Modified oligonucleotides and their use

Abstract

The inventors have now succeeded in developing modified lipid oligonucleotides comprising an oligonucleotide sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues. These compounds have the advantage of being active molecules on a proto-oncogenic targets, in particular proto-oncogenic targets such as KRAS and c-Myc. Such modified lipid oligonucleotides are thus of particular interest in medical treatments, in particular treatments involving antiproliferative activity against cancer cells. The present invention thus relates to modified lipid oligonucleotides comprising an oligonucleotide sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues, and their use in the treatment of cancer.

Description

[0001] MODIFIED OLIGONUCLEOTIDES AND THEIR USE

[0002] The present invention relates to modified lipid oligonucleotides comprising an oligonucleotide sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues, and their use in the treatment of cancer.

[0003] BACKGROUND OF THE INVENTION

[0004] Mutations of the KRAS gene are involved in different tumor types and due to its role in the development of cancers this oncogene is an attractive target for new anticancer drugs and strategies. Indeed, KRAS is an important element in the pathogenesis of several types of cancers and a primary target for anticancer drugs (Huang, L. et cd., Signal Transduct. Target. Ther. 2021, 6, 386). KRAS is the most frequently mutated oncogene in human cancer; i.e. mutated in more than 90% of pancreatic adenocarcinomas and in 50% of colorectal carcinomas, for example. If direct small-molecule inhibitors of KRAS are currently under investigation (Chen, H. et al., J. Med. Chem. 2020, 63, 14404-14424), new strategies are needed to specifically address this oncogenic target. MYC is one of the most common activated oncogene in human cancer, it is overexpressed in more than 40% of primary pancreatic cancers (Schleger, C. et al., Mod. Pathol. 2002, 15, 462-469; Allenson, K. et al., Ann. Oncol. 2017, 28, 741-747). Also, it was found that activation of MYC could convert pancreatic intraepithelial neoplasm into pancreatic cancer in mice (Sodir, N.M. et al. , Cancer Discov. 2020, 10, 588-607). It was observed in preclinical studies that inhibition of cMyc in resistant pancreatic cancer could improve the response to chemotherapies (Parasido, E. et al., Mol. Cancer Res. 2019, 17, 1815-1827).

[0005] Guanine-rich oligonucleotides (GROs) can self-assemble into four stranded G4 structures stabilized by 7t-7t stacking between G-quartets and via Hoogsteen hydrogen bonding. These G-4 quadruplexes are found for instance in telomeric and promoter regions, where they participate in a large diversity of biological processes. For example, the control region of the proto-oncogene KRAS contains a nuclease hypersensitive element (NHE), which can bind to nuclear proteins and is known to form G-quadruplex structures. Also, it is known that parallel-stranded G-quadruplex stabilized in the cMyc promoter region functions as a transcriptional repressor element (Seenisamy, J. et al. , J. Am. Chem. Soc. 2004, 126, 8702- 8709; Siddiqui-Jain, A. et al., Proc. Natl. Acad. Sci. 2002, 99, 11593-11598).

[0006] The expression of KRAS in pancreatic cancer cells can be downregulated by using decoy oligonucleotides mimicking one of the potential NHE quadruplexes (Cogoi, S. et al., Nucleic Acids Res. 2013, 41, 4049-4064; Cogoi, S. et al., Nucleic Acids Res. 2014, 42, 8379-8388; Miglietta, G. et al, ACS Med. Chem. Lett. 2015, 6, 1179-1183). In normal cells, KRAS transcription is blocked by the two G-quadruplexes, then it is activated when MAZ (a G4- binding protein) unfolds the G quadruplex structures, thus inducing the formation of transcription complexes. Thanks to a decoy strategy, it was demonstrated that synthetic G4 oligonucleotides (G4-Oligo) inhibit oncogenic KRAS in human cancer cells. However, this promising, “decoy approach”, which could be potentially extended to many G4 proteins involved in quadruplex binding and resolving (i.e. telomere and promoter regions, RNA quadruplexes or quadruplexes resolving helicases, etc) is facing major issues. Hence clinical applications of this decoy strategy are limited by: i) the cellular internalization of the decoy G4-Oligos (additional cationic transfection reagents are required, for example see Cogoi, S. et al., Nucleic Acids Res. 2013, 41, 4049-4064), ii) the chemical stability of G4-Oligos, and the iii) supramolecular stability G4-Oligos based quadruplexes in the adequate topology inside the cell. The latter is a major hurdle since the G4 supramolecular structures are required for the final biological activity. Hence, therapeutic approaches allowing the inhibition of both KRAS and c-MYC oncogenes expression would be highly desirable in the field of cancer.

[0007] Thus, there is a need for compounds having an activity on proto-oncogenic targets, in particular proto-oncogenic targets such as KRAS and c-MYC.

[0008] SUMMARY OF THE INVENTION

[0009] The inventors have now succeeded in developing modified lipid oligonucleotides comprising an oligonucleotide sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues. These compounds have the advantage of being active molecules on proto-oncogenic targets, in particular proto- oncogenic targets such as KRAS and c-MYC. Such modified lipid oligonucleotides are thus of particular interest in medical treatments, in particular treatments involving antiproliferative activity against cancer cells.

[0010] The invention therefore relates to a compound of Formula I, compounds of general Formula I, or their pharmaceutically acceptable salts thereof, as well as methods of use of such compounds of Formula I or compositions comprising such compounds of Formula I in medical treatments, in particular treatments involving antiproliferative activity against cancer cells, more particularly in the treatment of cancer.

[0011] The invention relates to a compound of Formula I: I, or a pharmaceutically acceptable salt thereof, wherein - ' is an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising more than 2 and less than 4 consecutive guanine residues; Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and - CH2-;

[0012] Z is O or S;

[0013] R1and R2are independently selected from the group consisting of H, halo, OH and Cl- C12-alkyl; B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;

[0014] L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H.

[0015] The present invention also concerns tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I as defined above, wherein each of the more than 2 and less than 4 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding.

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I as defined above or tetramolecular parallel G-quadruplexes as defined above, wherein the tetramolecular parallel G-quadruplexes self-assembled into micelles, and at least one pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant.

[0017] The invention also relates to a compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein is an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising at least 3 consecutive guanine residues;

[0018] Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and - CH2-;

[0019] Z is O or S;

[0020] R1and R2are independently selected from the group consisting of H, halo, OH and Cl- C12-alkyl;

[0021] B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;

[0022] L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H; or a tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I, wherein each of the at least 3 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding; for use in the treatment of cancer.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention relates to a compound of Formula I:

[0025] or a pharmaceutically acceptable salt thereof, wherein ] - ' is an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising more than 2 and less than 4 consecutive guanine residues;

[0026] Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and - CH2-;

[0027] Z is O or S; R1and R2are independently selected from the group consisting of H, halo, OH and Cl- C12-alkyl;

[0028] B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms; L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H.

[0029] As used herein, the term “oligonucleotide” refers to a nucleic acid sequence, 3 ’-5’ or 5 ’-3’ oriented, in particular 5 ’-3’ oriented, which may be single- or double-stranded, in particular single-stranded. The oligonucleotide used in the context of the invention may in particular be DNA or RNA, more particularly DNA.

[0030] The oligonucleotide used in the context of the invention may be further modified, preferably chemically modified, in order to increase the stability and / or therapeutic efficiency of the oligonucleotides in vivo. In particular, the oligonucleotide used in the context of the invention may comprise modified nucleotides.

[0031] Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and / or (iii) on the entire backbone structure of the oligonucleotide. For example, the oligonucleotides may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2 ’-Methoxy ethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective.

[0032] Additionally or alternatively, the oligonucleotide used in the context of the invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2’ position of the sugar, in particular with the following chemical modifications: O-methyl group (2’-0-Me) substitution, 2 -methoxy ethyl group (2’- O-MOE) substitution, fluoro group (2’-fluoro) substitution, chloro group (2’-Cl) substitution, bromo group (2’-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2’-CF3) substitution, OCF3 group (2’-OCF3) substitution, OCN group (2’-OCN) substitution, O-alkyl group (2’-O-alkyl) substitution, S-alkyl group (2’-S-alkyl) substitution, N-alkyl group (2’-N-akyl) substitution, O-alkenyl group (2’-O-alkenyl) substitution, S-alkenyl group (2’-S- alkenyl) substitution, N-alkenyl group (2’-N-alkenyl) substitution, SOCH3 group (2’- SOCH3) substitution, SO2CH3 group ^’-SCECEE) substitution, ONO2 group (2’-ONO2) substitution, NO2 group (2’-NO2) substitution, N3 group (2’-Ns) substitution and / or NH2 group (2’-NH2) substitution.

[0033] Additionally or alternatively, the oligonucleotide used in the context of the invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2’ oxygen and the 4’ carbon of the ribose, fixing it in the 3’-endo configuration. These constructs are extremely stable in biological medium, able to activate RNase H and form tight hybrids with complementary RNA and DNA.

[0034] Accordingly, in an embodiment, the oligonucleotide used in the context of the invention may comprise modified nucleotides selected from the group consisting of LNA, 2’-OMe analogs, 2’-phosphorothioate analogs, 2’-fluoro analogs, 2’-Cl analogs, 2’-Br analogs, 2’-CN analogs, 2’-CF3 analogs, 2’-OCF3 analogs, 2’-OCN analogs, 2’-O-alkyl analogs, 2’-S-alkyl analogs, 2’-N-alkyl analogs, 2’-O-alkenyl analogs, 2’-S-alkenyl analogs, 2’-N-alkenyl analogs, 2’-SOCH3 analogs, 2’-SO2CH3 analogs, 2’-ONO2 analogs, 2’-NO2 analogs, 2’-Ns analogs, 2’ -NEE analogs and combinations thereof. More preferably, the modified nucleotides are selected from the group consisting of LNA, 2’-OMe analogs, 2’- phosphorothioate analogs and 2’ -fluoro analogs.

