current medicinal chemistry, 763-771 1 antisense ... · current medicinal chemistry, 2005, 12,...

Current Medicinal Chemistry, 2005, 12, 763-771 1

Antisense Oligonucleotides: The State of the Art#

T. Aboul-Fadl*

Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut - Egypt

Abstract: The use of antisense oligonucleotides as therapeutic agents has generated considerable enthusiasmin the research and medical community. Antisense oligonucleotides as therapeutic agents were proposed as farback as in the 1970s when the antisense strategy was initially developed. Nonetheless, it has taken almost aquarter of a century for this potential to be realized. The principle of antisense technology is the sequence-specific binding of an antisense oligonucleotide to target mRNA, resulting in the prevention of genetranslation. The specificity of hybridization by Watson-Crick base pairing make antisense oligonucleotidesattractive as tools for targeted validation and functionalization, and as therapeutics to selectively modulate theexpression of genes involved in the pathogenesis of diseases. The last few years have seen a rapid increase inthe number of antisense molecules progressing past Phase I, II and III clinical trials. This review outlines thebasic concept of the antisense technology, its development and recent potential therapeutic applications.

Keywords: Antisense, oligonucleotides, anti-mRNA, ribozymes, RNA interference, Watson–Crick, RNase H, Triple-helix.

#Dedicated to Prof. Adel F. Youssef in celebration of this 70th birthday.

1. INTRODUCTION AS-ODs, thus, are unmodified or chemically modifiedssDNA, RNA or their analogs (1). They are 13–25nucleotides long and are specifically designed to hybridize tocorresponding RNA by Watson-Crick binding [4].

A quarter of a century since the first report that antisenseoligonucleotides (AS-ODs) could inhibit gene expression ina sequence-specific manner [1,2], antisense (AS) inhibition ofgene expression has reliesd primarily on the simple rules ofWatson-Crick base pairing of nucleic acids. A syntheticsmall single-stranded oligonucleotide (OD) that iscomplementary to a specific gene, via hybridization tocorresponding mRNA, inhibits the translation of that geneinto protein, Fig. (1), [3].

AS-ODs have been called “the next great wave of thebiotechnology revolution” and the “pharmacology of thefuture”. Certainly from a theoretical point of view, the ASapproach, in which the target pharmacophore is a specificsequence found in a specific mRNA, should have severaladvantages over traditional pharmaceuticals which targetproteins [5].

Among the characteristics that are essential for AS-ODsto be practically effective are their design and appropriatechemistry. These characteristics not only affect targethybridization, but also extra- and intracellular biologicalstability; their ability to avoid errant compartmentalization;and their efficient uptake and accumulation [6]. AStechnology has rapidly developed as a powerful research tool,resulting in enormous opportunities for its therapeuticapplication. Indeed, one AS drug already exists and severalmore are in advanced clinical trials [7].

The AS-OD technology represents a “newpharmacology”. The receptor, mRNA, has never before beenconsidered in the context of drug-receptor interactions. Beforethe advent of AS technology, no medicinal chemistry hadbeen practiced on the putative “drugs”, ODs. The basis ofthe drug-receptor interaction, Watson-Crick hybridization,had never been considered as a potential binding event fordrugs and put into a pharmacological context. Finally,postbinding events such as recruitment of nucleases todegrade the receptor RNA had never been considered fromPharmacological perspectives. A key to understanding AStechnology is to consider it in a pharmacological context. Itis essential to understand the structure, function, andmetabolism of the receptors for these drugs. It is essential toconsider the future in the context of advances in AS biologyand medicinal chemistry that result in improvedpharmacological behaviors [7].

Fig. (1). AS-ODs are designed to turn off certain genes bybinding to stretches of their mRNA [3].

*Address correspondence to this author at the Department ofPharmaceutical Medicinal Chemistry, Faculty of Pharmacy, AssiutUniversity, Assiut 71526, Egypt; E-mail: [email protected]

0929-8673/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

2 Current Medicinal Chemistry, 2005, Vol. 12, No. 3 T. Aboul-Fadl

O

YO

BO

PX O

O

YO

BHO

O

YOH

BO

PX On

5'

3'

(1)X = O, Phosphate; S, Phosphorothioate; Y = OH, H; B= nucleobase ( A, T, G or C).

2. HISTORY foundation for AS research and re-establish an interest inphosphate backbone modifications as approaches to improvethe properties of ODs [8]. Despite the observations of Miller,Ts’o and Zamecnik interest in AS, research was quitelimited until the late 1980s when advances in several areasprovided technical solutions to a number of impediments.As the design of AS-ODs require an understanding of thesequence of the RNA target, the explosive growth inavailability of viral and human genomic sequences providedthe information about which “receptor sequences” could beselected [8].

Prior to the evolution of effective transfection methodsand understanding of molecular biological techniques, DNAand RNA were administered as potential therapeutic agents.For example, DNA from several sources displayed antitumoractivity and the activity was reported to vary as a function ofsize, base composition and secondary structure [8,9].However, the molecular mechanisms by which DNA mightinduce antitumor effect were never defined and numerousother studies failed to demonstrate antitumor activities withDNA [8]. In contrast to studies on DNA as a therapeuticagent, substantially more work has been reported on RNAand polyribonucleotides. Much of the effort focused on theability of various polynucleotides to induce interferon, andthe most thoroughly studied polynucleotide in this regard ispolyriboinosine : polyribocytidine (poly rI:poly rC). PolyrI:poly rC was shown to have potent antiviral and antitumoractivities in vitro and in vivo, which were attributed tointerferon induction. The substantial toxicities of thispolyribonucleotide in both animal and humans, however,limited its utility [8,9].

Over the past decade, substantial development in ASscience, manufacturing, and development led to the approvalof the first AS drug Fomivirsen (VitraveneTM), (3), for thetreatment of AIDS-related CMV retinitis [10,11].Furthermore, in the mean time, up to 50 new AS-ODs haveentered phase I/II, and in some cases, phase III trials [7,12].

3. ANTI-mRNA STRATEGIES

Three types of anti-mRNA strategies can bedistinguished. First, the use of single stranded AS-OD;second, the triggering of RNA cleavage through catalyticallyactive ODs referred to as ribozymes; and finally, RNAinterference (RNAi) induced by small interfering RNA(siRNA) molecules, which are summarized in Fig. (2). Thefigure also demonstrates the difference between ASapproaches and conventional drugs, most of which bind toproteins and thereby modulate their function. In contrast, ASagents act at the mRNA level, preventing its translation intoprotein. AS-ODs pair with their complementary mRNA,whereas ribozymes and DNA enzymes are catalytically activeODs that not only bind, but can also cleave their targetRNA. In addition, RNAi has been established as a third,highly efficient method of suppressing gene expression inmammalian cells by the use of 21–23-mer siRNA molecules[13-16].

The other polyribonucleotide that has been studiedextensively is ampligen, a mismatched poly rI: poly rC12U.This polyribonucleotide has been shown to induce interferonand to activate 2’-5’ adenosine synthetase. Ampligen hassimilar properties to poly rI:poly rC, however, it has broaderactivities and lower toxicities and is still in development[8,9].

The first clear enunciation of the concept of exploitingAS-ODs as therapeutic agents was in the work of Zamecnikand Stephenson in 1978 [1,2]. They reported the synthesisof an oligodeoxyribonucleotide with 13 nucleotides long (2)that was complementary to a sequence in the respiratorysyncytial virus genome.

A-A-T-G-G-T-A-A-A-A-T-G-G

(2) Despite the seemingly simple idea to reduce translationby ODs complementary to an mRNA, several problems haveto be overcome for successful application. Accessible sites ofthe target RNA for OD binding have to be identified, AS

Though less precisely focused on the therapeuticpotential of AS-ODs, the work of Miller, Ts’o and theircollaborators during the same period, helped to establish the

Antisense Oligonucleotides Current Medicinal Chemistry, 2005, Vol. 12, No. 3 3

N

NHN

N

OP OS-

O O N

N

NH2

O

OPS- O

O N

NHN

NO

O

O

NH2

O

P OS-

O ON

NH

O

O

OPS- OO

H3C

ON

NH

O

O

H3 C

OPS- O

O ON

NH

O

O

H3C

OPS- O

O N

NHN

NO

O

NH2

OPO

OS-

HOO N

N

NH2

O

OPS- O

O O N

NH

O

O

H3 C

OP OS-

O O N

N

NH2

O

O

PS- O

O ON

NH

O

O

H3C

O

PS- OO O

N

NH

O

O

H3 C

OP OS-

O O N

N

NH2

O

OPS- OO O N

NH

O

O

H3C

OP

O

S- O

O N

NH

O

O

H3C

O

P

O

S- O

O N

N

NH2

O

ON

NH

O

O

O

PS- OH3C

O

N

NHN

NO

O

NH2

PS- OO

OPS- OO O N

N

NH2

O

OP OS-

O N

NHN

NO

O

NH2

OH

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(Na+)

(3)

agents have to be protected against nucleolytic attack, andtheir cellular uptake and correct intracellular localization haveto be achieved. In recent years, considerable progress has

been made towards the development of novel chemicalmodifications to stabilize ODs against nucleolyticdegradation and enhance their target affinity [13].

