Hybrid oligonucleotide containing phosphorothioate and/or phosphorodithioate communication, therapeutic pharmaceutical composition, the method of inhibiting gene expression

 

(57) Abstract:

Hybrid oligonucleotide refers to synthetic oligonucleotides used in the study of gene expression and for therapeutic purposes. Oligonucleotide contains phosphorothioate and/or phosphorodithioate communication and includes desoxyribonuclease, ribonucleotide or 2'-substituted ribonucleoside and phosphorothioate and/or phosphorodithioate mezhnukleotidnyh communication. When used in therapeutic compositions using an effective amount of the oligonucleotide having a sequence complementary to the sequence of a nucleic acid of a virus, a pathogenic organism or a cellular gene, and a pharmaceutically acceptable carrier. Oligonucleotide has phosphorothioate and/or phosphorodithioate communication and desoxyribonuclease, ribonucleotide or 2'-substituted ribonucleoside. The resulting hybrid oligonucleotide allows you to effectively regulate gene expression. 3 S. and 9 C.p. f-crystals, 4 Il., 3 table.

The invention relates to synthetic oligonucleotides that are useful in studies of gene expression, and use of antisense oligonucleotides for therapeutic purposes. The scale for such applications, resulting modifications of the sugar-phosphate backbone oligonucleotides.

The possibility of developing therapeutic applications of antisense oligonucleotides was first proposed in three articles published in 1977 and 1978, Paterson and others, Proc. Natl. Acad. Sci. USA 74: 4370-4374 (1987) reveals that translation of mRNA in a cell-free system can be ingibirovany binding of the oligonucleotide, complementary to the mRNA. Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75: 280-284 and 285-288 (1978) reveals that the 13-dimensional synthetic oligonucleotide, which is complementary to part of the genome of rous sarcoma virus (RSV), inhibits RSV replication in infected fibroblasts chickens and inhibits RSV - mediated transformation of primary fibroblasts Chicks in malignant sarcoma cells.

These early indications that synthetic oligonucleotides can be used for inhibition of viral replication and neoplasia, was accompanied by the use of synthetic oligonucleotides in the inhibition of many viruses. Goodchild and others, U.S. patent N 4806463 reveals inhibition of human immunodeficiency virus (HIV) synthetic oligodeoxynucleotide, complementary to different regions of the HIV genome. Leiter and others, Proc. Natl. A: 6268-6275 (1986) discloses the use of synthetic oligonucleotides in the inhibition of vesicular stomatitis virus (VSV). GaO and other Antimicrob. Agents Chem. 34: 808-812 (1990) discloses the inhibition of herpes simplex virus by synthetic oligonucleotides. Birg and other Nucleic Acids Res. 18: 2901-2908 (1990) discloses the inhibition of monkey virus (SV40) synthetic oligonucleotides. Storey and other Nucleic Acids Res. 19: 4109-4114 (1991) discloses the inhibition of human papillomavirus (HPV) synthetic oligonucleotides. The use of synthetic oligonucleotides and their analogues as antiviral agents has recently been described in detail Agrawal, Tibtech 10: 152-158 (1992).

In addition, synthetic oligonucleotides were used to inhibit a variety of non-viral pathogens, as well as selective inhibition of expression of some cellular genes.

Thus, the usefulness of synthetic oligonucleotides as agents in the inhibition of virus propagation, breeding non-viral pathogens and selective expression of cellular genes specifically installed. However, there is a need for improved oligonucleotides, which would be more effective in the inhibition of such viruses, pathogens and selective gene expression. The researchers attempted to obtain and test oligonucleotides with modifications in their mezhnukleotidnyh links. Some is counterparts. Sarin and others, Proc. Natl. Acad. Sci. USA 85: 7448-7451 (1988) indicates that methylphosphonate of oligodeoxynucleotide more active as inhibitors of HIV-1 than normal oligodeoxynucleotide. Agrawal and others, Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988) argues that phosphorothioate oligonucleotides and various phosphoramidate oligonucleotides more effective in the inhibition of HIV-1 than normal oligodeoxynucleotide. Agrawal and others, Proc. Natl. Acad. Sci. USA 86: 7790-7794 (1989) discloses the advantage of phosphorothioate oligonucleotides in the inhibition of HIV-1 in early and chronically infected cells.