[0035] The oligonucleotide used in the context of the invention may typically have from 3 to 50 nucleotides, in particular from 3 to 40 nucleotides, for example from 3 to 35 nucleotides, from 3 to 30, from 3 to 25, from 3 to 15, from 3 to 9 nucleotides.

[0036] Particular compounds of Formula I, or pharmaceutically acceptable salts or solvates thereof, ( ON ] are those wherein one or more of ' - ' , Y, Z, R1, R2, B, L1and L2are defined as follows: is an oligonucleotide comprising, in particular consisting of, a sequence from 3 to 50 nucleotides, said sequence comprising more than 2 and less than 4 consecutive guanine f ON ] residues; in particular ' - ' is an oligonucleotide comprising, in particular consisting of, a sequence from 3 to 40 nucleotides, for example from 3 to 35 nucleotides, from 3 to 30, from 3 to 25, from 3 to 15, from 3 to 9, said sequence comprising more than 2 and less than

[0037] 4 consecutive guanine residues; more particularly is a sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’), SEQ ID NO: 2 (5’-TGGGAGT- 3’) and SEQ ID NO: 3 (5’-AGGGCGGTGTGGGAAGAGGGA-3’); still more particularly is SEQ ID NO: 1 (5’-TGGGAG-3’) or SEQ ID NO: 2 (5’-TGGGAGT-3’); Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and -CH2- ; in particular Y is a divalent linker moiety selected from the group consisting of -O-, -S-, and -CH2-; more particularly Y is -O- or -S-; even more particularly Y is -O-;

[0038] Z is O or S; in particular Z is S;

[0039] R1and R2are independently selected from the group consisting of H, halo, OH and C1-C12- alkyl; in particular R1and R2are independently selected from the group consisting of H, halo and Cl-C6-alkyl; more particularly R1and R2are independently selected from the group consisting of H, F, Cl, Br and Cl-C4-alkyl; even more particularly R1and R2are independently selected from the group consisting of H, F, Cl and Cl-C2-alkyl; still more particularly R1and R2are independently selected from the group consisting of H, F, Cl and methyl; in a particular example R1and R2are H;

[0040] B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms; in particular B is a unsubstituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6-methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5- methylcytosine, 5-hydroxymethylcytosine and hypoxanthine; more particularly B is a unsubstituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine; still more particularly B is uracil;

[0041] L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; in particular L1and L2are independently selected from H and C6-C20-alkyl; more particularly L1and L2are independently selected from H and C10-C20 alkyl; still more particularly L1and L2are C12- C18-alkyl; even more particularly L1and L2are n-pentadecyl; with the proviso that L1and L2are not both H.

[0042] In one embodiment, the compounds of Formula I are those wherein L1and L2are C6-C20- alkyl; in particular L1and L2are C10-C20 alkyl; more particularly L1and L2are C12-C18- alkyl; still more particularly L1and L2are n-pentadecyl. In one embodiment, the compounds of Formula I are those wherein B is uracil.

[0043] In one embodiment, the compounds of Formula I are those wherein L1and L2are n- pentadecyl and B is uracil.

[0044] In one embodiment, the compounds of Formula I are those wherein quadruplex forming sequence. In particular, the G-quadruplex structure is a four-stranded nucleic acid structure. In other words, the G-quadruplex structure results from 4 strands of nucleic acid.

[0045] In one embodiment, the compounds of Formula I are those wherein sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’), SEQ ID NO: 2 (5’-TGGGAGT-3’) and SEQ ID NO: 3 (5’-AGGGCGGTGTGGGAAGAGGGA-3’).

[0046] In one embodiment, the compounds of Formula I are those wherein sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’) and SEQ ID NO: 2 (5’-TGGGAGT-3’).

[0047] In one embodiment, the compounds of Formula I are those wherein is SEQ ID

[0048] NO: 1 (5’-TGGGAG-3’).

[0049] In one embodiment, the compounds of Formula I are those wherein is SEQ ID NO: 2 (5’-TGGGAGT-3’).

[0050] In one embodiment, the compounds of Formula I are those wherein is SEQ ID NO: 3 (5’-AGGGCGGTGTGGGAAGAGGGA-3’).

[0051] Particularly preferred compounds of Formula I are those listed hereafter:

[0052]

[0053] The compound of Formula I, or any of its embodiments, can be prepared by different ways with reactions known by the person skilled in the art, in particular by using phosphoramidite chemistry with the compound of Formula I as starting material, as described by the examples.

[0054] In particular, the compound of formula I may be prepared by the following successive steps:

[0055] (i) synthesizing the oligonucleotide;

[0056] (ii) modifying the oligonucleotide obtained in step (i) by reaction with a suitable reactant comprising one or two saturated or unsaturated, in particular saturated, linear or branched, in particular linear, hydrocarbon chains comprising from 1 to

[0057] 22 carbon atoms, in particular from 6 to 20 carbon atoms, more particularly from 12 to 18 carbon atoms;

[0058] (iii) recovering the compound of Formula I.

[0059] Steps (i) and (ii) are generally carried out using a coupling methodology. According to a preferred embodiment, steps (i) and (ii) are carried out using the phosphoramidite methodology, which is well-known for the synthesis of oligonucleotides.

[0060] In particular, the reactant used in step (iii) comprises a phosphoramidite group. A “phosphoramidite group” refers to a monoamide of a phosphite diester moiety, which can be represented as follows: >N-P(-O-)2.

[0061] Step (ii) is generally carried out in the presence of a coupling agent, such as N- benzylthiotetrazole, which activates the phosphoramidite. The modified oligonucleotide synthesized in step (ii), advantageously comprising an OH 3’ end, is then added and the coupling occurs.

[0062] The compound of Formula I is then obtained according to usual procedures well-known in the framework of oligonucleotides synthesis.

[0063] Tetramolecular parallel G-quadruplex

[0064] As the compound of the invention comprises a nucleotide sequence comprising more than 2 and less than 4 consecutive guanine residues, the modified oligonucleotide of the invention can self-assemble into four stranded G4 structures stabilized by 7t-7t stacking between G- quartets and via Hoogsteen hydrogen bonding.

[0065] The present invention thus also concerns tetramolecular parallel G-quadruplex comprising 4 identical modified oligonucleotides or Formula I as defined above, wherein each of the more than 2 and less than 4 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quartet being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding.

[0066] By “tetramolecular parallel G-quadruplex” is meant herein a four-stranded nucleic acid structure, all four strands pointing in the same direction, comprising multiple stacked G- quartets, each of which consists of four guanine bases that associate in a cyclical manner through Hoogsteen hydrogen bonds and 7t-7t stacking, and may be further stabilized through coordination to a cation in the center.

[0067] In one embodiment, the tetramolecular parallel G-quadruplex of the invention further comprises a cation, which coordinates said G-quartets.

[0068] Said cation may be a monovalent or a divalent cation. Examples of suitable cations include K+and Mg2+. In a particular embodiment, said cation is a divalent cation, more particularly a Mg2+cation.

[0069] Composition

[0070] The present invention also provides a composition comprising tetramolecular parallel G- quadruplexes as defined above, wherein the tetramolecular parallel G-quadruplex selfassembled into micelles.

[0071] A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions of the molecules in contact with surrounding aqueous solvent, sequestering the hydrophobic “tail” regions of the molecules in the micelle center.

[0072] The tetramolecular parallel G-quadruplexes self-assemble into micelles having a core / shell structure, wherein the shell is hydrophilic and is formed of the oligonucleotide parts of the G-quadruplexes, and wherein the core is lipophilic and is formed of the lipid moiety of the modified oligonucleotides constituting the G-quadruplexes.

[0073] The composition may comprise up to 50% by weight of modified oligonucleotides, in particular from 0.1% to 40%, more particularly from 1% to 20%, still more particularly from 8% to 15% by weight of modified oligonucleotides, with respect to the total weight of the composition.

[0074] According to one embodiment, the composition as defined above may further comprise a hydrophobic active principle hosted in said micelles.

[0075] Said micelles may be used for the loading of hydrophobic drugs. Loading of such micelles with hydrophobic drug may vary from 10 nM to 2 mM.

[0076] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula I as defined above or tetramolecular parallel G-quadruplexes as defined above, wherein the tetramolecular parallel G-quadruplexes self-assembled into micelles, and at least one pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant. The present invention also relates to the pharmaceutical composition as defined above for use as a medicament.

[0077] APPLICATIONS

[0078] The compounds of the invention, which are modified oligonucleotides, and / or tetramolecul ar parallel G-quadruplexes comprising 4 identical compounds of the invention have an activity on proto-oncogenic targets, in particular proto-oncogenic targets such as, but not limited to, KRAS and c-MYC. In particular, the modified oligonucleotides of the invention and / or tetramolecular parallel G-quadruplexes comprising 4 identical modified oligonucleotides of the invention allow the inhibition of oncogenes expression, in particular oncogenes such as, but not limited to, KRAS and c-MYC oncogenes.

[0079] The modified oligonucleotides and / or tetramolecular parallel G-quadruplexes comprising 4 identical modified oligonucleotides of the invention of the invention are therefore useful in medical treatments, in particular treatments involving antiproliferative activity against cancer cells.

[0080] Thus, in one embodiment, there is provided a modified oligonucleotide of the present invention, or a pharmaceutically acceptable salt or solvate thereof, and / or tetramolecular parallel G-quadruplexes comprising 4 identical modified oligonucleotides of the invention of the invention, for use in treating cancer.

[0081] The invention thus also relates to a compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein is an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising at least 3 consecutive guanine residues;

[0082] Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and - CH2-;

[0083] Z is O or S;

[0084] R1and R2are independently selected from the group consisting of H, halo, OH and Cl- C12-alkyl;

[0085] B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;

[0086] L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H; or a tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I, wherein each of the at least 3 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding; for use in the treatment of cancer.