Fig. (2). Comparison of different antisense strategies [13].


Fig. (3). Blocking of translation by AS-ODs [22].

4. MECHANISM OF ACTION OF ANTISENSEOLIGONUCLEOTIDES

the three steps (initiation, elongation, and termination)required for translation has the potential to interrupt proteinsynthesis, Fig. (3), [20,21]. A general requirement for anAS-OD to be successful in mediating translational arrestappears to be a very high-affinity interaction between the ODand the targeted mRNA by Watson-Crick base pairing, thussterically blocking the translation of a transcript into aprotein.

Although there are multiple mechanisms by which anOD may terminate the activity of an RNA species to whichit binds, evidence has been reported for two majormechanisms which contribute to the AS activity of ODs[12,17-19]:

4.1. Translational Arrest by Blocking the Ribosome 4.2. Activation of RNase H

Prevention of binding of the protein translationalmachinery to the target mRNA by an AS-OD at any one of

The mechanism that was widely described is thedestruction of AS-mRNA hybrids by an enzyme which gets

Fig. (4). (1) The single-strand DNA OD passes through the cell membrane and enters the cytoplasm. (2) The OD enters the nucleus. (3)The OD binds (hybridizes) to the target mRNA, forming a sense-antisense duplex. (4) The formation of the duplex initiates therecruitment of the RNase H enzyme, an endogenous nuclease. (5) RNase H degrades the target mRNA, inhibiting target mRNAexpression [7].


activated, the RNase H [23,24]. RNase H is a ubiquitousenzyme that degrades the RNA strand of an RNA-DNAduplex. It has been identified in organisms as diverse asviruses and human cells [7]. The precise recognitionelements for RNase H are not known. However, ODs withDNA-like properties as short as tetramers can activate RNaseH [25]. It is possible to take advantage of chimeric ODsdesigned to activate RNase H, with greater affinity for theirRNA receptors, and to enhance specificity [26,27].Accordingly, most AS-ODs are designed to activate RNaseH, which cleaves the RNA moiety of a DNA.RNAheteroduplex and therefore leads to degradation of the targetmRNA, Fig. (4). This mechanism has resulted in the mostpotent AS-ODs and is the best understood.

RNA through transesterification or hydrolysis reactions thatresult in cleavage of phosphordiester bonds, Fig. (5), [29].

A variety of ribozymes, catalyzing intramolecularsplicing or cleavage reactions, has been found in lowereukaryotes, viruses and some bacteria. Different types ofribozymes and their mechanisms of action have beendescribed comprehensively [31-33].

The hammerhead ribozyme, which has been studied ingreat detail, is one of the most widely used catalytic RNAmolecules. The hammerhead ribozyme was isolated fromviroid RNA and its dissection into enzyme and substratestrands transformed this cis-cleaving molecule into a targetspecific trans-cleaving enzyme. It has a great potential forapplication in biological systems [34,35]. This minimizedhammerhead ribozyme is less than 40 nucleotides long andconsists of two substrate binding arms and a catalyticdomain, Fig. (6).

A therapeutic OD that will specifically bind to and cleavean RNA target should be attractive. In order to achievepotential therapeutic utility of ribozymes, two approacheshave been taken into account. The first approach, ribozyme-coding sequence has been incorporated into plasmids andadministered, which in effect uses a ribozyme gene therapy.Secondly, efforts are focused to identify the minimumribozyme structure combined with chemical modificationsthat retain ribozyme activity while enhancing stability tonucleases [35,37,38].

Substantial progress has been reported with regard to thesynthesis and testing of nuclease-resistant ribozyme drugs.Modifications including phosphorothioate (PS) andnucleoside analogs have been demonstrated to beincorporable in many sites in hammerhead ribozymes toincrease nuclease resistance and support retained ribozymeactivity [38-40]. Chemically stabilized ribozymes were takenup by cells in the synovial lining after intra-articularadministration and reduced the target mRNA. Highertransfection efficiencies can, however, usually be achievedwith delivery systems [41]. It is worthy to mention thatsome modified hammerhead ribozymes are currently inclinical trials, as is shown in section 13 [35,42,43].

6. RNA INTERFERENCE (RNAi)

The recent discovery of RNAi has revolutionizedbiological research and now holds promise as a potentialtherapy for a wide variety of human diseases [43-46]. RNAiis an innate cellular process that directs the degradation ofmRNA homologous to short dsRNA termed siRNA.Recognition of the target mRNA by siRNA is based onstandard Watson-Crick base pairing rules. Thus, it isanother mechanism of AS gene regulation. Since the firstdescription of this process in the nematode wormCaenorhabditis elegans, RNAi has become a powerful andwidely used tool for gene functionalization [47].

Fig. (5). Catalytic cleavage of RNA by ribozyme [30].

Other mechanisms of RNA inactivation which are similarto the traditional AS mechanisms mediated by ribozymesand the recently described RNAi will be discussed in thefollowing sections.

RNAi is initiated by long dsRNA molecules, which areprocessed into 21–23 nucleotide long RNAs by the Dicerenzyme, Fig. (7). This RNase III protein is thought to act asa dimer that cleaves both strands of dsRNAs and leaves two-nucleotide, 3’ overhanging ends. These small interferingsiRNAs are then incorporated into the RISC, a protein-RNA

5. RIBOZYMES

Ribozymes are RNA molecules that catalyze biochemicalreactions [28]. Ribozymes cleave single-stranded regions in


Fig. (6). Structure of hammerhead ribozymes targeting mRNA [36].

complex, and guide a nuclease which degrades the targetRNA [12,48].

however, its application to mammalian cells was hamperedby the fact that long dsRNA molecules induce an interferonresponse. This response triggers the degradation of mRNAby activation of RNase L and dsRNA-dependant protein

This conserved biochemical mechanism could be used tostudy gene functions in a variety of model organisms,

Fig. (7). (1) The double-strand OD passes through the cell membrane and enters the cytoplasm. (2) The helicase separates the sense andantisense strands of the OD. (3) The RISC complex, an endogenous conglomerate of functional components, associates with the AS-OD. (4) The antisense strand of the OD hybridizes to the target mRNA, forming a sense-antisense duplex. (5) The nuclease componentof RISC is an endogenous nuclease that degrades the target mRNA. This inhibits target mRNA expression [7].


Fig. (8). Triple-helix formation in the major groove between a polypyrimidine oligodeoxynucleotide (open pattern line) andpolypurine sequence in double-stranded (black line) DNA. Textural representation of this event is indicated below the cartoon [55].

kinase, which generalizes inhibition of protein synthesis andactivation of apoptotic pathway [49,50]. Fortunately, thisproblem can be resolved by using smaller dsRNAs such as21 nucleotide-long siRNA duplexes [51]. This finding hasled to an explosion in the use of RNAi in mammalian cells,as it is thought to provide a significantly higher potencycompared to traditional AS approaches. The first promisingin vivo experiments with siRNA have already been performedand further therapeutically important genes are expected to betargeted soon. No toxic reactions after siRNA applicationhave been observed in the studies performed to date,however, siRNAs may also exert off-target effects similar toPS-containing AS-ODs [12,52].

focus on the medicinal chemistry of ODs dates to perhaps acouple of years ago. The scope of medicinal chemistry hasexpanded enormously, but the biological data to supportconclusions about synthetic strategies are only beginning toemerge [7]. One of the major challenges for AS approaches isthe stabilization of ODs, as unmodifiedoligodeoxynucleotides are rapidly degraded in biologicalfluids by nucleases. The subjects of medicinal chemistryprograms focus on approaches to create stronger and moreselective affinity for RNA or duplex structures, to provide theability to cleave nucleic acid target to enhance nucleasestability, cellular uptake and distribution, in vivo tissuedistribution, metabolism, and clearance [12].

