siRNA Mediated Gene Silencing

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siRNA Mediated Gene Silencing: Hurdles, Strategies and Applications Prajakta Tambe, Pramod Kumar, Virendra Gajbhiye* and K.M. Paknikar* Center for Nanobioscience, Agharkar Research Institute, Pune411 004, India Abstract: Background: RNA interference (RNAi) has emerged as one of the most extensively explored areas of research that has promising applications in therapeutics. As an endogenous regulatory pathway, RNAi can be used for silencing targeted genes involved in the progression of diseases. Small interfering RNAs (siRNA) are 21-25 nucleotides in length and work on the principle of RNA interference. Owing to their high degree of safety, specificity, efficacy, easy synthesis and unrestricted choice of target, siRNAs are considered appropriate for gene silencing. Objective: This review discusses siRNA gene silencing mechanism, the challenges posed in siRNA delivery and the strategies developed to overcome some of these hurdles using nanocarriers. A few applications of siRNA in therapeutics are presented to highlight the utility of these strategies. Conclusion: In spite of advantages of siRNA technology, several shortcomings such as off-target effects, hurdles in delivering, endosomal trapping, stimulation of immune response and short half-life limit the practical use of siRNA. However, the situation has been improved considerably using nanotechnology based tools.

Keywords: Nanocarriers, RNA interference, siRNA, challenges, delivery strategies, non-viral vectors. 1. INTRODUCTION Nanomedicine, an emerging area of nanotechnology, is defined as governing, restoring and building human biological systems at the molecular level using engineered nanoconstructs and nanodevices [1]. Nanotechnology-based gene silencing through RNAi mechanism is one of the most widely explored methods for treatment of fatal diseases such as AIDS and cancer. Gene silencing is a method in which translation or transcription of the target gene is suppressed or interrupted. It consists of two levels; post-transcriptional gene silencing (PTGS) also known as RNA interference and Transcriptional gene silencing (TGS) [2]. RNA interference (RNAi) is a sequence specific PTGS cellular mechanism, wherein antisense RNA is used to degrade target mRNA *Address correspondence to these authors at the Center for Nanobioscience, Agharkar Research Institute, Pune-411 004, India; Tel:/Fax: + 91-020-25651542; E-mails: [email protected]; [email protected] 2211-7393/15 $58.00+.00

[3, 4]. Andrew Z. Fire and Craig C. Mello were joint recipients of the Nobel Prize in Physiology and Medicine in 2006 for their discovery of siRNA. They investigated the regulation of gene expression encoding a muscle protein in the nematode worm, Caenorhabditis elegans. When Fire and Mello injected sense and antisense mRNA sequences separately, encoding muscle protein at different times, they observed no effect in the worm behavior. However, when they injected sense and antisense mRNA simultaneously, worms showed strange and atypical twitching movements similar to those worms which lacked this gene completely. It was suggested that doublestranded RNA is formed when sense and antisense strands hybridize with each other. To test this hypothesis, Fire and Mello injected double-stranded RNA molecules with particular genetic codes for various other proteins present in the worm. Each time they found that no protein was formed from the gene having that particular code. Thus, Fire and Mello derived that double-stranded RNA can © 2015 Bentham Science Publishers

siRNA Mediated Gene Silencing: Hurdles

silence genes and termed this concept as RNA interference (RNAi) [5]. RNAi is thought to have developed as a protection mechanism against foreign genetic material (such as that from the viruses). It acts by preventing replication of such genetic material [6]. It is observed in both plants and animals. Simultaneous suppression of more than one gene was seen in transgenic plant Petunia, which showed suppression of both, existing genes and transgene [7]. 2. GENE SILENCING MECHANISM RNA interference mechanism silences gene expression in eukaryotes such as yeast and mammals [6]. Silencing is carried out in two steps, i.e., the initiator and effector step. In the initiator step, the double stranded RNA (dsRNA) is introduced into the cell that undergoes cleavage by an enzyme called Dicer, also referred as ‘molecular ruler’ [8]. It belongs to RNase III family that specifically cleaves dsRNA-producing fragments with 3’ overhangs of 2-3 nucleotides (nt) and is present in all eukaryotes [9]. Fragments formed are 21-25 nt long and are referred to as small interfering RNA (siRNA). In the next step, the siRNA fragments are transferred to the multi-nuclease effector complex, RNA-induced silencing complex (RISC), composed of members of the Argonaute protein family endowing ‘slicer’ activity to RISC. PAZ and PIWI are domains of argonaute protein. PAZ provides specificity, proper orientation and holds 3’ end of siRNA while PIWI domain provides an active site for cleavage [6, 10] and is the powerhouse for RISC. siRNA gets incorporated into the RISC complex and then interacts with argonaute-2 protein [11]. Of the two strands of siRNA, one acts as a guide strand serving as a template while the other strand called as ‘Passenger strand’ is degraded by argonaute-2 protein [6]. RISC directs recognition of target through intermolecular base pairing [12]. Argonaute-2 then selectively degrades mRNA (target) bound to the antisense strand, causing gene silencing [13]. Two conditions thought to be essential for silencing by RNAi is the establishment of phosphorylation of the antisense strand at 5’ and orientation of an antisense target mRNA duplex in the A-form helix. Heteroduplex formed between the siRNA antisense strand, and its target mRNA is stabilized by the A-form helix [6]. In addition to siRNA, other alternative forms of RNA, such as Micro (mi)

