WO1990015813A1 - Alpha anomer oligonucleotide compounds which inhibit retrovirus replication - Google Patents

Alpha anomer oligonucleotide compounds which inhibit retrovirus replication Download PDF

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Publication number
WO1990015813A1
WO1990015813A1 PCT/FR1990/000412 FR9000412W WO9015813A1 WO 1990015813 A1 WO1990015813 A1 WO 1990015813A1 FR 9000412 W FR9000412 W FR 9000412W WO 9015813 A1 WO9015813 A1 WO 9015813A1
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sequence
alpha
group
oligonucleotide
compounds
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PCT/FR1990/000412
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French (fr)
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Jean-Louis Imbach
Bernard Rayner
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Centre National De La Recherche Scientifique (Cnrs)
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • A61P31/18Antivirals for RNA viruses for HIV
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-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
    • 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/33Chemical structure of the base
    • C12N2310/337Chemical structure of the base in alpha-anomeric form

Definitions

  • the present invention relates to chemical compounds consisting of an oligonucleotide or an oligodeoxynucleotide comprising a chain of nucleotides having the non-natural anomeric configuration alpha as opposed to the natural anomeric configuration beta.
  • the present invention relates to such oligonucleotide compounds useful for inhibiting the replication of a retrovirus and, in particular, an oligonucleotide compound useful for inhibiting the replication of the HIV virus.
  • the compounds according to the invention consist of an oligoribonucleotide or oligodeoxyribonucleotide sequence of unnatural anomaly alpha, said sequence being complementary to a sequence included in the sequence called TBS or PBS (tRNA binding site or primer binding site) consisting of the binding site of the viral RNA replication tRNA primer or in a viral RNA sequence upstream of the TBS sequence.
  • TBS tRNA binding site or primer binding site
  • alpha oligonucleotide compounds according to the invention can optionally be linked coschreibly to an effector agent, such as an intercalating agent, in reactive or photoactivable chemical radical.
  • an effector agent such as an intercalating agent, in reactive or photoactivable chemical radical.
  • the radicals B can be identical or different and each represent a base of a nucleic acid possibly modified and attached to the glycosidic cycle according to an unnatural alpha anomeric configuration.
  • radicals B being complementary one to one of the bases of the target oligonucleotide sequence included in the TBS sequence or a sequence upstream of the proviral RNA;
  • the radicals X may be the same or different and each represents an oxoanion Q ⁇ , a thioanion S ⁇ , an alkyl group, an alkoxy group, aryloxy, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group;
  • R and R ' which may be the same or different, each represent a hydrogen atom or a group -Y-Z;
  • Y represents a straight or branched alkylene radical -alk- or a radical chosen from
  • E can have the same meanings as X;
  • J represents a hydrogen atom or a hydroxy group
  • Z is a radical corresponding to an effector
  • n is an integer including O
  • L represents an oxygen atom, a sulfur atom or a group
  • formula I represents a sequence of nucieotides which may be identical or different, n simply indicating the number of nucieotides included in the molecule; n is preferably a number between 1 and 30, and more preferably between 1 and 20 and depends on the size of the target sequence of the RNA of the retrovirus. In general, for example, the TBS sequences have about twenty nucieotides.
  • effector radicals correspond to effector agents which are compounds known in the techniques relating to nucleic acids. These are, for example, compounds capable of "intercalating" in the structure of DNAs or RNAs.
  • intercalating agents are especially constituted by polycyclic compounds having a plane configuration such as acridine, furocoumarin, daunomycin, 1,10-phenanthroline, phenanthridinium, prophyrins, dipyrido derivatives (1,2 -a: 3 ', 2'-d) imidazole, ellipticine or ieliipticinium and their derivatives.
  • effector agents can also be reactive chemical radicals such as chemical cleavage radicals, that is to say that these radicals can directly or indirectly react to split a chain of nucleotides.
  • these reactive chemical radicals will be activatable, for example chemically or photochemically.
  • the activatable cleavage reactive groups are, for example, derivatives of compounds such as:
  • the radical B consists of a base of a nucleic acid linked to the sugar part of the nucleotide by an alpha anomeric configuration. It can be thymine, adenine, cytosine, guanine or uracil. However, it is also possible to use modified nucleic acid bases, in particular halogenated or azidic, for example the
  • 5-bromo-uracil or 8-azidoadenine or amino derivatives such as 2-amino-adenine and its substituted derivatives, for example on the N by an aminoalkylene group or by an azidophenylalkyiene group; or guanine substituted on O ⁇ , for example by a ( ⁇ -alkylene) -9-acridine group or 8- ( ⁇ -aminoalkyl) -ammo-adenine and its derivatives substituted on NH 2 in u) by a group acridine.
  • the usual bases are therefore considered, as well as the possibility of introducing modified bases.
  • Des. "effector" bases capable of covalently binding to a complementary beta strand, may be introduced.
  • the functionalization of C or T by an aziridine group in position 4 leads to the formation of covending cross-links CH 2 -CH 2 between the two complementary strands on G and A respectively.
  • the radical B is chosen from thymine, adenine, cytosine, guanine, 4-azido-cytosine or 4-az ⁇ do-thymine and 8-azido-adenine, uracil, 5 bromo-uracil.
  • the radical X although it preferably represents an oxoanion, can have other meanings; when the radical X represents an alkyl group, it is preferably a C 1 to C 7 lower alkyl group and, for example, the ethyl, methyl or propyl groups; when the radical X represents an alkoxy group, it is preferably a C 1 to C 7 lower alkoxy group, for example the methoxy or ethoxy group; when X represents an amino-alkyl or amino-alkoxy group; it may be a mono-substituted, disubstituted amino-alkyl group or else an amino radical in the form of a quaternary ammonium salt.
  • the substituents are preferably lower alkyl radicals as defined above; as for the alkyl or alkoxy chain connecting the amino radical to the phosphorus, it is preferably a straight branched chain comprising from 1 to 10 carbon atoms.
  • the alkyl or alkoxy radical is substituted by a nitrogen heterocycle, it is in particular a saturated 5-6-membered ring comprising a nitrogen atom which may be quaternary.
  • the radical X is a thioalkyl radical, it is preferably a lower thioalkyl radical, that is to say which contains between 1 and 7 carbon atoms.
  • the radical -alk- is preferably a straight or branched alkylene radical having from 1 to 10 carbon atoms.
  • the effector can therefore be introduced via a chain
  • the preceding compounds are also concerned in the form of a salt with bases or acids, and the compounds in racemic form, or in the form of purified or mixed optical R or S isomers.
  • the preceding compounds are also concerned in the D or L series.
  • the subject of the present invention is also anti-sweat pharmaceutical compositions containing, as active substance, said compounds, and in particular compositions useful for the treatment of viral affections of RNA viruses, such as the HIV1 AIDS virus.
  • the subject of the present invention is the use of the compounds according to the invention for the preparation of antiviral pharmaceutical compositions in particular useful for the treatment of AIDS.
  • the application of the alpha oligonucleotide compounds according to the present invention consists in inhibiting the replication of retroviruses and in particular of the human immunodeficiency virus HIV1 from AIDS.
  • LAV iymphadenopathies
  • LAV HTLV III virus
  • ARV AIDS - related virus
  • DNA polymerase - dependent RNA also called reverse transcriptase or more commonly reverse transcriptase.
  • This enzyme copies into so-called complementary single-strand DNA (cDNA), viral RNA. This requires that it find an oligonucleotide primer paired at the 3 'end of the RNA template to be copied.
  • Reverse transcriptase is both necessary to establish the provirai state, to start the replication of the virus, and to possibly transform the cell.
  • the primer is a tRNA of cell origin present inside virions.
  • reverse transcriptase RT
  • the 3 'tRNA of lysine serves as the start of DNA which will be synthesized.
  • the synthesized strand is guided by the primer groove.
  • the DNA strand thus synthesized is then in the form of a single strand, following RNase activity linked to RT and a complementary DNA strand is synthesized via polymerase DNA activity dependent on RT.
  • the structure of a retrovirus before and after its integration into the DNA of a host cell can be schematized as follows: a) Integration of the retrovirus into the DNA of the host cell, viral RNA
  • RNA of a retrovirus is a single stranded RNA in which the coding regions are flanked by sequences essential for viral replication and expression. These sequences are, from the 5 'to 3' end:
  • tRNA binding site This is the site where the 3 'tRNA binds (which will serve as a primer during replication).
  • the complementary and antiparallel links will cover around 20 bps,
  • - coding region it is located in the center, it contains very few genes. Those are :
  • glyco for "group specifies antigen”
  • gene of the group antigen which codes for a polyprotein which, cut out, gives the proteins of the nuciéoid.
  • these internal proteins one of them carries the group's antigenicity
  • polymerase for "poiymerase” which codes for reverse transcriptase
  • env for "envelope” which codes for envelope giycoproteins.
  • env for "envelope”
  • coding region can also be found a gene coding for a viral protein (or a segment of protein) specific for the retrovirus (For example, in oncogenic retroviruses, there is an onc gene. This is not the case for the virus of AIDS which is not an oncogenic retrovirus, it does not cancerize the cells but destroys them).
