Eur. J. Biochem. 270, 1628–1644 (2003) Ó FEBS 2003
`
`doi:10.1046/j.1432-1033.2003.03555.x
`
`R E V I E W A R T I C L E
`
`Antisense technologies
`Improvement through novel chemical modifications
`
`Jens Kurreck
`
`Institut fu¨r Chemie-Biochemie, Freie Universita¨t Berlin, Germany
`
`Antisense agents are valuable tools to inhibit the expression
`of a target gene in a sequence-specific manner, and may be
`used for functional genomics, target validation and thera-
`peutic purposes. Three types of anti-mRNA strategies can be
`distinguished. Firstly, the use of single stranded antisense-
`oligonucleotides; secondly, the triggering of RNA cleavage
`through catalytically active oligonucleotides referred to as
`ribozymes; and thirdly, RNA interference induced by small
`interfering RNA molecules. Despite the seemingly simple
`idea to reduce translation by oligonucleotides complement-
`ary to an mRNA, several problems have to be overcome for
`successful application. Accessible sites of the target RNA for
`oligonucleotide binding have to be identified, antisense
`agents have to be protected against nucleolytic attack, and
`their cellular uptake and correct intracellular localization
`have to be achieved. Major disadvantages of commonly
`used phosphorothioate DNA oligonucleotides are their low
`
`affinity towards target RNA molecules and their toxic side-
`effects. Some of these problems have been solved in ‘second
`generation’ nucleotides with alkyl modifications at the
`2¢ position of the ribose. In recent years valuable progress has
`been achieved through the development of novel chemically
`modified nucleotides with improved properties such as
`enhanced serum stability, higher target affinity and low
`toxicity. In addition, RNA-cleaving ribozymes and deoxy-
`ribozymes, and the use of 21-mer double-stranded RNA
`molecules for RNA interference applications in mammalian
`cells offer highly efficient strategies to suppress the expression
`of a specific gene.
`
`Keywords:
`deoxyribozymes;
`antisense-oligonucleotides;
`DNA enzymes; locked nucleic acids; peptide nucleic acids;
`phosphorothioates; ribozymes; RNA interference; small
`interfering RNA.
`
`Introduction
`
`The potential of oligodeoxynucleotides to act as antisense
`agents that inhibit viral replication in cell culture was
`discovered by Zamecnik and Stephenson in 1978 [1]. Since
`then antisense technology has been developed as a powerful
`tool for target validation and therapeutic purposes. Theo-
`retically, antisense molecules could be used to cure any
`disease that is caused by the expression of a deleterious gene,
`e.g. viral
`infections, cancer growth and inflammatory
`diseases. Though rather elegant in theory, antisense approa-
`ches have proven to be challenging in practical applications.
`
`Correspondence to J. Kurreck, Institut fu¨ r Chemie-Biochemie,
`Freie Universita¨ t Berlin, Thielallee 63, 14195 Berlin, Germany.
`Fax: + 49 30 83 85 64 13, Tel.: + 49 30 83 85 69 69,
`E-mail: jkurreck@chemie.fu-berlin.de
`Abbreviations: AS, antisense; CeNA, cyclohexene nucleic acid; CMV,
`cytomegalovirus; FANA, 2¢-deoxy-2¢-fluoro-b-D-arabino nucleic acid;
`GFP, green fluorescence protein; HER, human epidermal growth
`factor; ICAM, intercellular adhesion molecule; LNA, locked nucleic
`acid; MF, morpholino; NP, N3¢-P5¢ phosphoroamidates; ON, oligo-
`nucleotide; PNA, peptide nucleic acid; PS, phosphorothioate;
`RISC, RNA-induced silencing complex; RNAi, RNA interference;
`shRNA, short hairpin RNA; siRNA, small interfering RNA;
`tc, tricyclo; TNF, tumor necrosis factor.