In addition, were obtained chimeric oligonucleotides having more than one type mezhnukleotidnyh communication within the oligonucleotide. Chimeric oligonucleotides contain only deoxyribonucleoside, but have areas containing different mezhnukleotidnyh communication. Pederson and others (U.S. patent N 5XXXXXX /Ser. N 07/480269 issued 12.24.91) discloses chimeric oligonucleotides having the core oligonucleotide with fosfomifira or phosphorothioate links, flanked by phosphoramidate, methylphosphonate or phosphoramidate oligonucleotides Furdon and other Nucleic Acids Res. 17: 9193-9204 (1989) discloses chimeric oligonucleotides having the area of postdifferentiated in addition to other specific oligonucleotides, with the field of fosfodiesterazu of the oligonucleotide and methylphosphonates of the oligonucleotide. Each of the above compounds uses phosphorothioate of deoxyribonucleotide, which reduce the stability of the duplex. Atabekov and other FEBS Letters 232: 9698 (1988) discloses chimeric oligonucleotides, in which all mezhnukleotidnyh links are phosphodieterase relationships, but in which the area of oligoribonucleotides and oligodeoxyribonucleotides mixed. Inoune, etc. FEBS Letters 215: 237-250 (1967) discloses chimeric oligonucleotides having only the phosphodiester bond, and oligodeoxyribonucleotides and 2'-OMe-ribonucleotides. None of these compounds have only phosphodiester bond, does not demonstrate the stability of either the endonuclease, exonuclease.

Many of these modified oligonucleotides have improved the potential efficacy of therapeutic aspect of antisense oligonucleotide. However, in the known oligonucleotides, there are certain disadvantages and these deficiencies can limit the effectiveness of such oligonucleotides as therapeutic agents. Wickstrom, J. Biochem. Biophys. Methods 13: 97-0102 (1986) indicates that oligonucletide with phosphodieterase links responsive to the action of nucleases. This osprey (1990) indicates, that phosphoramidate or methylphosphonate of the oligonucleotide by hybridization with RNA does not activate RNase H, the activation of which may be important for the function of antisense oligonucleotides. Agrawal and other Nucleosides and Nucleotides 8: 5-6 (1989) argues that phosphorothioate of oligodeoxyribonucleotide reduce the stability of the duplex upon hybridization with RNA.

There is therefore a need for improved oligonucleotides, which will be free from defects? known in the art. Ideally, these oligonucleotides must be resistant to nuclearities decay, should form stable duplexes with RNA, and must activate the RNase H in hybridization with RNA.

The invention provides a hybrid oligonucleotides that are resistant to nuclearities splitting, form stable duplexes with RNA or DNA, and activate RNase H when hybridization with RNA. The oligonucleotides in accordance with the invention, such characteristic features attached.t the presence of phosphorothioate or/and phosphorodithioate mezhnukleotidnyh links and segments from oligodeoxyribonucleotides, as well as segments from other oligoribonucleotides or 2'-substituted-oligoribonucleotides. For the purposes of the invention, the term "2' - amino, but not 2'-H, in which allyl, aryl or alkyl groups may be unsubstituted or substituted, for example halo-, hydroxy-, trifluoromethyl-, cyano-, nitro-, acyl, acyloxy-, alkoxy-, carboxy, carbalkoxy or amino groups.

The purpose of the invention to provide oligonucleotides that can be used to analyze and explain the importance of performance parameters and resistance to nuclease stability of the duplex and activation of RNase H in antimicrobic the oligonucleotides.

Another objective of the invention to provide oligonucleotides which are effective in regulating cellular pathogenic or viral gene expression at the mRNA level.

Another object of the invention is a therapeutic oligonucleotides, which are more effective.

The oligonucleotides according to the invention satisfy all of the goals and objectives of the invention.

In Fig. 1 shows ion-exchange analysis GHUR oligonucleotides treated with nuclease. Panel A presents the profiles A, B and C of oligonucleotides F, C and A, respectively, after splitting SVPD for 420 minutes

In Fig. 2 (panel B) A profile is nerissa is of SVPD.

In Fig. 3 and 4 presents the results of studies on the activation of RNase H oligonucleotides as described in example 4.

