Volume 33, Number 5/6, May/June 2006
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`and
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`in h.allh and dlsea •• H liu
`
`approach to therapeutics SH Koo and
`
`Frontiers in Research Reviews: CuUlng-Edge
`Molecular Approaches to Therapeutics
`Introductioo WSF Wong and AJ Melendez
`480
`482 Monoclonal antibodies as targeting and therapeutic
`agents: Prospects for liver transplantation,
`hepatitis
`and h.patoc.llular
`ca",inoma JM luk and K-F Wong
`489 Human embryonic stem cells: Technological
`challenges
`toward.
`Ih.rapy SKW Oh and ABH Choo
`496 DNAmicroarray technology for targelldenlilication
`validation M Jayapal and AJ Melendez
`Short inlelering RNA(.iRNA) a. a nov.l th.rap.utic
`PN Pushparaj and AJ Melendez
`Filarial nematode secreted product ES·62 is an
`anti-inflammatory
`agenl: Therapeutic potential of
`small molecule derivatives and ES-62 peptide mimetics
`W Harnett and MM Harnett
`regulatory T c.lI.
`519 C04'C025'
`and BP leung
`Pharmacogenetics
`EJO lee
`From design to therapeutic
`533 Antisense oligonucleotides:
`application JHP Chan, S lim and WSF Wnng
`Therapeutic vaccination for central nervous system repair
`BT Ang, G Xu and IC Xlao
`DNA vaccines and allergic diseases KY Chua, T Huangfu and
`IN Liew
`Art and science of photodynamic
`MOiivo
`Polymeric core-shell nanoparticles
`Y-Y Yang, Y Wang, R Poweli and P Chan
`Proteomics Technology and Therapeutics MLW Hong,
`N Jiang, S Gopinath and FT Chew
`Targeting tumours by adoptive transfer of immune cells
`PA MacAry, CT Too and X Oai
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`584
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`511
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`525
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`541
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`546
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`551
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`557
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`563
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`569
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`407
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`Brief Review
`Impact of obesity and insulin resistance on vasomotor
`lone: Nitric oxide and b.yond DW Stepp
`Original Articles
`r•• tor•• blood flow of
`415 Cytidin. 5'-dipho.phocholin.
`superior mesenteric and renal arteries and prolongs
`.urvival
`tim.
`In ha.morrhag.d
`anaesthetiz.d
`rat.
`MS Yilmaz, M Yalcln and Vsavel
`421 Blockade of p,- and Ilo-adr.noceptors delay. wound
`contraction and re-.pUhelialization in rat. BR SOUliJ,
`JS sanlOS and AMA Cosf!1
`431 Eff.cts of autonomic blockad. on non-Iin.ar
`cardiovascular
`variability
`indices in rats F Beckers,
`B VtIlheyden, D Ramaekers, B Swynghedauw and AE Aubert
`increases the gene expression
`448 Hyperinsulinaemia
`of .ndothelial nitric oxide syntha.e and Ih.
`pho.phatidylino.itoI3-k1na.e/Akt
`pathway in rat aorta
`H ToOO,E Gomyo, S Mlkl, TShimizu, A Yoshimura, R Inoue,
`N sawal, R Tsukamoto, J Asayama, M KoOOraand TNakala
`in rat aorta and
`44B Vasorelaxing eflects 01propranolol
`mesenteric artery: A role for nitric oxide and calcium
`entry blockad. FBM Privlero, CE TeiXEira,HAF Toque,
`MA Claudina, RC Webb, G De Nucci, A lanesco and E Anlunes
`·induced arterial hyperten.ion
`456 Ra" with inherited .tr ...
`(ISIAH.train) di.play .p.cilic quanlitativ.
`trail IDe;lor
`blood pr... ur. and lor body and kidney w.ight nn
`chromo.ome1
`OE Redina, NA Machanova, VM Elimov and
`AL Markei
`and aminophylline on tran.mitt.r
`465 Effect of th.ophyllin.
