review article lipid nanoparticles as carriers for rnai...

Review ArticleLipid Nanoparticles as Carriers for RNAi against ViralInfections Current Status and Future Perspectives

Josune Torrecilla Alicia Rodriacuteguez-GascoacutenMariacutea Aacutengeles Soliniacutes and Ana del Pozo-Rodriacuteguez

Pharmacokinetic Nanotechnology and Gene Therapy Group (PharmaNanoGene) Faculty of Pharmacy Centro de InvestigacionLascaray Ikergunea University of the Basque Country UPVEHU Paseo de la Universidad 7 01006 Vitoria-Gasteiz Spain

Correspondence should be addressed to Ana del Pozo-Rodrıguez anadelpozoehues

Received 25 February 2014 Revised 14 July 2014 Accepted 14 July 2014 Published 12 August 2014

Academic Editor Kamla Pathak

Copyright copy 2014 Josune Torrecilla et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans such ashepatitis C virus (HCV) hepatitis B virus (HBV) human immunodeficiency virus (HIV) human cytomegalovirus (HCMV)herpetic keratitis human papillomavirus or influenza virus Naked RNAimolecules are rapidly digested by nucleases in the serumand due to their negative surface charge entry into the cell cytoplasm is also hampered which makes necessary the use of deliverysystems to exploit the full potential of RNAi therapeutics Lipid nanoparticles (LNP) represent one of the most widely used deliverysystems for in vivo application of RNAi due to their relative safety and simplicity of production joint with the enhanced payloadand protection of encapsulated RNAs Moreover LNPmay be functionalized to reach target cells and theymay be used to combineRNAi molecules with conventional drug substances to reduce resistance or improve efficiency This review features the currentapplication of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in thisfield

1 Introduction

Gene therapy is a relatively recent approach in the man-agement of human diseases and has resulted in an increas-ingly interest as therapeutic strategy While traditional drugtherapies involve the administration of therapeutic chem-icals that have been synthesized outside the body genetherapy tries to direct patientrsquos cells to produce and delivera therapeutic agent or to knock down the production ofundesirable molecules Gene therapy was first defined asthe administration of genetic material into a human patientwith the intent of correcting a specific genetic defect [1]This definition appeared in the first gene therapy protocolsin the early 90s related to trials that aimed to correct theeffects of some monogenic recessive diseases Nowadays theEuropean Agency of Medicines (EMA) defines gene therapyas biological medicinal products which fulfils the followingtwo characteristics (a) the active substance contains orconsists of a recombinant nucleic acid applied to human

beings in order to regulate repair replace add or delete agenetic sequence (b) its beneficial effect relates directly to therecombinant nucleic acid sequence it contains or to the resultof genetic expression of this sequence [2]

Since the first FDA-approved gene therapy experiment in1990 [3 4]more than 1900 clinical trials have been performedusing a number of techniques for gene therapy up to January2014 [5] Table 1 collects the indications addressed by genetherapy clinical trials and the number of events related to eachindication As observed in this table infectious diseases are inthe third place in the ranking with a percentage with respectto total approved clinical trials similar to that of monogenicdiseases or cardiovascular diseases

Nucleic acid-based therapy has been traditionally focusedon the use of DNA as the active substance but since thediscovery of the RNA interference (RNAi) pathway [6] RNAhas also gained great interest Among the multiple possibleapplications of the RNAi-based therapy numerous studieshave demonstrated its potential in the control and treatment

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 161794 17 pageshttpdxdoiorg1011552014161794

2 BioMed Research International

Table 1 Indications addressed by gene therapy clinical trials up to January 2014

Ranking Indication Number of trials (119899) Percentage with respect to total gene therapy clinical trials ()1 Cancer diseases 1274 6382 Monogenic diseases 178 893 Infectious diseases 164 824 Cardiovascular diseases 162 815 Healthy volunteers 52 266 Gene marking 50 257 Neurological diseases 37 198 Other diseases 35 189 Ocular diseases 31 1610 Inflammatory diseases 13 07

of different viral infections in humans such as hepatitisC virus (HCV) hepatitis B virus (HBV) human immun-odeficiency virus (HIV) human cytomegalovirus (HCMV)herpetic keratitis human papillomavirus or influenza virus[7ndash13]

A key challenge to exploit the full potential of RNAitherapeutics is their efficient delivery to the target cells Onthe one hand naked RNAi molecules are rapidly digestedby nucleases in the serum after systemic administrationon the other hand due to the negative surface charge ofRNA the entry into the cell cytoplasm is hampered Anumber of techniques have been attempted to overcomethese problems physical methods such as electroporation orhydrodynamic injection [14 15] viral vectors [16] or lipidand polymeric nanoparticles [17 18] Among them lipidnanoparticles (LNP) represent one of themost widely studieddelivery systems for in vivo application of RNAi [19 20] Theaimof this review is to collect the state of the art and the futureperspectives of the utility LNP as RNAi vectors for humanviral infections

2 RNA Interference (RNAi)

RNAi is a naturally occurring process of gene regulationpresent in plants and mammalian cells The first evidenceof the existence of this mechanism appeared in 1998 whenFire et al [6] observed in Caenorhabditis elegans that double-stranded RNAs (dsRNAs) were the basis of sequence-specificinhibition of protein expression Subsequent works demon-strated that the molecules that induced RNAi were shortdsRNAs of 21 nucleotides in length called short interferingRNAs (siRNAs) and that siRNAs were able to start theRNAi process in mammalian cells [21] The RNAi responseis activated when the dsRNA is processed by a ribonucleaseIII-like enzyme called Dicer resulting in the formation ofa siRNA The siRNA is incorporated into the RNA inducedsilencing complex (RISC) where a helicase unwinds theduplex siRNAThe resulting antisense strand guides the RISCto its complementary mRNA which will be cleaved [22]Typically there are three different types of commonly usedRNAi molecules siRNA short-hairpin RNA (shRNA alsonamed expressed RNAi activators) or microRNA (miRNA)siRNAs as mentioned before are dsRNA molecules of about

19ndash23 base pair nucleotides in length able to mediate site-specific cleavage and destruction of the targeted mRNA[23] shRNAs consist of two complementary 19ndash22 bp RNAsequences linked by a short loop of 4ndash11 These RNAsare synthesized within the cell by DNA vector-mediatedproduction [24] shRNAs can be transcribed through eitherRNA polymerase II or III The first transcript generates ahairpin like stem-loop structure and then is processed inthe nucleus by a complex containing the RNase II enzymeDrosha The individual pre-shRNAs generated are finallytransported to the cytoplasm by exportin 5 Once in thecytoplasm the complex Dicer processes the loop of thehairpin to form a double-stranded siRNA shRNAs representan important tool in the assessment of gene function inmammals and are largely used as a research tool miRNAs aresingle stranded RNAs of about 20ndash24 nucleotides This kindof RNAs acts as endogenous posttranscriptional repressors todownregulate gene expression [25] miRNAs are transcribedfromDNA as primarymiRNA (pri-miRNA) in this case pri-miRNAs are processed into precursor miRNA (pre-miRNA)by two proteins Drosha and Pasha Pre-miRNAs are thentransported to the cytoplasm and after processing by Dicerand unwinding to obtain the miRNA the following stepsare identical to those that occur with siRNA and shRNA asit is illustrated in Figure 1 Recent observations have shownthat both miRNAs and siRNAs can suppress translation ofmRNAs (in the case of an imperfect match) and can cleavetarget RNAs (in the case of a perfect match) and play adecisive role in gene and genome regulation [26] It has beenalso observed that shRNA can act via miRNA- or siRNA-likemechanism It is believed that the miRNA-like mechanism isfaster than siRNA-like mechanism because the latter acts viaperfect complementarity for a target message [27]

The main advantage of synthesized siRNAs is that thesemolecules do not need to reach the nucleus to exert effectHowever modifications are necessary to increase their sta-bility resulting in some cases in loss of siRNA function[28] there must be a balance between stability and efficacywhenmodifications are introduced in siRNAWhenmiRNAsor shRNA expression plasmids are used they must initiallyreach the nucleus of cells to be processed The resultingmolecules are then transported to the cytoplasm and finallyincorporated into theRISC for activityTherefore a limitation

BioMed Research International 3

NUCL

shRNA plasmid

Nucleus

Cytoplasm

Pre-shRNA

Pre-miRNA

pri-miRNA

siRNA

Engineeredsynthetic siRNA

mRNA target

shRNA

Unwinding

miRNA

Pri-shRNA

Exportin 5

Drosha

Drosha

Pasha

Dicer

RISC

RISC

RISC

RISC

RISC

Dicer

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

59984005

998400

3998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

Figure 1 RNAi mechanism Differences between siRNA shRNA and miRNA as therapeutic tools

of miRNA and shRNA is the need to be delivered into the cellnucleus although they have a greater durability and highersilencing capacity than siRNAs [29]

RNAi seems to play an important role in the antiviraldefense mechanism in human cells suggesting its potentialuse as therapeutic in human infectious diseases For instanceHIV-1 shows higher replication capacity in cells that have suf-fered knockout of Dicer and Drosha expression [30] In thissense the mammalian stomatitis virus achieved increasedaccumulation in Caenorhabditis elegans with defective RNAimachinery [31] It has been also observed that the interferon(IFN) pathway works in coordination withmiRNA to controlviral infections IFN-120573 can induce the expression of severalcellular miRNAs that form almost perfect nucleotide basepairmatcheswith theHCVgenomeWhen thesemiRNAs areartificially introduced the antiviral effects of IFN-120573 in HCVare reproduced and the IFN response is lost when they areexperimentally removed [32]

As mentioned above to take advantage of the potential ofRNAi as antiviral therapy effective delivery is essential in thisregard lipid-based systems are being widely used as RNAi-delivery vectors

3 Lipid Nanoparticles (LNP)

Lipid-based systems have been increasingly recognized asone of the most promising delivery systems for RNAi Lipidcarriers may be available in solid semisolid or liquid statein the form of solid lipid nanoparticles nanostructuredlipid carriers lipid drug conjugate nanoparticles liposomesor nanoemulsions These lipid-based systems were initiallydesigned to address some of the challenges of conventionaldrug delivery systems such as the increase of bioavailabilityof poorly soluble drugs among others Nowadays the appli-cation of LNP in other fields such as gene therapy has gainedattraction

RNAi-lipid-based nanocarriers are able to provide pro-tection from serum nucleases and extended circulationwhich results in a higher access to the target tissue [38]Sometimes targeting is achieved by surface modification ofnanocarriers with specific ligands to target cell populationssuch as mannan-modified nanoparticles to direct vectorsto alveolar macrophages [39] Once in the target tissuethe RNA-delivery system will be internalized by the targetcell and upon receptor-mediated endocytosis will be ableto escape from the endosomal compartment into the cellcytoplasm where RNA machinery is located while avoidinglysosomal enzymes [40] When these delivery systems areapplied to the treatment of viral infections multiadministra-tion treatment modalities are possible for improved clinicaloutcomes [41] Moreover due to their biocompatibility andtheir ease of large-scale production large batches with repro-ducible specifications are possible

31 Solid Lipid Nanoparticles (SLNs) SLNs are considered tobe one of the most effective lipid-based colloidal carriersSLNs are in the submicron size range of 50ndash1000 nm andare composed of physiologically compatible lipids recognizedas safe which are in solid state at room temperature Theyconsist of a solid lipid core surrounded by a layer of sur-factants in an aqueous dispersion with multiple potentialcombinations of lipids and surfactants [42 43] The intereston SLNs has led to the development of different types ofproduction methods (ie high-pressure homogenization)successfully implemented in pharmaceutical industry TheSLNs obtainedwith these techniques show long-term stabilityand the possibility to be subjected to commercial sterilizationand lyophilized procedures [44ndash46]

SLNs are used not only as conventional drug deliverysystems but also as carriers for therapeutic peptides pro-teins or antigens and bioactive molecules MannosylatedSLNs loaded with hepatitis B surface antigen (HBsAg) were


subcutaneously administered in vivo in mice and sustainedantibody titer was obtained these results demonstrated thepotential of SLNs as carriers for vaccine delivery against HBV[59]

As gene delivery systems SLNs have been studied overthe last years for a large number of diseases [46 60 61]and different routes of administration [62] Cationic lipidsare used to prepare SLNs due to their positive surfacecharge that interacts electrostatically with the negative chargeof the nucleic acids Our research group has developednonviral vectors based on cationic SLNs [63 64] decoratedwith peptides [65 66] dextran [60] oligochitosans [67] orhyaluronic acid [68] able to transfect several cell lines andtissues both in vitro and in vivo [69ndash71] To date vectorsthat were developed for DNA delivery are being applied forsiRNA delivery In spite of their different physicochemicalproperties and the need to be transported to different parts ofthe cell (plasmid DNA needs to be transported into nucleusfor gene expression whereas siRNA reaches its target inthe cytoplasm) a number of publications describe the useof SLNs for delivery of both DNA [39 72ndash74] and RNAimediated molecules [75ndash78] with successful results

