Inverted chimeric and hybrid oligonucleotides

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DESCRIPTION

Chimeric oligonucleotides and inverted hybrids.

Background of the invention

Field of the invention

The invention relates to modified oligonucleotides that are useful for the study of gene expression and for antisense therapeutic strategy.

Summary of related matter

The possibilities of using oligonucleotides as inhibitors of the expression of specific genes in an antisense therapeutic strategy were first suggested in three articles published in 1977 and 1978. Paterson et al., Proc. Natl. Acad. Sci. USA. 74: 4370-4374 (1977) describes how translation of mRNA in cell-free medium can be inhibited by the binding of a complementary oligonucleotide to the mRNA. Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75: 280-284 and 285-288 (1978) describe how a 13-unit synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome can inhibit RSV replication in infected cell cultures and can inhibit RSV-mediated transformation of chicken primary fibroblasts into malignant sarcoma cells.

Since these first studies, the ability of antisense oligonucleotides to inhibit the spread of viruses has been firmly demonstrated. US Patent No. 4,806,463 describes the inhibition of the spread of the human immunodeficiency virus by oligonucleotides that are complementary to any of the various regions of the HIV genome. The US patent No. 5,194,428 describes the inhibition of influenza virus replication by oligonucleotides with phosphorothioate linkages complementary to the influenza virus polymerase 1 gene. Agrawal reviews, in Trends in Biotechnology 10: 152-158 (1992), of the use of antisense oligonucleotides as antiviral agents.

Antisense oligonucleotides have also been developed as antiparasitic agents. PCT Publication No. WO93 / 13740 describes the use of antisense oligonucleotides to inhibit the spread of drug resistant malaria parasites. Tao et al., Antisense Research and Development 5: 123-129 (1995), describe the inhibition of the spread of a schistosome parasite by antisense oligonucleotides.

More recently, antisense oligonucleotides have turned out to be promising candidates in therapeutic applications for diseases caused by cellular gene expression. PCT Publication No. WO95 / 09236 describes the neutralization of morphological abnormalities, caused by beta-amyloid in neuronal cell lines, by oligonucleotides that inhibit beta-amyloid expression. PCT Publication No. WO94 / 26887 describes the neutralization of aberrant splicing of a transcript of a globin gene by oligonucleotides complementary to certain portions of said transcript. PCT application no. PCT / US94 / 13685 describes the inhibition of tumorogenicity by oligonucleotides complementary to the gene encoding DNA methyltransferase.

The development of various antisense oligonucleotides as therapeutic and diagnostic agents has recently been reviewed by Agrawal and Iyer, Current Opinion in Biotechnology 6: 12-19 (1995).

With increasing interest in antisense therapeutic strategy, various efforts have been made to improve the pharmacokinetic properties of oligonucleotides by modifying the sugar-phosphate backbone. US Patent No. 5,149,797 describes traditional chimeric oligonucleotides having a phosphorothioate backbone interposed between methylphosphonate or phosphoramidate flanking regions. PCT Publication No. WO94 / 02498 describes traditional hybrid oligonucleotides having 2'-O-substituted ribonucleotide regions flanking a major DNA region.

Many discoveries are currently being made about the pharmacodynamic properties of oligonucleotides. Agrawal et al., Clinical Pharmacokinetics 28: 7-16 (1995) and Zhang et al., Clinical Pharmacology and Therapeutics 58: 44-53 (1995) describe the pharmacodynamics of anti-HIV oligonucleotides in human patients. Some of these new discoveries have made it possible to overcome new obstacles to optimizing oligonucleotides as therapeutic agents. For example, Kniep et al., Nature 374: 546-549 (1995) describes the in vivo mitogenic effect of oligonucleotides containing the CG dinucleotide flanked by certain sequences. Galbraith et al., Antisense Research and Development 4: 201-206 (1994) describe the activation of complement by oligonucleotides. Henry et al., Pharm. Res. 11: PPDM8082 (1994) describes possible oligonucleotide interference with blood coagulation. WO-A-94/08003, WO-A-95/02069 and WO-A-93/13114, as well as J. Biol. Chem. Vol. 268 (19), pp. 14514-22 (1993) describe oligonucleotides with a phosphorothioate internucleoside linkage region, flanked on both sides by 2'-O-substituted nucleoside regions.