7. TRIPLEX ANTISENSE TECHNOLOGY 8.1. Modifications of Antisense Oligonucleotides

A vast number of chemically modified nucleotides hasbeen studied in AS experiments. A dimer of an ODdepicting subunits that may be modified to enhance ASproperties is shown in Fig. (9). In general, three types ofmodifications of ribonucleotides can be distinguished:analogs with unnatural bases, modified sugars (especially atthe 2’ position of the ribose) or altered phosphate backbones[59].

In the face of all this progress, still newer technologiesare being developed based on concepts related to ASbiology. For example, it is known that ODs can, in certaininstances, bind to duplex DNA molecules through anunusual kind of base pairing, Fig. (8). Triplex-forming ODs,chemically similar to AS-ODs, associate with dsDNAthrough non-Watson-Crick base pairing (Hoogsteenbonding) that generally depends on polypurine-rich tracts[53,54].

A,C,G,T

X

Y

CH

HA,C,G,T

5'

Linkage

Pendants

X (O, S, C)

Y

O

O

Nucleobase

Sugar

Connection Sites (e.g., a)

Replace Sugar-Phosphate(e.g., amide l inkage, PNA)

2'-Position

5'

3'

4'

3'

In this triplex binding mode, ODs insert themselves intothe major groove of the DNA double helix on a reasonablyspecific basis determined by the nucleotide sequence of thetarget DNA [56]. This triplex technology provides theopportunity to reduce gene transcription itself, rather than todestroy mRNA once it is produced. Because the triplex ODscan be made to permanently alter the DNA after localizing tospecific target sites, the technology actually has the potentialto permanently silence genes [54]. This strategy has beenhindered by problems with both cell penetration and accessto the densely packed nuclear DNA. Despite these problems,this technology has been used to introduce mutations into agene in mice. This raises the interesting prospect of genetherapy to correct genetic abnormalities arising from pointmutations [55,57]. Furthermore, triplex technology mayhave important ramifications in the development of effectiveanticancer agents [54,58].

8. MEDICINAL CHEMISTRY OF ANTISENSEOLIGONUCLEOTIDES

Fig. (9). OD subunits showing different possible modificationsites.

8.1.1. Modifications of NucleobasesAt the core of any rational drug discovery program ismedicinal chemistry. Although the synthesis of modifiednucleic acids has been studied for some time, the intense

The nucleobase or heterocycles of nucleic acids providethe recognition points for the Watson-Crick base pairing


N

N

O

R

OH (NH2 )

N

N

N

R1OH (NH2)

NH2

N

N

O

N

O

H

N

NN

N

OH (NH2 )

N

R2

H

N

NN

N

NH2

NH2

N

N

O

N

O

H

ONH2

C C CH3C C CH3

C C CH2 NH2 C N

I

(CH2 )3NH2

H

N N

C C (CH2)3 CH3

R = R1 = R2 =

Fig. (10). Examples for modified nucleobases.

rules and any OD modification must maintain these specifichydrogen-bonding interactions. Therefore, the scope ofnucleobase modifications is rather limited. Nevertheless,there are a number of base modified ODs that have beenshown to be potentially useful. The relevant nucleobasemodifications can be grouped into 2 structuralclasses[12,60,61] :

AS-ODs, one of the non-bridging oxygen atoms in thephosphodiester bond is replaced by sulfur (4).

OB

HO

P

O

O S

(4)

1. Those that enhance base stacking by expanding the π-electron cloud are represented by lipophilicmodifications in the 5 position of pyrimidines and the7 position of 7-deaza-purines, Fig. (10).

2. Those that provide additional hydrogen bonding, arerepresented by 2-aminoadenine, Fig. (10). The introduction of PS linkages into ODs was primarily

intended to enhance their nuclease resistance. PS-DNAs havea half-life in human serum of approximately 9–10 hscompared to ~1 h for unmodified oligodeoxynucleotides[62,63]. In addition to nuclease resistance, PS-DNAs formregular Watson–Crick base pairs, activate RNase H, carrynegative charges for cell delivery and display attractivepharmacokinetic properties [64]. The major disadvantage ofPS-oligodeoxynucleotides, however, is their binding tocertain proteins, particularly those that interact withpolyanions such as heparin-binding proteins [65-68].

Nucleobase modifications providing a combination ofeffects are represented by 7-deaza-7-modified adenine and thetricyclic cytosine analogs having hydrogen-bondingcapabilities in the major groove of heteroduplex. All thesemodifications are positioned to lie in the major groove of theheteroduplex. These may also have cationic interactions thatstabilize duplex, do not greatly affect the sugar conformationof heteroduplex, provide little nuclease resistance, but willgenerally support an RNase H cleavage mechanism. Inconclusion, the modification of nucleobases to obtainnuclease resistance might not be a fruitful approach [9,12,13,61]. O

B

OCH3

O

O O

O

P

OB

OO

P

O

O O

OCH3

(5) (6)

8.1.2. Modifications of Sugar Moieties and PhosphateBackbones

The chemical modifications of ODs have mainly focusedon the phosphodiester backbone and/or the sugar moiety.Consequently, AS-ODs are classified into three generationsbased on variations of these modifications [13].

8.1.2.1. ‘First Generation’ Antisense Oligonucleotides8.1.2.2. ‘Second Generation’ Antisense OligonucleotidesPS-oligodeoxynucleotides are the major representatives of

first generation DNA analogs that are the best known andmost widely used AS-ODs to date [7,12]. In this class of

The problems associated with PS-oligodeoxynucleotidesare to some degree solved in second generation ODs


O BO

O H

PO

OS

O BO

O YP

O

OX

O BO

O YP

O

OXO B

O Y

PO

X

O B

O H

PO

S

O B

O Y

PO

OX

4- 5

Flank

Flank

Gap

X = S- or O- depending on nuclease resistance provided by 2'-Y

Fig. 11: Gapmer technology

Fig. (11). Gapmer technology.

containing nucleotides with alkyl modifications at the 2’position of the ribose. 2’-O-Methyl (5) and 2’-O-methoxyethyl RNA (6) are the most important members ofthis class [13,61].

In one form of this disease, a mutation in intron 2 of the β-globin gene causes aberrant splicing of the pre-mRNA and,as a consequence, β-globin deficiency. A PS 2’-O-methylOD that does not induce RNase H cleavage was targeted tothe aberrant splice site and restored correct splicing,generating correct β-globin mRNA and protein inmammalian cells [71].

AS-ODs made of these building blocks are less toxicthan PS-DNAs and have a slightly enhanced affinity towardstheir complementary RNAs [64,69]. These desirableproperties are, however, counterbalanced by the fact that 2’-O-alkyl RNA cannot induce RNase H cleavage of the targetRNA. Mechanistic studies of the RNase H reaction revealedthat the availability of the 2’-OH group of the RNA arerequired for efficient RNase H cleavage [70]. Because 2’-O-alkyl RNA ODs do not recruit RNase H, their AS effect canonly be due to a steric block of translation [13].

8.1.2.2.1. Gapmer Technology

For most AS approaches, however, target RNA cleavageby RNase H is desired in order to increase AS potency.Therefore, ‘chimeric strategy’ or ‘gapmer technology’ hasbeen developed [72]. Chimeric OD analogs bring togetherthe beneficial properties of the two types of chemistry. Ingeneral, they have two segments: one that contains an ODderivative capable of activating RNase H and another thatprovides increased binding affinity and fewer side effects.Accordingly, gapmers consist of a central stretch of DNA orPS-DNA monomers and modified nucleotides such as 2’-O-methyl RNA at each end, Fig. (11). The end blocks preventnucleolytic degradation of the AS-OD, and the contiguousstretch of at least four or five deoxy residues between flanking

Another approach, for which the OD must avoidactivation of RNase H, is an alteration of splicing. Incontrast to the typical role for AS-ODs in which they aresupposed to suppress protein expression, blocking of a splicesite with an AS-OD can increase the expression of analternatively spliced protein variant. This technique is beingdeveloped to treat the genetic blood disorder, β-thalassemia.


2’-O-methyl nucleotides was reported to be sufficient foractivation of Escherichia coli and human RNase H,respectively [73].

deoxyribose ring is replaced by a 3’-amino group (8). NPsexhibit both a high affinity towards a complementary RNAstrand and nuclease resistance [80].

