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Background

Two fundamental difficulties in the delivery of drugs to treat central nervous system (CNS) diseases are the systemic delivery of therapeutics across the bloodbrain-barrier (BBB), and the targeting of drugs to specific tissues or cells within the brain. With the advent and promise of RNA-based therapeutics that utilize RNA interference (RNAi) to trigger specific silencing of genes within diseased tissues, the necessity to surmount such obstacles has become even more urgent.

Objective

Most pre-clinical and clinical studies on delivery of RNAi to the CNS have utilized invasive, intra-cerebral delivery of RNA to the targeted tissue. Thus, methods need to be developed to facilitate delivery of therapeutically significant quantities of RNA to the CNS via the systemic route, and to elicit clinically significant RNAi effects within the CNS tissues.

Methods

Cell-penetrating-peptides (CPPs) are ‘molecular delivery vehicles’ that can traverse cell membranes and co-transport peptides or polynucleotides. The present invention examines 1) the utility of CPP-RNA duplexes for delivery of RNA to CNS tissues and, 2) cell-mediated release of the RNA payload once the CPP-RNA duplex is internalized by the CNS cells.

Conclusions

The invention and embodiments listed therein outline molecular tools that can be adapted for non-invasive, systemic delivery of therapeutic RNA to the CNS in a future clinical setting.

RNA interference (RNAi), an effective technique for regulating/silencing specific genes, can be applied to treat various diseases. Multiple clinical trials using RNAi are ongoing and molecular imaging can serve as a powerful tool in RNAi-based therapies. This brief review will highlight the current progress on in vivo imaging of RNAi delivery and silencing effects. Incorporation of suitable molecular imaging techniques into future RNAi-based clinical trials will provide more pieces of the puzzle, thus facilitating the transformation of RNAi into a powerful therapeutic modality in the clinic.

Within the past two decades we have become increasingly aware of the roles that RNAs play in regulation of gene expression. The RNA world was given a booster shot with the discovery of RNA interference (RNAi), a compendium of mechanisms involving small RNAs (less than 30 bases long) that regulate the expression of genes in a variety of eukaryotic organisms. Rapid progress in our understanding of RNAi-based mechanisms has led to applications of this powerful process in studies of gene function as well as in therapeutic applications for the treatment of disease. RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers. To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate. This article summarizes our current knowledge about the mechanisms and applications of RNAi.

Since the discovery that the triggers for RNA interference (RNAi), small interfering RNAs, could mediate silencing in mammalian cells without triggering a toxic response, RNAi has become the standard tool for sequence-specific knockdown of gene expression in molecular biology. This is due in part to the development of methods for promoter-based expression of RNAi triggers that can mediate stable silencing in mammalian cells. Numerous systems with slightly different characteristics exist, but despite incredible progress in a field that moves very rapidly, challenges still remain. The biggest challenge is to successfully and safely apply RNAi in vivo. Aside from potential issues of delivery, which is one of the most important considerations, successful application of short hairpin RNAs (shRNAs) in vivo requires expression systems that yield potent and specific knockdown of the target in the absence of toxicity. With a couple of exceptions, the current systems available for shRNA expression have not generally resulted in unexpected toxicities, while still providing strong knockdown of the intended targets; however, we do not know enough about how sequence-specific off-target effects will affect various cell and tissue types, or to what extent ectopic expression of RNAi triggers will perturb the endogenous RNAi mechanisms.

RNA interference (RNAi) is a collection of small RNA directed mechanisms that result in sequence specific inhibition of gene expression. The notion that RNAi could lead to a new class of therapeutics caught the attention of many investigators soon after its discovery. The field of applied RNAi therapeutics has moved very quickly from lab to bedside. The RNAi approach has been widely used for drug development and several phase I and II clinical trials are under way. However, there are still some concerns and challenges to overcome for therapeutic applications. These include the potential for off-target effects, triggering innate immune responses and most importantly obtaining specific delivery into the cytoplasm of target cells. This review focuses on the current status of RNAi-based therapeutics, the challenges it faces and how to overcome them.

Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients.1,2 Fire et al. first demonstrated that long, double stranded RNAs mediate RNAi in Caenorhabditis elegans,3 and Elbashir et al. opened the pathway to the use of RNAi for human therapy by showing that small interfering RNAs (siRNAs: ca. 21 base pair double stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response.4 We are currently conducting the first-in-human Phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumor biopsies from melanoma patients obtained after treatment reveal: (i) the presence of intracellularly-localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is a first for systemically delivered nanoparticles of any kind), and (ii) reduction in both the specific mRNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) when compared to pre-dosing tissue. Most importantly, we detect the presence of an mRNA fragment that demonstrates siRNA mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. These data when taken in total demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action.

With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising biotherapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near.

RNAi interference (RNAi) is a powerful gene silencing technology that has immense potential for treating a vast array of human ailments, for which suppressing disease-associated genes may provide clinical benefit. Here, we review the development of RNAi as a therapeutic modality for neurodegenerative diseases affecting the central nervous system (CNS). We overview promising preclinical data for the application of RNAi in the CNS and discuss key challenges (e.g. delivery and specificity) that remain as these approaches transition to the clinic.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

RNAi technology has brought a new category of treatments for various diseases including genetic diseases, viral diseases, and cancer. Despite the great versatility of RNAi that can down regulate almost any protein in the cells, the delicate and precise machinery used for silencing is the same. The major challenge indeed for RNAi-based therapy is the delivery system. In this review, we start with the uniqueness and mechanism of RNAi machinery and the utility of RNAi in therapeutics. Then we discuss the challenges in systemic siRNA delivery by dividing them into two categories--kinetic and physical barriers. At the end, we discuss different strategies to overcome these barriers, especially focusing on the step of endosome escape. Toxicity issues and current successful examples for lipid-based delivery are also included in the review.

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells.

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

Through a mechanism known as RNA interference (RNAi), small interfering RNA (siRNA) molecules can target complementary mRNA strands for degradation, thus specifically inhibiting gene expression. The ability of siRNAs to inhibit gene expression offers a mechanism that can be exploited for novel therapeutics. Indeed, over the past decade, at least 21 siRNA therapeutics have been developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders. Like other biological drugs, RNAi-based therapeutics often require a delivery vehicle to transport them to the targeted cells. Thus, the clinical advancement of numerous siRNA drugs has relied on the development of siRNA carriers including biodegradable nanoparticles, lipids, bacteria, and attenuated viruses. Most therapies permit systemic delivery of the siRNA drug, while others use ex vivo delivery by autologous cell therapy. For some of the drugs, advancements in bioengineering and nanotechnology have led to improved control of delivery and release of the siRNA. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules. Here, we provide an overview of siRNA therapeutics in clinical trials, including their clinical progress, the challenges they have encountered, and the future they hold in the treatment of human diseases.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip–mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

In recent years RNA interference (RNAi) has rapidly become the most widely used tool for gene knockdown due to its high specificity and potency. RNAi is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. In the past decade, hundreds of molecular targets have been identified for their roles in pain modulation. But most molecular targets are not readily druggable with small molecules. RNAi represents a therapeutic approach applicable to these non-druggable targets. There is a rapid increase in the number of studies that use small interfering RNAs (siRNAs) to validate new targets for pain regulation. In this review, we will discuss these pain-related RNAi studies (Table 1). We will also compare the advantages and disadvantages of RNAi with antisense knockdown (Table 2), because antisense oligodeoxynucleotides have been extensively used for target validation in pain research. Although in vivo delivery of siRNA remains to be a challenge, RNAi has a great potential to become a major therapeutic tool for pain management.

Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

Ewing's sarcoma tumors are associated with chromosomal translocation between the EWS gene and the ETS transcription factor gene. These unique target sequences provide opportunity for RNA interference(i)-based therapy. A summary of RNAi mechanism and therapeutically designed products including siRNA, shRNA and bi-shRNA are described. Comparison is made between each of these approaches. Systemic RNAi-based therapy, however, requires protected delivery to the Ewing's sarcoma tumor site for activity. Delivery systems which have been most effective in preclinical and clinical testing are reviewed, followed by preclinical assessment of various silencing strategies with demonstration of effectiveness to EWS/FLI-1 target sequences. It is concluded that RNAi-based therapeutics may have testable and achievable activity in management of Ewing's sarcoma.