`(Received 16 January 2003, revised 19 February 2003,
`accepted 4 March 2003)
`
`In the present review, three types of anti-mRNA strate-
`gies will be discussed, which are summarized in Fig. 1. This
`scheme also demonstrates the difference between antisense
`approaches and conventional drugs, most of which bind to
`proteins and thereby modulate their function. In contrast,
`antisense agents act at
`the mRNA level, preventing
`its
`translation into protein. Antisense-oligonucleotides
`(AS-ONs) pair with their complementary mRNA, whereas
`ribozymes and DNA enzymes are catalytically active ONs
`that not only bind, but can also cleave, their target RNA. In
`recent years, considerable progress has been made through
`the development of novel chemical modifications to stabilize
`ONs against nucleolytic degradation and enhance their
`target affinity. In addition, RNA interference has been
`established as a third, highly efficient method of suppressing
`gene expression in mammalian cells by the use of 21–23-mer
`small interfering RNA (siRNA) molecules [2].
`Efficient methods for gene silencing have been receiving
`increased attention in the era of functional genomics, since
`sequence analysis of the human genome and the genomes of
`several model organisms revealed numerous genes, whose
`function is not yet known. As Bennett and Cowsert pointed
`out in their review article [3] AS-ONs combine many desired
`properties such as broad applicability, direct utilization of
`sequence information, rapid development at low costs, high
`probability of success and high specificity compared to
`alternative technologies for gene functionalization and
`target validation. For example, the widely used approach
`to generate knock-out animals to gain information about
`
`MTX1061
`ModernaTX, Inc. v. CureVac AG
`IPR2017-02194
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`Ó FEBS 2003
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`Novel modifications of antisense-oligonucleotides (Eur. J. Biochem. 270) 1629
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`Fig. 1. Comparison of different antisense strategies. While most of the conventional drugs bind to proteins, antisense molecules pair with their
`complementary target RNA. Antisense-oligonucleotides block translation of the mRNA or induce its degradation by RNase H, while ribozymes
`and DNA enzymes possess catalytic activity and cleave their target RNA. RNA interference approaches are performed with siRNA molecules that
`are bound by the RISC and induce degradation of the target mRNA.
`
`activity. The first is that most AS-ONs are designed to
`activate RNase H, which cleaves the RNA moiety of a
`DNAÆRNA heteroduplex and therefore leads to degrada-
`tion of the target mRNA. In addition, AS-ONs that do not
`
`the function of genes in vivo is time-consuming, expensive,
`labor intensive and, in many cases, noninformative due to
`lethality during embryogenesis. In these cases, antisense
`technologies offer an attractive alternative to specifically
`knock down the expression of a target gene. Mouse
`E-cadherin (–/–) embryos, for example, fail to form the
`blastocoele, resulting in lethality in an early stage of
`embryogenesis, but AS-ONs, when administered in a later
`stage of development, were successfully employed to
`investigate a secondary role of E-cadherin [4]. Another
`advantage of the development of AS-ONs is the oppor-
`tunity to use molecules for therapeutic purposes, which have
`been proven to be successful in animal models.
`It should, however, be mentioned that it was questioned
`whether antisense strategies kept the promises made more
`than 20 years ago [5]. As will be described in detail below,
`problems such as the stability of ONs in vivo, efficient cellular
`uptake and toxicity hampered the use of AS agents in many
`cases and need to be solved for their successful application. In
`addition, nonantisense effects of ONs have led to misinter-
`pretations of data obtained from AS experiments. Therefore,
`appropriate controls to prove that any observed effect is due
`to a specific antisense inhibition of gene expression are
`another prerequisite for the proper use of AS molecules.
`
`Antisense-oligonucleotides
`
`AS-ONs usually consist of 15–20 nucleotides, which are
`complementary to their target mRNA. As illustrated in
`Fig. 2, two major mechanisms contribute to their antisense
`
`Fig. 2. Mechanisms of antisense activity. (A) RNase H cleavage
`induced by (chimeric) antisense-oligonucleotides. (B) Translational
`arrest by blocking the ribosome. See the text for details.
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`Ó FEBS 2003
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`induce RNase H cleavage can be used to inhibit translation
`by steric blockade of the ribosome. When the AS-ONs are
`targeted to the 5¢-terminus, binding and assembly of the
`translation machinery can be prevented. Furthermore, AS-
`ONs can be used to correct aberrant splicing (see below).