  • the transcribed double stranded DNA molecule is longer than the corresponding genomic DNA. Indeed, there is addition at the 2 ends of 2 sequences:
  • LTR long terminal repeat
  • the double stranded linear DNA becomes circular closed, then it is again opened to be integrated into the DNA of the host cell.
  • the integrated viral DNA is called "provirus".
  • the dinuciéotides (TT, AA) located at the ends of the transcribed DNA (and being part of U 3 or U 5 ) are eliminated.
  • direct repeat a direct repeated sequence
  • All CDs are 4 to 6 bps long. Their sequences are different. For the same provirus, moreover, there are different DRs in the DNA of a host cell. The site of integration of the virus is therefore not specific, the virus can be integrated into the genome of the host in several different places.
  • antisense oligonucleotides that is to say segments of DNA complementary to a portion of the messenger RNA of the virus which directly codes for the proteins to be synthesized. by the ribosome.
  • alpha oligonucleotides form heteroduplexes with their beta complementary oligonucleotides and that these are not substrates for the enzyme RNase H, it could be thought that the inhibition of the expression of proteins by the alpha oligonucleotide perhaps could be accomplished by preventing the ribosome from translating mRNA into viral protein.
  • the approach according to the present invention aimed at using oligonucleotides complementary to an initial sequence of the viral RNA and in particular of the initial lvsin tRNA binding site on provirai RNA, has been shown to be effective.
  • FIG. 1 represents the inhibition of the viral production of MT4 cell by the 5 'alpha d oligonucleotide (ACCGCGGGCTCTGTCCCTG) 3' (oligo alpha "PBS") by assaying the activity (cpm) of reverse transcriptase.
  • EXAMPLE 1 Cytotoxicity of alpha oligonucleotides
  • oligonucleotide whatever type of oligonucleotide is used, these end up more or less rapidly being degraded by cellular enzymes. It is shown below that the oligonucleotides alpha and their catabolites nucleosides al pha and nucleotide alpha do not exhibit cytotoxicity.
  • the oligonucleotide alpha-d (CCTCTCGTTCTTTAC) oligo alpha Tat (complementary) of the Tat sequence directed, a priori, against any site of the genome of the MT-4 cells, has a CD 50 greater than 250 ⁇ g / ml.
  • alpha oligothymidylates alpha- (dT) n (2 ⁇ n 12 12) have been found to be non-cytotoxic to the same cell line.
  • al pha nucleosides and alpha nucleotides which can result from the hydrolysis of oligonucleotides are also devoid of cytotoxicity against various cell lines (Hela, Vero, primary rabbit kidney cells. MT-4).
  • the evaluation is based on the study of the cytopathogenic effect of the HIV1 virus on the MT4 cell line, after infection with the HIV1 virus, the formation of syncitia is observed 4 to 6 days after infection, followed by production of viral particles, then by cell death.
  • T4 lymphocytes essential for immune defense are the first cause of the immune deficiency characteristic of HIV infection.
  • This virus kills cells by multiplying in them; when it escapes, it damages the cell membrane.
  • HIV may also kill T4 cells indirectly, by the gp l protein 20 present on the membrane of infected cells.
  • T4 lymphocytes carry a surface molecule, the CD4 receptor, to which the GP120 protein binds; healthy T4 cells attach to the protein and fuse with the infected cell.
  • the cell mass that forms is a "syncytiuni", unable to survive, and all the healthy cells that compose it die with the infected cell. HIV can also trigger normal immune responses against infected lymphocytes.
  • defense cytotoxic cells destroy an infected cell carrying viral proteins on its surface.
  • the protein gp120 sometimes circulates freely in solution in the blood of infected subjects; it binds to the CD4 receptor of healthy cells, simulating an infection and triggering a reaction of destruction of uninfected cells.
  • Syncytia are structures formed by several nuclei surrounded by a single cell membrane; These are signs of HIV infection in cell cultures; Syncytia are formed when infected cells that synthesize and transport the gp120 molecule on their surface merge with healthy cells carrying the CD4 molecule. b) Compound tested
  • the MT4 cells on day 2 after the last passage are pre-incubated for 1 hour at 37 ° C. with the successive dilutions of the compound at the rate of 3.10 cells per 100 ⁇ l of compound (in 96-well micropaque).
  • the infection is carried out in microwells by adding 100 ⁇ l of a 10 - 4 dilution of the HIV1 virus (the dilution of the virus was determined to induce the formation of syncitia in 4 days).
  • the infected MT4 cells are washed 5 times before being cultured at a concentration of 3.10 cells per ml, in a 24-well microplate, in the presence of the various dilutions chosen.
  • the cells are diluted 3 or 4 times and adjusted to the concentration of 3.10 cells / ml with RPM 1 medium 10% FCS 1% PSN 1% Gluta always in the presence of oligo alpha PBS.
  • FIG. 1 RT reverse transcriptase
  • FIG. 1 RT Monitoring of viral production of MT4 cells is carried out by assaying the activity of reverse transcriptase (FIG. 1 RT) in the culture every 3 or 4 days. Briefly, the enzymatic activity is tested using a synthetic primer and a H 3 radiolabelled substrate "in vitro". The quantity of radiolabelled material precipitated, expressed in counts per minute, is proportional to the activity of the reverse transcriptase itself proportional to the quantity of virus produced by the infected MT4 cells.
  • PBS used, the viral production remains lower than that obtained in the presence of the HIV1 virus, with a peak of delayed maximum viral production.
  • the oligonucleotide "alpha Tat” corresponds to the oligonucleotide alpha d 5 GTAAAAGTCTTAACCCAC 3 ' . It is complementary to the sequence of the Tat gene of the AIDS virus.
  • the oligo nucleotide "alpha random” corresponds to any olna nucleotide alpna d 5 ' ACTGACTGACTGACTGAC 3' .
  • the oligonucleotide "alpha PBS” shows an inhibition of viral production for concentrations of 100 ⁇ g / ml and 50 ⁇ g / mi.
  • oligonucleotide "alpha PBS” has an antiviral effect on the HIV1 virus at concentrations of 100 ⁇ g / ml and 50 ⁇ g / ML, while the oligonucleotide "alpha Tat" and the oligonucleotide " alpha Random "have no antiviral effect.

Abstract

The invention relates to oligonucleotide or oligodeoxynucleotide compounds with an alpha anomer structure and to their therapeutic applications. These compounds are used to inhibit RNA virus replication, in particular the alpha oligonucleotide d5'(ACCGCGGGCTTGTCCCTG)3' is used to inhibit the replication of AIDS virus HIV.

Description

COMPOSES OLIGONUCLEOTIDIQUES ANOMERES ALPHA INHIBANT LA REPLICATION DES RETROVIRUS . ALPHA ANOMERIC OLIGONUCLEOTIDE COMPOUNDS INHIBITING REPLICATION OF RETROVIRUSES.
La présente invention concerne des composés chimiques constitués par un oligonucleotide ou un oligodésoxynucléotide comportant un enchaînement de nucléotides possédant la configuration anomerique non naturelle alpha par opposition à la configuration anomerique naturelle bêta.  The present invention relates to chemical compounds consisting of an oligonucleotide or an oligodeoxynucleotide comprising a chain of nucleotides having the non-natural anomeric configuration alpha as opposed to the natural anomeric configuration beta.
De tels composés chimiques ont fait l'objet de demandes de brevets, en particulier les demandes FR S7 04339 (PCT WO 88/04301 ) et FR 88 12264 au nom de la demanderesse auxquelles il conviendra de se reporter en particulier pour y trouver la description de leurs procédés de préparation.  Such chemical compounds have been the subject of patent applications, in particular applications FR S7 04339 (PCT WO 88/04301) and FR 88 12264 in the name of the applicant to which reference should be made in particular to find the description. of their preparation processes.
Plus précisément, la présente invention concerne de tels composés oligonucléotidiques utiles pour inhiber la replication d'un rétrovirus et, en particulier, un composé oligonucléotidique utile pour inhiber la replication du virus VIH.  More specifically, the present invention relates to such oligonucleotide compounds useful for inhibiting the replication of a retrovirus and, in particular, an oligonucleotide compound useful for inhibiting the replication of the HIV virus.
Selon leurs caractéristiques essentielles, les composés selon l'invention consistent en une séquence d'oligoribonucléotide ou oligodésoxyribonuciéotide d'anomerie non naturelle alpha, ladite séquence étant complémentaire d'une séquence comprise dans la séquence dite TBS ou PBS (ARNt binding site ou primer binding site) consistant dans le site de fixation de l'amorce ARNt de replication de l'ARN viral ou dans une séquence de l'ARN viral en amont de la séquence TBS.  According to their essential characteristics, the compounds according to the invention consist of an oligoribonucleotide or oligodeoxyribonucleotide sequence of unnatural anomaly alpha, said sequence being complementary to a sequence included in the sequence called TBS or PBS (tRNA binding site or primer binding site) consisting of the binding site of the viral RNA replication tRNA primer or in a viral RNA sequence upstream of the TBS sequence.