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RNA, piwi-interacting (pi) RNA, Short hairpin (sh) RNA and small modulatory (sm) RNA have also been used in various applications. 2.1. shRNA shRNAs are 19-29 nt long sequences which contain sense, anti-sense and loop structure. In contrast to siRNA, shRNAs are synthesized in the nucleus and exported to the cytoplasm. In the cytoplasm, the hairpin loop is processed by enzyme dicer and TRBP/PACT [Tat–RNA-binding protein (TRBP)/ PACT (PKR activating protein)] that gives rise to double-stranded siRNA [6]. 2.2. siRNA As mentioned earlier, siRNAs are 21-25 nt long sequence and have been extensively used for gene silencing of target mRNA. siRNA are highly selective and perfectly complementary to the target mRNA. siRNA turns off gene expression by directing degradation of selective mRNAs. Elbashir et al. first discovered them in cell-free systems that showed efficient degradation of homologous mRNA [14]. Further experiments carried out by Zamore et al. (2000) using native, and processed siRNA revealed that siRNA are the true intermediates of RNAi [15]. These are 1000 times more effective in silencing the target gene as compared to antisense oligonucleotides (ODN). ODNs are DNA/RNA sequences, which hybridize with mRNA and RNase eventually degrade them [16]. Biosynthetic pathways for siRNA are different for plants and mammals. In plants, the silencing of genes is systemic, i.e. the reaction spreads from one place to another, mostly from the initiation site. However, this is not the case with animals, the differences being in the pre-Dicer, Dicer, and post-Dicer stage. siRNA can be custom synthesized specifically against the target gene of interest and with different silencing efficiencies at various regions of the same gene. These varying efficiencies were studied by many researchers via changing various parameters, e.g. sugar backbone, the length of nt chain and secondary structure of siRNA. siRNA with ~21 nucleotides length, Guanine-cytosine (GC) content of 30-70% and obtained from regions 50-100 bp downstream of start codon are thought to be of great potential [9]. Various studies show that siRNA not only works post-transcriptionally but at the transcriptional level as well [17]. Further, DNA methylation,

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heterochromatin formation and programmed elimination of DNA are thought to be the end products of siRNA -mediated gene silencing [9]. 3. CHALLENGES IN siRNA DELIVERY Although siRNA is highly promising for therapeutic applications, there are certain challenges that restrict their wide application. Controlled in vivo delivery of siRNA to the target cells is a major hurdle. Moreover, anionic backbone present in siRNA makes them hydrophilic, hampering the diffusion across cellular membranes [18]. Other extracellular and intracellular factors contributing to this are the large molecular weight and negative charges. Molecules larger than 13kDa are difficult to take up by the cells [11]. Furthermore, siRNAs are highly unstable due to their half-life that ranges from several minutes to an hour in serum, thus requiring repeated delivery for prolonged use [19]. Nucleases i.e. RNase degrades siRNA in serum and cause subsequent renal clearance. Serum inactivation can be avoided if these molecules are chemically modified on the ribose sugar of RNA, causing locking of nucleic acid conformation by methylene bridge formation in sugar rings. Modifications such as 2′-fluoro, 2′-O-methyl, and 2′amine conjugations cause nuclease stability and increased silencing potency [20, 21]. Similarly, phosphorothioate linkages (oxygen replaced by sulfur) also show increased resistance to nucleases and hence increase half-life of siRNA. Also, serum half-life can be improved by local or topical application and by the use of nanocarriers [11]. Many times the molecules do not reach the target cells due to reticuloendothelial system (RES) clearance, which results from binding of plasma proteins to the nanoparticles (NPs). Along with it, positive charges on the polymeric carrier also cause nonspecific interactions with non-target cells [20]. These non-specific interactions can be reduced by PEGylation; a process in which polyethylene glycol (PEG) chains are added to the polymer heads which then protects the charges on polymers. It also reduces phagocytosis and opsonization of NPs by RES [20].