[0087] Particular compounds for use of Formula I, or pharmaceutically acceptable salts or solvates thereof, are those wherein one or more of , Y, Z, R1, R2, B, L1and L2are defined as follows: is an oligonucleotide comprising, in particular consisting of, a sequence from 3 to 50 nucleotides, said sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues; in particular is an oligonucleotide comprising, in particular consisting of, a sequence from 3 to 40 nucleotides, for example from 3 to 35 nucleotides, from 3 to 30, from 3 to 25, from 3 to 15, from 3 to 9, said sequence comprising at least 3 consecutive guanine residues, in particular f more than 2 and less than 4 consecutive guanine residues; more particularly - ' is a sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’), SEQ ID NO: 2 (5’-TGGGAGT-3’) and SEQ ID NO: 3 (5’-AGGGCGGTGTGGGAAGAGGGA-3’); still more particularly is SEQ ID NO: 1 (5’-TGGGAG-3’) or SEQ ID NO: 2

[0088] (5’-TGGGAGT-3’);

[0089] Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and -CH2- ; in particular Y is a divalent linker moiety selected from the group consisting of -O-, -S-, and -CH2-; more particularly Y is -O- or -S-; even more particularly Y is -O-;

[0090] Z is O or S; in particular Z is S;

[0091] R1and R2are independently selected from the group consisting of H, halo, OH and C1-C12- alkyl; in particular R1and R2are independently selected from the group consisting of H, halo and Cl-C6-alkyl; more particularly R1and R2are independently selected from the group consisting of H, F, Cl, Br and Cl-C4-alkyl; even more particularly R1and R2are independently selected from the group consisting of H, F, Cl and Cl-C2-alkyl; still more particularly R1and R2are independently selected from the group consisting of H, F, Cl and methyl; in a particular example R1and R2are H;

[0092] B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms; in particular B is a unsubstituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6-methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5- methylcytosine, 5-hydroxymethylcytosine and hypoxanthine; more particularly B is a unsubstituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine; still more particularly B is uracil; L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; in particular L1and L2are independently selected from H and C6-C20-alkyl; more particularly L1and L2are independently selected from H and C10-C20 alkyl; still more particularly L1and L2are C12- C18-alkyl; even more particularly L1and L2are n-pentadecyl; with the proviso that L1and L2are not both H.

[0093] In one embodiment, the compounds for use of Formula I are those wherein L1and L2are C6-C20-alkyl; in particular L1and L2are C10-C20 alkyl; more particularly L1and L2are C12-C18-alkyl; still more particularly L1and L2are n-pentadecyl.

[0094] In one embodiment, the compounds for use of Formula I are those wherein B is uracil.

[0095] In one embodiment, the compounds for use of Formula I are those wherein L1and L2are n- pentadecyl and B is uracil.

[0096] In one embodiment, the compounds for use of Formula I are those wherein - ' is a

[0097] G-quadruplex forming sequence.

[0098] [

[0099] In one embodiment, the compounds for use of Formula I are those wherein ' - - ' is sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’), SEQ ID NO: 2 (5’-TGGGAGT-3’) and SEQ ID NO: 3 (5’-AGGGCGGTGTGGGAAGAGGGA-3’).

[0100] In one embodiment, the compounds for use of Formula I are those wherein is a sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGGGAG-3’) and SEQ ID NO: 2 (5’-TGGGAGT-3’).

[0101] In one embodiment, the compounds for use of Formula I are those wherein ' - - - ' is

[0102] SEQ ID NO: 1 (5’-TGGGAG-3’).

[0103] In one embodiment, the compounds for use of Formula I are those wherein ' - - - ' is

[0104] SEQ ID NO: 2 (5’-TGGGAGT-3’).

[0105] Particularly preferred compounds for use of Formula I are those listed hereafter:

[0106] In one embodiment, the invention relates to a compound of Formula I as defined above, for use in the treatment of cancer.

[0107] As the compound for use of the invention comprises a nucleotide sequence comprising at least 3 consecutive guanine residues, in particular more than 2 and less than 4 consecutive guanine residues, the compound for use of the invention can self-assemble into four stranded G4 structures stabilized by 7t-7t stacking between G-quartets and via Hoogsteen hydrogen bonding.

[0108] Thus, in one embodiment, the invention relates to a tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I as defined above, wherein each of the at least 3 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding, for use in the treatment of cancer. In one embodiment, the tetramolecular parallel G-quadruplex for use of the invention further comprises a cation, which coordinates said G-quartets.

[0109] Said cation may be a monovalent or a divalent cation. Examples of suitable cations include K+and Mg2+. In a particular embodiment, said cation is a divalent cation, more particularly a Mg2+cation.

[0110] In one embodiment, the cancer may be a cancer involving mutations in a gene selected from the group consisting of c-MYC, c-MYB, VEGF, VEGFR-2, BCL-2, c-KIT, KRAS, HRAS, PDGFR-P, MAPK12, NEAT1, EGFR and telomeric regions.

[0111] In particular, the cancer is selected from the group consisting of breast cancer, colon cancer, colorectal cancer, cervical cancer, leukemia, ovarian cancer, in particular ovarian epithelial carcinoma, pancreatic cancer, in particular pancreatic adenocarcinoma, gastrointestinal stromal tumors, melanoma, lung cancer, head and neck squamous cell carcinoma and bladder cancer.

[0112] In one embodiment, the cancer may be a cancer involving mutations in the KRAS gene and / or in the c-MYC gene.

[0113] In particular, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, breast cancer and cervical cancer.

[0114] In one embodiment, the cancer may be a cancer involving mutations in the KRAS gene.

[0115] In particular, the cancer is selected from the group consisting of pancreatic cancer, lung cancer and colon cancer.

[0116] In one embodiment, the cancer may be a cancer involving mutations in t the c-MYC gene.

[0117] In particular, the cancer is selected from the group consisting of breast cancer, colon cancer and cervical cancer.

[0118] In other terms, the invention also relates to a method of treating cancer, comprising the administration of a therapeutically effective amount of a compound of Formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof, to a patient in need of such treatment. Preferably the patient is a warm-blooded animal, more preferably a human. In particular, the cancer is as defined above.

[0119] The invention further provides the use of a compound of Formula I as defined above, or a pharmaceutically acceptable salt or solvates thereof, for the manufacture of a medicament for use in treating cancer. Preferably the patient is a warm-blooded animal, more preferably a human. In particular, the cancer is as defined above.

[0120] According to the present invention, the compound of the invention or the compound for use according to the invention may be administered as a pharmaceutical formulation in a therapeutically effective amount by any of the accepted modes of administration, preferably by intravenous or oral route.

[0121] Therapeutically effective amount ranges are typically from 0.1 to 50 000 pg / kg of body weight daily, preferably from 1 000 to 40 000 pg / kg of body weight daily, depending upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound, the route and the form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able in reliance upon personal knowledge, to ascertain a therapeutically effective amount of the anticancer agent of the present invention for a given cancer.

[0122] According to one embodiment, the compounds of the invention, or their pharmaceutical acceptable salts, may be administered as part of a combination therapy. Thus, are included within the scope of the present invention embodiments comprising co-administration of, and compositions and medicaments which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and / or active ingredients. Such multiple drug regimens, often referred to as combination therapy, may be used in the treatment of cancer, particularly those defined above.

[0123] Thus, the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of the invention, or their pharmaceutical acceptable salts thereof, in the form of monotherapy, but said methods and compositions may also be used in the form of multiple therapy in which one or more compounds of the invention or their pharmaceutically acceptable salts or solvates are co-administered in combination with one or more other therapeutic agents.

[0124] In one embodiment, the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of the present invention, or their pharmaceutical acceptable salts thereof, in combination with radiation therapy. According to this embodiment, the compounds of the invention, their pharmaceutical acceptable salts or solvates may be administered in combination with radiation therapy. Thus, there is provided a compound of Formula I and any of its embodiments, or any of its subformulae as defined above, for use in the treatment of cancers as defined above in combination with radiation therapy. Such radiation therapies include, but are not limited to, external beam radiation therapy, brachytherapy and systemic radioisotope therapy.

[0125] The invention also provides a pharmaceutical composition comprising a compound of the present invention, preferably a compound of Formula I and any of its embodiments, or any of its subformulae as defined above, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant. As indicated above, the invention also covers pharmaceutical compositions which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and / or active ingredients.

[0126] The invention also provides a compound of the invention, in particular a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in a therapeutic treatment in humans or animals, in particular in humans.

[0127] Another object of this invention is a medicament comprising at least one compound of the invention, or a pharmaceutically acceptable salt thereof, as active ingredient.

[0128] Generally, for pharmaceutical use, the compounds of the invention may be formulated as a pharmaceutical preparation comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant, and optionally one or more further pharmaceutically active compounds.

[0129] By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including ocular), cerebral administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington’s Pharmaceutical Sciences.

[0130] For example, the compound of the invention or a pharmaceutical composition comprising a compound of the invention can be administered orally in the form of tablets, coated tablets, pills, capsules, soft gelatin capsules, oral powders, granules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

[0131] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, a disintegrant such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, a binder such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia, a lubricant such as magnesium stearate, stearic acid, glyceryl behenate. Solid compositions of a similar type may also be employed as fillers in hard gelatin capsules. Preferred excipients in this regard include lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives or gelatin. Hard gelatin capsules may contain granules of the compound of the invention.

[0132] Soft gelatin capsules may be prepared with capsules containing the compound of the invention, vegetable oil, waxes, fat, or other suitable vehicle for soft gelatin capsules. As an example, the acceptable vehicle can be an oleaginous vehicle, such as a long chain triglyceride vegetable oil (e.g. corn oil).

