oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution,...

ANTISENSE & NUCLEIC ACID DRUG DEVELOPMENT 12:103–128 (2002)© Mary Ann Liebert, Inc.

Review

Oligonucleotide Conjugates as Potential Antisense Drugs with Improved Uptake, Biodistribution, Targeted Delivery,

and Mechanism of Action

MUTHIAH MANOHARAN

ABSTRACT

This review summarizes the effect of conjugating small molecules and large biomacromolecules to antisenseoligonucleotides to improve their therapeutic potential. In many cases, favorable changes in pharmacokineticand pharmacodynamic properties were observed. Opportunities exist to change the terminating mechanism ofantisense action or to enhance the RNase H mode of action via conjugate formation.

INTRODUCTION

ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS originate fromthe specific molecular recognition event between the

mRNA of the gene to be inhibited and the synthetic oligonu-cleotide drug. In addition to this key pharmacodynamic pro-cess, many pharmacokinetic processes must occur in order toreach the desired pharmacologic end point. Between deliveryinto the patient’s body and degradation or inhibition of transla-tion of the target mRNA, the success of this drug developmenttechnology relies on many receptor-ligand recognition pro-cesses.

The desired properties of antisense compounds are summa-rized in Figure 1. A potent antisense drug should have resis-tance to nucleases, high affinity for the target mRNA, and fa-vorable pharmacokinetics and should cause inactivation of thetarget mRNA either by RNase H-mediated cleavage or by non-RNase H mechanisms (Cook, 1998b; Crooke, 1998; Bennett,1999; Phillips, 2000). Many of these properties can be achievedby the choice of oligonucleotide modifications. Certain tetheredligands may improve the cellular delivery of oligomers and in-crease their affinity for the target gene and resistance to nucle-ases. Other modifications may play the role of synthetic nucle-ases. Furthermore, modifications to sugar or backbone or ligandconjugation can modulate the extent of protein binding either toenhance biodistribution or to lessen side effects due to nonspe-cific protein binding. Some ligands, such as folic acid, are capa-

ble of improving oral absorption properties of antisense oligo-nucleotides (Oleg Ketsenko, unpublished observations).

The first generation of antisense oligonucleotides with the29-deoxyoligonucleotide phosphorothioate modification has fa-vorable pharmacokinetic properties. However, these oligo-deoxynucleotides exhibit certain toxicities correlated with thephosphorothioate content in the backbone. Overcoming the toxicity without sacrificing their favorable pharmacokinetic orprotein-binding properties is a challenge that may be overcome using suitable oligonucleotide conjugates and appropriatechemical modifications. In this review, I summarize the differ-ent modifications that have been employed to synthesize de-signer antisense oligonucleotides that maximize the favorablerecognition events involved in delivery of antisense oligonu-cleotides to target mRNA.

Two earlier reviews (Goodchild, 1990; Manoharan, 1993)addressed the chemical methodology and applications of oligo-nucleotide conjugates for antisense therapeutics up to the year1993. An extensive study of the chemical strategies of attach-ment of reporter and conjugate groups to DNA was publishedby Beaucage (1999), and Lebedeva et al. (2000) recently re-viewed conjugates relevant to cellular delivery. In the presentreview, mainly conjugates that have been evaluated in cells orin vivo for antisense activity are covered. Antibody conjugates,polymer carrier conjugates, and other miscellaneous conjugatesof antisense oligonucleotides for altering therapeutic potentialare not included here. A more comprehensive review has been

Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc., Carlsbad, CA 92008.

103

published (Manoharan, 2001), with references to syntheticmethodology and applications including all those conjugatesthat have not yet been evaluated in cell-based assays.

OLIGONUCLEOTIDE TYPES MODIFIED BYLIGAND CONJUGATION

Conjugations have been made to antisense oligonucleotideswith the chemistries shown in Figure 2. Most of these conjuga-tions have been to oligonucleotides with either phosphorothioatemodification on the backbone or with 29 sugar modifications (29-O-(2-methoxyethyl), 29-O-MOE (Häner et al., 2000; Teplova etal., 1999a), 29-O-methyl, 29-O- Me) or both. Several reports ofphosphodiester 29-deoxyoligonucleotides conjugated to ligandshave appeared in the literature. Peptide nucleic acid (PNA)oligomers (Egholm et al., 1992; Nielsen, 2000a,b) with ligandconjugates have also been characterized. Conjugates of methylphosphonates (Miller, 1998) have been made and studied, but,surprisingly, there have been no reports of conjugates to otherclasses of modified antisense oligonucleotides (Cook, 1998a), forexample, morpholinos (Summerton and Weller, 1997), methyl-ene(methylimino) (MMI, 39-CH2-N(Me)-O-CH2-49) modification(Sanghvi et al., 1997), or phosphoramidates (Gryaznov, 1999).

Types of linkages used in conjugation methodology

In carrying out conjugation chemistry, many types of linkershave been used, including chemically and biologically stablelinkers (such as amide or carbamate linkers) and bioreversibledisulfide linkages (Fig. 3). Bioreversible linkages have an ad-vantage in that the ligand is detached from the oligo cargo in thecell so that the linker has no undesirable effects on further anti-sense oligonucleotide interactions with either target mRNA or aprotein (e.g., RNase H). If the goal is to increase the proteinbinding of antisense oligonucleotides for improving the phar-macokinetics, however, chemically and biologically stable link-ages are highly desired. Thus, the choice of linkers must bebased on the role of the ligand.

ENDOCYTOSIS, ENDOSOMAL ENTRAPMENT,ENDOSOMAL RELEASE, AND DIRECT

CELLULAR PERMEATION

To function as an antisense drug, the oligonucleotide shouldbe able to permeate the cellular membrane and must bind in thecytoplasm or nucleus or both to the target RNA for the appro-priate antisense mechanism (RNase H dependent or non-RNaseH dependent) to operate. A drug may enter the cell via endocy-

MANOHARAN104

FIG. 1. Desired properties of antisense drugs.

tosis or through a direct cellular permeation process (Fig. 4 andTable 1). If the drug molecule enters the cellular membrane bythe conventional endocytosis mode, association to the mem-brane or a membrane-bound receptor is involved. After inter-nalization into vesicles by an energy-dependent process, thedrug moves to endosomes and lysosomes, where it is either de-stroyed or recycled. In this case, cytoplasmic and nuclear deliv-ery is an inefficient process.

On the other hand, if the oligonucleotide is capable of perme-ating directly into the cell in an energy-independent process,thus reaching the cytoplasm directly, the amount of compound

available for antisense function will be greater than if perme-ation is through an endocytotic process. Direct permeation willalso be cell-type and tissue-type independent. Once the oligo-nucleotide is in the cytoplasm, it is capable of reaching the nu-cleus without much difficulty (Szoka et al., 1997; Marcusson etal., 1998). Oligonucleotides delivered by transfection agents toliving cells or cultured cells enter the nucleus efficiently. Al-though the purpose of conjugation chemistry is to achieve thisdirect permeation process, it has not been widely achieved, andonly a very few examples exist in the literature (Hughes et al.,2000).

OLIGO CONJUGATES 105

FIG. 2. First-generation and second-generation oligomers to which ligands have been conjugated.

In this review, I discuss how conjugation of ligand molecules(1) improves interaction between oligonucleotide and the cellmembrane to enhance cellular absorption and (2) provides analternative mechanism by which the oligonucleotides can reachthe cytoplasm directly.

CONJUGATES IMPROVING CELLULAR ASSOCIATION: LIPOPHILIC

MOLECULAR CONJUGATES

Conjugates to improve cellular uptake and alter pharmacokinetics

Oligonucleotides are hydrophilic molecules by virtue of theirphosphate (or modified phosphate) and sugar backbone. Al-though the nucleobase is hydrophobic, hydrophilicity domi-nates because of the extensive hydrogen bonding resulting fromthe phosphate and sugar residues. This intrinsic hydrophilicityis increased by the anionic nature of the backbone. The hy-drophilic character of the drug and the anionic backbone re-duces cellular permeation. However, simple elimination of the

anionic charges alone does not improve cellular uptake, as evi-denced by neutral backbone-containing oligomers, such asmethylphosphonates and PNA oligomers. Conjugation oflipophilic molecules is therefore, an alternate way to solve thecellular permeation problem.

Various lipophilic molecules have been conjugated to anti-sense oligonucleotides. The structures of the compounds areshown in Figure 5. Of these compounds, cholesterol is perhapsthe best characterized, having been studied by various groupsfor the past 11 years (Letsinger et al., 1989). It has been re-ported to increase binding of oligonucleotides to lipoprotein,thereby enhancing cellular association and transport (de Smidtet al., 1991; Krieg et al., 1993). Most of this section concen-trates on the considerable data available on cholesterol-conju-gated oligonucleotides. Data available on other lipophilic li-gands also are summarized.

Activity of cholesterol-conjugated antisenseoligonucleotides in cell-based assays

Antisense oligonucleotides conjugated to cholesterol havebeen evaluated in a number of cell-based systems. In the ab-

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FIG. 3. Chemical linkages used in making oligonucleotide conjugates.