Focusing on RNAi delivery Montana et al [78]demonstrated the utility of cationic SLNs produced bymicroemulsion using Compritol ATO 888 as matrix lipidPluronic F68 as tensioactive and dimethyldioctadecyl-ammonium bromide (DDAB) as cationic lipid as RNAcarriers In another study [76] SLNs composed ofcholesteryl ester triglyceride cholesterol dioleoylphos-phatidylethanolamine (DOPE) and 3beta-[N-(N1015840N1015840-dimethylaminoethane) carbamoyl]-cholesterol (DC-chol)were able to bind electrostatically siRNA conjugated withpolyethylene glycol (PEG) (siRNA-PEG) When comparedto polyethylenimine a commonly used transfectant polymerthe SLN-based system showed similar gene silencingefficiency and very low cytotoxicity in PC3 (human prostatecancer cell line) and MDAMB435 (human breast cancercell line) cells In a later work these SLNs containing asiRNA anti-c-Met (an oncogene overexpressed in a varietyof carcinomas) were evaluated in vivo in a glioblastomamultiform mouse model After intravenous administrationto mice the siRNA-PEGSLN system specifically crossedthe blood-brain barrier to the tumor site with no apparentsystemic toxicity Treatment significantly inhibited tumorgrowth in a dose dependent manner and a downregulationof c-Met was observed [77]

32 Nanostructured Lipid Carriers (NLCs) NLCs are solidlipid core carriers in the nanometer range composed of amixture of liquid and solid lipids that are spatially incom-patible leading to special structures with improved drugencapsulation and release properties NLCs were developedas a tool to overcome the drawbacks of SLNs and to increasethe oral bioavailability of poorly soluble compounds [79ndash83]

The choice of the lipid is critical to ensure the stabilityof the drug [79] and the structure of the lipid core matrixdetermines the classification of NLCs Imperfect NLCs aremade by mixing small amounts of liquid lipid and fatty acids

with different chain lengths as solid lipid The imperfectionsthat are generated in the lipid core due to the crystallizationincrease the drug load and reduce although not completelythe drug expulsion [84 85] Multiple NLCs are made bymixing solid lipids and an excess of liquid lipid generatingoily nanocompartments into the lipid matrix where thedrug is well accommodated [44] Finally in structurelessor also called amorphous NLCs special liquids are usedand the expulsion of the drug is avoided due to the lack ofcrystallization [85]

The application of NLCs to the treatment and preven-tion of infectious diseases has been attempted in variousstudies focused on the development of new antimalarialtreatments [86ndash88] or in the improvement of encapsulation ofhydrophilic antiretroviral drugs [89ndash91] Although there arefew reports about the application of NLCs as RNAi deliverysystems they could be an interesting alternative in codeliverystrategies with conventional drugs For instance Taratula etal [92] have recently described amultifunctionalNLC systemto improve the efficacy of anticancer drugs in lungs Thesystem was composed of an anticancer drug (doxorubicinor paclitaxel) joint with siRNA to avoid cellular resistance(siRNA targeted to MRP1 mRNA as a suppressor of pumpdrug resistance and siRNA targeted to BCL2 mRNA as asuppressor of nonpump cellular resistance) and an analog ofluteinizing hormone-releasing hormone as a targetingmoietyto lungs The inhalation of the NLCs by an orthotopic micemodel of human lung cancer resulted in efficient suppressionof tumor growth and prevention of adverse side effects onhealthy organs

33 Lipid Drug Conjugates (LDCs) LDCs are the mostaccepted lipid-based nanoparticle systems for the delivery ofhydrophilic drugs as they improve the drug loading capacityof SLNs and NLCs In LDC nanoparticles hydrophilic drugsare first conjugatedwith lipid components by covalent linkingbetween an amino group or a hydroxyl group of the drugand carboxyl groups of the lipid core to obtain a lipophiliccomplex [93] The water insoluble LDC is converted tonanoparticles by means of traditional methods used toprepare SLNs or NLCs [94 95]

LDCs have shown hopeful results as delivery systemfor hydrophilic antitrypanosomal drugs [96 97] for oralapplication of methotrexate [94] and more recently forchemotherapy agents [1 95] such as decitabine a drugthat shows low oral bioavailability [98] Neupane et al [99]developed LDC nanoparticles of decitabine to increase itspermeability and protect against chemical degradation LDCswere obtained by salt formation of DCB with stearic acid tobe formulated as LDC nanoparticles by cold high-pressurehomogenization after addition of surfactants Tween 80Poloxamer 188 and Labrasol Ex vivo gut permeation studiesproved that the drug in LDC nanoparticles showed nearlyfourfold increase in the apparent permeability coefficientswith respect to the plain decitabine

34 Cationic Emulsions Emulsions are dispersions of oneimmiscible liquid in another stabilized by a third component


the emulsifying agent [100] therefore they present in theircomposition three components oil water and surfactantsWhen cationic surfactants are used these dispersed sys-tems make them suitable for gene delivery The presenceof cationic surfactants causes the formation of positivelycharged droplets that promote strong electrostatic interac-tions between emulsion and the anionic nucleic acid phos-phate groups Cationic emulsions composed of cationic lipidsand core oil have been shown to be useful for gene delivery[101 102] The colipid DOPE is largely used to improve theability of cationic emulsions and liposomes to transfect cellsdue to its fusogenic propertiesThis can be partially explainedby the fact that the amine group of DOPE interacts withDNA phosphate groups thus weakening the binding affinitybetween cationic lipids and DNA [100]

In spite of the advantages of nanoemulsions for deliveryof nucleic acids only few attempts have been made to usethis new delivery system for RNAi For instance Kanedaet al [103] showed the potential application of cationicnanoemulsion preparedwithDOTAPDOPE and cholesterolfor siRNA delivery Transfection complexes with a meanparticle diameter of approximately 300 nm were able tosuppress endothelial cell expression of upregulated vascularadhesion molecules To the knowledge of the authors nostudies about cationic emulsions with RNAi molecules totreat viral infections have been published

35 Liposomes Liposomes are colloidal lipid- and surfactant-based delivery systems composed of a phospholipid bilayersurrounding an aqueous compartment They may present asspherical vesicles and can range in size from 20 nm to a fewmicrons Cationic lipid-based liposomes are able to complexwith negatively charged nucleic acid via electrostatic interac-tions resulting in complexes that offer biocompatibility lowtoxicity and the possibility of large-scale production requiredfor in vivo clinical applications [104] The lipid to RNAratio and overall lipid concentration used in forming thesecomplexes are very important for efficient gene delivery andvary with applications Liposomes can fuse with the plasmamembrane for uptake once inside the cell the liposomesare processed via the endocytic pathway and the geneticmaterial is then released from the endosomecarrier into thecytoplasm Compared to polymeric nanoparticles liposomeshave long been perceived as better drug delivery vehiclesbecause of their superior biocompatibility as liposomes arebasically analogues of biological membranes which can beprepared from both natural and synthetic phospholipids[104]

Neutral lipids are highly nontoxic and do not acti-vate an immune response [105] 12-Oleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPE are among the mostwidely used neutral lipids Simply mixing siRNA with DOPCresults in high encapsulation efficiency [106] However neu-tral liposomes yield relatively low transfection efficiencyCationic lipids such as 12-dioleoyl-3-trimethylammonium-propane (DOTAP) can complex electrostatically with siR-NAs and be used to create a more effective liposome as the

positively charged lipids provide enhanced cell entry andincreased protection against serum enzymes [107]

Although liposomes are one of the most commonly usedtransfection reagents in vitro safe and efficacious delivery invivo is more difficult to achieve due to toxicity nonspecificuptake and unwanted immune response Much of the non-specific response and toxicity is directly linked to the positivecharge on the surface of the particles necessary for the bind-ing of oligonucleotides In order to improve their behavior inrecent years significant effort has been dedicated to modify-ing the composition and chemical structure of liposomes Forinstance different additives such as hydrophobic cholesterolnonionic surfactants or PEG can be used to enhance invivo stability after exposure to blood components [108]It has been reported that the stability-enhanced liposomeshave much better transfection efficiencies especially underin vivo conditions [109] To increase specificity PEGylatedimmunoliposomes conjugated with targeting ligands havebeen developed [110]

Liposomes have been widely studied as RNAi carriers aspotential treatment of viral infections such as HCVHBV andVIH among others [33 34 48 49 111]

4 Lipid Nanoparticles as RNAi Carriersagainst Viral Infections

As mentioned above an effective delivery system will haveto be developed for exploiting the gene silencing by RNAinterference strategy for antiviral therapy The applicabilityof LNP for the treatment of viral infections is emerging inthe last years On the one hand the use of LNP as nonviralnanocarriers of gene material is the most studied approachIn this sense some researchers suggest the simultaneousadministration of different siRNAs or shRNAs to avoid theimportant payload of viral mutation escape [112] On theother hand the prophylaxis for viral infections using RNAihas been considered a promising strategy

The RNAi process requires a high specificity of genesequences and the choice of the target is the most crucialstep for a successful viral inhibition It is very important todesign siRNA against very highly conserved sequences ofthe viral genome in order to optimize efficacy in inhibitinga majority of virus straints In addition to its utility as astand-alone strategy RNAi may have expanded applicationsas an adjuvant in multipronged treatment settings AnotherRNAi adjuvant strategy is the use of dsRNA oligonucleotidesas immunostimulatory agonists alongside vaccines as in thecase of a RIG1 agonist to enhance the activity of a DNAvaccine against influenza [113]

41 Hepatitis C Virus (HCV) According to theWorld HealthOrganization (WHO) every year 3-4 million people areinfected with the HCV and about 150 million people arechronically infected which can lead to liver cirrhosis andorhepatocellular carcinoma (liver cancer) In consequencemore than 350000 people die from hepatitis C-related liverdiseases every year [114]


Combination of generic antiviral agents IFN-120572 andribavirin is the mainstay of current hepatitis C treatmentUnfortunately IFN-120572 is not available in some countriespeople do not always tolerate well this drug and manypeople do not finish their treatment In addition some virusgenotypes do not respond well to IFN-120572 [115] Recently newantiviral drugs telaprevir or boceprevir have been added toset up the so-called triple therapy the experience in patientswith chronic hepatitis C genotype 1 shows that this newcombination is superior to dual therapy in terms of sustainedvirologic response [116] However genotype independentalternatives should be more effective in the treatment andprevention of this liver infection In this regard the RNAitechnology is an attractive strategy

HCV is a RNA virus belonging to Flaviviridae familyThe single-stranded RNA genome of this virus acts alsoas mRNA which makes it an attractive target for RNAi-based therapy Several works show potent HCV replicationinhibition by using chemically synthesized siRNAs targetedagainst sequences in the protein-coding regions of core E2NS3 NS5B or NS4 [117ndash119] However these viral codingsequences suffer variations among different HCV genotypesTherefore highly conserved regions such as 51015840 untranslatedregions (51015840UTR) seem to be better targets for developing arational antiviral strategy [120] The replication of differentHCV genotypes has been inhibited by targeting 51015840UTR withsynthetic siRNA or shRNA expression [121ndash123] The mainlimitation of all these new strategies lies in the emergence ofresistant virus variants due to the high specificity of RNAi andthe prolonged treatment In order to prevent escape mutantssome approaches have been proposedThe internal ribosomeentry site (IRES) in the 51015840UTR is required for the synthesis ofviral proteins and therefore for viral replication Mutationsin these structures might lead to loss of function [9] makingIRES an excellent target for anti-HCV drugs which mightprevent viral escape it has also been targeted by the RNAitechnology [124] Another possibility to reduce resistantvariants is the combination of two or more RNAi moleculeswith different specificities [125] targeted to separated regionsof the HCV genome Targeting multiple sites of the HCVgenome and host genes necessary for virus replication hasalso been documented as a valid approach to prevent thedevelopment of resistance [126 127]

Among the RNAi delivery systems against HCV cationiclipid-based nanoparticles play an interesting role these lipid-based vectors are well characterized as nanocarriers forsystemic delivery of RNAi molecules to the liver due totheir safety profile and simplicity of production Table 2summarizes the strategies employed by different authors toimprove the efficacy of lipid nanosystems in RNAi-mediatedtherapies against HCV

Watanabe et al [33] designed lactosylated cationic lipo-somes to deliver siRNA targeting the 51015840-UTR and 31015840-UTR ofthe HCV genome into mouse liver hepatocytes the galactoseterminus of lactose is a ligand of the asialoglycoproteinreceptor which is specifically expressed in the surface ofhepatocytes Intravenous administration of the mentionedvectors into HCV-transgenic mice resulted in a decrease ofHCV core protein