[0133] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water may contain the active ingredient in a mixture with dispersing agents, wetting agents, and suspending agents and one or more preservatives. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0134] Liquid dosage forms for oral administration may include pharmaceutically acceptable, solutions, emulsions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or an oleaginous vehicle. Liquid dosage form may be presented as a dry product for constitution with water or other suitable vehicle before use. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, complexing agents such as 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin, and sweetening, flavoring, perfuming agents, coloring matter or dyes with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. These compositions may be preserved by the addition of an antioxidant such as butylated hydroxyanisol or alpha-tocopherol.

[0135] Finely divided powder of the compound of the invention may be prepared for example by micronisation or by processes known in the art. The compound of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types.

[0136] If the compound of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and / or by using infusion techniques.

[0137] The compound of the invention can be administered via the parenteral route with a readily available or a depot-type formulation.

[0138] The pharmaceutical compositions for the parenteral administration of a readily available formulation may be in the form of a sterile injectable aqueous or oleagenous solution or suspension in a non-toxic parenterally-acceptable diluent or solvent and may contain formulatory agents such as suspending, stabilizing dispersing, wetting and / or complexing agents such as cyclodextrin e.g. 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta- cyclodextrin.

[0139] The depot-type formulation for the parenteral administration may be prepared by conventional techniques with pharmaceutically acceptable excipient including without being limited to, biocompatible and biodegradable polymers (e.g. poly(P-caprolactone), polyethylene oxide), poly(gly colic acid), poly [(lactic acid)-co-(gly colic acid)...)], poly(lactic acid)...), non-biodegradable polymers (e.g. ethylene vinylacetate copolymer, polyurethane, polyester(amide), polyvinyl chloride...) aqueous and non-aqueous vehicles (e.g. water, sesame oil, cottonseed oil, soybean oil, castor oil, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils, propylene glycol, DMSO, THF, 2-pyrrolidone, N- methylpyrrolidinone, N-vinylpyrrolidinone... ).

[0140] Alternatively, the active ingredient may be in dry form such as a powder, crystalline or freeze-dried solid for constitution with a suitable vehicle. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0141] As indicated, the compound of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, (for example from Ineos Fluor), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch. For compositions suitable and / or adapted for inhaled administration, it is preferred that the compound or salt of formula I is in a particle-size-reduced form, and more preferably the size-reduced form is obtained or obtainable by micronisation. The preferable particle size of the size-reduced (e.g. micronized) compound or salt or solvate is defined by a D50 value of about 0.5 to about 50 microns (for example as measured using laser diffraction).

[0142] Alternatively, the compound of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compound of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. It may also be administered by the ocular route. For ophthalmic use, the compound can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, it may be formulated in an ointment such as petrolatum. For topical application to the skin, the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0143] DEFINITIONS

[0144] The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims.

[0145] Unless otherwise stated, any reference to compounds of the invention herein, means the compounds as such as well as their pharmaceutically acceptable salts and solvates.

[0146] When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise.

[0147] The term “alkyl” by itself or as part of another substituent refers to a hydrocarbyl group of Formula CnFhn+i wherein n is a number greater than or equal to 1. Alkyl groups may thus comprise 1 or more carbon atoms and generally, according to this invention comprise from 1 to 22, more preferably from 6 to 20 carbon atoms, still more preferably from 10 to 20 carbon atoms, and even more preferably from 12 to 18 carbon atoms. Alkyl groups within the meaning of the invention may be linear or branched. Examples of alkyl groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tertbutyl, n-pentyl, neopentyl, isopentyl, sec-pentyl, tert-pentyl, n-hexyl, neohexyl, isohexyl, sec-hexyl, tert-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n- pentadecyl, n-octadecyl. Particular examples of alkyl groups in the context of the invention include n-pentadecyl and n-octadecyl.

[0148] The compounds of the invention containing a basic functional group may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of the invention containing one or more basic functional groups include in particular the acid addition salts thereof. Suitable acid addition salts are formed from acids which form nontoxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bi sulphate / sulphate, borate, camsylate, cinnamate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

[0149] Pharmaceutically acceptable salts of compounds of Formula I and subformulae may for example be prepared as follows:

[0150] (i) reacting the compound of Formula I or any of its subformulae with the desired acid; or

[0151] (ii) converting one salt of the compound of Formula I or any of its subformulae to another by reaction with an appropriate acid or by means of a suitable ion exchange column.

[0152] All these reactions are typically carried out in solution. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost non-ionized.

[0153] The term “solvate” is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term “hydrate” is employed when said solvent is water.

[0154] The compounds of the invention include compounds of the invention as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) and isotopically-labelled compounds of the invention.

[0155] In addition, although generally, with respect to the salts of the compounds of the invention, pharmaceutically acceptable salts are preferred, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts, which may for example be used in the isolation and / or purification of the compounds of the invention. For example, salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of the invention.

[0156] The term “patient” refers to a warm-blooded animal, more preferably a human, who / which is awaiting or receiving medical care or is or will be the object of a medical procedure.

[0157] The term “human” refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.

[0158] The term “cancer” as used herein refers to the physiological condition in subjects that is characterized by unregulated or dysregulated cell growth with the potential to invade or spread to other parts of the body. The term “cancer” includes solid tumors and blood born tumors, whether malignant or benign.

[0159] Examples of cancer include, but are not limited to:

[0160] Acinar adenocarcinoma, acinar carcinoma, acral-lentiginous melanoma, actinic keratosis, adenocarcinoma, adenocystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenal rest tumor, adrenocortical carcinoma, aldosterone secreting carcinoma, alveolar soft part sarcoma, amelanotic melanoma, ameloblastic thyroid carcinoma, angiosarcoma, apocrine carcinoma, Askin’s tumor, astrocytoma, basal cell carcinoma, basaloid carcinoma, basosquamous cell carcinoma, biliary cancer, bladder cancer, bone cancer, bone marrow cancer, botryoid sarcoma, brain cancer, breast cancer, bronchioalveolar carcinoma, bronchogenic adenocarcinoma, bronchogenic carcinoma, carcinoma ex pleomorphic adenoma, cervical cancer, chloroma, cholangiocellular carcinoma, chondrosarcoma, choriocarcinoma, choroid plexus carcinoma, clear cell adenocarcinoma, colon cancer, colorectal cancer, comedocarcinoma, cortisol-producing carcinoma, cylindrical cell carcinoma, dedifferentiated liposarcoma, ductal adenocarcinoma of the prostate, ductal carcinoma, ductal carcinoma in situ, duodenal cancer, eccrine carcinoma, embryonal carcinoma, endometrial carcinoma, endometrial stromal carcinoma, epithelioid sarcoma, esophageal cancer, Ewing’s sarcoma, exophytic carcinoma, fibroblastic sarcoma, fibrocarcinoma, fibrolamellar carcinoma, fibrosarcoma, follicular thyroid carcinoma, gallbladder cancer, gastric adenocarcinoma, gastrointestinal stromal tumours, giant cell carcinoma, giant cell sarcoma, giant cell tumor of bone, glioma, glioblastoma or glioblastoma multiforme, granulose cell carcinoma, head & neck cancer, hemangioma, hemangiosarcoma, hepatoblastoma, hepatocellular carcinoma, Hurthle cell carcinoma, ileal cancer, infiltrating lobular carcinoma, inflammatory carcinoma of the breast, intraductal carcinoma, intraepidermal carcinoma, jejuna cancer, Kaposi’s sarcoma, Krukenberg’s tumor, Kulchitsky cell carcinoma, Kupffer cell sarcoma, large cell carcinoma, larynx cancer, lentigo maligna melanoma, leukaemia, liposarcoma, liver cancer, lobular carcinoma, lobular carcinoma in situ, lung cancer, lymphoepithelioma, lymphoepithelioma, lymphosarcoma, malignant melanoma, medullary carcinoma, medullary thyroid carcinoma, medulloblastoma, meningeal carcinoma, Merkel cell carcinoma, micropapillary carcinoma, mixed cell sarcoma, mucinous carcinoma, mucoepidermoid carcinoma, mucosal melanoma, myxoid liposarcoma, myxosarcoma, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, nodular melanoma, non-clear cell renal cancer, non-small cell lung cancer, oat cell carcinoma, ocular melanoma, oral cancer, osteoid carcinoma, osteosarcoma, ovarian cancer, Paget’ s carcinoma, pancreatic cancer, pancreatoblastoma, papillary adenocarcinoma, papillary carcinoma, papillary thyroid carcinoma, pelvic cancer, periampullary carcinoma, phyllodes tumor, pituitary cancer, pleomorphic liposarcoma, pleuropulmonary blastoma, primary intraosseous carcinoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, round cell liposarcoma, scar cancer, schistosomal bladder cancer, Schneiderian carcinoma, sebaceous carcinoma, signet-ring cell carcinoma, skin cancer, small cell lung cancer, small cell osteosarcoma, soft tissue sarcoma, splindle cell carcinoma, spindle cell sarcoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, synovial sarcoma, telangiectatic sarcoma, terminal duct carcinoma, testicular cancer, thyroid cancer, transitional cell carcinoma, tubular carcinoma, tumorigenic melanoma, undifferentiated carcinoma, urachal adenocarcinoma, urinary bladder cancer, uterine cancer, uterine corpus carcinoma, uveal melanoma, vaginal cancer, cerrucous carcinoma, villous carcinoma, well-differentiated liposarcoma, Wilm’s tubor or yolk sac tumor. Particular examples of cancer according to the invention include cancer, colon cancer, colorectal cancer, cervical cancer, leukemia, ovarian cancer, pancreatic cancer, gastrointestinal stromal tumors, melanoma, lung cancer, head and neck squamous cell carcinoma and bladder cancer. The present invention will be better understood with reference to the following examples and figures. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

[0161] FIGURES

[0162] Figure 1: DLS determined nano-object formation of lipid-conjugated oligonucleotides in physiological saline conditions. Light grey and dark grey graphs represent duplicates. (A) Lipid conjugated G4-2 oligonucleotides assemble into supramolecular structures that have a diameter of 223.9 nm and 26.02 nm in intracellular (upper graph) and extracellular conditions respectively. (B) Lipid G4-3 oligonucleotides form nano-objects with a diameter of 13.37 nm and 15.44 nm. (C) The native G quadruplex mimicking KRAS promoter sequence conjugated to lipid forms nano structures with a diameter of approximately 20 nm in both intracellular and extracellular buffers. (D) Agarose gel showing the resolved nanoobjects resulting from self-assembly of LONs.