`Long RNA molecules form complex secondary and
`tertiary structures and therefore the first task for a successful
`antisense approach is to identify accessible target sites of the
`mRNA. On average, only one in eight AS-ONs is thought
`to bind effectively and specifically to a certain target mRNA
`[6], but the percentage of active AS-ONs is known to vary
`from one target to the next. It is therefore possible to simply
`test a number of ONs for their antisense efficiency, but more
`sophisticated approaches are known for a systematic
`optimization of the antisense effect.
`Computer-based structure models of long RNA mole-
`cules are unlikely to represent the RNA structure inside a
`living cell, and to date are only of limited use for the
`design of efficient AS-ONs. Therefore, a variety of
`strategies have been developed for this purpose (reviewed
`in [7]). The use of random or semirandom ON libraries
`and RNase H, followed by primer extension, has been
`shown to reveal a comprehensive picture of the accessible
`sites [8,9]. A nonrandom variation of this strategy was
`developed in which target-specific AS-ONs were generated
`by digestion of the template DNA [10]. A rather simple
`and straightforward method providing comparable infor-
`mation about the structure of the target RNA is to screen
`a large number of specific ONs against the transcript in
`the presence of RNase H and to evaluate the extent of
`cleavage induced by individual ONs [11]. The most
`sophisticated approach reported so far is to design a
`DNA array to map an RNA for hybridization sites of
`ONs [12]. Because mRNA structures in biological systems
`in vitro
`are likely to differ
`from the structure of
`transcribed RNA molecules, and because RNA-binding
`proteins shield certain target sites inside cells, screening of
`ON efficiency in cell extracts [13] or in cell culture might
`be advantageous (e.g. [14,15]).
`When designing ONs for antisense experiments, several
`pitfalls should be avoided [6]. AS-ONs containing four
`contiguous guanosine residues should not be employed, as
`they might
`form G-quartets via Hoogsteen base-pair
`formation that can decrease the available ON concentration
`and might result in undesired side-effects. Modified guano-
`sines (for example 7-deazaguanosine, which cannot form
`Hoogsteen base pairs) may be used to overcome this
`problem.
`ONs containing CpG motifs should be excluded for
`in vivo experiments, because this motif is known to stimulate
`immune responses in mammalian systems. The CG dinu-
`cleotide is more frequently found in viral and bacterial
`DNA than in the human genome, suggesting that it is a
`marker for the immune system to signify infection. Coley
`Pharmaceuticals even makes use of CG-containing ONs as
`immune stimulants for treating cancer, asthma and infec-
`tious diseases in clinical trials [16].
`Another important step for the development of an
`antisense molecule is to perform a database search for each
`ON sequence to avoid significant homology with other
`mRNAs. Furthermore, control experiments should be
`carried out with great care in order to prove that any
`
`observed effect is due to a specific antisense knockdown of
`the target mRNA. A number of types of control ONs have
`been used for antisense experiments:
`random ONs,
`scrambled ONs with the same nucleotide composition as
`the AS-ON in random order, sense ONs, ONs with the
`inverted sequence or mismatch ONs, which differ from the
`AS-ON in a few positions only.
`In the following sections, properties of modified AS-ONs
`and recent advances obtained with novel DNA and RNA
`analogs will be discussed in more detail. Subsequently,
`strategies to mediate efficient cellular uptake of oligonucleo-
`tides and results of clinical trials will be described.
`
`Antisense-oligonucleotide modifications
`
`One of the major challenges for antisense approaches is the
`stabilization of ONs, as unmodified oligodeoxynucleotides
`are rapidly degraded in biological fluids by nucleases. A vast
`number of chemically modified nucleotides have been used
`in antisense experiments. In general, three types of modi-
`fications of ribonucleotides can be distinguished (Fig. 3):
`analogs with unnatural bases, modified sugars (especially at
`the 2¢ position of
`the ribose) or altered phosphate
`backbones.