Receptor

FIG. 4. Cartoon describing endocytosis vs. direct cell permeation of oligonucleotides.

sence of cationic lipids, oligonucleotides with 59-cholesteroltargeting the 39-untranslated region (39-UTR) of mouse inter-cellular adhesion molecule-1 (ICAM-1) inhibited ICAM-1 in adose-dependent manner with an IC50 of 2.5 mM, whereas un-conjugated oligonucleotide did not show any activity, even withhigh concentrations of oligonucleotide were used (M. Manoha-ran et al., unpublished results). The inhibition of protein expres-sion appeared to be target specific, as neither molecule showedsignificant inhibition of vascular cell adhesion molecule-1(VCAM-1) expression. In addition, a scrambled antisense oli-gonucleotide derived from ICAM-1 with or without cholesterolconjugation failed to show ICAM-1 inhibition (M. Manoharanet al., unpublished results).

Cholesterol-conjugated 29-deoxy and 29-O-MOE gapmer(chimeric 29-modified and 29-deoxyoligonucleotides that canelicit RNase H activity) oligonucleotide phosphorothioates tar-geted against protein kinase C-a (PKC-a) and C-raf mRNAhave been reported (Manoharan et al., 1997). As with theICAM-1 study, the cholesterol conjugates were active in the ab-sence of cationic lipids, whereas unconjugated oligonucleotideswere not.

Inhibition of expression of the multidrug resistance (MDR)-associated P-glycoprotein by 59-cholesterol-conjugated phos-phorothioate antisense oligonucleotides without the need forcationic lipids has been reported (Alahari et al., 1996). Treat-ment with cholesterol-conjugated antisense oligonucleotide en-hanced cellular accumulation of rhodamine-123, a well-knownsubstrate of the P-glycoprotein transporter. The effectiveness ofthe cholesterol-conjugated oligonucleotide appears to be due toits rapid and increased cellular uptake compared with unconju-gated oligonucleotides, as indicated in a flow cytometric andconfocal microscopic studies.

The effects of conjugating cholesterol to either or both endsof an oligonucleotide phosphorothioate were analyzed in termsof cellular uptake and antisense efficacy against the p75 nervegrowth factor receptor in differentiated PC12 cells, which express high levels of this protein (Epa et al., 1998). The 39-cholesterol analog was more active than the 59-analog, and abis-cholesterol (59 and 39)-conjugated oligonucleotide was the

most potent. At 1 mM, the bis-conjugate was as effective as ahigh dose of cycloheximide in decreasing the synthesis of p75.

Mechanism of action of cholesterol-conjugatedantisense oligonucleotides

The mechanism that causes uptake enhancement and im-proved efficacy of cholesterol-conjugated oligonucleotides hasnot been clearly determined, although a receptor-mediated pro-cess involving lipoproteins has been implicated (deSmidt et al.,1991; Krieg et al., 1993). The lipophilicity of the steroid skele-ton may also enhance cellular association by hydrophobic inter-actions. In addition, cholesterol conjugates may form micellarstructures that facilitate the transport of the conjugate within thecell. However, more mechanistic studies are needed to establishthe exact mode of action conclusively.

Other hydrophobic conjugates, such as adamantane, pyrene,eicosenoic acid, and C16-glyceride lipid nucleoside conjugates,were synthesized and incorporated into the ICAM-1 targetedoligonucleotide in the same fashion as for cholesterol (Manoha-ran et al., 1995). The relative lipophilicities of these conjugatesmeasured by reverse-phase HPLC were used as a measure ofthe interaction between the cell membrane and the antisenseoligonucleotide. A wide spectrum of lipophilicities was ob-served from the least lipophilic unconjugated oligonucleotide tothe glyceride lipid conjugate. In the antisense efficacy assays,without any added cationic lipid formulation, only the choles-terol conjugate inhibited ICAM-1 expression within the con-centration range of 1–10 mM (M. Manoharan et al., unpub-lished observations). Either the cholesterol conjugate has theoptimum lipophilicity, or it is interacting via a receptor-mediated process involving lipoproteins, as suggested earlier(deSmidt et al., 1991; Krieg et al., 1993).

Recently, LeDoan et al. (1999) reported interactions of oligo-nucleotide phosphodiesters conjugated to the cholesterol groupusing a disulfide linkage at an internal position of the choles-terol skeleton (3, 7, or 22 positions of cholesterol) to the 39-endof the oligodeoxynucleotide. The conjugates were assessed fortheir capacity to bind to cellular membranes, penetrate the cells,


TABLE 1. DIFFERENCES BETWEEN ENDOCYTOSIS AND DIRECT CELL PERMEATION INTO CELLS

(MUKHERJEE ET AL., 1997; LINDGREN ET AL., 2000)

Endocytosis Cell permeation

Absorption to the plasma membrane or Not receptor-dependenta membrane-bound receptor

Energy-dependent formation of a Energy-independent membranevesicle translocation

Destruction or recycling of the Conjugates delivered into the cytoplasmmolecules into compartments by the and nucleusendocytotic machinery

Release into the cytoplasm is Direct delivery into the cytoplasmproblematic

May be cell specific (receptor-mediated Not cell specificendocytosis)

Can be saturated Cannot be saturatedTemperature dependent Temperature independent, observed at both

37°C and 4°C

and partition in the cytoplasmic compartment of murine macro-phages. The results showed that these lipophilic conjugatesbind to cells much faster (t1/2 # 10 minutes) than the underiva-tized oligonucleotides. The cytosolic fraction of internalizedoligomers was studied by membrane permeabilization withdigitonin. Membrane binding and cell internalization correlatedwell with the hydrophobicity of the conjugates, as characterized

by their partition coefficients in a water-octanol system. How-ever, no pharmacologic end points were evaluated. This obser-vation suggests that the lipophilicity, rather than a receptor-mediated process, may be a dominant factor in determining theactivity of such conjugates.

Cellular uptake of 39-cholesterol-conjugated oligonucleo-tides has also been examined using real-time confocal laser mi-

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FIG. 5. Lipophilic molecules conjugated to oligonucleotides.

croscopy (Hanaki et al., 1997). The images revealed that the up-take of conjugate in the cytosol had started within the first 5minutes and that nearly 40 minutes were required for the endo-somal or lysosomal accumulations to appear. The histogram offluorescence intensity indicated that cytosolic uptake of theconjugate was 5-fold higher than that of oligonucleotide phos-phorothioates labeled with 59-fluorescein. However, the nuclearuptake of the cholesterol-fluorescein-oligonucleotide conjugatewas only 2-fold higher than that of the cholesterol-free com-pound. As fluorescein is also lipophilic, assessment of uptakeby fluorescein conjugation can be problematic. Nevertheless,these results are similar to those reported by Alahari et al.(1996), demonstrating a rapid and effective cellular uptake ofthe 39-cholesterol-59-fluorescein-conjugated MDR antisenseoligonucleotide, as indicated in flow cytometric and confocalmicroscopic studies.

Pharmacokinetics of cholesterol and other lipophilic conjugates

Biophysical and pharmacokinetic properties of severallipophilic analogs of the ICAM oligonucleotide have been eval-uated and reported (Crooke et al., 1996). Compared to the un-conjugated oligonucleotide, the three analogs with lipophilicconjugates, 59-octadecylamine, 59-cholesterol, and 39-choles-terol, were more lipophilic than unconjugated oligonucleotidebut had similar binding affinity for complementary RNA (asmeasured by thermal melting analysis). Certain lipophilic endmodifications can lead to significant increase in Tm (Bleczinskiand Clemens, 1999). Crooke et al. (1996) analyzed tissue distri-bution and half-life in mice using radioactively labeled oligonu-cleotide analogs. After bolus intravenous injection, the initialvolumes of distribution of the lipophilic conjugates were lower,and the initial clearance from plasma was slower than that of unconjugated phosphorothioates. Conjugation substantially in-creased the fraction of the dose accumulated by the liver. As dis-cussed in earlier sections, it is not clear if this change was due toan active transport of the lipophilic conjugates into the liver or ifthe effects observed were simply due to the increased lipophilic-ity of the conjugated phosphorothioate oligonucleotides. Therewas no distribution to the central nervous system (CNS).

The conjugates had metabolite patterns in plasma, liver, andkidney similar to those of oligonucleotide phosphorothioates.Neither the 59-cholesterol nor C18-amine modification signifi-cantly enhanced resistance to metabolism compared with un-derivatized oligonucleotides. However, the 39-cholesterol con-jugate was much more stable than the other oligonucleotidestested. The 39-hydroxyl group, which is involved in the nucle-ophilic attack of the adjacent phosphate bond when the exonu-clease enzyme forms a complex with the nucleic acid, is un-available in this analog.

