CA2223103A1 - Oligonucleotides having phosphorothioate linkages of high chiral purity - Google Patents

Oligonucleotides having phosphorothioate linkages of high chiral purity

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Publication number
CA2223103A1
CA2223103A1 CA 2223103 CA2223103A CA2223103A1 CA 2223103 A1 CA2223103 A1 CA 2223103A1 CA 2223103 CA2223103 CA 2223103 CA 2223103 A CA2223103 A CA 2223103A CA 2223103 A1 CA2223103 A1 CA 2223103A1
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CA
Canada
Prior art keywords
oligonucleotides
seq id
oligonucleotide
phosphorothioate
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2223103
Other languages
French (fr)
Inventor
Glenn Hoke
Phillip Dan Cook
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ionis Pharmaceuticals Inc
Original Assignee
Isis Pharmaceuticals Inc.
Glenn Hoke
Phillip Dan Cook
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US08/466,692 priority Critical patent/US5654284A/en
Priority to US08/471,966 priority
Priority to US08/469,851 priority patent/US5587361A/en
Priority to US08/471,966 priority patent/US5661134A/en
Priority to US08/467,597 priority
Priority to US08/468,447 priority patent/US5576302A/en
Priority to US08/470,129 priority
Priority to US08/466,692 priority
Priority to US08/467,597 priority patent/US5607923A/en
Priority to US08/468,447 priority
Priority to US08/469,851 priority
Priority to US08/468,569 priority patent/US5620963A/en
Priority to US08/471,967 priority patent/US5599797A/en
Priority to US08/468,569 priority
Priority to US08/470,129 priority patent/US5635488A/en
Priority to US08/471,967 priority
Application filed by Isis Pharmaceuticals Inc., Glenn Hoke, Phillip Dan Cook filed Critical Isis Pharmaceuticals Inc.
Publication of CA2223103A1 publication Critical patent/CA2223103A1/en
Application status is Abandoned legal-status Critical

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    • C12N15/1131Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
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    • C12N15/1133Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
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    • C12N15/1138Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/315Phosphorothioates

Abstract

Sequence-specific phosphorothioate oligonucleotides comprising nucleoside units which are joined together by either substantially all Sp or substantially all Rp phosphorothioate intersugar linkages are provided. Such sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are prepared by enzymatic or chemical synthesis.

Description

W O 96/39154 PCTrUS96/08757 OLI w Nu~EOTIDES HAVING PHOSPHOROTHIOATE LINKAGES
OF HIGH ~TR~T. PURITY

CROSS R~K~.~ TO RELATED APPLI QTIONS
This application is a continuation-in-part o~ U.S.
Patent application Serial Number 08/297,703, ~iled August 29, 1994, which is a continuation of U.S. Patent application Serial Number 07/777,007, ~iled October 16, 1991, now abandoned. This application also is a continuation-in-part o~ U.S. Patent application Serial Number 08/058,023, filed May 5, 1993, which is a divisional àpplication o~ U.S.
Patent application Serial Number 07/777,670, filed October 15, 1991 (issued as U.S. Patent 5,212,295, issue date May 18, 1993), which is a continuation-in-part o~ U.S. Patent application Serial Number 07/777,007. Each o~ the above-mentioned applications is commonly assigned with thisapplication, and the entire disclosures of each are herein incorporated by re~erence.

FIELD OF ~ Nv~NllON
This invention is directed to sequence-speci~ic phosphorothioate oligonucleotides comprising nucleosides joined by intersugar linkages, and to their synthesis and use. More particularly, the intersugar linkages linking the nucleosides of oligonucleotides o~ the present invention are substantially pure all Sp or all Rp chiral phosphorothioate linkages. Such oligonucleotides are prepared via chemical or enzymatic synthesis. They are especially well suited as diagnostics, therapeutics and research reagents.

~ =

R~R~u~DD OF I~DE lNv~NlloN
Oligonucleotides are known to hybridize to single-stranded RNA or single-stranded DNA. Hybridization is the sequence-specific base pair hydrogen bonding of bases of the oligonucleotides to bases of target RNA or DNA. Such base pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm)~ a characteristic physical property of double helices, denotes the temperature (~C) at which 50~ helical (hybridized) versus coil (unhy-bridized) forms are present. Tm is measured by using the Wspectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in W
absorption (hypochromicity). Consequently, a reduction in W absorption indicates a higher Tm~ The higher the Tm/ the greater the strength of the bonds between the strands.
Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme RNase H. The mechanism of such RNase H cleavage requires that a 2'-deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA
strand. Cleavage of the RNA strand destroys the normal function of the target RNA. Phosphorothioate oligo-nucleotides operate via this type of mechanism. However,for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary.

W O 96/39154 PCTrUS96/087~7 Several publications of Walder et al. describe the interaction of RNase H and oligonucleotides. Of particular ~ interest are: (1) Dagle et al., Nucleic Acids Research 1990, 18, 4751; (2) Dagle et al., Antisense Research And Development 1991, l, 11; (3) Eder et al., ~. Biol. Chem.
1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research 1991, 19, 1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.
As described in references (1), (2) and (4), to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2'-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. While the phosphoamidate-containing oligonucleotides were stabilized against exonuc-leases, in reference (4) the authors noted that each phos-phoramidate linkage resulted in a loss of 1.6~C in the measured Tm value of the phosphoramidate containing oligonu-cleotides. Such a decrease in the Tm value is indicative of a decrease in hybridization between the oligonucleotide and its target nucleic acid strand.
ApplicationS of oligonucleotides as diagnostics, research reagents, and therapeutic agents require that the oligonucleotides be transported across cell membranes or taken up by cells, appropriately hybridize to target RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend partly on the WO 96~9154 PCT~US96/08757 initial stability of oligonucleotides towards nuclease degradation. Further, these functions depend on specificity of the oligonucleotide for a target RNA or DNA molecule.
A serious deficiency of oligonucleotides for these purposes is their susceptibility to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly and extracellularly located. Unmodified, "wild type", oligonucleotides are not useful as therapeutic agents because they are rapidly degraded by nucleases.
Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been the primary focus of research directed towards the development of oligonucleotide therapeutics and diagnostics.
Modifications of oligonucleotides to enhance nuclease resistance has generally taken place on the sugar-phosphate backbone, particularly on the phosphorous atom.
Phosphorothioates have been reported to exhibit resistance to nucleases. In addition, phosphorothioate oligonucleotides are generally more chemically stable than natural phosphodiester oligonucleotides. Phosphorothioate oligonucleotides also exhibit solubility in aqueous media.
Further, phosphorothioate oligonucleotide-RNA heteroduplexes can serve as substrates for endogenous RNase H.
Additionally, phosphorothioate oligonucleotides exhibit high thermodynamic stability. However, while the ability of an oligonucleotide to bind to a target RNA or DNA with fidelity is critical for its hybridization to the target RNA or DNA, modifications at the phosphorous atom of the oligonucleotides, while exhibiting various degrees of nuclease resistance, have generally suffered from inferior hybridization properties [Cohen, J.S., Ed., Oligonucleotides: Antisense Inhibitors of Gene Expression (CRC Press, Inc., Boca Raton, FL, 1989].
One reason for this inferior hybridization may be the prochiral nature of the phosphorous atom. Modifications on the internal phosphorous atom of modified phosphorous oligonucleotides results in Rp and Sp stereoisomers.