Another possibility is the functionalization of LNP withapolipoproteins Kim et al [34] developed liver-specificsiRNA delivery vectors composed of cationic liposomes andapolipoprotein A-1 (apo A-1) derived from human plasmaThis protein component of high density lipoprotein (HDL)has been proposed as a targeting ligand to hepatocytes [48]After intravenous administration of the lipid-based systemscontaining HCV-core specific siRNA into an HCV mousemodel viral expression was inhibited by 65ndash75 in the liveron day 2 In the same work the chemical modification ofthe HCV-core specific siRNA to increase its serum stabilityresulted in gene silencing efficacy up to 95 for at least6 days In order to avoid safety problems related to therisk of pathogen contamination associated with the use ofplasma-derived protein in a posterior work [35] apo A-1was substituted by a recombinant human apo A-1 (rhapoA-1) This new apolipoprotein of low endotoxin grade wasexpressed and purified from an Escherichia coli expressionsystem The use of rhapo A-1 to deliver siRNA to the liverresulted as effective and selective as plasma-derived apo A-1without affecting the normal liver function

The choice of an adequate RNAi molecule is also cru-cial HCV replication has also been inhibited both in vitroand in vivo by delivery of different siRNAs with lipidnanosomes composed of the cationic lipidDOTAP the helperlipid cholesterol and the peptide protamine Nanosomesare nanosize unilamellar vesicles obtained by high-pressurehomogenization of lipid dispersions [36 128] In a firstwork nanovectors were optimized in terms of lipid-to-siRNAratio and favorable particle size depending on sonicationtime Cell viability was maintained about 90 and HCVinhibition reached approximately 85 [128] Later on thecationic lipid nanosomes were used to complex differentsiRNA targeted to the 51015840UTR of the HCV genome Chandraet al compared repeated treatments with two-siRNA versusa single siRNA treatment A reduction in the development ofresistant mutants to the siRNA therapy was observed whenthe combinatorial strategy was used and after systemic injec-tion in a liver tumor-xenotransplant mouse model of HCVsignificant inhibition of virus replication was obtained [36]More recently short synthetic shRNAs (sshRNAs) that targeta sequencewithin IREShave been formulated into LNPby theprocess of step-wise ethanol dilution and spontaneous vesicleformation sshRNA was dissolved in an aqueous solutioncontaining 30 ethanol and added to preequilibrated LNPat 35∘C After reaching the final sshRNA to lipid ratio themixture was incubated for further 30min at 35∘C to allowvesicle reorganization and encapsulation of the RNA FinallyLNP were dialyzed against PBS and filter sterilized through a02 120583m filterThe intravenous injection of this vector resultedin enough uptake by the hepatocytes to substantially suppressgene expression in a rapid and durable manner [37]

42 Hepatitis B Virus (HBV) WHO estimates that about600000 people die every year due to the consequences ofHBV infection (mainly cirrhosis of the liver and liver cancer)A vaccine against hepatitis B has been available since 1982Hepatitis B vaccine is 95 effective in preventing infection


Table 2 Studies carried out with lipid-based nanosystems as RNAi delivery vectors against hepatitis C (HCV)

Lipid nanosystem RNAi Targetingmolecule Culture cells In vivomodel Reference

Cationicliposomes

siRNAs against the51015840-UTR and 31015840-UTR ofthe HCV genome

Lactosylated-PE FLR3-1 andR6FLR-N cells CN2-29 transgenic mice [33]

Cationicliposomes

HCV-core specificsiRNA (siHCc) Apo A-I Huh7

HCV mouse model constructedby hydrodynamic injection ofDNA plasmid expressing viralproteins

[34]

Cationicliposomes

HCV-core specificsiRNA (siHCc)

Recombinanthuman apo A-I mdash

HCVmouse model constructedby hydrodynamic injection ofDNA plasmid expressing viralproteins

[35]

Cationicnanosomes

siRNAs against htestem-loop domainsIIndashIV of HCV 51015840UTR

mdash Huh-75 andR4-GFP cells

HCC tumor-xenograft micemodel for HCV [36]

Cationic LNP sshRNA targeting theHCV IRES mdash mdash

Reporter mice that express in theliver firefly luciferase under thecontrol of the HCV IRES

[37]

Lactosylated-PE lactosylated-phosphatidylethanolamine FLR3-1 cells HuH-7 cells bearing anHCV subgenomic replicon (genotype 1b) R6FLR-N cells HuH-7 cells bearing an HCV subgenomic replicon (genotype 1b) CN2-29 transgenic mice mice that carry an HCV transgene Apo A-I apolipoprotein A-I Huh7cells human hepatoma cell line Huh75 cells Huh7 cells that contain a mutation in RIG-I believed to be responsible for the improved replication of HCV R4-GFP cells IFN-120572-resistant HCV-GFP chimer replicon cell line HCC hepatocellular carcinoma LNP lipid nanoparticles IRES internal ribosome entry site

but offers scarce therapeutic benefit to chronic carriers[129] Some people with chronic hepatitis B are treated withdrugs including IFN nucleotidenucleoside analogues andimmunomodulators but there is no specific treatment foracute hepatitis B [130] Moreover current therapies havelimited efficacy are expensive produce side effects andare associated with viral resistance [131] The emergence ofresistance has been reduced by new antivirals (entecavir ortenofovir) but patients usually need to take these drugs forlife because interruption of treatment quickly reactivatesviral replication [132] Therefore there is need for findingeffective therapies againstHBVwith the use of RNAi being anattractive possibility RNAi has also been studied as a possiblevaccine against HBV [133] but its main application is thetreatment of chronic HBV infection

HBV viron contains a partly double-stranded relaxedcircular DNA (rcDNA) that is encapsidated by core proteinsand enveloped with S proteins and membrane lipids fromthe host to form viral particles [134] When a hepatocyte isinfected endogenous repair enzymes convert rcDNA in a fullydouble-stranded circular and supercoiled DNA (cccDNA)which serves as template for transcription of HBV RNAExpression of viral proteins and viral replication may bepotentially knocked down by RNAi-based therapeutics

RNAi activators used against HBV include expressedor engineered synthetic RNAi intermediates each classof silencing molecules has advantages and disadvantagesExpressed anti-HBV sequences as plasmids that producespecific siRNAs [135] or as shRNA [136ndash140] have demon-strated efficacy in vitro and in vivo and they achieve moresustained silencing effect whereas synthetic siRNAs requirerepeated administration to provide a long-term suppressionof HBV replication [141ndash143] However expressed RNAi

activators are complex in terms of delivery and dose controlwhich have led to the development of chemically modifiedsynthetic siRNAswith the aimof improving silencing efficacyspecificity for a particular target mRNA avoidance of innateimmunostimulation stability and delivery to target tissue[51 144]

The delivery of these RNAi molecules against HBV hasbeen addressed by several research groups by means ofcationic lipid-based systems recapitulated in Table 3 In anearly study Morrissey et al [47] entrapped a couple of chem-ically modified synthetic anti-HBV siRNAs in stable nucleic-acid-lipid particles (SNALPs) SNALPs consist of a lipidbilayer composed of cationic and fusogenic lipids which arecoated with a PEG-lipid Inner lipids enable cellular uptakeand endosomal release of the molecules payload whereasthe coating stabilizes the particles during formulation andshields the particles in vivo to avoid rapid systemic clearanceFollowing administration the PEG-lipid dissociates fromthe nanoparticles and the SNALPs become a transfection-competent entity [145] After intravenous administration ofSNALPs-siRNAvectors tomice carrying the replicating virusencapsulated siRNA presented a longer half-life in plasmaand liver than nonencapsulated Sustained specific reductionin HBV titers for up to 7 days and reduced toxic andimmunostimulatory side effects were achieved [47] Likein the case of HCV in order to improve liver-targetingapo A-1 has been combined with the cationic lipid DOTAPand the helper lipid cholesterol to form lipoplexes withsiRNAs against HBV [48] apo A-1 was incorporated into theformulation by reassembling the liposomes with a solution ofthe protein at 4∘C overnight Intravenous injection of theselipoplexes into a HBV mouse model significantly reducedviral protein expression during at least 8 days in only a single


Table 3 Studies carried out with lipid-based nanosystems as RNAi delivery vectors against hepatitis B (HBV)


SNALPHBV siRNAs chemicallystabilized for nucleaseresistance

mdash HBV-replicatingHepG2

HBV mouse model constructedby hydrodynamic injection ofHBV vector DNA

[47]

Cationic liposomes HBV-X specific siRNA(siHBV) Apo A-I HepG2 and Huh7

Acute HBV-infected mousemodel by hydrodynamicinjection of a plasmid

[48]

PEGylated cationicliposomes HBV specific siRNA mdash

Huh7 cellspreviouslytransfected withHBV replicationtarget plasmid

HBV transgenic mice [49]

DODAG 8 lipid HBV specific siRNA mdash mdash HBV transgenic mice [50]

Cationic liposomes Altriol modified HBV-XsiRNA Galactose

Huh7 cellspreviouslytransfected withHBV target DNAplasmid


SNALP stable nucleic acid lipid particle HepG2 liver hepatocellular carcinoma cells HuH7 human hepatoma cell line DODAG11987310158401198731015840-dioctadecyl-N-48-diaza-10-aminodecanoylglycine amide

treatment In two subsequent studies lipid-based systemsdemonstrated more effective or comparable inhibition ofviral proliferation than lamivudine a licensed HBV drug[49 50] In one case [49] lipoplexes were prepared by addingan aqueous solution of siRNA targeting conserved regionsof the HBV genome to a dispersion of lipid vesicles withconstant vortex mixing Thereafter a PEG-lipid was addedto the lipoplexes and the mixture was incubated for 16 hThese vectors were also lyophilized in presence of trehalosefor long-term storage In the second case [50] the siRNAtargeting conserved regions of the HBV genome was com-bined with the cationic lipid N1015840N1015840-dioctadecyl-N-48-diaza-10-aminodecanoylglycine amide (DODAG) under condi-tions of rapid vortex mixing to produce siRNA-DODAGnanoparticles More recently a liver-targeting cholesterolgalactoside was incorporated into lipoplexes to obtain analtriol-modified siRNA delivery system [51] The galactose-conjugated cholesterol was synthesized by a cooper-mediatedldquoclickrdquo reaction between the 2-propynylcarbamate derivativeof cholesterol and O-tetraacetate galactose azide This lipidwas mixed with a nucleic acid binding cholesterol derivativeand the helper lipid to obtain liposomes capable of bindingthe siRNA The improved hepatotropism and attenuatedimmunostimulatory properties of the vectors demonstratedthat galactose functionalization has also potential for deliveryof RNAs to hepatocytes in the treatment of hepatic viralinfections and other liver diseases

43 Human Immunodeficiency Virus (HIV) HIV continuesto be a major global public health issue having claimedmore than 36 million lives so far In 2012 approximately353 million people lived infected by HIV [146] Althougheffective treatment with antiretroviral drugs can control the

virus there is no cure for HIV infection therefore novel andmore promising strategies must be developed

HIV belongs to retroviruses which are viruses thatcontain RNA as genetic material Throughout its life cyclethe RNA of HIV is not protected during viral uncoatingand reverse transcription At this point therapeutic RNAimolecules may interact with viral RNA and blockage orreduce HIV infection Synthetic and expressed RNAi havebeen evaluated preclinically to target HIV-encoded RNAsuch as rev nef or integrase [147ndash149] or host factors mainlythe chemokine receptor 5 (CCR5) [111 150 151] a promisingtarget because it is apparently not important for human phys-iology [152] Some of these targets have also been evaluated inthe clinical practice by using lentiviral or ex vivo strategiesbut the drawbacks related to immunogenicity of the vectorsandor inconveniences for patients call for new deliverysystems [153ndash156] In addition as with other viruses whenonly one RNAi is targeted escape mutants can be generated[157] targeting multiple viral targets combinations of hostand viral proteins even combining host viral proteins andclinically approved antiretroviral drugs have demonstratedimproved efficacy against HIV [152 158ndash160]

In the field of vaccines against HIV RNAi may also playan important role On the one hand RNAi technology hasbeen proposed as a strategy to block genes related to thesuppression of immune responsemediated by DNA vaccineswhich are limited in nonhuman primates and humansprobably due to the relative brief duration of vaccine antigenexpression in vivo After intramuscular (im) administrationof plasmid DNA in mice an adaptive immune responsethat mediates the apoptotic destruction of vaccine antigen-expressing myocytes was detected [161] The use of a shRNAtargeted to caspase-12 a cell death mediator activated afterplasmid DNA vaccination resulted in increased HIV-gp120