Dizik et al. (1995) have also studied the pharmacokinetics of cholesterol-conjugated oligonucleotide phosphorothioates.They observed that conjugation of cholesterol to oligonucleo-tide phosphorothioates increased the plasma half-life. Sixtyminutes after injection into female mice, the levels of 39-choles-terol conjugates were nearly 4-fold higher than those of uncon-jugated oligonucleotides, and the levels of 59-cholesterol and59,39-cholesterol-conjugated oligonucleotides were nearly 7-fold higher.

Binding to serum proteins plays a key role in the pharmaco-kinetics of oligonucleotides and, in view of the negative effectsof phosphorothioates on clotting and complement activation, intheir toxicologic properties as well. As a model for proteinbinding to human serum albumin (HSA) in plasma, bindingconstants to bovine serum albumin (BSA) have been measured(Crooke et al., 1996). The affinities of the lipophilic conjugateswere greater at physiologic salt concentrations than the affinityof the 29-oligodeoxynucleotide phosphorothioates. The bindingaffinity of the conjugates was not salt dependent. The morelipophilic phosphorothioate conjugates may bind to more thanone type of site on BSA or may bind more tightly to the samesite as the unmodified phosphorothioate oligonucleotides.Srinivasan et al. (1995) evaluated interactions of cholesterol-conjugated oligonucleotides with BSA and HSA in vitro. WhenHSA-warfarin complex was incubated with a variety of oligo-nucleotides, a 59-cholesterol-conjugated 20-mer phosphoro-thioate displaced warfarin to a greater extent than the unconju-gated oligonucleotide. These two studies indicate that theassociation of cholesterol conjugates with serum albumin mayalso contribute to the improved uptake.

To gain insight into the mechanisms of the improved efficacyof cholesterol conjugates in vivo, Bijsterbosch et al. (2000) in-vestigated the disposition of the 39-cholesterol analog of theICAM-1-specific oligonucleotide phosphorothioate in rats. In-travenously injected tritium-labeled conjugate was cleared fromthe circulation with a t1/2 of 49.9 6 2.2 minutes (unconjugatedoligonucleotide, 23.3 6 3.8 minutes). Three hours after injec-tion, the liver contained 63.7% 6 3.3% of the dose of the con-jugated oligonucleotide, and the hepatic uptake was 2-foldhigher than the uptake of the control. Endothelial, Kupffer, andparenchymal cells accounted for 45.7% 6 5.7%, 33% 6 5.9%,and 21.3% 6 2.6% of the liver uptake of conjugated oligonu-cleotide, respectively, and intracellular concentrations of 2, 75,and 50 mM, respectively, could be reached in these cells (1 mg/kg dose). Cholesterol conjugation results in high accu-mulation of oligonucleotide phosphorothioates in various livercell types, which is likely to be advantageous for antisense ther-apy of liver-associated diseases. Preinjection with polyinosinicacid or polyadenylic acid reduced the hepatic uptake of the con-jugated oligonucleotide, suggesting the involvement of scav-enger receptors. Analysis of the oligonucleotides in rat plasmaindicated that the conjugated oligonucleotide bound high mo-lecular weight proteins more tightly and to a greater extent thanunmodified oligonucleotides. The authors conclude that thehigher affinity interaction of the conjugated oligonucleotidewith plasma proteins may explain their improved disposition inliver cells.

In vivo therapeutic efficacy of cholesterol-conjugatedoligonucleotides

The greater concentration in liver was correlated with thetherapeutic effect of the 59-cholesterol ICAM oligonucleotide,as measured by reduction of ICAM-1 mRNA levels in mouseliver in vivo. In this model, the expression of ICAM-1 mRNAwas induced by lipopolysaccharide (LPS) treatment. After in-travenous treatment of the mouse with oligonucleotides at adose of 10 mg/kg at 24 hours and 2 hours prior to polysaccha-ride treatment, we observed efficacy of the cholesterol conju-


gate, whereas the unmodified oligonucleotide had no effect atthe 10 mg/kg dose (M. Manoharan et al., unpublished observa-tions).

Iversen’s group (Desjardins et al., 1995) evaluated 59-choles-terol-conjugated oligonucleotide phosphorothioates with a se-quence complementary to the rat CYP2B1 mRNA for theirpharmacokinetic properties and ability to modulate CYP2B1 ex-pression in adult male Sprague-Dawley rats. Following intra-peritoneal administration of 35S-labeled oligonucleotides, thevolume of distribution was reduced to 33%, and the eliminationhalf-life was increased by 50% for the cholesterol-modifiedoligodeoxynucleotide relative to unconjugated controls. Hexo-barbital sleep times, a measure of CYP2B1 enzyme activity invivo, increased nearly 30% in cholesterol-oligodeoxynucleotide-treated animals.

Toxicity of oligonucleotide-cholesterol conjugates

The toxicologic properties of ICAM phosphorothioate andcholesterol analogs were examined in BALB/c mice (Henry etal., 1997). Oligonucleotides were administered at 50 mg/kg byintravenous bolus injection into the tail vein every other day for14 days. In general, the properties exhibited for underivitizedoligonucleotide and analogs were similar. Mice treated with thephosphorothioate were observed to have increases in livertransaminase levels and a decrease in triglycerides consistentwith results from previous studies performed in CD-1 mice.Spleen weights were also increased, but no histopathologic al-terations were noted. Alterations induced by the cholesterolconjugates were qualitatively similar but, in general, were morepronounced than for the control oligonucleotide. Greater reduc-tion in cholesterol and platelet counts and increases in bloodurea nitrogen (BUN) were observed relative to unconjugatedcontrol. Red blood cell (RBC) counts and level of hematocritwere also reduced in mice treated with conjugates relative tothe phosphorothioate treatment group. Kupffer cell hypertrophyand basophilic inclusions in Kupffer cells were observed inmice treated with cholesterol analogs. However, it is importantto consider the high dose involved in the study, as the goal ofthe study was to evaluate toxicity at these high doses. Des-jardins et al. (1995) observed some toxicity in their CYP2B1study with cholesterol conjugates and ascribed them to the link-ers used rather than to cholesterol.

Antisense applications of other lipophilic conjugates

Among steroid analogs, androstan-3-one (dihydrotestos-terone) has been covalently linked to PNA to target c-myc DNAin prostate cancer cell nuclei (Boffa et al., 2000). LNCaP cells,which express the androgen receptor gene, and DU145 cells, inwhich the androgen receptor gene is silent, were used in thisstudy. To allow visualization of the antisense compounds, boththe PNA conjugate and a control PNA were modified with arhodamine reporter group attached at the COOH-terminal posi-tion. The cellular uptake was monitored by confocal fluores-cence microscopy. PNA-rhodamine was detected only in thecytoplasm of both cell lines. The rhodamine signal of an-drostan-3-one-conjugated PNA was localized in the nuclei aswell as in the cytoplasm of LNCaP cells, which express the re-ceptor. In contrast, uptake of the conjugated PNA was minimalin DU145 cells and exclusively cytoplasmic. In LNCaP cells,

the myc protein level remained unchanged by exposure to thefree PNA, whereas a significant and persistent decrease was in-duced by the PNA-steroid conjugate. In DU145 cells, Myc ex-pression was unaltered by the PNA with or without the steroid.

To minimize the side effects associated with oligonucleotidephosphorothioates and to improve cellular uptake, Uhlmann’sgroup (Rait et al., 2000) used partially phosphorothioate-modi-fied oligonucleotides having a 39-hydrophobic tail derived from1-O-hexadecylglycerol. An 11-mer conjugate targeting the H-ras gene retained high sequence specificity and provided inhi-bition of ras p21 synthesis with minimal toxicity, even withoutthe use of a cellular uptake enhancer, such as cationic lipids.Moreover, treatment of T24 cells, a radiation-resistant humantumor cell line that carries a mutant ras gene, with the conju-gated oligonucleotide resulted in a reduction in the radiation resistance of the cells in vitro. The growth of RS504 (a humanc-Ha-ras-transformed NIH/3T3 cell line) mouse tumors wassignificantly inhibited by the combination of intratumoral injec-tion of the anti-ras conjugate and radiation treatment. Longeroligonucleotide conjugates (15-mer) did not show the free per-meability of the 12-mer, suggesting that the hydrophilicity/lipophilicity balance is dependent on the length of the oligonu-cleotide.

In the same H-ras system, Herdewijn’s group (Van Aerschotet al., 1995) observed that oligonucleotide phosphodiesters con-jugated with 1,3-propanediol reduced the growth of cells ex-pressing mutated Ha-ras. These conjugates also had improved39-exonucleolytic stability relative to unmodified phosphodi-ester oligonucleotides when treated with snake venom phospho-diesterase. Considering the simplicity of the ligand involved, it is also possible that this oligonucleotide phosphodiester adopted a sequence-dependent secondary structure that may have en-hanced its nucleate resistance.