[0163] Figure 2: Determination of G quadruplex formation. (A) Thioflavin T assay. Enhanced fluorescence of Thioflavin T is observed in the presence of g quadruplexes under bpth saline conditions. The threshold for G quadruplex formation is set at 1000 RFUs after normalization using the sequence length. (B) Ellipticities of unconjugated and lipid- modified G4 sequences under intracellular and extracellular conditions as determined by circular dichroism. Lipid conjugated G quadruplexes have a high ellipticity around 265 nm a characteristic of parallel G quadruplex structure. The topology is more stable under intracellular conditions due to the high concentration of K+ions.

[0164] Figure 3: Characterization of G4 formation and interaction with a G quadruplex binding protein. (A) Determination of the fluorescence threshold for G4 formation using Thioflavin assay. (B) CD melting experiment, from 5 to 90°C, of G-quadruplexes under intracellular and extracellular saline conditions. Lipid-conjugated sequences are more stable in comparison to quadruplexes tend to more stable than the KRAS native sequence.

[0165] Figure 4: Eletrophoretic mobility shift assay. Oligonucleotides are resolved in the absence (-) and presence (+) of GST tagged UP1. G quadruplexes interact with the protein hence retarded on the gel. The G quadruplexes, together with their protein interaction are visualized with the help of Thioflavin T fluorescence enhancement. Figure 5: G quadruplex decoys treatments inhibit pancreatic cancer cells growth in a dose dependent manner at 1 pM, 5 pM and 10 pM oligonucleotide concentration respectively. (A) Inhibition of the growth of AsPCl cells, which contain a KRAS G12D mutation on both alleles. (B) The survival of BxPC3 cells which have a KRAS wildtype gene. (C) Inhibited proliferation of HPAF II cells with a KRAS G12D mutation on one allele.

[0166] Figure 6: (A) KRAS protein reduced levels in pancreatic cancer cells treated with lOpM of lipid- conjugated oligonucleotides. Lipid G4-2 has the highest impact on KRAS downregulation in comparison to the native KRAS sequence and other oligonucleotides. (B) Intracellular MYC downregulation when the cells were treated with the mentioned oligonucleotides, with the highest knockdown level was observed with Lipid G4-3.

[0167] Figure 7: Overview of the G4 structures. (A) Drawing showing a tetramol ecular parallel G4 oligonucleotides formed by the sequences G4-2 and G4-3 formed in intracellular conditions. (B) Ketal Nucleolipid conjugates (LG4-2 and LG4-3) stabilizing the tetramolecular parallel G4 architecture observed in both extra and intracellular conditions. (C) Illustration of the micellar assemblies featuring a hydrophobic core (brown) surrounded by the parallel G4. These assemblies result from the self-aggregation of the tetramolecular lipid parallel G4 (LG4-2 and LG4-3) in both extra and intracellular conditions. (D) Chemical structure of the Ketal Nucleolipid (KNL) moiety inserted at the 5’ extremities via a 5’ -5’ PS linkage.

[0168] Figure 8: G4s decoys treatments prevent KRAS, cMYC and cKIT oncogenes expression in PDAC cells. (A) and (C) Representative images of western blots showing oncogenes protein levels in HPAFII (A) and AsPC-1 (C) PDAC cell lines after indicated treatments for 72 hours at 10 pM (CTRL = Control G4 Oligonucleotide). Actin was used as the house keeping gene. (B) and (D) Quantification of the western blots performed on 3 independent experiments, using Imaged software.

[0169] Figure 9: G quadruplex decoy treatments inhibit PDAC cells growth. (A) and (C) Inhibition of HPAF II (A) and AsPCl (C) cell growth when treated with the lipid-modified G4 oligonucleotides and controls at the concentration of 10 pM for 72h. (B) and (D) Representative western blot images reflecting Ki67 and BCL2 protein levels in HPAFII (B) and AsPCl (D) cells, treated with the indicated conditions at 10 pM. All the experiments were performed in independent triplicate.

[0170] Figure 10: NLG4-based treatments induce the reduction of BxPC3 PDAC cell survival, migration and growth with no effect on normal cells. (A) and (B) PDAC BxPC3 cell line (A) and normal human bronchial epithelium BEAS-2B cell line (B) survival after the administration of lipid-modified G4 oligonucleotides and controls (NT = Non Treated and CTRL= scrambled oligonucleotide) at the concentration of 10 pM for 72h. (C) BxPC3 cells migration capacities were evaluated using transwells, 72h after indicated treatments. Representative images of migrated cells stained with crystal violet are showed in the first line (n=3 transwells per treatment). For quantification, crystal violet was solubilized in ethanol and the absorbance at 555 nm was measured. (D) Spheroids diameters were measured 2 weeks after pre-treated BxPC3 cells were embedded in Matrigel™, using the Optika PRO View analysis software. Representative images of the spheroids are showed in the first line (scale bar = 100 pm). The quantification was performed on n=6 / 7 spheroids per conditions. (E) A viability assay was performed on BxPC3 cells treated with G4 oligonucleotides and controls (NT and CTRL) at 10 pM in combination (+GEM) or not (- GEM) with gemcitabine (GEM) chemotherapy at 5 pM for 3 days. (F) GEM-resistant BxPC3 cell line was treated with G4 oligonucleotide decoys and control conditions at 10 pM for 72h and the viability of the cells was measured using CellTiter Blue reagent. Statistically significant differences between the treatments were determined by analysis of variance (one-way ANOVA). p-values of Tukey's post-hoc analyses are presented. *P<0.05, **P<0.01 and ***P<0.001.

[0171] Figure 11: NLG4 treatments reduce critical oncogenic signaling pathway players in PDAC cells, modulating migration and spheroids growth abilities. (A) and (D) Representative western blots showing NF-kB, Akt / p-Akt and ERK1 / 2 protein levels in HPAFII (A) and AsPCl (D) cell lines, 72h after the indicated treatments. Western blot quantifications are represented with bar graphs following ImageJ software analyses in three independent experiments. (B) and (E) PDAC cells migration capacities were evaluated using transwells, 72h after the administration of the indicated treatments (NT= Non Treated cells). Representative images of HPAFII (B) and AsPCl (E) migrated cells stained with crystal violet are showed in the first line (n=3 transwells per treatment). For quantification (graph bars shown in second line), crystal violet was solubilized in ethanol and the absorbance at 555 nm was measured. (C) and (F) Spheroids diameters were measured 2 weeks after HPAFII (C) and AsPCl (F) cells were embedded in Matrigel™, using the Optika PRO View analysis software. Representative images of the spheroids are showed in the first line (scale bar = 100 pm). The quantification was performed on n=6 / 7 spheroids per conditions. Statistically significant differences between the treatments were determined by analysis of variance (one-way ANOVA). p-values of Tukey's post-hoc analyses are presented. *P<0.05, **P<0.01 and ***P<0.001.

[0172] Figure 12: G4 oligonucleotides conjugated with NLs treatments enable the sensitivity restoration of PDAC ells to chemotherapy. (A) and (B) Viability assays were performed on HPAFII (A) and AsPCl (B) cells treated with G4 oligonucleotides and controls (NT and CTRL) at 10 pM in combination (+GEM) or not (-GEM) with gemcitabine (GEM) chemotherapy at 5 pM for 3 days. (C) and (D) Gemcitabine (GEM) resistant PDAC cell lines were established by intermittent exposure to the chemotherapy. HPAFII (C) and AsPCl (D) GEM-resistant cell lines were treated with G4 oligonucleotide decoys and control conditions at 10 pM for 72h and the viability of the cells was measured using CellTiter Blue reagent. Statistically significant differences between the treatments were determined by analysis of variance (one-way ANOVA). p-values of Tukey's post-hoc analyses are presented. **P<0.01 and ***P<0.001.

[0173] EXAMPLES

[0174] ABBREVIATIONS

[0175] ACN: acetonitrile;

[0176] DMT or DMTr: 4,4'-dimethoxytrityl;

[0177] DMTC1: 4,4'-dimethoxytrityl chloride; eq.: equivalent;

[0178] ESI: electrospray ionization;

[0179] HPLC: high-performance liquid chromatography; HRMS: high resolution mass spectrometry;

[0180] LON: lipid oligonucleotide;NMR: nuclear magnetic resonance;

[0181] ON: oligonucleotide; ppm: parts per million; PTO: phosphorothioate;

[0182] RT : room temperature;

[0183] TEA: triethylamine;

[0184] TEAA: triethyl ammonium acetate;

[0185] THF : tetrahydrofurane; UV: ultraviolet.

[0186] Materials and Methods

[0187] Synthesis, purification and dosage of oligonucleotides and Lipid conjugated antisense oligonucleotides

[0188] The following oligonucleotides were synthetized: Table 1 The oligonucleotide synthesis step was performed on an automated Expedite 8909 DNA synthesizer at the pmol scale on 1000 A primer support (loading: 30-100 pmol / g, Link technologies, Synbase Control Pore Glass).

[0189] The cycled synthesis consisted of 4 steps: detrityl ati on, coupling, oxidation and capping.

[0190] The coupling of a double-chain nucleolipid (ketal-bis-Cis-Uridine) was performed by the Phosphoramidite methodology at the 5’ end of PTO-Oligonucleotides.

[0191] Oligonucleotides’ purification

[0192] Chromatographic analysis of purity

[0193] All oligonucleotides synthesized were analysed by using High Performance Liquid Chromatography (HPLC) on Elite LaChrom (VWR) system with a Diode detector at 260 nm and injection volume of 20 pL during 15 minutes.