Consequently, modified oligonucleotides are needed which retain the properties of inhibiting gene expression while producing fewer side effects than conventional oligonucleotides.

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Brief summary of the invention

The invention relates to modified oligonucleotides that are useful for gene expression studies and for antisense therapeutic strategy. The invention provides modified oligonucleotides that inhibit gene expression and produce fewer side effects than conventional oligonucleotides. In particular, the invention provides modified oligonucleotides that exhibit reduced mitogenicity, reduced complement activation, and reduced antithrombotic properties, compared to conventional oligonucleotides.

In a first aspect, the invention provides inverted hybrid oligonucleotides and material compositions for inhibiting the expression of specific genes with fewer side effects. Such inhibition of gene expression can be used as an alternative method to mutant analysis to determine the biological function of specific genes. Said inhibition of gene expression can also be used for the therapeutic treatment of diseases caused by the expression of the genes of a virus or a pathogen, or by the inappropriate expression of cellular genes.

The invention relates to inverted hybrid oligonucleotides having a 2'-O-substituted RNA region of 4-13 nucleosides between two regions, of oligodeoxyribonucleotides with phosphorothioate linkages, of 5-46 oligodeoxyribonucleosides. According to the invention, the 2'-O substituted RNA region lies between two oligodeoxyribonucleotide regions with phosphorothioate linkages, a structure that is "inverted" compared to traditional hybrid oligonucleotides.

The invention provides a method for the preparation of a medicament for modulating gene expression in a mammal, with fewer side effects. In the method according to this aspect of the invention, a composition of material according to the invention is administered to the mammal, wherein the oligonucleotide is complementary to a gene that is expressed in the mammal. Following administration of the material composition, one or more biological effect measurements selected from the group consisting of complement activation, mitogenesis, and inhibition of clot formation by thrombin are performed.

The invention provides a method for the preparation of a medicament for the therapeutic treatment, with fewer side effects, of a disease caused by aberrant gene expression, in which the material composition according to the invention is administered to an individual having the disease , wherein the oligonucleotide is complementary to an aberrantly expressed gene, wherein said aberrant expression causes disease. In this context, aberrant gene expression means expression in a host organism of a gene necessary for the propagation of a virus or a prokaryotic or eukaryotic pathogen, or the inappropriate expression of a host cellular gene. Inappropriate gene expression in the host cell includes the expression of a mutant allele of a cellular gene, or the under-expression or overexpression of a normal allele of a cellular gene, said disease being caused by said inappropriate gene expression in the host cell. Following administration of the material composition, one or more biological effect measurements selected from the group consisting of complement activation, mitogenesis, and inhibition of clot formation by thrombin are performed.

Brief description of the drawings

Figure 1 shows the inverted hybrid oligonucleotides, hybrid oligonucleotides and oligonucleotides with phosphodiester and phosphorothioate linkages used in the present studies. 2'-O-methylribonucleotides are outlined and nucleotides with phosphodiester bonds are underlined; the remainder are nucleotides linked with phosphorothioate bonds.

Comparative Figure 2 shows the mixed backbone, chimeric and inverted chimeric oligonucleotides used in the present studies. Nucleotides linked by methylphosphonate bonds are underlined; all the remainder are nucleotides linked with phosphorothioate bonds.

Figure 3 shows thymidine uptake by mouse splenocytes as a function of the concentration of phosphorothioate-linked oligonucleotides or any of the various inverted hybrid oligonucleotides.

Figure 4 shows the degree of complement-mediated inhibition of hemolysis observed when serum is treated with phosphorothioate-linked oligonucleotides or any of several inverted hybrid oligonucleotides.

Figure 5 shows the prolongation of aPTT obtained when normal human serum is treated with oligonucleotides with phosphorothioate linkages or with any of several inverted hybrid oligonucleotides.

Comparative Figure 6 shows thymidine uptake by mouse splenocytes as a function of the concentration of phosphorothioate-linked oligonucleotides or any of the various inverted chimeric oligonucleotides.

Comparative Figure 7 shows the degree of inhibition of complement-mediated hemolysis observed when serum is treated with phosphorothioate-linked oligonucleotides or any of several inverted chimeric oligonucleotides.