Modified phosphorus oligonucleotides obtained thus far, wherein the resulting molecule has nonsymmetrical substituents, have been racemic mixtures having 2n isomers, with n equal to the number of phosphorothioate intersugar linkages in the oligonucleotide. Thus, a 15-mer phosphorothioate oligonucleotide, containing 14 asymmetric centers has 214 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with sufficient affinity.
Chemically synthesized phosphorothioate oligo-nucleotides having chirally pure intersugar linkages had thus far been limited to molecules having only one or two diastereomeric intersugar linkages. Until recently, the effects of induced chirality in chemically synthesized racemic mixtures of sequence-specific phosphorothioate oligonucleotides had not been assessed since synthesis of oligonucleotides having chirally pure intersugar linkages had yet to be accomplished by automated synthesis. This was due to the non-stereospecific incorporation of sulfur during automated synthesis. For example, Stec et al., J.
Chromatography, 326:263 (1985), synthesized certain oligonucleotide phosphorothioates having racemic intersugar linkages, however, they were able to resolve only the diastereomers of certain small oligomers having one or, at most, two diastereomeric phosphorous intersugar linkages.
However, Stec et al. [Nucleic Acids Res., 19: 5883 (1991)] subsequently reported the automated stereocontrolled synthesis o~ oligonucleotides. The procedure described in the above-mentioned reference utilizes base-catalyzed nucleophilic substitution at a pentavalent phosphorothioyl center The synthesis o~ phosphorothioates having all Rp - intersugar linkages using enzymatic methods has been investigated by several authors [Burgers and Eckstein, J.
Biological Chemistry, 254:6889 (1979); Gupta et al ., ~.
Biol . Chem., 256:7689 (1982); Brody and Frey, Biochemistry, WO 96/3915~ PCT~US96/08757 20:1245 (1981); and Eckstein and Jovin, Biochemistry, 2:4546 tl983)]. Brody et al. tBioc~emistry, 21: 2570 (1982)] and Romaniuk and Eckstein, t~. Biol. Chem., 257:7684 (1982)]
enzymatically synthesized poly TpA and poly ApT
phosphorothioates, while Burgers and Eckstein [Proc. Natl.
Acad. Sci. U.S.A., 75: 4798 (1978)] enzymatically synthesized poly UpA phosphorothioates. Cruse et al. [~.
Mol. Biol., 192:891 (1986)] linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a h~mer.
The relative ability o~ an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature o~ a particular hybridization complex. The melting temperature (Tm)~ a characteristic physical property of double helixes, denotes the temperature (~C) at which 50~ helical versus coil (unhybridized) forms are present. Tm is measured by using the W spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in W
absorption (hypochromicity). Consequently a reduction in W
absorption indicates a higher Tm~ The higher the Tm~ the greater the strength of the binding of the strands. Non-Watson-Crick base pairing has a strong destabilizing e~fect on the Tm~
In a prel;~;n~y report [Stec, J.W., Oligonucleotides as Antisense Inhibitors of Gene Expression:
Therapeutic Implications, Meeting abstracts, June 18-21, 1989], thymidine homopolymer octamers having all but one linkage being modified phosphate linkages ("all except one") Rp stereocon~iguration or "all except one~ Sp stereoconfiguration in the intersugar linkages were ~ormed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester bond. It was noted that a Rp "all except one" methylphosphonate non-sequence-specific thymidine homooctamer, i. e. (dT) 8 having all but one Rp intersugar linkage, formed a WO 96/39154 PCT~US96/08757 thermodynamically more stable hybrid (Tm 38~C) with a 15-mer deoxyadenosine homopolymer, i.e. (dA)15~ than a hybrid formed ~ by a similar thymidine homopolymer having "all except one"
Sp configuration methylphosphonate linkages and of d(A) 15 (Tm u 5 ~0~C), i.e. a d(T)15 having all but one Sp intersugar linkage. A hybrid between (dT) 8 having natural phosphodiester linkages, i.e. octathymidylic acid, and d(A) 15 was reported to have a Tm of 14~C.
More recently, Ueda et al. [Nucleic Acids Research, 19:547 (1991)] enzymatically synthesized mRNAs intermittently incorporating Rp diastereomeric phosphoro-thioate linkages for use in translation systems. Ueda et al. employed T7 coliphane DNA having seventeen promoters and one termination site for T7 RNA polymerase. In vitro 15 synthesis by T7 RNA polymerase produced mRNAs having from several hundred to tens of thousands of nucleotides.
Backbone chirality may also affect the susceptibility of a phosphorothioate oligonucleotide-RNA
heteroduplex to RNase H activity. The ability to serve as a 20 template for RNAse H has significant therapeutic implications since it has been suggested that RNAse H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. With oligonucleotides containing racemic mixtures of Rp and Sp intersugar linkages, it is not known 25 if all phosphorothioate oligonucleotides can function equally as substrates for RNase H. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of 30 configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that ~ contain the correct chirality for m~; ml~m hybridization efficiency and termination of translation. Thus, increasing - the percentage of phosphorothioate oligonucleotides that can serve as substrates for RNAse H in a heteroduplex will likely lead to a more efficacious compound for antisense therapy.

CA 02223l03 l997-l2-02 WO 96~9154 PCTrUS96/08757 To enhance hybridization fidelty, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired. Further, such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages would lead to more efficacious therapeutic compounds. However, until now little success has been achieved in synthesizing such molecules. Therefore, simple methods of synthesizing phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are greatly desired.
It has been recognized that nuclease resistance of oligonucleotides and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Oligonucleotides possessing nuclease resistance are also desired as research reagents and diagnostic agents.

SllM ~ RY OF THE lNVhN'llON
In accordance with this invention, phosphorothioate oligonucleotides having all nucleoside units joined together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages are provided.
Preferably, the phosphorothioate oligonucleotides of the present invention are complementary to at least a portion of the sequence of a target RNA or DNA.
Further, in accordance with the present invention, chemical and enzymatic methods o~ synthesizing sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are provided wherein said phosphorothioate oligonucleotides are comprised of at least 10 nucleoside units joined together by either substantially all Rp or substantially all Sp intersugar linkages. Preferably, the phosphorothioate oligonucleotides are comprised of about 10 to about 50 nucleoside units joined by substantially chirally pure intersugar linkages.
More preferably, said phosphorothioate oligonucleotides are CA 02223l03 l997-l2-02 comprised of about 15 to about 30 nucleos1de units joined by substantially chirally pure intersugar linkages. Most ~preferably, said phosphorothioate oligonucleotides are comprised of about 17 to 21 nucleoside units joined together ~5 by substantially chirally pure intersugar linkages. Said methods comprise combining sequence primers, templates, and an excess of all four chirally pure nucleoside 5'-O-(1-thiotriphosphates~. Said methods further include synthesizing complementary oligonucleotides by the addition of polymerase followed by cleavage of the primer from the complementary oligonucleotides. In addition, said methods are comprised of disassociating said complementary oligonucleotides from said template.
In alternative embodiments of the present invention methods of synthesizing sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages, sequence primers, templates and racemic mixtures of nucleoside 5'-O-(1-thiotriphosphates) are combined. Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages and which are complementary to the template are synthesized by the addition of polymerase and a selected metal ion. Oligonucleotides thus synthesized are dissociated from the template and primer.
Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are useful for increasing the thermodynamic stability of heteroduplexes formed with target RNA and DNA and to elicit RNase H
activity. Further, oligonucleotides of the present invention are useful for modulating the activity of RNA.