The synthesis, as well as biochemical and biologic studies onoligodeoxynucleotide phosphodiesters bearing the lipophilicdimethoxytrityl group, placed as a phosphonate linkage eitherat the 59-end or the 39-end, has been reported (Misiura et al.,1998). These conjugates were reported to be totally resistant tonucleases present in human serum and able to activate cleavageof a complementary oligoribonucleotide by RNase H. In addi-tion, the modified oligonucleotides exhibited better antisenseinhibition of expression of plasminogen inhibitor type-1 (PAI-1) within endothelial cells than unconjugated oligonucleotidesat 2.5 mM concentration. Such an improvement is not observedfor deoxyphosphorothioate compounds. In this case, the ob-served trend may be simply due to some stabilization of phos-phodiester compounds by terminal dimethoxytrityl phosphonatelinkages. Although total nuclease resistance has been reported,some endonucleolytic degradation is expected, at least at thepyrimidine residues. In another example, a 59-dimethoxytrityl-conjugated, guanine-rich oligodeoxynucleotide with a phospho-diester linkage, 59-DMTrTGGGAGGGTGGGTCTG-3 9, hasbeen shown to exhibit potent sequence-specific inhibition ofHIV-1-induced cytopathic effect (Momota et al., 1997).

A series of conjugates of lipophilic alkyl groups at the 59-endof oligonucleotides targeted against human PAI-1 mRNA wassynthesized via the oxathiaphospholane approach (Kobylanskaet al., 1999b). The most active conjugates in cell culture con-tained menthyl or heptadecanyl groups. In this study also, anti-sense oligonucleotide phosphodiesters when conjugated to

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lipophilic alkyl residues were found to be more active than oli-gonucleotide phosphorothioates. The diesters exhibit only lim-ited anti-PAI-1 activity without conjugation. The cytotoxicityof anti-PAI-1 oligonucleotides and their conjugates were alsoevaluated in EA.hy 926 hybrid endothelial cells (Kobylanska etal., 1999a). Some cytotoxicity was found for cholesteryl andbornyl conjugates but at concentrations higher than those usedfor antisense inhibition experiments.

Enhancement of antiherpetic activity of oligonucleotidephosphorothioates targeting the immediate-early (IE) pre-

mRNA of herpes simplex virus-1 (HSV-1) has been observedwith another terpene alcohol, geraniol (Shoji et al., 1998). Cy-toplasmic distribution of a geranyl-oligonucleotide was con-firmed using confocal microscopy. Although some of the geranyl-conjugate was seen in the nucleus, the unmodifiedcompound had a punctate distribution in the cytoplasm, with lit-tle in the nucleus. These results suggested that geranyl modifi-cation enhanced antiherpetic activity by changing the subcellu-lar distribution of the oligonucleotides.

The preparation of lipophilic conjugates of oligodeoxynu-


FIG. 6. Representative cationic groups and polyamines conjugated to oligonucleotides at various conjugation sites.

cleotides and their interaction with low-density lipoprotein(LDL) has been described (Rump et al., 1998). The high ex-pression level of receptors for LDL on tumor cells makes LDLan attractive carrier for selective delivery of drugs to these cells.Rump et al. incorporated oncogene-directed antisense oligo-deoxynucleotides into the lipid moiety of LDL. The oligonu-cleotides conjugated with cholesterol, and the oleoyl esters oflithocholic and cholenic acid associated readily and nearlycompletely with LDL. Unmodified oligonucleotides did not as-sociate at all with LDL, and those conjugated with the dioleoylester of chenodeoxycholic acid associated incompletely. Freelithocholic acid and oleic acid are probably not sufficientlylipophilic to induce association with LDL, whereas the dioleoylester structure is perhaps too bulky and extended to allow parti-tioning into the lipid moiety of LDL.

Mishra et al. (1995) noted improvement in the leishmanicidaleffect (anti-infective effect against leishmaniasis) of phosphor-othioate antisense conjugates when delivery was LDL medi-ated. Phosphorothioate oligodeoxynucleotides were linked atthe 59-end to a palmityl group. This modification enabled theoligonucleotide to form a stable noncovalent complex withLDL through hydrophobic interactions. The antisense effect ofLDL-oligonucleotide complexes was assayed by targeting theminiexon sequence of Leishmania amazonensis in infectedmouse peritoneal macrophages. A 16-mer antisense oligonu-cleotide-palmitic acid conjugate-LDL complex exerted a morepronounced sequence-specific effect than the free oligomer.About 25% and 10% of infected macrophages were cured by a48-hour incubation in the presence of 2.5 mM of the complexedand the free oligomer, respectively.

Cytosine-phenoxazine (Flanagan et al., 1999a), a tricyclic 29-deoxycytidine analog, was designed to improve stacking inter-actions between heterocycles of oligonucleotide-RNA hybridsand to enhance cellular uptake. Although it is a nucleobasemodification, it is also a lipophilic intercalator molecule main-taining Watson-Crick hybridization properties of the cytidinesystem. Incorporation of four phenoxazine bases into a 7-meroligodeoxynucleotide phosphorothioate targeting SV40 large Tantigen enhanced in vitro binding affinity for the RNA target

compared with the unmodified oligonucleotide phosphorothio-ate. In the absence of the cationic lipid, the phenoxazine oligo-nucleotide accumulated in the nucleus of a variety of tissue culture cells and exhibited antisense activity at 3 mM concen-tration. However, the length of the oligonucleotide was in-creased, the extent of uptake in the absence of cationic lipid de-creased. More importantly, this modification also altered thesite of RNase H cleavage.

CATIONIC ZWITTERIONIC, ANDPOLYCATIONIC GROUPS

Cationic oligonucleotide (Letsinger et al., 1988) (with posi-tively charged backbones) and zwitterionic oligonucleotides(Manoharan et al., 1991) (with positively charged tethers) areimportant because these oligonucleotides are expected to havegood cellular permeation properties. Furthermore, because oftheir charge, they should have favorable binding properties to-ward RNA and single-stranded as well as double-strandedDNA. High-affinity binding is due to charge neutralization andfast on-rates of hybridization. Figure 6 shows some of thecationic modifications and polyamines conjugated to oligonu-cleotides and their sites of attachment (Ganesh et al., 1997;Prakash et al., 1994; Godzina et al., 1999; Nara et al., 1995;Potier et al., 1999; Sund et al., 1996, 1997; Heystek et al.,1998).

Several novel 29-cationic modifications giving rise to zwitte-rionic oligonucleotides have been developed and incorporatedinto oligonucleotides and evaluated for antisense properties(Fig. 7). The modified oligonucleotides exhibit very high nucle-ase resistance due to the charge effect. These oligonucleotidesmaintain high binding affinity to target RNA when the modifi-cations are dispersed throughout the oligonucleotide (Manoha-ran, 1999; Teplova et al., 1999b; T.P. Prakash et al., unpub-lished observations).

The G-clamp is a heterocycle modification (Flanagan et al.,1999b; Lin and Matteucci, 1998). It is a cytosine analog derived

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FIG. 7. Cationic 29-modifications in the protonated form at physiologic pH.

from phenoxazine that forms an additional hydrogen bond toguanine (Fig. 8). This modification enhanced oligonucleotideaffinity for RNA with a 6–18°C increase in Tm per modifica-tion. The broad range in change in Tm is due to the positionalpreference of to the G-clamp to achieve the additional stacking.The data suggest that an additional hydrogen bond is formedbetween the ammonium group on the G-clamp and the Hoog-steen face of guanine.

Dose-dependent, sequence-specific antisense inhibition wasobserved at nanomolar concentrations of the G-clamp oligonu-cleotide phosphorothioate against two different mRNA targets(Flanagan et al., 1999b; Lin and Matteucci, 1998). Incorpora-tion of a single G-clamp modification into a C-raf 20-mer phos-phorothioate oligonucleotide increased the potency 25-fold relative to the phosphorothioate (Flanagan et al., 1999b). Incell-free assays, G-clamp oligonucleotides activated RNase H-mediated cleavage of target RNA. This modification alsoprovides excellent specificity. In experiments targeting p27kip1,a single nucleotide mismatch between the oligonucleotide con-taining the G-clamp and the target mRNA reduced the potencyof the oligonucleotides by 5-fold (Flanagan et al., 1999b). Eval-uation of in vivo pharmacokinetics and pharmacology ofoligonucleotides with this modification is in progress.

Cationic groups also have been introduced into the backboneof oligonucleotides and into the backbone of PNA (Fig. 9).Modification of the PNA backbone with lysine alleviates someof the synthesis, purification, and aggregation problems associ-ated with neutral PNA, especially those that are rich in purine.Lysine residues also improve the target RNA binding affinityby approximately 1°C/modification.

PEG CONJUGATES

Polyethylene glycols (PEG) are amphipathic and are ex-pected to improve transport and cellular association propertiesof oligonucleotides. PEG are known to play an important role inthe pharmacokinetic behavior of therapeutic proteins. In addi-tion to serving as ligands themselves (Manoharan et al., 1995),they can also serve as linkers for conjugating other ligands(Wiederholt and McLaughlin, 1998) (Fig. 10).

