WO1994023026A1 - Staple and semi-staple oligonucleotides, method of preparation and applications - Google Patents

Staple and semi-staple oligonucleotides, method of preparation and applications Download PDF

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Publication number
WO1994023026A1
WO1994023026A1 PCT/FR1994/000336 FR9400336W WO9423026A1 WO 1994023026 A1 WO1994023026 A1 WO 1994023026A1 FR 9400336 W FR9400336 W FR 9400336W WO 9423026 A1 WO9423026 A1 WO 9423026A1
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sequence
staple
oligonucleotides
oligonucleotide
dna
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PCT/FR1994/000336
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French (fr)
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Marc Vasseur
Marta Blumenfeld
Saïd MEGUENNI
Bruno Poddevin
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Genset
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to a new type of oligonucleotide.
  • the oligonucleotides according to the invention are short synthetic DNA, RNA or mixed molecules.
  • the so-called "antisense" oligonucleotides have a sequence complementary to a target sequence belonging to a gene or to a messenger RNA whose expression it is desired to specifically block.
  • the antisense oligonucleotides can be directed against a messenger RNA sequence, or alternatively against a DNA sequence.
  • Antisense oligonucleotides hybridize to the sequence of which they are complementary and can thus block the expression of messenger RNA carrying this sequence.
  • oligonucleotide is used here generally to designate a polynucleotide from 2 to 100, and more generally from 5 to 50, nucleotides in ribo-, deoxyribo- or mixed series. When it is a question of a particular property linked to the use of a deoxyribo- or a ribo- series, the full name oligodeoxyribonucleotide or oligoribonucleotide will be used.
  • the term “apartment” or “hybridization” used herein means the formation of hydrogen bonds between complementary base pairs, such as guanine and cytosine forming three hydrogen bonds between them, and adenine and thymine or uracil in forming two.
  • the modified antisense oligonucleotides are synthesized chemically and frequently contain modifications which alter the very backbone of the molecule or carry additional reactive groups, located at their ends.
  • the purpose of these modifications introduced into the antisense oligonucleotides is either to increase the resistance of these molecules to nucleolytic degradation, or to promote their interactions with their targets, or to allow specific degradation / modification reactions of the targets, RNA or DNA, or to increase their intra-cellular penetration.
  • Antisense oligonucleotides are sensitive to nuclease degradation, and mainly to the action of exonucleases.
  • the nucleases are found in all compartments - cellular and extra-cellular, in particular in the serum - and cause a rapid degradation of these molecules.
  • Pharmacological use of antisense molecules involves solving these degradation problems in order to achieve satisfactory pharmacokinetics and therefore sufficient sustainability of the effects of these molecules. Numerous chemical modifications allow the antisense oligonucleotides to become resistant to nucleases.
  • RNA oligonucleotides All regions of a messenger RNA are not equally sensitive to the effects of an antisense oligonucleotide.
  • a messenger RNA is not a fixed linear molecule, but on the contrary a molecule comprising many secondary structures (complex intra-molecular hybridizations), tertiary (folds and particular conformations, pseudo-nodes), and interacting with structural and functional nucleoproteins (basic proteins, splicing complexes, polyadenylation, capping, translation complex for example).
  • the effective availability and accessibility of the different regions of a messenger RNA will depend on their commitments in these structures. Correlatively, the effectiveness of an inhibiting agent interacting with such and such a sequence will also depend on the engagement of this sequence in a particular function.
  • the target regions for the antisense molecules must be accessible to the oligonucleotide.
  • Antisense oligodeoxyribonucleotides can also be directed against certain regions of double-stranded DNA (homopurine / homopyrimidine sequences or rich in purines / pyrimidines) and thus form triple helices (Perroualt et al., 1990; Institut et al. (A), 1989; Institut et al. (B), 1989; Institut et al. (C), 1989; Wang et al., 1989; Maher et al., 1989; Sun et al., 1989; Boidot-Forget et al., 1988; Moser and Dervan, 1987; Dervan, 1986).
  • Oligonucleotides thus directed against DNA have been called "anti-gene” or "anti-code”.
  • the formation of a triple helix, at the level of a particular sequence, can block the binding of proteins involved in the expression of a gene and / or allow the introduction of irreversible damage in DNA if the oligonucleotide considered has a particular reactive group.
  • antisense oligonucleotides can become real artificial restriction endonucleases, directed on demand against specific sequences.
  • Hybridization between an antisense oligonucleotide and a target messenger RNA can block its expression in several ways, either sterically or pseudo-catalytically (Gagnor et al., 1989; Jessus et al., 1988; Markus-Sekura, 1987):
  • the interaction between the messenger RNA and a complementary antisense oligonucleotide can create a physical barrier preventing the fixation and / or the progression of proteins or protein complexes necessary for the translation, maturation, stabilization or transport of l 'Messenger RNA. This physical blockage will ultimately result in inhibition of expression of the targeted messenger RNA.
  • RNase H an enzyme present in all eukaryotic cells.
  • RNase H is an enzyme that specifically degrades RNA when it is hybridized to DNA.
  • the antisense oligonucleotides may contain reactive groups capable of directly producing irreversible damage in the target RNA molecules.
  • the antisense oligonucleotides directed against DNA they can act either by inhibiting the binding of a regulatory protein essential for the expression of the targeted gene (transcription factor for example), or by introducing irreversible damage (cuts , cross-links) in the DNA molecule, rendering it locally incapable of gene expression.
  • Antisense oligonucleotides make it possible to specifically block the expression of cellular messenger RNAs, for example messengers of the oncogenic type, (Tortora et al., 1990; Chang et al., 1989; Anfossi et al., 1989; Zheng et al.
  • Antisense oligonucleotides are therefore potential, powerful and highly specific pharmacological agents, making it possible to inhibit the expression of messengers coding for products having pathogenic effects.
  • oligonucleotides however encounters several problems of physiological type, in particular that of the intracellular delivery of these molecules and that of their sensitivity to nucleolytic degradation. Certain chemical modifications of the oligonucleotides make it possible to overcome the problem of sensitivity to nucleases but at the cost of a new problem, that of the possible toxicity of the chemical modifications introduced into the molecule.
  • modified antisense oligonucleotides pose toxicological problems. While some of the changes are said to be fairly neutral, most are not devoid of potential toxicity. Chemically modified antisense oligonucleotides can exhibit toxicity at several levels, either directly through the effects of the whole molecule, or indirectly via the effects of degradation products. Nucleotides carrying chemical modifications, and present in a cell at a high concentration can thus present a toxicity - and more particularly a genotoxicity - not negligible from the pharmacological point of view.
  • Nucleases are degradation enzymes capable of cutting the phosphodiester bonds of DNA or RNA, either by introducing internal cleavages on single- or double-stranded molecules, or by attacking these molecules from their ends. Enzymes attacking internally are called endonucleases and those attacking from the ends are called exonucleases.
  • endonucleases Enzymes attacking internally are called endonucleases and those attacking from the ends are called exonucleases.
  • the stability of antisense oligonucleotides - hence their efficiency - can be considerably increased by introducing various chemical modifications making them resistant to degradation as described above. It is established that it is the exonucleases which are the main cause of degradation of the antisense oligonucleotides in the serum and in the cell.
  • thermodynamic constraints governing the structure of nucleic acids are lower in an RNA molecule than in a DNA molecule.
  • RNA molecules unlike DNA molecules, RNA molecules generally spontaneously adopt secondary structures mediated by intramolecular apartments.
  • the secondary structure of RNA molecules results directly from the sequence of these molecules.
  • the target sequence can allow the spontaneous appearance of a secondary structure of the complementary molecule, which can in certain cases increase its resistance to nucleolytic degradation.
  • the short synthetic DNA fragments used as antisense agent more rigid, only rarely adopt secondary structures, and the stability of the adopted structures is generally weaker.
  • the aim of the present invention is to provide antisense oligonucleotides, in particular comprising only normal deoxyribonucleotides linked together by a natural phosphodiester bond, but nevertheless exhibiting resistance to degradation, without introducing chemical modifications capable of inducing toxicity, which may include any anti-sense sequence of interest.
  • the present invention provides an oligonucleotide molecule having at least one self-paired 3 'end, called a semi-staple or staple oligonucleotide in the case where the 5' end of this molecule is also self-paired.
  • the present invention relates to antisense oligonucleotides semi-staples or staples consisting of an oligonucleotide sequence whose 3 'end or both 3' and 5 'ends are engaged in an intracatenary self-pairing involving '' hybridization of at least the two terminal bases of the 3 'end or respectively of each 3' and 5 'end, with the same number of complementary consecutive bases located on the strand, so as to form a loop at the end 3 'or respectively a loop at each end framing a central sequence.
  • said central sequence comprises the entire sequence of interest complementary to a target sequence belonging to the DNA or the RNA messenger whose expression it is desired to block and, on the other hand, said loops have a sequence such that they do not hybridize with the strand of the target sequence.
  • oligonucleotides having the type of secondary structure according to the present invention are consequently stabilized in the media containing this type of enzymatic activities such as the serum or the intracellular medium.
  • said antisense sequence of interest does not overlap with said self-paired sequence because said antisense sequence of interest is entirely included in said central sequence.
  • This characteristic is particularly advantageous since the stability and the length of the self-paired structure are controlled independently of the antisense sequence, and therefore of the target. These two parameters are based solely on the characteristics of a fragment with a staple structure added at the 3 ′ end or at the two 3 ′ and 5 ′ ends.
  • the stability of the terminal clip structure depends on the nature of the bases involved in the intramolecular pairing and does not depend, in this embodiment, on the antisense sequence. It is therefore independent of the target sequence.
  • the stability of the secondary structure responsible for nuclease resistance is in principle limited.
  • an excessively stable intramolecular apartment would inhibit the formation of the target / antisense duplex.
  • the nature of the bases involved in the intramolecular apartment is partially imposed by the choice of the target sequence.
  • the bases "imposed" by the target sequence in the intracatenary apartment will be adenines or thymines, forming less stable apartments, the stability of the induced secondary structure will be weaker than if these bases were guanines or cytosines and, as a result, resistance to nucleases will be reduced.
  • the "imposed" bases are adenines or thymines, forming less stable apartments, it is necessary, in order to obtain sufficient stability of the secondary structure, to increase the length of the double-stranded segment, and consequently the length of the fragment added in 3 ′, and consequently the total length of the oligonucleotide. And, for equivalent secondary structure stabilities, a longer oligonucleotide will be more likely to pair nonspecifically and induce toxicity. Antisense oligonucleotides sometimes exhibit toxicity resulting from parasitic apartments with sequences partially homologous to the target sequence.
  • the stability of the secondary structures characterizing the staple or semi-staple oligonucleotides can be increased without limitation, thus ensuring maximum protection against nucleases, without harming the hybridization of the antisense oligonucleotide on his target.
  • the oligonucleotides according to the present invention can be used in their natural state but can however also contain nucleotides modified in their internucleotide bonds, in the osidic cycles or in the bases, with the aim of promoting cell penetration, and / or resistance to nucleases, and / or reinforce or stabilize their hybridization with a sequence complementary.
  • the oligonucleotides according to the invention may also comprise intercalating or reactive groups, or be physically associated with other molecules or macromolecules with the aim of enhancing their inhibition efficiency, their penetration, their affinity for targets, their cell targeting or intracellular, or to optimize any other property.
  • the molecules satisfying this definition can be subdivided into two categories, staple molecules and semi-staple molecules, and represented schematically as follows:
  • P 2 and P 3 represent the numbers of nucleotides constituting the loops and are variable from one oligonucleotide to another; n 1 ( n 2 and n 3 represent the numbers of nucleotides involved in the intracatenary apartments; they are variable from one oligonucleotide to another but necessarily greater than 2
  • nj at least two terminal bases involved in an intracatenary apartment are immediately followed or preceded by pi bases (at least two) not paired, forming a pin hinge area to hair. Said hinge zone is therefore adjacent to another zone of n;
  • the loop constituting the staples therefore comprises a hinge oligonucleotide sequence of at least 1, preferably from 3 to 25 nucleotides, and more preferably, from 4 to 20 nucleotides. In fact, if the hinge zone is too large, the stability of the intracatenary hybridization is weakened.
  • the staple oligonucleotide consists of a DNA or mixed DNA / RNA linear sequence of interest complementary to a target DNA or mRNA sequence the expression of which it is desired to block, 'end 3' or at the two ends 3 'and 5' to which a loop consisting of a DNA sequence or mixed DNA / RNA self-paired comprising 8 to 12 nucleotides has been added, said self-paired sequence being characterized by l 'pairing of its 3' end with its 5 'end and all of its nucleotides two by two with the exception of those constituting a so-called hinge sequence.
  • said self-paired loop or sequence is in "hairpin"
  • the 5 'end is paired at the 3' end and all the nucleotides not belonging to the so-called hinge area are paired two by two.
  • FIGS. 2, 4 and 5 of the present description An example of this type of fragment is presented in FIGS. 2, 4 and 5 of the present description.
  • this fragment is an octameric fragment whose intrinsic secondary structure, which is particularly stable, has been studied.
  • This fragment belongs to a family of fragments of variable stabilities, the advantage of which the present invention has validated as a stabilizing fragment of semi-staple oligonucleotides. It has been demonstrated, according to the present invention, that there is a correlation between the thermodynamic stability of the secondary structures of these fragments and the protection against nucleases which they induce when they constitute the hairpin of semi-staple oligonucleotides. .
  • an oligonucleotide constituted by an antisense sequence of interest comprising at its 3 ′ end or at both 3 ′ and 5 ′ ends a loop of 8 nucleotides in which the self-apartment is due to only 4 nucleotides, namely two GCs in 5 'and 3'.
  • the composition of the hinge sequence influences the stability of the loop and one of the sequences GCGAAAGC, GCTAAAGC, GCGAGAGC, GCGATAGC, GCGAATGC, GCGTTAGC is preferably used, which form very stable self-pairings.
  • Different nucleotides can enter into the formulation of staple or semi-staple oligonucleotides.
  • the staple and semi-staple oligonucleotides forming the subject of the present invention are composed by a nucleotide base sequence comprising in particular adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), linked together by internucleotide bonds, in particular natural, that is to say phosphodiesters.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • the staple and semi-staple oligonucleotides according to the invention may also contain rare nucleotides (Inosine, I, or ri for example) or modified nucleotides, either in deoxyribo- series or in ribo- series.
  • rare nucleotides Inosine, I, or ri for example
  • modified nucleotides either in deoxyribo- series or in ribo- series.
  • the staple and semi-staple oligonucleotides according to the invention may comprise reactive nucleotides, capable of establishing links with the sequence of the target molecule complementary to the oligonucleotide or, in another application, intramolecular links within the same staple or semi-staple oligonucleotide.
  • the staple and semi-staple oligonucleotides according to the invention can carry reactive groups grafted onto the nucleotides, such as for example psoralen groups, or other bridging agents or intercalating agents which can react with the sequence of the complementary target molecule to the oligonucleotide.
  • reactive groups grafted onto some of the nucleotides of the staple or semi-staple oligonucleotide may induce the formation of an intramolecular bridging within the molecule itself.
  • the oligonucleotide may have internal bonds produced by reactive agents belonging to or not belonging to the structure of the molecule itself.
  • staple or semi-staple oligonucleotides called chimeric, constituted by the covalent assembly of nucleotide and non-nucleotide fragments.
  • chimeric staple or semi-staple oligonucleotide the said hinge zone or zones will be of non-nucleotide nature.
  • staple or semi-staple antisense oligonucleotides coupled to molecules making it possible to increase their intracellular penetration, and in particular lipophilic groups, polypeptides or proteins.
  • the staple or semi-staple oligonucleotides described above and forming the subject of the invention are intended to provide new stable oligonucleotides capable of being internalized in cells and then of interacting with cellular or viral factors having an affinity for specific nucleic acid sequences.
  • the oligonucleotides according to the invention may comprise one or more lipophilic group (s) or any other molecular structure promoting the penetration, targeting, cellular or intra ⁇ cellular addressing of these oligonucleotides, or stabilizing the structure of these oligonucleotides.
  • the DNA / RNA duplexes are substrates of the RNAse H. This is why it may be advantageous if the said sequence of interest comprises a DNA or RNA sequence so as to form a DNA / RNA duplex with the target sequence mRNA or DNA respectively.
  • a mixed oligonucleotide with, for example, a DNA window framed by two RNA sequences or, conversely, constituting the staples. Concrete examples of some of these structures are shown in Figure 4.
  • oligonucleotides according to the invention can therefore be obtained by conventional methods of synthesis of natural oligonucleotides, chemically, biologically, or by approaches using combinations of the techniques of synthetic chemistry and molecular biology.
  • This approach begins with the condensation of a 3'-H-phosphonate deoxynucleoside with a deoxynucleoside coupled to a silica glass support. Successive condensation cycles lead to the synthesis of H-phosphonate oligonucleotides. These oligonucleotides are oxidized in one step to give the phophodiesters.
  • oligonucleotides having the desired primary structure are obtained.
  • This primary sequence is at the origin of the secondary structures which the molecule will spontaneously adopt according to its nucleotide sequence and the physico-chemical parameters of the solutions in which it will be found.
  • Obtaining a staple or semi-staple oligonucleotide will be done by the choice of a primary sequence inducing the formation at the 3 'end or at both 3' and 5 'ends of the molecule of the secondary structures characterizing the staple oligonucleotides or semi-staples according to the invention.
  • the staple and semi-staple oligonucleotides forming the subject of the invention described here may be used in all cases where it will be advantageous to have an oligonucleotide including a defined primary sequence, having increased resistance to exonucleases and a lower risk of apartment with sequences partially complementary to a linear oligonucleotide including the same primary sequence.
  • the staple and semi-staple oligonucleotides may in particular be used as antisense agents to act specifically on the transcription or translation of protein (s) whose expression level is to be modulated for research or therapeutic purposes.
  • the present invention therefore relates to the use of the oligonucleotides according to the invention, as a medicament.
  • inflammatory diseases such as atopic dermatitis or lupus erythematosus, keratinization diseases like ichthyosis and psoriasis, and tumor diseases like melanoma or cutaneous T lymphoma.
  • staple or semi-staple antisense oligonucleotides applied to dermatology can be directed against RNA messengers of inflammatory mediators such as interleukins, against RNA messengers of proteins implicated in epidermal cell proliferation disorders, or against messenger RNA coding for proteins possibly involved in phenotypic skin aging, such as, for example, collagenase, elastase, transglutaminases.
  • the staple and semi-staple oligonucleotides can, for example, be used as antiviral or antisense agents, whether for topical (dermatological) indications or for systemic indications.
  • such oligonucleotides can be used as anti-herpetic agents (HSV 1 and HSV 2, CMV, EBV), as anti-papillomavirus agents (cutaneous, genital, laryngeal or other HPV), as anti-hepatitis agents (HBV, HCV, HDV), as anti-HIV agents (HIV-1 and HIV-2), as anti-HTLV agent (HTLV-1 or HTLV-2), etc.
  • These staple or semi-staple oligonucleotides can also be used as agents for suppressing the expression of certain cellular proteins directly responsible for or involved in the etiology of diseases of cell proliferation and differentiation.
  • these staple or semi-staple oligonucleotides can be directed against the expression of hyper-expressed or uncontrolled cellular oncogenes in tumor cell types (RAS, ERB, NEU, SIS, MYC, MYB.etc .. .).
  • these natural staple or semi-staple oligonucleotides resistant to serum exonucleases can be used as an antisense agent directed against RNA messengers of oncogenes expressed in leukemic cells and involved in their proliferation.
  • the staple or semi-staple oligonucleotides can be used in the context of "ex vivo" applications. For these numerous indications, adequate dosage formulations must be established in order to optimize the delivery of these molecules to their target cells.
  • oligonucleotides staples or semi-staples can be encapsulated in liposomes, nanoparticles, LDL particles, or in any other type of micro ⁇ sphere allowing adequate preservation, and promoting targeting.
  • the staple or semi-staple oligonucleotide molecules can also be combined with cationic surfactants. It is obvious that these few examples are neither exhaustive nor limiting.
  • the staple or semi-staple oligonucleotides forming the subject of the invention described here are therefore capable of being included in all kinds of pharmaceutical, parapharmaceutical, cosmetological preparations, or intended for nucleic acid or diagnostic assays, varying concentrations as indicated, with appropriate excipients.
  • the present invention also relates to a method of modulating the genetic expression of a target sequence belonging to a messenger RNA or to a DNA, characterized in that the said target sequence is hybridized with an oligonucleotide according to the invention comprising a sequence complementary to said target sequence.
  • This type of process can be implemented for research, therapeutic or diagnostic purposes, under in vitro, in vivo or ex vivo conditions.