8.1.2.3. ‘Third Generation’ Antisense Oligonucleotides Their potency as AS molecules have already beendemonstrated in vivo, where a phosphoroamidate-OD wasused to specifically down regulate the expression of the c-myc gene [81]. Moreover, the phosphoroamidates were foundto be superior for the treatment of leukemia as compared toPS-ODs [82]. Because phosphoroamidates do not induceRNase H cleavage of the target RNA, they might proveuseful for specific applications, where RNA integrity needs tobe maintained, like modulation of splicing [13].

In recent years, a variety of modified nucleotides has beendeveloped to improve AS-OD properties such as targetaffinity, nuclease resistance and pharmacokinetics. Theconcept of conformational restriction has been used widely toenhance binding affinity and biostability. In analogy to theprevious terms, ‘first generation’ for PS-DNA and ‘secondgeneration’ for 2’-O-alkyl-RNA, these novel nucleotides willsubsequently be subsumed under the term ‘third generation’AS-ODs. DNA and RNA analogs with modified phosphatelinkages or riboses as well as nucleotides with a completelydifferent chemical moiety substituting the furanose ring havebeen developed. Promising examples of the vast body ofnovel modified nucleotides with improved properties will bediscussed here, although further modifications may prove tohave a great potential as AS molecules [13].

8.1.2.3.3. 2’-Deoxy-2’-Fluoro-β-D-Arabino Nucleic Acid(FANA)

ODs made of arabino nucleic acid, the 2’-epimer of RNA,or the corresponding FANA (9) were the first uniformlysugar-modified AS-ODs reported to induce RNase Hcleavage of a bound RNA molecule [83].

OB

O

P

O

O O

F

(9)

8.1.2.3.1. Peptide Nucleic Acids (PNAs)

PNAs belong to the first and most intensively studiedDNA analogs besides PS-DNA and 2’-O-alkyl-RNA [74-76]. In PNAs, the sugar-phosphate backbone is completelyreplaced with a peptide-based backbone (7), [77].

NNH

B

OO

(7)

FANA-RNA duplex revealed a similar helicalconformation to that of the corresponding DNA-RNA hybrid.The fluoro substituent is thought to project into the majorgroove of the helix, where it should not interfere with RNaseH. Full RNase H activation by PS–FANA, however, wasonly achieved with chimeric ODs containingdeoxyribonucleotides in the center, but the DNA stretchneeded for high enzyme activity was shorter than in 2’-O-methyl gapmers [84]. The chimeric FANA-DNA ODs werehighly potent in cell culture with a 30-fold lower IC50 thanthe corresponding PS-OD [13].

PNAs have favorable hybridization properties and highbiological stability, but do not elicit target RNA cleavage byRNase H. Additionally, as they are electrostatically neutralmolecules, solubility and cellular uptake are seriousproblems that have to be overcome for the usage of PNAs asAS agents to become practical. Improved intracellulardelivery could be obtained by coupling PNAs to negativelycharged oligomers, lipids, or certain peptides that areefficiently internalized by cells [74,78]. According to in vivostudies performed recently, PNAs seem to be nontoxic, sincethey are uncharged molecules with low affinity for proteinsthat normally bind nucleic acids. The greatest potential ofPNAs, however, might not be their use as AS agents, buttheir application to modulate gene expression by strandinvasion of chromosomal duplex DNA [76,79].

8.1.2.3.4. Locked Nucleic Acid

One of the most promising candidates of chemicallymodified nucleotides developed in the last few years is thelocked nucleic acid [85,86]. Locked nucleic acid, LNA (10),is a ribonucleotide containing a methylene bridge thatconnects the 2’-oxygen of the ribose with the 4’-carbon[75,87,88].

OB

NH

P

O

O O

(8)

OB

OO

P

O

O O

(10)

8.1.2.3.2. N3’-P5’ Phosphoroamidates (NPs)Introduction of LNA into a DNA-OD induces a

conformational change of the DNA-RNA duplex and preventsRNase H cleavage of the target RNA [89]. If degradation of

NPs are another example of a modified phosphatebackbone, in which the 3’-hydroxyl group of the 2’-


the mRNA is intended, a chimeric DNA-LNA gapmer thatcontains a stretch of 7–8 DNA monomers in the center toinduce RNase H activity should be used [64]. Chimeric 2’-O-methyl–LNA ODs that do not activate RNase H could beused as steric blocks to inhibit intracellular HIV-1 geneexpression [90].

intersubunit linkages are used instead of phosphodiesterbonds (12).

MF-ODs are promising AS molecules that possessfavorable hybridization, nuclease stability, and toxicityprofiles. They do not prevent gene expression by activationof RNase H, rather, they function by translational arrest [98-101].Chimeric DNA-LNA ODs reveal an enhanced stability

against nucleolytic degradation and an extraordinarily hightarget affinity [64,91,92]. This enhanced affinity towards thetarget RNA accelerates RNase H cleavage and leads to amuch higher potency of chimeric DNA-LNA ODs insuppressing gene expression in cell culture, compared to PS-DNAs or 2’-O-methyl modified [13].

8.1.2.3.6. Cyclohexene Nucleic Acids (CeNAs).

Replacement of the five-membered furanose ring by a six-membered ring is the basis for CeNAs (13), which arecharacterized by a high degree of conformational rigidity ofthe oligomers.

AS-ODs containing LNA were also directed againsthuman telomerase, which is an excellent AS target that isexpressed in tumor cells but not in adjacent normal tissue.Telomerase is a ribonucleoprotein with an RNA componentthat hybridizes to the telomere and should therefore beaccessible for AS-ODs. As RNA degradation is not necessaryto block the enzyme’s catalytic site, ODs unable to recruitRNase H should be suitable inhibitors of telomerasefunction. A comparative study revealed that LNAs have asignificantly higher potential to inhibit human telomerasethan PNAs [93].

B

O

P

O

O O

(13)

Full LNA-ODs were successfully used in vivo to blockthe translation of the large subunit of RNA polymerase II.These ODs inhibited tumor growth in a xenograft modelwith an effective concentration that was five times lower thanwas found previously for the corresponding PS-DNA [94].

They form stable duplexes with complementary DNA orRNA and protect ODs against nucleolytic degradation [102].In addition, CeNA-RNA hybrids have been reported toactivate RNase H, albeit with a 600-fold lower kcat comparedto a DNA-RNA duplex [103]. Therefore, the design of ODswith CeNA has a long way to go in order to obtain highlyefficient AS agents.O

B

OO

P

O

O O

(11)

8.1.2.3.7. Tricyclo-DNA (tcDNA)

Tricyclo-DNA (14) is another nucleotide with enhancedbinding to complementary sequences [104,105]. As withmost of the newly developed DNA and RNA analogs,tcDNA does not activate RNase H cleavage of the targetmRNA. It was, however, successfully used to correctaberrant splicing of a mutated β-globin mRNA with a 100-fold enhanced efficiency relative to 2’-O-methylphosphorothioate RNA [106].The most recently developed nucleosides as building

blocks for AS-ODs are 2’-O,4’-C-Ethylene-Bridged NucleicAcids (ENA),[95]. ENA (11) has a less-strained six-membered ring in contrast to the five-membered ring ofLNA. The corresponding ODs with ENA nucleosides retaina binding affinity to ssDNA and ssRNA as high as LNA andshow excellent triplex formation with dsDNA. They alsoexhibit much higher nuclease-resistance than LNA [96,97].

OH

O

B

P

O

OO

(14)

N

O B

O

PO N

O(12)

In summary, a great number of modified building blocksfor ODs has been developed during the last few years.Although not all of them could be discussed in the presentarticle, general features have been shown for some promisingexamples. Most of the newly synthesized nucleotides revealenhanced resistance against nucleolytic degradation inbiological fluids and stabilize the duplex between the AS-OD and the mRNA. A major inherent disadvantage ofnucleotides with modifications in the ribose moiety is theirinability to activate efficient RNase H cleavage of the target

8.1.2.3.5. Morpholino Oligonucleotides (MF-ODs)

MF-ODs are nonionic DNA analogs, in which the riboseis replaced by a morpholino moiety and phosphoroamidate


RNA. As a consequence, gapmers with a stretch ofunmodified or PS-DNA monomers in the center of the ODare widely used. Several of the third generation nucleotideshave already been used successfully in vivo, and a high ASpotency combined with low toxicity has been observed.Therefore, one might expect that recent advances innucleotide chemistry will soon lead to significantimprovements of the AS compounds for target validation andtherapeutic purposes.