In the first aspect of the invention provides oligonucleotides that are needed to study the parameters that are important for effective action of antisense oligonucleotides. For the purposes of the invention, the term oligonucleotide includes polymers of two or more ribonucleotides, deoxyribonucleotides, or both monomers of ribonucleotide and/or deoxyribonucleotide, connected via the 5' - 3' connection, which may include any of the links, known for antisense oligonucleotides. In addition, the term oligonucleotide includes such molecules, which have modified bases of nucleic acids and/or sugar, as well as molecules with additional substituents, such as diamines, cholesterol or other lipophilic groups. Some preferred combinations of monomers and mismanaging relations are discussed in detail below.

Usually it is believed that the antisense activity of the oligonucleotide depends on the binding of the oligonucleotide to the target nucleic acid that disrupts the function of the target or the stop of the hybridization or destruction of Zelazny to be two important parameters: the stability of the duplex and activation of RNase H. The stability of the duplex is important because it is assumed that the oligonucleotide must form a duplex (or triplex in the mechanism holsteinische mating) with the target nucleic acid, which is directed either to stop hybridization, or the destruction of the target by RNase H. Activation of RNase H (the ability to activate the RNase H in hybridization with the target RNA) occurs when the target nucleic acid is RNA, and this activation can result in effective destruction of molecules of target RNA. In addition to antisense oligonucleotide acted in vivo, it must long enough to continue to interact with the target nucleic acid. Given the fact that the environment in vivo is active endonuclease and ectonucleoside, hence there is the third option: namely that antisense oligonucleotide must be resistant to nuclearities splitting.

In order to analyze and explain the importance of each of these parameters for the effectiveness of antisense oligonucleotides, you need to have the oligonucleotides that changes in each of these settings. Properties of several well-known oligonucleotides shown in table. 1.

1.in comparison with DNA-RNA stability.

2. Comparison with DNA digestion by fosfodiesterasa).

3. Activation of RNase a duplex formed between the oligonucleotide and RNA.

Hybrid oligonucleotides according to the invention form more stable duplexes with complementary RNA than phosphorothioate of oligodeoxyribonucleotide. In addition, they are more resistant to endonucleases and ectonucleoside splitting than phosphorothioate of oligodeoxyribonucleotide, and they normally activate RNase H. Thus, the oligonucleotides according to the invention is complementary to the oligonucleotides shown in table.1 for the parameters involved in the effectiveness of antisense oligonucleotides.

With regard to the first aspect of the invention, the oligonucleotides according to the invention can have any sequence of the oligonucleotide, so as complementary oligonucleotides used in this study can be obtained from any sequence of the oligonucleotide. The oligonucleotides in accordance with this aspect of the invention are characterized by only the following characteristic features. First, at least some of mezhnukleotidnyh ties present in the oligonucleotides according to the invention are phosphorothioate and/or pasta can range from 1 to the figures mezhnukleotidnyh ties, which are present in the oligonucleotide. Thus, for the purposes of the invention, the term phosphorothioate and/or phosphorodithioate covers any such option. In a preferred embodiment, the number of nucleotides according to the invention will range from about 2 to about 50 nucleotides, and more preferably from about 6 to about 50 nucleotides. Thus, in this preferred embodiment, the oligonucleotides according to the invention will have from about 1 to 49 mezhnukleotidnyh of phosphorothioate and/or phosphorodithioate links.

The second feature of the oligonucleotides according to this aspect of the invention is the presence of deoxyribonucleoside. The oligonucleotides according to the invention contain at least one deoxyribonucleotide. Preferably the oligonucleotides according to the invention contain four or more deoxyribonucleotides in the adjacent block, in order to provide the trigger segment for RNase H. In certain preferred embodiments, such a trigger segment can be not one, but several. Such segments can be present at any position within the oligonucleotide. The oligonucleotides of the most may be deoxyribonucleoside. In fact, the nucleoside can be all but one of the nucleoside, Cove or better from 6 to 50 nucleosides, the number present deoxyribonucleosides will be from 1 to about 49 deoxyribonucleosides.

