Antisense oligonucleotides: modifications and clinical · PDF fileAntisense oligonucleotides: modifications and clinical trials Vivek K. Sharma,a Raman K. Sharmab and Sunil K. Singh*c
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Antisense oligon
DuiUAoDUjhlis
Chemistry, RSC Advances and NucAcids. His research interests areistry, multicomponent reactions an
aDepartment of Chemistry, University of DelbEvalueserve Pvt. Limited, Infospace (SEZ), GcDepartment of Chemistry, Kirori Mal Col
Vivek K. Sharma,a Raman K. Sharmab and Sunil K. Singh*c
There has been an upsurge in the number of clinical trials involving chemically modified oligonucleotide-
based drug candidates after the FDA approval of Vitravene, Macugen, and recently, Kynamro. Over the
years, different types of backbone, nucleobase and/or sugar-modified oligonucleotides have been
synthesized because natural DNA/RNA based oligonucleotides pose some limitations, such as poor
binding affinity, low degree of nuclease resistance, affecting their direct use in antisense therapeutics. In
this review article, we discuss in detail different modifications of nucleosides/oligonucleotides along with
the related clinical trials, which demonstrated their potential as drug candidates for antisense and related
nucleic acid based therapeutics.
Introduction
Most of the drugs present in the market interact withproteins; moreover, they oen bind to non-target proteins orexert an adverse effect through unknown interactions.1 Thedream of modern drug research to develop a therapeutictechnology that can act specically only on the targetresponsible for the disease has led to the development ofdrugs that can turn off genes by targeting directly the nucleic
r Vivek K. Sharma received hisndergraduate degree in chem-stry from Hansraj College,niversity of Delhi, in 2007.er receiving his masters inrganic chemistry from theepartment of Chemistry,niversity of Delhi, in 2009, heoined the same department foris PhD. To date, he has pub-ished 11 research papers innternationally reputed journalsuch as The Journal of Organicleosides, Nucleotides & Nucleicbiocatalysis, nucleoside chem-d heterocyclic chemistry.
hi, Delhi-110007, India
urgaon 122001, Haryana, India
lege, University of Delhi, Delhi-110007,
, 1454–1471
acids that code for the proteins. Antisense therapeuticswere introduced aer Paterson et al.2 in 1977 reportedthe utility of nucleic acids in modulating gene expression,and shortly aer, Zamecnik and Stephenson3 demonstratedthe inhibition of viral replication by modied oligonucleo-tides (ONs).4 In the quest of effective antisense candidates,various chemical modications of the natural ONs have beenstudied, such as modications in the phosphodiester back-bone, heterocyclic nucleobase and sugar moiety, which conferhigh affinity and specicity for their target nucleic acidsequences (Fig. 1).5
Dr Raman K. Sharma completedhis BSc from W.R.S. Govt.College Dehri, Kangra, H.P.India and his MSc fromG.N.D.U. Amritsar, PunjabIndia. During MSc he wasoffered the position of MedicinalResearch Chemist in the newdrug discovery division of Ran-baxy (now Sun Pharma), Gur-gaon, Haryana, India, where heworked for one and half yearsaer his MSc. He then joined the
research group of Prof. Ashok K. Prasad at the Department ofChemistry, University of Delhi, India for his PhD, and worked inthe area of biocatalysis and heterocyclic compounds. He haspublished eight research articles and is currently working as aProject Manager at IPRD Evalueserve Gurgaon, Haryana, India.
Fig. 1 An overview of different chemical modifications of antisense oligonucleotides (AONs); B ¼ nucleobase.
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Internucleoside linkage or backbonemodified AONs
These are also referred to as the rst generation of chemicallymodied antisense agents. They contain backbone
Dr Sunil K. Singh received hismasters degree in OrganicChemistry from the Departmentof Chemistry, University of Delhi,in 2004. He completed his PhD inChemistry from the samedepartment in 2010. Currently,he is working as an AssistantProfessor in Kirori Mal College,University of Delhi. His currentresearch focuses on the synthesisof nucleosides, biocatalytictransformations, multicompo-
nent one-pot synthesis, etc. To date, he has published 13 researchpapers in internationally reputed journals such as The Journal ofOrganic Chemistry, Nucleosides, Nucleotides & Nucleic Acids,Current Organic Chemistry, and Organic & BiomolecularChemistry.
(NP), S-methylthiourea, and guanidinium, and they havebeen designed and synthesized to circumvent the physicaland biological limitations of the natural phosphodiesterlinkage.5,6 These backbone modications can be broadlyclassied as neutral, anionic or cationic internucleosidelinkages (Fig. 2).
