Sudhir Agrawal

Antisense oligonucleotides as antiViralagents Antisense oligonucleotides are an attractive potential alternative to conventional drugs as antiviral agents. A major advantage is the relatively simple rational design of oligonucleotides which should bind only to specific nucleic acid sequences, compared with conventional drugs which are frequently targeted against sites of unknown structure in proteins. Progress to date provides hope for the development of a new class of antiviral chemotherapeutics

based on antisense oligo-

nucfeotides.

An antiscnsc oligonuclcotide may bind 50 its target nucleic acid cithcr by Watson-Crick base pairing or Hoogstccn base pairing. In Watsoll-Crick base pairing, hctrrocyclic bases ofan antiscnsc oligonucleotidc form hydrogen bonds with the hetcrocyclic bases of target single-stranded nucleic acids (RNA or singlcstranded DNA), whereas in Hoogstccn base pairing, the heterocyclic bases of target arc double-s:randcd nucleic acids (i.e. double-strmdcd DNA). Both thcsc models of binding by antisensc oligonucleotidcs have the potential to regulate gene expression, and their possible USCin modulating a wide range ofhuman and plant diseases is under investigation’-“. This 2rticlc focuses on the USC of antisense oligonucleotides to rc&te viral gcnc expression by interfering with single-stranded nucleic acid targets. It was shown in 1977 that cell-free translation of mRNA could bc inhibited by the binding ofan oligonuclcotidc cotnplementary to a short segtncnt of the mRNA” to form a duplex. The first cxarnplc of specific inhibition ofgcne expression i~rIliveby a synthetic oligodcoxynucleotidc was rcportzd by Zamccnik and Stcphcnson’~*. They dcmonstrarcd that a 13-mcr synthetic oligodeoxynucleotide, complementary to part pf the viral genomc, inhibited ROLE sarcoma virus replication in infected chicken fibroblasts. and also

inhibited transformation of primary chick iibroblasts into malignant sarcoma cells. Why consider antisense oligonucleotidcs as antiviral agents? There arc several potential advantszcs of antiscncc oligonuclcotidcs ;proachcs. an oligonucleotide incorporating unn~oditkd iwernude&de phosphates and phosphate analogs at predetcnnincd positions can be syntk sized”. Using automated synthesizers permits synt.xsis reactions varying widely in scale (%Z-l~JOo pool) to be perforrncd: this corresponds to a yield of -1 mg up to 3 gni of 20-mer oligonucleotide after purification. Oligonuclcotides containl::g a phosphav backbone can be purified using high-perfowlanre liquid chromatography (HFLC) or polyx-yknide gel electrophoresis (PAGE)‘“. The procedure used kx purification of oligonuclcotidcs containing nlodified phosphate backbolles depends on the nature of the mod&cation. Most oligonuclcotide analogs. contain chiral phosphates. thereby crekng diastsrereoitonlcrs at each intemucleotide linkage. Some oligonuclcotide

FigUrs 3 Synthesis of methylphosphonate d~godeoxyfibonucleotiis involve5 the use of a 50dimethoxybityideoxynucleoside3Wiis~r~~ methyQhosphonamidi intermedi&e 19, R = fCH,f, CHNK 1and 5 mi(d a&l catalyst. After eact coupling, the intermediate dtucleoside phospbite is oxidized with iodine to generate the methy!phcsphonate linkage 110).Becausethe methylphos&nate linkage is cleaved undo strongly bzsic con&&, Special precautions are required for deprotection. MB for abbrevlabons, see Fig. 2.

15h

mkws .----

-,.

I

?? r_z

2 R$l:$

Trsnslation -----+ Translation

product.

