Over the past 20 years, RNA interference (RNAi) technology has provided a new regulatory paradigm in biology. This technique can efficiently suppress target genes of interest in mammalian cells. Small non-coding RNAs play important roles in gene regulation, including both in post-transcriptional and in translational regulation. For in vivo experiments, continuous development has resulted in successful new ways of designing, identifying, and delivering small interfering RNAs (siRNAs). Proof-of-principle studies in vivo have clearly demonstrated that both viral and non-viral delivery methods can provide selective and potent target gene suppression without any clear toxic effects. There are also the persistent problems with off-target effects (OTEs), competition with cellular RNAi components, and effective delivery in vivo. Although recent researches and trials from a large number of animal model studies have confirmed that most OTEs are not dangerous, other important issues need to be addressed before RNAi-based drugs are ready for clinical use. Currently, RNAi may be harnessed as a new therapeutic modality for brain diseases. Finally, there are already several RNAi-based human clinical trials in progress. It is hoped that this technology will have also effective applications in human central nervous system (CNS)-related disease.
- RNAi therapy
- neurodegenerative disease
During developmental stage and in response to internal and external cellular stresses, small RNA molecules regulate gene expression . Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs . In most eukaryotic cells, RNA interference (RNAi) is a regulatory mechanism using small double-stranded RNA (dsRNA) molecules to direct homology-dependent control of gene activity [3, 4]. Small size [20–30 nucleotide (nt)] non-coding RNAs and associated proteins regulate the expression of genetic information . The discovery of RNAi phenomenon widened our understanding of gene regulation and revealed related pathways in small RNAs . As it processes, RNAi has been finding widespread in plants  and animals . Each small RNA associates with an Argonaute (AGO) family protein to form a sequence-specific complex. After then, gene-silencing ribonucleo-protein complex with specificity conferred by base pairing between the small RNA (guide RNA) and its target mRNA . The pathway is well known as the RNA-induced silencing complex (RISC), which gives a target mRNA silencing by degradation or transcriptional regression . Small interfering RNAs (siRNAs) loaded into RISC are double-stranded, and AGO-2, which having an active catalytic domain in human, cleaves and releases the “passenger” strand. RISC is activated with a single-stranded “guide RNA” molecule to impose the specificity recognizing the target by intermolecular base pairing .
MicroRNAs (miRNAs) are other endogenous substrates for the RNAi machinery, but the cellular origins of miRNA and siRNA are distinct. miRNAs are derived from the genome, whereas siRNAs may be endogenous or arise through viral infection or other exogenous sources . Typically, miRNAs are initially expressed in the nucleus with a transcript as long as primary miRNA (pri-miRNA), and the transcripts are at least over 1000 nt. Pri-miRNAs are processed by the microprocessor complex (histone deacetylase proteins) consisting in Drosha-DGCR8 [DiGeorge critical region 8 (a double cysteine-ligated Fe (III) heme protein)—DGCR8] in the nucleus [10, 11]. They are cleaved in the nucleus into 60–70 base pair (bp) hairpins, which are consisted in single-stranded 5′- and 3′-terminal overhangs and about 10-nt distal loops . In cytoplasm, the loop is further processed by the RNAse III Dicer, and one strand is loaded onto RISC. The mature miRNAs bind to the 3′ UTR of target mRNAs and then degrade the target . Despite their differing origins, these RNA processing pathways converge once either type of RNA assembles into the RISC.
With development of an efficient delivery system in various diseases, RNAi has been an emerging therapeutic approach for
This chapter introduced two of main themes. The first is the possibilities of therapeutics using RNAi principles and technique. The second is the challenges with siRNAs or miRNAs specifically in the area of brain disease. In addition, this chapter provided some prospects of siRNAs or miRNAs on disease prognosis, progress, and therapeutics in the present and future.
2. Principles of RNAi therapy
As far as it is true that siRNA has promising benefits, and, concomitantly, siRNA has still some of technological barriers to be widely used in clinical therapy, which generally due to the lack of efficient delivery tools. To success with siRNA therapies, an effective and safe carrier system is required that would overcome the inherent defects of siRNA and achieve maximum gene-silencing effect. There are many approaches that are being developed to achieve the efficient delivery of siRNA. In that, non-viral vectors have advantages of reproducibility, low immunogenicity, and relatively low production cost ; therefore, non-viral vectors made siRNA to be a potential therapeutic and nucleic acid–based drugs, such as plasmid DNAs or antisense oligonucleotides (ASOs) .
2.1. Advantages of RNAi
Theoretically, all disease-associated genes could be amenable to antisense-mediated RNAi suppression. RNAi can be a strategy for silencing of virtually all annotated protein-encoding genes in the human genome in large scale. The high specificity of siRNA lets targeting of disease-specific alleles that differ from the normal allele by only one or few nucleotide substitutions. This high fidelity and specificity of siRNAs are useful for targeting for some oncogenes, too.
The first advantage is the powerfulness of RNAi when compared with other antisense strategies, such as antisense DNA oligonucleotides and ribozymes . It is important fact that the effector molecules work at much lower concentration than any other antisense oligomers or ribozymes, suggesting that RNAi has higher potency. This is a critical point to set therapeutics.
The second is efficacy. The efficacy is generally presented by the half level of maximal inhibition or the value of IC50 against target site. The efficacy level is crucial for determining thermodynamic stability , targeted gene accessibility , or structure  of designed siRNA. For designing siRNA, the most important thing is end stability that is different from each end and is also meaning asymmetry and consistent with selected miRNA . However, to date, our knowledge of siRNA and the selection of targets are incomplete and being explored. The identification of “hyperfunctional” siRNAs, functioning at sub-nanomolar concentration, remains an elusive task.
2.2. Basic strategies for targeting-specific molecules
RNAi can be triggered by two different pathways: (1) a RNA-based approach, where the 21-nt long duplexed siRNA effectors are delivered to target cells, and (2) a DNA-based strategy, where the siRNA effectors are produced by intracellular processing of longer RNA hairpin transcripts . DNA-based strategy is based on short hairpin RNA (shRNA) synthesis in nucleus and transportation to the cytoplasm through miRNA machinery, which subsequently is processed by Dicer. Although the direct use of siRNA effectors is simple and effective way for gene silencing, the effect is transient. Therefore, it is costly for clinical usage due to the need of multiple large-scale application. In contrast, DNA-based RNAi drugs have the potential and stably introduced for application in a gene therapy. In principle, DNA-based RNAi allows a single treatment of viral vector that delivers shRNA genes to the targeted cells/or tissues.