Despite the significant improvements in the targetingefficiency in vitro of pendant ODs, only a limited number ofin vivo experiments has been performed. Nevertheless, thevalue of conjugation chemistry has been clearly demonstratedboth in vitro and in vivo [115,116].

8.2. Modifications of Ribozymes

In order to obtain therapeutic agents based on syntheticribozymes, it is mandatory to chemically modify thesesstructures. These modifications, as in the traditional AS-ODs, should confer resistance to nucleases, selectivity,proper hybridization and uptake characteristics. In the case ofribozymes, the design of new modified nucleotides becomesmore complex, since a proper folding of the nucleic acid isneeded in order to maintain the catalytic activity. Severalstudies (X-ray structure elucidation and mapping withmodified nucleotides) have shown that the presence of the 2'-hydroxyl group at specific positions in the catalytic core isessential for hydrolytic activity [61].

8.1.2.4. Modification of Backbone Linkage Sites (2’,5’-Oligonucleotides)

The 2’,5’-backbone modified OD systems (15) are ofinterest, as their AS effects are achieved through amechanism other than RNase H. They are a mediator of oneof the interferon pathways and activate RNase L [107,108]. Aserious limitation restricting the potential utility of theseAS-ODs is their rapid degradation by cellular nucleases[109]. However, it was reported that 2’,5’-linked 3’-deoxyoligonucleotides have selective affinity to singlestranded RNA with a markedly prolonged biological half lifecompared to 3’,5’-liked DNA [110]. In a comprehensive study, in which sequence–function

relationships of the hammerhead ribozyme were analyzed, agreat variety of modified nucleotides produced an optimizeddesign for a stabilized hammerhead ribozyme, which isalmost as active as its unmodified parent [13]. The nucleaseresistant ribozyme contains five unmodified ribonucleotides,a 2’-C-allyl uridine (16) at position 4 and a 2’-O-methylRNA at all remaining positions. In addition, the 3’ end wasprotected by an inverted thymidine (17). The serum half-lifeof the stabilized ribozyme is increased to more than 10 dayscompared to less than a 1 min half-life of the unmodifiedRNA ribozyme [40].

O

OY

BHO

O

O

O

O

Y

B

PO

O

OHY

BO

PO O

5'

n

2'

(15)Y= OH, H; B = nucleobase

OU

O

P

O

O O

OB

O

P

O

O O

OOH

N

HN

O

O

(16) (17)

These unique ODs are reported to inhibit the activities ofHIV-1 reverse transcriptase, DNA topoisomerase-I in HIV-1infected cells [111,112], and suppress the replication of RSV[113]. Furthermore, there have been some recent effortsdirected towards preparation of biologically stable analogswith a 2’,5’-internucleotide connection for potential clinicalapplications [113-115].

8.1.2.5. Pendant (Conjugated) OligonucleotidesA slightly improved version of this ribozyme with four

PS bonds in one substrate recognition arm and an inverted3’-3’ deoxy basic sugar led to the design presented in Fig.(12) that is now used for clinical trials [40].

In addition to the previously mentioned modifications,various molecules (pendants) have been attached (conjugated)to ODs to modify their pharmacokinetic properties. Otherpotential applications of pendants include increasedsolubility, lipophilicity, and ability to attach syntheticcleaver intercalaters (for improvement in binding affinity),cross-linking and alkylating groups [116-120]. Althoughconjugation of various functionalities (e.g. cholesterol, folicacid, fatty acids, bleomycins, etc.) to ODs has been reportedto achieve these objectives, the data supporting some of theclaims are limited and generalizations are not possible basedon the data presently available [9,116,118].

8.3. General Structure-Activity/Property Relationshipsof Antisense Oligonucleotides

The excitement of ODs as drugs stems from the fact that,unlike other drug discovery approaches, they areinformational materials. In other words, they are chemicalshaving a specific set of rules that clearly govern their binding


to a specific nucleic acid receptor [59]. Like mentionedbefore, they bind to their nucleic acid target via Watson-Crick base-pairing rules. Consequently, the first test todetermine whether a modified OD will be of interest ismaintaining the sequence specificity according to Watson-Crick rules. A newly modified OD not possessing thisfundamental specificity property is an immediate failure inthe SAR study. The next step in the SAR study is todetermine how tightly (binding affinity) the modified ODbinds to its target nucleic acid. This property relates to theresidence time that an OD is bound to its receptor. The nextessential property that an OD must possess is sufficientresistance to degradation by exo- and endonucleolytic plasmaand tissue nucleases [121].

extensively studied [12]. PS-ODs and chimeric 2’-modified/DNA PS-ODs rapidly distribute to whole tissuewith distribution half-lives ranging from 30 to 60 min invivo [123]. Distribution from plasma is largely due todistribution to peripheral tissues. In contrast, ODs that donot contain a PS linkage, such as PNA, appear to be rapidlyexcreted in the urine. This difference in tissue distributionappears to be largely due to interactions with plasmaproteins [124].

First and second-generation PS-ODs are broadlydistributed to all peripheral tissues. The highestconcentrations of ODs are found in the liver, kidney, spleen,lymph nodes and bone marrow with no measurabledistribution to the brain [125]. In general, the clearance ratesresult in half-lives of elimination ranging from 2 to 5 days inrodents and primates for first-generation ODs, with half livesincreasing 5–10-fold for second-generation ODs [123,126].The elimination half-lives for second-generation ODs suggestthat once weekly or even every two-week systemic dosing isfeasible. First and second-generation PS-ODs not onlydistribute to tissues but also accumulate within cells in thetissues [127]. At early time points after injection, PS-ODsappear to be associated with extracellular matrix and withincells; however, by 24 hs, almost all of the ODs are foundwithin cells in the tissues. The mechanism(s) by which ODsaccumulate within cells following parenteral administrationis(are) currently unknown. In summary, pharmacokineticstudies of PS-ODs demonstrate that they are well absorbedfrom parenteral sites, distribute broadly to all peripheraltissues, do not cross the blood–brain barrier, and areeliminated primarily by slow metabolism. In general, thepharmacokinetic properties of this class of compounds appearto be largely driven by chemistry rather than sequence [128].

10. TOXICITY OF ANTISENSE OLIGONUCLEO-TIDES

Fig. (12). Secondary structure model for a nuclease-resistanthammerhead ribozyme consists of 2’-O-methyl RNA (lowercase),five ribonucleotides (upper case), a 2’-C-allyluridin atposition 4, four PS linkages (s) and an inverted 3’-3’deoxabasic sugar. (H is any nucleotide except guanosine) [13].

There are two broad categories of potential toxicities forAS-ODs; toxicities due to exaggerated pharmacology andtoxicities due to non-AS effects of the OD. The formercategory of side effects results from the OD binding to thedesired target RNA or alternatively to non target RNA,producing an undesirable effect. Although this class oftoxicity has not been a major issue for the technology, thepotential for such toxicities can be further minimized bycareful selection of the drug target and homology searchesagainst human genomic databases [44].

Further biophysical and biochemical properties that maybe affected by OD modifications involve supporting ofendonucleolytic cleavage of the RNA of a heteroduplex,chemical stability, lipophilicity, solubility, protein bindingproperties, toxicological properties and pharmacologicalproperties [122].

The second category of potential toxicities, that is non-AS effects, has been documented at higher doses of ODs.This later category of toxicities is related to the chemicalclass of ODs and can also exhibit some sequence specificity.PS-ODs are the best characterized class of ODs with regardto potential toxicities, having been examined extensively ina full range of acute, chronic and reproductive studies inrodents, lagomorphs and primates. In addition, well over3000 patients have been exposed to PS-ODs in dozens ofclinical trials. PS-ODs have proven to be safer thanoriginally anticipated, yet, like any other drug, they mightproduce non-desirable effects at higher doses. The mostprobable mechanism of the observed toxicities is the bindingof ODs to proteins. It should be noted that different patternsof toxicity exist between species. In rodents, the primary

In the past few years, modifications at nearly everyposition in ODs have been attempted and numerouspotentially interesting analogs have been developed.Although it is far too early to determine which of themodifications may be most useful for particular purposes, itis clear that a wealth of new chemicals is available forsystemic evaluation and that these studies should provideimportant insights into the SAR of AS-ODs [7].