The effect of the different high molecular weight PEG chainson the biologic properties of the conjugated antisense oligonu-cleotides has been studied by Bonora et al. (1999). Of the twodifferent conjugates of an anti-HIV 12-mer oligonucleotidetested for antisense activity in MT-4 cells (Bonora et al., 1998),only the oligonucleotide conjugated to the linear monomethoxyPEG (MPEG) showed anti-HIV activity. The 12-mer, whenconjugated to a branched (MPEG)2, was inactive, as was the un-modified oligonucleotide.

A 20-mer oligonucleotide targeting mouse b-globin mRNAhas been conjugated at the 59-terminus to bis-aminoalkyl PEGusing acylimidazolides (Tullis, 1988). At 15 mM, the conjugateselectively inhibited protein synthesis in cultured Friend murineerythroleukemia cells by 95%.

An oligonucleotide that targets human ICAM-1 has beenconjugated to a series of PEG esters of average molecularweight 550, 2000, and 5000 (corresponding to 11, 44, and 110ethylene glycol residues). However, this study indicated thatPEG interfere with the cellular permeation of oligonucleotidesin vitro with or without cationic lipids present (Manoharan etal., 1995).


FIG. 8. (A) G-clamp and (B) the hypothetical four-bond base pairing with G.

CONJUGATES FOR MODULATING PROTEIN BINDING TO FACILITATE

TRANSPORT AND ABSORPTION

The interaction of antisense oligonucleotides with serum andcellular proteins determines their pharmacokinetic and pharma-codynamic properties and, hence, the eventual pharmacology(Crooke, 1998; Crooke et al., 1996b). First-generation antisensecompounds, the 29-deoxyoligonucleotide phosphorothioates(Fig. 2), have relatively avid binding to serum and cellular pro-

teins and, thus, favorable pharmacokinetic properties (Agrawalet al., 1998; Crooke, 1998; Crooke et al., 1996; Rykova et al.,1994; Srinivasan et al., 1995). However, these phosphorothio-ate oligonucleotides also bind to proteins, such as thrombin,factor IX, and factor H, contributing to the observed dose-limiting side effects, such as prolonged clotting time and com-plement activation (Gao et al., 1992; Levin et al., 1998). Chang-ing the phosphorothioate linkages to phosphodiester linkagesovercomes some of the side effects, but this change also causesloss of nuclease resistance. Modifications such as 29-O-MOE

MANOHARAN114

FIG. 9. Cationic backbone modifications: PNA with (A) internal and (B) terminal Lys groups.

FIG. 10. Some of the PEG ligands and PEG linker.

overcome the undesired sensitivity to nucleolytic degradation,but 29-O-MOE phosphodiester oligonucleotides have limiteddistribution to organs and fast urinary elimination presumablybecause of poorer affinity for serum proteins (Khatsenko et al.,1998; Geary et al., 2001). It would, therefore, be desirable toimprove binding affinity for HSA while reducing phosphoro-thioate linkage content via conjugation methodology. Unconju-gated free aspirin (Fig. 11) is known to affect the protein bind-ing of phosphorothioate oligonucleotides (Agrawal et al.,1998). The role of cholesterol in modulating serum proteinbinding of oligonucleotide phosphorothioates has been de-scribed (Bijsterbosch et al., 2000; Crooke et al., 1996). Other li-gands capable of affecting protein binding should be conju-gated and evaluated further.

ENHANCEMENT OF ABSORPTION VIARECEPTOR-MEDIATED PROCESSES

Vitamin conjugates

Cells and tissues have specific transport systems for nutri-ents, such as vitamins. Thus, conjugation of antisense oligonu-cleotides to vitamins is expected to improve transport into cells.Figure 12 shows structures of the vitamins that have been con-jugated to oligonucleotides and evaluated.

Folic acid conjugates. Folate-mediated targeting of thera-peutic and imaging agents to cancer cells has been reviewed re-cently (Reddy and Low, 1998). The vitamin folic acid enterscells either through a carrier protein, termed the reduced folatecarrier, or via folate-receptor-mediated endocytosis. Folate-drug conjugates are substrates for this receptor. Thus, they penetrate cells exclusively via folate-receptor-mediated endo-cytosis. When folic acid is covalently linked to a drug, folate-receptor binding affinity is not significantly compro-mised, and endocytosis proceeds relatively unhindered, pro-moting uptake of the attached drug by the folate-receptor-expressing cell. As folate-receptors are significantly overex-pressed in several human cancer cells (e.g., ovarian, lung,breast, endometrial, renal, and colon cancers and cancers ofmyeloid hematopoietic cells) but not on most normal tissue(Jansen, 1999; Kamen et al., 1988; Leamon et al., 1999; Lea-mon and Low, 1994; Reddy and Low, 1998), this methodologymay allow for the selective delivery of antisense oligonu-cleotides to tumor tissue.

Folate-mediated targeting of antisense oligonucleotides toepithelial ovarian cancer cells has been reported (Li et al.,

1998). A conjugate was prepared by the direct conjugation offolic acid to the 39-terminus of an anti-c-fos oligonucleotidewith a mixed phosphorothioate-phosphodiester backbone. Cel-lular uptake into FD2008 cells that overexpress folate receptorswas increased by about 8-fold relative to unconjugated oligonu-cleotide. In contrast, conjugation of folate to the oligonucleo-tide did not increase its uptake by CHO cells that do not expressfolate-receptor. The unmodified mixed backbone antisense oligonucleotide had some inhibitory effect on the growth ofFD2008 cells. However, conjugation with folic acid signifi-cantly increased activity. As further indication that increaseduptake and antisense activity were due to the folic acid conjuga-tion, the increase in the uptake and growth inhibition could be blocked by adding an excess amount of folic acid. Thelipophilicity of the pteridine ring may also contribute to the in-crease in uptake. Unfortunately, the low capacity of the folatereceptor may limit the activity of these conjugates, despite thehigh affinity of the receptor for the folate-oligonucleotide.

Vitamin E conjugates. 29-Deoxyoligonucleotide phosphoro-thioate conjugated to a-tocopherol (vitamin E) has been shownto inhibit the functioning of the dipeptide transporter system(DTS) in cultured Caco-2 intestinal cells. However, the effectwas mainly due to the lipophilicity of tocopherol, as 2-di-O-hexadecyl-3-glycerol conjugate produced a similar effect(Moore et al., 1997).

MULTIVALENT CARBOHYDRATE CLUSTERS

In their pioneering work, Wu and Wu (1988) demonstratedthat oligonucleotides complexed with polylysine covalentlylinked to asialoorosomucoid were taken up into human hepato-cellular carcinoma (HepG2) cells by the asialoglycoprotein(ASGP) receptor, a receptor found on parenchymal liver cells.Subsequently, Hangeland et al. (1995) reported a 20–40-foldenhancement in cellular uptake in HepG2 cells when methyl-phosphonate oligonucleotides were conjugated to the trianten-nary, N-acetylgalactosamine neoglycopeptide, Tyr-Glu-Glu-


FIG. 11. Examples of ligands capable of modulating protein binding.

FIG. 12. Two vitamins conjugated to oligonucleotides.

(aminohexyl-N-acetylgalactosamine)3, (YEE (ahGalNAc)3) (Fig.13). This is a glycotripeptide known to bind to Gal/GalNAc re-ceptor sites on hepatocytes with Kd of 7 nM. Using the same li-gand, Duff et al. (2000) showed that oligonucleotide phospho-rothioates conjugated to this glycotripeptide sequencespecifically suppressed (.90% inhibition) integrated hepatitisB virus (HBV) expression in hepatoma cells at 1 mM concen-tration. This was at least a 20-fold increase in efficacy com-pared with the unconjugated compound. Moreover, no toxicitywas observed over the entire concentration range studied (1–20 mM concentration). Uptake in cell culture was enhanced20–40-fold irrespective of the backbone charge, as both oligo-deoxyphosphorothioates and oligodeoxymethylphosphonateswere evaluated. In vivo experiments showed rapid and high up-take of the conjugate into mouse liver after tail vein injection.

Improved targeted delivery of oligonucleotides to parenchy-mal liver cells in vivo has been demonstrated by Biessen’s group(Biessen and Van Berkel, 1999; Biessen et al., 1999, 2000).They have conjugated oligonucleotide to the ligand L3G4 (Fig.

13) for the ASGP receptor. In vitro uptake studies and confocallaser scan microscopy studies demonstrated that the L3G4-conjugated oligonucleotide was far more efficiently bound to,and taken up by, parenchymal liver cells than was the underiva-tized oligonucleotide. Studies in rats showed that hepatic uptakewas greatly enhanced from 19% 6 1% for the unconjugated oli-gonucleotide to 77% 6 6% of the injected dose after glycocon-jugation. Importantly, accumulation into parenchymal liver cellswas improved almost 60-fold after derivatization with L3G4, anduptake has been attributed to the ASGP receptor.

Antisense oligonucleotides have been covalently attachedto ASGP via disulfide bond conjugation (Rajur et al., 1997).Multiple (approximately 6) oligonucleotides can be conju-gated to each ASGP molecule. The ASGP was used to deliverantisense oligonucleotide complementary to the mRNA ofthe interleukin-6 (IL-6) signal transduction protein (gp130)to HepG2 cells. These conjugates ultimately inhibited the cytokine-stimulated upregulation of the acute-phase proteinhaptoglobin.