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Comparative Figure 8 shows the prolongation of aPTT obtained when normal human serum is treated with oligonucleotides with phosphorothioate linkages or with any of the various inverted chimeric oligonucleotides. Detailed description of the preferred embodiments

The invention relates to modified oligonucleotides that are useful for gene expression studies and for antisense therapeutic strategy. The invention provides modified oligonucleotides that inhibit gene expression and produce fewer side effects than conventional oligonucleotides. In particular, the invention provides modified oligonucleotides that exhibit reduced mitogenicity, reduced complement activation, and reduced antithrombotic properties, compared to conventional oligonucleotides.

In a first aspect, the invention provides inverted hybrid oligonucleotides and material compositions for the inhibition of the expression of specific genes, with fewer side effects. Such inhibition of gene expression can be used as an alternative method to mutant analysis or gene "knockout" experiments to determine the biological function of specific genes. Said inhibition of gene expression can also be used for the therapeutic treatment of diseases caused by the expression of the genes of a virus or a pathogen, or by the inappropriate expression of cellular genes.

A composition of material for the inhibition of the expression of specific genes with fewer side effects comprises, according to this aspect of the invention, a modified oligonucleotide that is complementary to a portion of a genomic region or gene of which it is desired to inhibit the expression, or an RNA transcript obtained from said gene. The term "oligonucleotide" encompasses those polymers that have chemically modified bases or sugars and / or that have additional substituents, including, but not limited to, lipophilic groups, intercalating agents, diamines, and adamantane. Preferably, said oligonucleotides will have 5 to 50 nucleotides, most preferably 17 to 35 nucleotides. The term "complementary" means that it has the ability to hybridize with a genomic region or gene, or with its RNA transcript under physiological conditions. Such hybridization is usually the result of hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other forms of hydrogen bonding may also result in hybridization, as well as the stacking of bases. In practice, such hybridization can be deduced by observing the inhibition of the expression of specific genes. The sequence of the gene or the sequence of the RNA transcript, to which the sequence of the modified oligonucleotide is complementary, will depend on the biological effect that it is sought to modify. In some cases, the genomic region or gene, or its RNA transcript may come from a virus. Preferred viruses include, but are not limited to, human immunodeficiency virus (type 1 or 2), influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus, cytomegalovirus, respiratory virus. syncytium, influenza virus, hepatitis B virus, hepatitis C virus, and papilloma virus. In other cases, the genomic region or gene, or its RNA transcript may be derived from endogenous mammalian (including man) chromosomal DNA. Preferred examples of such regions, genomic genes, or their RNA transcripts include, but are not limited to, sequences encoding vascular endothelial growth factor (VEGF), beta-amyloid, DNAmethyltransferase, protein kinase A, ApoE4 protein. , p-glycoprotein, c-MYC protein, BCL-2 protein and CAPL. In still other cases, the genomic region or gene, or its RNA transcript may come from a prokaryotic or eukaryotic pathogen, including, but not limited to, Plasmodium falciparum, Plasmodium malarie, Plasmodium ovale, Schistosoma spp. and Mycobacterium tuberculosis.

In addition to the modified oligonucleotide according to the invention, the composition of material for the inhibition of gene expression, with fewer side effects, may optionally contain any of the well known pharmaceutically acceptable excipients or diluents. This composition of material may further contain one or more additional oligonucleotides according to the invention, said additional oligonucleotides may be an inverted hybrid oligonucleotide or an inverted chimeric oligonucleotide. Alternatively, this composition may contain one or more traditional antisense oligonucleotides, such as a phosphorothioate-linked oligonucleotide, a hybrid oligonucleotide, or a chimeric oligonucleotide, or it may contain any other pharmacologically active agent.