`A variety of heterocyclic modifications have been
`described, which can be introduced into AS-ONs to
`strengthen base-pairing and thus stabilize the duplex
`between AS-ONs and their target mRNAs. A comprehen-
`sive review dealing with base-modified ONs was published
`previously by Herdewijn [17]. Because only a relatively small
`number of these ONs have been investigated in vivo, little is
`known about their potential as antisense molecules and
`their possible toxic side-effects. Therefore, the present
`review will focus on ONs with modified sugar moieties
`and phosphate backbones.
`
`‘First generation’ antisense-oligonucleotides
`
`Phosphorothioate (PS) oligodeoxynucleotides are the major
`representatives of first generation DNA analogs that are the
`best known and most widely used AS-ONs to date
`(reviewed in [18]). In this class of ONs, one of the
`nonbridging oxygen atoms in the phophodiester bond is
`replaced by sulfur (Fig. 4). PS DNA ONs were first
`synthesized in the 1960s by Eckstein and colleagues [19]
`and were first used as AS-ONs for the inhibition of HIV
`
`Fig. 3. Sites for chemical modifications of ribonucleotides. B denotes
`one of the bases adenine, guanine, cytosine or thymine.
`
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`Novel modifications of antisense-oligonucleotides (Eur. J. Biochem. 270) 1631
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`Fig. 4. Nucleic acid analogs discussed in this review. B denotes one of the bases adenine, guanine, cytosine or thymine.
`
`replication by Matsukura and coworkers [20]. As described
`below, these ONs combine several desired properties for
`antisense experiments, but they also possess undesirable
`features.
`The introduction of phosphorothioate linkages into ONs
`was primarily intended to enhance their nuclease resistance.
`
`PS DNAs have a half-life in human serum of approximately
`9–10 h compared to  1 h for unmodified oligodeoxy-
`nucleotides [21–23]. In addition to nuclease resistance, PS
`DNAs form regular Watson–Crick base pairs, activate
`RNase H, carry negative charges for cell delivery and
`display attractive pharmacokinetic properties [24].
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`1632 J. Kurreck (Eur. J. Biochem. 270)
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`The major disadvantage of PS oligodeoxynucleotides is
`their binding to certain proteins, particularly those that
`interact with polyanions such as heparin-binding proteins
`(e.g. [25–27]). The reason for this nonspecific interaction is
`not yet fully understood, but it may cause cellular toxicity
`[reviewed in 28]. After PS DNA treatment of primates,
`serious acute toxicity was observed as a result of a transient
`activation of the complement cascade that has in some cases
`led to cardiovascular collapse and death. In addition, the
`clotting cascade was altered after the administration of PS
`DNA ONs. The lower doses of PS oligodeoxynucleotide
`used for clinical trials in humans, however, were generally
`well tolerated, as will be discussed below. Furthermore, the
`seemingly negative property of PS DNA ONs to interact
`with certain proteins proved to be advantageous for the
`pharmacokinetic profile. Their binding to plasma proteins
`protects them from filtration and is responsible for an
`increased serum half-life [28].
`Another shortcoming of PS DNAs is their slightly
`reduced affinity towards complementary RNA molecules
`in comparison to their corresponding phosphodiester oligo-
`deoxynucleotide. The melting temperature of a hetero-
`duplex is decreased by approximately 0.5 °C per nucleotide.
`This weakness is, in part, compensated by an enhanced
`specificity of hybridization found for PS ONs compared to
`unmodified DNA ONs [24].
`
`‘Second generation’ antisense-oligonucleotides
`
`The problems associated with phosphorothioate oligo-
`deoxynucleotides are to some degree solved in second
`generation ONs containing nucleotides with alkyl modifi-
`cations at the 2¢ position of the ribose. 2¢-O-methyl and
`2¢-O-methoxy-ethyl RNA (Fig. 4) are the most important
`members of this class. AS-ONs made of these building
`blocks are less toxic than phosphorothioate DNAs and have
`a slightly enhanced affinity towards their complementary
`RNAs [23,29].
`These desirable properties are, however, counterbalanced
`by the fact that 2¢-O-alkyl RNA cannot induce RNase H
`cleavage of the target RNA. Mechanistic studies of the
`RNase H reaction revealed that the correct width of the
`minor groove of the AS-ONÆRNA duplex (closer to A-type
`rather than B-type), flexibility of the AS-ON and availability
`of the 2¢-OH group of the RNA are required for efficient
`RNase H cleavage [30].