MANOHARAN116

FIG. 13. Carbohydrate clusters for liver cell-specific targeting.

CONJUGATES AIMED AT DIRECT DELIVERYOF OLIGONUCLEOTIDES IN THE

CYTOPLASM-PEPTIDE CONJUGATES

Another approach to the intracellular delivery of oligonu-cleotides is based on the use of several types of delivery pep-tides that seem to have the ability to transport large, polar mol-ecules, including peptides, oligonucleotides, and even proteins,across cell membranes (Schwarze et al., 1999, 2000; Astriab-Fisher et al., 2000). Two examples of such delivery peptides area 13-amino acid sequence (Tat) from the HIV Tat protein and a16-amino acid sequence (Ant) from the Drosophila antennape-dia homeotic protein. Antennapedia-type peptides have beenused to deliver oligonucleotides, including PNA, into neuronalcells (Langel et al., 1999), but their general applicability has yetto be completely studied. Other types of peptides containing hy-drophobic motifs and special recognition motifs have also beenused for antisense delivery (Table 2 and Fig. 14).

Astriab-Fisher et al. (2000) prepared Ant and Tat peptide-oligonucleotide conjugates to target MDR-1 P-glycoprotein, amembrane ATPase associated with multidrug resistance in tu-mor cells. The oligodeoxynucleotide phosphorothioate compo-nent of the conjugates was complementary to a site flanking theAUG of the message. Both types of peptide-antisense oligonu-cleotide conjugates, but not mismatched control conjugates,provided substantial inhibition (34%) of cell surface expressionof P-glycoprotein at submicromolar concentrations. The peptide-oligodeoxynucleotide conjugates were more potent in the pres-ence of serum than when used under serum-free conditions.This is in contrast to cationic lipid-based intracellular deliveryof nucleic acids. Flow cytometric profiles indicated that theconjugates accumulated in cells to a much greater degree thandid the free oligonucleotide. The conjugates reached the nu-cleus, whereas the free oligonucleotide had no intracellular flu-orescence.

In contrast to the above result, 18 conjugates of oligo-deoxynucleotide phosphorothioates to membrane translocationand nuclear localization peptides were prepared in good yieldand were thoroughly characterized with electrospray ionizationmass spectra (Antopolsky et al., 1999). When applied to cells,conjugates exhibiting membrane translocation and nuclear lo-calization properties displayed efficient intracellular penetra-tion but failed to show improved antisense effects. Further stud-ies on the intracellular distribution of the fluorescein-labeledconjugates revealed that the conjugates were trapped in endo-somes. As endosomal entrapment will lead to lysosomal degra-dation, cytoplasmic and nuclear delivery was reduced.

Langel’s group (Langel et al., 1999; Pooga et al., 1998a,b;Villa et al., 2000) demonstrated that conjugates of transporterpeptides to PNA showed improved delivery and were able toregulate galanin receptor levels and modify pain transmissionin vivo. A PNA antisense 21-mer to the human type 1 galaninreceptor was linked via a labile cysteine disulfide bond to thepeptides transportan and Ant (Table 2), known to impart cellmembrane permeation properties. The resulting conjugates im-proved internalization and downregulated the human galaninreceptor in Bowes cell line and in rat spinal cord in vivo. The in-trathecal administration of the peptide-PNA construct caused adecrease in galanin binding in the dorsal horn. Because of de-creased binding, galanin could not inhibit the C fiber stimula-tion-induced facilitation of the rat flexor reflex, demonstratingthat peptide-PNA constructs acted in vivo to suppress expres-sion of functional galanin receptors. These peptides have beendemonstrated to translocate across the plasma membrane of eu-karyotic cells by an energy-independent pathway (Lindgren etal., 2000).

The signal peptide Lys-Asp-Glu-Leu (KDEL) has been usedto transport oligonucleotides to the endoplasmic reticulum andfrom there to the cytosol and the nucleus, where their targets arelocated (Arar et al., 1995). These signal peptides are unique be-


TABLE 2. PEPTIDES USED FOR CELLULAR DELIVERY/IMPROVED UPTAKE OF ANTISENSE OLIGONUCLEOTIDES

No. Peptide sequence Source

1 Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met- Antennapedia helix 3 (43–58)Lys-Trp-Lys-Lys

2 Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro- HIV Tat fragment (48–60)Gln

3 Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly- TransportanLys-Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu

FIG. 14. Cell permeation peptide conjugates.

cause of their unique role in the targeting and translocation ofnearly all secreted proteins and many integral membrane pro-teins (Chou, 2001). A 59,39-modified 25-mer oligonucleotide,complementary to the translation initiation region of the gagmRNA of HIV, was coupled to a dodecapeptide containing aKDEL signal sequence. The anti-HIV activity of the oligonu-cleotide was compared with that of conjugates linked througheither a thioether bond or a disulfide bridge. The conjugate witha thioether bond had a higher antiviral activity than the peptide-free oligonucleotide or the conjugate linked via a disulfidebond.

CONJUGATES FOR IMPROVING RNase H ACTIVITY

Almost all the antisense compounds in clinical trials at pre-sent recruit RNase H to degrade the target mRNA. However, inaddition to unmodified DNA and 29-deoxyoligonucleotidephosphorothioates, only certain limited classes of modifiedoligonucleotides, such as 29-arabino-substituted oligonucleo-tides (Damha et al., 1998), boranophosphates (Rait and Shaw,1999), and cyclohexene oligomers (Wang et al., 2000), are ca-pable of activating RNase H for target RNA cleavage after thehybridization event. Other chemically modified oligonucleo-tides (29-ribo modifications, backbone modifications, othersugar modifications, and PNA) do not elicit RNase H activity,as the modifications affect conformation, required charge, hy-drogen bonds, hydration, steric bulk, and metal ion (Mg21 orMn21) coordination. The 29-ribo-modified oligonucleotides andbackbone-modified oligonucleotides can be used only as gap-mers (Cook, 1998a). In this regard, conjugates may play a dis-tinct role because conjugation of small molecules, especially atthe oligonucleotide termini, will not affect the factors requiredfor enzyme activity. Hélène’s group (Godard et al., 1995) dem-onstrated that cholesterol conjugation improved the RNase Henzymatic activity 3–5-fold. In this study, the 39-conjugate per-formed better than the 59-conjugate. Cholesterol-conjugatedoligonucleotides targeted to an mRNA fragment of Ha-rasoncogene were able to promote a higher extent of the targetRNA hydrolysis by RNase H compared with the nonconjugatedcounterpart (Godard et al., 1995). Acridine-conjugated oligonu-cleotides targeted to b-globin mRNA were shown to be more

potent inhibitors of b-globin synthesis than the unmodifiedoligonucleotides (Cazenave et al., 1987) in microinjected Xeno-pus oocytes due to RNase H activity.

More recently, Chattopadhyaya’s group evaluated the role ofpolycyclic aromatic chromophores, such as phenazine (PZN)and dipyridophenazine (DPPZ), in RNase H cleavage of the tar-get strand when conjugated to PZN and diphenazine ligands(Amirkhanov et al., 2001; Zamaratski et al., 2001a; Zamaratskiet al., 2001b). The 39-conjugated antisense oligomers promotedfaster hydrolysis of the target RNA than the unmodified com-pound. The enhancement was not dependent on the bindingaffinity, and a stabilizing interaction between the ligands andRNase H has been proposed (Fig. 15).

CONJUGATES WITH ALTERNATIVETERMINATION MECHANISMS

mRNA cleaving agents: synthetic ribonucleases

In an attempt to identify alternatives to the antisense oligonu-cleotide-RNase H-catalyzed degradation of target RNA, severallaboratories continue to search for efficient sequence-specificcleavage agents. Unlike oligodeoxynucleotide phosphoroth-ioates, most second-generation oligonucleotide modificationsare not substrates for RNase H, thus necessitating design andsynthesis of new RNA cleaving agents. A wide range of smallmolecules capable of hydrolytic cleavage of RNA under vari-ous conditions has been studied and reviewed (Vlassov et al.,1998; Häner et al., 1998; Häner and Hall, 1997). The cleavageeffectiveness of these reagents can be enhanced by couplingthem to molecules that bind to nucleic acids, such as intercala-tors and polycations (Fig. 16). For example, catalytic cleavermolecule-intercalator (e.g., imidazole-acridine) conjugateshave been synthesized and studied for their use as tRNA cleav-ing agents (Lorente et al., 1996).

Attachment of reactive groups to oligonucleotide comple-mentary to segments of the target RNA directs the hydrolyticcleaving groups to the desired RNA sites. Hydrolytic cleavinggroups include metallocomplexes, oligopeptides, amines, andmolecular constructs containing constituents of the active cen-ters of nucleases (imidazole, guanidinium, carboxylate, andamino groups). Design of small molecules capable of effective

MANOHARAN118

FIG. 15. RNase H activity enhancing aromatic ligand-conjugated oligonucleotides.

catalytic cleavage of RNA under physiologic conditions mayenable development of conjugates of antisense oligonucleotidesthat are active in cells.

