Oiigonuclestide therapeutics Jack S. Cohen Recent studies have focused on the ability of oligonucleotides to affect the genetic


of many organisms,

including viruses. Hence,


nucleotides may represent a future source of biotechnologically derived compounds of therapeutic importance for diseases such as cancer and AIDS. Most organisms fun&on through the trrrtt&r of genetic infom?:ttion front DNA to IIIRNA m proccin. In the cast ofretrovintscs, the itifortnntion origin~:cs in viral RNA. Novel :tpproachcs to thcrJpcutics, involving itttcrvrtttion iu thcec gcncTic procc::W.. usil tg oligonuclcotides, h;;vc been dcvclopcd in the F_>stfew years. Thew have the capability to supplcmcnt, if ttot. iti :hc long mu, to rcplncc the standard ztpprmcl: to the dcvrlopmtnt of p)iartilarcutic.?ls. In cff‘ccr, &is approach rcprescnts 3 conrplrtcly novel stratrby, that is in SOIIK ways intarmcdintc bctwccn ehc classical drug paradigm of s~nail r~?olcculc (lock-and-key) inhibitors, and gcnc thcmpy that involves the introindividuals. ductiutt :Jf whole gc:?cs into n&ctcd The distinct ticld ofoligormclcotide thcrapcutics cm be rcgardcd as a IKW subdivision ofbiotcchnology. It

is ijftcti r&m-cd To as ‘antiscnsc tcchnolo~z’. though this is a tnisnomcr, since that term dcscribcs only one of the subtopics within this field. Oligonuclm,tidc thcmpcutics’ dcvrlopmcnt has been driven la&y by the ahiliry TO synthesize oligonuctrotidcs, chcrmicnlly :Imdif;.cd oligonuclcotidc analo& and cottjugt:31ed oIigxuclcotides, in reasonable (tnilligrnm) quantity and of good purity, as a result .zf the now ready availability of oligodcosylluclcotide~ through mttomatcd synthesis using the phosphor-,4,,& anndttc mcrhod’. Suhscqum dctvrlo~~t~ l!y__....,

Q 1992. Elsewer Sxnce

Publishers Ltd IUKI

Otigonucteotide analogs Bccausc ttir nacur;il phosphodicctcr tivc

co dcgradxion


group is mlhin~~clcarc~,n;trur:tl nli#o-

mtclcotidcs must lx cheniically tnodificd CO rmde~ them rcrimnt to r?~~clras.cs that would d:grx!~: the:::: irr rifn. or cvrn ifr rim unless c3rc is taken to chonW appropriate condidons. Such dcgrad:ttion would bc .I significant intpcdimcnt to the USC ofx? oligmncr as a drug-like 11~olccu1cthat depends upon the integrity of

the scquwcc for its rccognirion specificity. Although rhc three co~nponcn:s ofnn oligonuclcotidcIhc bnw, sugar ;rnd phosphodtestcr bnckbonc - could, in principlc. lx ntodificd, chemical tnoditicationr. notably I?l~th~~~h~~S~hOII~t~ and pllosphorothjoat~ of the phosphwh~:stcr bxkbone have been used (Fig. 1). The t]letllylFhosphottate analog was dewloped ~CC:~LW ir is a non-ionic derivxivr, md hcncc is cxpccrcd to exhibit increased ccltul:lr uptake’. The phosphorotltioatc anato~ is ionic, and many applications have

been III;& with this an&~ ri (I. although it atso h:ts disadv;ili~~~#~~, such ns not,-scquencc-spcc-ific inhibitioni. ::. order to ovcrcontc the disndvantags oi thcsc analogy co-poiymcn \vith the natural phocphodicster linksgc have been tnadc and studied (Ref. 8: ;wd M. C&ash nnd J. S. Cohen, unpublished). Both the suliur and tncthyl tltono-~ubstictttcd an&gs c&hit sccrcoisontcristn, snd have rwo iwmcrs

88 reviews always bc destabilized, as found by reduced melting ;cmpcratures (M. Ghosh, J. S. Cohcu aud 0. Dahl,

unpublished), Antisense

0=6-o I x

HO Figure 1 Structure of an cjligodeoxynucleotide; nataral, X=0; [email protected] thioate, X=S; methylphosphonate, X-Me.

nt cad1 1' atom (R,, and s,,). Attempts to overcome this .~ppa~-cc;l~i probicm have been made, for example, using the achirnl phosphoro~thioatc, which contains two sulfllr substitutions for non-bridging oxygcns on the pho~;phodicstcr group. However, it is not clear that the ad-rantage of achirality outweighs the disadvancagus of this analog. Molecular simulatiorLs indicate that the R,, mono-~hosphorotlliontc isomer (with S pointing into the major groove) is destabilizer! relative to the S,, isomer”, rind, since the dithioatc always contain:, a sulfur in the R,, oricnmtion. it will

Novel compounds Applications

indicating reduced hybridization.


