WO1996034008A1 - Novel antisense nucleic acids directed against ras oncogenes, their preparation and use - Google Patents

Novel antisense nucleic acids directed against ras oncogenes, their preparation and use Download PDF

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WO1996034008A1
WO1996034008A1 PCT/FR1996/000652 FR9600652W WO9634008A1 WO 1996034008 A1 WO1996034008 A1 WO 1996034008A1 FR 9600652 W FR9600652 W FR 9600652W WO 9634008 A1 WO9634008 A1 WO 9634008A1
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nucleic acid
characterized
antisense
acid according
as4
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PCT/FR1996/000652
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French (fr)
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Claude Helene
Piet Herdewijn
Ester Saison-Behmoaras
Arthur Van Aerschot
Thanh Thuong Nguyen
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Centre National De La Recherche Scientifique (Cnrs)
Museum National D'histoire Naturelle
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Publication of WO1996034008A1 publication Critical patent/WO1996034008A1/en

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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/35Nature of the modification
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    • C12N2310/3511Conjugate intercalating or cleaving agent
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/35Nature of the modification
    • C12N2310/352Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3527Other alkyl chain

Abstract

The present invention concerns single-strand antisense nucleic acids complementing a region of an mRNA of a human RAS oncogene with localized mutation, comprising between 8 and 20 nucleotides and a group of the formula -(CHR)n-OH in position 3' and/or 5', in which n is an integer between 1 and 20 inclusive and R is a hydrogen atom or an OH group.

Description

ANTISENSE DIRECTED AGAINST NEW RAS- PREPARATION AND USES The present invention relates to antisense nucleic acids directed against the RNA messengers oncongènes ras, their preparation and their use, especially for the treatment of tumor diseases. It also relates to pharmaceutical compositions comprising said antisense nucleic acid, optionally adsorbed included or encapsulated in nanoparticles. It more specifically modified antisense nucleic acids able to specifically inhibit the expression of the mutated ras oncogenes. Various genes, called oncogenes and suppressor genes, are involved in the control of cell division. Of these, the ras genes and their products generally designated p21 proteins, play a key role in the control of cell proliferation in all eukaryotic organisms where they were searched. In particular, it was shown that certain specific modifications of these proteins cause them to lose their normal function and lead them to become oncogenic. Thus, a large number of human tumors have been associated with the presence of modified ras genes, and in particular ras genes with point mutations, most often at positions 12, 13 and 61. Similarly, overexpression of p21 protein can lead to a deregulation of cell proliferation. Understanding the exact role of these p21 proteins in cells, and the development of methods to inhibit their activity in tumor cells are therefore a major issue for the therapeutic approach to cancer.

One of the approaches proposed in the prior art to inhibit the activity of the ras oncogene is the use of antisense oligonucleotides. The regulation of expression of target gene by antisense oligonucleotides is indeed a therapeutic approach in increasing development. This approach relies on the ability of oligonucleotides to hybridize specifically with complementary regions of a nucleic acid and to thereby inhibit specifically the expression of particular genes. This inhibition may occur either at translational level (antisense oligonucleotide) or at the transcriptional level (anti-gene oligonucleotide). More particularly, the antisense nucleic acids are nucleic sequences capable of hybridizing selectively with target cell messenger RNAs to inhibit their translation into protein. These oligonucleotides form with the target mRNA, locally, double-stranded regions, by Watson-Crick interaction of conventional type. This may be for example of synthetic oligonucleotides, small size, complementary to cellular mRNAs and which are introduced into the target cells. Such oligonucleotides have been described for example in patent EP 92 574. It can also be antisense genes whose expression in the target cell generates complementary to cellular mRNAs. Such genes have been described for example in Patent No. EP 140 308. However, the in vivo use of antisense nucleic acids directed against the ras genes encounters a number of difficulties which limit up to now their therapeutic exploitation. First, the nucleic acids are highly sensitive to degradation by enzymes in the body, such as nucleases, which involves the use of large doses. In addition, they have low penetration in certain cell types and often inadequate intracellular distribution, which makes no therapeutic effect. Finally, it is important to have sufficiently stable and selective sequences to obtain a specific effect without altering other cellular functions.

In an attempt to solve some of these problems, it has been proposed to chemically modify the phosphodiester backbone of nucleic acids, to give rise to new classes of artificial oligonucleotides. Among these, there may be mentioned phosphonate oligonucleotides, phosphotriesters, phosphoramidates and phosphorothioates which are described, for example, in Patent Application WO94 / 08003, the oligonucleotides coupled to different agents such as cholesterol, a peptide, a cationic polymer etc. However, if some of these modified compounds exhibit good resistance to nucleases, their penetrating power and distribution in different cellular compartments is very low. Also their biological activity is generally not increased and they have certain side effects related to the presence of non-natural motifs in their structure. The present invention provides a particularly advantageous solution to these problems. The present invention indeed provides new antisense nucleic acids directed against ras, having a particular chemical modification. Particularly advantageously, the antisense according to the invention not only have a high nuclease resistance, but also good cell penetration followed by an appropriate distribution in the cells, a very high selectivity towards the target, and form particularly stable complexes with their targets.

A first object of the present invention resides in a single-stranded complementary nucleic acid antisense to a region of an mRNA of a human ras oncogene carrying a point mutation, characterized in that it comprises 8 to 20 nucleotides and a group of formula - (CHR) n -OH 3 'and or 5' wherein n represents an integer between 1 and 20 inclusive, and R represents a hydrogen atom or an OH group, R can vary from a group (CHR) to another.