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silencing to avoid lysosomal degradation. Cationic polymers demonstrate a typical property known as 'proton sponge effect’, which is an effective mechanism for endosomal escape. These polymers possess secondary and tertiary amine group, which facilitate protons influx in endosome from the cytosol. The simultaneous influx of chloride ions in endosomes increase ionic concentration causing an influx of water. Thus, endosomes swell and subsequently get ruptured, which results in the endosomal escape of the contents [22]. Moreover, neutral ion-pairs are formed when cationic moieties of complexes bind to anionic lipids of the endosome, destabilizing the endosomal membrane and subsequently release siRNA to the cytoplasm [20, 23]. Reduction of endosomal trapping and enhancement of cargo release can be achieved by introducing labile bonds in PEG. Also, pHresponsive mechanisms, fusogenic peptides, enzymatic reactions can reduce trapping and hence improve gene silencing [20]. 3.2. Immune Response Stimulation siRNA and some of the sequence motifs may initiate innate immune response [11]. Immune responses are elicited by the receptors expressed on the cell surface, cytosol and within endocytic vesicles. Receptors such as OAS1 (2’-5’oligoadenylate synthetase 1), PKR (Protein kinase R) and RIG-I (Retinoic acid-inducible gene 1) are present in the cytosol. They are expressed uniformly in all cell types and are induced by interferons aimed at combating viral pathogens. As opposed to cytosolic receptors, those expressed by cell surface and endosomal vesicles are expressed differently for each cell type [18]. TLR 3 receptors expressed by endosomes and cell surface recognize dsRNA while ssRNA has been recognized by TLR 7 and 8 expressed by endosomes. Sequence motifs present in siRNAs: GUCCUUCAA [24], UGUGU [25], UGU [22], UCA [26], GU-rich sequences [27], AU-rich sequences, and U-rich sequences stimulate immune response [18]. Stimulation of immune response can be reduced by chemical modification of siRNA backbone by phosphorothioate alteration.

3.1. Endosomal Trapping Although PEGylation shields the charges, they pose a problem in releasing cargo materials and thus trapping them in endosomes. Escaping the endosomes is an essential step for siRNA mediated

3.3. Off-target Effects A major drawback in siRNA based therapy is ‘off target effect’. The off-target effect is the binding of siRNA to non-target mRNA thus


silencing the genes other than those for which silencing is required [28], which may affect interferon response and toxicity. It is known that siRNA recognizes partial similarity with its sequence, silencing unwanted transcripts and miRNA-like targeting [11]. When numerous target matches are present within their 3’ untranslated regions (3′UTRs), the pairing of as little as eight nucleotides is enough for non-specific silencing. Off-target effects can also result due to nonspecific binding of antisense or sense strands to other non-targeted mRNAs [19, 29]. Optimizing selective loading by RISC complex may help to reduce the off-target effects to some level. Another reason that may cause off-target effects is the infrequent sense strand incorporation [19]. Hexamer regions that mimic the miRNA binding site in 3′ UTR of off-targets are often silenced [30]. Chemical modifications may reduce the chance of off targeting. Similarly, the design and selection of a specific siRNA that involves internal repeated sequence, secondary structure, GC content, siRNA length and specific base preference should be considered to avoid off-target effects. 3.4. Presence of Endogenous RNAs The siRNA mediated silencing depends on the mechanism of endogenous miRNA. Therefore, competition with endogenous miRNA is a major challenge to develop siRNA based therapies. Furthermore, siRNA also matches with miRNA precursors, which may result in the blockage of miRNA pathway [30]. Such blockage may lead to undesired adverse effect due to the high dosage of ectopic RNA. 4. NANOTECHNOLOGY-BASED STRATEGIES FOR IMPROVING siRNA DELIVERY Over a period, many strategies have been discovered to deliver siRNA to the target cells through vectors. Target cells can be transfected with siRNA using either viral vector or non-viral vectors. Viral vectors include retroviruses, lentiviruses, and adenoviruses while non-viral vectors include transfection using the nanocarriers. Viral vectors are one of the major vehicles that could be employed in gene silencing and adenoviruses and adeno-associated viruses are considered efficient in delivering shRNA [31]. Retroviruses such as murine leukemia virus (MLV) and lentivi-