`release at the mammalian neuromuscular
`junction is
`nol m.diat.d
`by cAMP TJ Nickels, AD SChwartz, DE Blevins,
`J7Drummond, GW Reed and DF Wilson
`471 Dissociation of blood pressure and sympathetic activation
`of renin release in slnoaortic-denervated
`rats MH Krieger,
`-ED Moreira, EM Diiveira, VLL Oliveira, EM Krieger and JE Krieger
`Short Communication
`Spironolactone
`further
`reduces urinary albumin
`excretion and plasma B-type nalrioretic peptide
`levle.
`in hyperten.lv.
`type IIdiab.t •• tr.aled wilh
`angiotensin-converting
`enzyme inhibitor 5 Ogawa,
`K Takeuchi, T Mori, K Nako and S lio
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`Clinical and Experimental Pharmacology
`
`and Physiology
`
`(2006) 33, 533-540
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Frontiers in Research Review: Cutting-Edge Molecular Approaches to Therapeutics
`
`FROM DESIGN TO
`ANTISENSE OLIGONUCLEOTIDES:
`THERAPEUTIC APPLICATION
`
`Jasmine HP Chan, Shuhui Lim and WS Fred Wong
`Department of Pharmacology,
`Yang Loa Lin School of Medicine and Immunology Program,
`National University of Singapore, Singapore
`
`SUMMARY
`
`strand
`is a short
`(ASO)
`oligonucleotide
`1. An antisense
`of deoxyribonucleotide
`analogue that hybridizes with the com-
`plementary mRNA in a sequence-specific manner via Watson-
`Crick base pairing. Formation of the ASQ-mRNA heteroduplex
`either triggers RNase H activity, leading to mRNA degradation,
`induces
`translatio_nal arrest by steric hindrance
`of ribosomal
`activity, interferes with mRNA maturation
`by inhibiting splicing
`or destabilizes
`pre-mRNA in the nucleus,
`resulting
`in down-
`regulation of target protein expression.
`tool in protein
`2. The ASO is not only a useful experimental
`target
`identification
`and validation, but also a highly selective
`therapeutic
`strategy
`for diseases with dysregulated
`protein
`expression.
`theoretical
`various
`review, we discuss
`3. In the present
`approaches
`to rational design of ASO, chemical modifications of
`ASO, ASO delivery systems and ASO-related toxicology. Finally,
`we survey ASO drugs in various current
`clinical studies.
`Key words:
`antisense oligonucleotide design, cell-penetrating
`peptide, dendrimer, gapmer antisense oligonucleotide,
`liposome,
`locked nucleic acid, peptide nucleic acid, phosphoroamidate
`morpholino oligomer, phosphorothioate, RNase H.
`
`INTRODUCTION
`An antisense oligonucleotide
`deoxyribo-
`(ASO) is a single-stranded
`nucleotide (typically 20 bp in length) that
`is complementary
`to the
`target mRNA Hybridization of ASO to the target mRNA via Watson-
`Crick base pairing can result in specific inhibition of gene expression
`by various mechanisms, depending on the chemical make-up of the
`ASO and location of hybridization,
`resulting in reduced levels of
`translation of the target
`transcript.' The ASO is not only a useful
`
`Correspondence:WS Fred Wong,Department of Pharmacology,YongLoo
`Lin School of Medicine, National Universityof Singapore, 1v1D218Medical
`Drive, Singapore 117597. Email: phcwongf@nus.edu.sg
`This paper has been peer reviewed.
`Received 18 October; revision 1 February 2006; accepted 5 February
`2006.
`e 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Asia Pty Ltd
`
`function and target validation, but
`tool for studies of loss-of-gene
`therapeutic
`strategy to treat any
`also highly valuable
`as a novel
`disease that
`is linked to dysregulated
`gene expression. Antisense
`oligonucleotide-induced
`protein knockdown is usually achieved by
`induction of RNase H endonuclease
`activity that cleaves the RNA-
`DNA heteroduplex. This leads to the degradation of target mRNA
`include
`while leaving the ASO intact.' Other ASO mechanisms
`translational
`arrest by steric hindrance
`of ribosomal
`activity,
`interference with mRNA maturation
`by inhibiting
`splicing and
`destabilization
`ofpre-mRNA in the nucleus'
`(Fig. 1).