Surprisingly, the Applicant has now shown that it was possible to obtain in vitro selective antiproliferative effects using short antisense nucleic acids are chemically modified by adding a group - (CHR) n -OH 3 'and / or 5 '. The results presented in the examples also show that antisense thus obtained have increased vis-à-vis the nuclease resistance and superior biological activity. Furthermore they induce no apparent cytotoxicity and have no significant side effects. The results confirm that these properties are quite unexpected in so far as the antisense of the invention are active at concentrations up to 500 times lower than those needed for other types of antisense (cholesterol, polymer, etc. ) including natural oligophosphodiesters.

As indicated above, the group of formula - (CHR) n -OH can be coupled 3 ', 5' or 3 'and 5' of the antisense nucleic acid. In general, the antisense and the group - (CHR) n -OH are synthesized separately and then assembled by standard chemical reaction. A method for grouping the coupling - (CHR) n -OH in each of these positions is indicated in the examples. Preferably, in the formula - (CHR) n -OH, n is an integer between 3 and 12 inclusive. Particularly interesting results have been obtained with antisense-modified dodecanediol group (n = 12), propanediol (n = 3), dimethyl-l, 3-propanediol (n = 5) or glycerol (n = 3).

The nucleic acids used in the context of the present invention advantageously have a length of between 8 and 20 nucleotides. The Applicant has indeed shown that it was better to obtain a combined selective effect good stability, the use of relatively short oligonucleotides. As illustrated in the examples, the selectivity of the anti-ras antisense nucleic acid is particularly important when the oligonucleotide has a length less than 14 bases. It is therefore particularly advantageous to use nucleic acids of size between 8 and 13 bases. Even more preferably, the nucleic acids used in the context of the present invention have a length between 10 and 13 nucleotides. Particularly good results were obtained with nucleic acids of 12 nucleotides. Examples include antisense nucleic acids of sequence SEQ ID No. 1 (AS2); SEQ ID No. 10 (AS4); SEQ ID NO: 14 or SEQ ID NO: 15 which have properties quite remarkable. The following modified antisense are particularly preferred for the purposes of the present invention: AS2-3'ml2OH; 5'ml2OH-AS2; AS2-3'm3OH; 5'm3OH-AS2; 5'ml2OH-AS2-3'ml2OH, AS4-3'ml2OH; 5'ml2OH-AS4; AS4-3'm3OH; 5'm3OH- AS4 and 5'ml2OH-AS4-3'ml2OH, AS2-3'Glycérol; AS4-3 'Glycerol. In a particularly preferred embodiment of the invention, the nucleic acids used are chosen so that the target point mutation of the ras oncogene is located in the center of the complementary region. The Applicant has indeed shown to be particularly advantageous that the antisense nucleic acid used is centered around the targeted mutation. As illustrated in the examples, when the nucleic acid is centered on the acid mutation, selective inhibition of 100% is obtained, while it is only 50% when the transfer is shifted only 2 bases. These particularly surprising results allow for a given target region, to prepare antisense nucleic acids much more effective. Moreover, to further improve the therapeutic efficacy of the antisense of the invention, other chemical modifications may be combined with the presence of the group (CHR) n -OH. In particular, the combination with an intercalating agent such as acridine, can further enhance the antisense potential (cf. examples). The intercalating agent may be introduced in the antisense or at one end, or both. Particularly good results were obtained with a modified antisense an acridine group 5 'and 3' (Acr-ASml2OH). The synthesis of this oligonucleotide was made by coupling a first step of dodecanediol group 3 'according to the techniques illustrated in the examples, then the acridine moiety at the 5' according to the techniques described by Helene in applications EP 117777 and EP 169787 by example, or using any other protocol known in the art. It is also possible to couple the first acridine group then dodecanediol group.

In a particular embodiment of the invention, antisense nucleic acids may be adsorbed, included or encapsulated in nanoparticles. The nanoparticles used in the context of the present invention can be any particle of small size, generally less than 500 nm, composed of polymer (s) biocompatible (s), capable of transporting or vectorize an active ingredient in the cells or in the bloodstream. Preferably, the nanoparticles of the invention are constituted by polymers having a majority of degradable units such as polylactic acid, optionally copolymerized with polyethylene glycol, or alkyl cyanoacrylates. Other polymers useful in achieving nanoparticles have been described in the prior art (see e.g. EP 275 796 and EP 520 889). For example, use may be made of nanoparticles of alkyl cyanoacrylate polymers (such as described in European patent EP 0007895 and the French patent 08172 8) or polymers of lactic acid or copolymers of lactic acid and glycolic acid (as described by G. Spenlehauer et al, J. Control Release (1988), 7, 217-229;.. EP 520 888; EP 520 889).

The nanoparticles of the invention generally have an average diameter in the range of 50 to 500 nanometers. Preferably, the nanoparticles used in the context of the present invention have an average diameter of about 150 nanometers. 96/34008

Of particular interest are the nanoparticles obtained by polymerization of methyl cyanoacrylate, ethyl, isobutyl or isohexyl or those obtained by copolymerization of lactic acid and glycolic acid.