Phosphorothioate oligonucleotides (PS-ONs) are the majorrepresentatives of this generation and have been used mostsuccessfully for gene silencing. The introduction of a PSlinkage into ONs confers sufficient resistance to nucleasedegradation, leading to higher bioavailability. In addition tonuclease resistance, PS-ONs form regular Watson–Crick basepairs, activate RNase H, carry negative charge for cell delivery,and display attractive pharmacokinetic properties and cellularuptake due to increased binding to plasma proteins and other
Fig. 3 Structure of phosphorothioate backbone modified drug Vitraven
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receptor sites as compared to natural phosphodiesters.4d,7
However, their proles for binding affinity to the targetoligonucleotide sequences and specicity are less satisfac-tory.8 Despite these disadvantages, the FDA approved the rstantisense drug Vitravene, a rst generation PS-modied AONfor the treatment of AIDS-related cytomegalovirus (CMV) reti-nitis (Fig. 3).9
Sugar modified AONs
In recent years, there has been a sudden leap in the synthesis ofconformationally constrained nucleoside analogues by modi-fying the sugar moiety in various ways. These include: (a)synthesis of nucleoside analogues containing an electronega-tive atom or substituent at the 20-position of sugar;10 (b)
Fig. 5 Structure of RNA like 20-substituted nucleosides; B ¼nucleobases.
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synthesis of bicyclic nucleoside analogues having an extra ringfused to the sugar moiety;11 (c) synthesis of nucleosideanalogues of varying sugar ring structures;12 and (d) synthesis ofspironucleosides containing a spirocyclic ring at differentpositions of the sugar ring (Fig. 4).13 The problems associatedwith PS-ONs, i.e. poor binding affinity to the target RNA, lack ofspecicity and low cellular uptake, are to some degree solved bythese second generation ONs containing a modied sugarmoiety. 20-O-Methyl (20-OMe), 20-O-methoxyethyl (20-OMOE) andlocked nucleic acid (LNA) are the most important members ofthis class (Fig. 4).
The structural difference between DNA and RNA includesthe 20-substitution on the furanose ring of RNA. Hence, theRNA binding behavior of AONs may be improved bymimicking RNA structures with 20-modied nucleosides(Fig. 5). Electronegative substituents like uorine and oxygeninuence the furanose sugar C 0
3-endo conformation14 due tothe preferred gauche orientation of the 20-substituent and thering oxygen (Fig. 5). As a result, RNA and 20-modied nucleo-sides are found predominantly in the C 0
3-endo conformationthat is exclusively present in A-type duplexes.15 Variousreported 20-substitutions have shown excellent results inantisense therapeutics as they provide high metabolic stabilityand high affinity to target mRNA; for example, 20-OMe-, 20-OMOE- and LNA-containing ONs have entered in humanclinical trials.15,16
The FDA in 2004 approved pegaptanib sodium (Macugen),an anti-vascular endothelial growth factor (anti-VEGF) RNAaptamer for the treatment of all types of neovascular age-related macular degeneration.17 Macugen consists of 20-F and20-OMe substituted sugar moieties (Fig. 6). Aptamers aresingle-stranded ONs (DNA/RNA) that form stable three-dimensional structures and are capable of binding withhigh affinity and specicity to a variety of molecular targets
Fig. 4 Structures of different types of sugar-modified constrainednucleoside analogues.
such as proteins and can modulate their functions. Becausethe targets are in the blood plasma or displayed on thesurface of cells, aptamers are likely to be degraded easily byserum nucleases. Therefore, unmodied aptamers haveshown half-lives in the blood as short as 2 minutes.18 Modi-cations such as the capping of ONs at the 30-terminus, oenfollowed by inverting the nucleotide at the 30-terminus, haveshown increased stability against endogenous serum nucle-ases (Fig. 6).19
Nucleobase modified AONs
Since the nucleobases provide the prime recognition site forWatson–Crick base pairing via specic hydrogen bondinginteractions, the scope of modication of the nucleobase isconned, which can only improve the binding affinity for thecomplementary ON but not the nuclease resistance.20 Althoughless common than backbone and sugar modications, chemi-cally modied heterocyclic nucleobases have also found appli-cations as AONs. Carefully designed nucleobase analogueswhen introduced into ONs can provide information on theimportance of specic functional groups in natural bases. Notethat even a subtle change can have a dramatic effect because ofthe change in size, electronic distribution, nucleoside sugarconformation, tautomeric structure or functional group pKa
values. Representative structures of several modied bases, i.e.pyrimidine and purine modication, and universal bases areshown in Fig. 7. The most attractive sites for substitution of thenucleoside bases are those positions that are exposed tosolvents in the major groove, i.e. the 4- and 5-positions ofpyrimidines and the 6- and 7-positions of purines (Fig. 7).Substitutions at these positions neither interfere with basepairing nor induce steric hindrance and inuence the generalgeometry of the double helix.5,21,22
Natural nucleobases display exquisite selectivity in recog-nizing complementary bases as given by Watson–Crick rules. Auniversal base is an analogue that can be substituted for any ofthe four natural bases in ONs without signicantly impairingthe duplex stability. In general, universal base analogues usearomatic ring stacking, instead of specic hydrogen bonds, tostabilize a duplex (Fig. 7).22
Fig. 6 Structure of the 20-sugar modified drug Macugen (Pegaptanib sodium).