  • FIGURE 1 represents the structure of the bases composing the oligonucleotides and the structure of the phosphodiester bond connecting the natural oligonucleotides between them.
  • FIGURE 2 represents examples of oligonucleotides adopting secondary structures in staple (a), in semi-staple (b and c) or in semi-staple "hairpin”.
  • FIGURE 3 represents examples of methods for obtaining semi-stapled oligonucleotides with in (a) obtaining a semi-stapled oligonucleotide by adding a self-paired oligonucleotide structure to the 3 'end of an oligonucleotide linear; and in (b) obtaining a semi-staple oligonucleotide by adding to the 3 'end of a linear oligonucleotide of another linear oligonucleotide whose terminal 3 'sequence is complementary to a fragment of the sequence of the first oligonucleotide.
  • FIGURE 4 shows in (a) the staple (HA12), semi-staple (SA 12, RAI 2, HA12 / ml, RA12 / ml) and linear (POLYA12, SA12 / ml, HA12 / m2) oligonucleotides used in the experiments degradation by serum enzymes; and in (b) anti-HSV-1 anti-sense oligonucleotides semi-staples (HMV10, SMV10) and linear (MV10, SMVIO / ml) used in the experiments of degradation by serum enzymes and the tests for inhibition of proliferation of herpes simplex virus type 1 (HSV-1).
  • FIGS. 5a to 5e represent electrophoresis under denaturing conditions of staple, semi-staple or linear oligonucleotides after incubation in the presence of 10% fetal calf serum.
  • FIGURES 6a and 6b show the half-lives of staple, semi-staple or linear oligonucleotides incubated in the presence of 10% fetal calf serum.
  • FIGURE 7 represents the inhibition of the proliferation of the herpes simplex virus type 1 (HSV-1) by an antisense oligonucleotide semi-staple (SMV10) and a linear antisense mutant targeted on the same sequence ( SMVIO / ml).
  • HSV-1 herpes simplex virus type 1
  • SMV10 antisense oligonucleotide semi-staple
  • SMVIO / ml linear antisense mutant targeted on the same sequence
  • the staple or semi-staple oligonucleotides exhibit a higher resistance to nucleolytic degradation than that of linear oligonucleotides of similar sequence when they are incubated in the presence of serum.
  • the first family was built from a linear homonucleotide polyadenylate sequence (POLYA12), from which a staple sequence (HA12) and semi-staple sequences (HA12 / ml, SA12, RA12, RA12 / ml) of different natures were built. Linear mutants of lengths equal to two of these molecules are designated respectively by HA12 / m2 and SA12 / ml.
  • POLYA12 linear homonucleotide polyadenylate sequence
  • SA12, RA12, RA12 / ml staple sequence
  • RA12 / ml semi-staple sequences
  • the second family was constructed from a linear heteronucleotide duodecamer (MV10) complementary to the splicing junction of an early messenger RNA of the herpes simplex virus type 1. Sequences in semi-staples of different natures and of different sizes have been synthesized on this linear basis: HMV10 (semi-clip) and SMV10 (semi-clip). A linear mutant of SMV10 has also been synthesized and studied: SMVIO / ml. The chemical formulas of all of these oligonucleotides and their secondary structure are shown in Figure 4.
  • MV10 linear heteronucleotide duodecamer
  • the degradation of the linear oligonucleotides is processive, and the appearance of degradation products of decreasing length is observed, becoming shorter and shorter as a function of time, which indicates that the degradation is mainly due to exonucleases, and in particular of 3 '-exonucleases;
  • staple or semi-staple oligonucleotides are systematically more resistant to nucleolysis by serum enzymes, and a non-negligible fraction of non-degraded oligonucleotide remains detectable even after several hours of incubation at 37 ° C .;
  • the oligonucleotides SMV10 and SMVIO / ml whose sequences are given in FIG. 6 were used in these experiments.
  • the SMV10 oligonucleotide comprises on the one hand the linear fragment MV10, complementary on 12 bases to the splicing junction of a messenger RNA early in the replication cycle of the HSV-1 virus and on the other hand a 3 ′ end extended by octameric semi-clip.
  • the mutant SMVIO / ml comprises the same antisense sequence MV10 as SMV10 but a mutation has been introduced into the terminal octameric sequence, a mutation which gives SMVIO / ml a linear structure.
  • oligonucleotides in solution in 2X culture medium free of serum are deposited on the cell monolayer.
  • 50 ⁇ l of viral solution are deposited 5 minutes later.
  • the cells are incubated for 1 hour at 37 ° C and shaken carefully every 15 minutes. After 1 hour of incubation, the 100 ml of medium are aspirated and 500 ml of complete medium are added to the cells. Incubation is continued for 24 hours before being stopped by freezing the plates in liquid nitrogen. All inhibition measurements are carried out in duplicate or triplicate.
  • Virus titration 50 ⁇ l of oligonucleotides in solution in 2X culture medium free of serum are deposited on the cell monolayer. 50 ⁇ l of viral solution are deposited 5 minutes later.
  • the cells are incubated for 1 hour at 37 ° C and shaken carefully every 15 minutes. After 1 hour of incubation, the 100 ml of medium are aspirated and 500 ml of complete medium are added to the cells. Incubation is continued for 24 hours before being stopped by
  • the viruses are recovered directly in the culture medium after 3 rapid cycles of freezing in liquid nitrogen - thawing at 37 ° C. They are then diluted in a serum-free medium to carry out the actual titration.
  • the indicator cells are seeded the previous day in complete medium at the rate of 250,000 cells per well of 2 cm 2 . The following day, the medium is aspirated and 100 ml of the various viral dilutions are deposited per well. After a one hour incubation at 37 ° C with shaking every 15 minutes, the medium is aspirated and the cells are covered with complete medium (2.5% in serum) containing 1.2% methyl cellulose for a 3-day incubation at 37 ° C.
  • the medium is eliminated, the cells are fixed with a PBS / 10% formalin solution for 20 minutes and then stained with a 2% crystal violet solution in PBS / 20% ethanol for 20 minutes. The plates are then rinsed and the areas counted by transparency on the X-ray viewer. Titrations are performed in triplicate for each point. The inhibition calculations are carried out relative to the viral titers observed in the absence of nucleotides.
  • SMVIO / ml for concentrations ranging from 10 ⁇ M to 30 ⁇ M is shown in FIG. 7. It clearly appears there that the semi-staple oligonucleotide is active at concentrations lower than its linear antisense mutant. SMV10 induces 70% viral inhibition at 10 ⁇ M while SMVIO / ml is inactive at this concentration.

Abstract

Staple and semi-staple oligonucleotides useful as anti-sense molecules in cosmetics, diagnosis and especially pharmacology, and methods of preparation of such molecules. The staple and semi-staple oligonucleotides of the present invention are oligonucleotide type molecules, the 3' terminal or the two 3' and 5' terminals being engaged in intramolecular hydrogen bindings between pairs of complementary bases. As a result,these molecules are better able to withstand exonuclease attacks and act with greater durability as an anti-sense factor than the corresponding nuclear oligonucleotides.

Description

OUGONUCLEOTIDES AGRAFES ET SEMI-AGRAFES, PROCEDE DE PREPARATION ET APPLICATIONS. STAPLED AND SEMI-STAPLED OUGONUCLEOTIDES, METHOD OF PREPARATION AND APPLICATIONS.
La présente invention concerne un nouveau type d'oligonucléotides. Les oligonucleotides selon l'invention sont de courtes molécules synthétiques d'ADN, d'ARN ou mixtes. En particulier les oligonucleotides dits "antisens" ont une séquence complémentaire à une séquence cible appartenant à un gène ou à un ARN messager dont on désire bloquer spécifiquement l'expression. Les oligonucleotides antisens peuvent être dirigés contre une séquence d'ARN messager, ou bien contre une séquence d'ADN. Les oligonucleotides antisens hybrident à la séquence dont ils sont complémentaires et peuvent ainsi bloquer l'expression de TARN messager portant cette séquence.The present invention relates to a new type of oligonucleotide. The oligonucleotides according to the invention are short synthetic DNA, RNA or mixed molecules. In particular, the so-called "antisense" oligonucleotides have a sequence complementary to a target sequence belonging to a gene or to a messenger RNA whose expression it is desired to specifically block. The antisense oligonucleotides can be directed against a messenger RNA sequence, or alternatively against a DNA sequence. Antisense oligonucleotides hybridize to the sequence of which they are complementary and can thus block the expression of messenger RNA carrying this sequence.
Le terme "oligonucléotide" est ici utilisé de façon générale pour désigner un polynucléotide de 2 à 100, et plus généralement de 5 à 50, nucléotides en série ribo-, désoxyribo- ou mixte. Lorsqu'il sera question qu'une propriété particulière liée à l'utilisation d'une série désoxyribo- ou d'une série ribo-, on utilisera la dénomination complète oligodésoxyribonucléotide ou oligoribonucléotide. Le terme "appartement" ou "hybridation" utilisé ici signifie la formation de liaisons hydrogènes entre des paires de bases complémentaires, telles que la guanine et la cytosine formant trois liens hydrogènes entre elles, et l'adénine et la thymine ou l'uracile en formant deux. I . ETAT DE LA TECHNIQUEThe term "oligonucleotide" is used here generally to designate a polynucleotide from 2 to 100, and more generally from 5 to 50, nucleotides in ribo-, deoxyribo- or mixed series. When it is a question of a particular property linked to the use of a deoxyribo- or a ribo- series, the full name oligodeoxyribonucleotide or oligoribonucleotide will be used. The term "apartment" or "hybridization" used herein means the formation of hydrogen bonds between complementary base pairs, such as guanine and cytosine forming three hydrogen bonds between them, and adenine and thymine or uracil in forming two. I. STATE OF THE ART
1 . 1 . Les oligonucleotides antisens modifiés Les oligonucleotides antisens sont synthétisés par voie chimique et comportent fréquemment des modifications altérant le squelette même de la molécule ou portent des groupement réactifs additionnels, localisés à leurs extrémités. Ces modifications introduites dans les oligonucleotides antisens ont pour buts soit d'augmenter la résistance de ces molécules à la dégradation nucléolytique, soit de favoriser leurs interactions avec leurs cibles, soit de permettre des réactions de dégradation/modification spécifiques des cibles, ARN ou ADN, soit d'accroître leur pénétration intra¬ cellulaire.1. 1. The modified antisense oligonucleotides The antisense oligonucleotides are synthesized chemically and frequently contain modifications which alter the very backbone of the molecule or carry additional reactive groups, located at their ends. The purpose of these modifications introduced into the antisense oligonucleotides is either to increase the resistance of these molecules to nucleolytic degradation, or to promote their interactions with their targets, or to allow specific degradation / modification reactions of the targets, RNA or DNA, or to increase their intra-cellular penetration.
Les oligonucleotides antisens sont sensibles aux dégradations nucléasiques, et principalement à l'action des exonucléases. Les nucléases se trouvent dans tous les compartiments -cellulaires et extra-cellulaires, en particulier dans le sérum- et provoquent une dégradation rapide de ces molécules. Une utilisation pharmacologique de molécules antisens implique la résolution de ces problèmes de dégradation afin d'arriver à une pharmacocinétique satisfaisante et donc à une pérennisation suffisante des effets de ces molécules. De nombreuses modifications chimiques permettent aux oligonucleotides antisens de devenir résistants aux nucléases.Antisense oligonucleotides are sensitive to nuclease degradation, and mainly to the action of exonucleases. The nucleases are found in all compartments - cellular and extra-cellular, in particular in the serum - and cause a rapid degradation of these molecules. Pharmacological use of antisense molecules involves solving these degradation problems in order to achieve satisfactory pharmacokinetics and therefore sufficient sustainability of the effects of these molecules. Numerous chemical modifications allow the antisense oligonucleotides to become resistant to nucleases.
Certaines modifications affectent directement la structure ou la nature de la liaison phosphodiester (méthylphosphonates, phosphorothioates, oligonucleotides alpha, phosphoramidates, pour citer quelques exemples), d'autres consistent en l'addition de groupements de blocage aux extrémités 3' et 5' des molécules (Perbost et al., 1989; Bertrand et al., 1989; Bazile et al., 1989; Andrus et al., 1989; Cazenave et al, 1989; Zon, 1988; Maher and Dolnick, 1988; Gagnor et al., 1987; Markus-Sekura, 1987). 1 . 2 . Ciblage des oligonucleotides antisens anti-ARN messager Toutes les régions d'un ARN messager ne sont pas sensibles de la même façon aux effets d'un oligonucléotide antisens. Un ARN messager n'est pas une molécule linéaire figée, mais au contraire une molécule comportant de nombreuses structurations secondaires (hybridations intra-moléculaires complexes), tertiaires (repliements et conformations particulières, pseudo-noeuds), et interagissant avec des nucléoprotéines structurales et fonctionnelles (protéines basiques, complexes d'épissage, de poly-adénylation, de capping, complexe de traduction par exemple). La disponibilité effective et l'accessibilité des différentes régions d'un ARN messager va dépendre de leurs engagements dans ces structurations. Corrélativement, l'efficacité d'un agent inhibiteur interagissant avec telle ou telle séquence va dépendre également de l'engagement de cette séquences dans une fonction particulière. Les régions cibles pour les molécules antisens doivent être accessibles à l'oligonucléotide.Certain modifications directly affect the structure or nature of the phosphodiester bond (methylphosphonates, phosphorothioates, alpha oligonucleotides, phosphoramidates, to cite a few examples), others consist of the addition of blocking groups at the 3 ′ and 5 ′ ends of the molecules. (Perbost et al., 1989; Bertrand et al., 1989; Bazile et al., 1989; Andrus et al., 1989; Cazenave et al, 1989; Zon, 1988; Maher and Dolnick, 1988; Gagnor et al., 1987; Markus-Sekura, 1987). 1. 2. Targeting of antisense anti-messenger RNA oligonucleotides All regions of a messenger RNA are not equally sensitive to the effects of an antisense oligonucleotide. A messenger RNA is not a fixed linear molecule, but on the contrary a molecule comprising many secondary structures (complex intra-molecular hybridizations), tertiary (folds and particular conformations, pseudo-nodes), and interacting with structural and functional nucleoproteins (basic proteins, splicing complexes, polyadenylation, capping, translation complex for example). The effective availability and accessibility of the different regions of a messenger RNA will depend on their commitments in these structures. Correlatively, the effectiveness of an inhibiting agent interacting with such and such a sequence will also depend on the engagement of this sequence in a particular function. The target regions for the antisense molecules must be accessible to the oligonucleotide.
L'utilisation de logiciels de prédiction de structures secondaires permet de prévoir des degrés d'accessibilité théoriques et donc d'orienter le choix de cibles pour des oligonucléotide antisens. Globalement, les régions les plus utilisées comme cibles sont les sites de démarrage de la traduction (région de l'AUG initiateur) ainsi que les sites d'épissage (jonctions SD/SA). De nombreuses autres séquences n'ayant pas de fonctionnalités particulières et non engagées dans des appartement intra- moléculaires se sont avérées également efficaces comme cible pour des oligonucleotides antisens (voir les exemples cités plus loin). 1 . 3 . Oligonucleotides antisens dirigés contre l'ADNThe use of secondary structure prediction software makes it possible to predict theoretical degrees of accessibility and therefore to orient the choice of targets for antisense oligonucleotides. Overall, the regions most used as targets are the translation start sites (initiating AUG region) as well as splicing sites (SD / SA junctions). Numerous other sequences having no particular functionalities and not engaged in intramolecular apartments have also proved to be effective as targets for antisense oligonucleotides (see the examples cited below). 1. 3. Antisense oligonucleotides directed against DNA
Les oligodésoxyribonucléotides antisens peuvent également être dirigés contre certaines régions d'ADN bicaténaire (séquences homopurines/homopyrimidines ou riches en purines/pyrimidines) et former ainsi des triples hélices (Perroualt et al., 1990; François et al. (A), 1989; François et al.(B), 1989; François et al.(C), 1989; Wang et al., 1989; Maher et al., 1989; Sun et al., 1989; Boidot-Forget et al., 1988; Moser and Dervan, 1987; Dervan, 1986). Des oligonucleotides dirigées ainsi contre l'ADN ont été appelés "anti-gène" ou encore "anti-code". La formation d'une triple hélice, au niveau d'une séquence particulière, peut bloquer la fixation de protéines intervenant dans l'expression d'un gène et/ou permettre d'introduire des dommages irréversibles dans l'ADN si l 'oligonucléotide considéré possède un groupement réactif particulier. De tels oligonucleotides antisens peuvent devenir de véritables endonucléases de restriction artificielles, dirigées à la demande contre des séquences spécifiques.Antisense oligodeoxyribonucleotides can also be directed against certain regions of double-stranded DNA (homopurine / homopyrimidine sequences or rich in purines / pyrimidines) and thus form triple helices (Perroualt et al., 1990; François et al. (A), 1989; François et al. (B), 1989; François et al. (C), 1989; Wang et al., 1989; Maher et al., 1989; Sun et al., 1989; Boidot-Forget et al., 1988; Moser and Dervan, 1987; Dervan, 1986). Oligonucleotides thus directed against DNA have been called "anti-gene" or "anti-code". The formation of a triple helix, at the level of a particular sequence, can block the binding of proteins involved in the expression of a gene and / or allow the introduction of irreversible damage in DNA if the oligonucleotide considered has a particular reactive group. Such antisense oligonucleotides can become real artificial restriction endonucleases, directed on demand against specific sequences.
1 . 4 . Mode d'action des oligonucleotides antisens L'hybridation entre un oligonucléotide antisens et un ARN messager cible peut en bloquer l'expression de plusieurs manières, soit de façon stérique, soit de façon pseudo-catalytique (Gagnor et al., 1989; Jessus et al., 1988; Markus-Sekura, 1987):1. 4. Mode of action of antisense oligonucleotides Hybridization between an antisense oligonucleotide and a target messenger RNA can block its expression in several ways, either sterically or pseudo-catalytically (Gagnor et al., 1989; Jessus et al., 1988; Markus-Sekura, 1987):
-l'interaction entre l'ARN messager et un oligonucléotide antisens complémentaire peut créer une barrière physique interdisant la fixation et/ou la progression de protéines ou de complexes protéiques nécessaire à la traduction, à la maturation, à la stabilisation ou au transport de l'ARN messager. Ce blocage physique aboutira finalement à une inhibition de l'expression de l'ARN messager ciblé.the interaction between the messenger RNA and a complementary antisense oligonucleotide can create a physical barrier preventing the fixation and / or the progression of proteins or protein complexes necessary for the translation, maturation, stabilization or transport of l 'Messenger RNA. This physical blockage will ultimately result in inhibition of expression of the targeted messenger RNA.
-l 'hybridation entre un ARN messager et un oligodésoxyribonucléotide antisens va créer un substrat pour la RNase H, enzyme présent dans toutes les cellules eucaryotes. La RNase H est un enzyme qui dégrade spécifiquement l'ARN lorsqu'il est hybride à de l'ADN.-the hybridization between a messenger RNA and an antisense oligodeoxyribonucleotide will create a substrate for RNase H, an enzyme present in all eukaryotic cells. RNase H is an enzyme that specifically degrades RNA when it is hybridized to DNA.
L'hybridation d'un oligonucléotide antisens ADN à un ARN cible aboutira donc à la coupure de cet ARN cible à l'emplacement de cette hybridation, et donc à son inactivation définitive. -Par ailleurs, comme indiqué plus haut, les oligonucleotides antisens peuvent comporter des groupement réactifs capables de produire directement des dommages irréversibles dans la molécules d'ARN cible. En ce qui concerne les oligonucleotides antisens dirigés contre l'ADN ils peuvent agir soit en inhibant la fixation d'une protéine de régulation indispensable à l'expression du gène ciblé (facteur de transcription par exemple), soit en introduisant des dommages irréversibles (coupures, cross-links) dans la molécule d'ADN, la rendant inapte, localement, pour l'expression génétique.Hybridization of an antisense DNA oligonucleotide to a target RNA will therefore result in the cleavage of this target RNA at the location of this hybridization, and therefore in its definitive inactivation. Furthermore, as indicated above, the antisense oligonucleotides may contain reactive groups capable of directly producing irreversible damage in the target RNA molecules. As regards the antisense oligonucleotides directed against DNA, they can act either by inhibiting the binding of a regulatory protein essential for the expression of the targeted gene (transcription factor for example), or by introducing irreversible damage (cuts , cross-links) in the DNA molecule, rendering it locally incapable of gene expression.