Moreover, prior to encapsulation, the nucleic acids of the invention are generally associated with cationic hydrophobic compounds The use of such compounds makes it possible to facilitate the association of nucleic acids with the nanoparticles. More particularly, the hydrophobic cationic compounds used in the compositions according to the invention permit the formation of ion pairs between the phosphorus derivatives of negatively charged nucleic chain and the hydrophobic cations and thus increase the lipophilicity and adsorption of nucleic acids to nanoparticles. In general, cationic hydrophobic compounds for use in preparing the compositions according to the invention are selected from tetraphenylphosphonium halides, preferably chloride; quaternary ammonium salts, more particularly chosen from halides trimethylalkylammonium such as chlorides or bromides, dodecyltrimethylammonium, tetradecyltrimethylammonium, of héxadécylriméthylammonium or cetyltrimethylammonium; fatty amines, such as D, L-dihydrosphingosine (D, L-dihydroxy-3-amino-2 octadecane), or oligopeptides, such as polylysine and oligopeptides of formula (lkkl) n, (lklk) n or (LRRL) n. Particularly advantageously is used in the context of the invention a salt of héxadécylriméthylammonium or oligopeptide

The nanoparticulate compositions of the invention may be obtained by any method for adsorbing, to include or encapsulating a nucleic acid, with or without a hydrophobic cationic compound, in a nanoparticle.

Typically, the nanoparticulate compositions of the invention can be obtained

- by bringing the nucleic acid and optionally cationic hydrophobic compound with the nanoparticles under conditions that allow a retention (or absorption) of the strong nucleic acid on the nanoparticles and easy achievement of target cells, - or by bringing the nucleic acid and optionally cationic hydrophobic compound with the monomer units, followed by polymerization,

- either by bringing the nucleic acid and optionally cationic hydrophobic compound with the polymer during the polymerization that is to say after the start of the polymerization but before the completion thereof.

Preferably, the compositions according to the invention are obtained by adsorption, inclusion or encapsulation of the nanoparticles already formed.

alkyl polycyanoacrylates of the nanoparticles can be prepared under the conditions described in European Patent EP 0007 895 and the French patent 81 08172 and those of polylactic-polyglycolic acid under the conditions described by G. Spenlehauer et al., J. Control . Release, (1988), 7, 217-229.

The adsorption of the nucleic acid or not complexed by cationic hydrophobic compounds on the nanoparticles is generally carried out by stirring a suspension of nanoparticles with the nucleic acid complexed with the cationic hydrophobic compound under specific conditions.

The thus transformed nanoparticles are generally separated by filtration or centrifugation and used to prepare therapeutically useful pharmaceutical compositions.

Typically, the adsorption is carried out in an aqueous medium whose pH is buffered to a value of about 7.

Can be used as buffer TRIS-HC1 buffer.

Also to stabilize the suspension, it may be useful to work in the presence of a stabilizing agent such as a synthetic copolymer of polyoxyethylene-polyoxypropylene nonionic as poloxamer 188 or a polyoxyethylene. This stabilizer serves in particular to reduce adhesion phenomena between particles leading to the formation of aggregates.

The adsorption is generally carried out at a temperature of 20 ° C and the reaction is complete after some hours of stirring.

It is particularly advantageous to use at the beginning of the adsorption operation, a nanoparticle concentration between 0.5 and 50 mg / cm 3 and preferably about 1 mg / cm3. The nucleic acid concentration is generally between 0.01 and 500 .mu.M. The selected nucleic acid concentration depends primarily on the nanoparticle concentration and the desired final concentration.

Furthermore, to increase the adsorption of nucleic acids on the nanoparticles, it may be advantageous to operate in the presence of an excess of cationic hydrophobic compound.

In general, the efficiency of adsorption of nucleic acids to the nanoparticles depends essentially on the length of the nucleotide chain and the charge density on the particle surface. Another object of the invention relates to pharmaceutical compositions comprising an antisense nucleic acid as defined above, in combination with one or more pharmaceutically acceptable diluents or adjuvants. The antisense can be encapsulated or not, given that even in free form, it has antiproliferative properties in vivo quite remarkable. The compositions according to the invention can be used in vitro, ex vivo or in vivo. In vitro, they can allow the transfer to cell lines of antisense nucleic acids, for example in order to study the regulation of ras proteins and towards ras-dependent signaling. Ex vivo, they can be used to transfer the therapeutic anti-ras antisense nucleic acids into a cell derived from an organism, in order to confer to said cell resistance ras oncogenes, prior to readministration to an organism. In vivo, they can be used to direct nucleic radministration anti-ras antisense acids. In particular, intra-tumoral administration is quite advantageous since it allows to deliver effectively and locally active composition. Preferably, the pharmaceutical compositions of the invention thus contain a pharmaceu-tically acceptable vehicle for an injectable formulation, especially for intratumoral injection. It may be in particular isotonic sterile solutions or dry compositions, in particular freeze-dried, which, by addition depending on the case of sterilized water or of physiological saline, allow the constitution of injectable solutions. The compositions according to the invention being capable of modulating the activity of oncogenic ras proteins, they allow to inhibit the development process and can thus be used for the treatment of various cancers. Many cancers have indeed been associated with the presence of oncogenic ras proteins. Among the cancers containing the most frequently mutated ras genes include particularly adenocarcinoma of the pancreas, 90% have an oncogene K-ras mutated on the twelfth codon (Almoguera et al., Cell 5_3 (1988) 549), the adenocarcinomas of the colon and thyroid cancers (50%), bladder cancer (40%, see Alcishi et al., Int. J. One. 4 (1994) 85), or carcinomas of the lung and myeloid leukaemias (30%, Bos, JL Cancer Res. 49 (1989) 4682).