`Because 2¢-O-alkyl RNA ONs do not recruit RNase H,
`their antisense effect can only be due to a steric block of
`translation (see above). The effectiveness of this mechanism
`was first shown in 1997, when the expression of the
`intercellular adhesion molecule 1 (ICAM-1) could be
`inhibited
`efficiently with
`an RNase H-independent
`2¢-O-methoxy-ethyl-modified AS-ON that was targeted
`against the 5¢-cap region [31]. This effect was probably
`due to selective interference with the formation of the 80S
`translation initiation complex.
`Another approach,
`for which the ON must avoid
`activation of RNase H, is an alteration of splicing. In
`contrast to the typical role for AS-ONs, in which they are
`supposed to suppress protein expression, blocking of a
`splice site with an AS-ON can increase the expression of
`an alternatively spliced protein variant. This technique is
`
`the genetic blood disorder
`being developed to treat
`b-thalassemia. In one form of this disease, a mutation
`in intron 2 of the b-globin gene causes aberrant splicing of
`the pre-mRNA and, as a consequence, b-globin defici-
`ency. A phosphorothioate 2¢-O-methyl oligoribonucleotide
`that does not induce RNase H cleavage was targeted to
`the aberrant splice site and restored correct splicing,
`generating correct b-globin mRNA and protein in mam-
`malian cells [32].
`For most antisense approaches, however, target RNA
`cleavage by RNase H is desired in order to increase
`antisense potency. Therefore,
`‘gapmer technology’ has
`been developed. Gapmers consist of a central stretch of
`DNA or phosphorothioate DNA monomers and modified
`such as 2¢-O-methyl RNA at each end
`nucleotides
`(indicated by red and yellow regions of
`the ON in
`Fig. 2B). The end blocks prevent nucleolytic degradation
`of the AS-ON and the contiguous stretch of at least four
`or five deoxy residues between flanking 2¢-O-methyl
`nucleotides was reported to be sufficient for activation of
`Escherichia coli and human RNase H,
`respectively
`[29,33,34].
`The use of gapmers has also been suggested as a solution
`for another problem associated with AS-ONs, the so-called
`‘irrelevant cleavage’ [5]. The specificity of an AS-ON is
`reduced by the fact that it nests a number of shorter
`sequences. A 15-mer, for example, can be viewed as eight
`overlapping 8-mers, which are sufficient
`to activate
`RNase H. Each of these 8-mers will occur several times
`in the genome and might bind to nontargeted mRNAs and
`induce their cleavage by RNase H. This theoretical calcu-
`lation became relevant for a 20-mer phosphorothioate
`targeting the 3¢-untranslated
`oligodeoxyribonucleotide
`region of PKC-a. Unexpectedly, PKC-f was codown-
`regulated by the ON, probably due to irrelevant cleavage
`caused by a contiguous 11-base match between the ON
`and the PKC-f mRNA. Gapmers with a central core of six
`to eight oligodeoxynucleotides and nucleotides unable to
`recruit RNase H at both ends can be employed to
`eliminate irrelevant cleavage, as they will only induce
`RNase H cleavage of one target sequence.
`
`‘Third generation’ antisense-oligonucleotides
`
`In recent years a variety of modified nucleotides have
`been developed (Fig. 4) to improve properties such as
`target affinity, nuclease resistance and pharmacokinetics.
`The concept of conformational restriction has been used
`widely to enhance binding affinity and biostability. In
`analogy to the previous terms ‘first generation’
`for
`phosphorothioate DNA and ‘second generation’ for 2¢-
`O-alkyl-RNA, these novel nucleotides will subsequently be
`subsumed under the term ‘third generation’ antisense
`agents. DNA and RNA analogs with modified phosphate
`linkages or
`riboses as well as nucleotides with a
`completely different chemical moiety substituting the
`furanose ring have been developed, as will be described
`below. Due to the limited space, only a few promising
`examples of the vast body of novel modified nucleotides
`with improved properties can be discussed here, although
`further modifications may prove to have a great potential
`as antisense molecules.