of expression

The most widely applied approach to interrupt the flow of genetic information is to USC oligodcoxy-= nuclcotides as inhibitors oftranslation, with the complcmentary or ‘antisense’ base sequence targeted to a specific ‘sense sequcncc in the mRNA. Thus, exprcssion ofa specific protein can be rcgulatcd or inhibited. The back~ound to the devclopmcnt ofthis approach, using antisensc mRNA, was rcccntly discussed in Scier~c”‘. It should bc noted that antisensc mRNAs occur naturally as a regulatory mcchanism”~lz. Figure 2 illustrates the development of the antiscnsc R&D field. Chemical synthesis is the driviug force, and is follow4 by characterization and resting. The most important measure of the maturitjr of the field is the appearance of successful irr tiitrc applications: suflicicnt numbers ofsuch application? have been reported in the antiscnse area (for reviews see Refs 13-l 5), that thcrapcutk candidates have reached the stage ofit) k’o testing. Mechanisms of antiscnsc inhibition include intcrfercncc with ribosomz binding and processing of niRNA’6, intcrkrel,cc with mRNA &m:onnation I7 or mRNA splicing’“, and RNasc-H activation of mRNA digestion 1‘)The preferred target for antiscusc inhibition is the Y-initiation codon. In gcncral, an oligonuclcotide of -1 S-20 bases is used for antiscnsc iuhibition, since this should apprcGmatc to only one unique tmjyt scqucnc~+ in the liumm g~:“iomP. Ii1 addition, an oligomcr of this length hybridizes well with its complementary target mRNA. III vitro applications that have been succcsrfui in the cancer area inclodc inhibition of cxprcssio?: of the following (proto-)oncogencs: c-rrtyr 21.‘12,N-rr~yc 23, 6~/-2’~, rrll& 3-i, and scvrl-al others, all of which have therapeutic relevance. Mauy applications to viral inhibition, including HIV iuhibition, hzve been reported’“. Transcription inhibition l3y targeting DNA, transcription arrest by au oligodeoxynuclcotidc would result iu direct inhibition

$ Cell uptake Labeled oligos Affinity chromatography

Figure 2 Flow diagram of antisense obgonucleotide R&T fkid. TIBTECH MARCH 1992 rdor 101

ofgcnc ~mction. However, the DNA duplex is very stable compared with single-stranded mRNA \vhich, alebough extensively folded, is readily acccssiblc. Use is thercforc made of the fact that a third srrand can bc accommodated in the major groove of the B-form DNA duplex to form a triplex structure (Fig. 3)27.2X. The fomlation of triplex has been shown to inhibit biuding of a transcription bccor”‘, and inhibition of c-III)~Cwas demonstrated in a cell-free systcnP. The first e::amp!c o:triplex inhibition in a cellular system, namely IL% gene expression in peripheral T lymphocytes, has recently been publishcdJ’. It should bc noted that the coding required to form the triplex structure is not Watson-Crick hydrogen bonding, but is in fact Hoogstecn and anti-Hoogsteen base pairing




C%? base triplet

TAT base triplet

Fipre 3 Schematic reoresentation of: (a) and (bl the maior base triplets; and (c) the binding of an oligodeoxynlicleotide [darker helix) in the major groove of duplex B-form DNA.

of the third base with the already formed base pairs (A.T or GC)“‘. Another approach to transcription arrest is to attempt to scqucster transcription factors that recognize specific base sequences by addition of this rccognition sequence as an oligomcr. It is anticipated that the oligomer will bc avidIy bound and will zaturatc the transcription 6ctor, thus preventing its normal fLnction. An example of the inhibition of transcription using a short duplex phosphorothioate oligomcr has been rcportcdJ3. Ribozymes A further area where cligo&nuclcotidcs arc being dcvelopcd as potential drugs is that of ribozymcs: in which a catalytic KNasc activity is flanked by antisense scqucnccs which permit recognition of the target sequcnrc (Fig. 4). While ribozymcs have received a great deal of attention, including au attempt to apply them to HIVJ4, there is less likelihood that thsy will be successful as therapeutic agents. As ribo-oligomm, they are more difXcult to synthesize and are susceptible to dcgradaiion by RNascs. They can be chcmitally modified (e.g. with phosphorothioates or with I’-O-methyl groups)““, but the effect of these modifications on their catalytic activity is uncertain. Protein epitope targeting (PET) A novel feature of oligonucleotidc synthesis is that it is possible deliberately to insert random base sequences in chosetl positions in an oligomer. Thr

syothcsizcr is progr:nnmcd to drlivcr qua1 propxtions ofthc chosen bases, sny 25% each. ofthc phmofA. T. C and C;. The rcstrltnnt mistxc phoraniditcs of oligorncn can then lx used, cithcr for the synthc sis of peptides with mixed amin.o-acid congmitions ;:t a given position in the polypcptidc chain, or for biirding to a [email protected] protein- 16.-li. The usefuh~css of this approach is that the variety of the oligomer mistorc is so grcnt (4 for II random positions), that challengi~~g