The present invention will be described in more detail with the following examples, which should be considered as illustrative and not restrictive. Lé2βnde figures

Figure 1: Position on their target (A) and hybridization efficiency (B) antisense 1-5, as measured by the induction of a cut RNA target for RNase H. Position on their target (C) and effectiveness hybridization (D) R1-R8 antisense measured as in (B). Materials and methods

Synthesis of single-stranded antisense nucleic acids The various nucleic acids used were synthesized using an automated solid-phase synthesizer (Applied Biosystems, Forster City, CA) according to the phosphoramidite chemistry. The nucleic acids were then precipitated twice with ethanol, washed in the presence of 75% ethanol, then dissolved in water. Cleavage by RNase H

cleavage experiments by RNase H from nuclear extracts of HeLa cells (HeLa Scribe Nuclear extract, Promega) were carried out in the reaction mixture (25 .mu.l) containing 40 mM Tris pH 7.9; 100 mM KC1; 2 raM MgC12; 0.8 .mu.l of HeLa nuclear extract, DNA 2μg carrier, 4 nM mRNA wild or mutated ras and 5 .mu.M of each nucleic acid to be tested. The reactions were carried out without premixing of the mRNA and nucleic acid to be tested. After treatment with phenol and precipitation, the cleavage products by RNase H were analyzed by electrophoresis polycarylamide sequencing gel (6%). The gels were then autoradiographs, and the amount of intact material and cleavage product was determined by densitometry. Cell Lines Used

- HBL100: Human epithelial cell line available from the ATCC

- HBLlOOrasl: This line is derived from human breast cell line HBL100 by transformation with a pSV2 plasmid carrying the human Ha-Ras oncogene EJ / T24. This line expresses the wild form of Ha-ras, and the form carrying the point mutation G -> U at position 12, encoding the amino acid valine instead of a glycine (Ha-ras Val12 (Lebeau et al. Oncogene 6 (1991) 1125).

- HBLlOOnéo: This line also derives from the human mammary cell line HBL100, by transformation with a plasmid pSV2 control. This line expresses only the wild form of Ha-ras. Preparation of nanoparticles

- Nanoparticles polyisohexylcyanoacrylate A nanoparticle suspension was prepared by adding, under magnetic stirring, 100 of μlitre isobutylcyanoacrylate or isohexylcyanoacrylate a polymerization medium composed of hydrochloric acid (1 mM, pH = 3 or 10 mM, pH = 2) and 1 g / 100 cm3 dextran. The polymerization of the cyanoacrylate monomers spontaneously carried out at a temperature of about 20 ° C. The polymerization is complete after 2 hours using the isobutylcyanoacrylate and after 6 hours using isohexylcyanoacrylate. After neutralization, 0.4 g / 100 cm 3 of poloxamer 188 is added to stabilize the nanoparticles. - nanoparticles of lactic acid-glycolic acid: 10 mg / cm3 of a lactic acid-glycolic acid copolymer containing 75% of lactic acid units and D L and 25% of glycol units are suspended in a medium containing 10 mM TRIS-HC1 at pH = 7 and 0.5% lecithin. After 12 hours of incubation, the nanoparticles can be separated from the suspension. Encapsulation of nucleic acids into nanoparticles The nanoparticles prepared above at a concentration of 0.5 mg cm3 were incubated with 500 microM nucleic acid 500 microM hydrophobic cation (e.g. CTAB), in Tris-HCl buffer (10 mM, pH = 7) without sodium chloride (Chavany et al, Pharmaceutical Res. 9 (1992) 441). After 6-8 hours of incubation, the nanoparticles can be separated from the suspension or the suspension may be used, after dilution, to test the efficacy of the nucleic acid adsorbed on the appropriate cells.

Generally, a few .mu.l of the diluted suspension (about 100 times in distilled water) are added to 100 .mu.l of the culture medium containing the cells, the number of .mu.l added is calculated to achieve the desired concentration of nucleic acid.

Examples

Example 1: Selection of antisense 1.1. Effect of length

This example shows that the length can have a significant impact on both the selectivity and affinity of the antisense and that, contrary to the general trend, the use of antisense limited size provides very important benefits. The following antisense were synthesized:

- 5'-CACACCGACGGC (SEQ ID NO: 1) - 12 Wed.

- 5'-CACACCGACGGCG (SEQ ID No. 2) - 13 Wed.

- 5'-CACACCGACGGCGC (SEQ ID NO: 3) - 14 Wed.

- 5'-CACACCGACGGCGCC (SEQ ID NO: 4) - 15 mer - 5'-CCACACCGACGGCGCC (SEQ ID NO: 5) - 16 Wed.

The position of these antisense vis-à-vis the target sequence (SEQ ID NO: 6) is shown in Figure 1A. Antisense corresponding directed against the normal flush Messenger have also been synthesized. These different antisense were incubated in the presence of nuclear extracts of HeLa cells, and their ability to hybridize with the ras mutated messenger was determined by RNase H cleavage as described in materials and methods. The results are shown in Figure IB. They show that the AS-Val antisense already produce a maximal response. They also show that a significant signal nonselective appears as soon as the length of the antisense than 13 nucleotides. These results show that the best ratio selectivity / affinity is achieved with antisense 12 to 13 Wed

1.2. Effect of the position

This example shows that, in addition to the length, the position of the antisense to the target sequence is important.