- DETATT~n DES ~ IPTION OF T ~ lNV~N-llON
The phosphorous atom in a phosphodiester linkage - of an oligonucleotide can be described as being "pro-chiral." Once a non-bonding oxygen atom of the phosphodi-ester linkage is replaced or modified, a chiral sugar-phosphate linkage is generated. The resulting intersugar linkage is either an Sp intersugar linkage or an Rp intersugar linkage. Replacement of a non-bonding oxygen atom of the natural phosphodiester linkage with sul~ur to obtain a phosphorothioate linkage results in the generation o~ a chiral center and af~ords Sp and Rp diastereomers.
Molecu~es wherein substantially all of the phosphorous atoms in the sugar backbone are either Sp or Rp are re~erred to herein as chirally pure.
Ribonucleoside- (NTP~S) and 2'-deoxyribonucleo-side-5'-0-(1-thiotriphosphates) (dNTP~S) have been synthesized as Sp and Rp racemic mixtures using the methodology of Ludwig and Eckstein [~. Org. Chem., 631 (1989)]. In this exemplary synthetic scheme, unprotected nucleosides can be reacted with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, which phosphitylates the 5'-hydroxyl group. Subsequent reaction with pyrophosphate yields cyclic triphosphate derivatives which are reactive to sulfur, yielding mixtures of Rp and Sp nucleoside 5'-0-(1-thiotriphosphates), i . e. ~-thiotriphosphates. The products can be puri~ied by DEAE-Sephadex chromatography and identi-~ied by NMR spectro~copy (by characteristic Rp or Sp chemical shifts).
As is shown in the examples below, pure Rp and Sp nucleoside-5'-0-(l-thiotriphosphates) diastereomers can be readily isolated on a preparative scale using, ~or example, reverse phase HPLC chromatography. Such HPLC-isolated nucleotide diastereomers can be further characterized by analytical HPLC comparisons with commercial samples o~ such Rp and Sp nucleoside 5'-0-(1-thiotripho~phates) diastereome-rs.
Enzymatic synthesis of sequence-speci~ic natural oligonucleotides, i.e. natural phosphodiester oligonucleotides, can be effected by the use of an appropriate nuclease in the presence of a template and primer. In a like manner, racemic mixtures o~
phosphorothioate oligonucleotides having chirally mixed intersugar linkages can be synthesized. According to the W O 96/39154 . PCT~US96/08757 present invention, such enzymatic synthesis can also be expanded to include the synthesis of sequence specific ~ phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages by utilizing ~ 5 enantiomerically pure all-Sp or all-Rp nucleoside 5'-O-(1-thiotriphosphates) as substrates for appropriate nucleases in the presence of a sequence-specific template and a primer. For example, commercially available DNA polymerase Sequenase~ (U.S. Biochemical, Inc., Cleveland, OH) may be used to synthesize phosphorothioate oligonucleotides using a phosphodiester oligonucleotide template and a racemic phosphorothioate oligonucleotide primer. Using this polymerase both phosphodiester and phosphorothioate primers may be extended.
Yields of enzymatically synthesized phosphoro-thioate oligonucleotides can be optimized by repetitive additions of template and primer, by repetitive additions of polymerase, by repetitive additions of nucleoside triphosphates or by combinations of some or all of these.
For instance, repetitive additions of template and primer results in m~; m; zing yields via an enzymatic cascade.
Further optimization can be achieved by pre-hybridization of template and primer together in system buffer, followed by cooling and addition of nucleoside triphosphates and polymerase.
A suitable polymerase may be selected to yield either DNA or RNA phosphorothioate oligonucleotides. Such polymerases include but are not necessarily limited to T7 DNA polymerase, modified T7 DNA polymerases such as the above referenced Sequenase~, E. coli DNA polymerase, DNA
poly Klenow fragment polymerase, M. luteus polymerase, T4 bacteriophage polymerase, modified T4 DNA polymerase, T7 RNA
polymerase and E. coli RNA polymerase.
- The enzymatic synthesis proceeds with inversion of configuration about the chiral center of the phosphorous atom. Thus, use of all Sp ~-thiotriphosphates yields substantially all Rp phosphorothioate oligonucleotides while WO 96/39154 PCTrUS96/08757 use of all Rp ~-thiotriphosphates yields substantially all Sp phosphorothioate oligonucleotides. In an alternate embodiment of the invention, phosphorothioate oligonucleotides may be synthesized from racemic mixtures of nucleoside-5'-0-(1-thiotriphosphates) utilizing metal ions in reaction solutions to promote preferential incorporation of one or the other of the chiral ~-thiotriphosphates. As noted above, polymerase synthesis of phosphorothioate oligonucleotides is accomplished with inversion of configuration about the chiral center of the precursor nucleoside-~-thiotriphosphate. While not wishing to be bound by theory, it is believed that optimization of an all Rp configuration may be accomplished by addition of a high concentration of magnesium ion in the reaction buffer utilizing, for instance, an E. coli polymerase. In a like manner, again while not wishing to be bound by theory, an all Sp configuration might be obtained by utilizing a high manganese ion concentration in the reaction buffer.
In accordance with the present invention, "substantially all" is meant to include all oligonucleotides in which at least 75~ of the intersugar linkages are chirally pure. More preferably, oligonucleotides having from about 85~ to about 100~ chirally pure intersugar linkages are substantially chirally pure. Most preferably, oligonucleotides having from about 95~ to about 100~
chirally pure intersugar linkages are substantially chirally pure.
In the context of this invention, the term "phosphorothioate oligonucleotide'~ includes phosphorothioate oligonucleotides formed from naturally occuring bases, sugars and phosphorothioate linkages. Naturally occuring bases include adenine, guanine, cytosine, thymine and uracil. Natural sugars include $-D-ribofuranosyl and ~-D-2'-deoxy-erythro-pentofuranosyl. To the extent that nucleoside-5'-0-(1-thiotriphosphate) analogs are substrates for suitable polymerases, "phosphorothioate oligonucleotides" also include modified bases or modified sugars incorporated within the phosphorothioate nucleotide units of the oligonucleotides. Modified bases of the oligonucleotides of this invention include a~n;ne, guanine, adenine, cytosine, uracil, thymine, xanthine, hypoxanthine, ~ 5 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl gll~n;nes, 8-hydroxyl guanine and other 8-substituted gll~n;nes, other aza and deaza uracils, other aza and deaza thymidines, other aza and deaza cytosines, other aza and deaza a~n;nes, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine. The sugar moiety may be deoxyribose or ribose. The oligonucleotides o~ the invention may also comprise modified nucleobases or nucleobases having other modifications consistent with the spirit of this invention, and in particular modifications that increase their nuclease resistance in order to facilitate their use as therapeutic, diagnostic or research reagents. Oligonucleotides of the invention can be utilized as diagnostics, therapeutics and as research reagents. They can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide of the invention to a suitable pharmaceutically acceptable diluent or carrier.
They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism can be contacted with an oligonucleo-tide of the invention having a sequence that is capable ofspecifically hybridizing with a strand of target nucleic - acid that codes for the undesirable protein.
The formulation of therapeutic compositions and - their subsequent administration is believed to be within the skill of those in the art. In general, for therapeutics, a patient in need of such therapy is administered an oligonucleotide in accordance with the invention, commonly WO96/391~4 PCTAJS9G,' in a pharmaceutically acceptable carrier, in doses ranging from 0.01 ~g to 100 g per kg of body weight depending on the age of the patient and the severity of the disease state being treated. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient, and may extend from once daily to once every several years. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, or if the disease state has been ablated.
In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo ~n;m~l models. In general, dosage is from 0.01 ~g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every several years.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is a~m~ n; ~~tered in maintenance doses, ranging from 0.01 ~g to 100 g per kg of body weight, once or more daily, to once every several years.
- The pharmaceutical compositions of the present invention may be administered in a number of ways depending - 5 upon whether local or systemic treatment is desired and upon the area to be treated. ~; n; stration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.
Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Phosphorothioate oligonucleotides of the present invention can be contrasted with both natural phosphodiester oligonucleotides and racemic phosphorothioate nucleotides as - to their effects on hybridization, nuclease resistance and RNAse H activity. In like manner, pure phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages may also be assessed for their ability CA 02223l03 l997-l2-02 WO 96/39154 PCT~US9Gi~'75 to increase effectiveness of therapy in in vivo test systems. Such increase in effectiveness of therapy might include attributes such as pharmacokinetics or metabolism, toxicology, disposition (i.e. absorption and distribution), and species comparisons.
Homopolymers having all Rp or all Sp intersugar linkages have been useful for initial studies of stability and other characteristics. However, these oligonucleotides have little use therapeutically as they are not specific for target molecules. Phosphorothioate oligonucleotides having specific sequences are necessary in order to specifically hybridize to target nucleic acids.
Sequence-specific phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are useful to increase the thermodynamic stability of heteroduplexes with target RNA and DNA and to elicit RNase H activity.
Radiolabeling can be used to assist in the identification of phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For racemic phosphorothioate oligonucleotides synthesized on an automated synthesizer, [35S] (radiolabeled elemental sulfur) can be used for oxidation of the hydrogen-phosphonate oligomers obtained from the synthesizer. Labeling of enzymatically synthesized phosphorothioate oligonucleotides can be accomplished with [~_32p] ATP and ligase or [~-35S] ATPs in the polymerase reaction. Also, radiolabeled nucleoside triphosphates can be used in probe and sequencing analysis.
Autoradiograms are prepared in standard manners.
Templates of the present invention are most preferably areas of nucleic acid sequence which direct synthesis of disease-potentiating proteins. Short oligonucleotides that base pair to a region of said template oligonucleotide act as primers which form the starting point for oligonucleotide synthesis by polymerases.
Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages may be - - -synthesized using a primer which may be selected to have a site thereon that is susceptible to nuclease cleavage, for example, restriction endonuclease cleavage. Said cleavage site may be located at the 3' end of said primer. Cleavage at said site by an appropriate restriction endonuclease results in oligonucleotides deriving a first 5' end nucleoside from said primer. Additional nucleosides of said phosphorothioate oligonucleotides of the present invention are those nucleoside chiral thiotriphosphates added via enzymatic means.
By selecting appropriate restriction nucleases in conjunction with selected primers, various 5'-terminal nucleosides of desired phosphorothioate oligonucleotides are appropriately positioned at the 5' end of a phosphorothioate nucleotide. Thus, any endonuclease recognition site can be designed as long as the staggered cut results in one nucleoside from the primer being the first 5' nucleoside of the newly synthesized sequence specific phosphorothioate oligonucleotide of the invention. This results in the generation of different nucleosides on 5' ends of enzymati-cally synthesized phosphorothioate oligonucleotides of the nvent ion .
Upon completion of enzymatic extension of said primer on an appropriate template of a desired sequence, phosphorothioate oligonucleotides of the invention may be released from said primer by use of appropriate nuclease.
For example, for incorporation of a guanosine nucleoside at the 5' end of desired phosphorothioate oligonucleotides, a primer having an CTGCAG sequence at its 3' terminal end may be used. Use of a Pst 1 restriction nuclease then may cleave the A-G linkage. The guanosine nucleoside component of this A-G linkage may thus incorporated as a 5' terminal nucleoside of desired phosphorothioate oligonucleotides.
Other restriction endonuclease include but are not limited to BamH1, Smal and HinD III restriction endonucleases.
Oligonucleotides still associated with said template may be dissociated from said template and then WO 96/39154 . PCTAUS96/08757 purified by gel electrophoresis and/or chromatography. For example, suitable purification can be accomplished utilizing standard polyacrylamide/urea gel electrophoresis coupled with SepPac (Millipore, Miford, MA) chromatography. Another useful chromatographic technique that may be employed is HPLC chromatography.
Chiral phosphorothioate oligonucleotides of the present invention may also be chemically synthesized via 1,3,2-oxathiaphospholane intermediates as described by Stec lO et al. [Nucleic Acids Res., 19:5883 (1991)] and Stec and Lesnikowski [Methods in Molecular Biology, S. Agrawal, Ed., Volume 20, p. 285, 1993].
Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages which are synthesized according to methods of the present invention may be analyzed by a number of methods. For example, configuration analysis of resulting sequence-specific phosphorothioate oligonucleotides having subtantially chirally pure all Sp or all Rp intersugar linkages may be determined by the use of [31p] NMR chemical shifts. Such chemical shifts have been used to identify the Rp epimer of a phosphorothioate dinucleotide [Ludwig and Eckstein, J.
Org. Chem., 631-635 (1989)].
The fidelity of sequences of phosphorothioate oligonucleotides of the invention can be determined using the sensitivities of heteroduplexes to S1 nuclease.
The sequence of the phosphorothioate oligonucleotides can be further substantiated by labeling the 3'hydroxyl groups of phosphorothioate oligonucleotides with [alpha-32P]cordycepin triphosphate, i. e. 3'-deoxyadeno-sine-5'-triphosphate. The resultant oligonucleotides may be subjected to enzymatic degradation.
The relative ability of phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages to bind to complementary strands is compared by determining the melting temperature of a hybridization complex of a phosphorothioate oligonucleotide WO 96/39154 PCTrUS96/08757 having substantially chirally pure intersugar linkages and its complementary strand. The melting temperature (Tm)~ a ~ characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50~ helical - 5 versus coiled (unhybridized) ~orms are present. Tm is measured by using the W spectrum to determine the ~ormation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in W absorption (hypochromicity). Consequently a reduction in W absorption indicates a higher Tm. The higher the Tm~ the greater the strength o~ the binding o~
the strands. Non Watson-Crick base pairing has a strong destabilizing e~ect on the Tm. Consequently, as close to optimal ~idelity o~ base pairing as possible is desired to have optimal binding o~ an oligonucleotide to its target RNA.
Phosphorothioate oligonucleotides of the invention can also be evaluated ~or their resistance to the degradative ability o~ a variety o~ exonucleases and endonucleases. Phosphorothioate oligonucleotides may be treated with nucleases and then analyzed, as ~or instance, by polyacrylamide gel electrophoresis (PAGE) ~ollowed by staining with a suitable stain such as Stains All~ (Sigma Chem. Co., St. Louis, M0). Degradation products may be quantitated using laser densitometry.
Fetal cal~ and human serum may be used to evaluate nucleolytic activity on phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages. For instance, a phosphorothioate oligonucleotide having substantially all Rp intersugar linkages may be evaluated in this manner. Testing on combinations o~ 3' or 5' end capped - (having one or several phosphorothioate linkages per cap) molecules may be used to establish a combination that yields ~ greatest nuclease stability. Capping can be e~ected by chemically synthesizing the cap portion of a sequence using puri~ied Rp monomers followed by incorporation o~ said cap into oligonucleotides on the DNA synthesizer. Analysis WO 96~9154 PCTAUS96/08757 involving capping can determine the importance of chirality on nucleolytic stability and the number o~ linkages required to obtain m~; ml~m stability.
The sensitivity of phosphorothioate oligonucleotide-RNA heteroduplexes to the catalytic activity of RNase H can also be assessed. A phosphorothioate oligo-nucleotide can be incubated with a radiolabeled target mRNA
(synthesized as for instance via T7 RNA polymerase) at various temperatures for hybridization. Heteroduplexes can then be incubated at 37~C with RNase H from E. col i according to the procedure of Minshull and Hunt [Nuc. Acid Res., 6433 (1986)]. Products may then be assessed for RNase H activity by Northern Blot analysis wherein products are electrophoresed on a 1. 2~ agarose/formaldehyde gel and transferred to nitrocellulose. Filters may then be probed using a random primer [32P]-labeled cDNA complementary to target mRNA and quantitated by autoradiography. Comparisons between different phosphorothioate analogs can be made to determine the impact of chirality on the ability to act as a substrate for RNase H when complexed to RNA.
Comparisons of the susceptibility of heteroduplexes to the catalytic action of E. col i RNase H
and m~mm~l ian RNAse H can be performed. Heteroduplexes can be incubated in rabbit reticulocyte lysates under conditions of translation and assayed via Northern blot analysis for catalytic cleavage of mRNA by endogenous RNase H. This allows for determination of the effects of chirality on m~mm~l ian RNAse H activity.
Phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can also be evaluated for inhibition o~ gene expression in cell culture model systems. To determine if a phosphorothioate oligonucleotide having substantially pure chirally pure intersugar linkages is more potent or a more specific inhibitor of gene-expression, a phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages designed to target reporter genes may be -synthesized and tested in cell culture models of gene expression. The use of the vector pSV2CAT has previously been described to measure antisense effects on gene expression [Henthorn et al ., Proc.Natl .Acad. Sci . U. S.A., 85:6342 ,(1988)]. This vector contains the bacterial chloramphenicol acetyl transferase gene under regulatory controls of the SV40 promoter. Utilizing a 15-mer phosphorothioate oligonucleotide having all Rp intersugar linkages of a sequence complementary to the initiation of translation of the CAT mRNA, pSV2CAT may be transfected into HeLa cells and, following treatment of the cells for 48 hr with a phosphorothioate oligonucleotide having all Rp intersugar linkages, CAT activity may then be assayed in the cells. The activity of a phosphorothioate having substantially chirally pure intersugar linkages in inhibition of gene expression may then be compared directly with a chemically synthesized random phosphorothioate having diastereomeric intersugar linkages and natural phosphodiester oligonucleotides of the same sequence.
The vector pSV2APAP [Marcus-Sekura et al., Nucleic Acids Research, 15:5749 (1987)] contains the m~mm~l ian placental alkaline phosphatase gene (PAP). This can also be used as a reporter for measuring antisense effects on gene expression. PAP has advantages over CAT as a reporter gene in that it is a m~mm~ 1 ian gene, rather than a bacterial gene that contains introns and other RNA processing signals. It is presently believed that PAP expression mimics more closely the events in natural m~mm~l ian gene expression. A
15-mer phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages as described above for the CAT mRNA can be ~m; n~d in parallel with chemically synthe-sized racemic phosphorothioate and natural phosphodiester oligonucleotides having similar sequences. The PAP and CAT
reporter constructs are used as controls in reciprocal experiments to test for non-specific effects on gene expression.