`algo-
`In the present
`review, we first discuss a few computational
`rithms in ASO design. There are other screening strategies to obtain
`potent ASO, such as mRNA walking," oligonucleotide
`array
`and
`RNase H mapping," but these approaches are more costly and labour
`intensive and require expensive automation equipment
`that many
`small laboratories may not be able to afford. Rational design of ASO
`that are freely available in the
`based on computational
`algorithms
`public domain is the most economical
`approach
`to ASO design
`and very often generates potent ASO from a handful of candidates.
`Because there is no stand-alone program in predicting highly potent
`ASO, one can increase the 'hit rate' using several computer software
`packages.· Unmodified ASOs
`are susceptible
`to degradation
`by
`nucleases. Therefore, different chemical modifications of ASO have
`been developed
`to decrease nuclease
`cleavage
`and increase
`the
`biostability
`and potency of the ASo. We then discuss
`the latest
`approaches
`for ASO delivery in vitro and in vivo. Owing to inherent
`ionic charges of ASO, it is difficult for the ASO to cross the plasma
`membrane
`efficiently. As such,
`the ASO needs to be coupled to a
`carrier
`for efficient membrane binding and internalization. Finally,
`toxicology and clinical studies of ASOs are discussed.
`
`ANTISENSE OLIGONUCLEOTIDE DESIGN
`The strength and stability of interactions between the ASO and com-
`plementary target mRNA depends on factors such as thermodynamic
`stability,
`the secondary structure of the target mRNA transcript and
`the proximity of the hybridization
`site to functional motifs on the
`designated transcript,
`such as the 5' CAP region or translational
`start
`site. We need to consider at least four parameters
`in ASO design in
`(i) prediction
`order
`to increase
`the 'hit
`rate':
`of the secondary
`structure of the RNA; (ii) identification of preferable RNA secondary
`local structures;
`(iii) motifs searching and GC content calculation;
`and (iv) binding energy (6.Go37) prediction.
`
`

`

`534
`
`List of abbreviations:
`
`ASO
`DOPE
`LNA
`2'-MOE
`
`Antisense oligonucleotide
`Dioleyphosphatidylethanolamine
`Locked nucleic acid
`2'~O-Methoxyethyl
`
`JHP Chan et aL
`
`2'-OMe
`PMO
`PNA
`PS
`
`2' -O·Methyl
`Phosphoroamidate morpholino oligomer
`Peptide nucleic acid
`Phosphorothioate
`
`Nucleus
`
`Pre.mRNA ""l'"
`
`,.1, 11!;' "#WV'!!
`
`'.)~'!-it')h'_'JJ),.
`
`Ii!;
`
`1 Transcription
`1Post-transcriptional
`
`modification
`3' poly(A)
`!III: I:! 11!!I'!!:! AhAA
`
`tail
`
`~~~~
`
`5' cap
`I,,!
`
`--....:::,~~+~"~&;,.!"",'*'F!'.,,Jb4~1JJ,~"tm••!
`1
`1
`
`(4)
`Inhibition of
`5'cap
`formation
`
`(5)
`Inhibition of
`RNA
`splicing
`
`(6)
`Activation
`of RNase H
`
`Hybridization
`"!!rII!!:!!!!!!!;!!!.AAAAA --....,;;::..---
`
`ITranslali":n-
`
`Target protein
`
`608 ribosomal
`• "(I;~"'~illlllllis~UjbUnit p.AAAA
`
`408 ribosomal
`sutiunit
`(1) Normal prolein
`translation
`
`AAAAA
`WII!!!!!!:!!!
`Formation of ASQ-mRNA heteroduplex
`
`::: ~,.
`
`~!!!! II:I:IIll!;!!