Antisense following 12-mer were synthesized:

- 5'-TGCCCACACCGA (RI: SEQ ID No. 7)

- 5 * -GCCCACACCGAC (R2: SEQ ID NO: 8)

- 5'-CCACACCGACGG (R3: SEQ ID NO: 9) - 5 * ACACCGACGGC -C (R4: SEQ ID NO: 1)

- 5'-CACCGACGGCGC (R5: SEQ ID NO: 10)

- 5'-CCGACGGCGCCC (R6: SEQ ID NO: 11)

- 5'-GACGGCGCCCAC (R7: SEQ ID NO: 12)

- 5'-ACGGCGCCCACC (R8: SEQ ID NO: 13)

The position of these antisense vis-à-vis the target sequence (SEQ ID NO: 6) is presented in Figure 1C. These different antisense were incubated in the presence of nuclear extracts of HeLa cells, and their ability to hybridize with the ras mutated messenger was determined by RNase H cleavage as described in materials and methods. The results are shown in Figure ID. They show that antisense centered around the mutated region of the target sequence (R4 and R5) have the best screw-selectivity RNA mutated oncogene, although they were not the most efficient great. Example 2: Preparation of 5'-HO (CH ι: CACACCGACGGC (5'-m-OH 12 AS2V 2.1) -Diméthoxytrityl- 1 1, 12-dodecanediol (Dmt-O- (CH -) 12ÛH) To a solution of 1,12-dodecanediol (15 mmol) and N-ethyl dimethylamine (6 mmol) in 20 cm3 of anhydrous pyridine is added at a temperature of 20 ° C and stirring dimethoxytrityl chloride (3 mmol) then continues stirring for 2 hours at a temperature of 20 ° C. to the reaction mixture was added 30 cm3 of an aqueous solution of sodium hydrogen carbonate 5% then the product is extracted with dichloromethane. the organic phase is dried over sodium sulfate. After filtration and concentration to dryness under reduced pressure, the product obtained is purified by chromatography on silica gel eluting with a dichloromethane-methanol mixture (97-3 by volume). There is thus obtained, with a yield of 75 %, DMT-O (CH2) i2θH whose characteristics are as follows: Rf = 0.65 ( Kieselgel 60F 254 Merck; dichloromethane-methanol (9-1 by volume)

Figure imgf000015_0001

The compound Dmt-O- (CH 2 -) ι 2 OH obtained above (1.2 mmol) was dried by co-evaporation under reduced pressure was treated with dry pyridine and then it is maintained under reduced pressure overnight. Product was solubilized in a solution of dimethylethyl amine (0.44 g) in dichloromethane (8 cm 3) and added slowly under argon atmosphere and under stirring, 2-cyanoethyl-N, N-chloro-phosphite dϋsopropylamino (0.7 g, 3 mmol). After 30 minutes of reaction, added to the reaction mixture 50 cm3 of ethyl acetate, followed by washing the organic solution with an aqueous 10% sodium hydrogen carbonate (2 times

80 cm3) and finally with saturated aqueous sodium chloride (20 cm3).

The organic phase is dried over sodium sulfate. After filtration and concentration to dryness under reduced pressure, the product is chromatographed on a silica gel column eluting with a mixture of ethyl acetate and triethylamine. Is thus obtained with a yield of 80% OCH 2 CH 2 CN Dmt-O- (CH 2 -) 2 OP ι whose characteristics are the following:

N (iPr) 2

Rf = 0.73 and 0.8 (stereoisomers) (Kieselgel 60F 254 Merck; ethyl acetate-triethylamine (9-1 by volume).

2.3) is carried dodécadésoxynucléotide assembly according to the phosphoramidite method on a DNA synthesizer using the GPC deoxycytidine (10 micromoles) and 50 micromoles of deoxyribonucleoside-3 '- (2-cyanoethyl) - dϋsopropylaminophosphoramidite per cycle. After detritylation the support is treated for 10 minutes under argon with a solution of the phosphoramidite prepared in Example 2.2 (1 cm3 0.1M in actonitrile) and tetrazole (3 cm3 0.5M in acetonitrile) . After removal of the liquid phase, the support is then treated with 10 cm 3 of a solution of iodine [0.01M of iodine in acetonitrile - water mixture collidine (65-30 by volume)]. the support and then isolating the treated with a concentrated ammonia solution for 7 hours at 55 ° C. After removing the medium by filtration, the solution is evaporated to dryness under reduced pressure and the residue was treated for 30 minutes at a temperature of 20 ° C, with acetic acid. acetic acid is removed by evaporation under reduced pressure. The product obtained is purified by FPLC. The purified product retention time is given in Table I.

Example 3 - Preparation of 5'CACACCGACGGC- (CH ι: OH f AS2-3'-OH1 m I2 3-1) 1 -Diméthoxytrityl- 12-succinyl-1, 12-dodecanediol

DMT-O- (CH 2 -) j 2 OC-CH 2 -CH 2 -COH

OO

Is reacted, at a temperature of 20 ° C with stirring for 18 hours, a solution of the product prepared according to Example 2.1 (2 mmol), dimethylaminopyridine (1.1 mmol) and succinic anhydride (1 , 6 mmol) in anhydrous pyridine (4 cm 3). the pyridine is removed under reduced pressure. The residue obtained is taken up in 15 cm3 of dichloromethane and then the organic solution is washed with an aqueous solution of 10% citric acid (2 x 10 cm3) then with water. The organic phase is dried and evaporated. The residue is dried under reduced pressure has the following characteristics:

Rf = 0.5 [Merck Kieselgel 60F 254; dichloromethane-methanol (9-1 by volume)]. 2) DMT-O- (CH 2 -) 12 OC-CH 2 -CH 2 -CNH (CH 2) 3 -Fractosil 500