Parmi ces séquences en amont de la séquence TBS ou PBS, on peut citer notamment la séquence CAP.  Among these sequences upstream of the TBS or PBS sequence, there may be mentioned in particular the CAP sequence.
Les composes oligonucléotidiques alpha selon l'invention peuvent éventuellement être liés de façon covaiente à un agent effecteur, tel qu'un agent intercalant, in radical chimique réactif ou photoactivable.  The alpha oligonucleotide compounds according to the invention can optionally be linked covaitly to an effector agent, such as an intercalating agent, in reactive or photoactivable chemical radical.
Dans un mode de réalisation des composés selon l'invention, ceux-ci présentent la formule suivante :  In one embodiment of the compounds according to the invention, they have the following formula:
Figure imgf000003_0001
dans laquelle :
Figure imgf000003_0001
in which :
Les radicaux B peuvent être identiques ou différents et représentent chacun une base d'un acide nucléique éventuellement modifiée et rattachée au cycle glycosidique selon une configuration anomerique alpha non naturelle.  The radicals B can be identical or different and each represent a base of a nucleic acid possibly modified and attached to the glycosidic cycle according to an unnatural alpha anomeric configuration.
Les radicaux B étant complémentaires un à un des bases de la séquence oligonucléotidiques cibles comprise dans la séquence TBS ou une séquence en amont de l'ARN proviral ;  The radicals B being complementary one to one of the bases of the target oligonucleotide sequence included in the TBS sequence or a sequence upstream of the proviral RNA;
Les radicaux X peuvent être identiques ou différents et représentent chacun un oxoanion Q , un thioanion S , un groupe alkyie, un groupe alcoxy, aryloxy, un groupe aminoalkyle, un groupe aminoalcoxy, un groupe thioalkyle ; The radicals X may be the same or different and each represents an oxoanion Q , a thioanion S , an alkyl group, an alkoxy group, aryloxy, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group;
R et R', qui peuvent être identiques ou différents, représentent chacun un atome d'hydrogène ou un groupement -Y-Z ;  R and R ', which may be the same or different, each represent a hydrogen atom or a group -Y-Z;
Y représente un radical alcoylène droit ou ramifié -alk- ou un radical choisi parmi  Y represents a straight or branched alkylene radical -alk- or a radical chosen from
Figure imgf000004_0001
Figure imgf000004_0001
avec U = O, N ou S  with U = O, N or S
ou bien un radical -Y"-O-Y' où Y" ou Y' peuvent avoir les significations données pour Y ;  or else a radical -Y "-O-Y 'where Y" or Y' may have the meanings given for Y;
E peut avoir les mêmes significations que X ;  E can have the same meanings as X;
J représente un atome d'hydrogène ou un groupe hydroxy ;  J represents a hydrogen atom or a hydroxy group;
Z est un radical correspondant à un agent effecteur ;  Z is a radical corresponding to an effector;
n est un nombre entier y compris O ;  n is an integer including O;
L représente un atome d'oxygène, un atome de soufre ou un groupe L represents an oxygen atom, a sulfur atom or a group
-NH-. Dans cette formule I, on utilise la représentation condensée des nucléosides suivante : -NH-. In this formula I, the following condensed representation of the nucleosides is used:
Figure imgf000005_0001
Figure imgf000005_0001
qui correspond à la formule développée :  which corresponds to the structural formula:
Figure imgf000005_0002
Figure imgf000005_0002
sur laquelle ont été mentionnées les extrémités (3') et (5'). on which the ends (3 ') and (5') have been mentioned.
Il convient de remarquer que la formule I représente un enchaînement de nuciéotides qui peuvent être identiques ou différents, n indiquant simplement le nombre de nuciéotides compris dans la molécule ; n est de préférence un nombre compris entre 1 et 30, et de préférence encore entre 1 et 20 et dépend de la taille de la séquence cible de l'ARN du rétrovirus. En général, par exemple, les séquences TBS ont une vingtaine de nuciéotides.  It should be noted that formula I represents a sequence of nucieotides which may be identical or different, n simply indicating the number of nucieotides included in the molecule; n is preferably a number between 1 and 30, and more preferably between 1 and 20 and depends on the size of the target sequence of the RNA of the retrovirus. In general, for example, the TBS sequences have about twenty nucieotides.
Les radicaux effecteurs correspondent à des agents effecteurs qui sont des composés connus dans les techniques touchant aux acides nucléiques. Il s'agit par exemple des composés capables de "s'intercaler" dans la structure des ADN ou des ARN.  The effector radicals correspond to effector agents which are compounds known in the techniques relating to nucleic acids. These are, for example, compounds capable of "intercalating" in the structure of DNAs or RNAs.
Ces agents d'intercalation sont notamment constitués par des composés polycycliques ayant une configuration plane tels que i'acridine, la furocoumarine, la daunomycine, la 1,10-phénanthroline, la phénanthridinium, les prophyrines, les dérivés de la dipyrido (1,2-a : 3', 2'-d) imidazole, l'ellipticine ou i'eliipticinium et leurs dérivés. Ces agents effecteurs peuvent aussi être des radicaux chimiques réactifs tels que des radicaux chimiques de scission, c'est-à-dire que ces radicaux peuvent directement ou indirectement réagir pour scinder une chaîne de nuciéotides. De préférence, ces radicaux chimiques réactifs seront activables, par exemple par voie chimique ou photochimique. These intercalating agents are especially constituted by polycyclic compounds having a plane configuration such as acridine, furocoumarin, daunomycin, 1,10-phenanthroline, phenanthridinium, prophyrins, dipyrido derivatives (1,2 -a: 3 ', 2'-d) imidazole, ellipticine or ieliipticinium and their derivatives. These effector agents can also be reactive chemical radicals such as chemical cleavage radicals, that is to say that these radicals can directly or indirectly react to split a chain of nucleotides. Preferably, these reactive chemical radicals will be activatable, for example chemically or photochemically.
Les groupes réactifs de scission activables sont, par exemple, des dérivés de composés tels que :  The activatable cleavage reactive groups are, for example, derivatives of compounds such as:
- l'acide éthylène-diamine-tétracétique,  - ethylene-diamine-tetracetic acid,
- l'acide diéthylène-triamine-pentaacétique,  - diethylene-triamine-pentaacetic acid,
- les porphyrines,  - porphyrins,
- la 1,10-phénanthroIine, le psoralène et autres groupes aromatiques absorbant les radiations du proche U.V. et du visible.  - 1,10-phenanthroine, psoralen and other aromatic groups absorbing near UV and visible radiation.
Ces groupements chimiquement activables en présence d'ions métalliques, d'oxygène et d'un agent réducteur, induisent des coupures dans des séquences d'acides nucléiques situées dans leur voisinage.  These chemically activatable groups in the presence of metal ions, oxygen and a reducing agent, induce cleavages in nucleic acid sequences located in their vicinity.
Le radical B est constitué par une base d'un acide nucléique liée à la partie sucre du nucléotide par une configuration anomerique alpha. Ce peut être la thymine, l'adénine, la cytosine, la guanine ou l'uracile. Mais il est également possible d'utiliser des bases d'acides nucléiques modifiées, notamment halogénées ou azidées, par exemple la The radical B consists of a base of a nucleic acid linked to the sugar part of the nucleotide by an alpha anomeric configuration. It can be thymine, adenine, cytosine, guanine or uracil. However, it is also possible to use modified nucleic acid bases, in particular halogenated or azidic, for example the
5-bromo-uracile ou la 8-azidoadénine ; ou des dérivés aminés comme la 2-amino-adénine et ses dérivés substitués, par exemple sur le N par un groupe aminoalkylène ou par un groupe azidophénylalkyiène ; ou la guanine substituée sur le O, par exemple par un groupe (ω-alkylène)-9-acridine ou la 8-(ω-aminoalkyl)-ammo-adénine et ses dérivés substitués sur le NH2 en u) par un groupe acridine. 5-bromo-uracil or 8-azidoadenine; or amino derivatives such as 2-amino-adenine and its substituted derivatives, for example on the N by an aminoalkylene group or by an azidophenylalkyiene group; or guanine substituted on O , for example by a (ω-alkylene) -9-acridine group or 8- (ω-aminoalkyl) -ammo-adenine and its derivatives substituted on NH 2 in u) by a group acridine.
Selon la présente invention, sont donc considérées les bases usuelles, ainsi que la possibilité d'introduction de bases modifiées. Des. bases "effectrices", susceptibles de se lier par covalence à un brin bêta complémentaire, pourront être introduites. Ainsi, la fonctionnalisation de C ou T par un groupement aziridine en position 4 conduit à la formation de pontages covaients CH2-CH2 entre les deux brins complémentaires sur respectivement G et A. Donc, plus particulièrement, le radical B est choisi parmi la thymine, l'adénine, la cytosine, la guanine, la 4-azido-cytosine ou la 4-azιdo-thymine et la 8-azido-adénine, l'uracile, la 5 bromo-uracile. According to the present invention, the usual bases are therefore considered, as well as the possibility of introducing modified bases. Des. "effector" bases, capable of covalently binding to a complementary beta strand, may be introduced. Thus, the functionalization of C or T by an aziridine group in position 4 leads to the formation of covaient cross-links CH 2 -CH 2 between the two complementary strands on G and A respectively. Therefore, more particularly, the radical B is chosen from thymine, adenine, cytosine, guanine, 4-azido-cytosine or 4-azιdo-thymine and 8-azido-adenine, uracil, 5 bromo-uracil.