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ruses mediated integration of viral genome into the host cell chromosomal DNA may be permanent, thus silencing the target genes. While retroviruses are used only for dividing cells, lentiviruses specifically can transduce non-dividing cells and thus are an efficient system for somatic and germ-line transduction and are suitable for carrying larger genes [30]. Although viral vectors are efficient, provide promising results in cancer therapy, carry large genes, there are some shortcomings, which limit their use in gene silencing. Retroviruses and lentiviruses induce oncogenesis in cells that are not desired in gene silencing. Not only do they induce oncogenesis but immunogenicity, insertional mutagenesis, and biohazards are of major concern [4]. Of all the viral vectors, adenovirus vectors are the most immunogenic and induce immunogenicity through cytotoxic T-lymphocyte response (CTL). On the other hand, non-viral vectors have various advantages over viral vectors. They are less toxic and immunogenic than the viral vectors. Nanocarriers can be loaded with siRNA that is subsequently delivered to a specific cell type for efficient gene silencing (Fig. 1). Also, non-viral vectors are easy to produce in a large amount and have the advantage of repeated administration. Cell or tissue specificity can be achieved by harnessing cell-specific functionality in the design of chemical or biological vectors while physical procedures can provide spatial precision. Although non-viral vectors are less efficient than viral vectors up to some extent, efforts have been taken to improve their efficiency [32]. Non-viral vectors include various nanotechnology-based delivery methods or nanocarriers e.g. cationic liposomes, cationic polymers such as dendrimers, and inorganic nanocarriers like carbon nanotubes, quantum dots, gold nanoparticles and mesoporous silica nanoparticles (MSNP). Nanotechnology-based siRNA delivery methods also include peptidesiRNA nanoconjugates and aptamer-siRNA nanoconjugates. These nanocarriers pose various advantages like site-specific delivery, protection from degradation, enhanced bioavailability, diminished immunogenicity, and increased efficiency. 4.1. Cationic Polymers for siRNA Delivery Cationic polymers including synthetic as well as natural polymers are being used for siRNA delivery. Examples of synthetic polymers are polyethyleneimine (PEI), poly-L-lysine (PLL), poly

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A

Nanoparticles loaded with siRNA

B Endosome breakage and siRNA release C

Endosome 5'

3' siRNA RISC complex D

Helicase

Nucleus

E

Passenger strand

Guide RNA F mRNA

Guide RNA mRNA mRNA clevage No protein

Fig. (1). Schematic illustration of the siRNA-mediated gene silencing mechanism using NPs. A. NPs loaded with siRNA are taken up by the cells through endocytosis. B. These particles are then trapped into the endosomes. C. Due to proton sponge effect or pH responsive mechanism, these NPs loaded with siRNA escape endosomes and release siRNA into the cytoplasm. D, E. Helicase present in the RISC complex removes the passenger strand leaving a guide siRNA strand intact. F. mRNA from nucleus binds to guide siRNA strand, thus causing mRNA degradation.

(β-aminoester) s (PAE) and polyarginine. Natural polymers such as atelocollagen and gelatin have also been used for siRNA delivery. In a study, low molecular weight PEI was complexed with siRNA and targeted to HER-2 expressing cell lines and then in a subcutaneous mouse tumor model. This study showed that PEI modification protects from nuclease degradation and efficiently delivers siRNA to the tumor [33]. In an another study, hypercholesterolemia was targeted by silencing apolipoprotein B (apo-B) using sixth generation PLL polymer (KG6) which was loaded with apo-B specific siRNA. The ability of these complexes to silence the apo-B expression was studied in mouse model C57BL/6. Their results demonstrated that KG6 is a potential carrier for siRNA delivery and can improve hypercholesterolemia [34]. Though these polymers possess some disadvantages, they are preferred to a certain extent due to their easy and cheap production, and high efficiency of com-

plexation with nucleic acid by electrostatic interaction to form polyplexes. 4.2. Liposomes for siRNA Delivery Liposomes are spherical vesicles prepared artificially using lamellar phase lipid bilayer and an aqueous core. Unilamellar or multilamellar liposomes interact with siRNA to form lipoplexes [4], which are stabilized by electrostatic interactions [11]. Based on lipids used liposomes can be categorized as cationic and anionic liposomes. Cationic liposomes pose high transfection efficiency and are formed by cationic lipids like DOTMA (N-[1-(2, 3-dioleyloxy) Propyl]- N,N, N trimethyl ammonium chloride), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), Transfectam (Promega) and 98N12-5 [4, 35]. Another type of liposomes formulated are the stable nucleic acid lipid particles (SNALPs) composed of