OOA a solution of product prepared according to Example 3.1 (0.5 mmol) in a mixture p.dioxane / pyridine (95/5 by volume; 2, 1 cm3) was added with stirring to a temperature of about 20 ° C, a solution of dicyclohexylcarbodiimide (1.25 mmol) in 300 μlitre of p.dioxane. Stirring is continued for one hour at a temperature of 20 ° C, dicyclohexylurea was removed by filtration. The solution obtained is treated with 1.5 g of aminopropyl-Fractosil 500 suspended in dimethylformamide and 0.38 cm3 of triethylamine for 18 hours at a temperature of about 20 ° C. The support is isolated by filtration and washed with acetonitrile and dried under reduced pressure. The capacity of the obtained carrier was determined by spectrophotometric determination of the amount of dimethoxytrityl cation released by a sample of the dry support acid treatment. In general the support has a capacity of 70 micromoles g. In order to acetylate the remaining amino functions, the support is taken up in 6 cm3 of pyridine and treated with 0.6 cm 3 of acetic anhydride in the presence of 30 mg of 4-dimethylaminopyridine. After one hour reaction, the support is isolated by filtration, washed with dichloromethane, methanol and ether and then dried under reduced pressure.

3-3) By using the carrier prepared according to Example 3.2 (10 micromoles), the assembly of the sequence of the dodecamer is performed automatically on synthéti¬ sor according to the phosphoramidite method via désoxyribonucléo- side-3 ' - (2-cyanoethyl) -dϋsopropylaminophosphoramidite (50 micromoles per cycle). Deprotection and purification of the dodecamer are performed according to the conditions described in Example 2.3. The purified product of the retention time is given in Table I. Example 4 - Preparation of 5 (CCACACCGA) 3 p (CH 2) 2 OH ι

Proceeding as in Example 3 was obtained nonanucleotide (SEQ ID NO: 14), the retention time is given in Table I. Example 5: Preparation of HO (CH 2) 12 5 pd (CACACCGACGGC) 3 'p (CH 2) 2 OH ι (5'ml2OH-AS2-3τnl2OF).

Proceeding as in Example 2 and replacing the deoxycytidine CPG by the support prepared according to Example 3 DODECAMER preparing substituted 3 'and 5' of the (CH 2) OH ι. Table I gives the retention time of the purified product.

TABLE 1

Example Retention time

(minutes) *

2 20.4

• - 20.1

4 19.2

5 23.2

* FPLC: Column Mono P HR 5/5 (Pharmacia); Solvent A: 0.01 M NaH 2 PO 4 in acetonitrile-water mixture (2-8 by volume; pH = 6.8) and solvent B: 0.01 M NaH 2 PO_ι, 1M NaCl in acetonitrile-water mixture (2 -8 volumes, pH = 6.8); linear gradient from 0 to 100% B; flow rate: 1 cm 3 / minute.

Example 6: Preparation of d5 (CACCGACGGCGC) p (CH 2) 3 OH

6.1) dimethoxytrityl-l, 3-propanediol

0.725 ml (10 mmol) of 1,3-propanediol one was dissolved in 20 ml of anhydrous pyridine and 4.07 g (12 mmol) of dimethoxytrityl chloride were added. The mixture was stirred 2h at room temperature, after which TLC (CH2C12-MeOH 99: 1) shows that the reaction is substantially complete. The reaction is then stopée by addition of an excess of MeOH and then, after 10 min, the mixture was concentrated. The concentrate is partitioned between CH2C12 phase and an aqueous 5% NaHCO3 (2x), then the organic phase is dried over Na2SO4. Purification on silica gel resulted in 1.7 g (4.47 mmol) of the expected product. Rf = 0.44 (Alugram Sil G / UN254, Macherey Νagel, CH2C12-MeOH 99: 1).

6.2) dimethoxytrityl-3-succinyl-1, 3-propanediol

A 1,62g of the product obtained above dissolved in 20 ml of anhydrous pyridine was added lg (10 mmol) of succinic anhydride and 1.22 g (100 mmol) of dimethylamino pyridine. The mixture was stirred 16 h at ambient temperature and then excess water was added to the reaction stoper. The mixture was concentrated and partitioned between cold ethyl acetate layer and a cold citric acid solution 10%. The organic phase was washed once again with cold citric acid solution then with brine. The organic phase is dried over Νa2SO4 then purified over silica gel on a gradient of MeOH (0-5%) in CH2C12 containing 0.25% triethylamine, leading to 1.3 g of the expected product. Rf = 0.48 (Alugram Sil G / UV254, Macherey Nagel, CH2C12-MeOH 95: 5).

6.3) By operating as in Example 3 was obtained decanucleotide AS4- 3'-m3OH (SEQ ID No. 10).

Example 7: Preparation of d5 (CACCGCCGGCGC) p (CH 2) 3 OH

Proceeding as in Example 6 was obtained dodécanucléotide (SEQ

ID NO: 15). SEQ ID NO: 15 sequence is complementary to normal mRNA ras.