Additionally, phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages can be evaluated as to their ability to act as inhibitors of RNA
translation in vivo. Various therapeutic areas can be targeted for such manipulation by oligonuclotides of the present invention. One therapeutic area is hepatitis caused by Hepatitis C virus (HCV). The following phosphorothioate oligonucleotides have application in the treatment of HCV
hepatitis: Oligo # 259, CCTTTCGCGACCCAACACTA (SEQ ID NO:1), Oligo # 260, GCCTTTCGCGACCCAACACT (SEQ ID NO:2), Oligo #
270, GTACCACAAGGCCTTTCGCG (SEQ ID NO:3), Oligo # 330, GTGCTCATGGTGCACGGTCT (SEQ ID NO:4) and Oligo # 340, TTTAGGATTCGTGCTCATGG (SEQ ID NO:5). Another therapeutic area inflammatory diseases mediated by intercellular cell adhesion molecule (ICAM-1). Oligonucleotides having application in the treatment of inflammatory diseases include: ISIS-2302, GCCCAAGCTGGCATCCGTCA (SEQ ID NO:6).
Another therapeutic area includes infections caused by cytomegalovirus (CMV). ISIS-2922 is a phosphorothioate oligonucleotide having application in the treatment of CMV
retinitis, and has the sequence GCGTTTGCTCTTCTTCTTGCG (SEQ
ID NO:7). Another therapeutic area includes cancers mediated by protein kinase C-~ (PKC-~). ISIS-3521 is a phosphorothioate oligonucleotide having application in the treatment of such cancers, and has the sequence GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8). A further therapeutic area includes C-raf kinase-mediated cancers. ISIS-5132 is a phosphorothioate oligonucleotide having application in the treatment of such cancers, and has the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9). A still further therapeutic area includes cancers mediated by Ha-ras or Ki-ras. The following phosphorothioate oligonucleotides have application in the treatment of such cancers: ISIS-2503, TCCGTCATCGCTCCTCAGGG (SEQ ID NO:10), ISIS-2570, CCACACCGACGGCGCCC (SEQ ID NO:11), and ISIS-6957, CAGTGCCTGCGCCGCGCTCG (SEQ ID NO:12). Another therapeutic area includes AIDS and other related infections mediated by W O 96~9154 PCT~US96,'~

HIV. ISIS-5320 is a phosphorothioate oligonucleotide having application in the treatment o~ AIDS, and has the sequence ~ TTGGGGTT (SEQ ID NO:17). In the above sequences, individual nucleotide units oi~ the oligonucleotides are listed in a 5' to 3' direction ~rom left to right.
In the context o~ this invention, "hybridization"
shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotide units. For example, a~enlne and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, also re~ers to sequence complementarity between two nucleotide units. For example, if a nucleotide unit at a certain position o~ an oligonucleotide is capable o~
hydrogen bonding with a nucleotide unit at the same position o~ a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA
are complementary to each other when a su~ficient number o~
corresponding positions in each molecule are occupied by nucleotide units which can hydrogen bond with each other.
Thus, I'speci~ically hybridizable" and "complementary" are terms which are used to indicate a su~ficient degree o~
complementarity such that stable and speci~ic binding occurs between the oligonucleotide and the target RNA or DNA. It is understood that an oligonucleotide need not be 100~
complementary to its target DNA sequence to be speci~ically hybridizable. An oligonucleotide is speci~ically hybridizable when binding o~ the oligonucleotide to the target RNA or DNA molecule inter~eres with the normal ~unction o~ the target RNA or DNA, and there is a su~icient - degree o~ complementarity to avoid non-speci~ic binding o~
the oligonucleotide to non-target sequences under conditions in which speci~ic binding is desired, i.e. under physiological conditions in the case o~ in vivo assays or therapeutic treatment, or in the case o~ in vi tro assays, under conditions in which the assays are per~ormed.

CA 02223l03 l997-l2-02 WO 96/39154 PCTAJS96'¢~/5 In particular, oligonucleotides of the invention may be used in therapeutics, as diagnostics, and for research as is specified in the following United States patent applications assigned to the assignee of this invention These applications are entitled: Compositions and Methods for Modulating RNA Activity, Serial No. 463,358, filed 1/11/90; Antisense Oligonucleotide Inhibitors of Papilloma Virus, Serial No. 445,196 Filed 12/4/89;
Oligonucleotide Therapies for Modulating the Effects of 10 Herpesvirus, Serial No. 485,297, Filed 2/26/90; Reagents and Methods for Modulating Gene Expression Through RNA Mimicry Serial No. 497,090, Filed 3/21/90; Oligonucleotide Modulation of Lipid Metabolism, Serial No. 516,969, Filed 4/30/90; Oligonucleotides for Modulating the Effects of 15 Cytomegalovirus Infections, Serial No. 568,366, Filed 8/16/90; Antisense Inhibitors of the Human Immunodeficiency Virus, Serial No. 521,907, Filed 5/11/90; Nuclease Resistant Pyrimidine Modified Oligonucleotides for Modulation of Gene Expression, Serial No. 558,806, Filed 7/27/90; Novel 20 Polyamine Conjugated Oligonucleotides, Serial No. 558,663, Filed 7/27/90; Modulation of Gene Expression Through Interference with RNA Secondary Structure, Serial No.
518,929, Filed 5/4/90; Oligonucleotide Modulation of Cell Adhesion, Serial No. 567,286, Filed 8/14/90; Inhibition of 25 Influenza Viruses, Serial No. 567,287, Filed 8/14/90;
Inhibition of Candida, Serial No. 568,672, Filed 8/16/90;
and Antisense Oligonucleotide Inhibitors of Papillomavirus, Serial No. PCT/US90/07067, Filed 12/3/90. These applications disclose a number of means whereby improved modulation of RNA and DNA activity may be accomplished through oligonucleotide interaction. In that the specific sequences disclosed therein may be used in conjunction with the present invention, the disclosures of the foregoing United States patent applications are incorporated herein by reference.
The following examples are illustrative and are not meant to be limiting of the present invention.
-ISOLATION- OF ALL Sp OR ALL Rp 5'-O-(1-THIOTRIPHOSPHATE) N-UCLEOSIDE
- 5'-O-(1-thiotriphosphate) deoxynucleosides and ribonucleosides are isolated using C-18 reverse phase high performance liquid chromatography (HPLC) using columns packed with ODS Hypersil (Shandon Southern, Runcon, UK) and eluted with an isocratic mixture of solvent A (30 mM
potassium phosphate containing 5 mM tetrabutylammonium ion, pH 7.0) and solvent B (5 mM tetrabutylammonuium hydroxide in methanol). Alternatively, effective separation is achieved using 100 mM triethylammonium bicarbonate, pH 7.5, containing a linear gradient of acetonitrile from 0~ to 15 over 20 minutes.
To establish the purity of such HPLC separated enantiomers the HPLC separated Sp and Rp deoxynucleotide enantiomers are compared to commercially available deoxy-nucleoside 5'-O-(1-thiotriphosphates) available from E.I.
Dupont, Wilmington, DE.