According to the invention, the composition of material comprises inverted hybrid oligonucleotides having a 4-13 nucleoside 2'-O-substituted RNA region between two oligodeoxyribonucleotide regions with 5-46 deoxyribonucleotide phosphorothioate linkages. The 2'-O-substituted RNA region is between two oligodeoxyribonucleotide regions with phosphorothioate linkages, a structure that is "inverted" compared to traditional hybrid oligonucleotides. Therefore, the oligonucleotides according to the invention are called inverted hybrid oligonucleotides. The 2'-O-substituted RNA region preferably has 4 to 13 2'-O-substituted nucleosides linked together by 5 'to 3' internucleoside linkages. Preferably, the total size of the inverted hybrid oligonucleotide will be 15 to 35 or 50 nucleotides. Most preferably, the 2'-O-substituted ribonucleosides are linked together via a 5 'to 3' phosphorothioate, phosphotriester, or phosphodiester linkage. For the purposes of the invention, the term "2'-O-substituted" means the substitution of the 2'-position of the pentose group with a -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein said alkyl, aryl or allyl group may be unsubstituted or may be substituted, for example, with halo, hydroxy, trifluoromethyl, cyano, nitro groups , acyl, acyloxy, alkoxy, carboxyl, carbalkoxy or amino; but not with a 2'-H group. The phosphorothioate flanked region (s) have 5 to 46 nucleosides linked to each other by 5 'to 3' phosphorothioate linkages, and preferably 5 to 26 such nucleosides linked by phosphorothioate linkages. Most preferably, the phosphorothioate regions have 5 to 15 nucleosides linked with phosphorothioate linkages. The

ES 2 201 198 T3 phosphorothioate linkages can be mixed Rp and Sp enantiomers, or they can be stereoregular or substantially stereoregular in Rp or Sp form (see Iyer et al., Tetrahedron Asymmetry 6: 1051-1054 (1995)).

The 2'-O-substituted ribonucleotide regions can include a nonionic internucleoside linkage or be formed entirely by nonionic internucleoside linkages. Furthermore, the oligonucleotides according to the invention may contain combinations of one or more 2'-O-substituted ribonucleotide regions and one or more non-ionic regions. (See Nucleosides & Nucleotides 14: 1031-1035 (1995) for relevant synthesis techniques.)

In a second aspect, the invention provides a method for the preparation of a medicament for modulating gene expression in a mammal, with fewer side effects. In the method according to this aspect of the invention, a composition of material according to the invention is provided for administration to the mammal, wherein the oligonucleotide is complementary to a gene that is expressed in the mammal. Preferably, said administration can be parenteral, oral, intranasal or intrarectal. Following administration of the material composition, one or more biological side effect measurements selected from the group consisting of complement activation, mitogenesis, and inhibition of clot formation by thrombin are made.

In a third aspect, the invention provides a method for the preparation of a drug for the therapeutic treatment, with fewer side effects, of a disease caused by aberrant gene expression, the method comprising supplying a composition of material, according to the invention , for administration to an individual with the disease, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein said aberrant expression causes the disease. In this context, aberrant gene expression means expression in a host organism of a gene necessary for the propagation of a virus or a prokaryotic or eukaryotic pathogen, or inappropriate expression of a host cellular gene. Inappropriate gene expression in the host cell includes the expression of a mutant allele of a cellular gene, or the under-expression or overexpression of a normal allele of a cellular gene, such that the disease is caused by such inappropriate gene expression in the host cell. . Preferably, said administration should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal. Administration of the therapeutic compositions can be accomplished using known procedures with dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered systemically, the therapeutic composition is preferably administered in a dosage sufficient to achieve a blood level of the oligonucleotide of from about 0.01 micromolar to about 10 micromolar. For localized administration, much lower concentrations than these can be effective, and much higher concentrations can be tolerated. Preferably, a total dosage of the oligonucleotide will range from about 0.1 mg of oligonucleotide per patient per day to about 200 mg of oligonucleotide per kg of body weight per day. It may be desirable to simultaneously or sequentially administer a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual, as a single episode of treatment. Following administration of the material composition, one or more biological effect measurements selected from the group consisting of complement activation, mitogenesis, and inhibition of clot formation by thrombin are performed.

The following examples serve to further illustrate certain preferred embodiments of the invention and are not intended to limit the scope of the invention.