`
`I]' I
`
`•
`
`RNase H
`
`(2) Activation of RNase H
`
`(3) Steric hinderance of
`ribosomal subunit binding
`
`In the absence of ASO, normal gene and protein expression is maintained. The ASQ is taken
`(ASOs).
`Fig. 1 Modes of action of antisense oligonucleotides
`up by cellular endocytosis and can hybridize with target mRNA in the cytoplasm. Formation ofanASo-mRNA
`heteroduplex induces (2) activation of RNase
`H, leading to selective degradation of bound mRNA or (3) steric interference of ribosomal assembly. Both actions will result
`in target protein knockdown.
`Alternatively,
`the ASO can enter the nucleus and regulate mRNA maturation
`by (4) inhibition of 5' cap formation,
`(5) inhibition ofmRNA splicing and
`(6) activation of RNase H. Theoretically,
`the ASO can selectively knock down any target gene and protein expression,
`leading to therapeutic benefit.
`
`Prediction
`
`of the secondary
`
`structnre
`
`of RNA
`
`that effective ASO design depends on
`accepted
`It is generally
`accurate prediction of the secondary structure of the RNAY How-
`ever, a truly reliable algorithm to predict any single rnRNA secondary
`structure and folding pattern is lacking. A widely used mfold program
`is available
`in the public domain
`(http://www.bioinfo.rpi.edu/
`applications/mfold)
`that predicts all possible optimal and suboptimal
`sequence of mRNA. The core algorithm
`structures of a particular
`predicts overall minimum free energy, aG, of different possible
`folding." Conversely, another commonly used computer algorithm
`is the sfold program (http://sfold.wardsworth.org/index.pl),
`which
`
`transcript."
`predicts only the best secondary structure of the target
`Using a combination of both mfold and sfold, one can determine the
`most frequently occurring secondary structure of the target mRNA
`with minimal overall free energy as a potential ASO target site.
`
`of preferable mRNA local secondary
`
`Identification
`structures
`An effective ASO should be designed at the regions where mRNA
`is accessible
`for hybridization.':"
`Local
`structures
`accessible
`to
`ASOs are those usually located at the terminal end, intemalloops,
`joint
`sequences, hairpins
`and bulges of 10 or more consecutive
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Asia Pry Ltd
`
`

`

`Antisense oligonucleotide
`
`535
`
`In conjunction with mfold, a new software (Target-
`nucleotides."
`Finder; http://www.bioit.org.cru.ao!targetfinder.com)
`has
`recently
`been developed to facilitate ASO target site selection based on the
`method of mRNA accessible
`site tagging (.MAST).13 Yang et at. 14
`have recently demonstrated that potentASO target sites can be found
`in highly conserved local motifs, whereas ASO targeting at variable
`local motifs may lead to non-sequence
`specific effects. Therefore,
`in order to increase the 'hit rate' of potent ASO design, one needs
`to look for locally conserved structures among various optimal and
`suboptimal mRNA predicted secondary structures.
`
`and GC content calcnlation
`Motifs determination
`the accessible
`conserved
`local
`secondary
`After confirmation
`of
`structures and the corresponding
`sequences of ASOs (approximately
`20 bp), one can settle on some well-defined activity enhancing motifs
`and discard those activity decreasing motifs in theASOs.
`15 Matveeva
`et al. 15 analysed data collected from> 1000 experiments using phos-
`phorothioate
`(PS)~modified ASOs and found a positive correlation
`between ASO-mediated
`mRNA knockdown
`and the presence
`of CCAC, TCCC, ACTC, GCCA and CTCT motifs in the ASOs.
`Conversely,
`the presence ofGGGO (O-quartets
`formation), ACTG,
`AAA and TAA motifs in ASOs weakened ASO activity. Although
`it is believed that
`the formation of the ASO-mRNA heteroduplex
`stimulates Rlqase H activity,
`leading to target mRNA degradation,
`Ho et al. J6 found that RNase H activity is sequence independent.