~YNl~SIS OF PHOSPHOROTHIOATE ~:X~ ~IoN HAVING SUBSTANTIALLY
ALL Rp IN-TERSUGAR T-TNRA~-C: OF A RACEMIC PHOSPHOROTHIOATB
OLI w Nu~EOTIDE
2S Enzymatic synthesis of an all Rp phosphorothioate extension of a racemic phosphorothioate oligonucleotide primer is effected using the modified T7 DNA polymerase I, Sequenase~ (U.S. Biochemicals Corp, Cleveland, OH). This T7 DNA polymerase is used to extend an 18 mer phosphorothioate - 30 oligonucleotide primer hybridized to a 21-mer natural phosphodiester oligonucleotide. 30 picomoles (pmol) of - primer and template in a lX Sequenase~ reaction buffer (U.S.
Biochemicals Corp., Cleveland, OH) (final vol 10 ~L) are heated for 5 minutes at 95~C and slowly cooled to room temperature. 180 pmol of deoxy 5'-[~-35S] cytidine WO 96~9154 . PCTAUS96/08757 triphosphate and Sequenase~ enzyme (U.S. Biochemicals Corp., Cleveland, OH) are added and incubated at 37~C for 20 minutes. The product is analyzed via polyacrylamide gel electrophoresis (PAGE) using a 20~ polyacrylamide/7 M urea denaturing gel. The autoradiograph of the product is compared to a control reaction absent primer/template. The final product is subjected to further characterization by, for example, enzymatic degradation. One such degradation is snake venom phosphatase degradation. A snake venom phospha-tase degradation of dinucleoside monophosphorothioatesynthesized using E. coli DNA polymerase I shows the dinucleoside to be of the Rp configuration.

~YN-1~SIS OF PHOSPHOROTHIOATE CGACTATGCAAGTAC (SEQ ID NO:13) OLI w NU~EOTIDE HAVING SUBSTANTIALLY PURE Rp IN~1~RSUGAR
T.TrnK~.~
A large scale enzymatic synthesis of sequence specific all Rp phosphorothioate oligonucleotide was effected utilizing a 55-mer natural phosphodiester template and a 41-mer natural phosphodiester primer. The template sequence was:
GTACTTG Q TAGTCGATCGGA~AATAGGGTTCTCATCTCCCGGGATTTGGTTGAG
(SEQ ID NO:14).
The primer sequence was:
CTCAACCA~ATCCCGGGAGATGAGAACCCTATTTTCCGATC (SEQ ID NO:15).
The template was selected to have a sequence complementary to a desired specific CGACTATGCAAGTAC (SEQ ID NO:13) sequence. A Sequenase~ buffer (U.S. Biochemicals Corp., Cleveland, OH) diluted from 5X to lX was used. The template and primer, both at concentrations of 20 nM are added to 40 ~L of this buffer. The template and primer were hybridized at 95~C for 5 minutes and cooled to room temperature. After cooling the buffer was adjusted to 7 mM DTT. 20 ~L of 1:8 diluted Sequenase~ enzyme and 320 ~M each of Sp GTP~S, CTP~S, ATP~S and TTP~S were then added. The reaction solution was adjusted to 140 ~L with H2O. It was incubated at 37~C for 18 hours. The reaction solution was extracted 2X with a like volume of phenol in a standard manner and precipated in a standard manner by treatment with 2.5 volumes of 100~ ethanol at -20~C, peltized, washed with 500 - 5 ~L of 70~ ethanol, peltized again and dried. The precipitate was suspended in 20 ~L H2O for 30 minutes then adjusted to 1 mM CaCl2, 25 mM Tris HCl pH 8.0 in 40 ~L H2O.
The solution was maintained at 95~C for 5 minutes and snap cooled, i.e. very quickly cooled with ice. The template and primer were removed from the synthesized oligonucleotide by the addition of 4.6 ~M DNase I and incubation at 37~C for 10 minutes. The reaction mixture was phenol extracted 2X and precipitated with ethanol as above. The precipate was resuspended in H2O and purfied using 20~ polyacrylamide/7 M
urea gel electrophoresis coupled with SepPak~ chromatography (Millipore, Milford, MA).
In an alternate synthesis, Pst 1 restriction nuclease ILi~e Technologies, Inc., Gaithersburg, MD) was used to cleave the primer-bound phosphorothioate oligonu-cleotide at the restriction site. The desiredCGACTATGCAAGTAC (SEQ ID NO:13) phosphorothioate oligonucleo-tide was purified using polyacrylamide/7 M urea gel electrophoresis coupled with SepPak~ chromatography (Milli-pore, Milford, MA). Yields were optimized using enzymatic cascade effected by repetitive template-primer addition throughout the reaction. The cascade augmented synthesis yielded 75 A260 units of the CGACTATGCAAGTAC (SEQ ID NO:13) all Rp configuration phosphorothioate oligonucleotide from a 20 mL reaction.

- ~YN-l~SIS OF PHOSPHOROTHIOATE OLI~ONu~EOTIDES HAVING A
RACEMIC Ml2~lu~cE OF INTERSUG~R T.Tl~RA~.~.~ USING AUTOMATED DNA
- ~YN l~;SIS .
Oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using hydrogenphosphonate chemistry in a standard manner [Agrawal WO 96~9154 PCT~US96/08757 et al., Proc. Natl. Acad. Sci. U.S.A., 85:7079 (1988)] .
After the final coupling step, the phosphorothioate linkages are generated by oxidizing the bound oligomer with sulfur in carbon disulfide/triethylamine/ pyridine. After sulfur oxidation, standard deblocking procedures with ammonium hydroxide are used to release the oligonucleotides from the support and remove base blocking groups. The phosphorothioate oligonucleotides are purified by oligonucleotide purification column (OPC; ABI, Foster City, CA) chromatography and HPLC, using a Beckman System Gold HPLC. The HPLC-purified oligonucleotides are then precipitated with ethanol and assessed for final purity by gel electrophoresis on 20~ acrylamide/7 M urea or by analytical HPLC. The authenticity of the oligonucleotide sequence was assessed by oxidation with iodine in pyridine/water and standard sequencing methods. These oligonucleotides contain a mixture of all possible combinations of Rp and Sp isomers at each phosphorous linkage.

EXAMPhE 5 ~YN~ SIS OF COMPLEMENTARY DNA OR RNA SE52u~Nc~;S USING T7 R
POLY~R~-~ FOR ~L~IJ~YNAMIC AND KlN~-llC RyRRTnIzATIoN
ANALYSIS.
The synthesis of short complementary DNA
oligonucleotides of natural phosphodiester linkages was performed utilizing s~andard automated synthesis on an ABI
model 380B DNA Synthesizer. The oligonucleotides of correct length were purified by HPLC and sequenced by standard techniques.
T7 RNA polymerase was use for the synthesis of short, complementary RNA oligonucleotides for hybridization analysis. A large amount of T7 RNA polymerase at high concentrations was needed for the many cycles of initiation required to synthesize short RNAs. Due to this requirement, the T7 RNA polymerase was derived from a strain of E. coli that contained a T7 RNA polymerase expression vector, BL21/pAR1219, obtained from Brookhaven National Laboratory (Upton, NY). The isolation yielded approximately 300,000 to 500,000 units of T7 RNA polymerase from 2 L of cells, absorbance value = 1.2 A600. This was sufficiently - 5 concentrated for synthesis of short (10-30 nucleotides) RNA
species. For synthesis, a T7 promoter and a template con-taining the complementary target sequence and T7 promoter hybridization sequence were synthesized using the ABI
synthesizer (ABI, Foster City, CA). Template and promoter were purified by HPLC to ensure that the correct species was present for enzymatic synthesis. Synthesized products were purified on a 20~ polyacrylamide/8 M urea gel and sequenced by standard procedures.

THERMAL DENATURATION
Oligonucleotides (either phosphorothioate oligonucleotides of the invention or otherwise) were incubated with either the complementary DNA or RNA
oligonucleotides at a standard concentration of 4 ~M for each oligonucleotide in 100 mM ionic strength buffer (89.8 mM NaCl, 10 mM Na-phosphate, pH 7.0, 0.2 mM EDTA). Samples were heated to 90~C and the initial absorbance taken using a Guilford Response II spectrophotometer (Corning). Samples were then slowly cooled to 15~C and the change in absorbance at 260 nm monitored during the heat denaturation procedure.
The temperature was elevated 1 degree/absorbance reading and the denaturation profile analyzed by taking the first derivative of the melting curve. Data was also analyzed using a two-state linear regression analysis to determine the Tm and delta G. The results of these tests are shown in Table 1.