A bis-diimidazole construction mimicking the active centerof RNase A has been conjugated to oligonucleotides comple-mentary to L. amazonensis miniexon and pre-miniexon se-quences (Yurchenko et al., 1997). The conjugates were shown

to cleave the target RNA at specific positions in cell-based as-says. In another report, the sequence-specific, hydrolytic cleav-age of mRNA from the HIV gag gene was observed using a terpyridyl Cu(II) complex (Bashkin et al., 1995). An oligonu-cleotide bearing a histamine group at the 39-end was able tocleave complementary RNA in a sequence-specific manner inthe presence of Zn21 ions at about 5% efficiency (Hovinen et


FIG. 16. RNA cleaver molecule functionalities.

al., 1995). Considering that this 5% is indeed above backgroundand it was sequence specific, this observation is still encourag-ing. Site-specific hydrolysis of a 25-mer RNA, at micromolarconcentration under physiologic conditions, by diethylenetri-amine (DETA)-conjugated 10-mer PNA has been reported byvan Boom’s group (Verheijen et al., 2000). Although none ofthese conjugates promote efficient cleavage of mRNA, theseexperiments indicate that progress toward chemically inducedcleavage of mRNA is being made.

Lanthanide complexes. Hall et al. (1994) have shown the se-quence-specific cleavage of a synthetic RNA using europium(Eu(III)) complexes linked to complementary oligonucleotides.The cleavage efficiency of the conjugate strongly depends onthe nature of the linker between the oligonucleotide and thecomplex. Nearly complete cleavage of the RNA target wasachieved within 16 hours at 37°C with 8–40 equivalents of theoligonucleotide conjugate.

Oligonucleotide conjugates of Eu(III) tetra-azamacrocycleswith pendent alcohol and amide groups promoted sequence-specific RNA cleavage (Huang et al., 2000). Two-fold excessconcentration of the Eu(III) macrocyclic-oligonucleotide conju-gate over RNA for 16 hours at 37°C produced 10%–15% hy-drolytic cleavage of a complementary oligoribonucleotide.Cleavage was not observed in the presence of Eu(III) conju-gates containing scrambled sequences or by free complexes.The extent of the cleavage observed was similar for conjugatescontaining either Eu(III) macrocyclic complex, despite the factthat one of the free macrocyclic complexes was more reactivethan the other.

An oligonucleotide-Eu(III) complex conjugate designed tocleave the 59-cap structure of the ICAM-1 transcript improvedthe antisense activity in cells (Baker et al., 1999). The 59-capstructure of cellular and viral mRNA synthesized by RNA poly-merase II is an N7-methylated guanosine residue that is linkedby a 59,59-triphosphate linkage to the 59-terminus of the

mRNA. Attachment of a Eu(THED)31 (THED 5 1,4,7,10-tetrakis-(2-hydroxyethyl)-1,4,7,10-tetra-azacyclododecane)analog to the 39-terminus of a 29-O-MOE oligonucleotide thattargets the 59-cap region of ICAM-1 mRNA improved the in-hibitory activity of the antisense oligonucleotide relative to theunconjugated oligonucleotide in cytokine-treated endothelialcells.

Based on a proposal by Hall’s group (Hüsken et al., 1996),Kuzuya and Komiyama (Komiyama et al., 1999; Kuzuya andKomiyama, 2000a,b) designed an intercalator-conjugated DNAto generate a bulge in the target RNA that will become a hotspot for the metal ion-mediated cleavage. In the first approach(Fig. 17A) using a ternary system, the target RNA is activatedto produce a bulge opposite the junction point where two DNAcomplementary strands are placed, an acridine-conjugatedDNA (18-mer) and unmodified DNA (17-mer), so that the36mer RNA is site-selectively hydrolyzed at this bulge by freeLu(III) ions. The nitrogen atoms of acridine may be involved inthe Lu(III) coordination, enhancing the cleavage. A 10-fold ex-cess of DNA strands and a 100-fold excess of metal ion wereemployed. At 37°C, the t1/2 of RNA was 13 hours. The site-specific cleavage was .30-fold more efficient than the uncon-jugated DNA1/DNA2/Lu(III) ternary system alone. In the sec-ond approach (Fig. 17B), a binary noncovalent system for site-selective RNA scission used a Lu(III) ion and a DNA bearingan acridine in the internal position. On formation of theDNA/RNA heteroduplex, the target phosphodiester linkagewas activated and preferentially hydrolyzed. The binary systemwas more active (about 20%) than the ternary system in termsof the target RNA cleaved.

Peptides as RNA cleavers. Oligonucleotide-peptide com-plexes offer another non-RNase H mechanism of sequence-specific, hydrolytic cleavage of mRNA. Oligonucleotide-peptide conjugates designed for mRNA cleavage have been ob-tained using several methods (Tung et al., 1991). Two oligonu-

MANOHARAN120

FIG. 17. Introduction of an RNA bulge via acridine conjugation in the complementary DNA. (A) Ternary system. (B) Binary system.

cleotide conjugates with peptide moieties that either mimic theactive site of RNase A (His-Gly-His motif) or contain a Cu(II)complexing metallopeptide (Gly-Gly-His motif) have been syn-thesized (Truffert et al., 1996).

Highly efficient endonucleolytic cleavage of single-strandedRNA by a 30-amino acid zinc-finger peptide was reported byLima and Crooke (1999). The peptide sequence corresponds toa single zinc-finger of the human male-associated ZFY protein,a transcription factor belonging to the Cys2His2 family of zinc-finger proteins. Interestingly, RNA cleavage was observed onlyin the absence of zinc, and the active structure was found to bea homodimeric form of the peptide. The catalytic activity wassingle-stranded RNA specific. Single-stranded DNA, double-stranded RNA and DNA, and 29-O-methyl-modified oligonu-cleotides were not degraded by the peptide. The initial rates ofcleavage (V0) observed for the finger peptide were comparableto rates observed for human RNases, and the catalytic rate (kcat)was comparable to rates observed for the group II intron ri-bozymes. Different chemical methods have been proposed toconjugate this peptide to antisense oligonucleotides (Lima etal., 2000).

Despite a wealth of information on synthesis and in vitro as-says, there are no published reports of any in vivo activity ofcleaving agent conjugates. More research is needed in this im-portant area to identify practical non-RNase H mechanisms ofmRNA cleavage.

CROSS-LINKING AGENTS

Psoralens

Psoralens are naturally occurring aromatic compounds con-sisting of a furan ring fused to a coumarin. They can be isolated

from plants or microorganisms, including fungi. Psoralens arevery effective interstrand DNA cross-linking agents. The mech-anism is a (2 1 2) cycloaddition involving the furan ring of thepsoralen and the 5,6-double bond of a thymidine (Fig. 18). Pso-ralens have demonstrated clinical value in the treatment of pso-riasis, vitiligo, cutaneous T cell lymphoma, and certain inflam-matory processes.

Inhibition of collagenase type I expression by psoralen anti-sense oligonucleotides in dermal fibroblasts has been docu-mented (Lin et al., 1995). Type I collagenase plays an importantrole in both tumor metastasis and the remodeling of connectivetissue in normal human skin during wound healing and mayparticipate in the pathophysiology of some dermatologic dis-eases, such as skin cancer and a chronic blistering diseasecalled recessive dystrophic epidermolysis bullosa. Antisenseoligodeoxynucleotide phosphorothioates, linked at the 59-endwith the photoreactive psoralen derivative 49-(hydroxyeth-oxymethyl)-4,59,8-trimethylpsoralen (HMT), targeting a regionoverlapping the initiation codon of the collagenase mRNA,were evaluated (Fig. 18). One oligonucleotide targeted theHMT moiety to a region with a 59TpA. This oligonucleotide-HMT conjugate was 50-fold better at cross-linking to its targetsequence after UVA irradiation in a cell-free system than theother, which did not target the preferred 59TpA site. Tissue cul-ture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in amonolayer or in a 3-dimensional dermal equivalent, showedlowered collagenase levels 24 hours after UVA irradiation ascompared with controls.

The synthesis of a derivatized cis-syn furan-side psoralenthymidine monoadduct that was incorporated into a DNA seg-ment, including a prototypical human TATA box sequence, hasbeen reported (Kobertz and Essigmann, 1997). In .90% yield,this oligonucleotide formed a cross-link with a complementary


FIG. 18. Cross-linking agents and their conjugation sites.

DNA strand after irradiation with 366 nm light. The cross-linkwas reversible either with 254 nm light or by heating in aqueousbase.

To selectively achieve mutation and inactivate specific genesin human cells, Glazer has developed a triplex strategy for geneknockout (Glazer, 2000). Triplex-forming psoralen-conjugatedoligonucleotides can direct DNA damage and, consequently,mutations to selected sites within mammalian cells.