Le radical X, bien qu'il représente de préférence un oxoanion, peut avoir d'autres significations ; lorsque le radical X représente un groupement alkyle, il s'agit de préférence d'un groupement alkyle inférieur en C 1 à C 7 et, par exemple, les groupements éthyle, méthyle ou propyle ; lorsque le radical X représente un groupe alcoxy, il s'agit de préférence d'un groupement alcoxy inférieur C 1 à C7, par exemple le groupement méthoxy ou éthoxy ; lorsque X représente un groupement amino-alkyle ou amino-alcoxy ; il peut s'agir d'un groupement amino-alkyle mono-substitué, disubstitué ou bien d'un radical amino sous forme de sel d'ammonium quaternaire. Dans ces conditions, les substituants sont, de préférence, des radicaux aikyles inférieurs comme définis précédemment ; quant à la chaîne alkyle ou alcoxy reliant le radical amino au phosphore, il s'agit de préférence d'une chaîne droite ramifiée comportant de 1 à 10 atomes de carbone. Lorsque le radical alkyle ou alcoxy est substitué par un hétérocycle azoté, il s'agit notamment de cycle saturé à 5-6 chaînons comportant un atome d'azote qui peut être quaternaire. Enfin, lorsque le radical X est un radical thioalkyle, il s'agit de préférence d'un radical thioalkyle inférieur, c'est-à-dire qui comporte entre 1 et 7 atomes de carbone. The radical X, although it preferably represents an oxoanion, can have other meanings; when the radical X represents an alkyl group, it is preferably a C 1 to C 7 lower alkyl group and, for example, the ethyl, methyl or propyl groups; when the radical X represents an alkoxy group, it is preferably a C 1 to C 7 lower alkoxy group, for example the methoxy or ethoxy group; when X represents an amino-alkyl or amino-alkoxy group; it may be a mono-substituted, disubstituted amino-alkyl group or else an amino radical in the form of a quaternary ammonium salt. Under these conditions, the substituents are preferably lower alkyl radicals as defined above; as for the alkyl or alkoxy chain connecting the amino radical to the phosphorus, it is preferably a straight branched chain comprising from 1 to 10 carbon atoms. When the alkyl or alkoxy radical is substituted by a nitrogen heterocycle, it is in particular a saturated 5-6-membered ring comprising a nitrogen atom which may be quaternary. Finally, when the radical X is a thioalkyl radical, it is preferably a lower thioalkyl radical, that is to say which contains between 1 and 7 carbon atoms.
Le radical -alk- est de préférence un radical alcoylène droit ou ramifié ayant de 1 à 10 atomes de carbone.  The radical -alk- is preferably a straight or branched alkylene radical having from 1 to 10 carbon atoms.
En particulier, à partir des fonctions alcool terminales 3' ou 5' de l'oligomère alpha, l'effecteur peut donc être introduit via une chaîne In particular, from the 3 ′ or 5 ′ terminal alcohol functions of the alpha oligomer, the effector can therefore be introduced via a chain
(CH2)n liée à une fonctionnalisation Z' de natures diverses à la partie osidique de l'oligomère. (CH 2 ) n linked to a functionalization Z ′ of various natures at the osidic part of the oligomer.
A partir de la formule  From the formula
3'  3 '
ou -O-Z'-(CH2)n-effecteur (Z) on obtiendra, selon ce que représente Z', par exemple or -O-Z '- (CH 2 ) n -effector (Z) we will obtain, depending on what Z 'represents, for example
- un phosphate ou méthyl phosphonate de formule  - a phosphate or methyl phosphonate of formula
Figure imgf000008_0001
Figure imgf000008_0001
U = O, N ou S U = O, N or S
- un éther de formule générale  - an ether of general formula
Figure imgf000008_0002
Figure imgf000008_0002
- un ester de formule générale
Figure imgf000008_0003
- an ester of general formula
Figure imgf000008_0003
ou encore or
- un carbamate de formule générale - a carbamate of general formula
Figure imgf000008_0004
Figure imgf000008_0004
Selon la présente invention sont concernés également les composés précédents sous forme de sel avec des bases ou des acides, et les composés sous forme racemique, ou sous forme d'isomères R ou S optiques purifiés ou en mélange.  According to the present invention, the preceding compounds are also concerned in the form of a salt with bases or acids, and the compounds in racemic form, or in the form of purified or mixed optical R or S isomers.
Selon la présente invention sont concernés également les composés précédents cans les séries D ou L.  According to the present invention, the preceding compounds are also concerned in the D or L series.
Dans un wode de réalisation de l'invention, on utilise des oiigodésoxynucléotides aipna. II s'agit des composés pour lesquels X = O; J, R et R' = H ; B est choisi parmi A, C, T et G, et L est un atome d'oxygène dans la f ormule I. In an embodiment of the invention, aipna oiodeodesynynucleotides are used. These are the compounds for which X = O ; J, R and R '= H; B is chosen from A, C, T and G, and L is an oxygen atom in the formula I.
La présente imention a également pour objet des compositions pharmaceutiques antiwraies contenant à titre de substance active lesdits composés, et notamment des compositions utiles pour le traitement des affections virales des virus à ARN, tels que le virus VIH1 du SIDA.  The subject of the present invention is also anti-sweat pharmaceutical compositions containing, as active substance, said compounds, and in particular compositions useful for the treatment of viral affections of RNA viruses, such as the HIV1 AIDS virus.
Enfin, la présente invention a pour objet l'utilisation des composés selon l'invention à la préparation de compositions pharmaceutiques antivirales notamment utiles pour le traitement du SIDA. Comme mentionné ci-dessus, l'application des composés oligonucléotidiques alpha selon la présente invention consiste à inhiber la replication des rétrovirus et notamment du virus de l'immunodéficience humaine VIH1 du SIDA. Finally, the subject of the present invention is the use of the compounds according to the invention for the preparation of antiviral pharmaceutical compositions in particular useful for the treatment of AIDS. As mentioned above, the application of the alpha oligonucleotide compounds according to the present invention consists in inhibiting the replication of retroviruses and in particular of the human immunodeficiency virus HIV1 from AIDS.
Le rétrovirus associé à des iymphadénopathies (LAV), également appelé virus HTLV III (HTLV - human T leukemia virus) ou AIDS - related virus (ARV) a été baptisé plus récemment VIHI et est en effet reconnu comme l'agent responsable du SIDA.  The retrovirus associated with iymphadenopathies (LAV), also called HTLV III virus (HTLV - human T leukemia virus) or AIDS - related virus (ARV) was more recently baptized HIVI and is indeed recognized as the agent responsible for AIDS.
Les virus à ARN et notamment le virus du SIDA ont un mécanisme de multiplication qui fait effectivement intervenir l'enzyme RNA viruses and in particular the AIDS virus have a multiplication mechanism which effectively involves the enzyme
ADN polymérase - ARN dépendante encore appelée transcriptase inverse ou plus communément réverse transcriptase. Cette enzyme copie en ADN monobrin dit complémentaire (ADNc), l'ARN viral. Il faut pour cela qu'elle trouve une amorce oligonucléotidique appariée à l'extrémité 3' de la matrice d'ARN à copier. DNA polymerase - dependent RNA also called reverse transcriptase or more commonly reverse transcriptase. This enzyme copies into so-called complementary single-strand DNA (cDNA), viral RNA. This requires that it find an oligonucleotide primer paired at the 3 'end of the RNA template to be copied.
La transcriptase réverse est à la fois nécesaire à l'établissement de l'état provirai, au démarrage de la replication du virus, et à l'éventuelle transformation de la cellule.  Reverse transcriptase is both necessary to establish the provirai state, to start the replication of the virus, and to possibly transform the cell.
Chez les rétrovirus, l'amorce est un ARNt d'origine cellulaire présent à l'intérieur des virions. Précisément, quand le virus infecte une cellule, la transcriptase reverse (RT) se fixe à l'ARN virale et l'ARNt-3' de la lysine sert de début à l'ADN qui sera synthétisé. Le brin synthétisé est guidé par le sillon d'amorce. Le brin d'ADN ainsi synthétisé est alors sous forme de monobrin, suite à l'activité RNasique liée à la RT et un brin complémentaire d'ADN est synthétisé via l'activité polymerasique DNA dépendante de la RT.  In retroviruses, the primer is a tRNA of cell origin present inside virions. Specifically, when the virus infects a cell, reverse transcriptase (RT) binds to viral RNA and the 3 'tRNA of lysine serves as the start of DNA which will be synthesized. The synthesized strand is guided by the primer groove. The DNA strand thus synthesized is then in the form of a single strand, following RNase activity linked to RT and a complementary DNA strand is synthesized via polymerase DNA activity dependent on RT.