2.3. Delivery routes for targeting
The effective delivery of siRNAs acts to be significant step in accelerating RNAi-based treatments. The instability of RNA and the relatively inefficient encapsulation process of siRNA remain critical issues toward the clinical translation of RNAi as a therapeutic tool. There are several obstacles for extracellular introduction of siRNA to deliver the target. Under normal physiological condition, the introduced molecules ought to have a positive charge to diffuse to cell membrane . It is the simplest way of naked nucleotides or transfecting siRNAs to deliver into cells . Another technique is microinjection and electroporation for direct delivery, but it has higher level of cellular toxicity . The delivery routes can be intra-peritoneal, intra-vascular, intra-muscular, intra-splenic, intra-cranial, and intra-tumoral injection. In addition, siRNAs can be delivered through subretinal, subcutaneous, mucosal, topical application, and oral ingestion to improve delivery . However, these transfection processes should be optimized for siRNA concentration, cell density, and ratio of transfection reagent to siRNA . Carriers for delivery of siRNA with cationic environment surrounding of siRNAs can be liposomes and dendrimers. These carriers reduce the nuclease activity and improve siRNA delivery into cells .
Microsponge is one of the mediators for siRNA delivery. Carrier and cargo combine and self-assemble into nanoscale pleated sheets of hairpin RNA. Subsequently, this complex forms sponge-like microspheres . The complex of siRNA and microsponges consists in cleavable RNA strands, and the stable hairpin RNA converts into working siRNA once cells uptake the complex. Therefore, it can provide a protection for siRNA during delivery and transport it to the cytoplasm. Single microsponge complex can deliver more than half a million copies of siRNA when uptaken into a cell .
2.4. Stabilizing the siRNA delivery
The stability of the siRNA complexes, penetrating into target cells without stimulating immune responses, is one of the limiting factors and the major bottleneck for developing siRNA therapeutic tools. It restricts the delivery of siRNA macromolecular complexes to the desired cell types, tissues, or organs. Usually, siRNAs do not easily penetrate the cellular membrane because of their negative charge and macromolecular size. Manipulation of nucleotide bases is needed to increase stability and protein interactions, which can harness to increase the structural improvement of siRNAs . The delivery systems for siRNA consist of four main methods, namely naked, lipid-based, peptide-based, and polymer-based delivery . Basically, polymer-based methods are similar to lipid-based methods in targeting, except some special triggers, such as temperature, pH, or pulse release .
Initial efforts to improve stability addressed above were focused on incorporating chemical modifications into the sugar backbone or bases of siRNA duplexes . The modified siRNA molecules increased stability, which effectively lowered the dose to achieve measurable and reproducible gene silencing . Several modifications were introduced. The thio (−SH), hydroxyl (−OH), or iodo (−I) can modify bases in specific sites or utilize the pseudouracil base in siRNA, which would augment potency of naked siRNA . There are three most popular chemical modification sites on siRNA structure containing the phosphodiester backbone, ribose 2′-hydroxyl group (R-2′-OH), and ribose ring. Endogenous cellular endonucleases can easily digest phosphodiester bond in RNA backbone . Alternative modification is oxygen bridges of RNA backbone that can be replaced with phosphorothioate, although it would increase toxicity and reduce silencing activity [33, 34]. Another alternative is boranophosphate linkages. These are more nuclease resistant and less toxic compared to phosphorothioate . Phosphonoacetate linkages are other candidates . The linkage is completely resistant to nuclease and is electrochemically neutral when they are esterified [36, 37]. Another modification is 2′-
On the other hand, alterations in sugar compartment of nucleotides reduced flexibility and nuclease sensitivity of siRNA structure [39, 40]. Binding of ribose 2′O into 1′C with methylene bridges, which finally produces oxetane, forms a locked conformation nucleic acid (locked nucleic acid—LNA) .
Generally, liposomes are classified into three classes: multilmellar vesicle (0.5~20 μm), small unilamellar vesicles (25~100 nm), and large unilamellar vesicles (100~500 nm) . Liposomes are developed for passive or active targeting mechanisms in different complexes of liposome and other interacting molecules, namely lipoplex (cationic liposome-pDNA complex), liposome polycationic DNA, mannose liposome, and so on [48, 49]. The siRNA with mannose (Man)-coated liposomes would be useful for treatment of some cancers, especially liver and brain cancers .
Dendrimers are hyper-branched, tree-shaped, and 3-D structures . Dendrimer can utilize broad spectrum, and the broad range of functional groups makes it possible to introduce dendrimers with extensive applications. There are different classes of cationic and anionic dendrimers, such as polyamidoamine (PAMAM), polypropylene imine (PPI), and polyethylene glycol (PEG)-grafted carbosilane . Specific dendritic polymers like PAMAM have been widely utilized in
2.4.3. Cationic polymers
Cationic polymers include chitosan, gelatin, cationic dextran, cationic cellulose, and cationic cyclodextrin and some synthetic biocompatible polyethyleneimine (PEI), PLL, poly(amidoamine)s (PAAs), poly(amino-co-ester), and poly(2-
2.4.4. Cationic peptides
CPPs are cationic peptides. CPPs interact covalently or non-covalently through disulfide or electrostatic–hydrogen interactions with siRNAs . Viral protein (VP22) , MPG (a peptide vector) , amphipathic peptide , and poly-arginine  were reported the same abilities. In addition, small cationic polypeptides (poly His, Lys, and Arg) coat and neutralize siRNA helping to pass through membrane .
For systemic delivery, a targeted nanocarrier-siRNA complex has been used. There are some studies that have experimentally condensed DNA or RNA into cancer-targeted nanoparticles with PEI, PLL, and cyclodextrin-containing polymers . PEI–PEG–arginine–glycine–aspartic acid (RGD) fusion was used to inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) expression . Angiogenesis can be inhibited by downregulation or silencing of VEGFR-2 expression . PEGylation of nanoparticles causes “muco-inert” properties, which enhances diffusion process through mucus and peptidoglycan barriers .
siRNAs can be coupled with aptamers or oligodeoxynucleotide through a disulfide bond. This releases actively into targeted cells siRNAs before cytosolic uptake. Conjugate of aptamer siRNA has suggested a novel therapeutics with widespread applications in medicine .
2.5.1. Competition with endogenous RNAs
In human brain diseases and normal brain development, RNAi potentiates the important role in normal neuronal function, although it is underestimated. When exogenous shRNA is introduced into the neuron, it might be considered whether the RNAi machinery perturbs normal physiologic condition of the system. Bioactive drugs that rely on cellular processing to exert their action face the risk of saturating such pathways and hence perturb the natural system. Sometimes, ectopically introduced RNAi does not trigger the silencing process because siRNA/shRNA activity may depend on the endogenous miRNA to achieve efficient target silencing. Mice that received liver-directed associate adeovirus (AAV)-encoded shRNAs were damaged in liver with dose-dependent manner. Within 2 months, the mice were killed by introducing high doses of AAV-encoded shRNAs. It was interpreted that the liver-specific miRNA was unexpectedly down-regulated by introducing shRNA . The enhanced expression of Exportin 5, the nuclear export component, increased RNAi efficacy, which was shown by competition assay .