The third characteristic feature of the oligonucleotides according to this aspect of the invention is the presence of ribonucleotides, 2'-substituted ribonucleosides or combinations thereof. For the purposes of the invention, the term "2'-substituted" means substitution of the 2' - OH in the ribose molecule, for example, 2' - OMe, 2'- allyl, 2'- aryl, 2' - alkyl, 2' - halo, or 2' - amino, but not with 2' - H, where allyl, aryl or alkyl groups may be unsubstituted or substituted, for example halo-, hydroxy-, trifluoromethyl-, cyano-, nitro-, acyl-, acyloxy, carboxyl-, carbalkoxy-, or amino groups. The oligonucleotides according to the invention contain at least one ribonucleotide and/or 2'-substituted ribonucleoside. In a preferred embodiment, such oligonucleotides have 6 or more ribonucleotides and/or 2'-substituted ribonucleosides to enhance the stability of the duplex. Such ribonucleotide and/or 2'-substituted ribonucleoside may be present as units, in pairs or in large contiguous segments, and can be in any position within the oligonucleotide or plural positions within the oligonucleotide. Such ribonucleotide and/or 2'-semidensities version number from 2 to 50 nucleosides, the number of ribonucleosides or 2'-substituted ribonucleosides will range from about 1 to about 49 deoxyribonucleosides.

The ability to change the number and position phosphorothioate and/or phosphorodithioate mezhnukleotidnyh clutches deoxyribonucleosides and ribonucleosides or 2'-substituted ribonucleosides allows the researcher to examine in detail how each of these variables affects the parameters of resistance to nuclease stability of the duplex and activate RNase H. the Ability to change the size of the oligonucleotide allows you to consider another option. In addition, a small oligo (e.g., dimers) can be used as building blocks for larger oligo. Thus, each such option, described above, is useful in such studies.

In the second aspect of the invention provides a hybrid oligonucleotides that are effective in the inhibition of viruses, pathogenic organisms, or the expression of cellular genes. The ability to inhibit such agents is very important for treatment of several diseases. Oligonucleotides according to this aspect of the invention share the characteristics of the above oligonucleotides, except that the sequence on which the notes of the virus, pathogenic organism or cell gene. Preferably, such oligonucleotides in length ranges from 6 to about 50 nucleotides. For the purposes of the invention, the term "sequence of the oligonucleotide, which is complementary nucleic acid sequence" means a sequence of the oligonucleotide (2 - about 50 nucleotides) that's hybrid with the nucleic acid sequence under physiological conditions, for example, by base pairing according to the Watson-Crick (interaction between oligonucleotide and single-stranded nucleic acid) or Justinussen by base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by other means. Such hybridization under physiological conditions is measured practically by the observation of interference with the function of nucleic acid sequence.

The sequence of the nucleic acid to which the invention oligonucleotide is complementary, will vary depending on the agent, which is retarded.

In many cases, the nucleic acid sequence is the sequence of viral nucleic acid. The use of antisense Oli is 10:152 - 158 (1992). Sequencing of viral nucleic acids that are complementary to effective antimuslim oligonucleotides, has been described for many viruses, including human immunodeficiency virus type I (U.S. patent N 4806463), herpes simplex virus (U.S. patent N 4689320), influenza virus (U.S. patent N 5XXXXXX ser N 07/516275 issued 30.06.92), human papilloma virus (Storey and others, Nucleic Acids Res. 19:4109 - 4114 (1991)). Sequence complementary to any sequences of a nucleic acid, can be used for oligonucleotides according to the invention, as the sequence of the oligonucleotide can be complementary sequences of nucleic acids from any other virus. Additional viruses that have a known nucleic acid sequence against which you can get the antisense oligonucleotides include the FMD virus (cm. Robertson and others, J. Virology 54:651 /1985/; Harris and others, J. Virology 36:659/1980/), yellow fever virus (cm. Rice and other Science 229:726 /1985/), varicella zoster virus (c m Davison and Scott, J. Gen. Virology 67:2279 /1986/), and cucumber mosaic virus (see Richards and others, Virology 89:395 /1978/).

In another embodiment, the oligonucleotides according to the invention can be oligonucleotide sequence, complementary sequence of nuclei the organisms, including the malaria pathogen, Plasmodium falciparum many pathogenic bacteria. Oligonucleotide sequences, complementary sequences, nucleic acids from any pathogenic organism, it is possible to use oligonucleotides according to the invention. Examples of pathogenic eukaryotes with a known nucleic acid sequence against which you can get the antisense oligonucleotides include Trypanosoma brucei gambience and Leishmania (see Campbell and others, Nature 311: 350 /1984/), Fasciola hepatica cm, Zurita, etc. Proc. Natl. Acad. Sci. USA 84: 2340 /1987/). Antifungal oligonucleotides can be prepared using hybridization region having the oligonucleotide sequence that is complementary to the nucleotide sequence, for example, gene chainsystems, and antibacterial oligonucleotides can be obtained by using, for example, gene laminaceae.