CA 02223l03 l997-l2-02 WO 96/39154 PCTAUS96i!~375 T~RLE 1 ~L~ ~L DENATURATION
Sequence SEQ ID NO: Com~lement Natural phosphodiester CGA CTA TGC AAG TAC 13 DNA 53.2 CGA CTA TGC AAG TAC 13 RNA 46.2 Phosphorothioate with racemic intersugar linkages CGA CTA TGC AAG TAC 13 DNA 46.0 10 CGA CTA TGC AAG TAC 13 RNA 3 6.5 Phosphorothioate with chirally pure intersugar linkages CGA CTA TGC AAG TAC 13 DNA 45.5 CGA CTA TGC AAG TAC 13 RNA 41.5 GA CTA TGC AAG TAC 16 DNA 44.5 GA CTA TGC AAG TAC1 16 RNA 40.0 ~SIS OF RADIoT~R~T~n OLI W Nu~EOTIDES
Filter binding assays are utilized to quantitate the binding stringencies of various phosphorothioate oligonucleotides, i.e. their tendencies to hybridize and form heteroduplexes with DNA or RNA. These assays require radiolabeled oligonucleotides.
Phosphorothioate oligonucleotides having all Rp intersugar linkages are synthesized by enzymatic methods from [35S]-monomers that have been purified from Sp monomers.
For automated synthesis of phosphorothioate oligonucleotides cont~;n;ng mixed chirality intersugar linkages, oligonucleotides are synthesized containing hydrogen phosphonates and then sul~urized in the presence of elemental [35S] in a pyridine/carbon disulfide mixture. The resulting radiolabeled phosphorothioate oligonucleotide can be purified by OPC chromatography and HPLC. Target mRNA are applied to nitrocellulose filters and baked at 80~C

W O 96/39154 PCT~US96/08757 for 2 hours, blocked and then hybridized with the radiolabeled phosphorothioate oligonucleotide. Binding stringency is assessed by quantitating radiolabeled oligonucleotide eluted from the filters after increases in - 5 temperature or increases in the ionic strength of an eluting buffer, as for instance, Tris NaCl buffer. Eluted oligonucleotides are also assessed for their mobility in an anion exchange HPLC protocol isocratically utilizing phosphate buffer. Results are compared to the mobility of standard oligonucleotides prepared having racemic mixtures of intersugar linkages.

NU~T.T~-~ DIGESTION
Determination of the rate of nuclease degradation of the phosphorothioate oligonucleotides in media containing 10~ fetal calf serum (FCS) was carried out in Dulbecco's Modified Essential Medium (DMEM) containing 10~ heat inactivated FCS. Heat inactivation of the FCS was carried out at 55~C for 1 hour prior to addition to media.
Oligonucleotides having racemic and chirally pure intersugar linkages were separately tested for resistance to nuclease digestion. 66 ~g/mL of each oligonucleotide were separately added to medium and incubated at 37~C, at the time intervals indicated in Table 2. 15 ~L Aliquots were removed and added to 15 ~L of 9 M urea in 0.1 M Tris-HCl (pH 8.3), 0.1 M boric acid and 2 mM EDTA. Aliquots were mixed by vortex and stored at -20~C. Polyacrylamide gel electrophoresis (PAGE) analysis was on 20~ polyacrylamide/7 M urea slab gels.
Following electrophoresis, gels were stained using "Stains All" (Sigma Chem. Co., St. Louis, MO). Following de-- st~;n;ng, gels were analyzed via laser densitometry using an UltraScan XL device (Pharmacia LKB Biotechnology, Uppsala, - Sweden). Integrations were performed and the data presented as the percentage decrease from full length (n) prior to incubation to n-1. These results are shown in Table 2 for WO 96/39154 PCT~US96/08757 the oligonucleotide sequence CGACTATGCAAGTAC (SEQ ID NO:6) having Rp chirally pure intersugar linkages.

NU~.R~.~R DIGESTION
5Tncllh~tion in 10~ fetal cal~ serum Digestion o~ oligonucleotide o~ length n to length n-1 Time (hours) Phosphorothioate with Phos~horothioat with with racemic intersuqar chirallY ~ure linkaqes intersuaar linkaqes As is evident ~rom Table 2, the phosphorothioate oligonucleotide having substantially chirally pure intersugar linkages showed greater resistance to nuclease degradation than did the phosphorothioate oligonucleotide having racemic intersugar linkages.

RNASE H ANALYSIS
Phosphorothioate oligonucleotides having racemic and substantially chirally pure intersugar linkages were analyzed ~or susceptibility to RNase H. Oligonucleotides (2-~old molar excess to RNA) and 5 ~g (3.1 kb) in vitro synthesized mRNA (using T7 RNA polymerase promoter) were incubated in 5 ~L RNase H hybridization buf~er ~or 30 minutes at 60~C. Samples were slowly cooled to room temperature and then adjusted to 3.7 mg/mL BSA, 20 units E.
coli RNase H (Promega), 142 mM DTT, 150 mM KCl, and 3 mM
MgCl2. Samples were incubated ~or 30 minutes at 37~C.
Samples were then extracted with phenol, precipitated with ethanol, and ana'yzed by electrophoresis on 1.2~ agarose gels ~ollowing ethidium bromide staining. Markers were run on gels concurrently with the samples to determine approximate length o~ RNA samples.

W O 96/39154 - PCT~US96/08757 E~MPLE 10 A patient suffering from hepatitis caused by HCV
is treated with Oligo # 259 (SEQ ID NO:1), Oligo # 260 (SEQ
ID NO:2), Oligo # 270 (SEQ ID NO:3), Oligo # 330 (SEQ ID
NO:4) or Oligo # 340 (SEQ ID NO:5), each of which are synthesized according to the procedure of Example 3 or Example 19. 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

E~MPLE 11 A patient suffering from an inflammatory disease mediated by ICAM-1 is treated with ISIS-2302, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence GCCCAAGCTGGCATCCGTCA
(SEQ ID NO:6). 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly.
Treatment may be repeated as necessary until the disease has been ablated.

EXP~MPLE 12 A patient suffering from retinitis caused by cytomegalovirus is treated with ISIS-2922, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence GCGTTTGCTCTTCTTCTTGCG
(SEQ ID NO:7). 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravitreally. Treatment may be repeated - 30 as necessary until the infection is ablated.

EX~MPLE 13 A patient suffering from PKC-~-mediated cancer is treated with ISIS-3521, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence GTTCTCGCTGGTGAGTTTCA (SEQ ID NO:8). 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

A patient suffering from C-raf kinase-mediated cancer is treated with ISIS-5132, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence TCCCGCCTGTGACATGCATT (SEQ ID NO:9). 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

A patient suffering from C-raf kinase-mediated cancer is treated with ISIS-2503 (SEQ ID NO:10), ISIS-2570 (SEQ ID NO:11) or ISIS-6957 (SEQ ID NO:12), each of which are synthesized according to the procedure of Example 3 or Example 19. 1-1000 ~g/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravenously or intramuscularly. Treatment may be repeated as necessary until the disease has been ablated.

25 Compounds 1, 2 and 3 of Examples 16, 17 and 18, respectively, are synthesized according to the procedure of Stec et al. [Nucleic Acids Res., 19:5883 (1991)] and Stec and Lesnikowski [Methods in Molecular Biology, S. Agrawal, Ed., Volume 20, p. 285, 1993].

~YNl~;SIS OF 2--~ T~oRo-l~ 3, 2--O~T~T~PHOSP~OT.~1~ (1) A mixture of pyridine (1 mol), benzene (400 mL), 2-mercaptoethanol (0.5 mol) and phosphorus trichloride (0.5 WO 96/39154 PCT~US96108757 mol) are stirred at room temperature for 30 minutes.
Pyridinium chloride is filtered off, solvent is evaporated under reduced pressure and crude product is purified by distillation under reduced pressure. The fraction boiling at 70-72~C/20 mm Hg is collected and characterized by 31p NMR.

~SIS OF N,N-DIISOPROPYLAMINO-l,3,2-Ox~T~T~PHOSP~
(2) Compound 1 (0.2 mol) is dissolved in n-pentane (300 mL) and diisopropylamine (0.4 mol) is added dropwise.
The reaction mixture is stirred at room temperature for 30 minutes, after which diisopropylamine hydrochloride is filtered off, solvent is evaporated under reduced pressure and crude product is purified by vacuum distillation.
Product 2 is obtained as the fraction boiling at 70~C/0.1 mm Hg and is characterized by 31p NMR and mass spectroscopy.