9. PHARMACOKINETICS OF ANTISENSE OLIGO-NUCLEOTIDES

The pharmacokinetics for PS-ODs (first generation ODs)and 2’-modified PS-OD (second generation ODs) have been


toxicities of first-generation ODs occur after prolongedexposure to the ODs at doses above 10 mg/kg and appear tobe a result of pro-inflammatory effects of the ODs. Theseeffects are characterized by splenomegaly, lymphoidhyperplasia and diffuse multiorgan mixed mononuclear cellinfiltrates [129].

identification and validation of new drug targets and thepotential therapeutic applications.

11.1. Application of Antisense Oligonucleotides inFunctional Genomics and Target Validation

Most PS-ODs produce some immune stimulation inrodents, however, ODs containing specific sequence motifscan exhibit profound immune stimulation [130]. Withcareful OD design, the immune stimulatory effects can beminimized. A major attribute of second generation ODs isdecreased immune stimulation in rodents [131]. In primates,the acute dose-limiting toxicities are a transient inhibition ofthe clotting cascade and the activation of the complementcascade [132]. Both of these toxicities are thought to berelated to the polyanionic nature of the molecules, and thebinding of these compounds to specific protein factors inplasma as such, relates to peak plasma concentration.Complement activation has been described primarily in non-human primates. To avoid the potential for complementactivation, most studies have increased infusion times, oradministered the drug by subcutaneous injection, with thegoal of keeping peak plasma concentrations below athreshold concentration [133].

The field of molecular biology has undergone arevolution in recent years, redefining the field as a data rich,as opposed to a data poor discipline. Two early and keysteps in the process of identifying appropriate targets for drugdiscovery are to identify specific gene functions and developan understanding of the role of a gene in contributingtowards or maintaining a disease pathology (targetvalidation). AS-ODs can be used to selectively manipulatethe expression of a chosen gene or genes. Since mRNAdetection and quantitation assays can be rapidly establishedfor any gene, the most widely used AS mechanisms that canaccomplish these goals are AS mechanisms that degradetarget mRNA through either an RNase H or an siRNA-dependant mechanism. Historically, most studies using ASapproaches have focused on identifying functions for one geneat a time. However, recent advances in ODs synthesis andthe application of high throughput cell-culture-based assayshave led to a more global, genome wide approach to genefunction studies [16,42,148-150].It should be noted that PS-ODs have been administered

by intravenous infusion to more than 3000 patients andvolunteers without any significant indication of activation ofthe alternative complement cascade. Second-generation ODsalso appear to have reduced propensity to activatecomplement in non-human primates [126].

The process of identifying AS-ODs for target validationstudies can result in a pharmacophore with a well-understoodmechanism of action, well-characterized distribution and asafe side effect profile, which could be used as a humantherapeutic. A significant advantage of AS approaches totraditional small-molecule-based drug development is theability to target any gene, irrespective of the proteinstructure. One example where this has been used to goodeffect is with the gene survivin. Survivin is a very attractivetarget for cancer therapeutics, as the protein regulates cellproliferation, apoptosis and is known to be highly expressedin many types of tumors [151]. Considerable efforts havegone into developing small molecule inhibitors againstsurvivin, however to date, no success has been reported [43].In contrast, an AS-OD inhibitor of survivin has beensuccessfully identified and characterized [152,153]. Inhibitionof survivin expression with this AS results in polyploidydue to a defect in cytokinesis and ultimately induction ofapoptosis. More recently, it has been shown that a survivinAS-OD suppresses growth of xenografted tumors. Theseresults have led to a decision to develop the survivin AS asa therapeutic for cancer [154].

11. PRACTICAL APPLICATION OF ANTISENSEOLIGONUCLEOTIDES

AS-ODs have the potential of applicability in differentareas. One of the major areas of applications is thefundamental research where the introduction of AS-ODs canhelp to identify and determine the role of a specific gene in aspecific physiological process [15,134-139]. The use of AS-ODs to control gene expression has long fascinatedresearchers because of the potential to rapidly generate potentand specific agents. In the past, AS technology has notalways kept pace with expectations, but recent advances indiverse areas are likely to make it a routine and trustedresearch tool [76].

AS-ODs and in particular PNAs (third generation),exhibit potential for use in detection of genetic mutations,determination of telomere size, nucleic acid purification,nucleic acid biosensors and several other diagnosticapplications [140-142]. PNAs may also find variousinteresting applications in chemistry and technology, e.g. aselectrochemical biosensors, and in optical data storage [143-145]. Furthermore, AS-ODs have provided novelopportunities to tailor the composition of plant-derivedproducts, so that they are optimized with respect to foodfunctionality and human dietary needs. In order to exploitthis new capability, it is essential for food scientists andnutritionists to define the compositions that would be mostdesirable for various purposes [146,147].

11.2. Potential Therapeutic Applications of AntisenseOligonucleotides

AS-ODs can serve as therapeutic molecules in their ownright. To this end, ODs have demonstrated pharmacologicalactivity in a number of animal models of human disease.They are being used in various in vitro and in vivo modelsand are being explored as potential therapeutics againstseveral diseases [155]. A huge number of publications andpatents disclosing potential therapeutic applications of AS-ODs has been published in the last few years. Although it isdifficult to cover all potential therapeutics in this article,major areas of AS-ODs therapeutic applications will beaddressed in which crossover may be observed.

Attention is being drawn to two important applicationsof AS-ODs, at least from the pharmaceutical point of view:


11.2.1. Antisense Oligonucleotides as Antiviral Agents growth and β-lactamase had been obtained by AS-ODs ofPNA (18 is a representative), [166].The use of AS-ODs has emerged as a powerful new

approach as antiviral agents. In fact, the initial therapeuticattempts of AS technology were intended to be as anantiviral agent [1,2]. The antiviral activity of AS-ODs isrelated to the complementation of an AS-OD to repeated orconsensus sequences of the genome of a negative strand RNAvirus, attached to an activator of RNase L [156]. However,the ribozymes were demonstrated to inhibit viral replicationup to 90% in cell; consequently, they were utilized indesigning new AS-ODs for therapeutic applications [34,35].The approval of the first antiviral AS-OD (Fomivirsen)significantly encouraged the initiation of a new era of rationaldrug design utilizing a chemistry that allows for theoptimization of treatment and opportunity to treat a widevariety of other viral infections. These viruses includedinfluenza A [157], HBV [158-160], HCV [100,161,162],CMV [157], HPV [163], RSV [157], HSV [157] and HIV[164]. Moreover, some of the antiviral AS-ODs are currentlyin clinical trials, as is shown in section 13.

HGCT- GTT-TC-Lys-NH2

(18)

In another study it was found that the mycolyltransferases and other mycobacterial genes are highlypromising targets for AS-ODs antitubercular therapy[167,168]. These results provide a proof of the concept thatAS-ODs can be used to develop gene-targeted, designedantibiotics against bacteria. Although much more work isneeded to obtain AS-ODs with adequately improved uptakein pathogenic bacteria.

11.2.3. Antisense Oligonucleotides in Apoptosis

Apoptosis is a process by which cells undergo acontrolled program of cell death that has been stronglyconserved during evolution to prevent uncontrolled cellproliferation [169]. Numerous proteins have been identifiedthat promote apoptosis or have the opposite effect, i.e.,protect cells from apoptosis. The upregulation of anti-apoptotic proteins in certain disease states, e.g., cancer, mayhave the serious effect of rendering the disease more resistantto therapeutic intervention. Conversely, the upregulation orpro-apoptotic proteins may also have serious consequencesleading to excessive cell loss in degenerative disorders.Several AS-ODs have been designed to target the nucleicacid sequences encoding apoptosis-related proteins. TheseAS-ODs may be useful in the treatment of cancer,autoimmune disease, viral infections and others. Arepresentative example of this is the inhibition of theexpression of nucleotide sequences encoding the human anti-apoptotic Bcl-2-related proteins, A-1, MCL-1 and MDM-2.

11.2.2. Antisense Oligonucleotides as AntibacterialAgents

Promising results have been recently suggested a role forAS-ODs in the regulation of bacterial growth [101,165]. Itwas found that AS-ODs targeted to either bacterialribonucleotide reductase or SecA sequences. The expressionof these proteins is essential for the growth of many commonbacteria. Ribonucleotide reductase provides a balancedsupply of deoxynucleotides for DNA synthesis, and SecA isone component of a multicomplex system responsible for thesecretion of proteins across the inner membrane of E coli.Significant inhibition of E coli β-galactosidase mediated cell

Fig. (13). Schematic representation of the signal pathways regulating the proliferation and life/death decision in cancer cells. The grayarrows indicate some of the most promising AS-ODs targeted key proteins in pathways [170].