cationic lipids, fusogenic lipids, and cholesterol, which have proven their utility in protection from Ebola virus [36]. They showed longer half-life in plasma and liver. These unilamellar liposomes, such as dioleoyl phosphatidylcholine encapsulate siRNA and protect them from endonucleases, enhancing their internalization [11]. Liposomes show versatility and flexibility in structure, enhance uptake by cells via the endosomal pathway, show efficient in vivo siRNA delivery, and downregulate target genes. However, some disadvantages, such as lack of tissue specificity, rapid liver clearance (RES sequester), induction of type 1 and 2–IFN response, dose-dependent toxicity and pulmonary inflammation limit their use. 4.3. Cationic Dendrimers for siRNA Delivery Dendrimers are spherical, highly branched polymeric molecules [4, 37, 38]. They possess a core, an inner shell and an outer shell (terminal functional groups), with the core determining the size and shape of dendrimer [39-41]. Dendrimers with cationic functionalization enable them to be loaded on or encapsulated with the negatively charged siRNA. Various cationic dendrimers such as polyamidoamine (PAMAM), polypropylenimine (PPI), poly-l- lysine (PLL) and carbosilane dendrimers have been explored widely. siRNA has been delivered to different cell lines using PAMAM dendrimers conjugated with various targeting ligands e.g. Luteinizing hormone-releasing hormone (LHRH), Epidermal growth factor (EGF), etc. The advantages of dendrimers are a large number of surface groups, multifunctionality, enhanced serum stability, efficient gene silencing, and specific targeting when conjugated with targeting ligand. Eventhough dendrimers are useful and have many advantages, shortcomings like hemolysis and lower efficiency to exert proton sponge effect are major hurdles in their use [42]. 4.4. Nanoparticles for siRNA Delivery NPs are nano-sized, ultra-small particles that have the property of natural diffusion in the target cells [43]. NPs protect bioactive molecules from physiological degradation as well as allow controlled release of the guest. Both, organic (polymeric) and inorganic NPs are being utilized for siRNA delivery. Polymeric NPs can be synthesized using natural as well as synthetic polymers.


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Chitosan is a natural, cationic and biodegradable polymer that easily forms complexes with siRNA. Properties such as low toxicity, biocompatibility makes it the most widely used polymer [44]. In a study, chitosan nanoparticles were conjugated to poly(D,L-lactic-co-glycolic acid) PLGA nanocarriers with a high siRNA loading capacity, which showed less initial burst release of nucleic acid [45]. Solid lipid NPs (SLNs) are lipid based submicron NPs that are used as colloidal carriers for siRNA delivery. They enhance serum stability and are efficient in target gene silencing. Largely, NPs are one of the most attractive nanocarriers to improve siRNA delivery to target cells due to their particular advantages [42]. Inorganic NPs e.g. calcium phosphate nanoparticles (CaP) are also widely used for delivery due to their property to easily de-assemble endosomes [42]. Mesoporous silica nanoparticles (MSNPs) are inorganic NPs, have been used extensively for drug and siRNA delivery to the target cells. MSNPs possess large uniform pores, mostly cylindrical thus having potential to carry many small molecules. Moreover, to enhance the delivery efficiency, MSNPs can be functionalized with different groups. Properties such as adjustable pore sizes (2-30 nm), modification of surface and encapsulation of guest molecules such as drugs, proteins, as well as biogenic molecules make MSNPs potential carriers for siRNAs delivery [46]. Not only do they encapsulate the molecules, but also the controllable release is achievable in the target cells. In addition, studies have shown that mesoporous particles have less cytotoxicity and are biocompatible with mammalian cells. In one of the studies, ‘clickable’ MSNPs were conjugated with PAMAM dendrimers and then complexed with plasmid DNA (pDNA) to study the cytotoxicity and transfection efficiency in vitro. Results demonstrated comparable transfection efficiency and low cytotoxicity in comparison to PEI [47]. Li et al. (2013) synthesized PEI coated MSNPs conjugated with fusogenic peptide and studied siRNA delivery efficiency of these NPs. The group demonstrated efficient delivery of siRNA, which silenced the VEGF, thus inhibiting tumor growth in vivo [48]. Despite the advantages mentioned above, some properties of MSNPs such as poor hemocompatibility limit their potential. Hemolysis occurs due to the presence of silanol groups on