[0194] For non-lipidic oligonucleotides, hydrophobic column Xbridge oligonucleotide BEH Cis (Waters) with particles’ size of 2.5 pm, 130 A of porosity and 4.6 x 50 mm of geometry was used. The mobile phase with 2.8 mL / min flow used was 70% of 95% of tri ethyl ammonium acetate (TEAA) at 100 mM + 5% of Acetonitrile (ACN) at pH 7) and 30% of 20% of TEAA 20 mM and 80% of ACN.

[0195] For lipid modified oligonucleotides, Nucleosil C4 column with 4 x 250 mm geometry and particles size of 5 pm, 300 A of porosity (Macherey Nagel) was used with 1.0 mL / min of 20% of TEAA 20 mM and 80% of ACN as mobile phase. Purification for lipid oligonucleotides was performed using preparative HPLC method with column XBridge Protein BEH C4 OBD Pre with 30 x 50 mm of geometry, particles size of 5 pM and porosity of 300 A. The mobile phase used was 20% of TEAA 20 mM and 80% of ACN at 56.25 mL / min flow. The run of analysis was 4 minutes

[0196] Dialysis method

[0197] A dialysis system was used to desalt the purified samples. The columns of Vivaspin Turbo 4 (Sartorius, cut-off 3.5 kDa, membrane Polyethersulfone) were used for oligonucleotides’ desalting. Membranes were rinsed with distilled water and then samples were added into the column before being centrifuged at 3000 rpm in 30 min. Three washings were made by adding 2 mL of distilled water into the superior part of the tube and then re-centrifuged as previously. 500 pL of distilled water was added on the membrane to re-suspend oligonucleotide and collect it. Then the membrane was rinsed 3 times with 500 pL of distilled water to limit the waste.

[0198] Oligonucleotides assay

[0199] The concentration of all ASOs and LASOs was determined by spectrophotometry Nanodrop® (Thermo Scientific™) at 260 nm with automatic oligonucleotide detection mode.

[0200] Mass analysis

[0201] ESI mass spectrometry analysis were carried out on a Thermo Fisher Q-Exactive. Oligonucleotide samples were prepared using 3.5 kD vivacon membrane (Sartorius) for dialysis against 50 mM ammonium acetate (Sigma-Aldrich). Spectra show multi-charged ions (M-z) / z obtained in the negative mode. Measured experimental monoisotopic masses were determined using the value of the first peak of the isotope distribution.

[0202] Dynamic light scattering (DLS) characterization

[0203] The size of LON’s objects was measured at room temperature using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). Size was measured in a specific cell ZEN 0040 (Malvern, France) for NPs and Zeta Potential in a DTS 1070 cell (Malvern, France). Measurement conditions were: material Protein (RI: 1.450; Absorption: 0.001), dispersant water (Viscosity: 0.8872 cP; RI: 1.330) temperature at 25°C or 37°C and equilibration time was 120s. Each test was triplicated.

[0204] LONs’ size of micelles was first determined in previously described conditions extracellular salt (145 mM Na+CF and 5 mM K+CF) (B. Vialet et al., Chem. Commun. 2017, 53, 11560- 11563), first at 30 pM and then compared to our 5 pM relevant concentration.

[0205] Dynamic Light Scattering

[0206] The size of particles was determined using the Zetasizer 3000 HAS MALVERN. Samples at a final concentration of 50 pM were prepared under extracellular (Phosphate buffer Na+200 mM pH 7.2, 1.45 M NaCl) and intracellular (Phosphate buffer K+200 mM pH 7.2, 1.4 M KC1,12O mM NaCl, 10 mM MgCh) conditions respectively in IX buffer and salt concentration, denatured at 90°C for 5 min and left at room temperature. Measurements were taken at 25°C.

[0207] Thioflavin T assay

[0208] Samples at a final concentration of 10 pM were prepared under extracellular (Phosphate buffer Na+ 200 mM pH 7.2, 1.45 M NaCl) and intracellular (Phosphate buffer K+ 200 mM pH 7.2, 1.4 M KC1,12O mM NaCl, 10 mM MgCh) conditions respectively in IX buffer and salt concentration, denatured at 90°C for 5 min and left at room temperature two hours. 20 pL of the sample were mixed with 20 pL of 400 nM Thioflavin T in a black 96 well plate and fluorescence readings were taken at an excitation and emission wavelength of 440 nm and 490 nm respectively. Fluorescence intensity was normalized by sequence length and analysis was done using GraphPad Prism.

[0209] Circular Dichroism

[0210] CD spectra and CD-monitored melting curves were recorded on a JASCO 1500 spectrometer equipped with a Peltier-type temperature control system. CD spectra were recorded from 220 nm to 335 nm at 25°C with a data pitch of 0.2 nm, a bandwidth of 2 nm, a response of 0.5 s, and a scanning speed of 100 nm / min. The resulting spectra were the averages of three accumulations. The oligonucleotide samples were prepared with 10 mM phosphate buffer at 10 pM at different saline concentrations. The sample solutions were annealed for 5 min at 90°C, then kept at RT for 2h before analysis.

[0211] CD melting experiments were performed overnight from 5°C to 90°C. Melting curves were extracted at 265 nm. The holder was the control sensor, and the cell was the monitor sensor. A temperature gradient of 0.5°C / min and a data pitch of 0.5 nm were used. The scanning speed was 200 nm / min.

[0212] Electrophoretic Mobility Shift Assay (EMSA)

[0213] Oligonucleotide samples at 100 pM concentration, were prepared under intracellular saline conditions for G quadruplex formation. A volume of 3 pL of the oligonucleotide, 3 pL unfolding protein 1 tagged to GST (UP1+ GST tag) at 100 pM and 2.6 pL of 5 mM 1,4 Dithiothreitol (DTT) were added in a well-labeled Eppendorf tube and incubated at room temperature for 30 minutes. Loading dye at a volume of 2 pL was mixed with the contents and resolved on 1% agarose gel in 0.5X Tris Borate (TB) buffer, containing 10 pM of Thioflavin T, at 50 V for 1 hour 40 min in the dark. The gel was then imaged using ChemiDoc™ (Bio-Rad)

[0214] Cell Culture

[0215] HPAF II cells (ATCC) were maintained in Minimum Essential Media (Gibco) supplemented with 10% fetal bovine serum (FBS). BxPC3 and AsPCl cell lines were purchased at the ATCC (CRL-1687™ and CRL-1682™ respectively) and cultured in RPMI (Gibco). BEAS- 2B non cancerous cell line was kindly provided by Dr Jeanne Leblond-Chain (ARNA Lab, Bordeaux FR) and cultured in DMEM (Gibco) supplemented with 10% FBS. The cells were cultured at 37°C and 5% CO2 under humified conditions.

[0216] Cell Viability

[0217] HPAF II cells were seeded in 96-well plates at a density of 2000 cells / well in 100 pL of culture media. After 24 hours, the cells were incubated in serum-free media containing oligonucleotides at the final concentrations of 1 pM, 5 pM, and 10 pM (transfection media), for 6 hours. The transfection media was then replaced by culture media (200 pL final volume). Three days later, HPAF II cell viability was measured using CellTiter® Blue cell viability assay (Promega) following the manufacturer’s protocol. Briefly, 20 pL of CellTiter® Blue reagent was added to each well and incubated at 37°C for two hours. Fluorescent readings were measured (Xexc= 560 nm, L-m = 590 nm, 10 nm bandwidth).

[0218] HPAF II, AsPCl and BEAS-2B cells were seeded in 96-well plates at a density of 5000 cells / well in 100 pL of culture media. BxPC3 cell line was seeded at 2500 cells / well. After 24 hours, the cells were incubated in serum-free MEM, DMEM or RPMI culture media containing oligonucleotides at the final concentrations of 1 pM, 5 pM, and 10 pM (transfection media), for 4 hours. Culture media containing 20% FBS was then added to each well, at the volume of 100 pL. Three days later, cell viability was measured using CellTiter® Blue cell viability assay (Promega) following the manufacturer’s protocol. Briefly, 40 pL of CellTiter® Blue reagent was added to each well and incubated at 37°C for two hours. Fluorescent readings were measured (Xexc= 560 nm, L-m = 590 nm, 10 nm bandwidth). Western blot

[0219] HPAF II, AsPCl and BEAS-2B cells were plated at the density of 200,000 cells per 10 cm culture plates in 10 mL of culture media for 24 hours. The culture media was replaced with 5 mL of transfection media at a final concentration of 10 pM oligonucleotide for 4 hours. Culture media supplemented with 20% FBS was then added to each plate. After 3 days, cells were harvested and lysed using Pierce™ RIPA Buffer (Thermo Scientific) following the manufacturer’s instructions. Protein quantification was measured using the bicinchoninic acid protein assay kit (Thermo Scientific) and 10 pg of protein per lane were loaded on 15% SDS PAGE gel. After migration, the proteins were transferred to a PVDF membrane. Blocking of the membranes with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) was done for one hour at room temperature. The membrane was incubated with primary antibodies, for 1-2 hours at room temperature, followed with secondary antibody incubations for 1 hour. SuperSignal™ West Pico PLUS chemiluminescence western blot substrate (Thermo Scientific) was used to reveal the blot. The following antibodies, purchased from Invitrogen, were used: KRAS recombinant rabbit monoclonal antibody (11H35L14, Invitrogen), cMyc recombinant rabbit monoclonal antibody ( 9E10, Invitrogen), c-Kit recombinant rabbit monoclonal antibody (NH34LC14, Invitrogen), Akt rabbit monoclonal antibody (D6G4, Cell Signaling Technology), Bcl2 recombinant rabbit monoclonal antibody (JF 104-8, Invitrogen), Actin recombinant rabbit monoclonal (JF47-01), and goat anti -rabbit poly HRP.