Mitomycin C and analogs

Mitomycin C (Fig. 18), a pyrrolo{1,2-a}indole derivative, isa bioreductive alkylating agent and a clinically used natural an-ticancer drug. Studies have shown that mitomycin derivativesselectively alkylate guanine residues at the N2 position withinduplex DNA with a remarkable preference for 5CpG se-quences. Huh et al. (1996) demonstrated antisense activity ofmitomycin C-conjugated oligonucleotides that target a segmentof the coding region of the human FGFR1 gene. The mitomycinC conjugates reduced the number of FGFR1 receptors at micro-molar concentration in human aortic smooth cells, suggestingdownregulation of FGFR1 gene expression. Further, these con-jugates inhibited cultured human aortic smooth muscle cell pro-liferation and were less cytotoxic than free porfiromycin. The

mechanism of action of the conjugate has yet to be completelyestablished, but these results suggest that antisense targeting ofmitomycin C may play a role in activity.

PORPHYRIN CONJUGATES

Porphyrin conjugation (Fig. 19) can have a number of favor-able effects on antisense activity. Porphyrin can function as ahydrolytic cleaving agent or as an oxidative or photoactivecleaving agent. The modification may also protect the oligonu-cleotides from nuclease digestion. These ligands are hydropho-bic and, thus, may alter oligonucleotide distribution in vivo. Fi-nally, as model heme ligands, they may also enhance uptakeinto certain liver cells.

Conjugates of the photosensitizer, meso(tetra-4-carboyx-phenyl) porphine (TPPC4), to oligonucleotides complementaryto rat actin mRNA were prepared (Seliger et al., 1998). Inter-nalization and phototoxicity of these conjugates were investi-gated in cultures of RR1022 cells. The conjugates were shownto accumulate in the cytoplasm, and this accumulation clearlydepended on the length and sequence of the oligonucleotidemoiety.

MANOHARAN122

FIG. 19. Porphyrins and their conjugation sites to oligonucleotides.

Porphyrin conjugates as RNA cleaving agents

Magda et al. (1994, 1997) reported a synthetic approach inwhich a dysprosium(III) texaphyrin (“porphyrin from Texas,”DyTx) metal complex was attached to the 59-end of oligo-deoxynucleotides. This synthetic methodology has been ex-tended to the preparation of analogs in which the complex is at-tached at an internal position within the DNA oligonucleotide.Under conditions of 25-fold conjugate excess, such 59-deriva-tized constructs were shown to effect the site-specific cleavageof a cognate RNA sequence in 6 hours at 37°C. A constrainedshort linker was more efficient than longer linkers. This modeof complex attachment affords the possibility of enhancing theRNA fragment product release because the number of basepairs joining the RNA to the DNA conjugate is reduced by afactor of approximately 2 on cleavage. In an effort to furtherimprove the efficacy of this system, kinetic and thermodynamicmeasurements were carried out using the Gd(III), Eu(III),Dy(III), and Lu(III) texaphyrin derivatives with single-strandedRNA (Black et al., 1997). Using titration and kinetic experi-ments, evidence for metal-porphyrin binding via a templated-stacking mode, as well as a binding affinity trend (Dy.Gd.

Eu.Lu), was observed. The dominant factor in the reactivity isthe coordination capability of the central lanthanide cation.

Sessler et al. (1996) reported a synthesis of sapphyrin-oligonu-cleotide conjugates. The sapphyrin-oligonucleotide conjugate pro-duces photodamage on a complementary oligonucleotide targetwhen irradiated at wavelengths above 620 nm. On piperidine treat-ment, guanine residues on the target strand in the vicinity of thesapphyrin macrocycle are cleaved more effectively than guaninesremote from the sapphyrin subunit. No sequence-specific photo-modification was observed when noncomplementary oligonu-cleotides were used as a target. The duplexes formed between thesapphyrin conjugate and complementary nucleic acids were foundto have higher Tm than analogous control systems consisting of un-modified oligonucleotides of the same sequence. Tm studies usingvariable salt concentrations and oligonucleotide targets indicatethat the binding enhancement is due to hydrophobic interactions.The sapphyrin unit attached to an oligonucleotide can thus serve adual role. It can act as a sequence-specific photomodification agentat irradiation at wavelengths .620 nm, and it can increase theaffinity of a sapphyrin-bearing oligonucleotide to a complemen-tary sequence. The mechanism of the degradation may involve di-rect electron transfer between purine bases (e.g., guanine residues)and the sapphyrin in either a triplet or singlet excited state. The sec-ond possibility is that a photoexcited sapphyrin in its triplet statereacts with molecular oxygen to form singlet oxygen, which reactsreadily with guanine (Maiya et al., 1990).


FIG. 20. Potential advantages of oligonucleotide conjugate drugs.

LESSONS LEARNED ANDLESSONS TO BE LEARNED

Oligonucleotide conjugation chemistry, attaching ligands tomodulate the biologic activity of oligonucleotides, has tremen-dous potential. In this review, I attempted to demonstrate thevalue of oligonucleotide conjugates for possible applications inimproving both the pharmacokinetic properties (cellular up-take, nuclease resistance, protein binding, and biodistribution)and the pharmacokinetic properties (binding affinity to the tar-get, improving the RNase H efficiency, or providing an alter-nate mechanism to degrade the target). It is clear from this re-view that oligonucleotide conjugates have been evaluated in awide range of cell culture and in vitro experiments, althoughonly a limited number of in vivo experiments have been per-formed. Nevertheless, the value of conjugation chemistry hasbeen clearly demonstrated by these studies. Pendent moleculesare definitely capable of altering the pharmacokinetics ofoligonucleotides and improving their pharmacology. The po-tential advantages of conjugation of ligands to antisense mole-cules are summarized in Figure 20. By constructing a designeroligonucleotide with all desired antisense properties, one cansynthesize, in theory, an ideal drug.

There could be some practical limitations in using conju-gates. Cost and ease of synthesis of some conjugates may bevalid concerns. However, when compared with the cost of thesome of the expensive building blocks used for some novel an-tisense oligomer backbone modifications, the cost of the linkersand simple ligands (such as cholesterol) is trivial. However, ifthe conjugate involves peptides or carbohydrate building blocksrequiring many protection and deprotection steps, cost may become too great. Although the synthesis of cholesterol orlipophilic molecules does not pose much of a synthetic chal-lenge, the synthesis of conjugates involving complex mole-cules, such as folic acid, carbohydrate clusters, and peptides,does create certain synthetic problems. These ligands have lim-ited solubility in appropriate solvents (for synthesis), requiredesign of orthogonal protecting strategies compatible with oli-gonucleotide synthesis, and need strategies that limit racemiza-tion of the chiral centers in the ligand during oligonucleotidedeprotection. Each of these problems depends on the chemistryof the antisense oligonucleotide and has to be addressed andovercome for each new ligand and linker.

Many biologic interactions relating to ligand conjugation re-main to be evaluated. How doe these pendent groups (smallmolecules, such as cholesterol or folic acid) or large molecules(e.g., peptides) interact with other proteins involved in the anti-sense action? What is their role in RNase H activation in termsof recruitment, stabilization of the intermediate catalytic step,or product release? How doe they interact with helicases? Howdo they affect the interaction of oligonucleotides with otherRNA regulatory elements? Do they play a role in the varioussteps involving mRNA editing? How do they affect translationregulation or recoding? Once some of these basic answers areknown, many medicinal chemistry experiments addressingstructure-activity relationships could be initiated based on ini-tial biologic results. These questions and others still remainunanswered. Unfortunately, the answers are being sought inonly a few laboratories. One can try to make intelligent guesses,

but only carefully planned and controlled experiments canbring the real answers.

As pointed out often in this review, the in vivo data availablefor oligonucleotide conjugates are very limited. It is very im-portant to carry out in vivo experiments in animals as early aspossible to evaluate distribution, pharmacology, and toxicol-ogy. If possible do the animal pharmacology experiment first!

In spite of these practical and knowledge-based limitations,antisense oligonucleotide conjugates offer incredible potentialfor improving antisense oligonucleotide technology. Amongthe chemical analogs of oligonucleotides evaluated to date, onlythe phosphorothioate (both 29-deoxy and 29-modified) analogsand some conjugates have clearly shown both valuable pharma-cologic and pharmacokinetic properties. This important experi-mental fact naturally demands enthusiastic preclinical and clinical evaluation of oligonucleotide conjugates for variousdisease targets.

ACKNOWLEDGMENTS

I thank Frank Bennett and Stan Crooke for many helpful dis-cussions and constructive comments. I am grateful to MarijaPrhavc, Andrei Guzaev, Martin Maier, T.P. Prakash, BruceRoss, K.G. Rajeev, and V. Mohan for their valuable help andsignificant contributions to this review and Nancy Meskan andWanda Schuelke for manuscript preparation.

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Address reprint requests to:Dr. Muthiah Manoharan

Department of Medicinal ChemistryIsis Pharmaceuticals, Inc.

2292 Faraday AvenueCarlsbad, CA 92008

E-mail: [email protected]

Received May 16, 2001; accepted in revised form August 31, 2001.

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