Table 1. Preclinical Applications of AS-ODs on Human Tumors

Target gene Biological end point Tumor type

Bcl-2 Apoptosis Lymphoma, melanoma, prostate, gastric and breast cancers

Bcl-2/Bcl-xL Apoptosis Breast and colon carcinomas

Clusterin Apoptosis Prostate, bladder and renal cancers

c-myb Proliferation Colon cancer and leukemia

PKA-I Proliferation Colon, breast, lung and prostate cancers

c-myc Proliferation/Apoptosis Melanoma

MDM2 Proliferation/Apoptosis Prostate, breast and colon cancers

An overexpression of the latter protects cells from p53-mediated apoptosis. Reducing hyperproliferation and treatinghyperproliferative conditions associated with overexpressionof MDM-2 such as cancer, psoriasis, fibrosis, atherosclerosisand restenosis [156]. Several AS-ODs that target apoptosisare currently in clinical trials, as is shown in section 13.

CNS diseases will address unmet medical needs; some ofthese diseases are summarized in Table 2 [173,177-181].

Table 2. Some Potential Therapeutic Applications of AS-ODs in CNS Diseases

Disease Gene target11.2.4. Antisense Oligonucleotides as Anticancer Agents

Neuropathic Pain β-arrestinParticular enthusiasm for AS therapeutic approach derivesfrom the identification of several cancer-associated proteins,which can represent potential targets for a selective anticancertherapy with less toxic side effects than conventionalchemotherapy. The preclinical development of several AS-ODs targeting cancer related genes has proceeded veryrapidly. Fig. (13) shows the key signal pathways thatregulate the proliferation and the life/death decision in cancercells [170].

Glutamate neurotoxicity c-fos

Oxidative cell injury CIRL-1 and -3

AD APP

Brain cancer PKCα and integrin αv

A significant number of studies, as well, suggested thatAS-ODs can be extremely valuable tools in the selectiveblock of the expression of neurotransmitter receptors. Thiswill lead to appearance of valuable therapeutic agents tomodify disorders involving these receptors. However, effortsshould be directed to improve the delivery of thesecompounds to their targets [182-184].

AS-ODs targeting cancer-related genes that interfere withboth of these pathways are also shown in Fig. (13). In fact,considering the multigenic defects of human tumors, thecombination of ‘agents’, targeting specific genes involved intumor progression, with antineoplastic drugs represents apromising strategy for improving cancer treatment [42]. 11.2.6. Antisense Oligonucleotides in Inflammation

TherapeuticsTargets for therapeutic AS-ODs include growth factorsand receptors, transcription factors, proto-oncogenes,cytokines, cyclin dependent kinase, protein kinases, DNAdemethylase and methyltransferase, telomerase, matrixmetalloproteinases, angiogenin, integrins, MDM 2, and Bcl-2 family members [171,172]. Promising results have beenachieved by AS-ODs treatment used as anticancer drugs.Table 1 reports some preclinical applications of AS-ODs onhuman tumors. Furthermore, several AS-ODs for thesetargets are currently in clinical trials, as is shown in section13.

With increased understanding of how the immune systemfunctions under normal conditions and a greater appreciationof how dysregulation of immune responses contributes to avariety of inflammatory diseases, a large number oftherapeutically useful molecular targets has been identified[185]. AS-ODs have been recently developed that havetherapeutic promise for inflammatory diseases. Severalinflammatory targets for AS-ODs have been identified and aresummarized in Table 3, [186,187]. Some of these AS-ODsare currently in clinical trials and others are likely to enterthe drug pipeline in the near future. Advances in the fieldwill be greatly facilitated by the genomic revolution[185,187,188].

11.2.5. Antisense Oligonucleotides in CNS Therapeutics

The potential of AS-ODs to treat CNS diseases isenormous, however, there are a number of obstacles to beovercome before AS-ODs can be used effectively in CNSdiseases. Basically, of concern is the transport of AS-ODsacross the BBB to their site of action. The only efficientCNS AS-ODs delivery method currently available is directadministration into the brain. Despite these problems, anumber of potential applications of AS-ODs in CNStherapeutics has been validated in vitro and, in some cases,in vivo [173-176]. The utility of AS-ODs in management of

11.2.7. Antisense Oligonucleotides in CardiovascularTherapeutics

AS-ODs in the field of cardiovascular disease would beuseful in the treatment of many diseases, includingmyocardial infarction [189], prevention of restenosis afterangioplasty [190,191], rejection in heart transplantation[192,193], hypertension and atherosclerosis [194,195].Representatives for the target genes involved are E2F,


Table 3. Some Gene Targets for AS-ODs and their Potential Therapeutic Applications

Target gene Therapeutic area

NF-κB p65 Colitis

NF-κB p50 Lupus

ICAM-1 Lung inflammation, skin inflammation, transplantation rejection, Reperfusion injury

A1 receptor Lung inflammation

IL-1 receptor Skin inflammation

IL-4, IL-5 Lung inflammation

TNF-α Lung inflammation, Rheumatoid Arthritis

VEGF Ocular disease

NFκB, ICAM-1 α and β myosin heavy chain gene. In spiteof the problems of AS-ODs delivery that have hindered theirclinical applications, two ODs are currently in clinical trials.Overall, AS therapy for cardiovascular disease is nowapparently not far from a reality, it is time to take a hardlook at practical issues that will determine the real clinicalpotential [191].

treatment, but it may require multiple applications of theAS-ODs [42].

12.2. Immunostimulatory Side Effects

Immunostimulatory side effects have been observed inanimals and humans with the use of the first generation AS-ODs, as was discussed in section 10 [130]. These problemscan be overcome by chemical modifications of AS-ODs asdiscussed in section 8.1. Typically, modification of the 2’position of the sugar moiety, produces a molecule withincreased affinity for RNA and stabilizes against nucleasedegradation but with fewer immunostimulatory and otherside effects [43,131].

11.2.8. Other Therapeutic Application potentials ofAntisense Oligonucleotides

As seen previously, there is tremendous interest in AS-ODs as potential therapeutic agents. In addition to the majortherapeutic categories mentioned above, AS-ODs have beenexplored as potential therapeutics for a variety of diseasesincluding diabetes [196-198], pain [184,199], psoriasis[200], myasthenia gravis [201] and even light affections suchas hair loss [202]. 12.3. Empirical Exercise for Antisense Oligonucleotides

DiscoveryThe most recent AS application as therapeutic tool is

aimed to treat SARS, caused by the corona virus, which hasimpacted the whole world [202]. With the sequencing of thehuman genome, it is envisaged that many more therapeuticpotentialities for AS-ODs will emerge.

The practical aspects of synthesizing and testing a seriesof AS-ODs should be considered as part of the AS approachfor target validation. Identifying the best sequence for ASactivity against a target mRNA is largely an empiricalexercise. Since not all areas of an mRNA molecule areequally amenable to AS hybridization and because of theinaccessibility of certain hybridization sites in targettranscripts, only few complementary ODs can successfullyhybridize to a target RNA and inhibit function. This may beattributed to inability to the predict accurate RNA structurein cells owing to, mainly, the secondary or tertiary structuresof mRNA or to the proteins bound to the mRNA. The bestway to obtain a highly potent AS inhibitor is still to designmany AS-ODs to the mRNA of the gene target. It isimportant to eliminate any ODs that have extensivecomplementarity to genes other than the desired target. Theresulting AS-ODs can then be used in a screen for the mosteffective inhibitor in a cell culture system. This processrequires some investment in the beginning, but it will betime well spent, however, more than one active nucleotidemay be yielded [203].