Example 1

Synthesis, deprotection and purification of oligonucleotides

Oligonucleotides with phosphorothioate linkages were synthesized using an automated DNA synthesizer (Model 8700, Biosearch, Bedford, MA.) Using beta-cyanoethylphosphoramidite techniques on a 10 micromole scale. To generate the phosphorothioate linkages, the intermediate phosphite linkage obtained after each coupling was oxidized using 3H-1,2-benzodithiol-3H-one-1,1-dioxide (See Beaucage, in Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Agrawal (editor), Humana Press, Totowa, NJ, pp. 33-62 (1993).) A similar synthesis was used to generate the phosphodiester linkages, except that a standard oxidation was performed using a standard iodine reagent. The inverted hybrid oligonucleotides were synthesized in a similar way, except that the segment containing the 2'-O-methylribonucleotides was prepared using 2'-O-methylribonucleosides with phosphoramidite bonds, followed by oxidation, to form a phosphorothioate bond or phosphodiester, as described above. The deprotection and purification of oligonucleotides was carried out according to a standard procedure, (See Padmapriya et al., Antisense Res. & Dev. 4: 185-199 (1994)),

Example 2

Reduction of complement activation in vitro by inverted hybrid oligonucleotides

To determine the relative effect of the inverted hybrid structure on oligonucleotide-mediated complement depletion, the following experiments were performed. Venous blood was drawn from healthy adult human volunteers. Serum for complement hemolytic activity assay was prepared by collecting blood in vacuum flasks (Becton Dickinson # 6430 Franklin Lakes, NJ) without commercial additives. Blood was allowed to clot at room temperature for 30 minutes, chilled on ice for 15 minutes, then centrifuged at 4 ° C to separate serum. The collected serum was kept on ice, if the test was carried out on the same day, or alternatively5

ES 2 201 198 T3 was stored at -70 ° C. The buffer, the phosphorothioate-linked oligonucleotide or the inverted hybrid oligonucleotide were then incubated with the serum. A standard CH50 assay was performed (see Kabat and Mayer (eds): Experimental Immunochemistry, 2nd Edition, Springfield, IL., CC Thomas (1961), p. 125) for complement-mediated lysis of sheep erythrocytes (Colorado Serum Co.) primed with antibody to sheep erythrocytes (hemolysin, Diamedix, Miami, FL.), Using duplicate determinations of at least five dilutions of each test serum, and then measuring the release of hemoglobin in the cell-free supernatants by spectrophotometry at 541 nm.

Example 3

Reduction of the in vitro mitogenicity of inverted hybrid oligonucleotides

To determine the relative effect of the inverted hybrid structure on oligonucleotide-mediated mitogenicity, the following experiments were performed. The spleen was removed from a male CD1 mouse (4-5 weeks, 20-22 g; Charles River, Wilmington, MA.). Single cell suspensions were prepared by carefully picking tissue with the ground edges of a slide. The cells were then cultured in complete RPMI medium [RPMI medium enriched with 10% fetal bovine serum (FBS), 50 micromolar 2-mercaptoethanol (2-ME), 100 U / ml penicillin, 100 micrograms / ml streptomycin, L -2 mM glutamine]. To minimize oligonucleotide degradation, the FBS was first heated for 30 minutes at 65 ° C (phosphodiester-linked oligonucleotides) or at 56 ° C (all remaining oligonucleotides). Cells were seeded in 96-well plates at a density of 100,000 cells per well (100 microliter volume / well). The oligonucleotides in 10 microliters of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were added to each well. After 44 hours of culture at 37 ° C, a tritiated thymidine microcurium (Amersham, Arlington Heights, IL.) In 20 microliters of RPMI medium was added over a 4 hour labeling period. The cells were then harvested on a cell automatic collector (Skatron, Sterling, VA.) And the filters were evaluated with a scintillation counter. In control experiments to assess mitogenicity, cells were treated identically, except that medium (negative control) or concanavalin A (positive control) was added to the cells instead of the oligonucleotides. The results of these studies are shown in Figure 1. All inverted hybrid oligonucleotides proved to be less immunogenic than oligonucleotides with phosphorothioate linkages. Inverted hybrid oligonucleotides having phosphodiester linkages in the 2'-O-methyl region were found to be somewhat less immunogenic than those containing phosphorothioate linkages in this region. No significant differences in mitogenicity were observed when the 2'-O-methylribonucleotide region was reduced from 13 to 11 or 9 nucleotides. These results indicate that incorporating the inverted hybrid backbone into an oligonucleotide can reduce its mitogenicity.