2.5.2. Stimulation of innate immune responses
RNAi therapy is importantly considered because of its potential for generating an adverse immune response, particularly in neurodegenerative diseases with affected brain. It has been already known as “heightened state of alert” to start chronic pro-inflammatory signaling cascades . All evolutionary conserved mechanisms aimed at combating against invading viral pathogens . In general, innate immune responses to non-virally delivered siRNAs are mediated by members of the toll-like receptor (TLR) family or by the two different dsRNA-sensing proteins: retinoic acid-inducible gene-1 and dsRNA-binding protein kinase . Certain siRNA sequence motifs invoked TLR7-dependent immune stimulation . The particular sequence motif (5′-GUCCUUCAA-3′) seems to be recognized by TLR7 in plasmacytoid dendritic cells and activates immune responses. The GU-rich regions, so-called “danger motifs,” stimulated innate immune responses and lead to secretion of inflammatory cytokines in a cell type and sequence-specific manner. As siRNA-mediated immune induction seems to rely on endosome-located TLR receptors (TLR7 and TLR8) , the delivery and compartmentalization of the siRNA significantly influence the cellular responses . These interactions can occur during endosomal or lysosomal compartments’ internalization or intracellular release of the siRNA molecule. It has a manner of dose and sequence dependence. Importantly, the chemically modified or nanoparticle-encased siRNA complexes avoid stimulation of immune response.
2.5.3. Suppression of off targets
Harmfulness of RNAi is “OTE.” Genome-wide sequencing analyses have clearly demonstrated that siRNA-treated cells show off-target silencing of a large number of genes . The research result suggests that siRNAs with a 2′-
The OTEs can also derive from non-specific changes in gene expression due to the activation of the interferon response (IR) . The OTEs can change another gene by binding either strand of the shRNA to partially complementary sequences rather than binding to the intended target gene . In case of dsRNA, it can result in a signaling cascade that culminates with the activation of interferon responsive genes and global translational repression . Nevertheless, IR activation was variable among the siRNAs used for each of these studies, and one recent report did not detect IR activation by siRNAs . In mice, injection of naked siRNA did not show detectable induction of an IR in one study while another study showed sequence-dependent induction of innate immunity [79, 80].
3. Applications of RNAi
RNAi has been used to generate model systems to identify novel molecular targets , to study gene function , and to create a new niche for clinical therapeutics . Many researchers reported that siRNAs have successfully been tested in various disease animal models. Recent reports reviewed the therapeutic potential of synthetic siRNAs in various human diseases and disorders .
3.1. Application for therapy with RNAi
Applications, such as gene function analysis, target identification and validation, and therapeutic agents, are the main spots of this new technology . Although RNAi is an efficient technique for
The administration route for siRNA, such as oral or intravenous, is not feasible and not efficiently delivered the siRNA into target cells. A single injection of naked siRNA into the brain parenchyma failed to good efficacy . A study reported that continuous infusion of siRNA into the ventricular CSF success with very high concentration . To penetrate the blood–brain barrier (BBB) and reach the target cells in the interesting site, receptor-specific pegylated immunoliposome (PIG) is used. PIGs encapsulate the plasmid vector–encoding siRNA or shRNA and are administered with peripheral route to the brain. This tool has been tried in brain cancer animal model and successfully worked . Another study showed effective and long-term knock down of endogenous tyrosine hydroxylase (TH) in rodent brain using shRNA-expressing adeno-associated virus (AAV) . There have been many successful
3.2. Application for therapy with RNAi in brain diseases
Many works using RNAi to suppress dominant disease genes have occurred primarily in cell culture models [99, 100]. Allele-specific silencing aims to suppress the disease gene without affecting any other normal genes. The possible therapeutic applications of RNAi for neurological diseases are broad, ranging from acquired diseases, such as viral infections, to purely genetic disorders.
Particularly, one attractive group of candidate diseases for RNAi therapy is the dominantly inherited neurodegenerative diseases, including polyglutamine disorders such as Huntington’s disease (HD) , amyotrophic lateral sclerosis (ALS) , familial Alzheimer’s disease (AD) , and frontotemporal dementia caused by tau mutations . HD has been approaching with animal model mimicking the human disease to provide some therapeutic clues with various ways. In the new preclinical study, single injection of a cholesterol conjugated-siRNA was targeting mutant Huntingtin (mhtt), and, subsequently, the pathologic symptoms containing behavioral dysfunction were improved .
The exciting recent works have taken place
Prion disease is one of the brain diseases that is invariably fatal, and no therapy is available. Once serious damage to the brain has already occurred, clinical symptoms manifest after the untreatable brain damage. Causing this reason, prion disease treatments have aimed not to cure the disease but to slow disease progression . Prion disease is caused by prions, in which a self-replicating, infectious protease resistant form of PrP (termed PrPSc), is the only essential component identified to date. PrPSc multiplies through conversion of the normal cellular PrP (PrPC) . Some reviews are presenting that lentivector-mediated anti-PrPC shRNA expression effectively suppressed prion replication in a murine neuroblastoma cell line, and researchers created chimeric mice using embryonic stem cells, which were transfected with a lentiviral vector carrying an anti-PrPC shRNA. Results showed that the survival time after prion inoculation was markedly prolonged [110, 111].
4. Prospects of RNA therapeutics in CNS disease
The current phamaceuticals required more knowledge to decipher potentials of the RNAi in spite of flourishing future. It is crucial that each disease has not only a unique pattern but also the understanding for pathogenesis relating pathways and activating or inhibiting factors . To introduce the DNA therapeutics into the CNS is much more complicated due to the BBB, which can be only permeable to lipophilic molecules of less than 400 Da . Using human viruses, DNA delivery system has been extensively trailed for over three decades. However, the results have been not satisfactory. Therefore, a critical goal for clinical neuroscience is to develop the efficient RNAi therapy to prevent the neuronal damage . We categorized the neurological disease containing cancers in below sections.
4.1. Genetic neuronal disease-familial neurological disease
The application of siRNA has been advanced in development of various incurable disease therapies, apart from the widespread usage of RNAi in fundamental biological application. Particularly, dominant inherited disorders are major application field. Among familial neurobiological diseases, HD has been tried to lots of therapies based on RNAi and may be beneficial effect from the therapy using siRNA. In the N171-82Q transgenic HD mouse model, a study using shRNA showed a 50–55% decrease in the N171-82Q mRNA when injected to striatum and a complete elimination of mHtt protein inclusions from the neuronal cells . There was also a rescue of motor dysfunctions. siRNAs against the “R6/2 huntingtin (htt) mRNA” reduced brain atrophy and neuronal inclusions in the R6/2 transgenic mouse model . With using a rAAV5 vector and administrating to the striatum, long-term expression of a mHtt-siRNA partially reduced in neuropathology condition .
Besides AAV, there is lentiviral vector that can be applied after onset of symptoms . Using lentivirus vector decreased htt protein expression by up to 35% and altered htt-related pathways but did not reduce cellular viability for at least 9 months after treatment. To enhance cellular uptake of siRNA, cholesterol-conjugated duplexes (cc-siRNA) have been applied to target htt mRNA . Allele-specific targeting of mhtt helped to overcome the side effects of RNAi where ASO or single nucleotide polymorphisms (SNPs) in the mHtt allele have been used to specifically target only the mutant gene product . Intra-cellular antibody fragments bind to abnormal aggregations, and allele-specific siRNA disrupts mhtt gene [120, 121]. Targeting of just three SNPs with five siRNAs covered most of the HD patients in the population studied .