~YNl~SIS OF 5'-O-DI~l~u~Yl~ITYLTHYMIDINE-3~-0[2-THIONO-1,3,2-O~T~T~PHOP.~OT.~N~] (3) 5'-O-Dimthoxytritylthymidine (10 mmol) and lH-tetrazole (11 mmol) are vacuum dried and dissolved in dichloromethane (25 mL). Compound 2 (11 mmol) is added to the solution and the reaction mixture is stirred at room temperature for 2 hours. Dried elemental sulfur (15 mmol) is added and the reaction mixture is stirred and left at room temperature for 16 hours. Unreacted sulfur is then filtered off and the filtrate is concentrated under reduced pressure. The residue is dissolved in chloroform (3 mL) and - 30 purified by silica gel (230-400 mesh) column chromatography, eluting first with chloroform, and next with chloroform:methanol (97:3). Individual diastereomeric species of compound 3 are obtained by column chromatography on silica gel. Compound 3 is dissolved in ethyl acetate and applied on a silica gel 60H column. Ethyl acetate is used WO 96~9154 PCT~U596i'~X7 as the eluting solvent, and elution is monitored by HPTLC
(silica gel 60, ethyl acetate as the developing solvent).
Fractions containing separated diastereomers of compound 3 are concentrated under reduced pressure and the residue is characterized by 31p NMR and HPLC (Lichrospher SilOO, 5 ~M, ethyl acetate as eluant, flow rate 3 mL/minute). The fast eluting fraction corresponds to the Sp diastereomer, and the slow eluting fraction is the Rp diastereomer.

STl;R~O~PECIFIC CONTROL OF REACTION ~ W~;~;N 5'-OH Nur~osIDEs YN'l'~SIS
The procedure of Stec et al. was followed using an Applied Biosystems (Foster City, CA) model 380B automated DNA synthesizer. The reaction between a 5'-OH nucleoside and diastereomerically pure nucleoside oxathiaphospholane, such as 3, requires the use of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) as the catalyst. Because the commercially available linker used for the attachment of oligonucleotide to support matrix in automated DNA synthesis is unstable to DBU, a modification is required in the linker. A suitable linker for oligonucleotide synthesis via the oxathiaphospholane method is a "succinic-sarcosinyl" linker that is resistant to DBU, and can be hydrolyzed by concentrated ammonium hydroxide at room temperature in less than 1 hour.

(A) Synthesis of 5'-0-dimethoxytritylnucleosides bound to solid matrix via "succinic-sarcosinyl~ linker:
(1) N-Fmoc-sarcosine (Bachem Bioscience, Inc., Philadelphia, PA) (1.6 mmol) is added to long chain alkyl amine-CPG (LCA-CPG, Sigma, St. Louis, MO) (2 g) and dried under vacuum. Anhydrous DMF (5 mL), pyridine (0.5 mL) and DCC (2.4 mmol) are added and the reaction mixture is shaken at room temperature for 12 hours. The solvent is then filtered off and the support is washed with W O 96/39154 PCT~US96/08757 methanol:acetonitrile:pyridine (1:1:1, 3X20 mL). The N-Fmoc protecting group is removed by treating the support with 10 mL o~ a 10~ solution o~ piperidine in pyridine. N-sarcosinylated LCA-CPG is washed with methanol:acetonitrile:pyridine (1:1:1, 3x20 mL) and dried under vacuum.
(2) 5'-O-Dimethoxytritylnucleoside is added to the sarcosinylated LCA-CPG obtained as described in (1) in the presence o~ DMF (2 mL), pyridine ( 0.2 mL) and DCC (50 mg). The reaction mixture is shaken at room temperature for 12 hours and then washed with methanol:acetonitrile:pyridine (1:1:1, 3x20 mL). A~ter drying, the support is treated with N-methylimidazole:THF (1 mL) and acetic anhydride/lutidine (1 mL) for 15 minutes. The support is then washed with methanol:acetonitrile:pyridine (1:1:1, 3XlO mL), ~ollowed by acetonitrile (3xlO mL), and then dried under vacuum.
(B) Diastereomerically pure activated nucleosides are subsequently added onto the oligonucleotide attached to the sarcosinyl LCA-CPG support in the presence o~ a 300-~old molar excess o~ DBU. The diastereomers o~ activated nucleosides are separated by column chromatography [silica gel 60H, ethyl acetate is used as the eluting solvent, elution is monitored by HPTLC (silica gel 60, ethyl acetate as the developing solvent)] prior to use in the coupling reaction. The synthetic protocol is shown in Table 3.

CA 02223l03 l997-l2-02 Table 3 Chemical steps ~or one synthesis cycle Reaaent or solventPurDose Time (minutes) 5 Trichloroacetic acid in Detritylation 1.5 dichloromethane (2:9 8) Acetonitrile Wash 2 Activated nucleoside (with Coupling 10 DBU) in acetonitrile 10 Acetonitrile Wash 2 Acetic anhydride/lutidine Capping in THF (1:1:8) and N-methylimidazole in THF
(4:21) 15 Acetonitrile Wash The diastereomeric purity o~ the phosphorothioate oligonucleotide can be determined by 31p NMR, by HPLC
(Lichrospher SilOO, 5 ~M, ethyl acetate as eluant, ~low rate 3 mL/minute), enzymatically or by electrophoretic methods.

EX~MPLE 20 TR~AT~DENT OF A DT~F~F STATE IN A H~n~AN PATI ~ T
The oligonucleotides of the invention may be used for treatment o~ various disease states. Treatment o~ a patient diagnosed with a particular disease state comprises administration oi~ an e~fective dose o~ the olignucleotide, in a pharmaceutically accepted ~ormulation, to the patient via an appropriate route. The e~ective oligonucleotide dose depends on the disease state being treated, the severity o~ the disease state and the age o~ the patient being treated. The e~ective dose of an oligonucleotide may be determined based on its IC50 and is a routine procedure ~or one of skill in the art. Alternatively, the e~fective dose o~ the oligomer may be determined by using the pharmacokinetics software program TopFit. For example, dosage o~ oligonucleotides may vary ~rom O.O1 ~g (~or WO 96~9154 PCT~US96/08757 ~ - 39 -children) to 100 g (for adults) per kg of body weight depending on progression of the disease state. Similarly, the frequency of dosing depends on the progression of the disease state and may vary from once or more daily to once every 20 years.
The route of oligonucleotide administration depends on the disease state being treated. For example, administration of an oligonucleotide to a patient being treated for an inflammatory disorder may be accomplished either via oral or rectal routes. For treatment of a patient afflicted with AIDS, the most effective method of oligonucleotide administration may be an oral route or by subcutaneous injection. Cancers such as breast cancer may be treated via subcutaneous injection, while colon cancer may be treated via oral or rectal administration of the oligonucleotide. Diseases or disorders of the central nervous system may best be treated by intrathecal or intraventricular administration for delivery of the oligonucleotide to the spinal column or the brain of the patient.
Following oligonucleotide administration, the patient may be monitored for alleviation of symptoms associated with the disease state. Subsequently, the dosage may be adjusted (increased or decreased) depending upon the severity and ~m~n~hility of the disease state to treatment.
It may be preferable to administer oligonucleotides of the invention in combination with other traditional therapeutics. The oligonucleotides may be administered in combination with drugs including, but not limited to, AZT
for the treatment of patients afflicted with AIDS, sulfasalazine for the treatment of an inflammatory disorder such as ulcerative colitis, and 5-fluorouracil for the treatment of colon cancer.
Also, it may be desirable to administer maintenance therapy to a patient who has been successfully treated for a disease state. The dosage and frequency of oligonucleotide administration as part of a maintenance regimen may vary CA 02223l03 l997-l2-02 WO 96~9154 PCTrUS96/08757 from 0.01 ~g to 100 g per kg of body weight, ranging from once or more daily to once every several years.

INTRAVENTRICULAR ADMINISTRATION OF OLI~Nu~Oll~S
Intraventricular drug administration, for the direct delivery of drug to the brain of a patient, may be desired for the treatment of patients with diseases afflicting the brain. To effect this mode of oligonucleotide administration, a silicon catheter is surgically introduced into a ventricle of the brain of a human patient, and is connected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis, MN) that has been surgically implanted in the abdominal region [ Cancer ~esearch, 44:1698 (1984)]. The pump is used to inject the oligonucleotides and allows precise dosage adjustments and variation in dosage schedules with the aid of an external programming device. The reservoir capacity of the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h. Depending on the frequency of i~rlm;n; stration, ranging from daily to monthly, and the dose of drug to be administered, ranging from 0.01 ~g to 100 g per kg of body weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump.

INTR~TR~ Q L ADMINISTRATION- OF OLIGONUCLEOTIDES
Intrathecal drug administration for the introduction of drug into the spinal column of a patient may be desired for the treatment of patients with diseases of the central nervous system. To effect this route of oligonucleotide administration, a silicon catheter is surgically implanted into the L3-4 lumbar spinal interspace of a human patient, and is connected to a subcutaneous infusion pump which has been surgically implanted in the CA 02223l03 l997-l2-02 W O 96~9154 PCTAUS96/08757 upper abdominal region [ The Annals of Pharmacotherapy, 27:912 (1993) and Cancer, 41:1270 (1993]. The pump is used to inject the oligonucleotides and allows precise dosage adjustments and variations in dose schedules with the aid of an external programming device. The reservoir capacity o~
the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/h to 1 mL/h. Depending on the frequency of drug administration, ranging from daily to monthly, and dosage of drug to be administered, ranging from 0.01 ~g to 100 g per kg of body weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by a single percutaneous puncture to the self-sealing septum of the pump.

A patient suffering from AIDS is treated with ISIS-5320, an oligonucleotide synthesized according to Example 3 or Example 19, and having the sequence TTGGGGTT (SEQ ID
NO:17). 1-1000 ~Lg/kg body weight of oligonucleotide is incorporated into a pharmaceutically acceptable carrier and administered intravitreally. Treatment may be repeated as necessary until the infection is ablated.