Substrate 5’





X X X x x X >! 7 Y Y Y X Y

llllllrl YYYYYYYY




Figure 4 Stucture of a ribozyme. The arrow shows the point of cleavage of complementary RNA substrate.


reviews Recycle

initiation partially random oligomcf p!us target protein




Purify Binding


I Amplify

+------I (FCR)

t Cloning/seqdencing Figure 5 Schematic of protein epitope targztitig (PET; or ‘aptamer’) by a partially random sequence oligodeoxynocleotide.

the protein is, in cfkct: similar to screening with runny thousmds ofsubstmccs. and will probnbl>, result in at Icast our cpitopc binding strongly .aud spcciiically (Fig. 5). The bound oligomcr cm then kx &t-cd from the protein and stquwccd to idrntie the specific r~ognition scqucncc. This approach has the xivmtagc that the rare recognition scqucnccs can bc amplifird rsing the poiynwrasc chain rcxtion ([‘CR) method. It should be cmphnsizrd chat this is a thcl-apmtic approach using +,onucleotidc~ that doe not involve genetic intcrvcntion or rcquirc cellular upt&e if the protciu target is cstraccllulnr. Hcnw it Las nanny ptc&l adwrt;~gcs. This approach has been r&rcd to as the ‘adaptmcr’ or ‘apmrci approach, bx ‘prowin cpitopc tnrgcting’ (PET j is a ~cicntificnlly mxc corrccc tam

Conclusion Oligonuc!eotides arc hcing dcvclopcd as potwcinl thmpcutics in scvrral novel ways ttr intcrvcnc lo the aberant cspt cssim oigcnrtir infomntitm in the cell.

Oligonucleotide therapeutics This artide introducas a mini-series to ba pubiished over the nsxt few issues of TISTECH. Although questions concerning optimat design and use of otiinudeotides remain, there are now many reports of their successful application as a specific means of maniputattng gene expression. The mini-se&s will investigate tha stat&-th*art of tha range of diverse technologies under devatopment as potential therapeutics.


&48CH1992 P&L 101



Recombinant haemoglobin in the development of red-blood-ceil substitutes

Jill E. Ogden

Increasing concern over viral contamination of blood is spurring the development of a blood substitute which can effectively replace the oxygencarrying capabilities of transfused erythrocytes. Solutions of r!?emically modiiied haemoglobin represent one option being evaluated for this role. More recently, recombinant-lINA techniques have enabled production of human haemoglobin in host expression systems, and progress is being made towards the creation of a genetically engineered molecule incorporating the properties required of a blood substitute. For many >;ears, there has been much interesr in dev& oping a sate and effective replacement for trausfuscd red blood celb (exyrhrocytcs) as a means of carrying oxygen. The starch for a blood substitute (or, more accurately, red-cell substitute, since such sohxtions canncf- replace all the fimctio;~ of blood) has iutcnsified because of increasing cowww over viral contamination of donated blood. A red-cell substitute is dcdrable for other reasons too: 0 Such a product could have a much longer shelf-Xc (potentially one year or more) than the 42 days of red cells; C As blood-group dctcrminants reside on the crythrocyte surface, a red-cell substitute would be biocontparible \vith ali iluud gruupj; (P Thcrc is increasing c\%dcnce, at least in the USA, of a diminishin~wpply nfdon3rz! b!c?z! f.rtr:&i:ic::“. This has been attributed. in part, to more selzctivc screening ofdonors and concerns ofpotcntial donoR; that blood donation itselfma~ cqwsc them to the risks ofviral infection. In addition to caqing oxygen, an imporrant role for a red-cell substitute is plasma expansion. Such a product could be us& in the emergency resuscitation of trauma victims, in some surgical procedures, in

org3n preservation, and in cwnour thtirapv. For thew indications, the potential market size has been csdmated at .&round USS? Siilion annuslly. The ukirwte goal is a product xtwh extended circulator\- h3lf-lifr (pc;sihIl; >30 daysj, which CM bc used clin&Hy as a complctc w&mntcJx I& red ceiis. for csampic in :bc trcatmcnt oichronic anactnia. In order to be an etktirc oxT,gcn-carr+Ilg aitcrnnrive to bl.Jod. a red-ccl1 substitute must GM 3 turnbcr ofrcquircmcnts: ?? It muX bc stcrilc. Grus-t&c. non-rosic. nonpyrogcnic and ~~on-in,~nrn~o~~~~ic: . It musr bc bioiompatible with atl blood group: ??It should have a ;wlg shePiifci_: 0 The product shouid lx:-; J sxishcto~- h&-htc 111 the circulation, and sho.lid k cleared thorn the circn.I^-gcn 3tlinity and oxygen-carrying cap3city to permit effiii~nt tksuc ok~gcnation.

Oligonucleotide therapeutics.

Recent studies have focused on the ability of oligonucleotides to affect the genetic processes of many organisms, including viruses. Hence, oligonucle...
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