Example 8: Encapsulation of Antisense 8.1. Antisense adsorbed onto the nanoparticles can be prepared as follows:

- a nanoparticle suspension is prepared by adding, under magnetic stirring, 100 of μlitre isobutylcyanoacrylate or isohexylcyanoacrylate a polymerization medium composed of hydrochloric acid (1 mM, pH = 3 or 10 mM, pH = 2) and 1 g / 100 cm3 dextran. The polymerization of the cyanoacrylate monomers spontaneously carried out at a temperature of about 20 ° C. The polymerization is complete after 2 hours using the isobutylcyanoacrylate and after 6 hours using isohexylcyanoacrylate. After neutralization, 0.4 g / 100 cm 3 of poloxamer 188 is added to stabilize the nanoparticles. - The nanoparticles thus obtained at a concentration of 0.5 mg / cm3 were incubated with 128 microM of antisense modified according to the invention, 500 .mu.M of a hydrophobic cation (e.g. CTAB), in Tris-HCl buffer (10 mM , pH = 7) without sodium chloride. After 12 hours of incubation, the nanoparticles can be separated from the suspension or the suspension may be used, after dilution, to test the effectiveness of the antisense adsorbed on the appropriate cells. Generally, a few .mu.l of the diluted suspension is added (about 100 times in distilled water) in 100 .mu.l of culture medium containing the cells, the number of .mu.l added is calculated to achieve the desired antisense concentration.

8.2. Antisense adsorbed onto the nanoparticles can also be prepared as follows: Suspend 10 mg / cm 3 of a lactic acid-glycolic acid copolymer containing 75% of lactic acid units and D L and 25% of units glycol in a medium containing 10 mM TRIS-HCl at pH = 7 and 0.5% lecithin. The nanoparticles were incubated with 2 .mu.M antisense, 1.6 mM cetyltrimethylammonium bromide. After 12 hours of incubation, the nanoparticles can be separated from the suspension.

The adsorption rate of the antisense nanoparticles is 82%.

Example 9: Inhibition of cell growth HBLlOOrasl

This example demonstrates the inhibitory properties quite remarkable antisense according to the invention.

HBLlOOrasl the cells were seeded in 96-well plates at a concentration of 4.10 3 cells per well in 50 .mu.l of medium supplemented with 7% fetal calf serum inactivated by heat, antibiotics and glutamine. When the cells adhere to culture wells (usually after 2 to 3 days), antisense are added in each well, at twice the final concentration in 50 .mu.l of culture medium resulting in a final volume of 100 .mu.l per well. After 72 hours of incubation at 37 ° C in a humidified atmosphere containing 5% CO2, the cells were counted using a hemocytometer. Cell viability, examined simultaneously by trypan blue staining, is greater than 95% for treated and untreated cells. The results are presented in Table 2 below. They are expressed as percent of cell proliferation relative to untreated cells, according to the following formula: 100 x (Nn-NO) / (Nn-N), wherein NO is the number of cells present initially, Nn is the number of untreated cells after n days of growth, and N is the number of cells treated after n days.

TABLE 2

HBL-100 cells rASL HBL 100 cells

Product Concentration% Concentration% Proliferation microM microM proliferation

AS4 (SEQ 10) 10 26 10 89

AS-GLY (SEQ 15) 20 86 20 95

AS2 (SEQ 1) 10 94 10 100

AS2 adsorbed 0.1 45 0.1 75

0.2 22 0.2 75

AS2-3'-mι 2 OH 2 92

(Example 3)

Figure imgf000021_0001
adsorbed 0.05 32

AS4-3' m-OH 3 0025 2 0025 100

(Example 6) 0.05 0.05 100 16

0,005 40 0,005 100

AS-GLY-3'-m 3 OH

(Example 7) 0.05 100

0,005 100 0,005 80

0.5 97 0.5 61 m3: - (CH 2) 3 - m 12 - (CH 2) i2 - The results show that:

- antisense (AS4-3'm OH) modified according to the invention induced inhibition of proliferation of HBLlOOrasl cells at very low concentrations. These concentrations are about 100 times lower than those to which the unmodified antisense is active.

- the antisense of the invention are specific for cells containing mutated ras since no effect is observed on HBLlOOnéo cells. Similarly, the antisense directed against the normal gene (AS-Gly) has no effect on the proliferation of cells HBLlOOrasl.

These results show that the composition of the invention show selectivity and a very important activity. The maximum effect for a single antisense injection was observed three days after addition of antisense culture.

Complementary results were obtained with another series of antisense (see Table 3). The results represent inhibition of cell proliferation

HBLlOOrasl cultured in the presence of the antisense indicated for 24 hours. The concentration required to inhibit 50% of cell proliferation are shown in Table 3.

These results confirm the selectivity and very important activity of the antisense of the invention. Particularly pronounced effects are obtained with 2,2-dimethyl-l, 3 propanediol and acridine-dodecanediol (Acr5'-AS4-3'ml2OH).

Example 12: Affinity Study

This example shows that the antisense according to the invention possess particularly high affinities for the target sequence ras substrate. For this, the antisense of the invention AS4 (SEQ ID NO: 10) modified at their 3 'end by various groups have been used, including antisense Acr5'-AS4-3'ml2OH. These antisense were placed in the presence of a target sequence ribonucleic radiolabeled with a length of 27 nucleotides. The gel shift analyzes to determine the concentrations required to secure 50% of the substrate. The results are presented in Table 4. Table 3