Example 4

Reduction of In Vitro Coagulation Inhibition by Inverted Hybrid Oligonucleotides

To determine the relative effect of the inverted hybrid structure on oligonucleotide-induced mitogenicity, the following experiments were performed. Venous blood was drawn from healthy adult human volunteers. Plasma for clotting time assay was prepared by collecting blood in siliconized vacuum vessels containing sodium citrate (Becton Dickinson # 367705), followed by two centrifugations at 4 ° C to prepare low platelet plasma. Aliquots of plasma were kept on ice, various test compounds were added, and either analyzed immediately or snap frozen on dry ice for later storage at -20 ° C prior to clotting assay. Activated partial thromboplastin time (aPTT) was measured in duplicate on an Electra 1000C device (Medical Laboratory Automation, Mount Vernon, NY) following the manufacturer's recommended instructions, using Actin FSL (Baxter Dade, Miami, FL.) And calcium. to initiate clot formation, which was measured by photometry. Prolongation of aPTT was considered an indication of coagulation inhibition produced, as a side effect, by the oligonucleotide. The results are shown in Figure 5, for inverted hybrid oligonucleotides (and in Comparative Figure 8 for inverted chimeric oligonucleotides). Traditional oligonucleotides with phosphorothioate linkages produce the greatest prolongation of aPTT, of all the oligonucleotides tested. Traditional hybrid oligonucleotides produced a somewhat reduced prolongation of aPTT. Compared to traditional oligonucleotides with phosphorothioate linkages or to traditional hybrid oligonucleotides, all inverted hybrid oligonucleotides tested produced significantly reduced prolongation of aPTT. The inverted hybrid oligonucleotides that had phosphodiester linkages in the region of 2'-O-substituted ribonucleotides showed the maximum reduction in this side effect, with one of these oligonucleotides having a region of 2'-O-methyl-RNA with phosphodiester linkages of 13 nucleotides very little prolongation of aPTT, even at oligonucleotide concentrations of 100 micrograms / ml. These results indicate that inverted hybrid oligonucleotides may have advantages in reducing the coagulation inhibition side effect when administered to modulate gene expression in vivo.

Example 5

Reduction in complement activation in vivo by inverted hybrid oligonucleotides

Rhesus monkeys (4-9 kg body weight) are acclimated to laboratory conditions for at least 7 days prior to the study. On the day of the study, light sedation is administered to each animal with ketamine-HCl (10 mg / kg).

ES 2 201 198 T3 and diazepam (0.5 mg / kg). Anesthesia is induced to the surgical level and is maintained during the procedure by continuous ketamine infusion. The phosphorothioate linked oligonucleotide or the inverted hybrid oligonucleotide is dissolved in normal saline and administered by intravenous infusion with a catheter into the cephalic vein, using a programmable infusion pump at a delivery rate of 0.42 ml / minute. For each oligonucleotide, oligonucleotide doses of 0, 0.5, 1, 2, 5 and 10 mg / kg are administered to two animals over a 10 minute infusion period. Arterial blood samples are taken 10 minutes before oligonucleotide administration and 2, 5, 10, 20, 40 and 60 minutes after the start of the infusion, as well as 24 hours after. Serum is used to determine CH50 complement, following the standard complement-dependent lysis procedure of sheep erythrocytes (see Kabat and Mayer, 1961, cited above). At the maximum dose, the phosphorothioate-linked oligonucleotide causes a decrease in serum CH50 complement beginning within 5 minutes of the start of the infusion. Inverted hybrid oligonucleotides are expected to show, under these conditions, a much smaller, or undetectable, decrease in serum CH50 complement.

Example 6

Reduction of the in vivo mitogenicity of inverted hybrid oligonudeotides

CD1 mice are injected intraperitoneally with a dose of 50 mg / kg body weight of oligonucleotides with phosphorothioate linkages or inverted hybrid oligonucleotides. Forty-eight hours later, the animals are sacrificed and the spleens are removed and weighed. Animals treated with inverted hybrid oligonucleotides are expected to show no significant increase in spleen weight, whereas those treated with phosphorothioate-linked oligonucleotides are expected to show slight increases in spleen weight.