In another embodiment, the oligonucleotides according to the invention can be oligonucleotide sequence, complementary to a cellular gene or gene transcript, the abnormal expression or product of which leads to a state of disease. Were described nucleotide sequence of several cellular genes, including prion, the various well-known oncogenes and proto-oncogenes such as c-myb, c-myc, c-abe and n-ras. In addition, oligonucleotides that inhibit the synthesis of structural proteins or enzymes, exclusively or significantly involved in spermatogenesis, sperm motility and associated sperm with the egg, or any other stage, affecting the viability of sperm, can be used as a contraceptive for men. Similarly contraceptives for women can be oligonucleotides that inhibit proteins or enzymes involved in ovulation, fertilization, implantation, or in the biosynthesis of hormones that are involved in these processes.

Hypertension can be controlled by oligodeoxynucleotide, which inhibit the synthesis of the enzyme that converts angiotensin or related enzymes in the system the renin/angiotensin; platelet aggregation can be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in miocarditis and cerebral circulatory disorders, heart attacks, atherosclerosis, embolism, and thrombosis; deposition of cholesterol in arterial walls can be slowed down by suppression of the synthesis of co-enzyme A: cholesterolcholesterol when arteriosklerose nervous disorders, for which the stop of the hybridization can be used to reduce or remove harmful disorders. For example, suppression of the synthesis of monoamine oxidase can be used in Parkinson's disease; suppression of catechol o-methyltransferase can be used in the treatment of depression; and the suppression of the indole N-methyltransferase can be used in the treatment of schizophrenia.

Suppression of selected enzymes in the cascade activation of arachidonic acid, which leads to prostaglandins and leukotrienes, may be useful in the control of platelet aggregation, allergies, inflammation, pain, and asthma.

Suppression of protein expressed by the gene of resistance to multiple drugs (mdr), which is responsible for the development of resistance to many anticancer drugs and is the main limiting factor in chemotherapy may be useful in the treatment of cancer. The sequence of the oligonucleotide, complementary to the nucleotide sequences of any of these genes can be used for oligonucleotides according to the invention, as the sequence of the oligonucleotide can be complementary to any other cellular gene or gene transcript, the abnormal expression or about Steny was described in U.S. patent N 5107065.

In the third aspect, the invention provides therapeutic pharmaceuticals oligonucleotides that are effective in the treatment of viral infections, infections caused by pathogenic organisms, or disease resulting from abnormal gene expression or gene expression product of an abnormal gene. Such pharmaceutical therapeutic agents include oligonucleotides in accordance with the second aspect of the invention in a pharmaceutically acceptable carrier.

In the fourth aspect of the invention provides a method for inhibition of gene expression of the virus, a pathogenic organism or a cellular gene, the method includes the step of the task of oligonucleotides to cells infected with a virus or pathogenic organism in the first two cases, or to cells in General in the latter case. Such methods are useful in studying gene expression and function of specific genes.

In the fifth aspect of the invention provides a method of treatment of the human or animal disease which is the result of a virus infection or pathogenic organism, or abnormal expression or abnormal production of the cellular gene. The method includes assigning therapeutic pharmaceuticeutical methods such assignments will include oral, intranasal, rectal and local destination. In such methods of treatment according to the invention, the oligonucleotides may be administered with other therapeutic agents, such as AZT in AIDS cases.

Many viral diseases can be treated by the method in accordance with the invention, including AIDS, ARC, oral or genital herpes, warts papillomavirus, influenza, foot and mouth disease, yellow fever, varicella, herpes zoster, leukemia, caused by the virus human T-cell leukemia and hepatitis. Among fungal diseases treatable by the method according to the invention, diseases such as candidiasis, cryptococcosis, histoplasmosis, blastomycosis, aspergillosis, sporotrichosis, chronomics, dermatophytes and Coccidioides. The method can also be used to treat diseases caused by Rickettsia (e.g., typhoid fever American tick ricketsiosis), and sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum. Many parasitic diseases can be treated by the method according to the invention, including amebiasis, Chagas disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis and pneumonia Pheumocystis carini, and helminthic infections, such as ascariasis, filariasis, trigonelline, regardless of whether it is called P. falciparum, P. vivax, P. orale or P. malarial.