Tuberous sclerosis is a common, dominantly inherited disorder caused by mutations in the tumor suppressor complex-1 (TSC1) or tumor suppressor complex-2 (TSC2) genes . The proteins hamartin (encoded by TSC1) and tuberin (encoded by TSC2) form a complex. This protein complex represses mTOR-S6K-4E-BP signaling pathway . Mutated TSC1 and TSC2 lead to loss of activity resulting in unchecked cell growth and hamartoma formation in the CNS. Recent studies propose that the target may be the GTPase Rheb . RNAi suppression of Rheb might respond the dysregulated cell proliferation in tuberous sclerosis.
Particularly, allele-specific silencing is apt for inherited neurological diseases. DYT1 is the most commonly inherited dystonia . Although the pathogenesis of DYT1 is unclear, several facts make DYT1 a good candidate to explore the therapeutic potential of RNAi . The three nucleotide difference between the wild type and the mutated gene has been enough to allow allele-specific silencing against mutant TA (the mutated protein in DYT1) in cultured cells using
Allelic discrimination has also been demonstrated for superoxide dismutase (SOD) mutations responsible for familial ALS , and also a mutation in an acetylcholine receptor subunit causes congenital myasthenia . In a tau mutation responsible for fronto-temporal dementia, siRNAs can act by discriminating between sequences differing by a single nucleotide .
An important role for RNAi in the brain is also presented for Fragile X syndrome (FXS) in human . FXS is the one of the most common forms of inherited mental retardation caused by mutations in Fragile X Mental Retardation Protein (FMRP), a protein influencing synaptic plasticity . FXS is stemmed from mutations in FMRP and is supported by the involvement of the RNAi process in human neurological disease . Increasing evidences from different studies support the view that FMRP regulates protein translation by regulating RNAi in neurons [128, 129].
4.2. Sporadic neurodegenerative diseases
Neurodegenerative diseases are age dependent, and many of them are inherited. However, non-genetic neurological diseases, such as sporadic AD or migraine, are much more common than diseases due to single-gene mutations.
The most common sporadic neurodegenerative disease, AD, is also the best studied with siRNA therapy. Many studies of AD pathogenesis investigate an essential role for β-amyloid (Aβ) in familial and sporadic forms of AD . Different RNAi strategies have been applied to regulate this pathogenic cascade. Researchers tried by directly silencing of amyloid precursor protein (APP) , by silencing of β-secretase (BACE1) that is one of two proteases required for Aβ production but not essential gene in mice , or by silencing of tau expression that is a component of the neurofibrillary tangles of AD neurons. Therapeutic use of RNAi is now being tested in animal models of AD targeting these proteins.
Migraine, one of the most common neurological disorders, is caused by diminished production of calcitonin gene-related peptide (CGPR) in the trigeminal system. CGPR can protect from migraine attacks . The CGPR-limited animals are normal, but the paroxysmal nature of this disorder necessitates to use promoters for CGPR. From the beginning of the pathogenic cascade, expression of the shRNA targeting CGPR can terminate the growing pain of this disease. This pain alleviating therapy for migraine is limited because of high threshold dose needed for RNAi .
4.3. Motor dysfunction disease
A viral delivery of shRNA was used to achieve a long-term RNAi in the CNS. In some reports, the delivery of shRNA-expressing lentivirus showed a rescue of spinal motor neurons with behavioral and histopathological phenotypes in a mouse model having dominant familial ALS .
Parkinson’s disease (PD) is the second most common neurodegenerative disease. Patient brain of PD is often littered with Lewy body, which is abnormal protein aggregate primarily of alpha-synuclein (α-syn) [135, 136]. parkinsonism is linked to hereditary to a single-point mutation in the α-syn as well as genetic duplication or triplication of the α-syn (SCNA) . The studies targeting the α-syn expression revealed RNAi as a therapeutic approach to PD [30, 137]. To date, conflicting results were reported. Regarding the effectiveness and tolerability, there is a report that nigrostriatal degeneration was detected after depleting the α-syn level in the brain . It can be inferred that RNAi approaches can be used to validate them in genetic and sporadic models of PD.
4.4. Neurovascular disease
RNAi can be applied to cardiovascular and cerebrovascular diseases. Cardiovascular disease results from the progressive occlusion of arteries, and it is most common in a process called atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke . It may be a trigger for the death of cardiac muscle cells or neurons . Although some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis in such cardiac myocytes and brain neurons [140, 141]. RNAi technology may be used to intervene in atherosclerosis or to reduce the damage of heart tissue and brain cells following a myocardial infarction or stroke .
Another vascular disease is an ocular disease. Representatively, there were two RNAi clinical trials. The trials performed direct intra-vitreal injection of siRNAs that are targeting VEGF or the VEGFR to test for the safety and efficacy in ocular diseases . siRNAs, targeting VEGF and VEGFR1, are currently in the early stages of clinical trials. The direct injection approach can also prove its usefulness for the other ocular diseases.
A chemo-resistance or radio resistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity have the potential to increase drug efficacy while reducing toxic effects on untargeted cells (144). Actually, oncogenes expressed at abnormally high levels are attractive targets for RNAi-based therapies against cancers , and such approaches have effectively inhibited tumor growth
In nasopharyngeal carcinoma, hyaluronan receptor (CD44) gene silencing resulted in profound reduction of malignant potential of the cells: tumorigenesis and metastasis of tumors in nude mice [105, 146]. It is also suggested a possible therapeutic effect of direct introduction of siRNA to CD44 into some human solid tumors with high expression of the CD44 gene . Although the role of epidermal growth factor receptor (EGFR) in altering tumor chemosensitivity has not yet been fully elucidated, selectively targeting EGFR supplies the reversal possibility of chemoresistance in many tumor types . Reduction of EGFR expression and increased chemosensitivity to docetaxel are emerging an effective strategy for the sensitization of cancer cells to taxane chemotherapy . siRNA-PG-Amine polyplexes can be systemically delivered to tumors in mice , and siRNA-nanocarrier system can efficiently inhibit expression of a specific gene in tumor cells. Once the intact siRNA molecule moves to the target, the gene of interest gets silenced. The PG-amine-based delivery system actually combines both tumor passive targeting with the sequence selectivity of siRNA .
The limiting point of targeted therapy is alternative pathway compensation by gene amplification. The “synthetic lethality” is proposed idea to overcome the above problem . This concept suggests that two genes may be considered to have a synthetically lethal relationship . When a mutation is existed either of the two genes alone has no effect on cell survival, but when mutations in both genes cell death is triggered at the same time. By genome-wide RNAi library screening, some synthetic lethal molecules have been discovered. Anaphase-promoting complex/cyclosome (APC/C) and polo-like kinase (PLK) are synthetically lethal with the RAS oncogene in colorectal cancer . The STK33 gene is also synthetic lethal interacting with a RAS mutation in multiple cancer cells from different tumor types . Modified EGFR (amplification or truncation) and hyperactivation of AKT play a major role in the development of glioblastoma, one of the extreme malignancies . There are approaches to develop the siRNA delivery efficiency such as the use of dsRNA-binding domain (DRBD) with a TAT peptide transduction domain (PTD) delivery peptide . These facilities are stable and efficient delivery of siRNAs into cells .