Dwarf Formula X 50% (microM) HBLlOOras cells Growth inhibition

R5 15

1, 3-propanediol -0- (CH2) 3-OH 0.042

OH

1, 2 Propanediol -0- (CH2) -CH-CH3 2.5

CH3

2,2-Dimethyl-l, 3- propanediol -O-CH2-C-CH2-OH 0.06

CH3

H

No propanediol friend -O-CH2-C-CH2-NH2 0.150

OH

Hexanediol -0- (CH2) 6-OH 0.5

Decanediol -0- (CH2) ιo-OH 0.25

Dodecanedi ol -0- (CH2) i2-OH 0.06

Acridine- Acridine, - (CH2) 12-OH 0.04 dodecanediol

Diethylenglycol -0- (CH2) 2-0- (CH2) 2OH 0.06

Glycerol -O-CH2-CHOH-CH2OH 0.04 Table 4

Name X Formula 50% (microM) Bindiπg

R5 0.19

1, 3-propanediol -0- (CH2) 3-OH 0.15

OH

1, 2 Propanediol 1

-0- (CH2) -CH-CH3 0.2

CH3

2, 2-Dimethyl-l, 3-Propanediol 1 -0-CH2-C-CH2-0H 0.19

1

CH3

H

Amino-Propanediol -O-CH2-C-CH2-NH2

0.75 OH

Hexanediol -0- (CH2) 5-OH 0.3

Decanediol -0- (CH2) ιo-OH 0.6

Dodecanediol -0- (CH2) i2-OH 2.0

Acridine- Acridine, - (CH2) 12 OH 0.7 dodecanediol

SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS 1 - antisense nucleic acid complementary single-stranded region of an mRNA of a human ras oncogene carrying a point mutation, characterized in that it comprises 8 to 20 nucleotides and a group of formula - (CHR) n -OH 3 'and / or 5', wherein n represents an integer between 1 and 20 inclusive, and R represents a hydrogen atom or an OH group, R can vary from a group (CHR) to another.
2. Nucleic acid according to claim 1 characterized in that n represents an integer between 3 and 12 inclusive.
3. Nucleic acid according to claim 1 or 2 characterized in that the group of formula - (CHR) n -OH is selected from dodecanediol, propanediol, dimethyl propanediol and glycerol.
4 - Nucleic acid according to one of claims 1 to 3 characterized in that it comprises from 10 to 13 nucleotides.
5 - Nucleic acid according to claim 4 characterized in that it comprises
12 nucleotides.
6. Nucleic acid according to one of claims 1 to 5 characterized in that the point mutation is located in the center of the complementary region.
7. Nucleic acid according to one of claims 1 to 6 characterized in that it is selected from AS2-3'ml2OH; 5'ml2OH-AS2; AS2-3'm3OH; 5'm3OH-AS2;
5'ml2OH-AS2-3'ml2OH, AS4-3'ml2OH; 5'ml2OH-AS4; AS4-3'm3OH; 5 * m3OH- AS4 and 5'ml20H-AS4-3'ml20H, AS2-3 'Glycerol; AS4-3 'Glycerol.
8. Nucleic acid according to one of claims 1 to 7 characterized in that it comprises an additional chemical modification.
9. Nucleic acid according to claim 8 characterized in that it comprises one or more intercalating agents, such as acridine.
10. Nucleic acid according to claim 8 characterized in that it is the antisense antisense Acr5'-AS4-3'ml2OH
11. Nucleic acid according to one of claims 1 to 10 characterized in that it is encapsulated, adsorbed or included in a nanopaπicule.
12 - Nucleic acid according to claim 1 1, characterized in that it is previously associated with cationic hydrophobic compounds.
13 - A pharmaceutical composition characterized in that it comprises one or more nucleic acids according to one of claims 1 to 12 in combination with one or more pharmaceutically acceptable diluents or adjuvants.
14. The pharmaceutical composition of claim 13 for intratumoral administration.
PCT/FR1996/000652 1995-04-28 1996-04-29 Novel antisense nucleic acids directed against ras oncogenes, their preparation and use WO1996034008A1 (en)

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US5985558A (en) * 1997-04-14 1999-11-16 Isis Pharmaceuticals Inc. Antisense oligonucleotide compositions and methods for the inibition of c-Jun and c-Fos
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Cited By (11)

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US6339066B1 (en) 1990-01-11 2002-01-15 Isis Pharmaceuticals, Inc. Antisense oligonucleotides which have phosphorothioate linkages of high chiral purity and which modulate βI, βII, γ, δ, Ε, ζ and η isoforms of human protein kinase C
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US5985558A (en) * 1997-04-14 1999-11-16 Isis Pharmaceuticals Inc. Antisense oligonucleotide compositions and methods for the inibition of c-Jun and c-Fos
US6312900B1 (en) 1997-04-14 2001-11-06 Isis Pharmaceuticals, Inc. Antisense oligonucleotide compositions and methods for the modulation of activating protein 1
US6887906B1 (en) 1997-07-01 2005-05-03 Isispharmaceuticals, Inc. Compositions and methods for the delivery of oligonucleotides via the alimentary canal
US8691785B2 (en) 1997-07-01 2014-04-08 Isis Pharmaceuticals, Inc. Compositions and methods for non-parenteral delivery of oligonucleotides
US6221850B1 (en) 1997-08-13 2001-04-24 Isis Pharmaceuticals Inc. Antisense oligonucleotide compositions and methods for the modulation of JNK proteins
US5877309A (en) * 1997-08-13 1999-03-02 Isis Pharmaceuticals, Inc. Antisense oligonucleotides against JNK
US6809193B2 (en) 1997-08-13 2004-10-26 Isis Pharmaceuticals, Inc. Antisense oligonucleotide compositions and methods for the modulation of JNK proteins
US6133246A (en) * 1997-08-13 2000-10-17 Isis Pharmaceuticals Inc. Antisense oligonucleotide compositions and methods for the modulation of JNK proteins
US8101585B2 (en) 2006-08-04 2012-01-24 Isis Pharmaceuticals, Inc. Compositions and methods for the modulation of JNK proteins

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