All infectious diseases, mentioned above, can be treated by the method according to the invention, because the infectious agents of these diseases are known and can be obtained oligonucleotides in accordance with the invention with such a nucleotide sequence that is complementary to the nucleotide sequence, which is essential for the multiplication of an infectious agent, such as an essential gene.

Other conditions are the diseases that are treated by the method according to the invention, are the result of abnormal expression or abnormal production of the cellular gene. These conditions can be treated by the appointment of oligonucleotides according to the invention and discussed in this disclosure above.

The oligonucleotides according to the invention can be synthesized by procedures well known in the art. Another preferred method of synthesis of these oligonucleotides is a method of using a approach H-phosphonate described in U.S. patent N 5XXXXXX, Ser. N 07/334679 issued 10.03.92, and Agrawal and Tang, Tetrahedron Lett. 31: 7541-7544 (1990). The oligonucleotides according to the invention can be made even more resistant to nuclearities splitting by adding TBE the ways of the invention and are not restrictive.

Example 1. Synthesis of hybrid oligonucleotides with phosphorothioate links.

Hybrid oligonucleotidic were synthesized on CPG 5-6 molar scale automated synthesizer/model 8700, Millipore, Milford, MA/ using H-phosphonate described in U.S. patent N 5XXXXXX (Ser. N 07/344679 issued 19.03.92 g), (H-phosphonates of deoxynucleoside were obtained from Millipore 2' - OMe H-phosphonates of ribonucleotide were synthesized by standard procedures. Segments of oligonucleotides containing 2'-OMe nucleotide, was collected using H-phosphonates 2'-OMe of ribonucleoside for the desired cycles. Similarly, the segments of oligonucleotides containing deoxyribonucleotide, was collected using H-phosphonates of deoxynucleoside for the desired cycles. After Assembly associated with CPC H-phosphonate of the oligonucleotide oxidized sulfur to form phosphorothioate communication. Then the oligonucleotides were subjected to unprotect in concentrated H4OH if 40oC 48 hours

The crude oligonucleotide (about A260units) were analyzed liquid chromatography low-pressure reversed-phase environment reversed phase C18. The DMT group was removed by treatment with 80% aqueous acetic acid, the relative resistance to nuclease hybrid oligonucleotides with phosphorothioate links.

To test the relative resistance to nuclease hybrid oligonucleotides with phosphorothioate links oligonucleotides were treated with snake venom phosphodiesterase (SVPD). About 0.2 A260units of oligonucleotides A, C and F were dissolved in 500 l of buffer (49 mm NH4CO3pH 0.4 + 20 mm MgCl2and mixed with 0.002 units SVPD. The mixture was incubated at 37oC 420 min After 0, 200, and 420 min aliquots were taken at 165 l and were analyzed using ion-exchange GHUR. The results are shown in Fig.1. Oligonucleotide F was very resistant to phosphodiesterase, while oligonucleotide A was cut almost completely and oligonucleotide C was cut by 50% (panel A).

Oligonucleotide with phosphodiesterase bonds was cut by about 80% within minutes using 1/10 part of the concentration SVPD.

These results show that the presence of 2'-OMe of ribonucleosides in phosphorothioate of the oligonucleotide increases the resistance to ectonucleoside splitting, and that this enhanced stability is increased when using a larger proportion of 2'-OMe of ribonucleotides. Given the similar nature and behavior of ribonucleotides, other 2'-substituted ribonucleotides and 2'-OMe of ribonucleotides, these results is whether phosphorodithioate hybrid oligonucleotide, having ribonucleotides, 2'-substituted ribonucleotides, or a mixture of ribonucleotides and 2'-substituted ribonucleotides.

Example 3. The relative stability of the hybrid duplex oligonucleotides with phosphorothioate links.

Oligonucleotides A - F were tested for the relative stability of the duplexes formed with complementary oligodeoxyribonucleotide, and with complementary oligoribonucleotide. In separate reactions, each oligonucleotide A - F was mixed with an equivalent amount (0.2 A260units) its complementary oligonucleotide 150 mm NaCl, 10 mm Na2PO4, 1 mm ATDC, pH 7. The mixture was heated to 85oC 5 min, then cooled to 30oC. Then the temperature was increased from 30 to 80oC with a speed of 1oC / minute and A260was recorded as function of temperature. The results are shown in table.3.