[0220] Migration assays

[0221] Migration assays were performed using Thermo Scientific™ Nunc™ polycarbonate cell culture inserts (membrane pore size of 8 pm) in 24 well plates. After treatments with G4 oligonucleotides at 10 pM, 75 000 cells / well were plated onto the transwell upper chamber in 200 pL of serum free culture media and 500 pl of complete media were added onto the bottom chamber. After 24 hours, the cells were washed twice in PBS, and fixed in PBS + 4% of paraformaldehyde (PF A) for 15 minutes at RT. After 2 washing steps in PB S, the nonmigrated cells on the upper surface of the transwells were gently removed with cotton swabs. The migrated cells on the bottom surface of the transwells were stained with 0.2% (w / v) crystal violet for 5 minutes. Migrated cells were observed with a phase-contrast microscope. For the quantification, crystal violet coloration was solubilized in ethanol for 5 minutes, transferred in clean 96 well plates and the optical density was read at 555 nm using a plate reader (Biorad).

[0222] Spheroids culture

[0223] Three days after the transfection with lOuM of oligonucleotides, pancreatic tumor HPAFII, AsPCl and BxPC3 cells were harvested, pelleted and suspended in Matrigel™ (Corning) at a final concentration of 50 000 cells / mL. The mixture cells / Matrigel™ was transferred into 96 well plates (100 pL / well) and left at 37°C for 15 minutes to allow the gelation of the matrices. Then, 200 pL of culture media were added on each well and refreshed twice a week. Pictures of the spheroids were taken three weeks after the cells were embedded in Matrigel™, with an inverted microscope (Magnification 10X - Optika). Spheroid diameters were measured two weeks after the seeding, using the Optika PRO View analysis software.

[0224] Statistical analysis

[0225] Results were analyzed and represented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc). The one-way ANOVA followed by Tukey -Kramer post-hoc test was used to compare the means between the different conditions of culture. Results with / J< 0.05 were considered statistically significant and are indicated by *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

[0226] NMR spectroscopy

[0227] NMR experiments were recorded on a Bruker Advance III 700 spectrometer equipped with a liquid TXI 1H / 13C / 15N / 2H probe. Samples (3 mm NMR tubes) were prepared in Kpi buffer 10 mM K2HPO4 / KH2PO4; 50 mM KC1, at pH 6.8. D2O (8 %). The water signal was suppressed using excitation sculpting with gradients (zgesgppe; dl=2sec; 512 scans; time domain=64k). NMR data sets were treated using TopSpin 4.1.

[0228] Results

[0229] Oligonucleotides synthesis

[0230] The oligonucleotides were modified at the 5 '-end with a Ketal NucleoLipid phosphorami dites according to the above procedure. The synthesized oligonucleotides sequences are listed in Table 2. For each sequence KNL modified and non-modified sequences were compared. Oligonucleotide sequences (‘G4 control’) were also synthesized as controls wherein the sequences did not form G4 structures. All the oligonucleotides were purified by HPLC and later characterized by ESI mass spectrometry. Table 2

[0231] COMPARATIVE EXAMPLE 1 (G4-2)

[0232] 5'- TGG GAG -3'

[0233] Comparative Example 1 was synthesized according to the general procedure for the synthesis of oligonucleotides.

[0234] ESI-MS: Calculated: 1951.241; Found.: 1951.183.

[0235] EXAMPLE 1 (Lipid G4-2)

[0236]

[0237] Example 1 was synthesized according to the general procedure for the synthesis of oligonucleotides

[0238] ESI-MS: Calculated: 2705.713; Found.: 2705.631. COMPARATIVE EXAMPLE 2 (G4-3)

[0239] 5'- TGG GAG T -3'

[0240] Comparative Example 2 was synthesized according to the general procedure for the synthesis of oligonucleotides.

[0241] ESI-MS: Calculated: 2271.264; Found.: 2271.193. EXAMPLE 2 (G4-3)

[0242]

[0243] Example 2 was synthesized according to the general procedure for the synthesis of oligonucleotides.

[0244] ESLMS: Calculated: 3025.736; Found.: 3025.646. EXAMPLE 3 (Lipid KRAS21R) Example 3 was synthesized according to the general procedure for the synthesis of oligonucleotides.

[0245] ESI-MS: Calculated: 7416.641; Found.: 7416.442.

[0246] Characterization of micellar systems

[0247] The oligonucleotides sequences, including KRAS21R (Marquevielle, J. etal., Nucleic Acids Res. 2020, 48, 9336-9345) and Hotoda’s sequence, were synthetized with phosphorothioate (PTO) backbone (Table 2). To facilitate their internalization in cells, oligonucleotides were conjugated at the 5 ’-end with a Ketal NucleoLipid phosphoramidite (KNL). For each oligonucleotide, KNL modified and non-modified sequences were compared.

[0248] Lipid modified oligonucleotide G4 self-assemble in micelles and larger objects. The mean size of micellar population of different sequences measured by Dynamic Light Scattering (DLS), in both extracellular (Phosphate buffer Na+200 mM pH 7.2, 1.45 M NaCl) and intracellular (Phosphate buffer K+200 mM pH 7.2, 1.4 M KC1, 120 mM NaCl, 1 OmM MgCh) conditions, ranged around 13-26 nm in diameter (Figure 1) with negative zeta potential as expected regarding polyanion structure of oligonucleotides. When the oligonucleotides are resolved in agarose gel, the LONs with guanine-rich sequences form clear bands with a higher molecular weight than their non-lipidic counterparts (Figure 1). There is no significant difference between the bands of the short LONs under high concentrations of potassium (intracellular condition) and sodium ions (extracellular condition). However, a clear difference in molecular weight is observed with the lipid 21R, with a retarded band at high potassium ion concentration as opposed to the one under high levels of sodium ions.

[0249] Conjugation of lipids to guanine-rich sequences facilitates the formation of G quadruplexes that have a parallel topology and are thermal stable

[0250] Thioflavin T assay was used to determine the formation of G-quadruplexes (G4s) in both LONs and unconjugated guanine-rich sequences. Thioflavin T, a cationic benzothiazole, binds specifically to G-quadruplexes resulting in enhanced fluorescence, while a lower fluorescence increase is observed for single and double-stranded sequences. The fluorescence of the short unconjugated oligonucleotides was lower in contrast to the self- complementary non-G-quadruplex forming sequence used as a control. With the threshold of fluorescence intensity set at 1000, short guanine-rich LONs and the lipid KRAS 21R had a higher fluorescence at both intracellular and extracellular conditions confirming the formation of g quadruplexes (Figure 2A). The short LONs formed tetra-molecular G- quadruplexes that resulted in a more than 200-fold increase in fluorescence intensity, compared to the unconjugated sequences.

[0251] Circular dichroism, CD, was carried out to confirm the formation and topology of G- quadruplexes. The non-lipidic oligonucleotides had a positive band at 255 nm regardless of the saline conditions, with their lipid-conjugated counterparts having a negative band at 240 nm and a positive one at 260 nm, a characteristic of parallel G quadruplexes (Figure 2B). Hence, lipid conjugation resulted in the formation of G-quadruplexes, in a parallel conformation. The lipid KRAS 21R sequence had a similar peak profile to the short guaninerich LONs. CD melting experiments show that lipid conjugation enhances the thermal stability of the G quadruplexes formed under both intracellular and extracellular conditions as all LONs maintain their peak profile under the different temperatures (Figure 3).

[0252] ANMR study of the G4 formation, which is characterized by several imino peaks in the 10.5 to 12 ppm region, was observed with the unmodified and KNL modified G4s at physiological saline conditions. Despite strong spectral-widths for the imino peaks, all samples show the formation of G4 fingerprint. In view of the data collected, a G4 architecture is proposed and presented in Figure 7.

[0253] In order to determine the possible interactions of the G4 structures electrophoretic mobility shift assays were performed in either the presence or absence of the UP1 protein, a transcription cofactor. In this experiment the oligonucleotides are incubated in the absence (-) and presence (+) of GST tagged UP1. As shown in Figure 4, the Lipid G-quadruplexes interact with the UP1 protein hence retarded on the gel.

[0254] Growth inhibition of pancreatic cancer cells and oncogene knockdown was higher in lipid-modified oligonucleotides

[0255] The anticancer activity of the oligonucleotides was assessed based on the level of cell growth inhibition. At 10 pM, the tetra-molecular G quadruplexes formed by the short LONs have a higher cell -killing effect than the lipid 21R sequence (Figure 5). Compared to the control oligonucleotide, the LONs had a growth inhibition effect. The unconjugated oligonucleotides had low to almost no effect on the proliferation of AsPCl, BxPC3 and HPAF II cells. The inhibition of the cancer cell growth by the oligonucleotides was observed to be in a dose-dependent manner. As the targeted oncogene is a key player in a signaling cascade that results in cancer cells proliferation, differentiation, survival, and metabolic remodeling, we sought to determine whether the cell growth inhibition was due to the oncogene knockdown. The protein levels of the oncogene were determined using the western blot technique (Figure 6). As observed with the cell viability assay, protein knockdown was observed with the LON decoys compared to the unconjugated oligonucleotides. This observation could be attributed to the lipid conjugation that not only does it facilitate the formation of stable G-quadruplexes, but also the internalization of oligonucleotides into the cells. Of the LONs, the decoys formed by the shorter sequences had better activity than the lipid 21R decoy.

[0256] NLRG4 treatments decrease KRAS, cMYC, and cKit key oncogene levels in pancreatic cancer cells.

[0257] The efficacy of G4 decoy oligonucleotides of the invention to inhibit the transcription of the oncogenes displaying G4’s structures in their promoter regions like KRAS, cMYC and cKit was then evaluated. It was found that in the HPAFII and AsPC-1 PDAC cell lines, the G4s oligonucleotides enable the reduction in key oncogenes protein production when conjugated with lipids (LG4.2 and LG4.3; Figure 8). In HPAFII cells, we observed a 60% decrease in KRAS, cMYC and cKIT protein levels after 72h of LG4.2 treatment (Figure 8 A and B). LG4-3 treatments induced a 20% reduction in KRAS and cKIT production and no significant decrease in cMYC protein levels in HPAFII cell line (Figure 8 A and B). In AsPCl PDAC cells, LG4-2 treatment induced a 50%, 70% and 20% decrease in KRAS, cMYC and cKit protein levels respectively (Figure 8C and D). In this cell line, LG4.3 oligonucleotide displayed a better efficacy compared to the HPAFII cells with a reduction of 40%, 70% and 25% in the key oncogenes production (Figure 8 C and D).