`Instead, GC content
`is strongly correlated with the thermodynamic
`stability of the ASO-mRNA duplexes and RNase H activity. Ho et al. 16
`observed strong ASO effects with a minimum of 11 G or C residues
`PC?Of inhibition was observed
`in 20 bp ASOs, whereas
`by ASOs
`having nine or fewer G or C residues.
`
`Binding energy (aGo 37) prediction
`
`to take into consideration
`also needs
`Successful ASO design
`thermodynamic
`energy. Software
`for calculating
`thermodynamic
`properties between the ASO and mRNA target sequence is available. 17
`The program OligoWalk from the package RNAstructure 3.5 (http://
`has been developed to calculate
`128. 151. 176.70/RNAstructure.html)
`binding energy of ASOI ASO and ASO/mRNA.
`To design a potent
`ASO, the binding energy between the ASO and mRNA should be
`AGo37~ -8 kcallmol, whereas the energy for binding betweenASOs
`should be LiO°37 ~ -1.1 kcallmol.
`J5 By using two large databases
`from ISIS Pharmaceuticals
`(Carlshad, CA, USA) and the puhlished
`literature, Matveeve et al. 18 showed that the hit rate of developing a
`potent and active ASO is six- and threefold higher,
`respectively,
`if
`the above criteria are met. In addition, by using this algorithm alone,
`Fei and Zhang" were able to design ASO for the downregulation
`of
`vascular endothelial growth factor protein expression with a success
`rate higher
`than 85%.
`a tedious ASO design approach using multiple
`To circumvent
`computer algorithms, a fast and handy ASO prediction based on a
`neural network has been developed
`using on a broad range of
`parameters,
`including base composition, RNA-ASO binding energy,
`RNA-ASO terminal properties, ASO-ASO binding properties
`and
`10 verified sequence motifs correlated with efficacy and Rnase H
`accessibilities." The prediction server interface is available at http://
`www.cgb.ki.se/AOpredict. Although this model can predict effective
`ASOs with> 50% gene-expression
`inhibition with a success rate of
`
`92%, some effective sequences may be missed because the selective
`there is a lack
`criteria of this program are too stringent. Furthermore,
`ofthermodynamic
`consideration in this network in correlating dimer
`energy with efficacy.
`algorithm to accu-
`In conclusion,
`there is no reliable stand-alone
`rately predict ASO. Inpractice, ASOs have to be tried and screened, so
`that some companies,
`such as ISIS Pharmaceuticals,
`have performed
`gene-walking and have screened hundreds of ASOs against one gene.
`Using this linear 'shot-gun' approach, only 2-5% ofthe oligonucleo-
`tides are generally found to be potent ASOs.4 However, by combining
`the above theoretical criteria using multiple computational algorithms,
`one can markedly increase the hit rate of highly potent ASOs.
`
`CHEMICAL MODIFICATIONS
`
`OF ASO
`
`An unmodified ASO is rapidly attacked by all types of nucleases in
`biological
`fluid and its overall charged property prevents it from
`penetrating through the cell membrane. Various chemical modifications
`have been developed to enhance nuclease resistance, prolong tissue
`half-life,
`increase
`affinity and potency and reduce non-sequence-
`specific toxicity (Fig. 2).
`
`First-generation ASOs
`
`First generation ASOs are those containing a PS-modified backbone,
`in which one of the non-bridging oxygen atoms in the phosphodiester
`bond is replaced by a sulphur atom." Phosphorothioate modification
`confers higher resistance to the ASO against nuclease degradation, lead-
`ing to higher bioavailability of the oligonucleotide. Phosphorothioate-
`modified ASOs promote RNase
`Il-mediated
`cleavage of target
`mRNA. However,
`this modification may slightly reduce the affinity
`of the ASO for its mRNA target because the melting temperature
`of the ASo-mRNA heteroduplex decreases by approximately 0.5°C
`per nucleotide."