Example 7

Reduction of In Vivo Coagulation Inhibition by Inverted Hybrid Oligonucleotides

Rhesus monkeys are treated as in Example 5. From the collected whole blood samples, plasma is prepared for the coagulation test, which is carried out as described in Example 4. It is expected that the prolongation of aPTT will be substantially reduced in the case of inverted hybrid oligonucleotides, compared to traditional oligonucleotides with phosphorothioate linkages.

Example 8

Effect of the inverted hybrid structure on the activity of RNase H

In order to determine the ability of inverted hybrid oligonucleotides to activate RNase H when bound to a complementary RNA molecule, the following experiments were performed. Each oligonucleotide with phosphorothioate linkages or inverted hybrid oligonucleotide was incubated together with a molar equivalent amount of complementary oligoribonucleotide (concentration of 0.266 micromolar of each), in a cuvette containing a final volume of 1 ml of RNase H buffer (Tris-HCl 20 mM, pH 7.5, 10 mM MgCl2, 0.1 M KCl, glycerol al

2%, 0.1 mM DTT). The samples were heated to 95 ° C, then gradually cooled to room temperature to allow hybridization and the formation of double-stranded structures. The hybridized double-stranded structures were incubated for 10 minutes at 37 ° C, then 5 units of RNase H were added and data collection was started over a period of three hours. Data was recorded using a GBC 920 spectrophotometer (GBC Scientific Equipment, Victoria, Australia) at 259 nm. The degradation by RNase H was determined by hyperchromic shift.

The results are shown in Table I below.

TABLE I

Oligonucleotide degradation by RNase H Oligo n ° (Characteristics) Half-life Oligo n ° (Characteristics) Half-life

<td>GEM91 (all PO)</td><td>8.8s</td><td>Hyb115 (5 'MP)</td><td>11.5s</td>

<td>GEM91 (all PS)</td><td>22.4s</td><td>Hyb116 (chimeric) *</td><td>9.7 s</td>

<td>GEM91H (hybrid)</td><td>32.7 s</td><td>Hyb117 (chimeric) *</td><td>8.1 s</td>

<td>Hyb108 (hyb inv.)</td><td>15.4s</td><td>Hyb118 (inv. Chem.)</td><td>11.5s</td>

<td>Hyb109 (hyb inv.)</td><td>7.9 s</td><td>Hyb119 (inv. Chem.)</td><td>14.4 s</td>

<td>Hyb110 (hyb inv.)</td><td>10.4 s</td><td>Hyb120 (inv. Chem.)</td><td>9.3 s</td>

<td>Hyb111 (inv.)</td><td>12.9 s</td><td>Hyb121 (3 'MP)</td><td>21.2s</td>

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TABLE I (continued)

<td>Oligo n ° (Characteristics)</td><td>Half-life</td><td>Oligo n ° (Characteristics)</td><td>Half-life</td>

<td>Hyb112 (hyb inv.)</td><td>12.5 s</td><td>Hyb122 (chimeric) *</td><td>23.0 s</td>

<td>Hyb113 (hyb inv.)</td><td>10.9 s</td><td>Hyb123 (chimeric) *</td><td>41.8 s</td>

<td>Hyb114 (hyb inv.)</td><td>20.3 s</td><td>Hyb124 (chimeric) *</td><td>did not detect.</td>

<td>* = comparative</td><td></td><td></td><td></td>

As expected, the phosphodiester-linked oligonucleotides performed as good cosubstrates for RNase H-mediated RNA degradation with a degradation half-life of 8.8 seconds. Oligonucleotides with phosphorothioate linkages produced an increase in half-life of 22.4 seconds. Introduction of a 2'-O-methylribonucleotide segment at either end of the oligonucleotide further impaired RNase H activity (half-life = 32.7 seconds). In contrast, the introduction of a 2'-O-methyl segment in the middle of the oligonucleotide (inverted hybrid structure) always led to an improvement in RNase H-mediated degradation. When a region of 13 2'-O-methylribonucleosides with phosphodiester bonds was flanked on both sides with DNA with phosphorothioate bonds, the maximum activity of RNase H was observed, with a half-life of 7.9 seconds. The introduction of large blocks of nucleosides linked by methylphosphonate bonds at the 3 'end of the oligonucleotide either produced no effect or caused further impairment of RNase H activity even when in a chimeric configuration. However, the introduction of nucleosides linked by methylphosphonate linkages at the 5 'end increased RNase H activity, particularly in the chimeric configuration with a single methylphosphonate linker at the 3' end (best half-life = 8.1 seconds) . All inverted chimeric oligonucleotides with methylphosphonate major regions (comparative examples) flanked by phosphorothioate regions gave good RNase results, with a half-life range of 9.3 to 14.4 seconds. These results indicate that the introduction of the inverted hybrid (or inverted chimeric) structure into phosphorothioate-containing oligonucleotides can restore part or all of the oligonucleotide's ability to act as a cosubstrate for RNase H, a potentially important feature for an effective antisense agent. Example 9