Small RNAs and non-coding small RNAs were important discovery for molecular cell biology; these small RNAs have a vital role in gene regulation that can be controlled by RNA interfering technology. Presently, attempts to integrate gene expression profiling and protein interaction mapping are the main research objectives. The proof-of-principle studies
Leung RK, Whittaker PA. RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol Ther. 2005;107:222–239. DOI: 10.1016/j.pharmthera.2005.03.004
Wilson RC, Doudna JA. Molecular mechanisms of RNA interference. Annual Review of Biophysics 2013;42:217–239. DOI: 10.1146/annurev-biophys-083012-130404
Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Advanced Drug Delivery Reviews 2007;59:75–86. DOI: 10.1016/j.addr.2007.03.005
Almeida R, Allshire RC. RNA silencing and genome regulation. Trends in Cell Biology 2005;15:251–258. DOI: 10.1016/j.tcb.2005.03.006
Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009;136:642–655. DOI: 10.1016/j.cell.2009.01.035
Hannon GJ. RNA interference. Nature 2002;418:244–251. DOI: 10.1038/418244a
Shabalina SA, Koonin EV. Origins and evolution of eukaryotic RNA interference. Trends in Ecology & Evolution 2008;23:578–587. DOI: 10.1016/j.tree.2008.06.005
Mittal V. Improving the efficiency of RNA interference in mammals. Nature Reviews Genetics 2004;5:355–365. DOI: 10.1038/nrg1323
Tang G. siRNA and miRNA: an insight into RISCs. Trends in Biochemical Sciences 2005;30:106–114. DOI: 10.1016/j.tibs.2004.12.007
Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 2003;425:415–419. DOI: 10.1038/nature01957
Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The Drosha-DGCR8 complex in primary microRNA processing. Genes & Development 2004;18:3016–3027. DOI: 10.1101/gad.1262504
Saini HK, Griffiths-Jones S, Enright AJ. Genomic analysis of human microRNA transcripts. Proceedings of the National Academy of Sciences of United States of America 2007;104:17719–17724. DOI: 10.1073/pnas.0703890104
Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 2005;122:553–563. DOI: 10.1016/j.cell.2005.07.031
Yuan X, Naguib S, Wu Z. Recent advances of siRNA delivery by nanoparticles. Expert Opinion on Drug Delivery 2011;8:521–536. DOI: 10.1517/17425247.2011.559223
Singha K, Namgung R, Kim WJ. Polymers in small-interfering RNA delivery. Nucleic Acid Therapeutics 2011;21:133–147. DOI: 10.1089/nat.2011.0293
Bertrand JR, Pottier M, Vekris A, Opolon P, Maksimenko A, Malvy C. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochemical and Biophysical Research Communications 2002;296:1000–1004. DOI: 10.1016/S0006-291X(02)02013-2
Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003;115:199–208. DOI: 10.1016/S0092-8674(03)00759-1
Heale BS, Soifer HS, Bowers C, Rossi JJ. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Research 2005;33:e30. DOI: 10.1093/nar/gni026
Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003;115:209–216. DOI: 10.1016/S0092-8674(03)00801-8
Gary DJ, Puri N, Won YY. Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. Journal of Controlled Release 2007;121:64–73. DOI: 10.1016/j.jconrel.2007.05.021
Lingor P, Michel U, Scholl U, Bahr M, Kugler S. Transfection of “naked” siRNA results in endosomal uptake and metabolic impairment in cultured neurons. Biochemical and Biophysical Research Communications 2004;315:1126–1133. DOI: 10.1016/j.bbrc.2004.01.170
McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nature Reviews Genetics 2002;3:737–747. DOI: 10.1038/nrg908
Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–553. DOI: 10.1126/science.1068999
Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery 2009;8:129–138. DOI: 10.1038/nrd2742
Lee JB, Hong J, Bonner DK, Poon Z, Hammond PT. Self-assembled RNA interference microsponges for efficient siRNA delivery. Nature Materials 2012;11:316–322. DOI: 10.1038/nmat3253
Borna H, Imani S, Iman M, Azimzadeh Jamalkandi S. Therapeutic face of RNAi: in vivo challenges. Expert Opinion on Biological Therapy 2015;15:269–285. DOI: 10.1517/14712598.2015.983070
Park TG, Jeong JH, Kim SW. Current status of polymeric gene delivery systems. Advanced Drug Delivery Reviews 2006;58:467–486. DOI: 10.1016/j.addr.2006.03.007
Ganta S, Devalapally H, Shahiwala A, Amiji M. A review of stimuli-responsive nanocarriers for drug and gene delivery. Journal of Controlled Release 2008;126:187–204. DOI: 10.1016/j.jconrel.2007.12.017
Behlke MA. Chemical modification of siRNAs for in vivo use. Oligonucleotides 2008;18:305–319. DOI: 10.1089/oli.2008.0164
Boudreau RL, Rodriguez-Lebron E, Davidson BL. RNAi medicine for the brain: progresses and challenges. Human Molecular Genetics 2011;20:R21–R27. DOI: 10.1093/hmg/ddr137
Peacock H, Kannan A, Beal PA, Burrows CJ. Chemical modification of siRNA bases to probe and enhance RNA interference. Journal of Organic Chemistry 2011;76:7295–7300. DOI: 10.1021/jo2012225
Kennedy S, Wang D, Ruvkun G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 2004;427:645–649.