1 . 5 . Exemples d'application des oligonucleotides antisens1. 5. Examples of application of antisense oligonucleotides
Les oligonucleotides antisens permettent de bloquer spécifiquement l'expression d'ARN messagers cellulaires, par exemple de messagers de type oncogénique, (Tortora et al., 1990; Chang et al., 1989; Anfossi et al., 1989; Zheng et al., 1989; Shuttleworth et al., 1988; Cope and Wille, 1989; Cazenave et al., 1989) et de nombreux différents types d'ARN messager viraux, provenant de virus aussi variés que le VSV (Degols et al., 1989; Leonetti et al., 1989), le SV40 (Westermann, et al., 1989), les virus influenza (Kabanov et al., 1990; Zerial et al., 1987), le virus de l'encéphalo- myocardite (Sankar et al., 1989), l'adénovirus (Miroshnichenko et al., 1989), le HSV (Gao et al., 1988) et le HIV (Matsukura et al., 1989; Stevenson et al., 1989; Matsukura et al., 1988; Goodchild et al., 1988). II . RÉSUMÉ DE L'INVENTION II . 1 . Problèmes soulevés par les applications pharmacologiques des antisensAntisense oligonucleotides make it possible to specifically block the expression of cellular messenger RNAs, for example messengers of the oncogenic type, (Tortora et al., 1990; Chang et al., 1989; Anfossi et al., 1989; Zheng et al. , 1989; Shuttleworth et al., 1988; Cope and Wille, 1989; Cazenave et al., 1989) and many different types of viral messenger RNA, from viruses as diverse as VSV (Degols et al., 1989; Leonetti et al., 1989), SV40 (Westermann, et al., 1989), influenza viruses (Kabanov et al., 1990; Zerial et al., 1987), encephalomyocarditis virus (Sankar and al., 1989), adenovirus (Miroshnichenko et al., 1989), HSV (Gao et al., 1988) and HIV (Matsukura et al., 1989; Stevenson et al., 1989; Matsukura et al. , 1988; Goodchild et al., 1988). II. SUMMARY OF THE INVENTION II. 1. Problems raised by pharmacological applications of antisense
Les oligonucleotides antisens sont donc des agents pharmacologiques potentiels, puissants et hautement spécifiques, permettant d'inhiber l'expression de messagers codant pour des produits exerçant des effets pathogènes.Antisense oligonucleotides are therefore potential, powerful and highly specific pharmacological agents, making it possible to inhibit the expression of messengers coding for products having pathogenic effects.
L'utilisation thérapeutique des oligonucleotides se heurte cependant à plusieurs problèmes de type physiologiques, en particulier celui de la délivrance intracellulaire de ces molécules et celui de leur sensibilité à la dégradation nucléolytique. Certaines modifications chimiques des oligonucleotides permettent de surmonter le problème de la sensibilité aux nucléases mais au prix d'un nouveau problème, celui de la toxicité éventuelle des modifications chimiques introduites dans la molécule.The therapeutic use of oligonucleotides however encounters several problems of physiological type, in particular that of the intracellular delivery of these molecules and that of their sensitivity to nucleolytic degradation. Certain chemical modifications of the oligonucleotides make it possible to overcome the problem of sensitivity to nucleases but at the cost of a new problem, that of the possible toxicity of the chemical modifications introduced into the molecule.
L'utilisation des oligonucleotides antisens modifiés pose en effet des problèmes de nature toxicologiques. Si certaines des modifications sont réputées plutôt neutres, la plupart ne sont pas dénuées de toxicité potentielle. Des oligonucleotides antisens modifiés chimiquement peuvent présenter une toxicité à plusieurs niveaux, soit directement par des effets de la molécule entière, soit indirectement via les effets des produits de dégradation. Des nucléotides porteurs de modifications chimiques, et présents dans une cellule à une concentration élevée peuvent ainsi présenter une toxicité -et plus particulièrement une génotoxicité- non négligeable au plan pharmacologique.The use of modified antisense oligonucleotides poses toxicological problems. While some of the changes are said to be fairly neutral, most are not devoid of potential toxicity. Chemically modified antisense oligonucleotides can exhibit toxicity at several levels, either directly through the effects of the whole molecule, or indirectly via the effects of degradation products. Nucleotides carrying chemical modifications, and present in a cell at a high concentration can thus present a toxicity - and more particularly a genotoxicity - not negligible from the pharmacological point of view.
Par exemple, de nombreux problèmes soulevés par l'emploi d'oligonucléotides antisens modifiés, en particulier des effets anti-viraux non séquence-spécifiques, semblent bien être dus à la nature de certaines des modifications chimiques introduites dans les oligonucleotides antisens pour les rendre résistants aux nucléases.For example, many of the problems raised by the use of modified antisense oligonucleotides, in particular non-sequence-specific anti-viral effects, seem to be due to the nature of some of the chemical modifications introduced into antisense oligonucleotides to make them resistant. nucleases.
Au plan toxicologique, il est donc évident que moins on modifie la structure naturelle de l'oligonucléotide, moins on risque d'être confronté à des problèmes pharmacologiques. Une molécule naturelle d'ADN ou d'ARN, ainsi que ses produits de dégradation, ne pose pas ou peu de problème de toxicologie et de pharmacocinétique, ce qui n'est pas le cas d'une structure modifiée pouvant donner après métabolisation des dérivés multiples et potentiellement toxiques. Les oligonucleotides antisens peuvent également dans certains cas présenter une toxicité spécifique liée à la séquence ciblée. En effet l'oligonucléotide peut s'apparier à des séquences possédant un certain degré d'homologie avec la séquence cible induisant de ce fait un effet antisens indésiré et toxique. II . 2 . Sensibilité nucléolytique des oligonucleotides antisens.From a toxicological point of view, it is therefore obvious that the less we modify the natural structure of the oligonucleotide, the less we are likely to be confronted with pharmacological problems. A natural molecule of DNA or RNA, as well as its degradation products, poses little or no problem of toxicology and pharmacokinetics, which is not the case of a modified structure which can give after metabolization of the derivatives multiple and potentially toxic. Antisense oligonucleotides may also in some cases exhibit specific toxicity linked to the targeted sequence. Indeed, the oligonucleotide can be paired with sequences having a certain degree of homology with the target sequence thereby inducing an undesired and toxic antisense effect. II. 2. Nucleolytic sensitivity of antisense oligonucleotides.
Dans les cellules, et plus encore dans un organisme, dans la circulation sanguine par exemple, les oligonucleotides antisens naturels sont sensibles aux nucléases.In cells, and even more in an organism, in the bloodstream for example, natural antisense oligonucleotides are sensitive to nucleases.
Les nucléases sont des enzymes de dégradation capables de couper les liaisons phosphodiester de l'ADN ou de l'ARN, soit en introduisant des coupures internes sur des molécules mono- ou bicaténaires, soit en attaquant ces molécules à partir de leurs extrémités. Les enzymes attaquant de façon interne sont appelées les endonucléases et celles attaquant par les extrémités sont appelés exonucléases. La stabilité des oligonucleotides antisens -donc leur efficacité- peut être considérablement augmentée en introduisant diverses modifications chimiques les rendant résistants à la dégradation comme décrit ci-dessus. Il est établi que ce sont les exonucléases qui sont la principale cause de dégradation des oligonucleotides antisens dans le sérum et dans la cellule.Nucleases are degradation enzymes capable of cutting the phosphodiester bonds of DNA or RNA, either by introducing internal cleavages on single- or double-stranded molecules, or by attacking these molecules from their ends. Enzymes attacking internally are called endonucleases and those attacking from the ends are called exonucleases. The stability of antisense oligonucleotides - hence their efficiency - can be considerably increased by introducing various chemical modifications making them resistant to degradation as described above. It is established that it is the exonucleases which are the main cause of degradation of the antisense oligonucleotides in the serum and in the cell.
Plus particulièrement, il semble que les exonucléases s'attaquant à l'extrémité 3 'OH soient surtout à incriminer dans ce phénomène. Des modifications apportées à la structure des extrémités des oligonucleotides antisens peuvent les protéger, bloquer les activités des exonucléases, et conférer aux oligonucleotides une stabilisation accrue.More particularly, it seems that the exonucleases attacking the 3 'OH end are especially to be accused of this phenomenon. Changes in the structure of the ends of the antisense oligonucleotides can protect them, block the activities of exonucleases, and give the oligonucleotides increased stabilization.
Les contraintes thermodynamiques régissant la structure des acides nucléiques sont plus faibles dans une molécule d'ARN que dans une molécule d'ADN. Ainsi, contrairement aux molécules d'ADN, les molécules d'ARN adoptent généralement spontanément des structures secondaires médiées par des appartements intramoléculaires. La structure secondaire des molécules d'ARN résulte directement de la séquence de ces molécules. Ainsi, dans le cas de molécules d'ARN antisens naturels, la séquence de la cible peut permettre l'apparition spontanée d'une structure secondaire de la molécule complémentaire, pouvant dans certains cas augmenter sa résistance à la dégradation nucléolytique. Compte tenu de contraintes thermodynamiques plus fortes, les courts fragments d'ADN synthétiques utilisés comme agent antisens, plus rigides, n'adoptent que plus rarement des structures secondaires, et la stabilité des structures adoptées est généralement plus faible. Ainsi, pou de nombreuses séquences cibles, l'apparition d'une structure secondaire incluant l'appariement intramoléculaire de l'extrémité 3' dans la molécule complémentaire est improbable. Le but de la présente invention est de fournir des oligonucleotides antisens, notamment ne comportant que des désoxyribonucléotides normaux reliés entre eux par une liaison phosphodiester naturelle, mais présentant cependant une résistance à la dégradation, sans introduire de modifications chimiques susceptibles d'induire une toxicité, pouvant comporter toute séquence anti-sens d'intérêt.The thermodynamic constraints governing the structure of nucleic acids are lower in an RNA molecule than in a DNA molecule. Thus, unlike DNA molecules, RNA molecules generally spontaneously adopt secondary structures mediated by intramolecular apartments. The secondary structure of RNA molecules results directly from the sequence of these molecules. Thus, in the case of natural antisense RNA molecules, the target sequence can allow the spontaneous appearance of a secondary structure of the complementary molecule, which can in certain cases increase its resistance to nucleolytic degradation. Taking into account higher thermodynamic constraints, the short synthetic DNA fragments used as antisense agent, more rigid, only rarely adopt secondary structures, and the stability of the adopted structures is generally weaker. Thus, for many target sequences, the appearance of a secondary structure including the intramolecular pairing of the 3 ′ end in the complementary molecule is improbable. The aim of the present invention is to provide antisense oligonucleotides, in particular comprising only normal deoxyribonucleotides linked together by a natural phosphodiester bond, but nevertheless exhibiting resistance to degradation, without introducing chemical modifications capable of inducing toxicity, which may include any anti-sense sequence of interest.
II . 3 . Oligonucleotides agrafes et semi-agrafesII. 3. Oligonucleotides staples and semi-staples
Pour ce faire la présente invention fournit une molécule d'oligonucléotide présentant au moins une extrémité 3' auto-appariée, appelée oligonucléotide semi-agrafe ou agrafe dans le cas où l'extrémité 5' de cette molécule est également auto-appariée. Plus précisément, la présente invention a pour objet des oligonucleotides anti-sens semi-agrafes ou agrafes constitués d'une séquence oligonucléotidique dont l'extrémité 3' ou les deux extrémités 3' et 5' sont engagées dans un auto-appariement intracaténaire impliquant l'hybridation d'au moins les deux bases terminales de l'extrémité 3' ou respectivement de chaque extrémité 3' et 5', avec un même nombre de bases consécutives complémentaires situées sur le brin, de manière à former une boucle à l'extrémité 3' ou respectivement une boucle à chaque extrémité encadrant une séquence centrale. Selon l'invention, d'une part, ladite séquence centrale comprend toute la séquence d'intérêt complémentaire d'une séquence cible appartenant à l'ADN ou l'ARNmessager dont on désire bloquer l'expression et, d'autre part, lesdites boucles ont une séquence telle qu'elles ne s'hybrident pas avec le brin de la séquence cible. Les oligonucleotides selon la présente invention, dont les extrémitésTo do this, the present invention provides an oligonucleotide molecule having at least one self-paired 3 'end, called a semi-staple or staple oligonucleotide in the case where the 5' end of this molecule is also self-paired. More specifically, the present invention relates to antisense oligonucleotides semi-staples or staples consisting of an oligonucleotide sequence whose 3 'end or both 3' and 5 'ends are engaged in an intracatenary self-pairing involving '' hybridization of at least the two terminal bases of the 3 'end or respectively of each 3' and 5 'end, with the same number of complementary consecutive bases located on the strand, so as to form a loop at the end 3 'or respectively a loop at each end framing a central sequence. According to the invention, on the one hand, said central sequence comprises the entire sequence of interest complementary to a target sequence belonging to the DNA or the RNA messenger whose expression it is desired to block and, on the other hand, said loops have a sequence such that they do not hybridize with the strand of the target sequence. The oligonucleotides according to the present invention, the ends of which
3' et/ou 5' engagées dans des liaisons d'hybridations intramoléculaires qui réservent un accès réduit aux exonucléases respectivement 3' et/ou 5' et par suite une sensibilité réduite et une protection partielle de ces oligonucleotides aux activités nucléolytiques de ces enzymes. Les oligonucleotides présentant le type de structure secondaire selon la présente invention sont par suite stabilisés dans les milieux contenant ce type d'activités enzymatiques comme le sérum ou le milieu intracellulaire.3 'and / or 5' engaged in intramolecular hybridization bonds which reserve reduced access to the exonucleases 3 'and / or 5' respectively and consequently a reduced sensitivity and a partial protection of these oligonucleotides to the nucleolytic activities of these enzymes. The oligonucleotides having the type of secondary structure according to the present invention are consequently stabilized in the media containing this type of enzymatic activities such as the serum or the intracellular medium.
Selon la présente invention, ladite séquence anti-sens d'intérêt ne se chevauche pas avec ladite séquence auto-appariée car ladite séquence anti-sens d'intérêt est entièrement comprise dans ladite séquence centrale. Cette caractéristique est particulièrement avantageuse car la stabilité et la longueur de la structure auto-appariée sont contrôlées indépendamment de la séquence anti-sens, et donc de la cible. Ces deux paramètres reposent uniquement sur les caractéristiques d'un fragment à structure en agrafe rajouté à l'extrémité 3' ou aux deux extrémités 3' et 5'. La stabilité de la structure en agrafe terminale dépend de la nature des bases engagées dans l'appariement intramoléculaire et ne dépend pas, dans ce mode de réalisation, de la séquence anti-sens. Elle est, par conséquent, indépendante de la séquence cible. Dans un mode de réalisation contraire à la présente invention, où la séquence anti-sens d'intérêt se chevauche avec la séquence auto- appariée, la stabilité de la structure secondaire responsable de la résistance aux nucléases est par principe limitée. D'une part, un appartement intramoléculaire trop stable inhiberait la formation du duplex cible/antisens. D'autre part, la nature des bases engagées dans l'appartement intramoléculaire est partiellement imposée par le choix de la séquence cible. Ainsi, par exemple, lorsque les bases "imposées" par la séquence cible dans l'appartement intracaténaire seront des adénines ou des thymines, formant des appartement moins stables, la stabilité de la structure secondaire induite sera plus faible que si ces bases étaient des guanines ou des cytosines et, par suite, la résistance aux nucléases s'en trouvera réduite. En outre, lorsque les bases "imposées" sont des adénines ou des thymines, formant des appartements moins stables, il est nécessaire, pour obtenir une stabilité suffisante de la structure secondaire, d'augmenter la longueur du segment double-brin, et par conséquent la longueur du fragment rajouté en 3', et par suite la longueur totale de l'oligonucléotide. Et, pour des stabilités de structures secondaires équivalentes, un oligonucléotide plus long aura davantage de chances de s'apparier de manière non spécifique et d'induire de la toxicité. Les oligonucleotides antisens présentent en effet parfois une toxicité résultant d'appartements parasites avec des séquences partiellement homologues à la séquence cible. Dans le mode de réalisation selon la présente invention, la stabilité des structures secondaires caractérisant les oligonucleotides agrafes ou semi-agrafes peut être augmentée sans limitation, assurant ainsi une protection maximale contre les nucléases, sans nuire à l'hybridation de l'oligonucléotide antisens sur sa cible.According to the present invention, said antisense sequence of interest does not overlap with said self-paired sequence because said antisense sequence of interest is entirely included in said central sequence. This characteristic is particularly advantageous since the stability and the length of the self-paired structure are controlled independently of the antisense sequence, and therefore of the target. These two parameters are based solely on the characteristics of a fragment with a staple structure added at the 3 ′ end or at the two 3 ′ and 5 ′ ends. The stability of the terminal clip structure depends on the nature of the bases involved in the intramolecular pairing and does not depend, in this embodiment, on the antisense sequence. It is therefore independent of the target sequence. In an embodiment contrary to the present invention, where the antisense sequence of interest overlaps with the self-paired sequence, the stability of the secondary structure responsible for nuclease resistance is in principle limited. On the one hand, an excessively stable intramolecular apartment would inhibit the formation of the target / antisense duplex. On the other hand, the nature of the bases involved in the intramolecular apartment is partially imposed by the choice of the target sequence. Thus, for example, when the bases "imposed" by the target sequence in the intracatenary apartment will be adenines or thymines, forming less stable apartments, the stability of the induced secondary structure will be weaker than if these bases were guanines or cytosines and, as a result, resistance to nucleases will be reduced. In addition, when the "imposed" bases are adenines or thymines, forming less stable apartments, it is necessary, in order to obtain sufficient stability of the secondary structure, to increase the length of the double-stranded segment, and consequently the length of the fragment added in 3 ′, and consequently the total length of the oligonucleotide. And, for equivalent secondary structure stabilities, a longer oligonucleotide will be more likely to pair nonspecifically and induce toxicity. Antisense oligonucleotides sometimes exhibit toxicity resulting from parasitic apartments with sequences partially homologous to the target sequence. In the embodiment according to the present invention, the stability of the secondary structures characterizing the staple or semi-staple oligonucleotides can be increased without limitation, thus ensuring maximum protection against nucleases, without harming the hybridization of the antisense oligonucleotide on his target.
Les oligonucleotides selon la présente invention peuvent être utilisés dans leur état naturel mais peuvent cependant comporter également des nucléotides modifiés dans leurs liaisons internucléotidiques, dans les cycles osidiques ou dans les bases, dans le but de favoriser la pénétration cellulaire, et/ou la résistance aux nucléases, et/ou renforcer ou stabiliser leur hybridation avec une séquence complémentaire. Les oligonucleotides suivant l'invention pouvant comporter en outre des groupements intercalants ou réactifs, ou être associés physiquement à d'autres molécules ou macromolécules dans le but de renforcer leur efficacité d'inhibition, leur pénétration, leur affinité pour les cibles, leur ciblage cellulaire ou intracellulaire, ou pour optimiser toute autre propriété.The oligonucleotides according to the present invention can be used in their natural state but can however also contain nucleotides modified in their internucleotide bonds, in the osidic cycles or in the bases, with the aim of promoting cell penetration, and / or resistance to nucleases, and / or reinforce or stabilize their hybridization with a sequence complementary. The oligonucleotides according to the invention may also comprise intercalating or reactive groups, or be physically associated with other molecules or macromolecules with the aim of enhancing their inhibition efficiency, their penetration, their affinity for targets, their cell targeting or intracellular, or to optimize any other property.
Sont donc comprises dans l'invention, toute molécule d'ADN, ou d'ARN, ou mixte ADN/ARN, monocaténaire, obtenue de façon biologique, chimique, ou par des procédés associant les techniques de la chimie de synthèse à celles de la biologie et de la biochimie, dont la séquence primaire des nucléotides qui la composent induit l'apparition d'une structure secondaire particulière définie par la présence au minimum de l'appariement des deux dernières bases situées à l'extrémité 3 ' de la séquence avec deux bases nucléotidiques consécutives situées au sein de la même molécule, et présentant une résistance aux exonucléases supérieure à celle d'un oligonucléotide de longueur et composition nucléotidique identique mais linéaire.Are therefore included in the invention, any DNA molecule, or RNA, or mixed DNA / RNA, single-stranded, obtained biologically, chemically, or by methods combining the techniques of synthetic chemistry with those of biology and biochemistry, whose primary sequence of the nucleotides which compose it induces the appearance of a particular secondary structure defined by the presence at least of the pairing of the last two bases located at the 3 'end of the sequence with two consecutive nucleotide bases located within the same molecule, and having a resistance to exonucleases greater than that of an oligonucleotide of identical length and nucleotide composition but linear.