`Phosphorothioate-modified
`ASOs have also been
`reported to produce non-specific
`effects by interactions with cell
`surface and intracellular proteins.' Despite these disadvantages, PS
`modification
`is the most widely performed
`chemical modification
`of ASOs for loss-of- function studies in vitro and in vivo for gene
`target identification and validation.
`Indeed,
`intravitreous
`fomivirsen,
`a 21 bp first generation PS-modifiedASO,
`is currently the only ASO
`drug approved for clinical use."
`
`Second-generation ASOs
`
`To further enhance nuclease resistance and increase binding affinity
`for target mRNA, second-generationASOs with 2'-alkyl modifications
`of the ribose were developed.
`2'-O-Methyl
`(2'-OMe)
`and 2'-0-
`Methoxyetbyl
`(2' -MOE) modifications ofPS-modified ASOs are the
`two most widely studied second-generation ASOs. I Unexpectedly,
`2' -OMe and 2'-MOE substitutions do not support RNase Il-mediated
`cleavage of target mRNA, which dampens the efficacy of the ASo. 24
`To circumvent
`this shortcoming,
`a chimeric ASO was developed in
`which
`a central
`'gap'
`region consisting
`of approximately
`10
`PS-modified
`2' -deoxynucleotides
`is flanked on both sides (5' and 3'
`directions) by approximately five nucleotide
`'wings'. The wings are
`composed of 2' -OMe or 2'-MOE PS-modified
`nucleotides." This
`chimeric 'gapmer' ASO allows RNase H to sit in the central gap to
`execute target-specific mRNA degradation; meanwhile,
`the 'wings'
`resist nuclease
`cleavage of ASO by 2' -alkyl modifications
`at both
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell Publishing Asia Pty Ltd
`
`

`

`JHP Chan et a1.
`
`2'.fluoro-arabino nucleic acid
`(FANA)
`
`-'(rB
`
`'"
`
`NI°I
`
`..-
`O=~-N <,
`
`0"
`Phosphoroamldale Morpholino
`(PMO)
`
`N3'·PS' Phosphoroamldate
`(NP)
`
`l;;;t'
`
`~ °
`
`O=p-o-
`
`I0
`
`"
`Locked
`
`nucjetc
`(LNA)
`
`acid
`
`·'~)::r
`
`"
`
`,
`"J+o~ase
`II',.~
`
`536
`
`'O~'
`
`H
`
`~
`
`0=)-8-
`0"
`PhosphorottlJoate
`(PS)
`
`DNA
`
`-cr~ ~
`
`0='1-0-
`
`CHs
`
`0"
`2'-D-methyl
`(OMe)
`
`RNA
`
`'~'"
`°
`
`I
`O=P-O-
`
`I0
`
`°
`
`'A
`
`O-CH~
`
`"
`2'-O·methoxy-ethyl
`(MOE)
`
`RNA
`
`~O=p-o-
`I0,
`
`nucleic acid
`Cycloheltene
`{CeNAl
`
`~
`Q=P-O-
`I°"
`
`Tricyclo-ONA
`(leONA)
`
`Peptide nucleic acid
`(PNA)
`
`Fig. 2 Chemical modifications of
`antisense oligonucleotides.