La structure d'un rétrovirus avant et après sont intégration dans le DNA d'une cellule-hôte peut se schématiser comme suit : a) Intégration du rétrovirus dans le DNA de la cellule-hôte ARN viralThe structure of a retrovirus before and after its integration into the DNA of a host cell can be schematized as follows: a) Integration of the retrovirus into the DNA of the host cell, viral RNA
Figure imgf000010_0001
transcriptase reverse
Figure imgf000010_0002
Figure imgf000010_0001
reverse transcriptase
Figure imgf000010_0002
DNA 2 brins 2 strand DNA
Figure imgf000010_0003
Figure imgf000010_0004
Figure imgf000010_0003
Figure imgf000010_0004
DNA cellule-hôte
Figure imgf000010_0005
Host cell DNA
Figure imgf000010_0005
Figure imgf000010_0006
Figure imgf000010_0006
b) Structure moléculaire du génome du rétrovirus durant les différentes phases de l'intégration  b) Molecular structure of the retrovirus genome during the different phases of integration
* ARN génomique  * Genomic RNA
5' R-U 5-UU - TBS- NC -région codante- NC - Pur -AA-U 3-R 3' 5 'RU 5 -UU - TBS- NC - coding region - NC - Pure -AA-U 3 -R 3'
(ARN)  (ARN)
Le ARN d'un rétrovirus est un ARN simple brin dans lequel les régions codantes sont flanquées de séquences essentielles pour la replication et l'expression virales. Ces séquences sont, de l'extrémité 5' vers 3' :  The RNA of a retrovirus is a single stranded RNA in which the coding regions are flanked by sequences essential for viral replication and expression. These sequences are, from the 5 'to 3' end:
- R (short "repeat") : une courte séquence à l'extrémité 5' qui comprend en début de séquence une séquence CAP nécessaire pour que puisse s'effectuer la traduction. (Les mêmes nuciéotides seront répétés à l'extrémité 3'),  - R (short "repeat"): a short sequence at the 5 'end which includes at the start of the sequence a CAP sequence necessary for translation to be carried out. (The same nuciéotides will be repeated at the 3 'end),
- U5 : séquence iinique de l'extrémité 5' (constituée d'environ 80 nuciéotides), - U 5 : linear sequence of the 5 ′ end (consisting of approximately 80 nuciéotides),
- UU : 2 nuciéotides à uracile,  - UU: 2 uracil nuciéotides,
- TBS ("tRNA binding site"). Il s'agit du site où se lie (par son extrémité 3') l'ARNt qui servira d'amorce lors de la replication. Les liaisons complémentaires et antiparallèles porteront sur 20 pdb environ,  - TBS ("tRNA binding site"). This is the site where the 3 'tRNA binds (which will serve as a primer during replication). The complementary and antiparallel links will cover around 20 bps,
- NC : une séquence non codante,  - NC: a non-coding sequence,
- région codante : elle est située au centre, elle contient très peu de gènes. Ce sont : - coding region: it is located in the center, it contains very few genes. Those are :
"gag", (pour "group spécifie antigen") ou gène de l'antigène de groupe, qui code pour une polyprotéine qui, découpée, donne les protéines du nuciéoîde. Parmi ces protéines internes, l'une d'entre elles porte i'antigénicité du groupe,  "gag", (for "group specifies antigen") or gene of the group antigen, which codes for a polyprotein which, cut out, gives the proteins of the nuciéoid. Among these internal proteins, one of them carries the group's antigenicity,
"pol" (pour "poiymérase") qui code pour la transcriptase réverse et "env" (pour "enveloppe") qui code pour les giycoprotéines de l'enveloppe. Une d'entre elles porte I'antigénicité de chaque sérotype viral.  "pol" (for "poiymerase") which codes for reverse transcriptase and "env" (for "envelope") which codes for envelope giycoproteins. One of them carries the antigenicity of each viral serotype.
Dans la région codante peut se trouver aussi un gène codant pour une protéine (ou un segment de protéine) virale spécifique du rétrovirus (Par exemple, chez les rétrovirus oncogenes, on trouve un gène onc. Ce n'est pas le cas du virus du SIDA qui n'est pas un rétrovirus oncogène, il ne cancérise pas les cellules mais les détruit).  In the coding region can also be found a gene coding for a viral protein (or a segment of protein) specific for the retrovirus (For example, in oncogenic retroviruses, there is an onc gene. This is not the case for the virus of AIDS which is not an oncogenic retrovirus, it does not cancerize the cells but destroys them).
- NC : une région non codante,  - NC: a non-coding region,
- Pur : une séquence riche en bases puriques,  - Pure: a sequence rich in purine bases,
- AA : 2 nuciéotides à adénine,  - AA: 2 nucieotides with adenine,
- U3 : séquence jjnique de l'extrémité 3' (environ 300 nuciéotides),- U 3 : logical sequence of the 3 ′ end (approximately 300 nucleotides),
- R : (short "repeat"), à l'extrémité 3'. - R: (short "repeat"), at the 3 'end.
* DNA virai après action de la transcriptase réverse : DNA viral non intégré  * DNA virai after action of reverse transcriptase: viral DNA not integrated
5' AA-U3-R-U5-TT-TBS-NC- région codante -NC-Pur-AA-U3-R-U5-TT 3' 5 'AA-U 3 -RU 5 -TT-TBS-NC- coding region -NC-Pur-AA-U 3 -RU 5 -TT 3'
(DNA)  (DNA)
3' TT-U3-R-U5-AA-TBS-NC- région codante-NC-Pyr-TT-U3-R-U _-AA 5' 3 'TT-U 3 -RU 5 -AA-TBS-NC- coding region-NC-Pyr-TT-U 3 -RU _-AA 5'
Après action de la transcriptase réverse, la molécule de DNA double brin transcrite est plus longue que le DNA génomique correspondant. En effet, il y a addition aux 2 extrémités de 2 séquences :  After the action of reverse transcriptase, the transcribed double stranded DNA molecule is longer than the corresponding genomic DNA. Indeed, there is addition at the 2 ends of 2 sequences:
- à l'extrémité 5' est ajoutée une séquence U3, - at the 5 'end is added a U 3 sequence,
- à l'extrémité 3' est ajoutée une séquence U5. - at the 3 'end is added a U 5 sequence.
On appelle "LTR" (long terminal repeat) l'ensemble : U3-R- U5.We call "LTR" (long terminal repeat) the set: U 3 -R- U 5 .
Il y a donc 1 LTR à chacune des 2 extrémités. * DNA viral intégré (provirus) '
Figure imgf000012_0001
There is therefore 1 LTR at each of the 2 ends. * Integrated viral DNA (provirus) '
Figure imgf000012_0001
Le DNA linéaire double brin devient circulaire clos, puis il est à nouveau ouvert pour être intégré dans le DNA de la cellule-hôte. On appelle "provirus" le DNA viral intégré. Lors de l'intégration, les dinuciéotides (TT, AA) situés aux extrémités du DNA transcrit (et faisant partie de U3 ou U5) sont éliminés. The double stranded linear DNA becomes circular closed, then it is again opened to be integrated into the DNA of the host cell. The integrated viral DNA is called "provirus". During integration, the dinuciéotides (TT, AA) located at the ends of the transcribed DNA (and being part of U 3 or U 5 ) are eliminated.
La jonction au niveau du site d'intégration implique :  Joining at the integration site involves:
- au niveau du DNA hôte, une séquence directe répétée : DR ("direct repeat"), à la jonction donc avec le provirus.  - at the level of the host DNA, a direct repeated sequence: DR ("direct repeat"), therefore at the junction with the provirus.
Toutes les DR sont longues de 4 à 6 pdb. Leurs séquences sont différentes. Pour un même provirus, on trouve d'ailleurs différentes DR dans le DNA d'une cellule-hôte. Le site d'intégration du virus n'est donc pas sepcifique, le virus peut être intégré dans le génome de l'hôte à plusieurs endroits différents.  All CDs are 4 to 6 bps long. Their sequences are different. For the same provirus, moreover, there are different DRs in the DNA of a host cell. The site of integration of the virus is therefore not specific, the virus can be integrated into the genome of the host in several different places.
- au niveau du DNA viral, une séquence inverse répétée ; IR ("inverted repeat") aux extrémités du rétrovirus. Deux séquences sont dites "inverses répétées" quand l'une est à la fois l'inverse et le complément de l'autre.  - at the viral DNA level, a repeated reverse sequence; IR ("inverted repeat") at the ends of the retrovirus. Two sequences are said to be "repeated inverses" when one is both the reverse and the complement of the other.
On a essayé de lutter contre la replication du virus du SIDA, avec des oligonucléotides antisens, c'est-à-dire des segments d'ADN complémentaires d'une portion de l'ARN messager du virus qui code directement les protéines devant être synthétisées par le ribosome.  We tried to fight against the replication of the AIDS virus, with antisense oligonucleotides, that is to say segments of DNA complementary to a portion of the messenger RNA of the virus which directly codes for the proteins to be synthesized. by the ribosome.