Tribolium resembles C. elegans in showing a robust systemic RNAi response, but does not have C. elegans-type RNAi mechanisms; insect systemic RNAi probably uses a different mechanism.

Background

RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects.

Results

We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans.

Conclusion

Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

Author Summary

RNAi interference pathways play fundamental roles in eukaryotes and provide important methods for the analysis of gene function. Occasionally RNAi has been lost, precluding its use as a tool, as well as raising the question of what forces could lead to loss of such a key pathway. Genomic and functional studies previously showed that within trypanosomatids protozoans RNAi was absent in both Leishmania major and Trypanosoma cruzi. The genome of L. braziliensis, a member of the early diverging Leishmania subgenus Viannia, retained key genes required for RNAi such as an Argonaute. We demonstrated that in fact L. braziliensis shows strong RNAi activity with reporter and endogenous genes affecting flagellar function. These data suggest that RNAi may be productively applied for functional genomic studies in L. braziliensis. We mapped the evolutionary point at which RNAi was lost in lineage leading to Leishmania and Crithidia, and establish that RNAi must have been lost at least twice in the trypanosomatids, once on the lineage leading to T. cruzi and independently following the divergence of the Viannia subgenus from other Leishmania species. Lastly, we discuss hypotheses concerning the forces leading to the loss of RNAi in Leishmania evolution, including viral invasion, increased genome plasticity, and altered virulence.

Worldwide, approximately one and a half million new cases of lung cancer are diagnosed each year, and about 85% of lung cancer are non-small cell lung cancer (NSCLC). As the molecular pathogenesis underlying NSCLC is understood, new molecular targeting agents can be developed. However, current therapies are not sufficient to cure or manage the patients with distant metastasis, and novel strategies are necessary to be developed to cure the patients with advanced NSCLC.

RNA interference (RNAi) is a phenomenon of sequence-specific gene silencing in mammalian cells and its discovery has lead to its wide application as a powerful tool in post-genomic research. Recently, short interfering RNA (siRNA), which induces RNAi, has been experimentally introduced as a cancer therapy and is expected to be developed as a nucleic acid-based medicine. Recently, several clinical trials of RNAi therapies against cancers are ongoing. In this article, we discuss the most recent findings concerning the administration of siRNA against polo-like kinase-1 (PLK-1) to liver metastatic NSCLC. PLK-1 regulates the mitotic process in mammalian cells. These promising results demonstrate that PLK-1 is a suitable target for advanced NSCLC therapy.

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites.

RNA interference (RNAi) is a post-transcriptional pathway in which double-stranded RNA (dsRNA) triggers the degradation of complementary mRNA in the cytoplasm of eukaryotic cells. In plants and in some animals, including Caenorhabditis elegans, initiation of RNAi in one cell can lead to sequence-specific RNA silencing in another cell, a phenomenon referred to as non-cell-autonomous RNAi. Until recently, this phenomenon had not been observed in mammalian cells. Here, we review emerging data demonstrating that non-cell-autonomous RNAi occurs in cultured mammalian cells. We discuss possible mechanisms for the transfer of RNAi between mammalian cells and highlight the implications of this phenomenon for the development of in vivo cell-based RNAi delivery.

RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.

RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for hepatocellular carcinoma and cirrhosis. Current treatment regimens, which include interferon-α and nucleoside/nucleotide analogs, are only partially effective and new treatment methods remain an important objective. Harnessing the RNA interference (RNAi) pathway to achieve post-transcriptional silencing of rogue genetic elements is an exciting avenue for development of novel therapeutic strategies. The specific and potent suppression of HBV gene expression and replication is an attractive option as a novel and effective approach for the treatment of chronic HBV infection. However, despite significant and rapid progress, existing RNAi technologies require further refinement before clinical applications can be realized. Here, we review current efforts aimed at improving the efficiency of anti-HBV RNAi-based delivery systems, at limiting the toxicities associated with RNAi modalities and at preventing reactivation of viral replication. We discuss the progress towards clinical implementation of anti-HBV RNAi therapies.