[0258] Pancreatic cancer cells growth is reduced after lipid-conjugated tetra molecular G quadruplexes treatment

[0259] The impact of lipid-G4 oligonucleotides of the invention (LG4.2 and LG4.3) on PDAC cell viability and survival markers Ki67 and BCL2 levels was then evaluated. The viability of HPAFII, AsPCl and BxPC3 PDAC cell lines after G4 decoys treatment for 72h at 10 pM. The growth of the three cell lines was significantly reduced in LG4.2 and LG4.3 treated cells (Figure 9 A and C, Figure 10A). LG4.2 treatment induced a 35%, 50% and 45% inhibition of HPAFII, AsPCl and BxPC3 cells viability respectively. Significant decreased cell viability was also observed in PDAC cells after LG4.3 treatments illustrated by a 60%, 38% and 25% reduction in HPAFII, AsPCl and BxPC3 cell lines growth respectively (Figure 9 A and C; Figure 10A). Consistent with these observations, LG4.2 and LG4.3 treated PDAC cells expressed low levels of the cell proliferation Ki67 and anti-apoptotic BCL2 markers (Figure 9 B and D). Altogether, these results demonstrate that our G4 decoy strategy prevent the transcription and production of key oncogenes, well described to have pivotal role in PDAC progression and drug resistance, leading to the decrease in cancer cell viability and proliferation rates. Interestingly, no or very small impact of LG4.2 and LG4.3 treatments in BEAS-2B normal cell line growth was found (Figure 10B).

[0260] Key markers of canonical oncogenic signaling pathways are reduced in NLG4 treated PDAC cells, leading to the decrease of their migratory and proliferative capacities.

[0261] NF-kB, Ras / Raf / MEK / ERK and PI3K / Akt signaling are critical for the development and progression of PDAC tumors. The deregulation of these key oncogenic pathways is associated with poor prognosis and drug resistance in PDAC patients. Oncogenes activate these signaling pathways to promote malignant transformation, tumor cell growth, proliferation, migration and apoptosis resistance. The expression levels of key players involve in oncogenic pathways transduction was thus interrogated. In HPAFII cells, LG4-2 treatment reduced by half the levels of NFkB, phospho-Akt (pAkt) and ERK1 / 2 (Figure 11 A). Despite significant reduction in LG4-3 treated HPAFII cell viability, no important changes in oncogenes and signaling key effectors protein levels were observed (Figure 8A and 11 A). In AsPCl cells both NLG4 treatments, LG4-2 and LG4-3, downregulated NFkB, pAkt, Akt and ERK1 / 2 protein levels (Figure 11D). To further assess the impact of NLG4 treatments on PDAC cells migratory capacities and growth, migration assays and cultured HPAFII, AsPCl and BxPC3 cell lines in Matrigel™ were performed, 72h after the indicated treatments. G4 decoys conjugated with NLs, LG4-2 and LG4-3, significantly reduced HPAF II, AsPCl and BxPC3 cells migration in comparison to the controls (Figure 11 B and E, Figure 10C). The cell growth abilities of PDAC cells, treated with NLG4 and control oligonucleotides, were interrogated measuring the spheroid diameters 2 weeks after they were embedded in Matrigel™. The average spheroid diameters of lipid conjugated G4 oligonucleotides were significantly decreased in PDAC cells when compared to the nonconjugated G4 oligonucleotide counterparts (Figure 11 C and F; Figure 10D). This last result show that NLG4 based treatments impact on PDAC spheroid growth can last for up to 2 weeks.

[0262] NLG4 treatments restore PDAC cells sensitivity to gemcitabine-based chemotherapy.

[0263] Gemcitabine (GEM) is the standard-of-care chemotherapeutic agent to treat advanced / metastatic pancreatic cancer; however, its clinical benefits are limited due to resistance occurrence. As NLG4-based therapeutics inhibit signaling pathways associated with chemoresistance promotion, we evaluate their ability to restore the PDAC cells sensitivity to gemcitabine. HPAFII, AsPCl and BxPC-3 cell lines were first treated with G4 oligonucleotides in combination with gemcitabine at 5 pM. The administration of gemcitabine only displayed small effect on G4 controls treated HPAF II cells, with a 15 to 20% decrease in cell viability (Figure 12A). Interestingly, the efficacy of gemcitabine was significantly increased when combined with NLG4-based therapies, leading to additional reduction of 25 and 20% in cell viability following LG4.2 and LG4.3 treatments respectively (Figure 12 A). AsPCl cells were more sensitive to gemcitabine with a cell viability of 30% in the non-treated (NT) and scrambled oligonucleotide (CTRL) conditions (Figure 1 IB). However, the combination of G4 decoys with gemcitabine enhanced the chemotherapy cytotoxicity, notably when AsPCl cells were co-treated with LG4.2 and LG4.3 (Figure 12B). Same results were found in BxPC3 cell line where the combinatorial effect of NLG4- based treatments and gemcitabine led to important reduction of tumour cell viability (Figure 10E). The ability of G4 decoys to reduce PDAC cells viability in gemcitabine-resistant cell lines was then investigated. Chemoresistance to HPAFII, AsPCl and BxPC3 was induced by treating them with increased concentrations of gemcitabine for 3 months. Once the PDAC cells were growing under the chemotherapy pressure, they were treated with G4 oligonucleotides and controls to assess cell viability 72h after the indicated treatments. In HPAFII cells, both LG4.2 and LG4.3-based therapies restored gemcitabine efficacy by reducing significantly cell viability (Figure 12C). In AsPCl and BxPC3 cell lines, LG4.3 treatments enabled the restoration of gemcitabine sensitivity and LG4.2 had limited impact on cell viability (Figure 12D; Figure 10F).

Claims

CLAIMS1. A compound of F ormula I :I, or a pharmaceutically acceptable salt thereof; wherein' - ' is an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising more than 2 and less than 4 consecutive guanine residues;Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and -CH2-;Z is O or S;R1and R2are independently selected from the group consisting of H, halo, OH and Cl-C12-alkyl;B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H.

2. The compound according to claim 1, wherein Y is -O-.

3. The compound according to claim 1 or 2, wherein R1and R2are H.

4. The compound according to any one of claims 1 to 3, wherein L1and L2are independently selected from H and C6-C20-alkyl, in particular L1and L2are independently selected from H and C10-C20 alkyl, more particularly L1and L2are Cl 2- C18-alkyl, still more particularly L1and L2are n-pentadecyl.

5. The compound according to any one of claims 1 to 4, wherein B is uracil. f6. The compound according to any one of claims 1 to 5, wherein ' - ' is an oligonucleotide consisting of a sequence selected from the group consisting of SEQ ID NO: 1 (5’-TGG-GAG-3’), SEQ ID NO:2 (5’-TGG-GAG-T-3’) and SEQ ID NO:3 (5’- AGG-GCG-GTG-TGG-GAA-GAG-GGA-3 ’ ).

7. The compound according to claim 1, selected from the group consisting of:

8. A tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I as defined in any one of claims 1 to 7, wherein each of the more than 2 and less than 4 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding.

9. The tetramol ecul ar parallel G-quadruplex according to claim 8, wherein said tetramol ecular parallel G-quadruplex comprises a divalent cation which coordinates said G-quartets.

10. The tetramol ecul ar parallel G-quadruplex according to claim 9, wherein said divalent cation is Mg2+.

11. A pharmaceutical composition comprising a compound of Formula I according to any one of claims 1 to 7 or tetramolecular parallel G-quadruplexes according to any one of claims 8 to 10, wherein the tetramolecular parallel G-quadruplexes self-assembled into micelles, and at least one pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant.

12. The pharmaceutical composition according to claim 11, for use as a medicament.

13. A compound of Formula I:or a pharmaceutically acceptable salt thereof, whereinis an oligonucleotide sequence comprising from 3 to 50 nucleotides, said sequence comprising at least 3 consecutive guanine residues;Y is a divalent linker moiety selected from the group consisting of -O-, -S-, -NH- and -CH2-;Z is O or S;R1and R2are independently selected from the group consisting of H, halo, OH and Cl-C12-alkyl;B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;L1and L2are independently selected from H and a saturated or unsaturated, linear or branched, hydrocarbon chain comprising from 1 to 22 carbon atoms; with the proviso that L1and L2are not both H; or a tetramolecular parallel G-quadruplex comprising 4 identical compounds of Formula I, wherein each of the at least 3 consecutive guanine residues included in the sequence of each nucleotide respectively form G-quartets with the corresponding guanine residues of the other 3 oligonucleotides, said G-quarted being stabilized by 7t-7t stacking and Hoogsteen hydrogen bonding; for use in the treatment of cancer.

14. The compound or the tetramolecular parallel G-quadruplex for use according to claim 13, wherein the cancer is a cancer involving mutations in a gene selected from the group consisting of c-MYC, c-MYB, VEGF, VEGFR-2, BCL-2, c-KIT, KRAS, HRAS, PDGFR-P, MAPK12, NEAT1, EGFR and telomeric regions, preferably the cancer is a cancer involving mutations in the KRAS gene and / or in the c-MYC gene.

15. The compound or the tetramolecular parallel G-quadruplex for use according to claim 13 or 14, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, colorectal cancer, cervical cancer, leukemia, ovarian cancer, pancreatic cancer, gastrointestinal stromal tumors, melanoma, lung cancer, head and neck squamous cell carcinoma and bladder cancer.

Images