Compte tenu du fait que les oligonucléotides alpha forment des hétéroduplex avec leurs oligonucléotides complémentaires bêta et que ceux-ci ne sont pas substrats pour l'enzyme RNase H, on pouvait penser que l'inhibition de l'expression de protéines par l'oligonucléotide alpha pouvait peut-être s'effectuer par un empêchement du ribosome à traduire l'ARNm en protéine virale.  Given the fact that the alpha oligonucleotides form heteroduplexes with their beta complementary oligonucleotides and that these are not substrates for the enzyme RNase H, it could be thought that the inhibition of the expression of proteins by the alpha oligonucleotide perhaps could be accomplished by preventing the ribosome from translating mRNA into viral protein.
Toutefois, les expériences qui ont été entreprises en ce sens n'ont pas été concluantes.  However, the experiments which have been undertaken in this direction have not been conclusive.
En revanche, l'approche selon la présente invention visant à utiliser des oligonucléotides complémentaires d'une séquence initiale de l'ARN viral et notamment du site initial de fixation de l'ARNt de la lvsine sur l'ARN provirai, s'est avérée efficace. On the other hand, the approach according to the present invention aimed at using oligonucleotides complementary to an initial sequence of the viral RNA and in particular of the initial lvsin tRNA binding site on provirai RNA, has been shown to be effective.
Ainsi, la présente invention a pour objet un oligonucleotide alpha dont ia séquence est complémentaire du site 182- 199 de la séquence PBS de l'ARN proviral du virus VIH, soit l'oiigodésoxynucleotide alpha d5' (ACCGCGGGCTTGTCCCTG)3' ou plus généralement un oligonucleotide alpha de formule ACCGCGGGCXXGXCCCXG avec X = T pour un oligodésoxyribonuciéotide alpha ou X = U pour un oligoribonucléotide alpha ou une séquence complémentaire en interchangeant X et A d'une part et C et G d'autre part. Thus, the present invention relates to an alpha oligonucleotide whose sequence is complementary ia Site 182- 199 PBS sequence of proviral HIV RNA virus or the alpha oiigodésoxynucleotide d 5 (ACCGCGGGCTTGTCCCTG) 3 'or more generally an alpha oligonucleotide of formula ACCGCGGGCXXGXCCCXG with X = T for an oligodeoxyribonucleotide alpha or X = U for an oligoribonucleotide alpha or a complementary sequence by interchanging X and A on the one hand and C and G on the other hand.
D'autres caractéristiques et avantages de la présente invention apparaîtront à la lumière des exemples qui vont suivre.  Other characteristics and advantages of the present invention will become apparent in the light of the examples which follow.
La figure 1 représente l'inhibition de la production virale de cellule MT4 par l'oligonucléotide alpha d5'(ACCGCGGGCTTGTCCCTG)3 ' (oligo alpha "PBS") par dosage de l'activité (cpm) de transcriptase inverse. EXEMPLE 1 : Cytotoxicité des oligonucléotides alpha FIG. 1 represents the inhibition of the viral production of MT4 cell by the 5 'alpha d oligonucleotide (ACCGCGGGCTCTGTCCCTG) 3' (oligo alpha "PBS") by assaying the activity (cpm) of reverse transcriptase. EXAMPLE 1 Cytotoxicity of alpha oligonucleotides
Quel que soit le type d 'oligonucleotide utilisé, ceux-ci finissent plus ou moins rapidement par être dégradés par les enzymes cellulaires. On montre ci-après que les oligonucléotides alpha et leur catabolites nucléosides al pha et nucléotide alpha ne présentent pas de cytotoxicité.  Whatever type of oligonucleotide is used, these end up more or less rapidly being degraded by cellular enzymes. It is shown below that the oligonucleotides alpha and their catabolites nucleosides al pha and nucleotide alpha do not exhibit cytotoxicity.
Ainsi, l 'ol igonucléotide alpha-d(CCTCTCGTTCTTTAC) oligo alpha Tat (complémentaire) de la séquence Tat dirigé, à priori, contre aucun site du génome des cellules MT-4, présente une CD50 supérieure à 250 μg/ml. De même, des alpha oligothymidylates alpha-(dT)n (2≤ n≤ 12) se sont révélés non cytotoxiques vis-à-vis de la même lignée cellulaire. Thus, the oligonucleotide alpha-d (CCTCTCGTTCTTTAC) oligo alpha Tat (complementary) of the Tat sequence directed, a priori, against any site of the genome of the MT-4 cells, has a CD 50 greater than 250 μg / ml. Likewise, alpha oligothymidylates alpha- (dT) n (2 ≤ n 12 12) have been found to be non-cytotoxic to the same cell line.
Enfin, des al pha nucléosides et alpha nuciéotides pouvant résulter de l'hydrol yse len te d'oligonucleotides sont également dépourvus de cytotoxicité contre diverses lignées cellulaires (Hela, Vero, cellules primaire de rein de lapin. MT-4).
Figure imgf000014_0001
Finally, al pha nucleosides and alpha nucleotides which can result from the hydrolysis of oligonucleotides are also devoid of cytotoxicity against various cell lines (Hela, Vero, primary rabbit kidney cells. MT-4).
Figure imgf000014_0001
EXEMPLE 2 : Evaluation de l'effet antiviral de l'oligo alpha EXAMPLE 2 Evaluation of the antiviral effect of oligo alpha
_ d_5'(ACCGCGGGCTTGTCCCTG)3' _ d_ 5 ' (ACCGCGGGCTTGTCCCTG) 3'
1. Protocole  1. Protocol
a) méthode  a) method
L'évaluation est basée sur l'étude de l'effet cytopathogène du virus VIH1 sur la lignée cellulaire MT4, après infection par le virus VIH1, la formation des syncitia est observée 4 à 6 jours après l'infection, suivie par une production de particules virales, puis par la mort des cellules.  The evaluation is based on the study of the cytopathogenic effect of the HIV1 virus on the MT4 cell line, after infection with the HIV1 virus, the formation of syncitia is observed 4 to 6 days after infection, followed by production of viral particles, then by cell death.
La destruction de lymphocytes T4 indispensables à la défense immunitaire est la première cause de la déficience immunitaire caractéristique de l'infection par le VIH. On sait que ce virus tue les cellules en s'y multipliant ; lorsqu'il s'en échappe, il endommage la membrane cellulaire. Le VIH tue peut-être aussi les lymphocytes T4 indirectement, par la protéine gp l 20 présente sur la membrane des cellules infectées. En effet, les lymphocytes T4 portent une molécule de surface, le récepteur CD4, à laquelle la protéine GP120 se lie ; les lymphocytes T4 sains se fixent à la protéine et fusionnent avec la cellule infectée. L'amas cellulaire qui se forme est un "syncytiuni", incapable de survivre, et toutes les cellules saines qui le composent meurent avec la cellule infectée. Le VIH peut aussi déclencher des réactions immunitaires normales contre les lymphocytes infectés. Avec ou sans l'aide des anticorps, les cellules cytotoxiques de défense détruisent une cellule infectée portant à sa surface des protéines virales. Enfin, la protéine gp120 circule parfois librement en solution dans le sang des sujets infectés ; elle se lie au récepteur CD4 des cellules saines, simulant une infection et déclenchant une réaction de destruction de cellules non infectées.  The destruction of T4 lymphocytes essential for immune defense is the first cause of the immune deficiency characteristic of HIV infection. We know that this virus kills cells by multiplying in them; when it escapes, it damages the cell membrane. HIV may also kill T4 cells indirectly, by the gp l protein 20 present on the membrane of infected cells. In fact, T4 lymphocytes carry a surface molecule, the CD4 receptor, to which the GP120 protein binds; healthy T4 cells attach to the protein and fuse with the infected cell. The cell mass that forms is a "syncytiuni", unable to survive, and all the healthy cells that compose it die with the infected cell. HIV can also trigger normal immune responses against infected lymphocytes. With or without the help of antibodies, defense cytotoxic cells destroy an infected cell carrying viral proteins on its surface. Finally, the protein gp120 sometimes circulates freely in solution in the blood of infected subjects; it binds to the CD4 receptor of healthy cells, simulating an infection and triggering a reaction of destruction of uninfected cells.
Les syncytia sont des structures formées de plusieurs noyaux entourés par une seule membrane cellulaire ; Ce sont des signes d'infection par le VIH dans les cultures cellulaires; Les syncytia se forment lorsque les cellules infectées qui synthétisent la molécule gp120 et la transportent à leur surface fusionnent avec des cellules saines portant la molécule CD4. b) Composé testé Syncytia are structures formed by several nuclei surrounded by a single cell membrane; These are signs of HIV infection in cell cultures; Syncytia are formed when infected cells that synthesize and transport the gp120 molecule on their surface merge with healthy cells carrying the CD4 molecule. b) Compound tested
alpha-d5' ACCGCGGGCTTGTCCCTG 0,54 U alpha-d 5 'ACCGCGGGCTTGTCCCTG 0.54 U
solution mère à 1 mg/ml en eau bidistillée stérile et conservée à-80°C. stock solution at 1 mg / ml in sterile double-distilled water and stored at -80 ° C.
c) Test MT4  c) MT4 test
Les cellules MT4 au jour 2 après le dernier passage sont pré-incubées 1 heure à 37°C avec les dilutions successives du composé à raison de 3.10 cellules pour 100 μl de composé (en micropaque à 96 puits).  The MT4 cells on day 2 after the last passage are pre-incubated for 1 hour at 37 ° C. with the successive dilutions of the compound at the rate of 3.10 cells per 100 μl of compound (in 96-well micropaque).