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MSNPs that interact with the phospholipids on RBCs. 4.5. Carbon Nanotubes for siRNA Delivery Carbon nanotubes (CNTs) are allotropes of carbon with a diameter of ~1-2 nm and length of 50100 nm [49]. These hydrophobic nanotubes have large loading capacity for genes, drugs and other small molecules due to high aspect ratio. Carbon nanotubes have emerged as carriers in therapeutic applications, owing to their many attractive properties. These include huge surface area, ultrahigh functionalization and loading capacities, high penetration potential to biological barriers, ideal electrical and thermal conductivities, good mechanical strength, and encapsulation and depository functions in the delivery of molecules [50]. Efficient delivery of siRNA is possible with CNTs if they are functionalized with different functional moieties and conjugated with various targeting ligands [49]. In a study, Single-walled carbon nanotubes (SWCNTs) functionalized with PEI-succinic acid (PEI-SA) were used by Siu et al. (2014) for topical siRNA delivery in murine models. Herein, they found significant gene silencing in the tumor tissue in vivo [51]. 4.6. Peptide-based Nanoparticles for siRNA Delivery The peptide or protein-based nanoparticles have been used recently for delivery of siRNA, where peptide molecules are directly conjugated to siRNA [52]. These nanoconjugates can be modified with ligands such as antibodies that enhance the uptake by target cells. Antibodies bind to the antigens present on target cells and are internalized by endocytosis, which then release their cargo i.e. siRNA into cytoplasm causing gene silencing. Another type of peptide-based delivery vehicles are the cell-penetrating peptides (CPPs). CPPs are small chain peptides composing positively charged amino acids so that they can bind to negatively charged nucleic acids. CPPs can improve intracellular routing of cargos, which are mostly attached by chemical cross-linking. Numerous amphipathic peptide-based strategies were developed for efficient siRNA in vivo [52]. Tat(48-60) (GRKKKRRQRRRPPQ) and penetratin® (RQIKIWFQNRRMKWKKC) are the two most used cell penetrating peptides that act by binding

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to the 3’ end of sense strand of siRNA [4, 53]. After incorporation into the cells, these peptides are removed due to reducing environment and the presence of disulfide bonds between siRNA and peptide [54], thus releasing siRNA into the cytoplasm. 4.7. Aptamer-siRNA Nanoconjugates for siRNA Delivery Aptamers are short nucleic acids sequences that are produced by a method known as Systematic Evolution of Ligands via Exponential Enrichment (SELEX). Aptamers are very precise and have a stable three-dimensional structure that provides specific binding with target [4]. In the peptidebased delivery, peptides are attached to siRNA through disulfide bonds while, in aptamer-siRNA complex, they are attached by direct conjugation. Small-sized aptamers are preferred because they show negligible immunogenicity in vivo, can be synthesized chemically and rapid in vitro selection makes them good candidates for delivery. Many researchers have exploited their targeting ability. McNamara et al. (2006) conjugated aptamers to siRNA in order to target prostate specific membrane antigen (PSMA). The internalized chimera caused cell death in vitro due to siRNA that targeted anti-apoptotic gene (Plk1). Further experiments in xenograft model demonstrated significant tumor regression [55]. Aptamers have also been used to silence an anaplastic lymphoma kinase (ALK) gene in anaplastic large cell lymphoma (ALCL) cells using PEI as a carrier. The results showed that these complexes containing siRNAs, aptamers, and PEI are specific to cell type and cancerous gene in ALCL cells [56]. Researchers have reported that modifications (e.g. PEGylation) are required to reduce their faster excretion and susceptibility to serum degradation. 5. APPLICATIONS OF siRNA TECHNOLOGY For decades, diseases were treated using various chemically synthesized drugs. Though these drugs are popular and are extensively used, the side effects they cause are the major limitations. Researchers have been striving to discover alternative ways to treat diseases using various mechanisms and genetic modifications. One of the concepts taking root in the recent years is silencing of


