing human therapeutic fields, including gene and antibody therapy, there has been a cooling off after initial enthusiasm.

This was driven by a realistic understanding of the various complex milestones that needed to be reached for efficient RNAi-based therapy, before eventual approval for use in human therapy. This trend is now reversing as a result of the excitement generated by the advent of nanomedicine as a potential way of overcoming the challenges in this field.

In recent years, there has been an increase in the numbers of preclinical and clinical RNAi-based trials being undertaken. Early proof-of-principle studies in animal models have strengthened the usefulness of RNAi as specific and powerful inhibitors of gene expression without significant toxicity. Translation of animal studies has in some cases progressed to early human trials. In particular, the research interest is strongest when the aim is targeting genes with single nucleotide polymorphism mutations, as found in dominant-negative disorders, genes specific for pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases.

In these cases, RNAi could represent the only entities providing an opportunity for a potent and specific approach. The relevant clinical trials have addressed many kinds of disease, including retinal degeneration, dominantly inherited brain and skin diseases, viral infections, respiratory disorders, cancer, and metabolic disease. However, large-scale Phase III clinical trials and regulatory approvals remain distant. Use of RNAi for respiratory tract and neurological disorders, metabolic disease such as hypercholesterolemia , and hepatic cancer has been widely revised in recent times.

Of note, a recent study has showed that the efficacy of antivascular endothelial growth factor siRNA in the eye is not due to specific gene silencing, but is actually caused by nonspecific stimulation of the TLR-3 pathway, which can reduce angiogenesis. Another important success has been achieved with a formulation targeting the nucleocapsid N gene of respiratory syncytial virus, a major cause of respiratory disease in infants and young children. In July , the complete data from a Phase II study in adult lung transplant patients naturally infected with RSV were reported, documenting the significant antiviral efficacy, safety, and tolerability of this formulation.

This study aims to repeat and extend the results already seen in this patient population. No adverse events were reported during the trial or during a 3-month washout period. This study was an example of siRNA application in a clinical setting to target a mutant gene or a genetic disorder, and the first to use siRNA targeted to human skin. Recent reports have discussed other RNAi clinical trials in detail. Although many hindrances remain for applying these technologies in the treatment of cancer, early clinical results are exciting, and suggest that we are moving forward quickly and also highlight how far we have already come.

An example of the clinical feasibility of siRNA application for cancer therapy is represented by chronic myeloid leukemia. This abnormality is due to fusion between the Abelson Abl tyrosine kinase gene at chromosome 9 and the breakpoint cluster Bcr gene at chromosome 22, resulting in the chimeric oncogene Bcr-Abl and a constitutively active Bcr-Abl tyrosine kinase implicated in the pathogenesis of chronic myeloid leukemia.

On the basis of these observations, an attempt was made to exploit RNAi for inhibition of tumor-specific genes to hit cancer cells in a selective manner. To this end, the Bcr-Abl clinical trial addressed safety and efficacy issues that have now been published. However, effective knockdown of the target gene in circulating leukemic cells was difficult to assess due to concomitant treatment.

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