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The stacking interactions between the planar heterocycles ofnucleic acids are largely responsible for the stability of DNA andRNA duplexes. Maximizing stacking interactions throughchemical modication provides a means of creating duplexhelices of greater stability, e.g. tricyclic phenoxazine and G-clamp cytosine derivatives have been shown to enhance stack-ing.23 A tricyclic phenoxazine (Fig. 8) serves as a rigid scaffoldfor the attachment of groups designed to interact with theHoogsteen binding face of a complementary base pairedguanine. Appending an arm with strong hydrogen bond donor,i.e. an aminoethyloxy tether to the phenoxazine, recognizesboth the Watson–Crick and the Hoogsteen sites of guanine;hence, it is termed as a G-clamp (Fig. 8).23
The G-clamp-containing AON displayed dramaticallyenhanced stability. The greatly increased affinity and specicity
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of the base-modied G-clamp was also conrmed by in vivostudies.23 However, the acyclic derivative lacks the conforma-tional restriction and hence does not demonstrate enhancedaffinity. The G-clamp0s affinity for the complementary guanineis due to the appropriate positioning of the strong hydrogenbond donors (Fig. 8).
Other advanced modified AONs
Although AONs made of only sugar modied building blocksare less toxic than PS-AONs and have slightly enhanced affinitytowards their complementary RNAs, their efficiency to induceRNase H cleavage of the target RNA is a matter of concern.24
Since RNase H cleavage is the most desirable mechanism forthe antisense effect and 20-O-alkyl modications are desirable
Fig. 7 Structures of different types of nucleobasemodified nucleosideanalogues.
Fig. 8 Cytosine nucleobase modified analogues used in hybridizationexperiments and interaction of the G-clamp with guanosine.
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for nuclease resistance and high binding affinity, a hybrid ONconstruct incorporating both characteristics has appeared inthe form of the ‘gapmer’ antisense oligonucleotide.25 A gapmercontains a central ‘gap’ of deoxynucleotides sufficient toinduce RNase H cleavage anked by blocks of 20-O-modiedribonucleotide ‘wings’ that protect the internal block fromnuclease degradation, e.g. 20-OMOE sugar modied nucleo-sides can be further combined with a phosphorothioate (PS)linkage as in Kynamro,26 which is the second antisense drugapproved by the FDA to reduce low density lipoprotein-cholesterol (LDL-C), apolipoprotein-B, total cholesterol andnon-high density lipoprotein–cholesterol in patients withhomozygous familial hypercholesterolemia (HoFH) (Fig. 9).Kynamro also represents the rst systemic antisense drug andis given as a 200 mg weekly subcutaneous injection as anadjunct therapy to lipid-lowering medications and a controlleddiet. Some serious side effects such as liver toxicity have beenencountered with Kynamro; hence, it is available with awarning on the package citing the risk of hepatic toxicity. Thecommon adverse reactions to Kynamro include injection sitereactions, increased alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) levels, u-like symptoms,and abnormal liver function test results.27 Numerous modiedONs are being tested in multiple clinical trials to explorewhether this ‘gapmer’ type chimera has improved therapeuticproperties (Table 1).