the target gene(s) responsible for a particular disease. siRNA technology is proving its potential in various fields, primarily in nanomedicine to treat various diseases such as cancer, ocular, brain, viral, heart, and many other diseases. Some of the diseases, which are a potential target for siRNA therapy, are briefly discussed below. 5.1. Cancer Therapy siRNA provides an opportunity to treat cancers in a better way as compared to the chemotherapeutic agents, due to their low inherent toxicity and high specificity. A number of studies reporting treatment of different cancers (e.g. melanoma, myeloma, neuroblastoma, solid tumors, chronic myeloid leukemia, pancreatic cancer, liver cancer, non-small cell lung carcinoma, and prostate cancer) have appeared in the literature. In one such study, liposomes (lipid-based vector) were conjugated to siRNA (referred as Atu027 drug) and used for the treatment of lymph node metastases in orthotopic mouse tumor models of prostate and pancreatic cancer. Treatment with Atu027 drug showed a reduction in tumor growth and lymph node metastasis suggesting that it is a potential, novel siRNA formulation [57]. Solid tumors are tumors that do not contain cysts or liquid areas [58]. Alnylam Pharmaceuticals have been performing phase I trials for SNALP mediated siRNA delivery (ALN-VSP02) to treat solid tumors by targeting kinesin spindle protein (KSP) and vascular endothelial growth factor (VEGF). Interim data showed that ALN-VSP02 was well tolerated in initial 28 patients at the highest dose (1.25 mg/kg) [59]. TKM-080301 is another phase I trial drug that targets Polo-like kinase 1 (PLK1) using siRNA conjugated to lipid nanoparticles to treat multiple cancers in adult patients. Apart from lipid nanoparticles, cyclodextrin-siRNA complexes have also been employed for cancer therapy. CALAA01 drug which is a cyclodextrin-based polymeric nanoparticles, target M2 subunits of ribonucleotide reductase (RRM2) [60]. 5.2. Brain Diseases Epilepsy, Schizophrenia, Parkinson and Alzheimer's disease are brain-related disorders that are potential targets for siRNA based therapy. Epilepsy, a neurodegenerative disorder has received less attention, which can now be targeted using


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siRNA mechanism. Ion channels such as voltagegated sodium channel might be some of the promising targets for epilepsy [61]. In Alzheimer disease, β -secretase (BACE1) increases due to neurodegeneration and accumulation of amyloid precursor protein (APP) thus forming plaques containing beta-amyloid (Aβ). Singer et al. (2005) carried out a study to silence genes involved in Alzheimer disease by using lentiviral vectors with siRNA targeting BACE1. Their aim was to either reduce or enhance cleavage of APP. They observed reduced level of amyloid production and behavioral deficits in mice [62]. 5.3. Viral Diseases Viral infections are a major concern affecting a large human population. RNAi mechanism has potential to treat viral infection more effectively than the drugs already present in the market. Viral infections such as Human immunodeficiency virus (HIV), Hepatitis C, Hepatitis B and Ebola virus infection have been studied using siRNA mechanism. Acquired immunodeficiency syndrome (AIDS) caused due to HIV can be targeted using siRNA, which silences genes involved in early and late steps of HIV-1 replication [63]. Liver diseases like liver cirrhosis and hepatocellular carcinoma are caused due to Hepatitis C virus (HCV). In a study, siRNA was administered into the target cells expressing Fas receptor that prevented the liver disease in mice. Fas protein expression reduced in the mice models and remained up to 10 days [64]. Apart from HCV, Hepatitis B virus infection (HBV) was also targeted using siRNA mechanism [65]. Two target sequences of HBV genome were selected from which core region (C) target was seen to be effective in inhibiting HBeAg levels (a serological marker of HBV) on a transient basis in mice models [66]. 5.4. Ocular Diseases Eye related diseases such as age-related macular degeneration (AMD), optic atrophy, nonarteritic anterior ischemic optic neuropathy, ocular pain, and dry eye syndrome have been tested using siRNA mechanism. The fibrotic eye in which corneal haze, subconjunctival scarring and inflammation occur can be reduced with the siRNA mechanism by targeting TGF-β2, reducing the inflammatory response. TβRII specific siRNA