These results reveal that if a complementary oligonucleotide is oligoribonucleotide, the presence of 2'-OMe of ribonucleotides increases the stability of the duplex, and that the gain increases with increasing proportions of 2'-OMe of ribonucleosides. These results should similarly be attributed to phosphorothioate and/or phosphorodithioate guy and 2'-substituted ribonucleotides. Thus, phosphorothioate and/or phosphorodithioate hybrid oligonucleotide according to the invention should bind viral RNA or virus, pathogenic organism or cell mRNA with greater affinity than normal phosphorothioate of oligodeoxynucleotide.

Example 4. Activation of RNase H in hybrid oligonucleotides with phosphorothioate links

Oligonucleotidic and various hybrid oligonucleotidic were investigated for their properties activate RNase h Oligonucleotide A (PL. 2), phosphorothioate of the oligonucleotide, which is known to activate the RNase H was used as control. Oligonucleotide F/2'-OMe analog phosphorothioate of the oligonucleotide and oligonucleotide C, B and E, hybrid oligonucleotides were analyzed for the ability to activate the RNase H.

For the experiment, complementary 32-dimensional oligoribonucleotide was synthesized (Fig. 3) and were treated with kinase 5'-end,32P-labeled 32-dimensional RNA /0.003 A260units; 0.01 g / and oligonucleotides /0.0635 A260units; 1.9 g / mixed in 20 l of buffer /0.15 M NaCl, 0.01 MgCl2, 0.01 M chloride Tris, pH 7.9/ containing 0.001 M DTT. The mixture was treated with 6 units of RNase H /E. coli/ at 37oC. Aliquots of 4.5 l uberalles A) showed site-specific cleavage of RNA by RNase H. Oligonucleotide F/2'-OMe analog; Duplex B/ showed no cleavage of RNA in the presence of RNase H. the Hybrid oligonucleotides B, C, and E /Duplexes C, D, E, respectively/ showed site-specific cleavage of RNA by RNase H. Duplex F, which was investigated incompatible phosphorothioate of the oligonucleotide showed no cleavage of RNA. Row C shows that in the presence of RNase H, RNA was not split.

Example 5. Inhibition of HIV hybrid oligonucleotides with phosphorothioate links

Hybrid oligonucleotidic were tested for their ability to inhibit HIV-1 in tissue culture, H9 cells were infected with virions of HIV-1 /=0.01 - 0.1 TCID50/ cell/ 1H at 37oC. After 1 h needsomeone varioni were washed and infected cells were divided into holes 24 holes tablet. To the infected cells were added to the appropriate concentration (initial solution) of the oligonucleotide to obtain the desired concentration in the environment 2 ml positive control experiment was added to the ddC or AZT. Then the cells were cultured for 3 days. At the end of three days the supernatant layer from the infected culture was collected and measured on the expression of p 24 enzyme-linked immunosorbent assay (ELISA). Level cyclotide.

All tested hybrid oligonucleotidic showed a significant inhibition of the expression of p 24 g g/ml concentrations without significant cytotoxicity (data not shown). These results indicate that phosphorothioate hybrid oligonucleotide containing 2'-OMe the ribonucleotides, effective as inhibitors of gene expression. Similar effectiveness to be expected from phosphorothioate and/or phosphorodithioate of the oligonucleotide containing ribonucleotide, 2'-substituted ribonucleoside or a mixture of ribonucleotides and 2'-substituted ribonucleosides.

1. Hybrid oligonucleotide containing phosphorothioate and/or phosphorodithioate communication and includes desoxyribonuclease, ribonucleotide or 2'-substituted ribonucleoside and phosphorothioate and/or phosphorodithioate mezhnukleotidnyh link.

2. Oligonucleotide under item 1, characterized in that desoxyribonuclease is present in the segment of at least four adjacent deoxyribonucleosides.

3. Oligonucleotide under item 1, characterized in that ribonucleotide or 2'-substituted ribonucleotide is present in the segment of the at least two adjacent ribonucleotides and/or 2'-substituted ribonucleosides.

4. Oligonucletides posledowatelxnosti virus pathogenic organism or cell gene.

5. Oligonucleotide under item 2, characterized in that it has the sequence of the oligonucleotide, which is complementary to the nucleotide sequence of a virus, a pathogenic organism or a cellular gene.

6. Oligonucleotide under item 3, characterized in that it has the sequence of the oligonucleotide, which is complementary to the nucleotide sequence of a virus, a pathogenic organism or a cellular gene.