Env antigen expression and higher CD8 T cell and antibodyresponses [162] On the other hand siRNA can be usedin a prophylactic manner For instance siRNAs designedto knock down CCR5 andor viral genes in CD4+ T cellsmacrophages and dendritic cells have shown protectioncapacity against HIV vaginal transmission when appliedintravaginally to humanized mice [163] In this regard LNPmay be the delivery system of choice as they have demon-strated adjuvant properties for HIV vaccine Anionic SLNsprepared with an emulsifying wax and coated with the HIV-Tat (transactivator of transcription) protein and administeredsubcutaneously twice to mice at an interval of 2 weekselicited IgG and IgM responses similar to the commonly usedadjuvant Alum and a higher release of IFN-120574 in splenocytes[164] In addition anti-Tat IgG titers obtained with Alumcarrying Tat were lower than those obtained with a reduceddose of the peptide adjuvanted with the SLN [165] Otheradministration routes have also been explored Intradermaland intranasal administration in mice of carnauba wax basedSLN coated with HIV gp140 antigen and toll-like receptor-9(TLR-9) yielded higher systemic andmucosal immunity thanthe antigen alone [166] In another work liposomes bearinganti-CCR5 siRNA were functionalized with the monoclonalantibody (mAbd) against lymphocyte function-associatedantigen-1 (LFA-1) integrin After intravenous administrationinto humanized mice leukocyte-specific gene silencing wasobtained during 10 days and when challenged with HIVplasma viral load and loss of CD4 T cells were reduced [111]

44 Other Viral Infections Although HCV HBV and HIVare the most studied RNAi with lipid formulations hasalso been applied to the potential treatment of other viralinfections such as herpes simplex virus Ebola virus humanpapillomavirus (HPV) or rabies virus among others

Herpes simplex virus-2 (HSV-2) infection causes sig-nificant morbidity and is an important cofactor for thetransmission of HIV infection In a study carried out byPalliser et al [167] seven siRNAs targeting three essentialHSV-2 genes (UL5 (a component of the helicase-primasecomplex) UL27 (envelope glycoprotein B) and UL29 (aDNA-binding protein)) were prepared and assayed for viralprotection siRNAs lipoplexes were efficiently taken up byepithelial and lamina propria cells and silenced gene expres-sion in the mouse vagina and ectocervix for at least ninedays Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes was well tolerated did not induceIFN-responsive genes or cause inflammation and protectedmice when administered before andor after lethal HSV-2challenge In a later study [168] one of the viral siRNAswas combined with a siRNA targeting the HSV-2 receptornectin-1 Cholesterol-conjugated- (chol-) siRNAs silencedgene expression in the vagina without causing inflammationor inducing IFNs The viral siRNA prevented transmissionwithin a day of challenge whereas the siRNA targeting theHSV-2 receptor nectin-1 protected for a week but protectionis delayed for a few days until the receptor is downmodulatedCombining siRNAs targeting a viral and host gene protected

mice from HSV-2 for a week irrespective of the time ofchallenge

Ebola virus (EBOV) infection causes a frequently fatalhemorrhagic fever that is refractory to treatment with cur-rently available antiviral therapeutics Geisbert et al [169]prepared four siRNAs targeting the polymerase gene of theZaire species of EBOV (ZEBOV) and complexed them withpolyethylenimine or formulated them in SNALPs Guineapigs were treated with these siRNAs either before or afterlethal ZEBOVchallenge Treatment of guinea pigswith a poolof the L gene-specific siRNAs delivered by polyethyleniminepolyplexes reduced plasma viremia levels and partially pro-tected the animals from death when administered shortlybefore the ZEBOV challenge Evaluation of the same poolof siRNAs delivered using SNALPs proved that this systemwas more efficacious as it completely protected guinea pigsagainst viremia and death when administered shortly afterthe ZEBOV challenge Additional experiments showed that 1of the 4 siRNAs alone could completely protect guinea pigsfrom a lethal ZEBOV challenge In a later study [170] thesame research group assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal nonhumanprimate model of ZEBOV haemorrhagic fever Two (66)of three rhesus monkeys given four postexposure treatmentsof the pooled anti-ZEBOV siRNAs were protected fromlethal ZEBOV infection whereas all macaques given sevenpostexposure treatments were protected The treatment waswell toleratedwithminor changes in liver enzymes thatmighthave been related to viral infection From results obtainedin these studies in January 2014 a Phase I clinical trialcommenced as it will be mentioned in Section 5 of thisreview

The human papillomavirus (HPV) is the etiologic agentof cervical cancerThe E7 gene is a promising target for RNAiapplication in the management of HPV infection [171] Itplays an important role in cell-cycle regulation Moreover E7inhibits the therapeutic effect of antiviral agents (such as IFN-120572) and suppresses local immunity through functional inhibi-tion of antigen-presenting cells and cytotoxic T lymphocytesE7 gene silencing in high risk HPV types such as HPV-16 and HPV-18 by siRNAs suppressed cell growth in vitroand in vivo siRNA duplexes or shRNA-expressing plasmidstargeting the E7 genes of HPV-6b or HPV-11 were inoculatedinto cultured E7-expressing cells via cationic liposomes orinto E7 gene-expressing mouse tumor models intratumorallyor intravenously [170] Both siRNAs and shRNA-expressingplasmids reduced in vitro the mRNA levels of HPV-6b orHPV-11 E7 to 20ndash40 E7mRNAexpression in tumormodelswas reduced to 45ndash50 after three intratumoral injectionsIntratumoral injections of RNAi effectors induced greaterinhibition than did intravenous injections

Rabies virus (RABV) infection continues to be a globalthreat to human and animal health yet no curative ther-apy has been developed siRNAs that target the conservedregion of the RABV challenge virus standard (CVS)-11strain nucleoprotein gene represent a promising approachfor treating RABV infections [172] Using a plasmid-basedtransient expression model these siRNAs were capable ofsignificantly inhibiting viral replication in vitro and in vivo


They effectively suppressed RABV expression in infectedbaby hamster kidney-21 (BHK-21) cells as evidenced bydirect immunofluorescence assay viral titer measurementsreal-time PCR and Western blotting In addition liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival compared to anonliposome-mediated delivery method

siRNA immunoliposomes have also been shown as atherapeutic agent against H5N1 influenza virus infectionIn a recent study [173] siRNA specific for influenza virusnucleoprotein (NP) mRNAwas employed as the key antiviralagent to inhibit viral replication A humanized single-chainFv antibody (huscFv) against the hemagglutinin (HA) ofH5N1 highly pathogenic avian influenza virus (HPAI) wasused as the targeting molecule to HA of H5N1 virus whichis abundantly expressed on the surface of infected cells(the HA target cells) The immunoliposomes were shown tospecifically bind HA-expressing Sf9 cells and demonstratedenhanced siRNA transfection efficiency Furthermore thesiRNA silencing effect was more pronounced when theimmunoliposomes were administered 6 to 12 h after H5N1infection in MDCK cells compared with the nontargetedliposomes

In a recent study [174] liposomes were used to deliver aself-amplifying RNA vaccine for respiratory syncytial virus(RSV) The vaccine potently induced neutralizing antibodiesin cotton rats as well as antigen-specific IFN-120574-producingCD4+ and CD8+ T cells in mice These responses werecomparable to or exceeding those elicited by RNA deliveredby viral particles or electroporation of pDNA and providedprotection against subsequent RSV infection

5 Clinical Development of LNP-RNAi forViral Infections

The exhaustive work undertaken in preclinical studies (bothin vitro in culture cells and in vivo in animals) has shown thatRNAi therapeutic against viral infections is highly effective atreducing virus replication and also useful as a tool to rapidlyidentify novel antiviral drug targets via large-scale screens fora number of viral infections [175] Several novel viral targetshave been identified and are the subject of intense researchand development but definitive evidence is lacking fromwell-controlled studies that demonstrate the effectiveness inthese infection diseases [176] Therefore the next logical stepmust consist of human clinical trials that depict the realisticpossibilities of this kind of therapy in viral infections

The number of RNAi-based clinical trials for viral infec-tions has grown over the past several years and has includedstudies against respiratory syncytial virus (RSV) HCV HBVHIV and Ebola virus [177] some of them using lipid-basedsystems as vectors Table 4 collects the clinical trials evolvingthe application of RNAi molecules to the treatment of viralinfections

Alnylam Pharmaceuticals Inc has carried out clinicaltrials with a naked siRNA against RSV nucleocapsid (ALN-RSV01) A Phase IIb study in adult lung transplant patients

showed that this candidate is a promising alternative for RSV-induced bronchiolitis obliterans syndrome (BOS) that causessignificant morbidity and mortality in this group of patientsmoreover the treatment with the siRNA was safe and welltolerated and it was associatedwithmore than 50 reductionin the incidence of new or progressive BOS at days 90 and 180[52]

The international clinical-stage biopharmaceutical com-pany Santaris Pharma AS has developed an anti-miRNAdrug candidate currently in clinical testing (Phase II) fortreatment of HCV infections (Miravirsen SPC3649) Thisdrug acts against MiR-122 a liver-specific miRNA that theHCV requires for replication [53] Data from the Phase IIatrial showed that Miravirsen was well tolerated by patientswith chronic HCV genotype 1 given as weekly subcutaneousinjections over 4 weeks Antiviral activity was continued andprolonged well beyond the end of active therapy These dataprovide clinical evidence about the potential of Miravirsen asonce weekly monotherapy for chronic HCV [54] Althoughin a less advanced state Benitec Biopharma Ltd with itssubsidiary Tacere Therapeutics has designed a shRNAi-based multicassette vector called TT-034 which targets 3well-conserved regions ofHCV simultaneously thus prevent-ing generation of drug-resistant mutants In addition thetargeted regions are conserved across all genotypes RecentlyBenitec Biopharma Ltd has announced that the first patientenrolled in the ldquofirst in manrdquo Phase III clinical trial for TT-034 has received the first dose [55]

The biopharmaceutical company Arrowhead ResearchCorporation has recently presented data on the Phase Iclinical study of ARC-520 the companyrsquos clinical candidatefor the treatment of chronic HBV infection ARC-520 is asiRNA-based therapeutic composed of a hepatocyte-targetedpeptide (NAG-MLP) that promotes the endosomal escape ofthe liver tropic HBV cholesterol-siRNA Although additionalblinding results are still missing initial results in 36 healthyvolunteers receiving different doses indicate that ARC-520 iswell tolerated at doses expected to be efficacious in patientswith chronic HBV [178] and a Phase IIa clinical trial hasrecently begun [56] The study is planned to enroll up to16 chronic HBV patients in two-dose cohorts with patientsreceiving either ARC-520 or placebo in combination withentecavir The study is designed to evaluate the depth andduration of hepatitis B surface antigen (HBsAg) declineamong other measures in response to a single dose of ARC-520

In the case of HIV an ex vivo silencing approach has beendeveloped by the company Calimmune by using shRNAlicensed by Benitec Biopharma T cells are extracted fromHIV patients the gene that codes for the CCR5 receptorprotein is silenced ex vivo and re-injecting the modified cellsresistance to HIV is conferred to the patients In its Phase lllclinical trial of the treatment the enrolment of the first cohortof patients which corresponds to the group that will receivemodified CD4+ T cells and CD34+ stem cells is about to becompleted [57]

Finally siRNA delivery against Ebola virus is also beingunder subject of a Phase I clinical trial recently initiated Tek-mira Pharmaceuticals has developed a therapeutic product


Table 4 Clinical trials evolving the application of RNAi in the treatment of viral infections

Clinical candidate Targeting virus RNAimolecule Clinical phase Company Reference

ALN-RSV01 RSV siRNA IIb Alnylam Pharmaceuticals Inc [52]Miravirsen SPC3649 HCV antimiRNA IIa Santaris Pharma AS [53 54]TT-034 HCV shRNA IIIa Benitec Biopharma Ltd [55]

ARC-520 HBV siRNA II Arrowhead ResearchCorporation [56]

CCR5 negative cells(generated ex vivo) HIV shRNA III Calimmune and Benitec

Biopharma Ltd [57]

TKM-Ebola Ebola virus siRNAs I Tekmira Pharmaceuticals [58]CCR5 chemokine receptor 5 RSV respiratory syncytial virus HCV hepatitis C virus HBV hepatitis B virus HIV human immunodeficiency virus siRNAshort interfering RNA miRNA microRNA shRNA short hairpin RNA

composed of a combination of modified siRNAs targetingthe Zaire Ebola polymerase viral protein (VP) 24 and VP35formulated in LNP (TKM-Ebola) The Ebola Phase I clinicaltrial is a randomized single-blind placebo-controlled studyinvolving single ascending doses and multiple ascendingdoses of the lipid nanoparticle based formulation (TKM-Ebola) The study will assess the safety tolerability andpharmacokinetics of administering TKM-Ebola to healthyadult subjects [58]

6 Conclusions and Future Perspectives

As summarized in this review RNAi is a promising ther-apeutic strategy due to its ability to silence any gene witha known sequence However the use of RNAi in the clinicis limited by the complexity of effective and well-controlleddelivery in vivo Naked RNAs have potential toxicities suchas saturation of the innate RNAi machinery stimulationof the immune response and off-target effects Moreoversystemic administration of RNAs encounters several obsta-cles that reduce their therapeutic efficacy they are highlyunstable intravascularly with a short half-life due to theirsusceptibility to serum nucleases and rapid renal clearanceConsequently RNAs do not accumulate in target tissues andcannot readily cross target cell membranes to access theircytoplasmic site of action In addition in the field of viralinfections it is necessary to find out new specific and effectivetargets and alternative combined targets to avoid mutantescapes