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`Novel modifications of antisense-oligonucleotides (Eur. J. Biochem. 270) 1633
`
`Peptide nucleic acids (PNAs). Peptide nucleic acids
`(PNAs) belong to the first and most intensively studied
`DNA analogs besides phosphorothioate DNA and 2¢-O-
`alkyl RNA [reviewed in 35–37]. In PNAs the deoxyribose
`phosphate backbone is replaced by polyamide linkages.
`PNA was first introduced by Nielsen and coworkers in 1991
`[38] and can now be obtained commercially, e.g. from
`Applied Biosystems (Foster City, CA, USA). PNAs have
`favorable hybridization properties and high biological
`stability, but do not elicit
`target RNA cleavage by
`RNase H. Additionally, as
`they are electrostatically
`neutral molecules, solubility and cellular uptake are
`serious problems that have to be overcome for the usage
`of PNAs as antisense agents to become practical. Improved
`intracellular delivery could be obtained by coupling PNAs to
`negatively charged oligomers, lipids or certain peptides that
`are efficiently internalized by cells [summarized in 35,37].
`the latest and most convincing in vivo
`In one of
`studies, PNAs (as well as several other modified ONs)
`were used to correct aberrant splicing in a transgenic
`mouse model
`[39]. The ONs were directed against a
`the human b-globin gene
`mutated intron of
`that
`interrupted the gene encoding enhanced green fluorescent
`protein (GFP). Only in the presence of systemically
`delivered AS-ONs was the functional GFP expressed.
`Interestingly, PNAs
`linked to four
`lysines at
`the
`C-terminus were the most effective of
`the AS-ONs
`investigated, whereas a 2¢-O-methoxy-ethyl ON had a
`slightly lower activity in all
`tissues except
`the small
`intestine. Morpholino (MF) ONs were significantly less
`effective while PNA with only one lysine was completely
`inactive, indicating that the four-lysine tail is essential for
`antisense activity of PNAs in vivo.
`According to the in vivo studies performed to date, PNAs
`seem to be nontoxic, as they are uncharged molecules with
`low affinity for proteins that normally bind nucleic acids.
`The greatest potential of PNAs, however, might not be their
`use as antisense agents but their application to modulate
`gene expression by strand invasion of chromosomal duplex
`DNA [37].
`
`N3¢-P5¢ phosphoroamidates (NPs). N3¢-P5¢ phosphoro-
`amidates (NPs) are another example of a modified
`phosphate backbone, in which the 3¢-hydroxyl group of
`the 2¢-deoxyribose ring is replaced by a 3¢-amino group. NPs
`exhibit both a high affinity towards a complementary RNA
`strand and nuclease resistance [40]. Their potency as AS
`molecules has already been demonstrated in vivo, where a
`phosphoroamidate ON was used to specifically down-
`regulate the expression of the c-myc gene [41]. As a
`consequence, severe combined immunodeficiency mice
`that were injected with myeloid leukemia cells had a
`reduced peripheral blood leukemic load. Animals treated
`with the AS agent had significantly prolonged survival
`compared to those treated with mismatch ONs. Moreover,
`the phosphoroamidates were found to be superior for the
`treatment of
`leukemia compared to phosphorothioate
`oligodeoxynucleotides. The sequence specificity of phospho-
`roamidate-mediated antisense effects by steric blocking of
`translation initiation could be demonstrated in cell culture,
`and in vivo with a system in which the target sequence was
`present just upstream of the firefly luciferase initiation
`
`codon [42]. Because phosphoroamidates do not induce
`RNase H cleavage of the target RNA, they might prove
`useful for special applications, where RNA integrity needs
`to be maintained, like modulation of splicing.
`
`2¢-Deoxy-2¢-fluoro-b-D-arabino nucleic acid (FANA).