12. LIMITATIONS OF PRACTICAL APPLICATIONSOF ANTISENSE OLIGONUCLEOTIDES

Although the AS-ODs technologies offer different choicesin terms of their applicability for target validation and genefunctionalization and thereby hold tremendous and advantageover other traditional methods of drug discovery, they alsohave certain limitations. Some of these issues are:

12.1. Protein Half-Life

As AS-ODs target RNA and not protein, the biologicalconsequences of the inhibition of a particular gene productare dependent on the normal degradation rate (i.e. half-life) ofthe already translated protein product. Often, this is not asignificant concern, as the half-lives of most proteins are fromfew hours to a day, but some proteins last up to severaldays. Therefore, consideration should be given for lag timesin such cases, and the most attractive AS targets fordetermining gene function are those that either induce orencode proteins with a short to moderate half-life. Proteinsthat have long half-lives can be reduced by AS-ODs

12.4. Cellular and In Vivo Delivery

An important hurdle that needs to be overcome forsuccessful AS applications is the cellular uptake of themolecules. In cultured cells, internalization of naked DNA is


usually inefficient due to the charged ODs having to cross ahydrophobic cell membrane. A number of methods hastherefore been developed for in vitro and in vivo delivery ofODs [18,204-206]. By far, the most common and successfuldelivery systems used are liposomes and charged lipids,which can either encapsulate nucleic acids within theiraqueous center or form lipid–nucleic acid complexes as aresult of opposing charges. These complexes are usuallyinternalized by endocytosis. For efficient release of the ODsfrom the endosomal compartment, many transfection reagentscontain helper lipids that disrupt the endosomal membraneand help to set the ODs free. A number of macromolardelivery systems has been developed recently that mediate ahighly efficient cellular uptake and protect the bound ODsagainst degradation in biological fluids [18].

[7,12]. In 1996, only a handful of AS molecules was inclinical trials [207]. Of these six trials, five were only inPhase I. However, the past few years have seen an explosivegrowth in the number of AS-related clinical trials. Currently,there are nearly 50 AS-ODs in trials for various diseases; upto 10 of these are in phase III, with an additional 20 in PhaseII, and representatives of these AS-ODs are given in Table 4[7,12,35,208]. The majority of the drugs in clinical trials arefirst-generation PS-ODs that are designed to inhibit geneexpression through an RNase H mechanism.

The clinical experience to date should be considered partof the beginning of the story of AS treatment, with moreclinical trials of new AS drugs expected in the near future.The fuller story, yet to be written, promises to be rich.

14. CONCLUSIONDespite these successful applications of free AS

molecules, higher levels of cellular uptake can usually beachieved by the use of transfection agents. Therefore, thedevelopment of delivery systems that mediate efficientcellular uptake and sustained release of the drugs remains oneof the major challenges in the AS field.

The AS-ODs have emerged as a valid approach toselectively modulate gene expression by adhering to a strictset of specific rules. A significant advantage of AS-ODs totraditional small-molecule-based drugs development is theability to target any gene, irrespective of the proteinstructure. Consequently, AS technologies have gainedincreasing attention in recent years. Major improvementshave been achieved by the development of modifiednucleotides that provide high target affinity, enhancedbiostability and low toxicity. Since most of the new DNA

13. CLINICAL TRIALS OF ANTISENSE OLIGONU-CLEOTIDES

To date, one AS-OD (Fomivirsen, 3) has been approvedby the FDA for local administration to treat CMV retinitis

Table 4. Representatives of AS-ODs Approved or in Clinical Trialsa

Product Chemistry Target Disease Rout of Administration Status (Phase)

Vitravene™ PS IE2 CMV Retinitis Intravitreal On Market

Affintak™ PS PKC-α Cancer–NSCLC, others Parenteral III

Alicaforsen™ PS ICAM-1 Crohn's Disease Parenteral III

ISIS 2302 PS ICAM-1 Topical Psoriasis Topical II

ISIS 2302 PS ICAM-1 Ulcerative Colitus Enema II

ISIS 2503 PS H-ras Cancer-pancreatic, others Parenteral II

ISIS 14803 PS-DNA Antiviral Hepatitis C Parenteral II

ISIS 104838 Chimeric PS(2nd Gen.)

TNF-α Rheumatoid Arthritis Parenteral/Oral II

ISIS 104838 Chimeric PS(2nd Gen.)

TNF-α Psoriasis Topical II

OGX-011 N/A Clusterin Cancer Parenteral I

Genasense™ PS Bcl-2 Cancer Intravenous II/III

E2F Decoy N/A E2F Atherosclerosis Ex-vivo II/III

Resten-NG MF(3ed Gen.)

c-myc Restenosis Intravenous III

Heptazyme™ RNA/DNA HCV Hepatitis C Intravenous II

Product R PNA(3ed Gen.)

CCR5 HIV Unknown I/II

1018-ISS PS Immune Response Hepatitis B Intravenous II/III

1018-ISS PS Immune Response Asthma Intravenous I/II

a: completion based on references 7,12,35,208N/A: Not Available or Company will not disclose. Gen.: Generation


analogs do not induce RNase H cleavage, the design of AS-ODs has to be adjusted according to whether the targetmRNA must remain intact, e.g. for alteration of splicing, orshould be degraded (gapmer technology). Stable ribozymeswith high catalytic activity were obtained by systematicallymodifying naturally occurring ribozymes or by in vitroselection techniques. A major breakthrough was thediscovery that short dsRNA molecules can be used to silencegene expression specifically in mammalian cells. Thismethod has a significantly higher efficiency compared totraditional AS approaches.

MCL = Myeloid Cell Leukemia

MF = Morpholino

MeTase = Methyl Transferase

NSCLC = Non-Small Cell Lung Cancer

NF-κB = Nuclear Factor kappa B

NPs = N3'-P5' Phosphoroamidates

OD = Oligonucleotide

ODs = OligonucleotidesIn spite of the number of AS-ODs and ribozymes

currently in Phase I, II and III clinical trials, and the alreadyapproved AS-OD “Fomivirsen”, which demonstrate theireffectiveness as therapeutic agents, there are still significanthurdles to be overcome. The main barrier is achievingsystematic delivery of the AS-ODs to the correct target,within the desired time frame, to achieve functional downregulation of the target gene. These issues are currently beingactively addressed and new research will hopefully continueto shed light on ways to increase therapeutic efficacy andspecificity. Accordingly, AS-ODs can be expected to bewidely used for studies of genes with unknown function, fortarget validation in drug development and finally fortherapeutic purposes of course. The promise of AS-basedtechnology is therefore stronger than ever.

PKA = Protein Kinase A

PKCα = Protein Kinase CαPKD = Polycystic Kidney Disease

PNA = Peptide Nucleic Acid

PS = Phosphorothioate

RISC = RNA-Induced Silencing Complex

RNase H = Ribonuclease H

RNase L = Ribonuclease L

RNAi = RNA Interference

RNR = Ribonucleotide Reductase

RSV = Respiratory Syncytial Virus

15. ABBREVIATIONSSARS = Severe Acute Respiratory Syndrome

siRNA = small interfering Ribonucleic AcidA-1 = Adeinosine 1 ssDNA = single-stranded Deoxynucleic AcidAIDS = Acquired Immunodeficiency Syndrome tcDNA = Tricyclo-DNAAD = Alzheimer’s disease TGFβ2 = Tumor Growth Factor-α 2APP = Amyloid Precursor Protein TNF-α = Tumor Necrosis Factor-αAS = Antisense VEGF = Vascular Endothelial Growth FactorAS-OD = Antisense Oligonucleotide VEGFR = Vascular Endothelial Growth Factor Receptor.AS-ODs = Antisense Oligonucleotides

16. REFERENCESCIRL = Calcium-Independent Receptors for α-Latrotoxin

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FANA = 2'-Deoxy-2'-Fluoro-β-D- Arabino Nucleic Acid

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HCV = Hepatitis C Virus

HIV = Human Immunodeficiency Virus [9] Crooke, S. T. In Burger’s Medicinal Chemistry and DrugDiscovery 6th Edition; Abraham D. J., Ed.; John Wiley & Sons,Inc.: New Jersey, 2003; Vol. 2, pp. 115-166.

HPV = Human Papilloma Virus

ICAM = Intercellular Adhesion Molecule [10] Marwick, C. JAMA, 1998, 280, 871.[11] Crooke, S. T. Antisense Nucleic Acid Drug Dev., 1998, 8, 7.

IL = interleukin [12] Bennett, C. F.; Swayze, E.; Geary, R.; Levin, A. A.; Mehta, R.;Teng, C-L.; Tillman, L.; Hardee, G. In Gene and Cell Therapy,Therapeutic mechanisms and Strategies 2nd Edition, Templeton N.S., Ed.; Marcel Dekker, Inc. : New York, 2004; pp. 347-374.

LNA = Locked Nucleic Acid

Lys = Lysine


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