Chiu YL, Rana TM. siRNA function in RNAi: a chemical modification analysis. RNA 2003;9:1034–1048. DOI: 10.1261/rna.5103703
Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Research 2003;31:589–595. DOI: 10.1093/nar/gkg147
Hall AH, Wan J, Shaughnessy EE, Ramsay Shaw B, Alexander KA. RNA interference using boranophosphate siRNAs: structure-activity relationships. Nucleic Acids Research 2004;32:5991–6000. DOI: 10.1093/nar/gkh936
Sheehan D, Lunstad B, Yamada CM, Stell BG, Caruthers MH, Dellinger DJ. Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides. Nucleic Acids Research 2003;31:4109–4018. DOI: 10.1093/nar/gkg439
Yamada CM, Dellinger DJ, Caruthers MH. Synthesis and biological activity of phosphonocarboxylate DNA. Nucleosides, Nucleotides & Nucleic Acids 2007;26:539–546. DOI: 10.1080/15257770701489896
Odadzic D, Bramsen JB, Smicius R, Bus C, Kjems J, Engels JW. Synthesis of 2′-O-modified adenosine building blocks and application for RNA interference. Bioorganic & Medicinal Chemistry 2008;16:518–529. DOI: 10.1016/j.bmc.2007.09.019
Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Langkjaer N, et al. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Research 2009;37:2867–2881. DOI: 10.1093/nar/gkp106
Wengel J, Petersen M, Nielsen KE, Jensen GA, Hakansson AE, Kumar R, et al. LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets. Nucleosides, Nucleotides & Nucleic Acids 2001;20:389–396. DOI: 10.1081/NCN-100002312
Trang P, Medina PP, Wiggins JF, Ruffino L, Kelnar K, Omotola M, et al. Regression of murine lung tumors by the let-7 microRNA. Oncogene 2010;29:1580–1587. DOI: 10.1038/onc.2009.445
Glud SZ, Bramsen JB, Dagnaes-Hansen F, Wengel J, Howard KA, Nyengaard JR, et al. Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration. Oligonucleotides 2009;19:163–168. DOI: 10.1089/oli.2008.0175
Vaish N, Chen F, Seth S, Fosnaugh K, Liu Y, Adami R, et al. Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Research 2011;39:1823–1832. DOI: 10.1093/nar/gkq961
Nauwelaerts K, Fisher M, Froeyen M, Lescrinier E, Aerschot AV, Xu D, et al. Structural characterization and biological evaluation of small interfering RNAs containing cyclohexenyl nucleosides. Journal of the American Chemical Society 2007;129:9340–9348. DOI: 10.1021/ja067047q
Fisher M, Abramov M, Van Aerschot A, Xu D, Juliano RL, Herdewijn P. Inhibition of MDR1 expression with altritol-modified siRNAs. Nucleic Acids Research 2007;35:1064–1074. DOI: 10.1093/nar/gkl1126
Bonoiu AC, Mahajan SD, Ding H, Roy I, Yong KT, Kumar R, et al. Nanotechnology approach for drug addiction therapy: gene silencing using delivery of gold nanorod-siRNA nanoplex in dopaminergic neurons. Proceedings of the National Academy of Sciences of United States of America 2009;106:5546–5550. DOI: 10.1073/pnas.0901715106
Lasic DD. Novel applications of liposomes. Trends in Biotechnology 1998;16:307–321. DOI: 10.1016/S0167-7799(98)01220-7
Lavigne C, Slater K, Gajanayaka N, Duguay C, Arnau Peyrotte E, Fortier G, et al. Influence of lipoplex surface charge on siRNA delivery: application to the in vitro downregulation of CXCR4 HIV-1 co-receptor. Expert Opinion on Biological Therapy 2013;13:973–985. DOI: 10.1517/14712598.2013.743526
Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O, et al. Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid. Gene Therapy 2001;8:677–686. DOI: 10.1038/sj.gt.3301460
Kawakami S, Sato A, Nishikawa M, Yamashita F, Hashida M. Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes. Gene Therapy 2000;7:292–299. DOI: 10.1038/sj.gt.3301089
Kang H, DeLong R, Fisher MH, Juliano RL. Tat-conjugated PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides. Pharmaceutical Research 2005;22:2099–2106. DOI: 10.1007/s11095-005-8330-5
Liu M, Frechet JM. Designing dendrimers for drug delivery. Pharmaceutical Science & Technology Today 1999;2:393–401.
Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochemical Society Transactions 2007;35:61–67. DOI: 10.1042/BST0350061
Kaminskas LM, Kelly BD, McLeod VM, Sberna G, Boyd BJ, Owen DJ, et al. Capping methotrexate alpha-carboxyl groups enhances systemic exposure and retains the cytotoxicity of drug conjugated PEGylated polylysine dendrimers. Molecular Pharmaceutics 2011;8:338–349. DOI: 10.1021/mp1001872
Samal SK, Dash M, Van Vlierberghe S, Kaplan DL, Chiellini E, van Blitterswijk C, et al. Cationic polymers and their therapeutic potential. Chemical Society Reviews 2012;41:7147–7194. DOI: 10.1039/c2cs35094g
Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Advanced Drug Delivery Reviews 2005;57:579–596. DOI: 10.1016/j.addr.2004.10.005
Elliott G, O’Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 1997;88:223–233. DOI: 10.1016/S0092-8674(00)81843-7
Morris MC, Vidal P, Chaloin L, Heitz F, Divita G. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Research 1997;25:2730–2736.
Oehlke J, Scheller A, Wiesner B, Krause E, Beyermann M, Klauschenz E, et al. Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochimica et Biophysica Acta 1998;1414:127–139.
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. Journal of Biological Chemistry 2001;276:5836–5840. DOI: 10.1074/jbc.M007540200
El-Aneed A. An overview of current delivery systems in cancer gene therapy. Journal of Controlled Release 2004;94:1–14. DOI: 10.1016/j.jconrel.2003.09.013
Pack DW, Hoffman AS, Pun S, Stayton PS. Design and development of polymers for gene delivery. Nature Reviews Drug Discovery 2005;4:581–593. DOI: 10.1038/nrd1775
Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Research 2004;32:e149. DOI: 10.1093/nar/gnh140
Feng Y, Hu J, Ma J, Feng K, Zhang X, Yang S, et al. RNAi-mediated silencing of VEGF-C inhibits non-small cell lung cancer progression by simultaneously down-regulating the CXCR4, CCR7, VEGFR-2 and VEGFR-3-dependent axes-induced ERK, p38 and AKT signalling pathways. European Journal of Cancer 2011;47:2353–2363. DOI: 10.1016/j.ejca.2011.05.006
Suk JS, Lai SK, Boylan NJ, Dawson MR, Boyle MP, Hanes J. Rapid transport of muco-inert nanoparticles in cystic fibrosis sputum treated with N-acetyl cysteine. Nanomedicine 2011;6:365–375. DOI: 10.2217/nnm.10.123
McNamara JO, 2nd, Andrechek ER, Wang Y, Viles KD, Rempel RE, Gilboa E, et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nature Biotechnology 2006;24:1005–1015. DOI: 10.1038/nbt1223
Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006;441:537–541. DOI: 10.1038/nature04791
Yi R, Doehle BP, Qin Y, Macara IG, Cullen BR. Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 2005;11:220–226. DOI: 10.1261/rna.7233305
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–498. DOI: 10.1038/35078107
Robbins M, Judge A, MacLachlan I. siRNA and innate immunity. Oligonucleotides 2009;19:89–102. DOI: 10.1089/oli.2009.0180
Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, et al. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nature Medicine 2005;11:263–270. DOI: 10.1038/nm1191
Marques JT, Williams BR. Activation of the mammalian immune system by siRNAs. Nature Biotechnology 2005;23:1399–1405. DOI: 10.1038/nbt1161
Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 2003;21:635–637. DOI: 10.1038/nbt831
Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 2006;12:1197–205. DOI: 10.1261/rna.30706
Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005;433:769–773. DOI: 10.1038/nature03315
Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, Lim L, et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 2006;12:1179–1187. DOI: 10.1261/rna.25706
Miller VM, Paulson HL, Gonzalez-Alegre P. RNA interference in neuroscience: progress and challenges. Cellular and Molecular Neurobiology 2005;25:1195–1207. DOI: 10.1007/s10571-005-8447-4
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annual Review of Biochemistry 1998;67:227–264. DOI: 10.1146/annurev.biochem.67.1.227
Heidel JD, Hu S, Liu XF, Triche TJ, Davis ME. Lack of interferon response in animals to naked siRNAs. Nature Biotechnology 2004;22:1579–1582. DOI: 10.1038/nbt1038
Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology 2005;23:457–462. DOI: 10.1038/nbt1081
Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Advances in Genetics 2005;54:117–142. DOI: 10.1016/S0065-2660(05)54006-9
Xie FY, Woodle MC, Lu PY. Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discovery Today 2006;11:67–73. DOI: 10.1016/S1359-6446(05)03668-8
Martin SE, Caplen NJ. Applications of RNA interference in mammalian systems. Annual Review of Genomics and Human Genetics 2007;8:81–108. DOI: 10.1146/annurev.genom.8.080706.092424
Pushparaj PN, Melendez AJ. Short interfering RNA (siRNA) as a novel therapeutic. Clinical and Experimental Pharmacology & Physiology 2006;33:504–510. DOI: 10.1111/j.1440-1681.2006.04399.x
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nature Biotechnology 2004;22:326–330. DOI: 10.1038/nbt936
Nishikawa M, Huang L. Nonviral vectors in the new millennium: delivery barriers in gene transfer. Human Gene Therapy 2001;12:861–870. DOI: 10.1089/104303401750195836
Du Q, Thonberg H, Wang J, Wahlestedt C, Liang Z. A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Research 2005;33:1671–1677. DOI: 10.1093/nar/gki312
Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Research 2004;32(Web Server issue):W124–W129. DOI: 10.1093/nar/gkh442
Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. Journal of Virology 1998;72:9873–9880.