In order to further enhance target affinity, nuclease resis-tance, biostability and pharmacokinetics, an advanced thirdgeneration of AONs was developed mainly by modications ofthe furanose ring of the nucleotide. Peptide nucleic acid (PNA)and phosphorodiamidate morpholino oligomer (PMO) are themost well studied third-generation AONs.28
Peptide nucleic acid (PNA) is a non-charged nucleotideanalogue in which the phosphodiester backbone is replaced bya exible pseudopeptide polymer N-(2-aminoethyl)glycine andthe nucleobases are attached to the backbone via methyl-enecarbonyl-linkage (Fig. 1). PNAs can hybridize withcomplementary DNA or RNA strands with higher affinity andspecicity than natural oligonucleotides. PNA is not asubstrate for RNaseH and exerts its antisense effect by forminga sequence-specic duplex with mRNA, causing sterichindrance of translational machinery, leading to proteinknockdown.29
In phosphorodiamidate morpholino oligomer (PMO), theribose sugar is replaced by a six-membered morpholino ring,whereas the phosphodiester bond is replaced by a phosphor-odiamidate linkage (Fig. 1).30 Like PNAs, this modication alsodoes not activate RNase H; hence, it acts only as a steric blockerfor specic inhibition of gene expression. PMO provide excel-lent nuclease stability in comparison to that of the unmodiedAONs. PMO has demonstrated antisense efficacy in animalmodels in vivo and in human clinical trials.31–34
Clinical trials of modifiedoligonucleotides
The last 35 years have witnessed an explosive growth in thenumber of modied ON-related clinical trials. We havecollated the data for 76 oligonucleotide drug candidates thathave been tested in the clinical trials for treatment of variousdiseases, and the majority of them have shown promisingpotential. Please note that we have considered only those ONdrugs that have been tested in a minimum of phase I clinicaltrials or onwards. Most of these chemically modied ONsinvolve phosphorothioate (PS) chimera and are designed tospecic inhibition of gene expression through an antisensemechanism (Table 1). Antisense technology led to the foun-dation of ON based therapeutics and has now been con-junctured for use in more potent strategies, e.g. antigene, RNAinterfering (RNAi), aptamer, ribozyme and decoy ON, all ofwhich utilise the knowledge gained from the difficult effortsmade in developing the antisense technology.112 These ON-based approaches target different sites in the central dogmaof molecular biology in order to exert their therapeutic effects.Antigene and decoy ONs bind to DNA and hence block thetranscription process. Antisense, ribozyme and RNAi inhibitprotein synthesis (transcription) by blocking the
From the Table 1, it is quite clear that ISIS pharmaceuticals,which is a pioneer in antisense technology, has contributedabout �20% of all modied ON based drugs that are in clinicaltrials. Sarepta Therapeutics had six drug candidates in clinicaltrials involving PMO chemistry. A brief representation of thenumber of drugs in clinical trials for different assignees is
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provided in Fig. 11. Please note that assignees having one or twodrugs are grouped together as ‘others’ assignees in the graph.
To see the pattern in the number of drug candidates thatentered Phase 1 clinical trials over time (2 year intervals), weretrieved the corresponding data for these drug candidates fromvarious sources113 and prepared a graph to showcase thispattern (Fig. 12). From the graph, it is clear that aer the rsttwo drug candidates entered clinical trials in 1997–1998 (both
Fig. 11 Number of modified ON drugs in clinical trials according todifferent assignees.
Fig. 10 General functional representation of different oligonucleotide based therapeutic approaches in the central dogma of molecular biology.
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drugs in 1998), there was an increase in the number of drugcandidates entering clinical trials and a maximum of 34 drugcandidates entered between 2005–2008. From then, there was adecrease in the number of drug candidates, and only two drugsentered clinical trials in 2011–2012. A maximum of 18 drugcandidates entered clinical trials in 2007–2008, which was fol-lowed by 16 drugs in 2005–2006. Out of these, a maximum of 12drug candidates entered Phase 1 clinical trials in 2005.
Fig. 12 Number of modified ON drugs in clinical trials in different years
The recent approval of Kynamro by the FDA has added a muchneeded boost to research on antisense based therapeutics,which since 1998 was thought to be directionless and futile.Chemical manipulations of natural oligonucleotides arerequired as the direct use of these nucleotides as therapeuticagent suffers from some limitations such as low binding affinityto the complementary nucleic acid and poor nuclease stability.Hence, in the search for suitable antisense drug candidates,vast number of modications have been carried out, e.g. back-bone, nucleobase and sugar moiety modication of the naturalDNA/RNA, leading to the development of three FDA-approveddrugs. Furthermore, with persistent promising clinical trialsinvolving these modied oligonucleotides, it can be anticipatedthat more new potent antisense drugs may appear in the nearfuture.
Acknowledgements
VKS thanks CSIR, New Delhi, for award the Junior/SeniorResearch Fellowships.
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