The efforts to achieve a realistic clinical application ofRNAi as therapeutic for viral infections should be focusedmainly on three areas reducing toxicity of RNAs whileincreasing stability avoiding mutant escapes and deliveryto target tissues RNA design and chemical modificationsincrease stability reduce immunogenicity and may help toreduce viral escapes however even modified naked RNAshave poor cellular uptake due to their small size net negativecharge renal clearance and hydrophilicity A range of deliv-ery vectors such as liposomes polymers and nanoparticleshave been developed to facilitate efficient cellular uptakeand target site accumulation as well as to provide a degreeof protection Among all delivery platforms safe and easily

manufactured LNP are good candidates which can addi-tionally be functionalized to achieve appropriate tropismespecially when the RNAi molecules are not tissue-specificIn addition the availability of different types of LNP favorsthe chance of attaining synergistic effects between RNAimolecules and conventional drugs which may be greatlyuseful in the case of drugs that develop resistances or donot result effective All these considerations joint with datafrom ongoing clinical trials suggest that in an early futurethe delivery of RNAi therapeutics by LNP could be a first-linetreatment in several human diseases such as viral infections

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the Basque GovernmentrsquosDepartment of Education Universities and Investigation (IT-341-10)

References

[1] United States Congress ldquoOffice of Technology Assessment[OTA] Human Gene Therapy-Background Paperrdquo Washing-ton DC USA

[2] Committee for Advanced Therapies (CAT) ldquoReflection paperon classification of advanced therapy medicinal productsrdquoEMACAT6002802010 2012

[3] S A Rosenberg P Aebersold K Cornetta et al ldquoGene trans-fer into humansmdashimmunotherapy of patients with advancedmelanoma using tumor-infiltrating lymphocytes modified byretroviral gene transductionrdquoNew England Journal of Medicinevol 323 no 9 pp 570ndash578 1990

[4] C Sheridan ldquoGene therapy finds its nicherdquo Nature Biotechnol-ogy vol 29 no 2 pp 121ndash128 2011

[5] The Journal of Gene Medicine Clinical Trial sitehttpwwwabediacomwileyindicationsphp


[6] A Fire S Xu M K Montgomery S A Kostas S E Driver andC CMello ldquoPotent and specific genetic interference by double-stranded RNA in caenorhabditis elegansrdquo Nature vol 391 no6669 pp 806ndash811 1998

[7] B Kim Q Tang P S Biswas et al ldquoInhibition of ocularangiogenesis by siRNA targeting vascular endothelial growthfactor pathway genes therapeutic strategy for herpetic stromalkeratitisrdquoThe American Journal of Pathology vol 165 no 6 pp2177ndash2185 2004

[8] M S Weinberg and P Arbuthnot ldquoProgress in the use of RNAinterference as a therapy for chronic hepatitis B virus infectionrdquoGenome Medicine vol 2 no 4 pp 28ndash34 2010

[9] M Motavaf S Safari and S M Alavian ldquoTherapeutic potentialof RNA interference a new molecular approach to antiviraltreatment for hepatitis Crdquo Journal of Viral Hepatitis vol 19 no11 pp 757ndash765 2012

[10] E Xiaofei B M Stadler M Debatis S Wang S Luand T F Kowalik ldquoRNA interference-mediated targeting ofhuman cytomegalovirus immediate-early or early gene prod-ucts inhibits viral replication with differential effects on cellularfunctionsrdquo Journal of Virology vol 86 no 10 pp 5660ndash56732012

[11] R Singhania N Khairuddin D Clarke and N A McMillanldquoRNA interference for the treatment of papillomavirus diseaserdquoThe Open Virology Journal vol 6 pp 204ndash215 2012

[12] D Vlachakis G Tsiliki A Pavlopoulou M G RoubelakisS Champeris Tsaniras and S Kossida ldquoAntiviral stratagemsagainst HIV-1 using RNA interference (RNAi) technologyrdquoEvolutionary Bioinformatics vol 9 pp 203ndash213 2013

[13] T Betakova and P Svancarova ldquoRole and application of RNAinterference in replication of influenza virusesrdquoActa Virologicavol 57 no 2 pp 97ndash104 2013

[14] D Ovcharenko R Jarvis S Hunicke-Smith K Kelnar and DBrown ldquoHigh-throughput RNAi screening ldquoin vitrordquo from celllines to primary cellsrdquo RNA vol 11 no 6 pp 985ndash993 2005

[15] V Dyer A Ely K Bloom M Weinberg and P ArbuthnotldquoTRNALys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAsrdquo Biochem-ical and Biophysical Research Communications vol 398 no 4pp 640ndash646 2010

[16] M Machitani F Sakurai K Katayama et al ldquoImprovingadenovirus vector-mediated RNAi efficiency by lacking theexpression of virus-associated RNAsrdquo Virus Research vol 178no 2 pp 357ndash363 2013

[17] T I Novobrantseva A Borodovsky J Wong et al ldquoSystemicRNAi-mediated gene silencing in nonhuman primate androdent myeloid cellsrdquo Molecular Therapy-Nucleic Acids vol 1article e4 2012

[18] H Arima A Yoshimatsu H Ikeda et al ldquoFolate-PEG-appended dendrimer conjugate with 120572-cyclodextrin as a novelcancer cell-selective siRNA delivery carrierrdquo Molecular Phar-maceutics vol 9 pp 2591ndash2604 2012

[19] A R de Fougerolles ldquoDelivery vehicles for small interferingRNA in vivordquo Human Gene Therapy vol 19 no 2 pp 125ndash1322008

[20] K A Whitehead R Langer and D G Anderson ldquoKnockingdown barriers advances in siRNA deliveryrdquo Nature ReviewsDrug Discovery vol 8 no 2 pp 129ndash138 2009

[21] S M Elbashir W Lendeckel and T Tuschl ldquoRNA interferenceis mediated by 21- and 22-nucleotide RNAsrdquo Genes and Devel-opment vol 15 no 2 pp 188ndash200 2001

[22] S M Hammond ldquoDicing and slicing the core machinery of theRNA interference pathwayrdquo FEBS Letters vol 579 no 26 pp5822ndash5829 2005

[23] M R Lares J J Rossi and D L Ouellet ldquoRNAi and smallinterfering RNAs in human disease therapeutic applicationsrdquoTrends in Biotechnology vol 28 no 11 pp 570ndash579 2010

[24] C B Moore E H Guthrie M T Huang and D J TaxmanldquoShort hairpin RNA (shRNA) design delivery and assessmentof gene knockdownrdquoMethods inMolecular Biology vol 629 pp141ndash158 2010

[25] Y Zhang Z Wang and R A Gemeinhart ldquoProgress inmicroRNA deliveryrdquo Journal of Controlled Release vol 172 no3 pp 962ndash974 2013

[26] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010

[27] D D Rao J S Vorhies N Senzer and J Nemunaitis ldquosiRNAversus shRNA similarities and differencesrdquo Advanced DrugDelivery Reviews vol 61 no 9 pp 746ndash759 2009

[28] P R Romano D E McCallus and C J Pachuk ldquoRNAinterference-mediated prevention and therapy for hepatocellu-lar carcinomardquo Oncogene vol 25 no 27 pp 3857ndash3865 2006

[29] D D Rao J S Vorhies N Senzer and J Nemunaitis ldquosiRNA vsshRNA similarities and differencesrdquo Advanced Drug DeliveryReviews vol 61 no 9 pp 746ndash759 2009

[30] R Triboulet BMari Y L Lin et al ldquoSuppression ofmicroRNA-silencing pathway by HIV-1 during virus replicationrdquo Sciencevol 315 no 5818 pp 1579ndash1582 2007

[31] C Wilkins R Dishongh S C Moore M A Whitt M Chowand K Machaca ldquoRNA interference is an antiviral defencemechanism in Caenorhabditis elegansrdquo Nature vol 436 no7053 pp 1044ndash1047 2005

[32] I M Pedersen G Cheng S Wieland et al ldquoInterferonmodulation of cellular microRNAs as an antiviral mechanismrdquoNature vol 449 no 7164 pp 919ndash922 2007

[33] TWatanabe T Umehara F Yasui et al ldquoLiver target delivery ofsmall interfering RNA to the HCV gene by lactosylated cationicliposomerdquo Journal of Hepatology vol 47 no 6 pp 744ndash7502007

[34] S I Kim D Shin H Lee B Ahn Y Yoon and M Kim ldquoTar-geted delivery of siRNA against hepatitis C virus by apolipopro-tein A-I-bound cationic liposomesrdquo Journal of Hepatology vol50 no 3 pp 479ndash488 2009

[35] H Lee S I Kim D Shin et al ldquoHepatic siRNA delivery usingrecombinant human apolipoprotein A-I in micerdquo Biochemicaland Biophysical Research Communications vol 378 no 2 pp192ndash196 2009

[36] P K Chandra A K Kundu S Hazari et al ldquoInhibition ofhepatitis C virus replication by intracellular delivery of multiplesiRNAs by nanosomesrdquo Molecular Therapy vol 20 no 9 pp1724ndash1736 2012

[37] A Dallas H Ilves J Shorenstein A Judge R Spitler andC Conta ldquoMinimal-length synthetic shRNAs formulated withlipid nanoparticles are potent inhibitors of hepatitis C virusIRES-linked gene expression in micerdquo Molecular TherapymdashNucleic Acids vol 2 p e123 2013


[38] L C Gomes-da-Silva N A Fonseca V Moura M C Pedrosode Lima S Simoes and J N Moreira ldquoLipid-based nanopar-ticles for siRNA delivery in cancer therapy paradigms andchallengesrdquo Accounts of Chemical Research vol 45 no 7 pp1163ndash1171 2012

[39] W Yu C Liu Y Liu N Zhang and W Xu ldquoMannan-modifiedsolid lipid nanoparticles for targeted gene delivery to alveolarmacrophagesrdquo Pharmaceutical Research vol 27 no 8 pp 1584ndash1596 2010

[40] J N Moreira A Santos V Moura M C P de Uma andS Simoes ldquoNon-viral lipid-based nanoparticles for targetedcancer systemic gene silencingrdquo Journal of Nanoscience andNanotechnology vol 8 no 5 pp 2187ndash2204 2008

[41] A K Vaishnaw J Gollob C Gamba-Vitalo et al ldquoA statusreport on RNAi therapeuticsrdquo Silence vol 1 no 1 article 142010

[42] E Vighi B Ruozi MMontanari R Battini and E Leo ldquopDNAcondensation capacity and in vitro gene delivery propertiesof cationic solid lipid nanoparticlesrdquo International Journal ofPharmaceutics vol 389 no 1-2 pp 254ndash261 2010

[43] R Cortesi M Campioni L Ravani M Drechsler M Pinottiand E Esposito ldquoCationic lipid nanosystems as carriers fornucleic acidsrdquoNew Biotechnology vol 31 no 1 pp 44ndash54 2014

[44] R H Muller M Radtke and S A Wissing ldquoSolid lipidnanoparticles (SLN) and nanostructured lipid carriers (NLC)in cosmetic and dermatological preparationsrdquo Advanced DrugDelivery Reviews vol 54 supplement 1 pp S131ndashS155 2002

[45] A del Pozo-Rodrıguez M A Solinıs A R Gascon and JL Pedraz ldquoShort- and long-term stability study of lyophilizedsolid lipid nanoparticles for gene therapyrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 71 no 2 pp 181ndash1892009

[46] A del Pozo-Rodrıguez D Delgado A R Gascon and M ASolinıs ldquoLipid nanoparticles as druggene delivery systems tothe retinardquo Journal of Ocular Pharmacology and Therapeuticsvol 29 no 2 pp 173ndash188 2013

[47] D V Morrissey J A Lockridge L Shaw et al ldquoPotent andpersistent in vivo anti-HBV activity of chemically modifiedsiRNAsrdquo Nature Biotechnology vol 23 no 8 pp 1002ndash10072005

[48] S I Kim D Shin T H Choi et al ldquoSystemic and specificdelivery of small interfering RNAs to the liver mediated byapolipoprotein A-IrdquoMolecular Therapy vol 15 no 6 pp 1145ndash1152 2007

[49] S Carmona M R Jorgensen S Kolli et al ldquoControllingHBV replication in vivo by intravenous administration oftriggered PEGylated siRNA-nanoparticlesrdquoMolecular Pharma-ceutics vol 6 no 3 pp 706ndash717 2009

[50] M Mevel N Kamaly S Carmona et al ldquoDODAG a versatilenew cationic lipid that mediates efficient delivery of pDNA andsiRNArdquo Journal of Controlled Release vol 143 no 2 pp 222ndash232 2010

[51] J Hean C Crowther A Ely et al ldquoInhibition of hepatitis Bvirus replication ldquoin vivordquo using lipoplexes containing altritol-modified antiviral siRNAsrdquo Artificial DNA PNA and XNA vol1 no 1 pp 17ndash26 2010