Les molécules satisfaisant à cette définition peuvent être subdivisées en deux catégories, les molécules agrafes et les molécules semi-agrafes, et représentées schématiquement de la manière suivante:The molecules satisfying this definition can be subdivided into two categories, staple molecules and semi-staple molecules, and represented schematically as follows:
Figure imgf000011_0001
Figure imgf000011_0001
OUGONUCLEOTIDE AGRAFE OUGONUCLEOTIDE SEMI-AGRAFESTAPLED OUGONUCLEOTIDE SEMI-STAPED OUGONUCLEOTIDE
°ù Pi» P 2 et P3 représentent les nombres de nucléotides constituant les boucles et sont variables d'un oligonucléotide à l'autre; n1 ( n2 et n3 représentent les nombres de nucléotides impliqués dans les appartements intracaténaires; ils sont variables d'un oligonucléotide à l'autre mais nécessairement supérieurs à 2° ù Pi » P 2 and P 3 represent the numbers of nucleotides constituting the loops and are variable from one oligonucleotide to another; n 1 ( n 2 and n 3 represent the numbers of nucleotides involved in the intracatenary apartments; they are variable from one oligonucleotide to another but necessarily greater than 2
Il résulte de cette structure qu'à l'extrémité, ou aux deux extrémités 3' et 5', les nj (au moins deux) bases terminales impliquées dans un appartement intracaténaire sont immédiatement suivies ou précédées de pi bases (au moins deux) non appariées, constituant une zone charnière en épingle à cheveux. La dite zone charnière est donc adjacente à une autre zone de n;It follows from this structure that at the end, or at both 3 'and 5' ends, the nj (at least two) terminal bases involved in an intracatenary apartment are immediately followed or preceded by pi bases (at least two) not paired, forming a pin hinge area to hair. Said hinge zone is therefore adjacent to another zone of n;
(au moins deux) nucléotides consécutifs complémentaires des ni dites bases terminales.(at least two) consecutive nucleotides complementary to neither said terminal bases.
La boucle constituant les agrafes comporte donc une séquence oligonucléotidique charnière d'au moins 1, de préférence de 3 à 25 nucléotides, et de préférence encore, de 4 à 20 nucléotides. En effet, si la zone charnière est de taille trop importante, la stabilité de l'hybridation intracaténaire se trouve fragilisée.The loop constituting the staples therefore comprises a hinge oligonucleotide sequence of at least 1, preferably from 3 to 25 nucleotides, and more preferably, from 4 to 20 nucleotides. In fact, if the hinge zone is too large, the stability of the intracatenary hybridization is weakened.
Dans un mode de réalisation, l'oligonucléotide agrafe est constitué par une séquence d'intérêt d'ADN ou mixte ADN/ARN linéaire complémentaire d'une séquence cible d'ADN ou d'ARNm dont on désire bloquer l'expression, à l'extrémité 3' ou aux deux extrémités 3' et 5' de laquelle on a rajouté une boucle consistant en une séquence d'ADN ou mixte ADN/ARN auto-appariée comportant 8 à 12 nucléotides, ladite séquence auto-appariée étant caractérisée par l'appariement de son extrémité 3' avec son extrémité 5' et de la totalité de ses nucléotides deux à deux à l'exception de ceux constituant une séquence dite charnière. Dans ce cas particulier d'oligonucléotide semi-agrafe, ladite boucle ou séquence auto-appariée est en "épingle à cheveux", l'extrémité 5' est appariée à l'extrémité 3' et la totalité des nucléotides n'appartenant pas à la zone dite charnière sont appariés deux à deux.In one embodiment, the staple oligonucleotide consists of a DNA or mixed DNA / RNA linear sequence of interest complementary to a target DNA or mRNA sequence the expression of which it is desired to block, 'end 3' or at the two ends 3 'and 5' to which a loop consisting of a DNA sequence or mixed DNA / RNA self-paired comprising 8 to 12 nucleotides has been added, said self-paired sequence being characterized by l 'pairing of its 3' end with its 5 'end and all of its nucleotides two by two with the exception of those constituting a so-called hinge sequence. In this particular case of semi-staple oligonucleotide, said self-paired loop or sequence is in "hairpin", the 5 'end is paired at the 3' end and all the nucleotides not belonging to the so-called hinge area are paired two by two.
Un exemple de ce type de fragment est présenté sur les figures 2, 4 et 5 de la présente description. Il s'agit, dans ce cas, d'un fragment octamérique dont la structure secondaire intrinsèque, particulièrement stable, a été étudiée. Ce fragment appartient à une famille de fragments de stabilités variables, dont la présente invention a validé l'intérêt en tant que fragment stabilisateur d'oligonucléotides semi-agrafes. On a démontré, selon la présente invention, qu'il existe une corrélation entre la stabilité thermodynamique des structures secondaires de ces fragments et la protection contre les nucléases qu'ils induisent lorsqu'ils constituent l'épingle à cheveux d'oligonucléotides semi-agrafes.An example of this type of fragment is presented in FIGS. 2, 4 and 5 of the present description. In this case, it is an octameric fragment whose intrinsic secondary structure, which is particularly stable, has been studied. This fragment belongs to a family of fragments of variable stabilities, the advantage of which the present invention has validated as a stabilizing fragment of semi-staple oligonucleotides. It has been demonstrated, according to the present invention, that there is a correlation between the thermodynamic stability of the secondary structures of these fragments and the protection against nucleases which they induce when they constitute the hairpin of semi-staple oligonucleotides. .
On cite en particulier un oligonucléotide constitué par une séquence anti-sens d'intérêt comportant à son extrémité 3' ou aux deux extrémités 3' et 5' une boucle de 8 nucléotides dans laquelle l'auto- appartement est dû à 4 nucléotides seulement, à savoir deux GC en 5' et 3'. Dans ce cas, la composition de la séquence charnière influe sur la stabilité de la boucle et on utilise de préférence l'une des séquences GCGAAAGC, GCTAAAGC, GCGAGAGC, GCGATAGC, GCGAATGC, GCGTTAGC, qui forment des auto-appariements très stables. Différents nucléotides peuvent rentrer dans la formulation d'oligonucléotides agrafes ou semi- agrafes. Les oligonucleotides agrafes et semi-agrafes faisant l'objet de la présente invention sont composés par une séquence de bases nucléotidiques comportant notamment de l'adénine (A), de la guanine (G), de la cytosine (C), de la thymine (T) et de l'uracile (U), reliées entre elles par des liaisons internucléotidiques, notamment naturelles c'est-à-dire phosphodiesters. Les formules générales de ces bases sont présentées en figure 1.We cite in particular an oligonucleotide constituted by an antisense sequence of interest comprising at its 3 ′ end or at both 3 ′ and 5 ′ ends a loop of 8 nucleotides in which the self-apartment is due to only 4 nucleotides, namely two GCs in 5 'and 3'. In this case, the composition of the hinge sequence influences the stability of the loop and one of the sequences GCGAAAGC, GCTAAAGC, GCGAGAGC, GCGATAGC, GCGAATGC, GCGTTAGC is preferably used, which form very stable self-pairings. Different nucleotides can enter into the formulation of staple or semi-staple oligonucleotides. The staple and semi-staple oligonucleotides forming the subject of the present invention are composed by a nucleotide base sequence comprising in particular adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), linked together by internucleotide bonds, in particular natural, that is to say phosphodiesters. The general formulas of these bases are presented in figure 1.
Les oligonucleotides agrafes et semi-agrafes selon l'invention peuvent également comporter des nucléotides rares (Inosine, I, ou ri par exemple) ou des nucléotides modifiés, soit en série désoxyribo- soit en série ribo-.The staple and semi-staple oligonucleotides according to the invention may also contain rare nucleotides (Inosine, I, or ri for example) or modified nucleotides, either in deoxyribo- series or in ribo- series.
Les oligonucleotides agrafes et semi-agrafes selon l'invention peuvent comporter des nucléotides réactifs, capables d'établir des liens avec la séquence de la molécule cible complémentaire à l'oligonucléotide ou, dans une autre application, des liens intramoléculaires au sein même de l'oligonucléotide agrafe ou semi-agrafe.The staple and semi-staple oligonucleotides according to the invention may comprise reactive nucleotides, capable of establishing links with the sequence of the target molecule complementary to the oligonucleotide or, in another application, intramolecular links within the same staple or semi-staple oligonucleotide.
Ainsi, les oligonucleotides agrafes et semi-agrafes selon l'invention peuvent porter des groupements réactifs greffés sur les nucléotides, comme par exemple des groupements psoralènes, ou d'autres agents de pontage ou agents intercalant pouvant réagir avec la séquence de la molécule cible complémentaire à l'oligonucléotide. Dans un cas particulièrement intéressant des groupements réactifs greffés sur certains des nucléotides de l'oligonucléotide agrafe ou semi-agrafe pourront induire la formation d'un pontage intramoléculaire au sein même de la molécule.Thus, the staple and semi-staple oligonucleotides according to the invention can carry reactive groups grafted onto the nucleotides, such as for example psoralen groups, or other bridging agents or intercalating agents which can react with the sequence of the complementary target molecule to the oligonucleotide. In a particularly interesting case, reactive groups grafted onto some of the nucleotides of the staple or semi-staple oligonucleotide may induce the formation of an intramolecular bridging within the molecule itself.
L'oligonucléotide pourra comporter des liaisons internes produites par des agents réactifs appartenant ou n'appartenant pas à la structure de la molécule elle-même.The oligonucleotide may have internal bonds produced by reactive agents belonging to or not belonging to the structure of the molecule itself.
Font également partie de l'invention des oligonucleotides agrafes ou semi-agrafes, dits chimériques, constitués par l'assemblage covalent de fragments nucléotides et non-nucléotides. Dans un cas particulièrement intéressant d'oligonucléotide agrafe ou semi-agrafe chimérique la ou les dites zones charnière seront de nature non-nucléotidique.Also part of the invention are staple or semi-staple oligonucleotides, called chimeric, constituted by the covalent assembly of nucleotide and non-nucleotide fragments. In a particularly interesting case of chimeric staple or semi-staple oligonucleotide, the said hinge zone or zones will be of non-nucleotide nature.
Font également partie de l'invention des oligonucleotides antisens agrafes ou semi-agrafes couplés à des molécules permettant d'accroître leur pénétration intra-cellulaire, et en particulier des groupement lipophiles, des polypeptides ou des protéines.Also included in the invention are staple or semi-staple antisense oligonucleotides coupled to molecules making it possible to increase their intracellular penetration, and in particular lipophilic groups, polypeptides or proteins.
D'une façon générale les oligonucleotides agrafes ou semi-agrafes décrits précédemment et faisant l'objet de l'invention sont destinés à fournir de nouveaux oligonucleotides stables capables d'être internalisés dans les cellules et d'interagir ensuite avec des facteurs cellulaires ou viraux présentant une affinité pour des séquences spécifiques d'acides nucléiques.In general, the staple or semi-staple oligonucleotides described above and forming the subject of the invention are intended to provide new stable oligonucleotides capable of being internalized in cells and then of interacting with cellular or viral factors having an affinity for specific nucleic acid sequences.
Ainsi, les oligonucleotides selon l'invention peuvent comporter un ou des groupement s) lipophile(s) ou toute autre structure moléculaire favorisant la pénétration, le ciblage, l'adressage cellulaire ou intra¬ cellulaire de ces oligonucleotides, ou stabilisant la structure de ces oligonucleotides.Thus, the oligonucleotides according to the invention may comprise one or more lipophilic group (s) or any other molecular structure promoting the penetration, targeting, cellular or intra¬ cellular addressing of these oligonucleotides, or stabilizing the structure of these oligonucleotides.
Les duplexes ADN/ARN sont substrats de la RNAse H. C'est pourquoi il peut être avantageux que la dite séquence d'intérêt comporte une séquence d'ADN ou d'ARN de manière à former un duplex ADN/ARN avec la séquence cible d'ARNm ou ADN respectivement.The DNA / RNA duplexes are substrates of the RNAse H. This is why it may be advantageous if the said sequence of interest comprises a DNA or RNA sequence so as to form a DNA / RNA duplex with the target sequence mRNA or DNA respectively.
Dans ce cas, il peut être approprié d'avoir recours à un oligonucléotide mixte avec, par exemple, une fenêtre d'ADN encadrée par deux séquences d'ARN ou, inversement, constituant les agrafes. Des exemples concrets de certaines de ces structures sont représentées en figure 4.In this case, it may be appropriate to use a mixed oligonucleotide with, for example, a DNA window framed by two RNA sequences or, conversely, constituting the staples. Concrete examples of some of these structures are shown in Figure 4.
Les oligonucleotides suivant l'invention peuvent par conséquent être obtenus par les méthodes conventionnelles de synthèse des oligonucleotides naturels, par voie chimique, biologique, ou par des approches faisant appel à des combinaisons des techniques de la chimie de synthèse et de la biologie moléculaire.The oligonucleotides according to the invention can therefore be obtained by conventional methods of synthesis of natural oligonucleotides, chemically, biologically, or by approaches using combinations of the techniques of synthetic chemistry and molecular biology.
Parmi les méthodes qualifiées de conventionnelles, diverses méthodes de synthèse chimique d'oligonucléotides naturels ont été développées et sont bien connu des spécialistes travaillant dans le domaine. Par exemple, une méthode consiste à utiliser un support solide ditAmong the methods qualified as conventional, various methods of chemical synthesis of natural oligonucleotides have been developed and are well known to specialists working in the field. For example, one method is to use a solid support called
CPG (controlled pore glass) sur lequel le premier nucléoside est fixé covalemment par un bras de couplage, par l'intermédiaire de son extrémité 3 'OH. L'extrémité 5 'OH du nucléoside est protégé par un groupe di-p-méthoxytrityl acido-labile. Cette approche, utilisant la chimie des phosphites triesters, et dans laquelle des désoxynucléosides 3'phosphoramidites sont utilisés comme synthons est appelée la méthode des phosphoramidites (Caruthers, 1985). Cette approche est la plus utilisée actuellement et présente l'avantage d'être entièrement automatique.CPG (controlled pore glass) on which the first nucleoside is attached covalently by a coupling arm, via its 3 'OH end. The 5 'OH end of the nucleoside is protected by an acid labile di-p-methoxytrityl group. This approach, using the chemistry of triester phosphites, and in which 3'phosphoramidite deoxynucleosides are used as synthons is called the phosphoramidite method (Caruthers, 1985). This approach is the most used today and has the advantage of being fully automatic.
Pour citer un second exemple, une autre approche utilisée pour la synthèse d'oligonucléotides est celle de la chimie des phosphonates (Froehler et al, 1986).To cite a second example, another approach used for the synthesis of oligonucleotides is that of phosphonate chemistry (Froehler et al, 1986).
Cette approche commence par la condensation d'un désoxynucléoside 3'-H-phosphonate avec un deoxynucléoside couplé à un support de verre de silice. Des cycles de condensation successifs conduisent à la synthèse d'oligonucléotides H-phosphonates. Ces oligonucleotides sont oxidés en une étape pour donner les phophodiesters.This approach begins with the condensation of a 3'-H-phosphonate deoxynucleoside with a deoxynucleoside coupled to a silica glass support. Successive condensation cycles lead to the synthesis of H-phosphonate oligonucleotides. These oligonucleotides are oxidized in one step to give the phophodiesters.
En utilisant l'une ou l'autre de ces techniques, ou tout autre procédure séquentielle permettant la synthèse de chaînes polynucléotidiques de séquence déterminée à l'avance, on obtient des oligonucleotides présentant la structure primaire désirée. Cette séquence primaire est à l'origine des structures secondaires qu'adoptera spontanément la molécule en fonction de sa séquence nucléotidique et des paramétres physico-chimiques des solutions dans lesquelles elle se trouvera.By using either of these techniques, or any other sequential procedure allowing the synthesis of polynucleotide chains of sequence determined in advance, oligonucleotides having the desired primary structure are obtained. This primary sequence is at the origin of the secondary structures which the molecule will spontaneously adopt according to its nucleotide sequence and the physico-chemical parameters of the solutions in which it will be found.
L'obtention d'un oligonucléotide agrafe ou semi-agrafe se fera par le choix d'une séquence primaire induisant la formation à l'extrémité 3' ou aux deux extrémités 3' et 5' de la molécule des structures secondaires caractérisant les oligonucleotides agrafes ou semi-agrafes selon l'invention.Obtaining a staple or semi-staple oligonucleotide will be done by the choice of a primary sequence inducing the formation at the 3 'end or at both 3' and 5 'ends of the molecule of the secondary structures characterizing the staple oligonucleotides or semi-staples according to the invention.
On prépare une séquence comportant ladite séquence anti-sens d'intérêt qui elle-même pourrait éventuellement adopter un auto- appartement, et on ajoute à l'extrémité 3' ou aux extrémités 3' et 5' de cette séquence anti-sens d'intérêt une séquence nucléotidique supplémentaire dont la nature des bases et la longueur permettent un auto-appariement stable de ladite séquence supplémentaire impliquant au moins ses deux bases terminales en 3' pour la séquence supplémentaire à l'extrémité 3', et en 5' pour la séquence supplémentaire à l'extrémité 5' le cas échéant. Les oligonucleotides agrafes et semi-agrafes faisant l'objet de l'invention décrite ici pourront être utilisés dans tous les cas ou il sera avantageux de disposer d'un oligonucléotide incluant une séquence primaire définie, présentant une résistance accrue aux exonucléases et un risque moindre d'appartement avec des séquences partiellement complémentaires par rapport à un oligonucléotide linéaire incluant la même séquence primaire.We prepare a sequence comprising said antisense sequence of interest which itself could possibly adopt a self-apartment, and we add to the 3 'end or to the 3' and 5 'ends of this antisense sequence of' interest an additional nucleotide sequence whose base nature and length allow a stable self-pairing of said additional sequence involving at least its two terminal bases in 3 'for the additional sequence at the 3' end, and in 5 'for the additional sequence at the 5 'end if necessary. The staple and semi-staple oligonucleotides forming the subject of the invention described here may be used in all cases where it will be advantageous to have an oligonucleotide including a defined primary sequence, having increased resistance to exonucleases and a lower risk of apartment with sequences partially complementary to a linear oligonucleotide including the same primary sequence.
Les oligonucleotides agrafes et semi-agrafes pourront en particulier être utilisés comme agents antisens pour agir de façon spécifique sur la transcription ou la traduction de protéine(s) dont on désire moduler le niveau d'expression dans un but de recherche ou de thérapeutique. La présente invention a donc pour objet l'utilisation des oligonucleotides selon l'invention, à titre de médicament.The staple and semi-staple oligonucleotides may in particular be used as antisense agents to act specifically on the transcription or translation of protein (s) whose expression level is to be modulated for research or therapeutic purposes. The present invention therefore relates to the use of the oligonucleotides according to the invention, as a medicament.
Les quelques applications possibles qui sont données ci-dessous ne sont que des exemples non-exhaustifs et en aucun cas limitatifs de situations dans lesquelles une approche de type antisens pourrait être utile, et où l'utilisation d'oligonucléotides naturels résistants aux exonucléases et présentant une toxicité des plus réduites offre un avantage. De façon générale, les oligonucleotides agrafes et semi-agrafes trouvent des terrains d'applications particulièrement adaptés dans le domaine de la dermatologie en raison de l'accessibilité des cibles à traiter, et de la toxicité mineure ou inexistante de ces composés. Toutes les affections dermatologiques, pouvant relever d'un mécanisme de dysfonctionnement génétique pour lesquelles on peut identifier un facteur étiologique, en connaître la séquence du gène, et/ou de l'ARN messager permet d'appliquer une approche anti-sens.The few possible applications which are given below are only non-exhaustive examples and in no way limitative of situations in which an antisense approach could be useful, and where the use of natural oligonucleotides resistant to exonucleases and having reduced toxicity offers an advantage. In general, staple and semi-staple oligonucleotides find areas of application particularly suitable in the field of dermatology due to the accessibility of the targets to be treated, and the minor or non-existent toxicity of these compounds. All the dermatological affections, being able to raise from a mechanism of genetic dysfunction for which one can identify an etiological factor, to know the sequence of the gene, and / or messenger RNA allows to apply an anti-sense approach.
L'utilisation d'oligonucléotides phosphodiesters naturels, présentant la toxicité la plus réduite, permet d'envisager la possibilité de ce genre d'approche pour des affections graves ou mineures, et même éventuellement pour des utilisations de type cosmétologiques, c'est-à-dire sur peau saine, et dans un domaine d'application ou les toxicités des produits doivent êtres nulles.The use of natural phosphodiester oligonucleotides, presenting the most reduced toxicity, makes it possible to envisage the possibility of this kind of approach for serious or minor affections, and even possibly for uses of cosmetic type, that is to say -to say on healthy skin, and in a field of application where the toxicities of the products must be zero.