Isacson R, Kull B, Salmi P, Wahlestedt C. Lack of efficacy of ‘naked’ small interfering RNA applied directly to rat brain. Acta Physiologica Scandinavica 2003;179:173–177. DOI: 10.1046/j.1365-201X.2003.01188.x
Thakker DR, Natt F, Husken D, Maier R, Muller M, van der Putten H, et al. Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference. Proceedings of the National Academy of Sciences of United States of America 2004;101:17270–17275. DOI: 10.1073/pnas.0406214101
Pardridge WM. Intravenous, non-viral RNAi gene therapy of brain cancer. Expert Opinion on Biological Therapy 2004;4:1103–1113. DOI: 10.1517/14712518.104.22.1683
Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. Local gene knockdown in the brain using viral-mediated RNA interference. Nature Medicine 2003;9:1539–1544. DOI: 10.1038/nm964
Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Medicine 2003;9:347–351. DOI: 10.1038/nm828
McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002;418:38–39. DOI: 10.1038/418038a
Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J. Inhibition of influenza virus production in virus-infected mice by RNA interference. Proceedings of the National Academy of Sciences of United States of America 2004;101:8676–8681. DOI: 10.1073/pnas.0402486101
Bitko V, Musiyenko A, Shulyayeva O, Barik S. Inhibition of respiratory viruses by nasally administered siRNA. Nature Medicine 2005;11:50–55. DOI: 10.1038/nm1164
Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT, Knipe DM, et al. An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 2006;439:89–94. DOI: 10.1038/nature04263
Miller VM, Xia H, Marrs GL, Gouvion CM, Lee G, Davidson BL, et al. Allele-specific silencing of dominant disease genes. Proceedings of the National Academy of Sciences of United States of America 2003;100:71957–72000. DOI: 10.1073/pnas.1231012100
Maxwell MM, Pasinelli P, Kazantsev AG, Brown RH, Jr. RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells. Proceedings of the National Academy of Sciences of United States of America 2004;101:3178–3183. DOI: 10.1073/pnas.0308726100
Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT, et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nature Medicine 2004;10:816–820. DOI: 10.1038/nm1076
Xia XG, Zhou H, Zhou S, Yu Y, Wu R, Xu Z. An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase. Journal of Neurochemistry 2005;92:362–367. DOI: 10.1111/j.1471-4159.2004.02860.x
Liu Z, Li S, Liang Z, Zhao Y, Zhang Y, Yang Y, et al. Targeting beta-secretase with RNAi in neural stem cells for Alzheimer’s disease therapy. Neural Regeneration Research 2013;8:3095–3106. DOI: 10.3969/j.issn.1673-5374.2013.33.003
Hardy J. Genetic analysis of pathways to Parkinson disease. Neuron 2010;68:201–206. DOI: 10.1016/j.neuron.2010.10.014
Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD. siRNA, miRNA, and shRNA: in vivo applications. Journal of Dental Research 2008;87:992–1003. DOI: 10.1177/154405910808701109
Keiser MS, Boudreau RL, Davidson BL. Broad therapeutic benefit after RNAi expression vector delivery to deep cerebellar nuclei: implications for spinocerebellar ataxia type 1 therapy. Molecular Therapy 2014;22:588–595. DOI: 10.1038/mt.2013.279
Gonzalez-Alegre P, Miller VM, Davidson BL, Paulson HL. Toward therapy for DYT1 dystonia: allele-specific silencing of mutant TorsinA. Annals of Neurology 2003;53:781–787. DOI: 10.1002/ana.10548
White MD, Mallucci GR. RNAi for the treatment of prion disease: a window for intervention in neurodegeneration? CNS & Neurological Disorders – Drug Targets 2009;8:342–352.
Kong Q. RNAi: a novel strategy for the treatment of prion diseases. Journal of Clinical Investigation 2006;116:3101–3103. DOI: 10.1172/JCI30663
White MD, Farmer M, Mirabile I, Brandner S, Collinge J, Mallucci GR. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease. Proceedings of the National Academy of Sciences of United States of America 2008;105:10238–10243. DOI: 10.1073/pnas.0802759105
Caughey B, Caughey WS, Kocisko DA, Lee KS, Silveira JR, Morrey JD. Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: a common mechanism for anti-TSE compounds? Accounts of Chemical Research 2006;39:646–653. DOI: 10.1021/ar050068p
Rider TH, Zook CE, Boettcher TL, Wick ST, Pancoast JS, Zusman BD. Broad-spectrum antiviral therapeutics. PLoS One 2011;6:e22572. DOI: 10.1371/journal.pone.0022572
Boado RJ. Blood-brain barrier transport of non-viral gene and RNAi therapeutics. Pharmaceutical Research 2007;24:1772–1787. DOI: 10.1007/s11095-007-9321-5
Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proceedings of the National Academy of Sciences of United States of America 2005;102:5820–5825. DOI: 10.1073/pnas.0501507102
Wang YL, Liu W, Wada E, Murata M, Wada K, Kanazawa I. Clinico-pathological rescue of a model mouse of Huntington’s disease by siRNA. Neuroscience Research 2005;53:241–249. DOI: 10.1016/j.neures.2005.06.021
Rodriguez-Lebron E, Denovan-Wright EM, Nash K, Lewin AS, Mandel RJ. Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington’s disease transgenic mice. Molecular Therapy 2005;12:618–633. DOI: 10.1016/j.ymthe.2005.05.006
Drouet V, Perrin V, Hassig R, Dufour N, Auregan G, Alves S, et al. Sustained effects of nonallele-specific Huntingtin silencing. Annals of Neurology 2009;65:276–285. DOI: 10.1002/ana.21569
DiFiglia M, Sena-Esteves M, Chase K, Sapp E, Pfister E, Sass M, et al. Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proceedings of the National Academy of Sciences of United States of America 2007;104:17204–17209. DOI: 10.1073/pnas.0708285104
Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annual Review of Pharmacology and Toxicology 2010;50:259–293. DOI: 10.1146/annurev.pharmtox.010909.105654
Southwell AL, Ko J, Patterson PH. Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington’s disease. Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2009;29:13589–13602. DOI: 10.1523/JNEUROSCI.4286-09.2009
Southwell AL, Khoshnan A, Dunn DE, Bugg CW, Lo DC, Patterson PH. Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. Journal of Neuroscience: The Official Journal of the Society for Neuroscience 2008;28:9013–9020. DOI: 10.1523/JNEUROSCI.2747-08.2008
Pfister EL, Kennington L, Straubhaar J, Wagh S, Liu W, DiFiglia M, et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Current Biology 2009;19:774–778. DOI: 10.1016/j.cub.2009.03.030
Davidson BL, Paulson HL. Molecular medicine for the brain: silencing of disease genes with RNA interference. Lancet Neurology 2004;3:145–149. DOI: 10.1016/S1474-4422(04)00678-7
Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Molecular Cell 2003;11:1457–1466. DOI: 10.1016/S1097-2765(03)00220-X
Fahn S, Bressman SB, Marsden CD. Classification of dystonia. Advances in Neurology 1998;78:1–10.