7. Therapeutic pharmaceutical composition comprising an effective amount of the oligonucleotide having a sequence complementary to the sequence of a nucleic acid of a virus, a pathogenic organism or a cellular gene, and a pharmaceutically acceptable carrier, characterized in that the said oligonucleotide is an oligonucleotide under item 1.

8. The composition according to p. 7, characterized in that desoxyribonuclease is present in the segment of at least four adjacent deoxyribonucleosides.

9. The composition according to p. 7, characterized in that ribonucleotide or 2'-substituted ribonucleotide is present in the segment of the at least two adjacent ribonucleotides and/or 2'-substituted ri is knogo gas, comprising contacting cells infected with the said virus or containing the said organism or gene, are resistant to nuclease, single-strand, which is not found in the nature of the oligonucleotide molecule comprising desoxyribonuclease, ribonucleotide or 2'-substituted ribonucleoside, and the nucleotide sequence of the above-mentioned molecule complementary to the nucleic acid sequence of a virus, a pathogenic organism or a cellular gene, wherein the contacting of the cells is performed with the molecule of the oligonucleotide containing phosphorothioate and/or phosphorodithioate mezhnukleotidnyh link.

11. The method according to p. 10, characterized in that desoxyribonuclease is present in the segment of at least four adjacent deoxyribonucleosides.

12. The method according to p. 10, characterized in that ribonucleotide or 2'-substituted ribonucleotide is present in the segment of the at least two adjacent ribonucleotides and/or 2'-substituted ribonucleosides.

 

Same patents:

The invention relates to medicine and biology and relates to methods of obtaining hybrids, DNA-RNA, having biological activity

The invention relates to biochemistry and bacteriology and can be used to obtain nucleic acids from anthrax and other spore-forming bacilli
The invention relates to medicine, namely to methods of treatment of male infertility

The invention relates to medicine, in particular to pharmacology and pharmacy and concerns the means for the correction of disorders in the disorders caused by various factors
The invention relates to biotechnology, molecular biology, medical diagnostics and can be used to identify the studied biological material, in particular when hybridization analysis of nucleic acids, including viral, using the detector avidin-bitenova (streptavidin-bitenova) system
The invention relates to medicine, namely to the treatment of inflammatory diseases of the mucous membranes, such as upper respiratory tract

d-arabinofuranosyl)-n-purine, method for their preparation and use and pharmaceutical composition" target="_blank">

The invention relates to mono-, di - or tri-esters of 2-amino-6-(C1-C5-alkoxy)-9-(-D-arabinofuranosyl)-N-purine General formula (I)

< / BR>
where arabinofuranosyl residue substituted for 2'-, 3'- or 5'-positions, and esters formed by carboxylic acids, in which decarbonising part selected from n-propyl, tert-butyl, n-butyl, methoxymethyl, benzyl, phenoxymethyl, phenyl, methanesulfonyl and succinyl
The invention relates to medicine, namely the subject of the invention are tablets based on partially or fully water-soluble natural and/or synthetic polymers selected from rubbers, alginates, Cartagena, starch, pectin and gelatin containing poly-(dimethyl-siloxanes) (Dimethicone, simethicone), and the method of their manufacture
The invention relates to medicine, in particular to methods for treating obesity

The invention relates to oligonucleotides and their derivatives to suppress the expression of isoprenylation, pharmaceutical compositions containing such compounds and to the use of such compositions of oligonucleotides for the treatment or therapy of the human or animal diseases caused by abnormal and/or unregulated proliferation

d-arabinofuranosyl-2-amino-6-methoxy - n-purines" target="_blank">

The invention relates to 9--D-arabinofuranosyl-2-amino-6-methoxy-N-purine and its derivatives and 9--D-arabinofuranosyl-2-amino-6-methoxy-N-purine and its derivatives and pharmaceutical compositions comprising these derivatives of purine, which is active against viral infections in humans caused by the virus stays zoster (VZV)

The invention relates to a substance which can be used for a new purpose, namely as a drug against HIV-1

FIELD: medicine.

SUBSTANCE: method involves carrying out hernia removal in intralaminar way. Posterior longitudinal ligament defect is covered with Tacho-Comb plate after having done disk cavity curettage. Subcutaneous fat fragment on feeding pedicle is brought to dorsal surface of radix and dural sac.

EFFECT: enhanced effectiveness of treatment; reduced risk of traumatic complications.

1 dwg

Up!