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downregulated its expression in C57BL6 mouse models thus lessening the inflammation and matrix deposition [67]. Another eye related disease; Retinitis pigmentosa (RP) caused due to degenerating photoreceptors can be reduced by silencing mRNA of rhodopsin through siRNA. Pathological advancement of autosomal-dominant RP (adRP) has been reduced by directing siRNA against adRP mutant transcripts in COS-7 (green monkey fibroblast) cells. Results showed efficient silencing as compared to the wild-type transcript [68]. AMD is a disease that results in irreversible blindness caused due to the aberrant invasion of the blood vessel into the retina. VEGF and its receptor VEGFR1 are involved in this pathogenesis process. Reich et al. (2003) carried out an experiment to reduce the growth and vascular permeability by targeting VEGF. siRNA against human VEGF was co-injected with viruses carrying hVEGF cDNAs subretinally in mice. Results demonstrated a reduction in the hypoxia-induced VEGF levels. In addition, siRNA injected against murine VEGF, inhibited choroidal neovascularization [69]. Furthermore, siRNA targeting caspase-2 was demonstrated to treat optic nerve crush by protecting retinal ganglion cell. siRNA was administered intravitreally in adult rats after optic nerve injury thus inhibiting caspase-2 expression [70]. 5.5. Heart Related Diseases Heart diseases are leading cause of death in human population and affect both males and females. Hypercholesterolemia is a disorder in which there is an increase in the level of cholesterol leading to atherosclerosis. Lipid nanoparticles conjugated with siRNA were targeted to proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, which increases low-density lipoprotein (LDL) receptors thus reducing LDL in circulation. Specific silencing of PCSK9 mRNA and its 90% reduction in serum was observed in non-human primates during preclinical studies [71]. Restenosis is re-occurrence of narrowing blood vessels i.e. stenosis. Vessels become renarrowed after receiving treatment for a blockage in artery or blood vessels. The immune reaction between artery and stent leads to further narrowing. Sun et al. (2012) studied the effect of STAT-3 siRNA on intimal thickening in veins. STAT-3, a signal transducer and activator of transcription-3 are thought to be involved in the growth of vascular smooth muscle

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cells (VSMCs) and angiotensin-II/platelet-derived growth factor (PDGF)-induced proliferation. In vitro in rat VSMCs and in vivo studies in Wistar rats showed inhibition of proliferation and reduction in neointimal production [72]. In another study, a metal stent coated with cationized pullulan hydrogel was conjugated with the siRNA against metalloproteinase (MMP2) gene responsible for restenosis. It was then studied in vascular cells and implanted into a rabbit balloon-injured carotid artery in vivo. The results demonstrated a significant reduction in pro-MMP2 [73]. 5.6. Miscellaneous Applications Apart from pathologies briefly discussed here, many other illnesses can also be treated using siRNA technology. Transthyretin (TTR) mediated amyloidosis (ATTR) is a threatening disease caused due to mutations in TTR gene involved in carrying the retinol binding protein. These mutations lead to the formation of amyloid fibers, which subsequently deposit in heart, liver, etc. Alnylam Pharmaceuticals has been developing complexes using lipid nanoparticles carrying siRNA for the treatment of ATTR. A dosedependent reduction of TTR was observed in the serum of ATTR patients [4]. Asthma, a disease in which the patient suffers from chest tightness, coughing is caused due to environmental and genetic factors that lead to blocking of airways in the lungs. Various efforts have been taken to treat asthma among which treatment using siRNA has now taken roots. ZaBeCor has taken an initiative to develop a drug called Excellair® currently under phase II trial, which targets syk kinase using naked siRNA. The study is still ongoing [4]. Diseases like chronic obstructive pulmonary disease (COPD), cicatrix scar prevention, pachyonychia congenita, delayed graft function and acute kidney injury are also potential targets of siRNA treatment and are under clinical trials [4]. 6. CONCLUSION Development of appropriate treatment strategies for diseases that are difficult to cure is an unmet medical need and a global challenge. Existing therapies are inadequate to achieve a complete cure, especially in diseases that have a genetic basis. siRNA based silencing has now provided a way to study and silence gene function. Silencing


genes implicated in the progression of diseases may lead to cures for various cardiovascular, liver, lung, viral, eye diseases as well as cancer. The proven potential of siRNA can be realized in practice provided their delivery to the target cells is achieved. This problem has now been addressed, especially by using nano-sized delivery agents. Among these, viral delivery vehicles are found to be useful as siRNA delivery agents. However, their immunogenic, mutagenic and toxicity potential limits many practical uses. Over this backdrop, various non-viral, i.e., nanotechnologybased vectors, as discussed above, appear highly promising as efficient siRNA delivery agents. Nanocarriers based siRNA delivery techniques are still under clinical trials. Consorted efforts to resolve issues such as off-target effects, immune stimulation, endosomal trapping are necessary to bring these therapies in the clinics. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS

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Revised: December 12, 2015

Accepted: January 04, 2016