[52] A Simon V Karsten J Cehelsky et al ldquoResults of a phase 2bmulti-center trial of ALN-RSV01 in respiratory syncytial virus

(RSV)-infected lung transplant patientsrdquo European RespiratoryJournal vol 40 p 267s 2012

[53] L F Gebert M A Rebhan S E Crivelli R Denzler M Stoffeland J Hall ldquoMiravirsen (SPC3649) can inhibit the biogenesisof miR-122rdquo Nucleic Acids Research vol 42 no 1 pp 609ndash6212014

[54] H L Janssen H W Reesink S Zeuzem et al ldquoRandomizeddouble-blind placebo-controlled safety anti-viral proof ofconcept study of Miravirsen an oligonucleotide targeting miR-122 in treatment-naıve patients with genotype 1 chronic HCVinfectionrdquo in Proceedings of the Liver Meeting by the AASLDmdash(62nd Annual Meeting) Program Number LB-6 2011

[55] httpwwwbeniteccomdocuments140529HEPATITISCPAT-GCOMMENCEDFINALpdf

[56] httpwwwarrowheadresearchcompress-releasesarrowhead-begins-dosing-phase-2a-trial-rnai-therapeutic-arc-520-chron-ic-hepatitis-b

[57] 2014 httpwwwbeniteccomdocuments1311 CalimmunePresn to SISpdf

[58] Y C Tam S Chen and P R Cullis ldquoAdvances in lipidnanoparticles for siRNA deliveryrdquo Pharmaceutics vol 5 no 3pp 498ndash507 2013

[59] H Mishra D Mishra P K Mishra M Nahar V Dubey andN K Jain ldquoEvaluation of solid lipid nanoparticles as carriersfor delivery of hepatitis b surface antigen for vaccination usingsubcutaneous routerdquo Journal of Pharmacy and PharmaceuticalSciences vol 13 no 4 pp 495ndash509 2010

[60] A P Ruiz de Garibay D Delgado A del Pozo-Rodrıguez MA Solinıs and A R Gascon ldquoMulticomponent nanoparticlesas nonviral vectors for the treatment of fabry disease by genetherapyrdquo Drug Design Development and Therapy vol 6 pp303ndash310 2012

[61] DDoroudAVatanara F Zahedifard et al ldquoCationic solid lipidnanoparticles loaded by cystein proteinase genes as a novel anti-leishmaniasis DNA vaccine delivery system characterizationand in vitro evaluationsrdquo Journal of Controlled Release vol 148no 1 pp e105ndashe106 2010

[62] L Priano D Esposti R Esposti et al ldquoSolid lipid nanoparticlesincorporating melatonin as new model for sustained oraland transdermal delivery systemsrdquo Journal of Nanoscience andNanotechnology vol 7 no 10 pp 3596ndash3601 2007

[63] A Rodrıguez-Gascon M A Solinıs A del Pozo-Rodrıguez DDelgado and J L Pedraz ldquoLipid nanoparticles for gene therapyrdquoUS 20120183589 A1 2012

[64] A Rodrıguez-Gascon M A Solinıs A del Pozo-RodrıguezD Delgado and E Fernandez ldquoLipid nanoparticles for treatingocular diseasesrdquo WO 2012085318 A1 2012

[65] A del Pozo-Rodrıguez S Pujals D Delgado et al ldquoAproline-rich peptide improves cell transfection of solid lipidnanoparticle-based non-viral vectorsrdquo Journal of ControlledRelease vol 133 no 1 pp 52ndash59 2009

[66] D Delgado A Del Pozo-Rodrıguez M A Solinıs and ARodrıguez-Gascon ldquoUnderstanding the mechanism of pro-tamine in solid lipid nanoparticle-based lipofection the impor-tance of the entry pathwayrdquo European Journal of Pharmaceuticsand Biopharmaceutics vol 79 no 3 pp 495ndash502 2011

[67] D Delgado A del Pozo-Rodrıguez M A Solinıs ABartkowiak and A Rodrıguez-Gascon ldquoNew gene delivery


system based on oligochitosan and solid lipid nanoparticlesldquoin vitrordquo and ldquoin vivordquo evaluationrdquo European Journal ofPharmaceutical Sciences vol 50 no 3-4 pp 484ndash491 2013

[68] P S Apaolaza D Delgado A del Pozo-Rodrıguez A RGascon and M A Solinıs ldquoA novel gene therapy vector basedon hyaluronic acid and solid lipid nanoparticles for oculardiseasesrdquo International Journal of Pharmaceutics vol 465 no1-2 pp 413ndash426 2014

[69] A del Pozo-Rodrıguez D Delgado M A Solinıs et alldquoSolid lipid nanoparticles as potential tools for gene therapyldquoin vivordquo protein expression after intravenous administrationrdquoInternational Journal of Pharmaceutics vol 385 no 1-2 pp 157ndash162 2010

[70] D Delgado A R Gascon A del Pozo-Rodr120593guez et alldquoDextran-protamine-solid lipid nanoparticles as a non-viralvector for gene therapy ldquoin vitrordquo characterization and ldquoinvivordquo transfection after intravenous administration to micerdquoInternational Journal of Pharmaceutics vol 425 no 1-2 pp 35ndash43 2012

[71] D Delgado A del Pozo-Rodrıguez M A Solinıs et al ldquoDex-tran and protamine-based solid lipid nanoparticles as potentialvectors for the treatment of X-linked juvenile retinoschisisrdquoHuman Gene Therapy vol 23 no 4 pp 345ndash355 2012

[72] A del Pozo-Rodrıguez D Delgado M A Solinıs and A RGascon ldquoLipid nanoparticles as vehicles for macromoleculesnucleic acids and peptidesrdquo Recent Patents on Drug Delivery ampFormulation vol 5 no 3 pp 214ndash226 2011

[73] W Yu C Liu J Ye W Zou N Zhang and W Xu ldquoNovelcationic SLN containing a synthesized single-tailed lipid as amodifier for gene deliveryrdquo Nanotechnology vol 20 no 21Article ID 215102 2009

[74] N Pedersen S Hansen A V Heydenreich H G KristensenandH S Poulsen ldquoSolid lipid nanoparticles can effectively bindDNA streptavidin and biotinylated ligandsrdquo European Journalof Pharmaceutics and Biopharmaceutics vol 62 no 2 pp 155ndash162 2006

[75] E Miele G P Spinelli E D Fabrizio E Ferretti S Tomao andA Gulino ldquoNanoparticle-based delivery of small interferingRNA challenges for cancer therapyrdquo International Journal ofNanomedicine vol 7 pp 3637ndash3657 2012

[76] H R Kim I K Kim K H Bae S H Lee Y Lee and TG Park ldquoCationic solid lipid nanoparticles reconstituted fromlow density lipoprotein components for delivery of siRNArdquoMolecular Pharmaceutics vol 5 no 4 pp 622ndash631 2008

[77] J Jin K H Bae H Yang et al ldquoIn vivo specific delivery of c-MetsiRNA to glioblastoma using cationic solid lipid nanoparticlesrdquoBioconjugate Chemistry vol 22 no 12 pp 2568ndash2572 2011

[78] G Montana M L Bondı R Carrotta et al ldquoEmployment ofcationic solid-lipid nanoparticles as RNA carriersrdquoBioconjugateChemistry vol 18 no 2 pp 302ndash308 2007

[79] A Puri K Loomis B Smith et al ldquoLipid-based nanoparticles aspharmaceutical drug carriers from concepts to clinicrdquo CriticalReviews in Therapeutic Drug Carrier Systems vol 26 no 6 pp523ndash580 2009

[80] S Das W K Ng and R B H Tan ldquoAre nanostructured lipidcarriers (NLCs) better than solid lipid nanoparticles (SLNs)development characterizations and comparative evaluationsof clotrimazole-loaded SLNs and NLCsrdquo European Journal ofPharmaceutical Sciences vol 47 no 1 pp 139ndash151 2012

[81] C L Fang S A Al-Suwayeh and J Y Fang ldquoNanostructuredlipid carriers (NLCs) for drug delivery and targetingrdquo RecentPatents on Nanotechnology vol 7 no 1 pp 41ndash55 2013

[82] M A Iqbal S Md J K Sahni S Baboota S Dang and J AlildquoNanostructured lipid carriers system recent advances in drugdeliveryrdquo Journal of Drug Targeting vol 20 no 10 pp 813ndash8302012

[83] A Beloqui M A Solinıs A Delgado C Evora A Isla andA Rodrıguez-Gascon ldquoFate of nanostructured lipid carriers(NLCs) following the oral route design pharmacokinetics andbiodistributionrdquo Journal ofMicroencapsulation vol 31 no 1 pp1ndash8 2014

[84] V Teeranachaideekul R H Muller and V B JunyaprasertldquoEncapsulation of ascorbyl palmitate in nanostructured lipidcarriers (NLC)-effects of formulation parameters on physico-chemical stabilityrdquo International Journal of Pharmaceutics vol340 no 1-2 pp 198ndash206 2007

[85] M Radtke E B Souto and R H Muller ldquoNanostructuredlipid carriers a novel generation of solid lipid drug carriersrdquoPharmaceutical Technology Europe vol 17 no 4 pp 45ndash502005

[86] M Joshi S Pathak S Sharma and V Patravale ldquoDesignand in vivo pharmacodynamic evaluation of nanostructuredlipid carriers for parenteral delivery of artemether nanojectrdquoInternational Journal of Pharmaceutics vol 364 no 1 pp 119ndash126 2008

[87] A P Nayak W Tiyaboonchai S Patankar B Madhusudhanand E B Souto ldquoCurcuminoids-loaded lipid nanoparticlesnovel approach towards malaria treatmentrdquo Colloids and Sur-faces B Biointerfaces vol 81 no 1 pp 263ndash273 2010

[88] N P Aditya S Patankar B Madhusudhan R S R Murthy andE B Souto ldquoArthemeter-loaded lipid nanoparticles producedby modified thin-film hydration pharmacokinetics toxicolog-ical and ldquoin vivordquo anti-malarial activityrdquo European Journal ofPharmaceutical Sciences vol 40 no 5 pp 448ndash455 2010

[89] K W Kasongo J Pardeike R H Muller and R B WalkerldquoSelection and characterization of suitable lipid excipientsfor use in the manufacture of didanosine-loaded solid lipidnanoparticles and nanostructured lipid carriersrdquo Journal ofPharmaceutical Sciences vol 100 no 12 pp 5185ndash5196 2011

[90] A Beloqui M A Solinıs A R Gascon A del Pozo-Rodrıguez A des Rieux and V Preat ldquoMechanism of transportof saquinavir-loaded nanostructured lipid carriers across theintestinal barrierrdquo Journal of Controlled Release vol 166 no 2pp 115ndash123 2013

[91] A Beloqui M A Solinıs A des Rieux V Preat andA Rodrıguez-Gascon ldquoDextran-protamine coated nanostruc-tured lipid carriers as mucus-penetrating nanoparticles forlipophilic drugsrdquo International Journal of Pharmaceutics vol468 no 1-2 pp 105ndash111 2014

[92] O Taratula A Kuzmov M Shah O B Garbuzenko andT Minko ldquoNanostructured lipid carriers as multifunctionalnanomedicine platform for pulmonary co-delivery of anti-cancer drugs and siRNArdquo Journal of Controlled Release vol 71no 3 pp 349ndash357 2013

[93] R H Muller and C Olbrich ldquoLipid matrix-drug conjugatesparticles for controlled release of active ingredientrdquo US67702992004

[94] R Paliwal S Rai and S P Vyas ldquoLipid drug conjugate(LDC) nanoparticles as autolymphotrophs for oral delivery of


methotrexaterdquo Journal of Biomedical Nanotechnology vol 7 no1 pp 130ndash131 2011

[95] P Sharma B Dube and K Sawant ldquoSynthesis of cytarabinelipid drug conjugate for treatment of meningeal leukemiadevelopment characterization and in vitro cell line studiesrdquoJournal of Biomedical Nanotechnology vol 8 no 6 pp 928ndash9372012

[96] C Olbrich A Gessner O Kayser and R H Muller ldquoLipid-drug-conjugate (LDC) nanoparticles as novel carrier systemfor the hydrophilic antitrypanosomal drug diminazenediacetu-raterdquo Journal of Drug Targeting vol 10 no 5 pp 387ndash396 2002

[97] C Olbrich A Gessner W Schroder O Kayser and R HMuller ldquoLipid-drug conjugate nanoparticles of the hydrophilicdrug diminazenemdashcytotoxicity testing and mouse serumadsorptionrdquo Journal of Controlled Release vol 96 no 3 pp 425ndash435 2004

[98] D Liu ldquoRecent advances in myelodysplasia update from 2011ASH annual meetingrdquo Journal of Hematology amp Oncology vol5 supplement 1 p A4 2012