A côté des cibles virales définies plus loin, de nombreuses maladies dermatologiques peuvent êtres traitées par des oligonucleotides agrafes ou semi-agrafes résistants aux exonucléases. Parmi les cibles potentielles de telles approches, on peut noter les maladies inflammatoires telles que les dermatites atopiques ou le lupus érythémateux, les maladies de la kératinisation comme l'ichtyose et le psoriasis, et les maladies tumorales comme le mélanome ou le lymphome T cutané. Ainsi par exemple, des oligonucleotides antisens agrafes ou semi-agrafes appliqués à la dermatologie peuvent être dirigés contre des ARN messagers de médiateurs de l'inflamation comme des interleukines, contre des RNA messagers de protéines impliquées dans les troubles de la prolifération des cellules épidermiques, ou bien contre des ARN messagers codant pour des protéines impliquées éventuellement dans le vieillissement cutané phénotypique, comme par exemple la collagénase, l'élastase, les transglutaminases.In addition to the viral targets defined below, many dermatological diseases can be treated with staple or semi-staple oligonucleotides resistant to exonucleases. Among the potential targets of such approaches, we can note inflammatory diseases such as atopic dermatitis or lupus erythematosus, keratinization diseases like ichthyosis and psoriasis, and tumor diseases like melanoma or cutaneous T lymphoma. Thus, for example, staple or semi-staple antisense oligonucleotides applied to dermatology can be directed against RNA messengers of inflammatory mediators such as interleukins, against RNA messengers of proteins implicated in epidermal cell proliferation disorders, or against messenger RNA coding for proteins possibly involved in phenotypic skin aging, such as, for example, collagenase, elastase, transglutaminases.
De façon plus générale, les oligonucleotides agrafes et semi-agrafes peuvent par exemple être utilisés comme agents antiviraux, antisens, que ce soit pour des indications topiques (dermatologiques) ou pour des indications systémiques. Par exemple, de tels oligonucleotides peuvent êtres utilisés comme agents anti-herpétiques (HSV 1 et HSV 2, CMV, EBV), comme agents anti-papillomavirus (HPV cutanés, génitaux, laryngés ou autres), comme agents anti-hépatites (HBV, HCV, HDV), comme agents anti- HIV (HIV-1 et HIV-2), comme agent anti-HTLV (HTLV-1 ou HTLV-2), etc.. Ces oligonucleotides agrafes ou semi-agrafes peuvent également être utilisés comme agents de répression de l'expression de certaines protéines cellulaires directement responsables ou impliquées dans l'étiologie de maladies de la prolifération et de la différenciation cellulaire. Par exemple, ces oligonucleotides agrafes ou semi-agrafes peuvent être dirigés contre l'expression d'oncogènes cellulaires hyper- exprimés ou exprimés de façon incontrôlés dans des types cellulaires tumoraux (RAS, ERB, NEU, SIS, MYC, MYB.etc...).More generally, the staple and semi-staple oligonucleotides can, for example, be used as antiviral or antisense agents, whether for topical (dermatological) indications or for systemic indications. For example, such oligonucleotides can be used as anti-herpetic agents (HSV 1 and HSV 2, CMV, EBV), as anti-papillomavirus agents (cutaneous, genital, laryngeal or other HPV), as anti-hepatitis agents (HBV, HCV, HDV), as anti-HIV agents (HIV-1 and HIV-2), as anti-HTLV agent (HTLV-1 or HTLV-2), etc. These staple or semi-staple oligonucleotides can also be used as agents for suppressing the expression of certain cellular proteins directly responsible for or involved in the etiology of diseases of cell proliferation and differentiation. For example, these staple or semi-staple oligonucleotides can be directed against the expression of hyper-expressed or uncontrolled cellular oncogenes in tumor cell types (RAS, ERB, NEU, SIS, MYC, MYB.etc .. .).
En particulier, ces oligonucleotides agrafes ou semi-agrafes naturels résistants aux exonucléases sériques peuvent êtres utilisés comme agent antisens dirigés contre des ARN messagers d'oncogènes exprimés dans des cellules leucémiques et impliqués dans leur prolifération. Dans le cadre du traitement de certaines leucémies, pour des greffes de moelle, les oligonucleotides agrafes ou semi-agrafes peuvent êtres utilisés dans le cadre d'applications "ex vivo". Pour ces nombreuses indications, des formulations galéniques adéquates doivent être établies afin d'optimiser la délivrance de ces molécules à leurs cellules cibles. Ainsi, par exemple, des oligonucleotides agrafes ou semi-agrafes pourront être encapsulés dans des liposomes, des nano-particules, des particules LDL, ou dans tout autre type de micro¬ sphère permettant une conservation adéquate, et favorisant le ciblage. Les molécules oligonucléotidiques agrafes ou semi-agrafes peuvent également être associées à des agents surfactants cationiques. Il est bien évident que ces quelques exemples ne sont ni exhaustifs ni limitatifs.In particular, these natural staple or semi-staple oligonucleotides resistant to serum exonucleases can be used as an antisense agent directed against RNA messengers of oncogenes expressed in leukemic cells and involved in their proliferation. In the context of the treatment of certain leukemias, for bone marrow transplants, the staple or semi-staple oligonucleotides can be used in the context of "ex vivo" applications. For these numerous indications, adequate dosage formulations must be established in order to optimize the delivery of these molecules to their target cells. So, for example, oligonucleotides staples or semi-staples can be encapsulated in liposomes, nanoparticles, LDL particles, or in any other type of micro¬ sphere allowing adequate preservation, and promoting targeting. The staple or semi-staple oligonucleotide molecules can also be combined with cationic surfactants. It is obvious that these few examples are neither exhaustive nor limiting.
Les oligonucleotides agrafes ou semi-agrafes faisant l'objet de l'invention décrite ici, sont donc susceptibles d'être inclus dans toutes sortes de préparations pharmaceutiques, parapharmaceutiques, cosmétologiques, ou destinées à des dosages d'acides nucléiques ou de diagnostic, à des concentrations variables selon les indications, avec les excipients appropriés.The staple or semi-staple oligonucleotides forming the subject of the invention described here are therefore capable of being included in all kinds of pharmaceutical, parapharmaceutical, cosmetological preparations, or intended for nucleic acid or diagnostic assays, varying concentrations as indicated, with appropriate excipients.
La présente invention a aussi pour objet un procédé de modulation de l'expression génétique d'une séquence cible appartenant à un ARN messager ou à un ADN, caractérisé en ce qu'on hybride la dite séquence cible avec un oligonucléotide selon l'invention comportant une séquence complémentaire à la dite séquence cible.The present invention also relates to a method of modulating the genetic expression of a target sequence belonging to a messenger RNA or to a DNA, characterized in that the said target sequence is hybridized with an oligonucleotide according to the invention comprising a sequence complementary to said target sequence.
Ce type de procédé peut être mis en oeuvre à des fins de recherche, thérapeutique ou diagnostic, dans des conditions in vitro, in vivo ou ex vivo.This type of process can be implemented for research, therapeutic or diagnostic purposes, under in vitro, in vivo or ex vivo conditions.
III . EXEMPLES DE RÉALISATIONS DE L'INVENTIONIII. EXAMPLES OF EMBODIMENTS OF THE INVENTION
D'autres caractéristiques et avantages de la présente invention apparaîtront à la lumière des exemples qui vont suivre en référence aux figures 1 à 7. La FIGURE 1 représente la structure des bases composant les oligonucleotides et la structure de la liaison phosphodiester reliant les oligonucleotides naturels entre eux.Other characteristics and advantages of the present invention will become apparent in the light of the examples which will follow with reference to FIGS. 1 to 7. FIGURE 1 represents the structure of the bases composing the oligonucleotides and the structure of the phosphodiester bond connecting the natural oligonucleotides between them.
La FIGURE 2 représente des exemples d'oligonucléotides adoptant des structures secondaires en agrafe (a), en semi-agrafe (b et c) ou en semi-agrafe "épingle à cheveux".FIGURE 2 represents examples of oligonucleotides adopting secondary structures in staple (a), in semi-staple (b and c) or in semi-staple "hairpin".
La FIGURE 3 représente des exemples de méthodes d'obtention oligonucleotides semi-agrafes avec en (a) l'obtention d'un oligonucléotide semi-agrafe par addition d'une structure oligonucléotidique auto-appariée à l'extrémité 3' d'un oligonucléotide linéaire; et en (b) l'obtention d'un oligonucléotide semi-agrafe par addition à l'extrémité 3' d'un oligonucléotide linéaire d'un autre oligonucléotide linéaire dont la séquence 3' terminale est complémentaire à un fragment de la séquence du premier oligonucléotide.FIGURE 3 represents examples of methods for obtaining semi-stapled oligonucleotides with in (a) obtaining a semi-stapled oligonucleotide by adding a self-paired oligonucleotide structure to the 3 'end of an oligonucleotide linear; and in (b) obtaining a semi-staple oligonucleotide by adding to the 3 'end of a linear oligonucleotide of another linear oligonucleotide whose terminal 3 'sequence is complementary to a fragment of the sequence of the first oligonucleotide.
La FIGURE 4 représente en (a) les oligonucleotides agrafes (HA12), semi-agrafes (SA 12, RAI 2, HA12/ml, RA12/ml) et linéaires (POLYA12, SA12/ml, HA12/m2) utilisés dans les expériences de dégradation par les enzymes sériques; et en (b) des oligonucleotides anti-sens anti-HSV-1 semi-agrafes (HMV10, SMV10) et linéaires (MV10, SMVIO/ml ) utilisés dans les expériences de dégradation par les enzymes sériques et les tests d'inhibition de la prolifération du virus de l'herpès simplex de type 1 (HSV-1 ).FIGURE 4 shows in (a) the staple (HA12), semi-staple (SA 12, RAI 2, HA12 / ml, RA12 / ml) and linear (POLYA12, SA12 / ml, HA12 / m2) oligonucleotides used in the experiments degradation by serum enzymes; and in (b) anti-HSV-1 anti-sense oligonucleotides semi-staples (HMV10, SMV10) and linear (MV10, SMVIO / ml) used in the experiments of degradation by serum enzymes and the tests for inhibition of proliferation of herpes simplex virus type 1 (HSV-1).
Le s FIGURES 5a à 5e représentent les électrophorèses en conditions dénaturantes d'oligonucléotides agrafes, semi-agrafes ou linéaires après incubation en présence de 10% de sérum de veau foetal. L e s FIGURES 6a et 6 b représentent les demi-vies d'oligonucléotides agrafes, semi-agrafes ou linéaires incubés en présence de 10% de sérum de veau foetal.FIGS. 5a to 5e represent electrophoresis under denaturing conditions of staple, semi-staple or linear oligonucleotides after incubation in the presence of 10% fetal calf serum. FIGURES 6a and 6b show the half-lives of staple, semi-staple or linear oligonucleotides incubated in the presence of 10% fetal calf serum.
La FIGURE 7 représente l'inhibition de la prolifération du virus de l'herpès simplex de type 1 (HSV-1 ) par un oligonucléotide anti-sens semi- agrafe (SMV10) et un mutant anti-sens linéaire ciblé sur la même séquence (SMVIO/ml).FIGURE 7 represents the inhibition of the proliferation of the herpes simplex virus type 1 (HSV-1) by an antisense oligonucleotide semi-staple (SMV10) and a linear antisense mutant targeted on the same sequence ( SMVIO / ml).
Les exemples expérimentaux sont donnés ci-dessous pour illustrer les avantages d'un oligonucléotide agrafe ou semi-agrafe incluant une séquence nucléotidique linéaire donnée sur des oligonucleotides incluant la même séquence linéaire mais ne présentant de structure secondaire ni en agrafe, ni en semi-agrafe. Pour chaque comparaison un premier oligonucléotide phosphodiester linéaire a été synthétisé.The experimental examples are given below to illustrate the advantages of a staple or semi-staple oligonucleotide including a linear nucleotide sequence given on oligonucleotides including the same linear sequence but having no secondary structure in staple or in semi-staple . For each comparison, a first linear phosphodiester oligonucleotide was synthesized.
A partir de cette séquence linéaire des oligonucleotides plus longs ont été synthétisés constitués de la même séquence linéaire prolongée à son extrémité 3', ou à ses deux extrémités 3' et 5' par des séquences oligonucléotidiques induisant l'apparition d'une structure secondaire en agrafe ou en semi-agrafe. Des mutants de ces molécules ont aussi été synthétisés par modification d'un nombre minimum de nucléotides permettant la déstabilisation des structures secondaires en agrafe ou semi- agrafe . Ces mutants sont ainsi des molécules linéaires de longueur égale à celle des molécules en agrafe ou semi-agrafe et incluant la même séquence linéaire initiale. Exemple 1 : Résistance des oligonucleotides agrafes et semi-agrafe aux nucléases sériquesFrom this linear sequence, longer oligonucleotides were synthesized consisting of the same linear sequence extended at its 3 'end, or at its two 3' and 5 'ends by oligonucleotide sequences inducing the appearance of a secondary structure in staple or semi-staple. Mutants of these molecules have also been synthesized by modification of a minimum number of nucleotides allowing the destabilization of secondary structures in staple or semi-staple. These mutants are thus linear molecules of length equal to that of staple or semi-staple molecules and including the same initial linear sequence. EXAMPLE 1 Resistance of the staple and semi-staple oligonucleotides to serum nucleases
Les oligonucleotides agrafes ou semi-agrafes présentent une résistance à la dégradation nucléolytique supérieure à celle d'oligonucléotides linéaires de séquence voisine lorsqu'ils sont incubés en présence de sérum.The staple or semi-staple oligonucleotides exhibit a higher resistance to nucleolytic degradation than that of linear oligonucleotides of similar sequence when they are incubated in the presence of serum.
Pour ces expériences deux familles d'oligonucléotides ont été synthétisés conformément à la description ci-dessus. La première famille a été construite à partir d'une séquence linéaire homonucléotidique polyadénylate (POLYA12), à partir de laquelle une séquence en agrafe (HA12) et des séquences en semi-agrafes (HA12/ml, SA12, RA12, RA12/ml ) de natures différentes ont été construites. Des mutants linéaires de longueurs égales à deux de ces molécules sont désignés respectivement par HA12/m2 et SA12/ml . En guise de contrôle supplémentaire des polyadénylates de longueurs égales à HAÏ 2, SA 12 et RAI 2 ont également été synthétisés et étudiés. La deuxième famille a été construite à partir d'un duodécamère hétéronucléotidique linéaire (MV10) complémentaire à la jonction d'épissage d'un ARN messager précoce du virus de l'herpès simplex de type 1. Des séquences en semi-agrafes de différentes natures et de différentes tailles ont été synthétisés sur cette base linéaire : HMV10 (semi-agrafe) et SMV10 (semi-agrafe). Un mutant linéaire de SMV10 a également été synthétisé et étudié : SMVIO/ml. Les formules chimiques de l'ensemble de ces oligonucleotides et leur structure secondaire sont indiquées sur la figure 4.For these experiments two families of oligonucleotides were synthesized according to the description above. The first family was built from a linear homonucleotide polyadenylate sequence (POLYA12), from which a staple sequence (HA12) and semi-staple sequences (HA12 / ml, SA12, RA12, RA12 / ml) of different natures were built. Linear mutants of lengths equal to two of these molecules are designated respectively by HA12 / m2 and SA12 / ml. As additional control polyadenylates of length equal to HAÏ 2, SA 12 and RAI 2 were also synthesized and studied. The second family was constructed from a linear heteronucleotide duodecamer (MV10) complementary to the splicing junction of an early messenger RNA of the herpes simplex virus type 1. Sequences in semi-staples of different natures and of different sizes have been synthesized on this linear basis: HMV10 (semi-clip) and SMV10 (semi-clip). A linear mutant of SMV10 has also been synthesized and studied: SMVIO / ml. The chemical formulas of all of these oligonucleotides and their secondary structure are shown in Figure 4.
Pour ces expériences 1 μg de chacun des oligonucleotides 5'- 2P est incubé à 37°C dans 10 μl de milieu MEM contenant 10% de sérum de veau foetal. La cinétique de la réaction est suivie jusqu'à 10 heures et les prélèvements effectués au cours du temps analysés sur gel de polyacrylamide 20% en conditions dénaturantes. Les autoradiographies des gels de dégradation sont présentées en figure 7. Les bandes correspondants à la longueur initiale de chaque oligonucléotide sont excisées du gel et leur radioactivité mesurée par effet Cerenkov sur un compteur de radioactivité bêta. Cette mesure permet la quantification en fonction du temps de la proportion d'oligonucléotide non dégradé et par suite une estimation chiffrée de la cinétique de sa dégradation en terme de demi-vie. Les demi- vies des différents oligonucleotides évaluées dans les conditions décrites ci-dessus sont récapitulées dans le tableau de la figure 6.For these experiments, 1 μg of each of the 5'- 2 P oligonucleotides is incubated at 37 ° C. in 10 μl of MEM medium containing 10% fetal calf serum. The kinetics of the reaction are monitored up to 10 hours and the samples taken over time analyzed on 20% polyacrylamide gel under denaturing conditions. The autoradiographs of the degradation gels are presented in FIG. 7. The bands corresponding to the initial length of each oligonucleotide are excised from the gel and their radioactivity measured by the Cerenkov effect on a beta radioactivity counter. This measurement allows the quantification as a function of time of the proportion of non-degraded oligonucleotide and therefore a quantified estimate of the kinetics of its degradation in terms of half-life. The half-lives of the different oligonucleotides evaluated under the conditions described above are summarized in the table in FIG. 6.
Ces expériences permettent de tirer les conclusions suivantes :These experiments lead to the following conclusions:
- les oligonucleotides linéaires, indépendamment de leur taille et de leur séquence sont dégradés rapidement par les nucléases du sérum; leur dégradation intervient dans les premières minutes de l'incubation et est complète après quelques heures;- linear oligonucleotides, regardless of their size and sequence, are rapidly degraded by nucleases in serum; their degradation occurs in the first minutes of incubation and is complete after a few hours;
- le temps de demi-vie d'un oligonucléotide linéaire, indépendamment de sa taille et de sa séquence en présence de 10% de sérum de veau foetal est compris entre 30 et 60 minutes;- The half-life time of a linear oligonucleotide, regardless of its size and its sequence in the presence of 10% fetal calf serum is between 30 and 60 minutes;
- la dégradation des oligonucleotides linéaires est processive, et on observe l'apparition de produits de dégradation de longueur décroissante, devenant de plus en plus courts en fonction du temps, ce qui indique que la dégradation est principalement le fait d'exonucléases, et en particulier de 3 '-exonucléases;the degradation of the linear oligonucleotides is processive, and the appearance of degradation products of decreasing length is observed, becoming shorter and shorter as a function of time, which indicates that the degradation is mainly due to exonucleases, and in particular of 3 '-exonucleases;
- en revanche les oligonucleotides agrafes ou semi-agrafes sont systématiquement plus résistants à la nucléolyse par les enzymes sériques, et une fraction non négligeable d'oligonucléotide non dégradé reste détectable même après plusieurs heures d'incubation à 37°C;- on the other hand, staple or semi-staple oligonucleotides are systematically more resistant to nucleolysis by serum enzymes, and a non-negligible fraction of non-degraded oligonucleotide remains detectable even after several hours of incubation at 37 ° C .;
- les temps de demi-vie des oligonucleotides agrafes et semi-agrafes étudiés varient d'un minimum de 2 heures à un maximum de l'ordre de 8 heures et sont en bonne corrélation avec les stabilités respectives théoriques des structures secondaires en semi-agrafes dans lesquelles sont engagées leur extrémité 3' (Fig.6).- the half-life times of the staple and semi-staple oligonucleotides studied vary from a minimum of 2 hours to a maximum of the order of 8 hours and are in good correlation with the respective theoretical stabilities of the secondary structures in semi-staples in which are engaged their 3 'end (Fig. 6).