Effect of inverted hybrid structure on melting temperature

To determine the effect of the inverted hybrid structure on the stability of the double-stranded structure formed between an antisense oligonucleotide and a target molecule, the following experiments were performed. Thermal denaturation (Tm) data was recorded using a GBC 920 spectrophotometer, which had six 10mm cuvettes mounted on a double carousel. In the Tm experiments, the temperature was adjusted and controlled by means of a Peltier effect temperature controller connected to a computer, using the software supplied by GBC, following the manufacturer's instructions. The Tm data were analyzed by the first derivative method and by the midpoint method, with the help of the software. Tm experiments were performed in a buffer containing 10 mM PIPES, pH 7.0, 1 mM EDTA, 1 M NaCl. A VWR 1166 chilled bath (VWR, Boston, MA.) Was connected to the Peltier effect temperature controller to absorb heat. The concentration of oligonucleotide strands was determined using the absorbance values ​​at 260 nm, taking into account the extinction coefficients.

The results are shown in Table II below.

TABLE II

Stability of the double-stranded structure of oligonucleotides

<td>Oligo n ° (Characteristics)</td><td>Tm (° C) *</td><td>Oligo n ° (Characteristics)</td><td>Tm (° C) *</td>

<td>GEM91 (all PO)</td><td> 72,0</td><td>Hyb115 (5 'MP)</td><td> 61,8</td>

<td>GEM91 (all PS)</td><td> 63,6</td><td>Hyb116 (chimeric) *</td><td> 61,0</td>

<td>GEM91H (hybrid)</td><td> 67,0</td><td>Hyb117 (chimeric) *</td><td> 60,5</td>

<td>Hyb108 (hyb inv.)</td><td> 76,4</td><td>Hyb118 (inv. Chem.)</td><td> 57,9</td>

<td>Hyb109 (hyb inv.)</td><td> 80,0</td><td>Hyb119 (inv. Chem.)</td><td> 57,7</td>

<td>Hyb110 (hyb inv.)</td><td> 74,2</td><td>Hyb120 (inv. Chem.)</td><td> 56,8</td>

<td>Hyb111 (inv.)</td><td> 76,9</td><td>Hyb121 (3 'MP)</td><td> 60,7</td>

<td>Hyb112 (hyb inv.)</td><td> 72,1</td><td>Hyb122 (chimeric) *</td><td> 60,5</td>

<td>Hyb113 (hyb inv.)</td><td> 74,3</td><td>Hyb123 (chimeric) *</td><td> 59,0</td>

<td>Hyb114 (hyb inv.)</td><td> 71,3</td><td>Hyb124 (chimeric) *</td><td>did not detect.</td>

<td>* = comparative</td><td></td><td></td><td></td>

* = with complementary RNA

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These results demonstrate that the conversion of an oligonucleotide with phosphodiester linkages to an oligonucleotide with phosphorothioate linkages results in a reduction in the stability of the double chain structure, and that the introduction of methyl phosphonate linkages further reduces the stability of the chain structure. double. The stability of the double-stranded structure can be restored by adding 2'-O-methylribonucleotides, and can exceed that of the phosphodiester-linked oligonucleotide when an inverted hybrid structure is used. Conversely, the use of an inverted chimeric structure results in the lowest melting temperatures observed for any of the hybridizing methylphosphonate oligonucleotides, although the stability of the double-stranded structure was maintained even well above physiological temperatures. . Taken together, these results suggest that the inverted hybrid structure or the inverted chimeric structure can be used for custom oligonucleotide design to obtain particular desired stabilities of the double-stranded structure in particular experimental or therapeutic applications.