`
`ends. Extensive studies have been performed in vivo to assess the
`stability and toxicity of these modified ASOS.1,26
`
`Third-generation ASOs
`
`To further enhance target affinity, nuclease resistance, biostability
`and pharmacokinetics,
`a third generation of ASO was developed
`mainly by chemical modifications of the furanose ring of the
`nucleotide. Peptide nucleic acid (PNA), locked nucleic acid (LNA)
`and phosphoroamidate morpholino oligomer
`(PMO) are the three
`most studied third-generation ASOS.3,27
`Peptide nucleic acid is a synthetic DNA mimic in which the
`phosphodiester backbone is replaced with a flexible pseudopeptide
`polymer
`(N-(2-aminoethyl)glycine)
`and nucleobases are attached to
`the backbone via methylene carbonyl bnkage.":"
`Peptide nucleic
`acid is a non-charged
`nucleotide
`analogue
`that can hybridize
`complementary DNA or RNA with higher affinity and specificity
`than unmodified DNA-DNA and DNA-RNA duplexes. Inaddition,
`PNA demonstrates high biostability in biological
`fluid because it is
`not degraded by nucleases or peptidases. Peptide nucleic acid exerts its
`antisense effect by fanning a sequence-specific duplex with mRNA,
`which mainly causes steric hindrance of translational machinery
`leading to protein knockdown because it is not a substrate for RNase
`H. Furthermore, PNA can elicit anti gene effects by hybridizing with
`double-stranded DNA in four possible configurations,
`including
`triplex, triplex invasion, duplex invasion and double duplex invasion,29,3o
`resulting in transcriptional arrest. Substantial data have revealed the
`effectiveness of PNA in gene silencing in various ex vivo models
`and in genetic and cytogenetic analyses,":"
`but its efficacy in vivo
`remains to be determined.
`restricted nucleotide
`Locked nucleic acid is a conformationally
`containing a 2'-0,4' -C-methylene bridge in the j3-D-ribofuranosyl
`configuration. This modification greatly enhances its hybridization
`affinity towards -target mRNA and DNA, with a substantial
`increase
`
`in the thermal stability of the duplexes." Inaddition, LNA is resistant
`to nuclease degradation. Like any 2'-0 ribose modification, LNA is not
`a substrate for RNase H. Notwithstanding, LNA monomer can be freely
`incorporated into RNA and DNA to form chimeric oligonucleotides
`resulting in restoration of RNase H-mediated cleavage of mRNA.
`It has been shown that the chimeric LNAIDNA/LNA gapmer with
`seven to 10 PS-modified DNA central gaps flanked by three to four
`LNA oligomers on both ends provides highly efficient mRNA cleavage,
`in addition to high ASO potency,
`target accessibility and nuclease
`resistance." Among the nine members of the LNA molecular
`family,
`u-L-LNA is the stereoisomer
`of j3-D-LN~ and has been shown
`to demonstrate
`the highest efficacy in rnRNA knockdown
`in both
`in vitro and in vivo studies, making it one of the most promising
`LNA antisense agents.":"
`Phosphoroamidate morpholino oligomer represents a non-charged
`ASO agent in which the ribose sugar is replaced by a six-membered
`morpholino ring and the phosphodiester bond is replaced by a phos-
`phoroamidate
`linkage."
`Phosphoroamidate morpholino
`oligomer
`does not support RNaes H activity,
`such that
`its ASO effect
`is
`primarily mediated by steric interference
`of ribosomal
`assembly
`resulting -in translational
`arrest. This chemical modification
`also
`confers excellent resistance to nucleases and proteases in biological
`fluid. Phosphoroamidate morpholino oligomer does not readily enter
`mammalian cells in culture, but a recent study using an arginine-rich
`peptide (ARP) conjugation to PMO markedly enhanced its cellular
`uptake and antisense potency by increasing the thermal stability of
`theARP-PMo-mRNA
`heteroduplex." Phosphoroamidate morpholino
`oligomer has demonstrated
`antisense
`efficacy in animal models
`in vivo and in human clinical
`trials.40,41
`
`DELIVERY OF ASO
`
`Unmodified naked ASO has a net negative charge and can barely
`penetrate the plasma membrane. Cellular uptake of ASO is primarily
`
`© 2006 The Authors
`Journal compilation © 2006 Blackwell PublishingAsia Pty Ltd
`
`

`

`Antisense oligonucleotide
`
`537
`
`an adsorptive endocytosis process." Phosphorothioate modification
`of ASOs not only enhances nuclease resistance, but also promotes
`adsorption
`of ASOs to cell surface proteins,
`resulting
`in higher
`internalization of the ASOs. Peptide nucleic acid and PMO are non-
`charged oligonucleotides
`that do not interact well with cell surface
`proteins, making them even more difficult for adsorptive endocytosis.