`ONs made of arabino nucleic acid, the 2¢ epimer of
`RNA, or the corresponding 2¢-deoxy-2¢-fluoro-b-D-arabi-
`no nucleic acid analogue (FANA) were the first uni-
`formly sugar-modified AS-ONs
`reported to induce
`RNase H cleavage of a bound RNA molecule [43]. The
`circular dichroic spectrum of a FANAÆRNA duplex
`closely resembled that of the corresponding DNAÆRNA
`hybrid,
`indicating similar helical conformations. The
`fluoro substituent is thought to project into the major
`groove of the helix, where it should not interfere with
`RNase H. Full RNase H activation by phosphorothio-
`ate–FANA, however, was only achieved with chimeric
`ONs containing deoxyribonucleotides in the center, but
`the DNA stretch needed for high enzyme activity was
`shorter than in 2¢-O-methyl gapmers [44]. The chimeric
`FANAÆDNA ONs were highly potent in cell culture with
`a 30-fold lower IC50 than the corresponding phosphoro-
`thioate DNA ON.
`
`Locked nucleic acid (LNA). One of the most promising
`candidates of chemically modified nucleotides developed in
`the last
`few years is locked nucleic acid (LNA), a
`ribonucleotide
`containing
`a methylene bridge
`that
`connects the 2¢-oxygen of the ribose with the 4¢-carbon
`[reviewed in 36,45,46]. ONs containing LNA were first
`synthesized in the Wengel [47,48] and Imanishi laboratories
`[49] and are commercially available from Proligo (Paris,
`France and Boulder, CO, USA).
`Introduction of LNA into a DNA ON induces a
`conformational change of the DNAÆRNA duplex towards
`the A-type helix [50] and therefore prevents RNase H
`cleavage of the target RNA. If degradation of the mRNA is
`intended, a chimeric DNAÆLNA gapmer that contains
`a stretch of 7–8 DNA monomers in the center to
`induce RNase H activity should be used [23]. Chimeric
`2¢-O-methyl–LNA ONs that do not activate RNase H
`could, however, be used as steric blocks to inhibit intracel-
`lular HIV-1 Tat-dependent trans activation and hence
`suppress gene expression [51]. LNAs and LNAÆDNA
`chimeras efficiently inhibited gene expression when targeted
`to a variety of regions (5¢ untranslated region, region of the
`start codon or coding region) within the luciferase mRNA
`[52].
`Chimeric DNAÆLNA ONs reveal an enhanced stability
`against nucleolytic degradation [23,53] and an extraordin-
`arily high target affinity. An increase of the melting
`temperature of up to 9.6 °C per LNA introduced into an
`ON has been reported [50]. This enhanced affinity towards
`the target RNA accelerates RNase H cleavage [23] and
`leads to a much higher potency of chimeric DNAÆLNA
`ONs in suppressing gene expression in cell culture, com-
`pared to phosphorothioate DNAs or 2¢-O-methyl modified
`gapmers [A. Gru¨ nweller, E. Wyszko, V. A. Erdmann and
`
`J. Kurreck, unpublished results]. Whether the high target
`affinity of LNAs results in a reduced sequence specificity will
`need to be investigated. If unspecific side-effects of LNA
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`1634 J. Kurreck (Eur. J. Biochem. 270)
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`ONs are observed, their length would have to be decreased
`to find an optimum for target affinity and specificity.
`AS-ONs containing LNA were also directed against
`human telomerase, which is an excellent antisense target
`that is expressed in tumor cells but not in adjacent normal
`tissue. Telomerase is a ribonucleoprotein with an RNA
`component that hybridizes to the telomere and should
`therefore be accessible for AS-ONs. As RNA degradation is
`not necessary to block the enzyme’s catalytic site, ONs
`unable to recruit RNase H should be suitable inhibitors of
`telomerase function. A comparative study revealed that
`LNAs have a significantly higher potential to inhibit human
`telomerase than PNAs [54]. Due to their high affinity for
`their complementary sequence, LNA ONs as short as eight
`nucleotides long were efficient inhibitors in cell extracts.