Ces résultats confirment que la dégradation des oligonucleotides phosphodiesters dans le sérum est principalement le fait d'exonucléases et non pas d'endonucléases et mettent en évidence la résistance à l'activité nucléolytique des enymes sériques des structures en agrafe ou en semi- agrafe. Exemple 2 : Inhibition de la prolifération du virus de l'herpès simplex de type I (HSV-1) par un oligonucléotide antisens semi-agrafeThese results confirm that the degradation of phosphodiester oligonucleotides in serum is mainly due to exonucleases and not endonucleases and demonstrate the resistance to the nucleolytic activity of serum enzymes of clip or semi-clip structures. Example 2 Inhibition of the proliferation of the herpes simplex virus type I (HSV-1) by a semi-staple antisense oligonucleotide
Les oligonucleotides SMV10 et SMVIO/ml dont les séquences sont données en figure 6 ont été utilisées dans ces expériences. L'oligonucléotide SMV10 comprend d'une part le fragment linéaire MV10, complémentaire sur 12 bases de la jonction d'épissage d'un ARN messager précoce du cycle de réplication du virus HSV-1 et d'autre part une extrémité 3' prolongée en semi-agrafe octamèrique. Le mutant SMVIO/ml comprend la même séquence antisens MV10 que SMV10 mais une mutation à été introduite dans la séquence octamèrique terminale, mutation qui confère à SMVIO/ml une structure linéaire.The oligonucleotides SMV10 and SMVIO / ml whose sequences are given in FIG. 6 were used in these experiments. The SMV10 oligonucleotide comprises on the one hand the linear fragment MV10, complementary on 12 bases to the splicing junction of a messenger RNA early in the replication cycle of the HSV-1 virus and on the other hand a 3 ′ end extended by octameric semi-clip. The mutant SMVIO / ml comprises the same antisense sequence MV10 as SMV10 but a mutation has been introduced into the terminal octameric sequence, a mutation which gives SMVIO / ml a linear structure.
Infection des cellules en présence ou en l'absence d'oligonucléotides Les cellules (cellules Véro ATCC cultivées en milieu Gibco-MEM complémenté de 5% de sérum de veau foetal, de L-glutamine, d'acides aminés non-essentiels et de pénistrepto) sont ensemencées la veille de l'infection à une densité de 50 000 cellules par puits de 2 cm 16 à 24 heures plus tard, les cellules sont infectées avec HSV-1 à une multiplicité d'infection de 3 pfu/cellule en présence ou en l'absence d'oligonucléotides.Infection of cells in the presence or absence of oligonucleotides Cells (Vero ATCC cells grown in Gibco-MEM medium supplemented with 5% fetal calf serum, L-glutamine, non-essential amino acids and penistrepto ) are seeded the day before infection at a density of 50,000 cells per well of 2 cm 16 to 24 hours later, the cells are infected with HSV-1 at a multiplicity of infection of 3 pfu / cell in the presence or in the absence of oligonucleotides.
50 μl d'oligonucléotides en solution dans du milieu de culture 2X exempt de sérum sont déposés sur la monocouche cellulaire. 50 μl de solution virale sont déposés 5 minutes plus tard. Les cellules sont incubées pendant 1 heure à 37°C et agitées précautioneusement toutes les 15 minutes. Après 1 heure d'incubation, les 100 ml de milieu sont aspirés et 500 ml de milieu complet sont ajoutés sur les cellules. L'incubation est poursuivie 24 heures avant d'être arrêtée par congélation des plaques dans l'azote liquide. Toutes les mesures d'inhibition sont effectuées en duplicat ou en triplicat. Titration du virus50 μl of oligonucleotides in solution in 2X culture medium free of serum are deposited on the cell monolayer. 50 μl of viral solution are deposited 5 minutes later. The cells are incubated for 1 hour at 37 ° C and shaken carefully every 15 minutes. After 1 hour of incubation, the 100 ml of medium are aspirated and 500 ml of complete medium are added to the cells. Incubation is continued for 24 hours before being stopped by freezing the plates in liquid nitrogen. All inhibition measurements are carried out in duplicate or triplicate. Virus titration
Les virus sont récupérés directement dans le milieu de culture après 3 cycles rapides de congélation dans l'azote liquide - décongélation à 37°C. Ils sont ensuite dilués en milieu sans sérum pour effectuer la titration proprement dite. Les cellules indicatrices sont ensemencées la veille en milieu complet à raison de 250 000 cellules par puits de 2 cm 2. Le lendemain, le milieu est aspiré et on dépose 100 ml des différentes dilutions virales par puits. Après une incubation d'une heure à 37°C avec agitation toutes les 15 minutes, le milieu est aspiré et les cellules sont recouvertes par du milieu complet (2.5% en sérum) contenant 1.2% de méthyl-cellulose pour une incubation de 3 jours à 37°C.The viruses are recovered directly in the culture medium after 3 rapid cycles of freezing in liquid nitrogen - thawing at 37 ° C. They are then diluted in a serum-free medium to carry out the actual titration. The indicator cells are seeded the previous day in complete medium at the rate of 250,000 cells per well of 2 cm 2 . The following day, the medium is aspirated and 100 ml of the various viral dilutions are deposited per well. After a one hour incubation at 37 ° C with shaking every 15 minutes, the medium is aspirated and the cells are covered with complete medium (2.5% in serum) containing 1.2% methyl cellulose for a 3-day incubation at 37 ° C.
Après 3 jours, le milieu est éliminé, les cellules sont fixées avec une solution de PBS/10% formol pendant 20 minutes puis colorées par une solution 2% cristal violet dans du PBS/20% éthanol pendant 20 minutes. Les plaques sont ensuite rincées et les plages comptées par transparence sur le négatoscope. Les titrations sont effectuées en triple pour chaque point. Les calculs d'inhibition sont effectués par rapport aux titres viraux observés en l'absence de nucléotides. RésultatsAfter 3 days, the medium is eliminated, the cells are fixed with a PBS / 10% formalin solution for 20 minutes and then stained with a 2% crystal violet solution in PBS / 20% ethanol for 20 minutes. The plates are then rinsed and the areas counted by transparency on the X-ray viewer. Titrations are performed in triplicate for each point. The inhibition calculations are carried out relative to the viral titers observed in the absence of nucleotides. Results
En expériences préliminaires, la cytotoxicité de SMV10 et SMVIO/ml a été testée sur les cellules Véro. Pour des incubations de 1 heure à 37°C et des concentrations en oligonucleotides atteignant 50 μM aucune toxicité cellulaire n'a été détectée. L'inhibition de la prolifération virale induite par SMV10 etIn preliminary experiments, the cytotoxicity of SMV10 and SMVIO / ml was tested on Vero cells. For 1 hour incubations at 37 ° C and concentrations of oligonucleotides reaching 50 μM, no cellular toxicity was detected. Inhibition of viral proliferation induced by SMV10 and
SMVIO/ml, pour des concentrations allant de 10 μM à 30 μM est reportée sur la figure 7. Il y apparait clairement que l'oligonucléotide semi-agrafe est actif à des concentrations inférieures à son mutant antisens linéaire. SMV10 induit 70% d'inhibition virale à 10 μM tandis que SMVIO/ml est inactif à cette concentration.SMVIO / ml, for concentrations ranging from 10 μM to 30 μM is shown in FIG. 7. It clearly appears there that the semi-staple oligonucleotide is active at concentrations lower than its linear antisense mutant. SMV10 induces 70% viral inhibition at 10 μM while SMVIO / ml is inactive at this concentration.
Ce résultat confirme l'avantage, par rapport à une structure linéaire, du type de structure nucléotidique faisant l'objet de l'invention, dans le cadre de son utilisation comme agent antisens.This result confirms the advantage, compared to a linear structure, of the type of nucleotide structure which is the subject of the invention, in the context of its use as an antisense agent.
Exemple 3 : Corrélation entre la stabilité thermodynamique des structures secondaires impliquées dans les oligonucleotides semi-agrafes et leur résistance à la dégradation par les nucléases sériquesExample 3 Correlation between the thermodynamic stability of the secondary structures involved in the semi-staple oligonucleotides and their resistance to degradation by serum nucleases
Dans ces expériences, différentes structures octamériques ayant la capacité d'adopter spontanément des structures secondaires en épingle à cheveux ont été utilisées pour prolonger, en 3', l'oligonucléotide antisens MV10 (ici GT3022) décrit dans les Exemples 1 et 2. Onze structures octamériques de stabilités variables ont été étudiées. Ces structures ont été sélectionnées suivant deux critères: 1 ) leur capacité à former spontanément une épingle à cheveux octamèrique en se repliant sur elle- même; 2) leur incapacité à venir s'hybrider partiellement à la séquence de GT3022, évitant ainsi l'apparition d'une structure secondaire alternative dans laquelle une partie de la séquence antisens serait engagée.In these experiments, different octameric structures having the capacity to spontaneously adopt secondary hairpin structures were used to extend, in 3 ', the antisense oligonucleotide MV10 (here GT3022) described in Examples 1 and 2. Eleven structures octamerics of varying stabilities have been studied. These structures were selected according to two criteria: 1) their capacity to spontaneously form an octameric hairpin by folding back on itself; 2) their inability to partially hybridize to the GT3022 sequence, thus avoiding the appearance of an alternative secondary structure in which part of the antisense sequence would be engaged.
La mesure des températures de fusion de chacune de ces structures octamériques a été rapportée par Hirao et al., Nucleic Acid Research, 20 ( 15), 3891-3896, 1992. Les températures de fusion de ces structures caractérisent leur stabilité thermodynamique. Elles sont présentées dans le Tableau 1. Elles varient de 33°C pour la plus instable, extrémité 3' de GT3516, à 76.5°C pour la plus stable, extrémité 3' de SMV10 (ici GT3500). Deux d'entre elles n'ont pas de température de fusion, ce qui indique que leur structure secondaire présumée ne se forme vraisemblablement pas et que les extrémités 3' des oligonucleotides (GT3517, GT3518) dont elles constituent l'extrémité sont linéaires. Il est intéressant de noter que, dans cet exemple, toutes les épingles à cheveux sont formées par l'appariement intra-moléculaire d'une paire GC; ainsi, dans cet exemple, la stabilité thermodynamique des structures en épingle à cheveux est modulée par la composition de la boucle non-appariée de la structure.The measurement of the melting temperatures of each of these octameric structures has been reported by Hirao et al., Nucleic Acid Research, 20 (15), 3891-3896, 1992. The melting temperatures of these structures characterize their thermodynamic stability. They are presented in Table 1. They vary from 33 ° C for the most unstable, 3 'end of GT3516, to 76.5 ° C for the most stable, 3' end of SMV10 (here GT3500). Two of them do not have a melting temperature, which indicates that their presumed secondary structure is unlikely to form and that the 3 'ends of the oligonucleotides (GT3517, GT3518) of which they constitute the end are linear. It is interesting to note that, in this example, all of the hairpins are formed by the intra-molecular pairing of a GC pair; thus, in this example, the thermodynamic stability of the hairpin structures is modulated by the composition of the unpaired loop of the structure.
La résistance à la dégradation par les nucléases sériques de ces oligonucleotides a été analysée suivant le protocole décrit dans l'exemple 1. La demi-vie dans le sérum de chacun de ces oligonucleotides est reportée dans le Tableau 1.The resistance to degradation by serum nucleases of these oligonucleotides was analyzed according to the protocol described in Example 1. The half-life in serum of each of these oligonucleotides is reported in Table 1.
L'ensemble des données présentées dans le Tableau 1 confirme que la présence d'une structure auto-appariée à l'extrémité 3' d'un oligonucléotide augmente de manière significative sa résistance aux nucléases sériques. Cette résistance est proportionnelle à la stabilité thermodynamique des dites structures auto-appariées.
Figure imgf000025_0001
All the data presented in Table 1 confirms that the presence of a self-paired structure at the 3 ′ end of an oligonucleotide significantly increases its resistance to serum nucleases. This resistance is proportional to the thermodynamic stability of said self-paired structures.
Figure imgf000025_0001
REFERENCES BIBLIOGRAPHIQUES CITEES DANS LE TEXTEBIBLIOGRAPHICAL REFERENCES CITED IN THE TEXT
Andrus, A.; Geiser, T.; Zon, G. ( 1989), Nucleosides Nucléotides, 8, 5-6, 967-8Andrus, A .; Geiser, T .; Zon, G. (1989), Nucleosides Nucleotides, 8, 5-6, 967-8
Anfossi, Giovanni; Gewirtz, Alan M.; Calabretta, Bruno, ( 1989), Proc. Natl. Acad. Sci. U.S.A., 86, 3379-83Anfossi, Giovanni; Gewirtz, Alan M .; Calabretta, Bruno, (1989), Proc. Natl. Acad. Sci. U.S.A., 86, 3379-83
Bazile D; Gautier C; Rayner B; Imbach JL; Paoletti C; Paoletti J, ( 1989),Bazile D; Gautier C; Rayner B; Imbach JL; Paoletti C; Paoletti J, (1989),
Nucleic Acids Res, 17, 7749-59Nucleic Acids Res, 17, 7749-59
Bertrand JR; Imbach JL; Paoletti C; Malvy C, (1989), Biochem Biophys ResBertrand JR; Imbach JL; Paoletti C; Malvy C, (1989), Biochem Biophys Res
Commun, 164, 311-8 Caruthers, M. H. ( 1985) Sciences, 230, 281Commun, 164, 311-8 Caruthers, M. H. (1985) Sciences, 230, 281
Cazenave, C; Stein, C. A.; Loreau, N.; Thuong, N. T.; Neckers, L. M.;Cazenave, C; Stein, C. A .; Loreau, N .; Thuong, N. T .; Neckers, L. M .;
Subasinghe, C; Hélène, C; Cohen, J. S.; Toulme, J. J., ( 1989), Nucleic AcidsSubasinghe, C; Hélène, C; Cohen, J. S .; Toulme, J. J., (1989), Nucleic Acids
Res., 17, 4255-73Res., 17, 4255-73
Chang, E. H.; Yu, Z.; Shinozuka, K.; Zon, G.; Wilson, W. D.; Strekowska, A., (1989), Anti-Cancer Drug Des., 4, 221-32Chang, E. H .; Yu, Z .; Shinozuka, K .; Zon, G .; Wilson, W. D .; Strekowska, A., (1989), Anti-Cancer Drug Des., 4, 221-32
Cope, F. O.; Wille, J. J., (1989), Proc. Nati. Acad. Sci. U.S.A., 86, 5590-4Cope, F. O .; Wille, J. J., (1989), Proc. Nati. Acad. Sci. U.S.A., 86, 5590-4
Degols, G.; Leonetti, J.P.; Gagnor, C; Lemaitre, M.; Lebleu, B., ( 1989),Degols, G .; Leonetti, J.P .; Gagnor, C; Lemaitre, M .; Lebleu, B., (1989),
Nucleic Acids Res., 17, 9341-50Nucleic Acids Res., 17, 9341-50
De Vroom E., Broxterman, H.J., Sliedregt, L.A.J., Van der Marel, G.A., Van Boom, J.H., (1988), Nucl. Ac. Res., 16, 4607-4620De Vroom E., Broxterman, H.J., Sliedregt, L.A.J., Van der Marel, G.A., Van Boom, J.H., (1988), Nucl. Ac. Res., 16, 4607-4620
Dervan PB, ( 1986), Science, 232, 464-71Dervan PB, (1986), Science, 232, 464-71
François JC; Saison-Behmoaras T; Barbier C; Chassignol M; Thuong NT;François JC; Season-Behmoaras T; Barber C; Chassignol M; Thuong NT;
Hélène C, ( 1989), Proc Natl Acad Sci, 86, 9702-9706Hélène C, (1989), Proc Natl Acad Sci, 86, 9702-9706
François JC; Saison-Behmoaras T; Chassignol M; Thuong NT; Hélène C, (1989), JBiol Chem, 264 5891-5898François JC; Season-Behmoaras T; Chassignol M; Thuong NT; Hélène C, (1989), JBiol Chem, 264 5891-5898
François JC; Saison-Behmoaras T; Hélène C, ( 1988), Nucleic Acids Res, 16,François JC; Season-Behmoaras T; Hélène C, (1988), Nucleic Acids Res, 16,
11431-1144011431-11440
François JC; Saison-Behmoaras T; Thuong NT; Hélène C, ( 1989),François JC; Season-Behmoaras T; Thuong NT; Hélène C, (1989),
Biochemistry, 28, 9617-9619 Froehler, B.C., Ng, P. and Matteucci, M.D. ( 1986) Nucl. Acid Res., 14, 5399Biochemistry, 28, 9617-9619 Froehler, B.C., Ng, P. and Matteucci, M.D. (1986) Nucl. Acid Res., 14, 5399
Gagnor, C, Bertrand, J.R., Thenet, S., Lemaitre, M., Morvan, F., Rayner, B.,Gagnor, C, Bertrand, J.R., Thenet, S., Lemaitre, M., Morvan, F., Rayner, B.,
Malvy, C, Lebleu, B.,Imbach, J.L, Paoletti, C.,( 1987). Nucleic Acids Res., 15,Malvy, C, Lebleu, B., Imbach, J.L, Paoletti, C., (1987). Nucleic Acids Res., 15,
10419-3610419-36
Gao, W., Stein, C.A., Cohen, J.S., Dutchmann, G. and Cheng, Y.C., ( 1988). J. Biol. Chem., 264, 11521-11532 Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun,Gao, W., Stein, CA, Cohen, JS, Dutchmann, G. and Cheng, YC, (1988). J. Biol. Chem., 264, 11521-11532 Goodchild, John; Agrawal, Sudhir; Civeira, Maria P .; Sarin, Prem S .; Sun,
Daisy; Zamecnik, Paul C, ( 1988), Proc. Natl. Acad. Sci. U.S.A., 85, 5507-11Daisy; Zamecnik, Paul C, (1988), Proc. Natl. Acad. Sci. U.S.A., 85, 5507-11
Jessus C; Chevrier M; Ozon R; Hélène C; Cazenave C, ( 1989), Gène, 72, 311-Jessus C; Chevrier M; Ozon R; Hélène C; Cazenave C, (1989), Gene, 72, 311-
312 Kabanov, A. V.; Vinogradov, S. V.; Ovcharenko, A. V.; Krivonos, A. V.;312 Kabanov, A. V .; Vinogradov, S. V .; Ovcharenko, A. V .; Krivonos, A. V .;
Melik-Nubarov, N. S.; Kiselev, V. I.; Severin, E. S., ( 1990), FEBS Lett., 259,Melik-Nubarov, N. S .; Kiselev, V. I .; Severin, E. S., (1990), FEBS Lett., 259,
327-30327-30
Leonetti, J.P, Degols, G., Milhaud, P., Gagnor, C, Lemaitre, M., Lebleu, B.,Leonetti, J.P, Degols, G., Milhaud, P., Gagnor, C, Lemaitre, M., Lebleu, B.,
( 1989), Nucleosides Nucléotides, 8, 5-6, 825-8 Maher, Louis J, III; Dolnick, Bruce J., ( 1988), Nucleic Acids Res., 16, 3341-(1989), Nucleosides Nucleotides, 8, 5-6, 825-8 Maher, Louis J, III; Dolnick, Bruce J., (1988), Nucleic Acids Res., 16, 3341-
5858
Markus-Sekura, Carol J.; Woerner, Amy M.; Shinozuka, Kazuo; Zon,Markus-Sekura, Carol J .; Woerner, Amy M .; Shinozuka, Kazuo; Zon,
Gerald; Quinnan, Gerald V., Jr., (1987), Nucleic Acids Res., 15, 5749-63Gerald; Quinnan, Gerald V., Jr., (1987), Nucleic Acids Res., 15, 5749-63
Matsukura, Makoto; Zon, Gerald; Shinozuka, Kazuo;Robert-Guroff, Miroshnichenko, O. I.; Ponomareva, T. I.; Tikhonenko, T. I., ( 1989), Gène,Matsukura, Makoto; Zon, Gerald; Shinozuka, Kazuo; Robert-Guroff, Miroshnichenko, O. I .; Ponomareva, T. I .; Tikhonenko, T. I., (1989), Gene,
84, 83-984, 83-9
Perbost M; Lucas M; Chavis C; Pompon A; Baumgartner H; Rayner B;Perbost M; Lucas M; Chavis C; Pompon A; Baumgartner H; Rayner B;
Griengl H; Imbach JL, ( 1989), Biochem Biophys Res Commun, 165, 742-7Griengl H; Imbach JL, (1989), Biochem Biophys Res Commun, 165, 742-7
Perroualt, L., Asseline, U., Rivalle.C, Thuong, N.Y., Bisagni, E., Sankar, Sabita; Cheah, Keat Chye; Porter, Alan G., (1989), Eur. J. Biochem. 184, 39-Perroualt, L., Asseline, U., Rivalle.C, Thuong, N.Y., Bisagni, E., Sankar, Sabita; Cheah, Keat Chye; Porter, Alan G., (1989), Eur. J. Biochem. 184, 39-
4545
Shuttleworth, John; Matthews, Glenn; Dale, Les; Baker, Chris; Colman,Shuttleworth, John; Matthews, Glenn; Dale, Les; Baker, Chris; Colman,
Alan, ( 1988), Gène;, 72,1-2, 267-75Alan, (1988), Gene; 72.1-2.267-75
Stevenson, Mario; Iversen, Patrick L, (1989), J. Gen. Virol., 70, 2673-82 Sun JS; François JC; Montenay-Garestier T; Saison-Behmoaras T; Roig V;Stevenson, Mario; Iversen, Patrick L, (1989), J. Gen. Virol., 70, 2673-82 Sun JS; François JC; Montenay-Garestier T; Season-Behmoaras T; Roig V;
Thuong NT; Hélène C, (1989) Proc Nati Acad Sci 86, 9198-9202Thuong NT; Hélène C, (1989) Proc Nati Acad Sci 86, 9198-9202
Tortora, Giampaolo; Clair, Timothy; Cho-Chung, Yoon Sang, ( 1990), Proc.Tortora, Giampaolo; Clair, Timothy; Cho-Chung, Yoon Sang, (1990), Proc.