[99] Y R Neupane M D Sabir N Ahmad M Ali and K KohlildquoLipid drug conjugate nanoparticle as a novel lipid nanocarrierfor the oral delivery of decitabine ldquoex vivordquo gut permeationstudiesrdquoNanotechnology vol 24 no 41 Article ID 415102 2013

[100] L M Verissimo L F Agnez Lima L C Monte Egito A G deOliveira and E S T do Egito ldquoPharmaceutical emulsions anew approach for gene therapyrdquo Journal of Drug Targeting vol18 no 5 pp 333ndash342 2010

[101] S W Yi T Y Yune T W Kim et al ldquoA cationic lipidemulsionDNA complex as a physically stable and serum-resistant gene delivery systemrdquo Pharmaceutical Research vol 17no 3 pp 314ndash320 2000

[102] H S Yoo O Mazda H Y Lee et al ldquoIn vivo gene therapy oftype I diabetic mellitus using a cationic emulsion containingan Epstein Barr Virus (EBV) based plasmid vectorrdquo Journal ofControlled Release vol 112 no 1 pp 139ndash144 2006

[103] M M Kaneda Y Sasaki G M Lanza J Milbrandt and S AWickline ldquoMechanisms of nucleotide trafficking during siRNAdelivery to endothelial cells using perfluorocarbon nanoemul-sionsrdquo Biomaterials vol 31 no 11 pp 3079ndash3086 2010

[104] A Rodrıguez-Gascon A del Pozo-Rodrıguez and M ASolinıs ldquoDevelopment of nucleic acid vaccines use of self-amplifying RNA in lipid nanoparticlesrdquo International Journal ofNanomedicine vol 9 no 1 pp 1833ndash1843 2014

[105] K Gavrilov and W M Saltzman ldquoTherapeutic siRNA prin-ciples challenges and strategiesrdquo Yale Journal of Biology andMedicine vol 85 no 2 pp 187ndash200 2012

[106] C N Landen Jr A Chavez-Reyes C Bucana et al ldquoTherapeu-tic EphA2 gene targeting in vivo using neutral liposomal smallinterfering RNA deliveryrdquo Cancer Research vol 65 no 15 pp6910ndash6918 2005

[107] C Zhang N Tang X Liu W Liang W Xu and V PTorchilin ldquosiRNA-containing liposomes modified with pol-yarginine effectively silence the targeted generdquo Journal ofControlled Release vol 112 no 2 pp 229ndash239 2006

[108] S M Kwon H Y Nam T Nam et al ldquoIn vivo time-dependentgene expression of cationic lipid-based emulsion as a stableand biocompatible non-viral gene carrierrdquo Journal of ControlledRelease vol 128 no 1 pp 89ndash97 2008

[109] Z Hyvonen S Ronkko MR Toppinen I Jaaskeainen APlotniece andAUrtti ldquoDioleoyl phosphatidylethnolamine andPEG-lipid conjugates modify DNA delivery medicated by 14-dihydropyridine amphiphilesrdquo Journal of Controlled Releasevol 99 no 1 pp 177ndash190 2004

[110] L Deng Y Zhang L Ma et al ldquoComparison of anti-EGFR-Fab1015840 conjugated immunoliposomesmodifiedwith two differentconjugation linkers for siRNA delivery in SMMC-7721 cellsrdquoInternational Journal of Nanomedicine vol 8 pp 3271ndash32832013

[111] S Kim D Peer P Kumar et al ldquoRNAi-mediated CCR5 silenc-ing by LFA-1-targeted nanoparticles prevents HIV infection inBLT micerdquoMolecular Therapy vol 18 no 2 pp 370ndash376 2010

[112] Q Pan H W Tilanus H L A Janssen and L J W Van DerLaan ldquoProspects of RNAi and microRNA-based therapies forhepatitis Crdquo Expert Opinion on Biological Therapy vol 9 no 6pp 713ndash724 2009

[113] JM Luke G G Simon J Soderholm et al ldquoCoexpressed RIG-Iagonist enhances humoral immune response to influenza virusDNA vaccinerdquo Journal of Virology vol 85 no 3 pp 1370ndash13832011

[114] Hepatitis C Fact sheet N∘ 164 httpwwwwhointmediacen-trefactsheetsfs164en

[115] H J Hnatyszyn ldquoChronic hepatitis C and genotyping Theclinical significance of determining HCV genotypesrdquo AntiviralTherapy vol 10 no 1 pp 1ndash11 2005

[116] C Park S Jiang and K A Lawson ldquoEfficacy and safety oftelaprevir and boceprevir in patients with hepatitis C genotype1 a meta-analysisrdquo Journal of Clinical Pharmacy and Therapeu-tics vol 39 no 1 pp 14ndash24 2014

[117] Y Takigawa M Nagano-Fujii L Deng et al ldquoSuppression ofhepatitis C virus replicon by RNA interference directed againstthe NS3 and NS5B regions of the viral genomerdquo Microbiologyand Immunology vol 48 no 8 pp 591ndash598 2004

[118] R Prabhu P Vittal Q Yin et al ldquoSmall interfering RNAeffectively inhibits protein expression and negative strand RNAsynthesis from a full-length hepatitis C virus clonerdquo Journal ofMedical Virology vol 76 no 4 pp 511ndash519 2005

[119] M Kim D Shin S I Kim andM Park ldquoInhibition of hepatitisC virus gene expression by small interfering RNAs using atri-cistronic full-length viral replicon and a transient mousemodelrdquo Virus Research vol 122 no 1-2 pp 1ndash10 2006

[120] R Prabhu R F Garry and S Dash ldquoSmall interfering RNAtargeted to stem-loop II of the 51015840 untranslated region effectivelyinhibits expression of sixHCVgenotypesrdquoVirology Journal vol3 article 100 2006

[121] T Yokota N Sakamoto N Enomoto et al ldquoInhibition ofintracellular hepatitis C virus replication by synthetic andvector-derived small interferring RNAsrdquo EMBO Reports vol 4no 6 pp 602ndash608 2003

[122] T Kanda R Steele R Ray and R B Ray ldquoSmall interferingRNA targeted to hepatitis C virus 51015840 nontranslated region exertspotent antiviral effectrdquo Journal of Virology vol 81 no 2 pp669ndash676 2007

[123] A R N Zekri A A Bahnassy H M A El-Din and H MSalama ldquoConsensus siRNA for inhibition of HCV genotype-4replicationrdquo Virology Journal vol 6 article 13 2009


[124] R B Ray and T Kanda ldquoInhibition of HCV replication by smallinterfering RNArdquo Methods in Molecular Biology vol 510 pp251ndash262 2009

[125] R de Francesco andGMigliaccio ldquoChallenges and successes indeveloping new therapies for hepatitis Crdquo Nature vol 436 no7053 pp 953ndash960 2005

[126] L Zender S Hutker C Liedtke et al ldquoCaspase 8 small inter-fering RNA prevents acute liver failure in micerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 13 pp 7797ndash7802 2003

[127] S D Henry P van derWegen H J Metselaar H W Tilanus BJ Scholte and L J W van der Laan ldquoSimultaneous targeting ofHCV replication and viral binding with a single lentiviral vectorcontaining multiple RNA interference expression cassettesrdquoMolecular Therapy vol 14 no 4 pp 485ndash493 2006

[128] A K Kundu P K Chandra S Hazari Y V Pramar S Dash andT K Mandal ldquoDevelopment and optimization of nanosomalformulations for siRNA delivery to the liverrdquo European Journalof Pharmaceutics and Biopharmaceutics vol 80 no 2 pp 257ndash267 2012

[129] D Ivacik A Ely and P Arbuthnot ldquoCountering hepatitis Bvirus infection using RNAi How far are we from the clinicrdquoReviews in Medical Virology vol 21 no 6 pp 383ndash396 2011

[130] B Hepatitis Fact sheet No 204 2013 httpwwwwhointmediacentrefactsheetsfs204en

[131] E B Keeffe D T Dieterich J Pawlotsky and Y BenhamouldquoChronic hepatitis B preventing detecting andmanaging viralresistancerdquo Clinical Gastroenterology and Hepatology vol 6 no3 pp 268ndash274 2008

[132] F Zoulim ldquoHepatitis B virus resistance to antiviral drugs whereare we goingrdquo Liver International vol 31 supplement 1 pp 111ndash116 2011

[133] W Jiang ldquoBlockade of B7-H1 enhances dendritic cell-mediatedT cell response and antiviral immunity in HBV transgenicmicerdquo Vaccine vol 30 no 4 pp 758ndash766 2012

[134] C I Wooddell D B Rozema M Hossbach et al ldquoHepatocyte-targeted RNAi therapeutics for the treatment of chronic hepati-tis B virus infectionrdquoMolecular Therapy vol 21 no 5 pp 973ndash985 2013

[135] A Shlomai and Y Shaul ldquoInhibition of hepatitis B virusexpression and replication by RNA interferencerdquo Hepatologyvol 37 no 4 pp 764ndash770 2003

[136] S L Uprichard B Boyd A Althage and F V ChisarildquoClearance of hepatitis B virus from the liver of transgenic miceby short hairpin RNAsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 no 3 pp 773ndash778 2005

[137] S Carmona A Ely C Crowther et al ldquoEffective inhibitionof HBV replication in vivo by anti-HBx short hairpin RNAsrdquoMolecular Therapy vol 13 no 2 pp 411ndash421 2006

[138] D Sun C Rosler K Kidd-Ljunggren and M Nassal ldquoQuanti-tative assessment of the antiviral potencies of 21 shRNA vectorstargeting conserved including structured hepatitis B virussitesrdquo Hepatology vol 52 no 6 pp 817ndash826 2010

[139] F He E Chen L Liu et al ldquoInhibition of hepatitis B Virusreplication by hepatocyte nuclear factor 4-alpha specific shorthairpin RNArdquo Liver International vol 32 no 5 pp 742ndash7512012

[140] X J Wang Y Li H Huang et al ldquoA simple and robust vector-based shRNA expression system used for RNA interferencerdquoPLoS ONE vol 8 no 2 Article ID e56110 2013

[141] H Giladi M Ketzinel-Gilad L Rivkin Y Felig O Nussbaumand E Galun ldquoSmall interfering RNA inhibits hepatitis B virusreplication in micerdquo Molecular Therapy vol 8 no 5 pp 769ndash776 2003

[142] M D Moore M J McGarvey R A Russell B R Cullen andM O McClure ldquoStable inhibition of hepatitis B virus proteinsby small interfering RNA expressed from viral vectorsrdquo Journalof Gene Medicine vol 7 no 7 pp 918ndash925 2005

[143] K Wu X Zhang J Zhang et al ldquoInhibition of Hepatitis Bvirus gene expression by single and dual small interfering RNAtreatmentrdquo Virus Research vol 112 no 1-2 pp 100ndash107 2005

[144] MDMarimani A ElyM C R Buff S Bernhardt JW Engelsand P Arbuthnot ldquoInhibition of hepatitis B virus replication incultured cells and in vivo using 21015840-119874-guanidinopropylmodifiedsiRNAsrdquoBioorganic andMedicinal Chemistry vol 21 no 20 pp6145ndash6155 2013

[145] E Ambegia S Ansell P Cullis J Heyes L Palmer andI MacLachlan ldquoStabilized plasmid-lipid particles containingPEG-diacylglycerols exhibit extended circulation lifetimes andtumor selective gene expressionrdquo Biochimica et Biophysica ActaBiomembranes vol 1669 no 2 pp 155ndash163 2005

[146] HIVAIDS Fact sheet No360 2013 httpwwwwhointme-diacentrefactsheetsfs360en

[147] N S Lee T Dohjima G Bauer et al ldquoExpression of smallinterfering RNAs targeted against HIV-1 rev transcripts inhuman cellsrdquo Nature Biotechnology vol 20 no 5 pp 500ndash5052002

[148] Y Naito K Nohtomi T Onogi et al ldquoOptimal design andvalidation of antiviral siRNA for targetingHIV-1rdquoRetrovirologyvol 4 article 80 2007

[149] O ter Brake N Legrand K J von Eije et al et al ldquoEvaluation ofsafety and efficacy of RNAi againstHIV-1 in the human immunesystem (Rag-2(--)gammac(--)) mouse modelrdquo Gene Therapyvol 16 no 1 pp 148ndash153 2009

[150] D S An R E Donahue M Kamata et al ldquoStable reductionof CCR5 by RNAi through hematopoietic stem cell transplantin non-human primatesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 104 no 32 pp13110ndash13115 2007

[151] J S Anderson J Walker J A Nolta and G Bauer ldquoSpecifictransduction of hiv-susceptible cells for CCR5 knockdown andresistance to HIV infection a novel method for targeted genetherapy and intracellular immunizationrdquo Journal of AcquiredImmune Deficiency Syndromes vol 52 no 2 pp 152ndash161 2009

[152] F Boutimah J J M Eekels Y P Liu and B Berkhout ldquoAntiviralstrategies combining antiretroviral drugs with RNAi-mediatedattack on HIV-1 and cellular co-factorsrdquo Antiviral Research vol98 no 1 pp 121ndash129 2013