CA 02223l03 l997-l2-02 -~u~-~ LISTING
(1) GENER~L INFORMATION:
(i) APPLICANTS: ISIS Phar~lce~ticals, Inc.,et al.
(ii) TITLE OF lNv~NllON: Oligonucleotides Having Phosphorothioate Linkages Of High Chiral Purity (iii) NUMBER OF ~yU~N~S: 17 (iV) C~ ON1~'N~ ; Anm?~.qs:
~'A' Ann~.qs~: Woodcock Washburn Kurtz Mackiewicz ~ Norris B STREET: one Liberty Place - 46th Floor ~C~ CITY: Ph;l~ h;~
~D STATE: PA
~E~ C~UN'L~Y: U . S . A.
,F, ZIP: 19103 (v) COMPUTER READABLE FORM:
~A) MEDIUM TYPE: 3.5 inch disk, 720 Kb ~:B) COMPUTER: IBM PC compatible ~C) OPERATING SYSTEM: PC-DOS/MS-DOS
~D) SOFTWARE: WordPerfect 6 1 (Vi ) ~UKR~'N'L APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/08757 (B) FILING DATE: 05-JUN-1996 (C) CLASSIFICATION: N/A
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/471,967 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/467,597 (B) FILING DATE: 06-~UN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/468,447 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/468,569 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/466,692 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/471,966 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/469,851 (B) FILING DATE: 06-JUN-1995 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:08/470,129 (B) FILING DATE: 06-JUN-1995 (viii) Al l'~KN~ Y /AGENT INFORMATION:
(A) NAME: Joseph Lucci (B) REGISTRATION NUMBER: 33,307 (C) K ~ K N~/DOCKET NUMBER: ISIS-2298 S~il 111 ITE SHEET (P~U~

CA 02223l03 l997-l2-02 W 0 96/39154 PCTrUS96/08757 (ix) TFT~RC~MMUNICATION INFORMATION:
(A) TELEPHONE: 215-568-3100 (B) TELEFAX: 215-568-3439 (2) INFORMATION FOR SEQ ID NO:1:
( i ) ~yU~N~ CHARACTERISTICS:
'A' LENGTH: 20 base pairs ~B TYPE: nucleic acid .C~ STRAN~ )N~ S: single ;D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~yu~:Nc~ DESCRIPTION: SEQ ID NO:1:
C~LLlC~CGA CCCAACACTA 20 (2) INFORMATION FOR SEQ ID NO: 2:
( i ) S~YU~N~ CHARACTERISTICS:
lA LENGTH: 20 ~B TYPE: nucleic acid C~ STRAN~ N~:~S: single ,D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:2:
GC~l-l-LCQCG ACCCAACACT 20 (2) INFORMATION FOR SEQ ID NO: 3:
( i ) ~yU~N~ CHARACTERISTICS:
A'~ LENGTH: 20 Bl TYPE: nucleic acid C STRAN~ N~S: single ~,D, TOPOLOGY: linear ( i i ) MoT ~F-CTTT ~F TYPE: DNA (genomic) (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:3:
GTACCACAAG GC~lllCGCG 20 (2) lN~ 0~ ~ATION FOR SEQ ID NO: 4:
( i ) ~yU~N~ CHARACTERISTICS:
~A~ LENGTH: 20 ~B~ TYPE: nucleic acid ~C~ STRAN~ N~:~S: single ~,D, TOPOLOGY: linear (ii) MnT~R~JT~T2 TYPE: DNA (genomic) (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO: 5:
( i ) ~yU~N~ CHARACTERISTICS:
~A~ LENGTH: 20 B TYPE: nucleic acid ~,C~ ST~N~ )N~ S: single ~DJ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi ) ~yU~:N~ DESCRIPTION: SEQ ID NO:5:

TTTAGGATTC ~l~l~ATGG 20 (2) INFORMATION FOR SEQ ID NO: 6:
( i ) ~yU~N~ CHARACTERISTICS:
~A~ LENGTH: 20 ~B, TYPE: nucleic acid ~C STR~N~ N~:~S: single ;D) TOPOLOGY: linear (ii) M~T~FCUT~F TYPE: DNA (genomic) SUb~ill I lJTE SHEET (RULE 2B) WO 96/39154 PCT~US96/08757 (xi) ~yu~:N~ DESCRIPTION: SEQ ID NO:6:
GCCCAAGCTG GCAl CC~L~A 20 (2) INFORMATION FOR SEQ ID NO: 7:
(i) ~yu~N-~: CHARACTERISTICS:
~A:~ LENGTH: 2l B TYPE: nucleic acid C STRPN~ JN~:C,c: single ~:D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:7:
GC~lllG~l~' 1 L~l lUL l~C G 2l (2) INFORMATION FOR SEQ ID NO: 8:
(i) S~YU~N~ CHARACTERISTICS:
'A' LENGTH: 20 B TYPE: nucleic acid C:~ STRAN1)~:l)N~:CS: single ~,D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:8:
~1' L cLCGCTG GTGAGTTTCA 20 (2) INFORMATION FOR SEQ ID NO: 9:
(i) ~yu~N~ CHARACTERISTICS:
(A~ LENGTH: 20 B l TYPE: nucleic acid C~ STR~N~ ) N ~: C s: single ,DJ TOPOLOGY: linear (ii) MOT~RCUT~ TYPE: DNA (genomic) (xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO: lO:
(i) ~Qu~Nch CHARACTERISTICS:
~A,~ LENGTH: 20 B TYPE: nucleic acid C~ STR~N~ N~-~.C: single ~D,~ TOPOLOGY: linear (ii) M~T~R~T.R TYPE: DNA (genomic) (xi) ~yU~N~ DESCRIPTION: SEQ ID NO:lO:
LCC~-L~ATCG ~LC~l~GGG 20 (2) INFORMATION FOR SEQ ID NO: ll:
(i) ~yu~N~ CHARACTERISTICS:
~A LENGTH: l7 B TYPE: nucleic acid ~C~ ST~N~ )N~:-C~: single ~D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:ll:
cc~rcr~r GGCGCCC 17 (2) LN~O~L~TION FOR SEQ ID NO: 12:
(i) ~UU~N~ CHARACTERISTICS:
A' LENGTH: 20 B l TYPE: nucleic acid C~ STR~N~ N~:cS: single ~D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RU1 E 26~
-CA 02223l03 l997-l2-02 WO 96139154 PCT~US96/08757 (xi) S~yu~N~ DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO: 13:
( i ) S~yU~NC~ CHARACTERISTICS:
(A LENGTH: 15 ~B TYPE: nucleic acid ~C STRAN~ )N~ q: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO: 14:
( i ) ~yU~NC~ CHARACTERISTICS:
'A LENGTH: 56 B. TYPE: nucleic acid TT~ ~NI)~:~)N~:qS: single ~,D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO: 15:
(i) ~yU~N~ CHARACTERISTICS:
~ ~A'~ LENGTH: 41 ~B TYPE: nucleic acid ~C~ STT~NnT~nN~S single D, TOPOLOGY: linear (ii) M~T.R~JT.~ TYPE: DNA (genomic) (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:15:
CTCAACCAAA lCCCGG~AGA TGAGAACCCT AlllLCC~AT C 41 (2) INFORMATION FOR SEQ ID NO: 16:
(i) ~yU~N~ CHARACTERISTICS:
(A LENGTH: 14 :B TYPE: nucleic acid C STRANn~nNR~S: single D, TOPOLOGY: linear (ii) MOT.T~JT.T~ TYPE: DNA (genomic) (Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO: 17:
( i ) ~yU~N~ CHARACTERISTICS:
~'A' LENGTH: 8 B TYPE: nucleic acid C~ STR~NI-~:l)N~:cs: single ~D~ TOPOLOGY: linear (ii) ~T- - ~TT-T' TYPE: DNA (genomic) (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:17:
~ LL~GG~l-L 8 S I~STITU~E SHEEr (RULE 2~)

Claims (6)

1. An oligonucleotide represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:17 wherein at least 75% of the nucleoside units are joined together by Sp phosphorothioate 3' to 5' linkages.
2. The oligonucleotide of claim 1 wherein all of the nucleoside units are joined together by Sp phosphorothioate 3' to 5' linkages.
3. A composition containing an oligonucleotide of claim 1 and an acceptable carrier.
4. An oligonucleotide represented by SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:17 wherein at least 75% of the nucleoside units are joined together by Rp phosphorothioate 3' to 5' linkages.
5. The oligonucleotide of claim 4 wherein all of the nucleoside units are joined together by Rp phosphorothioate 3' to 5' linkages.
6. A composition containing an oligonucleotide of claim 4 and an acceptable carrier.
CA 2223103 1991-10-15 1996-06-05 Oligonucleotides having phosphorothioate linkages of high chiral purity Abandoned CA2223103A1 (en)

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US08/471,966 1995-06-06
US08/469,851 US5587361A (en) 1991-10-15 1995-06-06 Oligonucleotides having phosphorothioate linkages of high chiral purity
US08/471,966 US5661134A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating Ha-ras or Ki-ras having phosphorothioate linkages of high chiral purity
US08/467,597 1995-06-06
US08/468,447 US5576302A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating hepatitis C virus having phosphorothioate linkages of high chiral purity
US08/470,129 1995-06-06
US08/466,692 1995-06-06
US08/467,597 US5607923A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating cytomegalovirus having phosphorothioate linkages of high chiral purity
US08/468,447 1995-06-06
US08/469,851 1995-06-06
US08/468,569 US5620963A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating protein kinase C having phosphorothioate linkages of high chiral purity
US08/471,967 US5599797A (en) 1991-10-15 1995-06-06 Oligonucleotides having phosphorothioate linkages of high chiral purity
US08/468,569 1995-06-06
US08/470,129 US5635488A (en) 1991-10-15 1995-06-06 Compounds having phosphorodithioate linkages of high chiral purity
US08/471,967 1995-06-06
US08/466,692 US5654284A (en) 1991-10-15 1995-06-06 Oligonucleotides for modulating RAF kinase having phosphorothioate linkages of high chiral purity

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