Nati. Acad. Sci. U.S.A., 87, 705-8Nati. Acad. Sci. U.S.A., 87, 705-8
Wang AH; Cottens S; Dervan PB; Yesinowski JP; van der Marel GA; van Boom JH, ( 1989), J Biomol Struct Dyn, 7, 101-17Wang AH; Cottens S; Dervan PB; Yesinowski JP; van der Marel GA; van Boom JH, (1989), J Biomol Struct Dyn, 7, 101-17
Westermann, P.; Gross, B.; Hoinkis, G., (1989), Biomed. Biochim. Acta, 48,Westermann, P .; Gross, B .; Hoinkis, G., (1989), Biomed. Biochim. Acta, 48,
85-9385-93
Zerial A; Thuong NT; Hélène C, ( 1987), Nucleic Acids Res, 15, 9909-9919Zerial A; Thuong NT; Hélène C, (1987), Nucleic Acids Res, 15, 9909-9919
Zheng, H., Sahai, Béni M., Kilgannon, P., Fotedar, A., Green, D. R., ( 1989), Proc. Natl. Acad. Sci. U.S.A., 86, 3758-62 IDENTIFICATEUR DE SEQUENCE (1) INFORMATIONS GENERALES :Zheng, H., Sahai, Béni M., Kilgannon, P., Fotedar, A., Green, DR, (1989), Proc. Natl. Acad. Sci. USA, 86, 3758-62 SEQUENCE IDENTIFIER (1) GENERAL INFORMATION:
(i) DEPOSANT: (A) NOM: GENSET(i) DEPOSITOR: (A) NAME: GENSET
(B) RUE: 1 PASSAGE ETIENNE DELAUNAY(B) STREET: 1 PASSAGE ETIENNE DELAUNAY
(C) VILLE : PARIS(C) CITY: PARIS
(E) PAYS : FRANCE(E) COUNTRY: FRANCE
(F) CODE POSTAL: 75011(F) POSTAL CODE: 75011
(G) TELEPHONE: 33(1)43565900 (H) TELEFAX: 33(1)43562625(G) TELEPHONE: 33 (1) 43565900 (H) TELEFAX: 33 (1) 43562625
(ii) TITRE DE L'INVENTION "OLIGONUCLEOTIDES AGRAFES ET SEMI-AGRAFES, PROCEDE DE PREPA¬ RATION ET APPLICATIONS"(ii) TITLE OF THE INVENTION "STAPLE AND SEMI-STAPLE OLIGONUCLEOTIDES, PREPARATION PROCESS AND APPLICATIONS"
( iii ) NOMBRE DE SEQUENCES : 23(iii) NUMBER OF SEQUENCES: 23
(iv) FORME DECHIFFRABLE PAR ORDINATEUR : ( A ) TYPE DE SUPPORT : DISQUETTE(iv) COMPUTER-DETACHABLE FORM: (A) TYPE OF MEDIUM: DISK
(B) ORDINATEUR: MACINTOSH(B) COMPUTER: MACINTOSH
(C) SYSTEME D'EXPLOITATION : MAC OS - SYSTEME 7(C) OPERATING SYSTEM: MAC OS - SYSTEM 7
(D) LOGICIEL : WORD PERFECT VERSION 2.0.(D) SOFTWARE: WORD PERFECT VERSION 2.0.
(v) DATE DE LA DEMANDE ACTUELLE(v) DATE OF CURRENT REQUEST
(A) NUMERO DE LA DEMANDE(A) REQUEST NUMBER
(B) DATE DU DEPOT:(B) DATE OF DEPOSIT:
(vi) DATE DE LA DEMANDE ANTERIEURE :(vi) DATE OF PREVIOUS APPLICATION:
(A) NUMERO DE LA DEMANDE : 9303514(A) REQUEST NUMBER: 9303514
(B) DATEDE DEPOT: 26 MARS 1993(B) DEPOSIT DATE: MARCH 26, 1993
(2) INFORMATIONS POUR LA SEQID N° : 1 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(2) INFORMATION FOR SEQID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 12 NUCLEOTIDES(A) LENGTH: 12 NUCLEOTIDES
(B) TYPE: ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION: LINEAIRE(D) CONFIGURATION: LINEAR
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
(iv) TYPE DE FRAGMENT : SEQUENCE POLYADENYLATE(iv) TYPE OF FRAGMENT: POLYADENYLATE SEQUENCE
( v ) CARACTERISTIQUE : (A) NOM/CLE: POLYA12(v) CHARACTERISTIC: (A) NAME / KEY: POLYA12
(vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 1 : AAAAAAAAAA AA 12 (3) INFORMATIONS POUR LA SEQUENCE N° : 2 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 1: AAAAAAAAAA AA 12 (3) INFORMATION FOR SEQUENCE N °: 2: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B ) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO- APPARIEE A L'EXTREMITE 3'(D) CONFIGURATION: SELF-MATCHED SECONDARY STRUCTURE AT THE 3 'END
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
( iv) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
(v) CARACTERISTIQUE : (A) NOM/CLE : SA12(v) CHARACTERISTICS: (A) NAME / KEY: SA12
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 2 AAAAAAAAAA AAGCGAAAGC 20(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 2 AAAAAAAAAA AAGCGAAAGC 20
(4) INFORMATIONS POUR LA SEQUENCE N° : 3 ( i ) CARACTERISTIQUES DE LA SEQUENCE(4) INFORMATION FOR SEQUENCE NO: 3 (i) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR : 28 NUCLEOTIDES(A) LENGTH: 28 NUCLEOTIDES
( B) TYPE : ACIDE NUCLEIQUE(B) TYPE: NUCLEIC ACID
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO- APPARIEE AUX EXTREMITES 3' ET 5'(D) CONFIGURATION: SELF-MATCHING SECONDARY STRUCTURE AT THE 3 'AND 5' END
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
(iv) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
(v) CARACTERISTIQUE : (A) NOM/CLE : HA12(v) CHARACTERISTIC: (A) NAME / KEY: HA12
(vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 3 GCGAAAGCAA AAAAAAAAAA GCGAAAGC 28(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 3 GCGAAAGCAA AAAAAAAAAA GCGAAAGC 28
(5 ) INFORMATIONS POUR LA SEQUENCE N° : 4 :(5) INFORMATION FOR SEQUENCE N °: 4:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 34 NUCLEOTIDES(A) LENGTH: 34 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO-(D) CONFIGURATION: SELF-SUPPORTING STRUCTURE
APPARIEE A L'EXTREMITE 3' ( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUEMATCHED AT THE 3 'END (ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
( iv ) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
(v) CARACTERISTIQUE : (A) NOM/CLE : RA12(v) CHARACTERISTICS: (A) NAME / KEY: RA12
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 4 : AAAAAAAAAA AAGCTTCCTC CTGCGGGCGA AAGC .34(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 4: AAAAAAAAAA AAGCTTCCTC CTGCGGGCGA AAGC .34
(6) INFORMATIONS POUR LA SEQUENCE N° : 5 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(6) INFORMATION FOR SEQUENCE NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
( iv ) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
( v ) CARACTERISTIQUE : (A) NOM/CLE : SA12/ml(v) CHARACTERISTIC: (A) NAME / KEY: SA12 / ml
( B ) AUTRES RENSEIGNEMENTS: MUTANT UNEAIRE SA 12 ( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 5 : AAAAAAAAAA AATAGAAAGC 20(B) OTHER INFORMATION: MUTANT UNEAIRE SA 12 (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 5: AAAAAAAAAA AATAGAAAGC 20
(7) INFORMATIONS POUR LA SEQUENCE N° : 6 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(7) INFORMATION FOR SEQUENCE N °: 6: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 28 NUCLEOTIDES(A) LENGTH: 28 NUCLEOTIDES
( B ) TYPE • ADN(B) TYPE • DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDS
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO- APPARIEE EN 3'(D) CONFIGURATION: 3 'SELF-MATCHING SECONDARY STRUCTURE
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
( iv ) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE (v) CARACTERISTIQUE : (A) NOM/CLE : HA12/ml ( B ) AUTRES RENSEIGNEMENTS: MUTANT UNEAIRE EN 5'(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE (v) CHARACTERISTIC: (A) NAME / KEY: HA12 / ml (B) OTHER INFORMATION: MUTANT UNEAIRE IN 5 '
DE HAÏ 2FROM HAI 2
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 6 : CGGAAAGCAA AAAAAAAAAA GCGAAAGC 28(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 6: CGGAAAGCAA AAAAAAAAAA GCGAAAGC 28
(8) INFORMATIONS POUR LA SEQUENCE N° : 7 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(8) INFORMATION FOR SEQUENCE N °: 7: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 28 NUCLEOTIDES(A) LENGTH: 28 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
(iv) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
( v ) C ARACTERISTIQUE : (A) NOM/CLE : HA12/m2 ( B ) AUTRES RENSEIGNEMENTS: MUTANT UNEAIRE EN ET 3'(v) CHARACTERISTICS: (A) NAME / KEY: HA12 / m2 (B) OTHER INFORMATION: MUTANT UNION IN AND 3 '
(vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 7 CGGAAAGCAA AAAAAAAAAA TAGAAAGC 28(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 7 CGGAAAGCAA AAAAAAAAAA TAGAAAGC 28
(9) INFORMATIONS POUR LA SEQUENCE N° : 8 ( i ) CARACTERISTIQUES DE LA SEQUENCE :(9) INFORMATION FOR SEQUENCE N °: 8 (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 34 NUCLEOTIDES(A) LENGTH: 34 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : STRUCRURE SECONDAIRE AUTO- APPARIEE EN 3'(D) CONFIGURATION: 3 'SELF-MATCHING SECONDARY STRUCTURE
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : NON(iii) ANTI-SENSE: NO
(iv) TYPE DE FRAGMENT : SEQUENCE POLYADENYLEE(iv) TYPE OF FRAGMENT: POLYADENYLATED SEQUENCE
(v) CARACTERISTIQUE : (A) NOM/CLE : RA12/ml ( B ) AUTRES RENSEIGNEMENTS: MUTANT DE RAI 2 (vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 8 : AAAAAAAAAA AACGAACCTC CTGCGGGCGA AAGC 34(v) CHARACTERISTIC: (A) NAME / KEY: RA12 / ml (B) OTHER INFORMATION: MUTANT FROM RAI 2 (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 8: AAAAAAAAAA AACGAACCTC CTGCGGGCGA AAGC 34
( 10) INFORMATIONS POUR LA SEQUENCE N° : 9 : ( i ) CARACTERISTIQUES DE LA SEQUENCE :(10) INFORMATION FOR SEQUENCE N °: 9: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 12 NUCLEOTIDES(A) LENGTH: 12 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDED STRANDS
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNmAT THE SPLICE JUNCTION OF AN mRNA
PRECOCE DU VIRUS HSV1EARLY HSV1 VIRUS
(v) CARACTERISTIQUE : (A) NOM/CLE : MV10 ou: GT 3022 (vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 9 : TTCCTCCTGCGG 12(v) CHARACTERISTIC: (A) NAME / KEY: MV10 or: GT 3022 (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 9: TTCCTCCTGCGG 12
( 11 ) INFORMATIONS POUR LA SEQUENCE N° : 10 :(11) INFORMATION FOR SEQUENCE N °: 10:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO-(D) CONFIGURATION: SELF-SUPPORTING STRUCTURE
APPARIEE EN 3'MATCHED IN 3 '
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNmAT THE SPLICE JUNCTION OF AN mRNA
PRECOCE DU VIRUS HSV1EARLY HSV1 VIRUS
(v) CARACTERISTIQUE (A) NOM/CLE : SMV10(v) CHARACTERISTIC (A) NAME / KEY: SMV10
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 10 : TTCCTCCTGC GGGCGAAAGC 20 ( 12) INFORMATIONS POUR LA SEQUENCE N° : 1 1 ( i ) CARACTERISTIQUES DE LA SEQUENCE :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 10: TTCCTCCTGC GGGCGAAAGC 20 (12) INFORMATION FOR THE SEQUENCE N °: 1 1 (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS MONOBRIN(C) NUMBER OF SINGLE-STRANDS
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE ( iii) ANTI-SENS : OUI(ii) TYPE OF MOLECULE: NUCLEIC ACID (iii) ANTI-SENSE: YES
( iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
(v) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE : SMVIO/ml(A) NAME / KEY: SMVIO / ml
( B) AUTRES RENSEIGNEMENTS: MUTANT UNEAIRE(B) OTHER INFORMATION: MUTANT UNEAIRE
DE SMV10FROM SMV10
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 11 TTCCTCCTGC GGTAGAAAGC 20(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 11 TTCCTCCTGC GGTAGAAAGC 20
( 13) INFORMATIONS POUR LA SEQUENCE N° : 12 :(13) INFORMATION FOR SEQUENCE N °: 12:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 28 NUCLEOTIDES(A) LENGTH: 28 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE AUTO-(D) CONFIGURATION: SELF-SUPPORTING STRUCTURE
APPARIEE EN 3'MATCHED IN 3 '
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNmAT THE SPLICE JUNCTION OF AN mRNA
PRECOCE DU VIRUS HSV1EARLY HSV1 VIRUS
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE : HMV10(A) NAME / KEY: HMV10
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
EN 3 ' PAR UNE SEQUENCE INDUISANT UN AUTO-APPA- RIEMENT ET EN 5' PAR UNE SEQUENCE UNEAIREIN 3 'BY A SEQUENCE INDUCING A SELF-PAIRING AND IN 5' BY A ONE-SEQUENCE
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 12 GCGAAAGCTT CCTCCTGCGG GCGAAAGC 28 ( 14) INFORMATIONS POUR LA SEQUENCE N° : 13 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 12 GCGAAAGCTT CCTCCTGCGG GCGAAAGC 28 (14) INFORMATION FOR SEQUENCE N °: 13:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 34 NUCLEOTIDES(A) LENGTH: 34 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE (ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(D) CONFIGURATION: SECONDARY STRUCTURE (ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
( iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
(v ) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE PAR(v) OTHER INFORMATION: SEQUENCE MVIO EXTENDED BY
DES EXTREMITES 5' ET 3 'APPARIEES ENTRE ELLESMatching 5 'and 3' ends between them
(vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 13 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 13:
GCGAAAGCTT CCTCCTGCGG GAGGAAGCTT TCGC 34GCGAAAGCTT CCTCCTGCGG GAGGAAGCTT TCGC 34
( 15) INFORMATIONS POUR LA SEQUENCE N° : 14 :(15) INFORMATION FOR SEQUENCE N °: 14:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
(B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) C ARACTERISΗQUE :(v) C ARACTERISΗQUE:
(A) NOM/CLE GT3514(A) NAME / KEY GT3514
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
PAR UNE SEQUENCE A STRUC¬ TURE SECONDAIRE AUTO- APPARIEEBY A SEQUENCE WITH SELF-MATCHING SECONDARY STRUCTURE
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 14 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 14:
TTCCTCOGC OÎGCCAAAGC 20 ( 16) INFORMATIONS POUR LA SEQUENCE N° : 15 :TTCCTCOGC OÎGCCAAAGC 20 (16) INFORMATION FOR SEQUENCE N °: 15:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
( ii ) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
(v) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3515(A) NAME / KEY GT3515
( B) AUTRK RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) AUTRK INFORMATION: EXTENDED MVIO SEQUENCE
PAR UNE SEQUENCE A STRUC¬ TURE SECONDAIRE AUTO- APPARIEEBY A SEQUENCE WITH SELF-MATCHING SECONDARY STRUCTURE
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 15 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 15:
TTCCTCCir GGGCTAAAGC 20TTCCTCCir GGGCTAAAGC 20
( 17) INFORMATIONS POUR LA SEQUENCE N° : 16 :(17) INFORMATION FOR SEQUENCE N °: 16:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3516(A) NAME / KEY GT3516
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
PAR UNE SEQUENCE A STRUC¬ TURE SECONDAIRE AUTO- APPARIEE (vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 16 :BY A SEQUENCE WITH SELF-MATCHING SECONDARY STRUCTURE (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 16:
T c ccTGCGGα rπTGC 20T c ccTGCGGα rπTGC 20
( 18) INFORMATIONS POUR LA SEQUENCE N° : 17 :(18) INFORMATION FOR SEQUENCE N °: 17:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
( ii ) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
( iv ) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3517(A) NAME / KEY GT3517
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE NON AUTO- APPARIEEAT THE 3 'END BY A NON-SELF-MATCHED SEQUENCE
(vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 17 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 17:
TTCCTCCir GGGCGTTTGC 20TTCCTCCir GGGCGTTTGC 20
( 19) INFORMATIONS POUR LA SEQUENCE N° : 18 :(19) INFORMATION FOR SEQUENCE N °: 18:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
(B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : LINEAIRE(D) CONFIGURATION: LINEAR
( ii ) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3518(A) NAME / KEY GT3518
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE NON AUTO- APPARIEE (vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 18 : TTCCTCCTGC GGGCAAAAGC 20AT THE 3 'END BY A NON-SELF-MATCHED SEQUENCE (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 18: TTCCTCCTGC GGGCAAAAGC 20
(20) INFORMATIONS POUR LA SEQUENCE N° : 19 :(20) INFORMATION FOR SEQUENCE N °: 19:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
( ii ) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
(v) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3519(A) NAME / KEY GT3519
(B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE AUTO-APPARIEEAT THE 3 'END BY A SELF-MATCHING SEQUENCE
( vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 19 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 19:
TICCTCCTGC GGGCGTAAGC 20TICCTCCTGC GGGCGTAAGC 20
(21) INFORMATIONS POUR LA SEQUENCE N° : 20 :(21) INFORMATION FOR SEQUENCE N °: 20:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
(B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
( ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) C ARACTERISΗQUE :(v) C ARACTERISΗQUE:
(A) NOM/CLE GT3520(A) NAME / KEY GT3520
(B) AUTRES RENSEIGNEMENTS: SEQUENCE MV10 PROLONGEE(B) OTHER INFORMATION: EXTENDED MV10 SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE AUTO-APPARIEE (vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 20 : TTCCTCCTGC GGGCGAGAGC 20AT THE 3 'END BY A SELF-MATCHING SEQUENCE (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 20: TTCCTCCTGC GGGCGAGAGC 20
(22) INFORMATIONS POUR LA SEQUENCE N° : 21 :(22) INFORMATION FOR SEQUENCE N °: 21:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
( iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3521(A) NAME / KEY GT3521
(B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE AUTO-APPARIEEAT THE 3 'END BY A SELF-MATCHING SEQUENCE
( vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 21 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 21:
TTCCTCCTGC GGGCGATAGC 20TTCCTCCTGC GGGCGATAGC 20
(23) INFORMATIONS POUR LA SEQUENCE N° : 22 :(23) INFORMATION FOR SEQUENCE N °: 22:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
(ii) TYPE DE MOLECULE : ACIDE NUCLEIQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
( iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
(v) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3522(A) NAME / KEY GT3522
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MV10 PROLONGEE(B) OTHER INFORMATION: EXTENDED MV10 SEQUENCE
A L'EXTREMITE 3* PAR UNE SEQUENCE AUTO-APPARIEE (vi ) DESCRIPTION DE LA SEQUENCE : SEQID N° : 22 : TTCCrcCTCC GGGCGAATGC 20AT THE 3 * END BY A SELF-MATCHING SEQUENCE (vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 22: TTCCrcCTCC GGGCGAATGC 20
(24) INFORMATIONS POUR LA SEQUENCE N° : 23 :(24) INFORMATION FOR SEQUENCE N °: 23:
( i ) CARACTERISTIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 20 NUCLEOTIDES(A) LENGTH: 20 NUCLEOTIDES
( B) TYPE : ADN(B) TYPE: DNA
(C) NOMBRE DE BRINS : MONOBRIN(C) NUMBER OF STRANDS: SINGLE-STRAND
(D) CONFIGURATION : STRUCTURE SECONDAIRE A L'EXTREMITE(D) CONFIGURATION: SECONDARY STRUCTURE AT THE END
3' AUTO-APPARIEE3 'SELF-MATCHING
( ii) TYPE DE MOLECULE : ACIDE NUC LOQUE(ii) TYPE OF MOLECULE: NUCLEIC ACID
(iii) ANTI-SENS : OUI(iii) ANTI-SENSE: YES
(iv) TYPE DE FRAGMENT : SEQUENCE COMPLEMENTAIRE(iv) TYPE OF FRAGMENT: COMPLEMENTARY SEQUENCE
A LA JONCTION D'EPISSAGE D'UN ARNm PRECOCE DU VIRUS HSV1AT THE SPLICE JUNCTION OF AN EARLY HSV1 VIRUS mRNA
( v ) CARACTERISTIQUE :(v) CHARACTERISTIC:
(A) NOM/CLE GT3523(A) NAME / KEY GT3523
( B) AUTRES RENSEIGNEMENTS: SEQUENCE MVIO PROLONGEE(B) OTHER INFORMATION: EXTENDED MVIO SEQUENCE
A L'EXTREMITE 3' PAR UNE SEQUENCE AUTO-APPARIEEAT THE 3 'END BY A SELF-MATCHING SEQUENCE
( vi) DESCRIPTION DE LA SEQUENCE : SEQID N° : 23 :(vi) DESCRIPTION OF THE SEQUENCE: SEQID N °: 23:
TTCCTCCTGCGC<ÎCGTTAGC 20 TTCCTCCTGCGC <ÎCGTTAGC 20

Claims

REVENDICATIONS
1. Oligonucléotide anti-sens semi-agrafe ou agrafe constitué d'une séquence oligonucléotidique dont l'extrémité 3' ou les deux extrémités 3' et 5' sont engagées dans un auto-appariement intracaténaire stable impliquant l'hybridation d'au moins les deux bases terminales de l'extrémité 3' ou respectivement de chaque extrémité 3' et 5', avec un même nombre de bases consécutives complémentaires situées sur le même brin, de manière à former une boucle à l'extrémité 3' ou respectivement une boucle à chaque extrémité 3' et 5' encadrant une séquence centrale, ladite séquence centrale comprenant une séquence anti-sens d'intérêt complémentaire d'une séquence cible appartenant à un ADN ou un ARN messager dont on désire bloquer spécifiquement l'expression et lesdites boucles ne comportant pas de séquences susceptibles de s'hybrider avec le brin d'ADN ou d'ARNm portant la séquence cible.1. Anti-sense semi-staple or staple oligonucleotide consisting of an oligonucleotide sequence whose 3 ′ end or both 3 ′ and 5 ′ ends are engaged in a stable intracatenary self-pairing involving the hybridization of at least the two terminal bases at the 3 'end or respectively at each 3' and 5 'end, with the same number of complementary consecutive bases located on the same strand, so as to form a loop at the 3' end or respectively a loop at each 3 ′ and 5 ′ end framing a central sequence, said central sequence comprising an antisense sequence of interest complementary to a target sequence belonging to a DNA or a messenger RNA whose expression and said loops are specifically desired to be blocked not containing sequences capable of hybridizing with the DNA or mRNA strand carrying the target sequence.