[153] R G Amado R T Mitsuyasu J D Rosenblatt et al ldquoAnti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study myeloid and lym-phoid reconstitution in human immunodeficiency virus type-1-infected patientsrdquo Human Gene Therapy vol 15 no 3 pp 251ndash262 2004

[154] B L Levine L M Humeau J Boyer et al ldquoGene transferin humans using a conditionally replicating lentiviral vectorrdquo


Proceedings of the National Academy of Sciences of the UnitedStates of America vol 103 no 46 pp 17372ndash17377 2006

[155] D L DiGiusto A Krishnan L Li et al ldquoRNA-based genetherapy for HIV with lentiviral vector-modified CD34+ cellsin patients undergoing transplantation for AIDS-related lym-phomardquo Science TranslationalMedicine vol 2 no 36 pp 36ndash432010

[156] S J Zeller and P Kumar ldquoRNA-based Gene therapy for thetreatment and prevention of HIV from bench to bedsiderdquo TheYale Journal of Biology and Medicine vol 84 no 3 pp 301ndash3092011

[157] X Lu Q Yu G K Binder et al ldquoAntisense-mediated inhibitionof human immunodeficiency virus (HIV) replication by useof an HIV type 1-based vector results in severely attenuatedmutants incapable of developing resistancerdquo Journal of Virologyvol 78 no 13 pp 7079ndash7088 2004

[158] O ter Brake P Konstantinova M Ceylan and B BerkhoutldquoSilencing of HIV-1 with RNA interference a multiple shRNAapproachrdquoMolecular Therapy vol 14 no 6 pp 883ndash892 2006

[159] P Kumar H Ban S Kim et al ldquoT cell-specific siRNA deliverysuppresses HIV-1 infection in humanized micerdquo Cell vol 134no 4 pp 577ndash586 2008

[160] Y P Liu J Haasnoot O ter Brake B Berkhout and P Kon-stantinova ldquoInhibition of HIV-1 by multiple siRNAs expressedfrom a single microRNA polycistronrdquo Nucleic Acids Researchvol 36 no 9 pp 2811ndash2824 2008

[161] J R Greenland R Geiben S Ghosh W A Pastor and NL Letvin ldquoPlasmid DNA vaccine-elicited cellular immuneresponses limit in vivo vaccine antigen expression through Fas-mediated apoptosisrdquo Journal of Immunology vol 178 no 9 pp5652ndash5658 2007

[162] R Geiben-Lynn K Frimpong-Boateng and N L LetvinldquoModulation of plasmid DNA vaccine antigen clearance bycaspase 12 RNA interference potentiates vaccinationrdquo Clinicaland Vaccine Immunology vol 18 no 4 pp 533ndash538 2011

[163] L AWheeler R Trifonova VVrbanac et al ldquoInhibition ofHIVtransmission in human cervicovaginal explants and humanizedmice using CD4 aptamer-siRNA chimerasrdquo Journal of ClinicalInvestigation vol 121 no 6 pp 2401ndash2412 2011

[164] Z Cui J Patel M Tuzova et al ldquoStrong T cell type-1 immuneresponses to HIV-1 Tat (1ndash72) protein-coated nanoparticlesrdquoVaccine vol 22 no 20 pp 2631ndash2640 2004

[165] J Patel D Galey J Jones et al ldquoHIV-1 Tat-coated nanoparticlesresult in enhanced humoral immune responses andneutralizingantibodies compared to alum adjuvantrdquo Vaccine vol 24 no 17pp 3564ndash3573 2006

[166] M A Arias A Loxley C Eatmon et al ldquoCarnauba waxnanoparticles enhance strong systemic and mucosal cellularand humoral immune responses to HIV-gp140 antigenrdquo Vac-cine vol 29 no 6 pp 1258ndash1269 2011

[167] D Palliser D Chowdhury Q Wang et al ldquoAn siRNA-basedmicrobicide protects mice from lethal herpes simplex virus 2infectionrdquo Nature vol 439 no 7072 pp 89ndash94 2006

[168] Y Wu F Navarro A Lal et al ldquoDurable protection fromHerpes Simplex Virus-2 transmission following intravaginalapplication of siRNAs targeting both a viral and host generdquo CellHost and Microbe vol 5 no 1 pp 84ndash94 2009

[169] T W Geisbert L E Hensley E Kagan et al ldquoPostexposureprotection of guinea pigs against a lethal ebola virus challenge is

conferred by RNA interferencerdquo Journal of Infectious Diseasesvol 193 no 12 pp 1650ndash1657 2006

[170] T W Geisbert A C Lee M Robbins et al ldquoPostexposureprotection of non-human primates against a lethal Ebola viruschallengewith RNA interference a proof-of-concept studyrdquoTheLancet vol 375 no 9729 pp 1896ndash1905 2010

[171] X Z Chen K J Zhu Y Xu et al ldquoRNA interference silencesthe human papillomavirus 6b11 early gene E7 in vitro and invivordquo Clinical and Experimental Dermatology vol 35 no 5 pp509ndash515 2010

[172] Y J Yang P S Zhao T Zhang et al ldquoSmall interfering RNAstargeting the rabies virus nucleoprotein generdquo Virus Researchvol 169 no 1 pp 169ndash174 2012

[173] K Khantasup P Kopermsub K Chaichoun and T DharakulldquoTargeted small interfering RNA-immunoliposomes as apromising therapeutic agent against highly pathogenic AvianInfluenza A (H5N1) virus infectionrdquo Antimicrobial Agents andChemotherapy vol 58 no 5 pp 2816ndash2824 2014

[174] A J Geall A Verma G R Otten et al ldquoNonviral deliveryof self-amplifying RNA vaccinesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no36 pp 14604ndash14609 2012

[175] S J Blake F F Bokhari andN AMcMillan ldquoRNA interferencefor viral infectionsrdquo Current Drug Targets vol 13 no 11 pp1411ndash1420 2012

[176] J DeVincenzo R Lambkin-Williams T Wilkinson et alldquoA randomized double-blind placebo-controlled study of anRNAi-based therapy directed against respiratory syncytialvirusrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 19 pp 8800ndash8805 2010

[177] R Kanasty J R Dorkin A Vegas and D Anderson ldquoCurrentprospects for RNA interference-based therapies and deliverymaterials for siRNA therapeuticsrdquo Nature Materials vol 12 pp967ndash977 2013

[178] T Schluep L Kalinoski C Wooddell D Lewis R Gish andJ Lickliter ldquoA Phase I first in human clinical trial of ARC-520 an siRNA-based therapeutic for the treatment of chronichepatitis B virus infection in normal healthy volunteersrdquoGlobalAntiviral Journal vol 9 suplement 2 p 57 2013 HEP DART

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Table 1 Indications addressed by gene therapy clinical trials up to January 2014

Ranking Indication Number of trials (119899) Percentage with respect to total gene therapy clinical trials ()1 Cancer diseases 1274 6382 Monogenic diseases 178 893 Infectious diseases 164 824 Cardiovascular diseases 162 815 Healthy volunteers 52 266 Gene marking 50 257 Neurological diseases 37 198 Other diseases 35 189 Ocular diseases 31 1610 Inflammatory diseases 13 07

of different viral infections in humans such as hepatitisC virus (HCV) hepatitis B virus (HBV) human immun-odeficiency virus (HIV) human cytomegalovirus (HCMV)herpetic keratitis human papillomavirus or influenza virus[7ndash13]

A key challenge to exploit the full potential of RNAitherapeutics is their efficient delivery to the target cells Onthe one hand naked RNAi molecules are rapidly digestedby nucleases in the serum after systemic administrationon the other hand due to the negative surface charge ofRNA the entry into the cell cytoplasm is hampered Anumber of techniques have been attempted to overcomethese problems physical methods such as electroporation orhydrodynamic injection [14 15] viral vectors [16] or lipidand polymeric nanoparticles [17 18] Among them lipidnanoparticles (LNP) represent one of themost widely studieddelivery systems for in vivo application of RNAi [19 20] Theaimof this review is to collect the state of the art and the futureperspectives of the utility LNP as RNAi vectors for humanviral infections

2 RNA Interference (RNAi)

RNAi is a naturally occurring process of gene regulationpresent in plants and mammalian cells The first evidenceof the existence of this mechanism appeared in 1998 whenFire et al [6] observed in Caenorhabditis elegans that double-stranded RNAs (dsRNAs) were the basis of sequence-specificinhibition of protein expression Subsequent works demon-strated that the molecules that induced RNAi were shortdsRNAs of 21 nucleotides in length called short interferingRNAs (siRNAs) and that siRNAs were able to start theRNAi process in mammalian cells [21] The RNAi responseis activated when the dsRNA is processed by a ribonucleaseIII-like enzyme called Dicer resulting in the formation ofa siRNA The siRNA is incorporated into the RNA inducedsilencing complex (RISC) where a helicase unwinds theduplex siRNAThe resulting antisense strand guides the RISCto its complementary mRNA which will be cleaved [22]Typically there are three different types of commonly usedRNAi molecules siRNA short-hairpin RNA (shRNA alsonamed expressed RNAi activators) or microRNA (miRNA)siRNAs as mentioned before are dsRNA molecules of about

19ndash23 base pair nucleotides in length able to mediate site-specific cleavage and destruction of the targeted mRNA[23] shRNAs consist of two complementary 19ndash22 bp RNAsequences linked by a short loop of 4ndash11 These RNAsare synthesized within the cell by DNA vector-mediatedproduction [24] shRNAs can be transcribed through eitherRNA polymerase II or III The first transcript generates ahairpin like stem-loop structure and then is processed inthe nucleus by a complex containing the RNase II enzymeDrosha The individual pre-shRNAs generated are finallytransported to the cytoplasm by exportin 5 Once in thecytoplasm the complex Dicer processes the loop of thehairpin to form a double-stranded siRNA shRNAs representan important tool in the assessment of gene function inmammals and are largely used as a research tool miRNAs aresingle stranded RNAs of about 20ndash24 nucleotides This kindof RNAs acts as endogenous posttranscriptional repressors todownregulate gene expression [25] miRNAs are transcribedfromDNA as primarymiRNA (pri-miRNA) in this case pri-miRNAs are processed into precursor miRNA (pre-miRNA)by two proteins Drosha and Pasha Pre-miRNAs are thentransported to the cytoplasm and after processing by Dicerand unwinding to obtain the miRNA the following stepsare identical to those that occur with siRNA and shRNA asit is illustrated in Figure 1 Recent observations have shownthat both miRNAs and siRNAs can suppress translation ofmRNAs (in the case of an imperfect match) and can cleavetarget RNAs (in the case of a perfect match) and play adecisive role in gene and genome regulation [26] It has beenalso observed that shRNA can act via miRNA- or siRNA-likemechanism It is believed that the miRNA-like mechanism isfaster than siRNA-like mechanism because the latter acts viaperfect complementarity for a target message [27]

The main advantage of synthesized siRNAs is that thesemolecules do not need to reach the nucleus to exert effectHowever modifications are necessary to increase their sta-bility resulting in some cases in loss of siRNA function[28] there must be a balance between stability and efficacywhenmodifications are introduced in siRNAWhenmiRNAsor shRNA expression plasmids are used they must initiallyreach the nucleus of cells to be processed The resultingmolecules are then transported to the cytoplasm and finallyincorporated into theRISC for activityTherefore a limitation


NUCL

shRNA plasmid

Nucleus

Cytoplasm

Pre-shRNA

Pre-miRNA

pri-miRNA

siRNA


mRNA target

shRNA

Unwinding

miRNA

Pri-shRNA

Exportin 5

Drosha

Drosha

Pasha

Dicer

RISC

RISC

RISC

RISC

RISC

Dicer

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

59984005

998400

3998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400












































Cationicliposomes



Cationicliposomes



[34]

Cationicliposomes




[35]

Cationicnanosomes






[37]













[47]



[48]









































Biopharma Ltd [57]









Acknowledgment


References


































































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


NUCL

shRNA plasmid

Nucleus

Cytoplasm

Pre-shRNA

Pre-miRNA

pri-miRNA

siRNA


mRNA target

shRNA

Unwinding

miRNA

Pri-shRNA

Exportin 5

Drosha

Drosha

Pasha

Dicer

RISC

RISC

RISC

RISC

RISC

Dicer

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

59984005

998400

3998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400












































Cationicliposomes



Cationicliposomes



[34]

Cationicliposomes




[35]

Cationicnanosomes






[37]













[47]



[48]









































Biopharma Ltd [57]









Acknowledgment


References


































































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



































Cationicliposomes



Cationicliposomes



[34]

Cationicliposomes




[35]

Cationicnanosomes






[37]













[47]



[48]









































Biopharma Ltd [57]









Acknowledgment


References


































































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







[47]



[48]









































Biopharma Ltd [57]









Acknowledgment


References


































































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




























Biopharma Ltd [57]









Acknowledgment


References


































































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























































































































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

review article lipid nanoparticles as carriers for rnai...

Documents