I

Antisense

oligonucleotide

Antisense

qligonucleotide

Antisense

oligonlicleoltde

+-

Antiselise

oligonucleotlde

Figure 4 Translation of mRNA may be blocked bv the binding of a complementary oligonucleotide. There are two possible mechanisms by which this can occur: (1) by base-specific hybridizstton, thus preventing access by the translation machinery, i.e. ‘hybridization arrest’; or (2, by form ing an RNA-DNA duplex which is recognized by the intracellular nuclease RNase H, specific for digesting RNA in an RNA-DNA duplex. Difierent types of chemical modification of the oligonucleotides can result in three different modes of action. A, B and C. (A) The antisense oligonucleotide (red) binds the target by base-specific hybridization, causing both hybndization arrest and RNase H activation. Degradation of mRNA by RNase H releases the oligonucleotide, which can then bind to other copies of the target mRNA. The susceptibility of the oligonucleotide to cleavage by other nucleases (i.e. the in viva half-life of the antisense oligonucleotide) is therefore a major parameter affecting this ‘catcl.ytic’ mode of degradation. Unmodified phosphodiester oligonucleotides and their phosphorothioate analogs fait into this categcry. (B) The antisense oligonucleotide (pink) binds to the target by base-specific hybridization causing translation arrest, but does not activate RNase H. Oligoribonucleotides and their analogs, and oligodeoxyribonucleotides containing methylphosphonate, phosyhoramidate and tiarious non-phosphate internucleotide linkages fali In this category. These oligonucleorides are nuclease resistant, and are effective in inhibiting translation, but are generally required in higher molar concentrations than those which activate RNase H. NE) The oligonucleotides in this group combine the features of (A) and (BI: the oligonucleotide coiltains internucleotide linkages which activate RNase H (e.g. phosphrdiester, phosphorothioate), flanked by nuclease-resistant intemuc!eotide linkages which do not activate RNase H (e.g. methytphosphoni te, phosphoramidafes, non.phosphate internucleotide linkages etc. 22~1~52) Digestion of the mRNA target in the RNA-DNA duplex releases the o!igonucleotide, rhich because of Its nuclease resistance and, hence, longer rn viva half-life, is more effective than oligonucleotides in category (A). Some oligorUeotides in category K!) are hybrids of Jigodeoxyribonucleotides (central region; red) and either oligoribonucleotides or their analogs (flanking regions: Ipink).

viral activity against several viruses (Table 2). Their nuclcase susceptibility’“, however, limits their potcnti31 for use iir r~iW. iJlctliylphospholl~it~ analogs 3rc nuclcasc rcsist:mt24 , anid xc mule active 3s antiviral agents than their unmodifcd phosphodiestcr counterparts”*‘QC~(and CC rcfcxnxs cited in Itch. 23). Mcthytphosphonatc nnalogs probably have antiviral activity due to hybridization xrcst, since the d~~plcs of IncthylphospbrJllatc DI’JA with RNA does not xtivatc IZN;W Hz’. Pt~osphnntuiciatc ;~nalogs have :m antiviral c&t 5inlil:lr k) the tl,cthytptiosyhc,n,ltc :m;rtog9. t’tlost)tlc.:l.ottlto~~t~ oliR(‘Jc’o”yribollrtrlcotides, which h:ivc conibi~~d tktufcs of 11uc1c~ reistanccl” and ;accivatiun of I\Nxc Hz?, xc found to bc the most accivc autivirah. ‘1hry arc cffcctivc in inhibiting HIV”,’ I,2x*2”,HSV.“‘, H1’V3’ and influenza viru+3i, in the cokxxntriltion rang lO-x--l (I-‘, M, without noticcabtc cytotoxicity up co 1W M conccntration. Otigonuclco~idc analogs carrying conjugates

157 reviews Table 2. Properties 04 ofiaonucleatides and their analogs F_

---

ztrw

;$_-iiGi_

Refs

-I

Oligodeoxyribonucleotide (phosphz:;; - - Yes Oligodeoxyribonucleotide phosphorothioate Lower + Yes 22,41,42 Oligodeoxyribonucleotide selenoate Lower + Not known 43 Oligodeoxyribonucleotide phosphoramidate Lower ++-t NO Oligoribonucleotide (phosphate) X, Higher -%45 Oligoribonucleotide phosphorothioate X, Higher + 1: 44:45 8gX:O: X, 2’-OMe-Oligoribonucleotide (phosphate) Higher + No X, 2’-OMe-Oligori9onucleohde (phosphorothioate) Higher ++ 8h X = S, No :: 10 Oligodeoxyribonucleotide mf~thylphosphonate Lower t++ No 47 _. .“__.._ ,..,““l.l aNumbers8 and 10 correspondto structuresin Figs 2 and 3, respectively. bDuplexstabilityof oligonucleotideto complementaryoligoribonucleotide underphysiologicalconditions , c?Tgared to DNA-RNAstzbi!i!y. cComparedfrom DNA (phosphodiesterasedig&on). dActivation of ANase H by the duplex formed between oligonucleotide and RNA. 8a X = 8b X = 8c X = 8d X = ;;;r:

0, S, Se, NR,,

x, x, x, X,

= FI =H =H =H = OH = OH = OMe = OMe

poly-~-lysinc~“~~7 and acridin@)

(such 3s cholsstcrol”“,

have shown UllCOlljl~g:ltCd

1t1 uivo

improved

antiviral activity ovct COlllltC~M~tS in SOlllC C:IS(:S.

appiication

their

ofoligonucleotides

crudies carried OLX in mice and rats using rin unmodified oligonuclcotidc an.! its p!:osphorothioatc analog (both are 30-mcrs) showed tha; a single intrapcritoncal dose of 150 mg kgg’ can be tolcratcd’“. Rcpcakd doses of 100 mg kg-1 in mice I’rcliminary

toxicity

for 14 days, tither intraperitoncally or subcutaneously, caused no mortality. Phannacokinctic studies carried out in mice show that the rate of oligonuclcotidc cxcrction depends on the nature ofthe intcrnucleotidc backbone: Y!!% of an unmoditicd oligonuclccddc containing four phosphorothioate linkages was cxcrcted in I2 hourP; and 75% of a mcthylphosphali;rte analog was cxcrctcd in two hours”‘. In contclst, a phosphorothioatc nnnlog WRS cxcrctrd only up to 30% in the first 34 houn, with an additional 2S’%, CscrctCd in t!-c following I 24 hours”. The phosphorothioarc ohgonuclcotidc was found to be distributed among most of ~hc organs, with the highest conccntration present in kidney and liver tissues. Phosphorothioate and mctl~ylpl~ospl~ot~:~tc oligonuclcntidcs

did not appear in the brain in significant ;u~mun~P~~‘~ and both Fhosphorothioatc nnd mcthylphosphollatc otigonuclc.kdcs wcrc quite stablP. Ikgradation in the blood and in tissues occurred prcdominnntly due to the action

of 3+-csonuclcaics3’J.

Developing antisense pharmaceuticals must be considered? In summary, rhc an&i& propcrtics

-what

of antisonsc oliRur,~lclcotidcr and their vxious nil;llOg, togcthcr with their np~:~.~ntly low toxicity and bvorablc pham~acokinetic, in mic:: and rats, yrovidc ;~n initial indication that they may bc suitable candidates for piurmaccutical dcvulopment. Although, to date, there have been no published reports ofeficrcy ofantisrnsc oligonuclcotidcs against viral discascs in animal model ‘, systems, unpubh:hcd results from s~cr;J !abontorics arc promising.

Thcrc arc scvcral issues :;riII unrcsolvctl which arc critical to thr dcvclopmcilt JF aneiscnsc oligonuclcotidcs PS :rnrivir~l opts. TIIC cost of synthcsizing oligonuclcotides at prcscnt may impede further development for which multigram quantities .Lrc required. The cost is driven by a combination of the scale of synthcsis, choice of approach (solid- or solution-phase) and puriticarion ccchniqucs. The cost will decrease dramatically with the further dcvcioymcnt of &sting technolo$rs or cvcn new chcmisrry for pilot-scale production, combmcd with a WCstep purification procedure. To &tc, tosicity and pharmacokinctic studies have been carried out by administering

the oligonuclcotid:
r his consr&nt tupp~rt, encouragement a;ld guidance. I 2111 thankfi~l to Dr Thoru Pcdcrson, for crikllly rmiing the manuscrip; and making May helpful sugg:cctions; and also to Dr David Knipc (Harvard Medical School) for discussions and suggestions. Rcscarch

cited from

audior’s laboratory was supported by NIAID co-opcrntivc drug discovery grant UO1 A12Wk5, and by :; gt:nt from the G. Harold and LciIa Y. Mathcrs FOLIW da&m. I thank ,Grs Carol Turcic for processing tbc manuscript and for cspcrt sccrctarkll nssistanrc.

..__.. -___-. .-I._--. TIBTECHMPY 1992 IVOLlOl

15x

reviews

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