2. Oligonucléotide semi-agrafe ou agrafe selon la revendication 1 , caractérisé en ce que la (ou lesdites) boucle(s) comporte(nt) une séquence de nucléotides charnière non appariés intracaténairement, de 3 à 25, de préférence de 4 à 20 nucléotides.2. Semi-staple or staple oligonucleotide according to claim 1, characterized in that the (or said) loop (s) comprise (s) a sequence of hinge nucleotides unmatched intracatenarily, from 3 to 25, preferably from 4 to 20 nucleotides.
3. Oligonucléotide agrafe ou semi-agrafe selon l'une des revendications 1 ou 2, caractérisé en ce qu'il est constitué par une séquence d'intérêt d'ADN ou mixte ADN/ARN linéaire complémentaire d'une séquence cible d'ADN ou d'ARNm dont on désire bloquer l'expression, à l'extrémité 3' ou aux deux extrémités 3' et 5' de laquelle on a rajouté une boucle conistant en une séquence d'ADN ou mixte ADN/ARN auto-appariée comportant 8 à 12 nucléotides, ladite séquence auto- appariée étant caractérisée par l'appariement de son extrémité 3' avec son extrémité 5' et de la totalité de ses nucléotides deux à deux à l'exception de ceux constituant une séquence dite charnière.3. clip or semi-clip oligonucleotide according to one of claims 1 or 2, characterized in that it consists of a DNA sequence of interest or mixed DNA / linear RNA complementary to a target DNA sequence or of mRNA whose expression it is desired to block, at the 3 ′ end or at both 3 ′ and 5 ′ ends to which a loop consisting of a DNA or mixed DNA / RNA self-paired sequence comprising 8 to 12 nucleotides, said self-paired sequence being characterized by the pairing of its 3 ′ end with its 5 ′ end and of all of its nucleotides two by two with the exception of those constituting a so-called hinge sequence.
4. Oligonucléotide agrafe ou semi-agrafe selon l'une des revendications 1 ou 2, caractérisé en ce que les liaisons inter- nucléotidiques sont des liaisons phosphodiesters naturelles. 4. Clip or semi-clip oligonucleotide according to one of claims 1 or 2, characterized in that the inter-nucleotide bonds are natural phosphodiester bonds.
5. Oligonucléotide selon l'une des revendications 1 à 4 caractérisé en ce que ladite séquence anti-sens comporte une séquence d'ADN ou mixte ADN/ARN de manière à former un duplex ADN/ARN avec la séquence cible ARNm ou ADN respectivement.5. Oligonucleotide according to one of claims 1 to 4 characterized in that said antisense sequence comprises a DNA or mixed DNA / RNA sequence so as to form a DNA / RNA duplex with the target sequence RNA or DNA respectively.
6. Oligonucléotide selon l'une des revendications 1 à 5, caractérisé en ce qu'il comporte une partie non-nucléotidique pouvant être un (ou des groupements) lipophile(s), ou tout autre structure moléculaire favorisant la pénétration, le ciblage ou l'adressage cellulaire ou intra- cellulaire ou stabilisant la structure de ces oligonucleotides.6. Oligonucleotide according to one of claims 1 to 5, characterized in that it comprises a non-nucleotide part which may be a lipophilic group (s), or any other molecular structure favoring penetration, targeting or cellular or intra-cellular addressing or stabilizing the structure of these oligonucleotides.
7. Oligonucléotide selon la revendication 6, caractérisé en ce que la zone charnière de la boucle est constituée par un fragment non- nucléotidique.7. Oligonucleotide according to claim 6, characterized in that the hinge zone of the loop consists of a non-nucleotide fragment.
8. Oligonucléotide selon l'une des revendications 1 à 7, caractérisé en ce qu'il comporte dans sa séquence des nucléotides modifiés soit en série ribo- soit en série désoxyribo-.8. Oligonucleotide according to one of claims 1 to 7, characterized in that in its sequence nucleotides modified either in ribo- series or in deoxyribo- series.
9. Oligonucléotide selon l'une des revendications 1 à 8 caractérisé en ce qu'il comporte des groupements réactifs greffés sur ses nucléotides, comme par exemple des groupements psoralènes, ou d'autres agents de pontage ou agents intercalants pouvant réagir avec la séquence de la molécule cible complémentaire à l'oligonucléotide ou induire la formation de pontage intramoléculaire au sein même de la molécule.9. Oligonucleotide according to one of claims 1 to 8 characterized in that it comprises reactive groups grafted onto its nucleotides, such as for example psoralen groups, or other bridging agents or intercalating agents which can react with the sequence of the target molecule complementary to the oligonucleotide or induce the formation of intramolecular bridging within the molecule itself.
10. Oligonucléotide selon l'une des revendications 1 à 8 caractérisé en ce qu'il comporte des liaisons internes produites par des agents réactifs appartenant ou n'appartenant pas à la structure de la molécule elle-même.10. Oligonucleotide according to one of claims 1 to 8 characterized in that it comprises internal bonds produced by reactive agents belonging to or not belonging to the structure of the molecule itself.
11. Oligonucléotide selon l'une des revendications 1 à 10 caractérisé en ce qu'il est constitué par une séquence anti-sens d'intérêt comportant à son extrémité 3' ou aux deux extrémités 3' et 5' une des séquences GCGAAAGC, CGCAAGC, GCGAGAGC, GCGATAGC, GCGAATGC, GCGTTAGC. 11. Oligonucleotide according to one of claims 1 to 10 characterized in that it consists of an antisense sequence of interest comprising at its 3 ′ end or at the 3 ′ and 5 ′ ends one of the sequences GCGAAAGC, CGCAAGC , GCGAGAGC, GCGATAGC, GCGAATGC, GCGTTAGC.
12. Procédé de préparation d'un oligonucléotide selon l'une des revendications 1 à 10, caractérisé en ce qu'on prépare une séquence comportant ladit séquence d'intérêt à l'extrémité 3' ou aux deux extrémités 3 ' et 5 ' de laquelle on ajoute aux séquences nucléotidiques supplémentaires dont la nature et la longueur permettent un auto- appariement intra-caténaire stable de ladite séquence supplémentaire impliquant au moins ses deux bases terminales à l'extrémité 3' pour la séquence supplémentaire à l'extrémité 3 ' et l'extrémité 5 ' pour la séquence supplémentaire à l'extrémité 5', de manière à adopter la structure en semi-agrafe ou agrafe.12. Method for preparing an oligonucleotide according to one of claims 1 to 10, characterized in that a sequence is prepared comprising said sequence of interest at the 3 'end or at the 3' and 5 'ends of which is added to the additional nucleotide sequences whose nature and length allow a stable intra-catenary self-pairing of said additional sequence involving at least its two terminal bases at the 3 'end for the additional sequence at the 3' end and the 5 'end for the additional sequence at the 5' end, so as to adopt the semi-staple or staple structure.
13. Utilisation des oligonucleotides selon l'une des revendications 1 à 11 , à titre de médicament.13. Use of the oligonucleotides according to one of claims 1 to 11, as a medicament.
14. Composition pharmaceutique destinée à des utilisations à visée thérapeutique et, en particulier, à des indications en dermatologie et en virologie ou en cancérologie comportant, à titre de principe actif, un (ou plusieurs) oligonucléotide(s) selon l'une des revendications 1 à 11 avec des excipients pharmaceutiques appropriés.14. Pharmaceutical composition intended for therapeutic uses and, in particular, for indications in dermatology and virology or oncology comprising, as active principle, one (or more) oligonucleotide (s) according to one of claims 1 to 11 with appropriate pharmaceutical excipients.
15. Utilisation des oligonucleotides selon l'une des revendications 1 a l l a des fins de recherche de cosmétique ou de diagnostic.15. Use of the oligonucleotides according to one of claims 1 to 1 for cosmetic research or diagnostic purposes.
16. Composition destinée à des usages para-pharmaceutiques et, en particulier, à des usages cosmétiques comportant à titre de principe actif, un (ou plusieurs) oligonucléotide(s) selon l'une des revendications 1 à 11 avec des excipients appropriés.16. Composition intended for para-pharmaceutical uses and, in particular, for cosmetic uses comprising, as active principle, one (or more) oligonucleotide (s) according to one of claims 1 to 11 with appropriate excipients.
17. Composition, matériel ou kit destiné(e) à être utilisé(e) à des fins de dosage d'acides nucléiques ou de diagnostic, comportant un ou plusieurs oligonucléotide(s) selon l'une des revendications 1 à 11 avec des excipients appropriés. 17. Composition, material or kit intended to be used for nucleic acid assay or diagnostic purposes, comprising one or more oligonucleotide (s) according to one of claims 1 to 11 with excipients appropriate.
18. Procédé de modulation de l'expression génétique d'une séquence cible appartenant à un ARN messager ou à un ADN, caractérisé en ce qu'on hybride ladite séquence cible avec un oligonucléotide selon l'une des revendications 1 à 11, comportant une séquence complémentaire à ladite séquence cible. 18. A method of modulating the genetic expression of a target sequence belonging to a messenger RNA or to a DNA, characterized in that said target sequence is hybridized with an oligonucleotide according to one of claims 1 to 11, comprising a sequence complementary to said target sequence.
PCT/FR1994/000336 1993-03-26 1994-03-25 Staple and semi-staple oligonucleotides, method of preparation and applications WO1994023026A1 (en)

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032473A1 (en) * 1995-04-13 1996-10-17 Genset Dumbbell sense oligonucleotide for inhibiting herpes simplex virus (hsv)
WO1998005770A2 (en) * 1996-08-07 1998-02-12 Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts Antisense rna with a secondary structure
WO2000022114A1 (en) * 1998-10-09 2000-04-20 Ingene, Inc. PRODUCTION OF ssDNA $i(IN VIVO)
WO2000022113A1 (en) * 1998-10-09 2000-04-20 Ingene, Inc. ENZYMATIC SYNTHESIS OF ssDNA
WO2000053745A1 (en) * 1999-03-09 2000-09-14 Bioalliance Pharma (S.A.) Oligonucleotides containing an antisense sequence stabilised by a secondary structure and pharmaceutical compositions containing same
JP2004512020A (en) * 2000-06-23 2004-04-22 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Recombinant constructs and methods for using same in reducing gene expression
WO2004044135A2 (en) * 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Structural motifs and oligomeric compounds and their use in gene modulation
WO2005030960A1 (en) * 2003-09-30 2005-04-07 Anges Mg, Inc. Staple type oligonucleotide and drug comprising the same
WO2007095387A2 (en) 2006-02-17 2007-08-23 Dharmacon, Inc. Compositions and methods for inhibiting gene silencing by rna interference
EP1838875A2 (en) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions and methods for modulating gene expression using self-protected oligonucleotides
US7358066B2 (en) 2001-07-24 2008-04-15 Serono Genetics Institute S.A. Variants and exons of the GlyT1 transporter
US7419964B2 (en) 1999-09-16 2008-09-02 Cytogenix, Inc. Treatment of HSV-related pathologies using ssDNA
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7696345B2 (en) 2002-11-05 2010-04-13 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
US8124745B2 (en) 2002-11-05 2012-02-28 Isis Pharmaceuticals, Inc Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US9150606B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
EP3018207A1 (en) * 1999-01-30 2016-05-11 Alnylam Europe AG Oligoribonucucleotide for inhibiting the expression of a predefined gene

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427074A2 (en) * 1989-11-09 1991-05-15 Miles Inc. Nucleic acid amplification employing transcribable hairpin probe
WO1991017246A1 (en) * 1990-05-04 1991-11-14 Isis Pharmaceuticals, Inc. Modulation of gene expression through interference with rna secondary structure
WO1992002641A1 (en) * 1990-08-09 1992-02-20 Genta Incorporated Psoralen conjugated methylphosphonate oligonucleotides as therapeutic agents for chronic myelogenous leukemia
WO1992019732A1 (en) * 1991-04-25 1992-11-12 Genset Closed sense and antisense oligonucleotides and uses thereof
WO1994001550A1 (en) * 1992-07-02 1994-01-20 Hybridon, Inc. Self-stabilized oligonucleotides as therapeutic agents
WO1994012633A1 (en) * 1992-11-24 1994-06-09 Stiefel Laboratories, Inc. Stable oligonucleotides

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2008096A1 (en) * 1989-01-19 1990-07-19 Samuel Rose Nucleic acid amplification using single primer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427074A2 (en) * 1989-11-09 1991-05-15 Miles Inc. Nucleic acid amplification employing transcribable hairpin probe
WO1991017246A1 (en) * 1990-05-04 1991-11-14 Isis Pharmaceuticals, Inc. Modulation of gene expression through interference with rna secondary structure
WO1992002641A1 (en) * 1990-08-09 1992-02-20 Genta Incorporated Psoralen conjugated methylphosphonate oligonucleotides as therapeutic agents for chronic myelogenous leukemia
WO1992019732A1 (en) * 1991-04-25 1992-11-12 Genset Closed sense and antisense oligonucleotides and uses thereof
WO1994001550A1 (en) * 1992-07-02 1994-01-20 Hybridon, Inc. Self-stabilized oligonucleotides as therapeutic agents
WO1994012633A1 (en) * 1992-11-24 1994-06-09 Stiefel Laboratories, Inc. Stable oligonucleotides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
C. CASE ET AL., EMBO JOURNAL., vol. 8, no. 13, 1989, EYNSHAM, OXFORD GB, pages 4297 - 4305 *
J. JACQUES ET AL., NUCLEIC ACIDS RESEARCH, vol. 19, no. 11, 1991, ARLINGTON, VIRGINIA US, pages 2971 - 2977 *
T. HJALT ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, no. 24, 1992, ARLINGTON, VIRGINIA US, pages 6723 - 6732 *

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Publication number Priority date Publication date Assignee Title
WO1996032473A1 (en) * 1995-04-13 1996-10-17 Genset Dumbbell sense oligonucleotide for inhibiting herpes simplex virus (hsv)
FR2732971A1 (en) * 1995-04-13 1996-10-18 Genset Sa OLIGONUCLEOTIDE SENSE INHIBITOR OF HERPES SIMPLEX VIRUS (HSV) WITH HALTERED STRUCTURE
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US7919612B2 (en) 1996-06-06 2011-04-05 Isis Pharmaceuticals, Inc. 2′-substituted oligomeric compounds and compositions for use in gene modulations
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
WO1998005770A2 (en) * 1996-08-07 1998-02-12 Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts Antisense rna with a secondary structure
WO1998005770A3 (en) * 1996-08-07 1998-03-26 Deutches Krebsforschungszentru Antisense rna with a secondary structure
WO2000022114A1 (en) * 1998-10-09 2000-04-20 Ingene, Inc. PRODUCTION OF ssDNA $i(IN VIVO)
WO2000022113A1 (en) * 1998-10-09 2000-04-20 Ingene, Inc. ENZYMATIC SYNTHESIS OF ssDNA
EP3018207A1 (en) * 1999-01-30 2016-05-11 Alnylam Europe AG Oligoribonucucleotide for inhibiting the expression of a predefined gene
JP2004500018A (en) * 1999-03-09 2004-01-08 バイオオーリアンス　ファーマ（エス．エー．） Oligonucleotide containing antisense sequence stabilized by secondary structure and pharmaceutical composition containing the same
FR2790757A1 (en) * 1999-03-09 2000-09-15 Bioalliance Pharma OLIGONUCLEOTIDES CONTAINING AN ANTISENSE SEQUENCE STABILIZED BY A SECONDARY STRUCTURE AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME.
WO2000053745A1 (en) * 1999-03-09 2000-09-14 Bioalliance Pharma (S.A.) Oligonucleotides containing an antisense sequence stabilised by a secondary structure and pharmaceutical compositions containing same
US7022832B2 (en) 1999-03-09 2006-04-04 Bioalliance Pharma (S.A.) Oligonucleotides containing an antisense sequence stabilized by a secondary structure, pharmaceutical compositions containing them and method of blocking gene expression using them
US7419964B2 (en) 1999-09-16 2008-09-02 Cytogenix, Inc. Treatment of HSV-related pathologies using ssDNA
JP2004512020A (en) * 2000-06-23 2004-04-22 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Recombinant constructs and methods for using same in reducing gene expression
US7358066B2 (en) 2001-07-24 2008-04-15 Serono Genetics Institute S.A. Variants and exons of the GlyT1 transporter
WO2004044135A2 (en) * 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Structural motifs and oligomeric compounds and their use in gene modulation
US9150606B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US8604183B2 (en) 2002-11-05 2013-12-10 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US9150605B2 (en) 2002-11-05 2015-10-06 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US7696345B2 (en) 2002-11-05 2010-04-13 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
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US8124745B2 (en) 2002-11-05 2012-02-28 Isis Pharmaceuticals, Inc Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7595301B2 (en) * 2003-09-30 2009-09-29 Anges Mg, Inc. Staple type oligonucleotide and drug comprising the same
WO2005030960A1 (en) * 2003-09-30 2005-04-07 Anges Mg, Inc. Staple type oligonucleotide and drug comprising the same
JPWO2005030960A1 (en) * 2003-09-30 2006-12-07 アンジェスＭｇ株式会社 Staple oligonucleotide and pharmaceutical comprising the same
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
EP1838875A4 (en) * 2004-12-30 2010-08-25 Todd M Hauser Compositions and methods for modulating gene expression using self-protected oligonucleotides
EP1838875A2 (en) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions and methods for modulating gene expression using self-protected oligonucleotides
WO2007095387A2 (en) 2006-02-17 2007-08-23 Dharmacon, Inc. Compositions and methods for inhibiting gene silencing by rna interference
EP1986697A2 (en) * 2006-02-17 2008-11-05 Dharmacon, Inc. Compositions and methods for inhibiting gene silencing by rna interference
EP1986697B1 (en) * 2006-02-17 2016-06-29 GE Healthcare Dharmacon, Inc. Compositions and methods for inhibiting gene silencing by rna interference

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