`The amount of ASO that enters cells is so low that a variety of delivery
`strategies has been devised to enhance
`cellular uptake of ASOs
`and the ensuing mRNA knockdown
`(for a review, see Lysik and
`Wu-Pong"). Mechanical
`techniques,
`like electroporation
`and micro-
`injection, are very useful in delivering ASOs into cell cultures in vitro,
`but are impractical
`for in vivo studies. In contrast, chemical-mediated
`ASO delivery has been tested extensively in both in vitro and in vivo
`studies. Cationic lipid carriers
`like N-[I-(2,3-dioleoyloxy)propyl]-
`N,N,N-trimethylammonium chloride
`(DOTMA)
`and N-[I-(2,3-
`dioleoyloxy)propyl]-N,N,N-trimethylammonium
`methyl
`sulphate
`(DOTAP) are the most widely used vectors for ASO internalization.
`Upon entry into the intracellular milieu in the form of an endosome,
`the ASO needs
`to escape
`from the endosomal
`vesicles
`to avoid
`lysosomal degradation so as to interact directly with target mRNA
`in the cytosol and in the nucleus. Dioleylphosphatidylethanolamine
`(DOPE) has been added into the lipid carrier formulation to promote
`endosomal membrane destabilization,
`leading to release of the ASO
`into the cytosol.":"
`Indeed,
`a pl-l-sensitive
`fusogenic
`liposome
`preparation
`consisting
`of DOPE and an amphipathic
`lipid, such
`as cholesteyl
`hemisuccinate,
`supports
`fusion of the liposomal
`and endosomal membranes at a pH below 5.5, resulting inASO escape
`and enhanced mRNA knockdown efficacy." The use of microparticles,
`such as biodegradable
`copolymer
`poly(n,L-lactide-coglycolide)
`in sustained-release ASO delivery has also been investigated. The
`ASOs, encapsulated in microspheres
`ranging from 10 to 60 IJ.m, are
`released gradually with enhanced serum stability, increased cytosolic
`and nuclear delivery and prolonged duration of ASO action in both
`in vitro and in vivo models.S'"
`Covalent conjugation of ASO to a macromolecule like dendrimer'v"
`and to cell-penetrating
`peptides (CPP)46 has been shown to promote
`cellular .....uptake of the ASO. Dendrimers
`are spherical
`and highly
`branched polymers with cationic polyamidoamine moieties capable
`of forming a covalent complex with the ASO.
`In contrast with the
`liposome formulation,
`the dendrimer-ASO complex is stable and
`active in the presence of serum. It enhances ASO cellular delivery
`into the cytosol and nucleus
`and increases
`the retention time of
`ASO in the cells. Conversely, CPP is a short peptide sequence « 30
`amino acids) with net positive charge that allows rapid translocation
`of a large molecule
`like the ASO through the cell membrane
`via
`an energy
`dependent
`pathway. Commonly
`used CPP include
`penetratin (RQIKIWFQNRRMKWKK),
`HlV TAT peptide 48--60
`(GRKKRRQRRRPPQ)
`and transportan (GWTLNSAGYLLGKIN-
`LKALAALAKKIL-amide).
`The ASOs can be directly conjugated
`to any of these CPP via formation of disulphide bridge. By far, PNA
`is the most frequently usedASO in evaluating CPP-mediated mRNA
`knockdown in both in vitro and in vivo studies.":":"
`in liposome technology,
`Despite the advances
`the most critical
`challenge
`for the ASO to be an effective
`therapeutic
`is for it to
`be delivered to the site of action and to produce expected efficacy
`in vivo. There is a new trend of using topical application of ASOs
`Infact, the first clinically
`as the most popular mode of administration.
`approved ASO, formivirsen,
`is administered
`intravitreously, More
`recent studies have revealed that alicaforsen,
`a PS-modified inter-
`
`cellular adhesion molecule- I ASO, produced promising acute and
`long-term benefits
`in ulcerative colitis patients when given locally
`in an enema preparation," AP 12009,
`a PS-modified
`trans