L'infection est réalisée en micropuits en ajoutant 100 μl d'une dilution 10- 4 du virus VIH1 (la dilution de virus a été déterminée pour induire la formation de syncitia en 4 jours). The infection is carried out in microwells by adding 100 μl of a 10 - 4 dilution of the HIV1 virus (the dilution of the virus was determined to induce the formation of syncitia in 4 days).
Après une incubation de 1 heure à 37°C, les cellules MT4 infectées sont lavées 5 fois avant d'être mises en cultures à la concentration de 3.10 cellules par ml, en microplaque à 24 puits, en présence des différentes dilutions choisies.  After an incubation of 1 hour at 37 ° C, the infected MT4 cells are washed 5 times before being cultured at a concentration of 3.10 cells per ml, in a 24-well microplate, in the presence of the various dilutions chosen.
Tous les 3 ou 4 jours, les cellules sont diluées 3 ou 4 fois et ajustées à la concentration de 3.10 cellules/ml avec le milieu RPM 1 10 % FCS 1 % PSN 1 % Gluta toujours en présence de l'oligo alpha PBS.  Every 3 or 4 days, the cells are diluted 3 or 4 times and adjusted to the concentration of 3.10 cells / ml with RPM 1 medium 10% FCS 1% PSN 1% Gluta always in the presence of oligo alpha PBS.
Tous les 2 ou 3 jours, l'apparition des syncitia est observée dans les cultures.  Every 2 or 3 days, the appearance of syncitia is observed in cultures.
d) Dosage de la transcriptase inverse  d) Determination of reverse transcriptase
Le suivi de la production virale des cellules MT4 est réalisé par dosage de l'activité de la transcriptase inverse (figure 1 RT) dans la culture tous les 3 ou 4 jours. Brièvement, l'activité enzymatique est testée en utilisant une amorce synthétique et un substrat radiomarque au H 3 "in vitro". La quantité de matériel radiomarque précipité, exprimée en coups par minute, est proportionnelle à l'activité de la transcriptase inverse elle-même proportionnelle à la quantité de virus produit par les cellules MT4 infectées. Monitoring of viral production of MT4 cells is carried out by assaying the activity of reverse transcriptase (FIG. 1 RT) in the culture every 3 or 4 days. Briefly, the enzymatic activity is tested using a synthetic primer and a H 3 radiolabelled substrate "in vitro". The quantity of radiolabelled material precipitated, expressed in counts per minute, is proportional to the activity of the reverse transcriptase itself proportional to the quantity of virus produced by the infected MT4 cells.
2. Résultats EVALUATION DE L'INHIBITION DE L'EFFET 2. Results EVALUATION OF INHIBITION OF EFFECT
DU VIRUS VIH 1 SUR LA L IGNEE MT4 PAR L'OLIGONUCLEOTIDE "ALPHA PBS"  OF HIV 1 VIRUS ON THE IGNEE MT4 BY THE "ALPHA PBS" OLIGONUCLEOTIDE
J3 J 4 J5 J7 J10 J11 J12 J14 J18J3 J 4 J5 J7 J10 J11 J12 J14 J18
100 μg/ml - - - - - - - - - (+) - (-) (+) ++ ++ T100 μg / ml - - - - - - - - - (+) - (-) (+) ++ ++ T
25 μg/ml (+) - (+) - (+) - (+) (+) ( +) ++ + ++ + -+ ++ ++ T T25 μg / ml (+) - (+) - (+) - (+) (+) (+) ++ + ++ + - + ++ ++ T T
10 μg/ml - (+) ( 4 ) ( 4 ) + + + ++ ++ ++ ++ + + ++ ++ ++ ++ ++ T T10 μg / ml - (+) (4) (4) + + + ++ ++ ++ ++ + + ++ ++ ++ ++ ++ ++ T T
5 μg/ml (+)+(+) + + ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++5 μg / ml (+) + (+) + + ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
I μg/ml (+) (+) (+) (+) + (+) ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++I μg / ml (+) (+) (+) (+) + (+) ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
100 ng/ml (+) (+) (+) ++ + ++ +- ++ ++ ++ ++ ++ ++ ++ ++ ++ T ++100 ng / ml (+) (+) (+) ++ + ++ + - ++ ++ ++ ++ ++ ++ ++ ++ ++ T ++
50 ng/ml (+) (+) + + + + + + + + + ++ + + ++ ++ + + ++ ++ ++ ++ T50 ng / ml (+) (+) + + + + + + + + + + ++ + + ++ ++ + + ++ ++ ++ ++ T
VIH 1 (+) (+) + + ++ + + ++ ++ + + ++ ++ ++ ++ ++ ++ ++ T T HIV 1 (+) (+) + + ++ + + ++ ++ + + ++ ++ ++ ++ ++ ++ ++ T T
Légende : Caption:
- : absence de syncitia -: absence of syncitia
( + ) : syncitia en formation (+): syncitia in training
+ : apparition de syncitia +: appearance of syncitia
+ + : syncitia + +: syncitia
T : cel lules tuées T: cells killed
3. Conclusions 3. Conclusions
Lors de l'évaluation de l'inhibition de l'effet cytopathogène du virus VIH1 sur la lignée MT4 par l'oligo alpha "PBS", un retard dans la formation des syncitia est observé pour les doses non toxiques de 100 μg/ml à 10 μg/ml, ce retard pouvant atteindre le 12ème jour après l'infection à la concentration de 100 μg/ml.  When evaluating the inhibition of the cytopathogenic effect of the HIV1 virus on the MT4 line by the oligo alpha "PBS", a delay in the formation of syncitia is observed for the non-toxic doses of 100 μg / ml to 10 μg / ml, this delay being able to reach the 12th day after infection at the concentration of 100 μg / ml.
Parallèlement, le suivi de la production virale par les cellules At the same time, monitoring of viral production by cells
MT4 infectées montre (figure 1 ) que quelques soient les doses d'oligo alphaMT4 infected shows (figure 1) that whatever the doses of oligo alpha
"PBS" utilisées, la production virale reste inférieure à celle obtenue en présence du virus VIH1 , avec un pic de production virale maximale retardée. "PBS" used, the viral production remains lower than that obtained in the presence of the HIV1 virus, with a peak of delayed maximum viral production.
L'ensemble de ces résultats indiquent que l'oligo alpha PBS présente un effet antiviral sur le virus VIHI .  All of these results indicate that the oligo alpha PBS has an antiviral effect on the HIVI virus.
EXEMPLE 3 : Comparaison de l'effet antiviral d'oligonucleotides "alpha  EXAMPLE 3 Comparison of the Antiviral Effect of "alpha oligonucleotides
PBS", "alpha Tat", "alpha random"  PBS "," alpha Tat "," alpha random "
La production de particules virales par les cellules CEM, infectées par VIH1 BRU et cultivées en présence d'oligonucleotides, est suivie par dosage de la Reverse Transcriptase (cpm).  The production of viral particles by CEM cells, infected with HIV1 BRU and cultured in the presence of oligonucleotides, is monitored by assaying the Reverse Transcriptase (cpm).
L'oligonucléotide "alpha Tat" correspond à l'oligonucléotide alpha d5 GTAAAAGTCTTAACCCAC3'. Il est complémentaire de la séquence du gène Tat du virus du SIDA. The oligonucleotide "alpha Tat" corresponds to the oligonucleotide alpha d 5 GTAAAAGTCTTAACCCAC 3 ' . It is complementary to the sequence of the Tat gene of the AIDS virus.
L'oligo nucléotide "alpha random" correspond à un oligonucléotide quelconque alpna d5'ACTGACTGACTGACTGAC3'. The oligo nucleotide "alpha random" corresponds to any olna nucleotide alpna d 5 ' ACTGACTGACTGACTGAC 3' .
Les résultats de la Reverse Transcriptase en coups par minute comparés à un contrôle Random sont les suivants. The results of Reverse Transcriptase in counts per minute compared to a Random control are as follows.
Figure imgf000019_0001
Figure imgf000019_0001
L'oligonucléotide "alpha PBS" montre une inhibition de la production virale pour des concentrations de 100 μg/ml et 50 μg/mi. The oligonucleotide "alpha PBS" shows an inhibition of viral production for concentrations of 100 μg / ml and 50 μg / mi.
A la concentration de 6,25 μ/ml on remarque que l'oligonucléotide "alpha Tat" ne présente pas de production virale au jour 1 1. Il en est de même pour le Random à 3,1.  At the concentration of 6.25 μ / ml it is noted that the oligonucleotide "alpha Tat" does not show viral production on day 1 1. It is the same for the Random at 3.1.
L'ensemble de ces résultats indique l'oligonucléotide "alpha PBS" présente un effet antiviral sur le virus HIV1 à des concentrations de 100 μg/ml et 50 μg/ML, alors que l'oligonucléotide "alpha Tat" et l'oligonucléotide "alpha Random" ne présentent aucun effet antiviral.  All of these results indicate that the oligonucleotide "alpha PBS" has an antiviral effect on the HIV1 virus at concentrations of 100 μg / ml and 50 μg / ML, while the oligonucleotide "alpha Tat" and the oligonucleotide " alpha Random "have no antiviral effect.