`In addition to target affinity, improved cellular uptake of
`ONs consisting of 2¢-O-methyl RNA and LNA, compared
`to an all 2¢-O-methyl RNA oligomer, was suggested to
`account for high antisense potency of LNA [51]. In the first
`in vivo study reported for an LNA, an efficient knock-down
`of the rat delta opioid receptor was achieved in the absence
`of any detectable toxic reactions in rat brain [53]. Subse-
`quently, full LNA ONs were successfully used in vivo to
`block the translation of the large subunit of RNA poly-
`merase II [55]. These ONs inhibited tumor growth in a
`xenograft model with an effective concentration that was
`five times lower than was found previously for the
`corresponding phosphorothioate DNA. Again, the LNA
`ONs appeared to be nontoxic in the optimal dosage.
`Therefore, full LNA and chimeric DNAÆLNA ONs seem to
`offer an attractive set of properties, such as stability against
`nucleolytic degradation, high target affinity, potent biolo-
`gical activity and apparent lack of acute toxicity.
`
`Morpholino oligonucleotides (MF). Morpholino ONs are
`nonionic DNA analogs, in which the ribose is replaced by a
`morpholino moiety and phosphoroamidate intersubunit
`linkages are used instead of phosphodiester bonds. They are
`commercially available from Gene Tools LLC (Corvallis,
`OR, USA). Recently, the success and limitations of their
`usage have been reviewed comprehensively, with particular
`focus on developmental biology [56] as most published work
`on morpholino compounds has been carried out using
`zebrafish embryos. An entire issue of Genesis (volume 30,
`issue 3, 2001) has been devoted to the study of gene function
`using this technique.
`MFs do not activate RNase H and, if inhibition of gene
`expression is desired, they should therefore be targeted to
`the 5¢ untranslated region or to the first 25 bases
`downstream of the start codon to block translation by
`preventing ribosomes from binding. Because their backbone
`is uncharged, MFs are unlikely to form unwanted interac-
`tions with nucleic acid-binding proteins. Their target affinity
`is similar to that of isosequential DNA ONs, but lower than
`the strength of RNA binding achieved with many of the
`other modifications described in this section.
`Effective gene knockdown in all cells of zebrafish
`embryos was achieved with MFs against GFP in a
`ubiquitous GFP transgene [57]. In this study, equivalents
`of known genetic mutants as well as models for human
`diseases were developed and new gene functions were
`determined by the use of MFs. A potential therapeutic
`
`application was reported for MFs that corrected aberrant
`splicing of mutant b-globin precursor mRNA [58]. Treat-
`ment of erythroid progenitors from peripheral blood of
`thalassemic patients with ONs antisense to aberrant splice
`sites restored correct splicing and increased the hemoglobin
`A synthesis. Due to the limited cellular uptake of MFs,
`however, these experiments required high ON concentra-
`tions and mechanical disturbance of the cell membrane.
`Another relevant question that has to be answered is the
`reason for unspecific side-effects that have been observed in
`several studies (summarized in [56]).
`
`Cyclohexene nucleic acids (CeNA). Replacement of the
`five-membered furanose ring by a six-membered ring is the
`basis for cyclohexene nucleic acids (CeNAs), which are
`characterized by a high degree of conformational rigidity of
`the
`oligomers. They
`form stable
`duplexes with
`complementary DNA or RNA and protect ONs against
`nucleolytic degradation [59]. In addition, CeNAÆRNA
`hybrids have been reported to activate RNase H, albeit
`with a 600-fold lower kcat compared to a DNAÆRNA duplex
`[60]. Therefore, the design of ONs with CeNA has a long
`way to go in order to obtain highly efficient AS agents.
`
`Tricyclo-DNA (tcDNA). Tricyclo-DNA (tcDNA)
`is
`another nucleotide with enhanced binding to comple-
`mentary sequences, which was first
`synthesized by
`Leumann and coworkers [61,62]. As with most of the
`newly developed DNA and RNA analogs, tcDNA does not
`activate RNase H cleavage of the target mRNA. It was,
`however, successfully used to correct aberrant splicing of a
`mutated b-globin mRNA with a 100-fold enhanced
`2¢-O-methyl-
`efficiency
`relative
`to an isosequential
`phosphorothioate RNA [63].
`In summary, a great number of modified building blocks
`for ONs have been developed during the last few years.
`Although not all of them could be discussed in the present
`review, general
`features have been shown for some
`promising examples. Most of the newly synthesized nucleo-
`tides rev