Abdelgany A, Wood M, Beeson D. Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference. Human Molecular Genetics 2003;12:2637–2644. DOI: 10.1093/hmg/ddg280
Jin P, Zarnescu DC, Ceman S, Nakamoto M, Mowrey J, Jongens TA, et al. Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nature Neuroscience 2004;7:113–117. DOI: 10.1038/nn1174
Jin P, Alisch RS, Warren ST. RNA and microRNAs in fragile X mental retardation. Nature Cell Biology 2004;6:1048–1053. DOI: 10.1038/ncb1104-1048
Caudy AA, Myers M, Hannon GJ, Hammond SM. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes & Development 2002;16:2491–2496. DOI: 10.1101/gad.1025202
Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–356. DOI: 10.1126/science.1072994
Miller VM, Gouvion CM, Davidson BL, Paulson HL. Targeting Alzheimer’s disease genes with RNA interference: an efficient strategy for silencing mutant alleles. Nucleic Acids Research 2004;32:661–668. DOI: 10.1093/nar/gkh208
Kao SC, Krichevsky AM, Kosik KS, Tsai LH. BACE1 suppression by RNA interference in primary cortical neurons. Journal of Biological Chemistry 2004;279:1942–1949. DOI: 10.1074/jbc.M309219200
Lipton RB, Dodick DW. CGRP antagonists in the acute treatment of migraine. Lancet Neurology 2004;3:332. DOI: 10.1016/S1474-4422(04)00764-1
Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DC, et al. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nature Medicine 2005;11:429–433. DOI: 10.1038/nm1205
Halliday GM, McCann H. The progression of pathology in Parkinson’s disease. Annals of the New York Academy of Sciences 2010;1184:188–195. DOI: 10.1111/j.1749-6632.2009.05118.x
Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha-synuclein in Lewy bodies. Nature 1997;388:839–840. DOI: 10.1038/42166
Sapru MK, Yates JW, Hogan S, Jiang L, Halter J, Bohn MC. Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral-mediated RNAi. Experimental Neurology 2006;198:382–390. DOI: 10.1016/j.expneurol.2005.12.024
Gorbatyuk OS, Li S, Nash K, Gorbatyuk M, Lewin AS, Sullivan LF, et al. In vivo RNAi-mediated alpha-synuclein silencing induces nigrostriatal degeneration. Molecular Therapy 2010;18(8):1450–1457. DOI: 10.1038/mt.2010.115
Angaji SA, Hedayati SS, Poor RH, Madani S, Poor SS, Panahi S. Application of RNA interference in treating human diseases. Journal of Genetics 2010;89:527–537.
Mattson MP. Apoptosis in neurodegenerative disorders. Nature Reviews Molecular Cell Biology 2000;1:120–129. DOI: 10.1038/35040009
Zhao ZQ, Vinten-Johansen J. Myocardial apoptosis and ischemic preconditioning. Cardiovascular Research 2002;55:438–455. DOI: 10.1016/S0008-6363(02)00442-X
Reddy KS. India wakes up to the threat of cardiovascular diseases. Journal of the American College of Cardiology 2007;50:1370–1372. DOI: 10.1016/j.jacc.2007.04.097
Kim DH, Rossi JJ. Strategies for silencing human disease using RNA interference. Nature Reviews Genetics 2007;8:173–184. DOI: 10.1038/nrg2006
Michiue H, Eguchi A, Scadeng M, Dowdy SF. Induction of in vivo synthetic lethal RNAi responses to treat glioblastoma. Cancer Biology & Therapy 2009;8:2306–2313.
Pai SI, Lin YY, Macaes B, Meneshian A, Hung CF, Wu TC. Prospects of RNA interference therapy for cancer. Gene Therapy 2006;13:464–477. DOI: 10.1038/sj.gt.3302694
Shi Y, Tian Y, Zhou YQ, Ju JY, Qu L, Chen SL, et al. Inhibition of malignant activities of nasopharyngeal carcinoma cells with high expression of CD44 by siRNA. Oncology Reports 2007;18:397–403.
Dickerson EB, Blackburn WH, Smith MH, Kapa LB, Lyon LA, McDonald JF. Chemosensitization of cancer cells by siRNA using targeted nanogel delivery. BMC Cancer 2010;10:10. DOI: 10.1186/1471-2407-10-10
Ofek P, Fischer W, Calderon M, Haag R, Satchi-Fainaro R. In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers. FASEB Journal 2010;24:3122–3134. DOI: 10.1096/fj.09-149641
Dai B, Fang B, Roth JA. RNAi-induced synthetic lethality in cancer therapy. Cancer Biology & Therapy 2009;8:2314–2316.
Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH. Integrating genetic approaches into the discovery of anticancer drugs. Science 1997;278:1064–1068. DOI: 10.1126/science.278.5340.1064
Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook TF, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 2009;137:835–848. DOI: 10.1016/j.cell.2009.02.024
Scholl C, Frohling S, Dunn IF, Schinzel AC, Barbie DA, Kim SY, et al. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 2009;137:821–834. DOI: 10.1016/j.cell.2009.03.017
Hulleman E, Helin K. Molecular mechanisms in gliomagenesis. Advances in Cancer Research 2005;94:1–27. DOI: 10.1016/S0065-230X(05)94001-3
Gump JM, Dowdy SF. TAT transduction: the molecular mechanism and therapeutic prospects. Trends in Molecular Medicine 2007;13:443–438. DOI: 10.1016/j.molmed.2007.08.002
Prochiantz A. Protein and peptide transduction, twenty years later a happy birthday. Advanced Drug Delivery Reviews 2008;60:448–451. DOI: 10.1016/j.addr.2007.08.040