Claims

REVENDICATIONS
1. Composé oiigonucléotidique utile pour inhiber la replication d'un rétrovirus, caractérisé en ce qu'il consiste en une séquence d'oligoribonucléotide ou oligodésoxyribonucléotide d'anomerie non naturelle alpha, ladite séquence étant complémentaire d'une séquence initiale de l'ARN viral comprise dans la séquence TBS ou une séquence en amont de l'ARN viral, notamment la séquence CAP. 1. Oiigonucleotide compound useful for inhibiting the replication of a retrovirus, characterized in that it consists of an oligoribonucleotide or oligodeoxyribonucleotide sequence of unnatural anomaly alpha, said sequence being complementary to an initial sequence of viral RNA included in the TBS sequence or a sequence upstream of the viral RNA, in particular the CAP sequence.
2. Composé oiigonucléotidique selon la revendication 1, caractérisé en ce qu'il est en outre lié de façon covalente à un agent effecteur. 2. Oiigonucleotide compound according to claim 1, characterized in that it is also covalently linked to an effector.
3. Composé oiigonucléotidique selon l'une des revendications I ou 2, caractérisé en ce que les composés ont pour formule 3. Oiigonucleotide compound according to one of claims I or 2, characterized in that the compounds have the formula
Figure imgf000021_0001
dans laquelle :
Figure imgf000021_0001
in which :
Les radicaux B peuvent être identiques ou différents et représentent chacun une base d'un acide nucléique éventuellement modifiée et rattachée au cycle glycosidique selon une configuration anomerique alpha non naturelle,  The radicals B can be identical or different and each represents a base of a nucleic acid possibly modified and attached to the glycosidic cycle according to an unnatural anomeric alpha configuration,
les radicaux B étant complémentaires un à un des bases de la séquence oligonucléotidiques cibles de la séquence TBS ou d'un séquence en amont de l'ARN viral ;  the radicals B being complementary one to one of the bases of the target oligonucleotide sequence of the TBS sequence or of a sequence upstream of the viral RNA;
Les radicaux X peuvent être identiques ou différents et représentent chacun un oxoanion O , un thioanion S , un groupe alkyle, un groupe alcoxy, aryloxy, un groupe aminoalkyle, un groupe aminoalcoxy, un groupe thioalkyle ; R et R', qui peuvent être identiques ou différents, représentent chacun un atome d'hydrogène ou un groupement -Y-Z ; The radicals X may be the same or different and each represents an oxoanion O , a thioanion S , an alkyl group, an alkoxy group, aryloxy, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group; R and R ', which may be the same or different, each represent a hydrogen atom or a group -YZ;
Y représente un radical alcoylène droit ou ramifié -alk- ou un radical choisi parmi
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
Figure imgf000022_0004
et
Y represents a straight or branched alkylene radical -alk- or a radical chosen from
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
Figure imgf000022_0004
and
Figure imgf000022_0005
Figure imgf000022_0006
Figure imgf000022_0007
Figure imgf000022_0005
Figure imgf000022_0006
Figure imgf000022_0007
avec U = O, N ou S  with U = O, N or S
ou bien un radical -Y"-O-Y' où Y" ou Y' peuvent avoir les significations données pour Y ;  or else a radical -Y "-O-Y 'where Y" or Y' may have the meanings given for Y;
E peut avoir les mêmes significations que X ;  E can have the same meanings as X;
J représente un atome d'hydrogène ou un groupe hydroxy ;  J represents a hydrogen atom or a hydroxy group;
Z est un radical correspondant à un agent effecteur ;  Z is a radical corresponding to an effector;
n est un nombre entier y compris O ;  n is an integer including O;
L représente un atome d'oxygène, un atome de soufre ou un groupe -NH-.  L represents an oxygen atom, a sulfur atom or a group -NH-.
4. Composé selon l'une des revendications précédentes utile pour l'inhibition de la replication du virus du SIDA VIH dont la séquence est complémentaire du site 182-199 de l'ARN proviral du virus, soit comporte l'oligonucléotide alpha ACCGCGGGCXXGXCCCXG avec X = T ou U ou sa séquence complémentaire en interchangeant X et A d'une part, et C et G d'autre part. 4. Compound according to one of the preceding claims useful for the inhibition of the replication of the HIV AIDS virus, the sequence of which is complementary to the site 182-199 of the viral proviral RNA, or comprises the alpha oligonucleotide ACCGCGGGCXXGXCCCXG with X = T or U or its complementary sequence by interchanging X and A on the one hand, and C and G on the other.
5. Composé selon l'une des revendications précédentes, caractérisé en ce que les composés sont des oligodésoxynucléotides alpha pour lesquels dans la formule I X = O; 3, R, R' = H ; B est choisi parmi A,5. Compound according to one of the preceding claims, characterized in that the compounds are alpha oligodeoxynucleotides for which in the formula IX = O ; 3, R, R '= H; B is chosen from A,
C, T et G ; et L est un atome d'oxygène. C, T and G; and L is an oxygen atom.
6. Composition pharmaceutique antivirale caractérisée en ce qu'elle comporte à titre de substance active un composé selon l'une des revendications précédentes. 6. An antiviral pharmaceutical composition characterized in that it comprises, as active substance, a compound according to one of the preceding claims.
7. Composition pharmaceutique utile pour le traitement du7. Pharmaceutical composition useful for the treatment of
SIDA caractérisée en ce qu'elle comporte à titre de substance active l'oligonucléotide d'anomerie alpha d5'(ACCGCGGGCTTGTCCCTG)3'. SIDA characterized in that it comprises, as active substance, the oligonucleotide of alpha d 5 '(ACCGCGGGCTTGTCCCTG) alpha 3 '.
8. Utilisation des composés selon l'une des revendications précédentes pour la préparation d'une composition pharmaceutique utile pour le traitement des affections virales des virus à ARN contenant lesdits composés à titre de substance active. 8. Use of the compounds according to one of the preceding claims for the preparation of a pharmaceutical composition useful for the treatment of viral affections of RNA viruses containing said compounds as active substance.
9. Utilisation selon la revendication précédente pour la préparation d'une composition pharmaceutique utile pour le traitement du9. Use according to the preceding claim for the preparation of a pharmaceutical composition useful for the treatment of
SIDA. AIDS.
10. Utilisation de l'oligonucléotide alpha 10. Use of the alpha oligonucleotide
d5'(ACCGCG GGGGCCTTTTGGTTCCCCCCTTGG)3' pour la préparation d'une composition pharmaceutique utile pour le traitement du SIDA d 5 '(ACCGCG GGGGCCTTTTGGTTCCCCCCTTGG) 3 ' for the preparation of a pharmaceutical composition useful for the treatment of AIDS
PCT/FR1990/000412 1989-06-13 1990-06-12 Alpha anomer oligonucleotide compounds which inhibit retrovirus replication WO1990015813A1 (en)

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EP0527916A1 (en) * 1990-05-11 1993-02-24 Isis Pharmaceuticals, Inc. Antisense inhibitors of the human immunodeficiency virus
FR2717081A1 (en) * 1994-03-14 1995-09-15 Centre Nat Rech Scient Retropeptides, antibodies against them, and their uses for vaccination and in vitro diagnosis.
EP1016715A1 (en) * 1992-09-29 2000-07-05 Isis Pharmaceuticals, Inc. Oligonucleotides having a conserved G4 core sequence
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
WO2008037924A2 (en) 2006-09-28 2008-04-03 Biomerieux Novel labelled oligonucleotide
US8663923B2 (en) 2008-07-04 2014-03-04 Biomerieux Detection probe

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0527916A1 (en) * 1990-05-11 1993-02-24 Isis Pharmaceuticals, Inc. Antisense inhibitors of the human immunodeficiency virus
EP0527916A4 (en) * 1990-05-11 1994-04-06 Isis Pharmaceuticals, Inc.
EP1016715A1 (en) * 1992-09-29 2000-07-05 Isis Pharmaceuticals, Inc. Oligonucleotides having a conserved G4 core sequence
FR2717081A1 (en) * 1994-03-14 1995-09-15 Centre Nat Rech Scient Retropeptides, antibodies against them, and their uses for vaccination and in vitro diagnosis.
WO1995024916A1 (en) * 1994-03-14 1995-09-21 Centre National De La Recherche Scientifique Retropeptides, antibodies thereto, and uses thereof for vaccination and in vitro diagnosis
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
WO2008037924A2 (en) 2006-09-28 2008-04-03 Biomerieux Novel labelled oligonucleotide
FR2906532A1 (en) * 2006-09-28 2008-04-04 Biomerieux Sa NEW OLIGONUCLEOTIDE BRAND
WO2008037924A3 (en) * 2006-09-28 2008-07-31 Biomerieux Sa Novel labelled oligonucleotide
US8158345B2 (en) 2006-09-28 2012-04-17 Biomerieux Labeled oligonucleotide
US8663923B2 (en) 2008-07-04 2014-03-04 Biomerieux Detection probe

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