Microrna mimic for use in cancer treatment

Abstract

The invention relates to a microRNA mimic or a combination of microRNA mimics, for use in the prevention and / or treatment of cancer in a patient, wherein the microRNA mimic is selected from a mimic of miR323a, miR3127 and / or miR3161, alone or in combination.

Description

[0001] MicroRNA mimetic for use in cancer treatment

[0002] The invention relates to a microRNA mimetic or a combination of microRNA mimetics, for its use in the prevention and / or treatment of cancer in a patient.

[0003] STATE OF THE ART

[0004] MicroRNAs (or miRNAs) are small non-coding RNAs, typically composed of 18 to 25 nucleotides, that participate in the post-translational regulation of gene expression through RNA interference. They play a crucial role in regulating target genes by binding to complementary regions of mRNAs to repress their translation or regulate their degradation. Often, a single miRNA can target multiple mRNAs, and an mRNA can be regulated by multiple miRNAs targeting different 3' UTR regions. Once bound to an mRNA, the miRNA can modulate gene expression and protein production by affecting, for example, mRNA translation and stability. Each microRNA has the potential to regulate approximately 200 target genes. Therefore, microRNA-mediated gene regulation is now considered to play a significant role in biological processes.

[0005] During tumorigenesis, microRNAs frequently undergo aberrant expression. Used as biomarkers for the diagnosis and therapeutic monitoring of various cancer types, microRNAs can be used to induce the upregulation of targeted mRNAs. Another way to upregulate targeted mRNAs is through the use of synthetic molecules designed to mimic natural microRNAs. These are known as microRNA mimetics. Recently, further research has identified a combination of miR-17 and miR-340 microRNA mimetics for the prevention and / or treatment of glioblastomas (international patent application WO2023 / 052372). Despite progress in understanding and using microRNA mimetics in oncology, there remains a need for new combinations of microRNA mimetics for the prevention and / or treatment of cancers.

[0006] SUMMARY OF THE INVENTION

[0007] The inventors are now proposing specific microRNA mimetics, useful in oncology.

[0008] The invention thus provides a microRNA mimetic or a combination of microRNA mimetics, for use in the prevention and / or treatment of cancer in a patient, said microRNA mimetic being selected from a miR323a, miR3127 and / or miR3161 mimetic, alone or in combination.

[0009] More specifically, the cancer is chosen from pancreatic cancer, breast cancer, melanoma, liver cancer, brain tumor, preferably glioblastoma, lung cancer, preferably small cell lung cancer, even more preferably squamous cell carcinoma of the lung (SCC).

[0010] DESCRIPTION OF THE FIGURES

[0011] Figure 1 shows the viability of cancer cells transfected with miR-3127, miR-323a, and miR-3161 mimetics. The viability of cancer cells from: (A) melanoma (A475), (BC) breast cancer (MCF7 and MDA-MB-231), (DE) pancreatic cancer (PANC1 and MIA-PACA), and (F) lung cancer (HCC95) was assessed using the CellTiterFluor kit (Promega) 7 days after miRNA transfection. Data represent mean ± SEM (*p<0.05, **p<0.01, and ***p<0.001), ns = not significant (n=3-4).

[0012] Figure 2 shows the viability of cancer cells transfected with combinations of miRNA mimetics. The combination designated "Combo 1" corresponds to the combination of miRNA mimetics miR-30d, miR-3161, and miR-660. The combination designated "Combo 2" corresponds to the combination of miRNA mimetics miR-3127, miR-323a, and miR-708. The viability of cancer cells from: (A) melanoma (A475), (BC) breast cancer (MCF7 and MDA-MB-231), (DE) pancreatic cancer (PANC1 and MIA-PACA), (F) lung cancer (HCC95), and (G) fibroblast cells (HFF) was evaluated using the CellTiterFluor kit (Promega), 7 days after miRNA transfection. The data represent the mean ± SEM (*p<0.05, **p<0.01 and ***p<0.001), ns= not significant (n=3-4).

[0013] Figure 3 shows the migration of cancer cells transfected with miR-3127, miR-323a, and miR-3161 mimetics, alone or in combination. The migration of cancer cells from (A) lung cancer (HCC95), (B) breast cancer (MCF7), and (C) pancreatic cancer (MIA-PACA) was assessed using the scratch assay, 3 days after miRNA transfection. The data represent the results of a triplicate experiment.

[0014] Figure 4 shows the apoptosis of cancer cells transfected with miR-3127, miR-323a, and miR-3161 mimetics, alone or in combination. Apoptosis of cancer cells from (A) breast cancer (MCF7), (B) pancreatic cancer (MIA-PACA), and (C) lung cancer (HCC95) was assessed by flow cytometry using Annexin V and propidium iodide staining (Life Technologies), 3 days after miRNA transfection. Data represent mean ± SEM (*p<0.05, **p<0.01, and ***p<0.001), ns = not significant. (n = 3–4).

[0015] Figure 5 shows the modulation of gene expression involved in different cellular processes. Gene expression was assessed by quantitative RT-PCR in cancer cells from (A) breast cancer (MCF7), (B) pancreatic cancer (MIA-PACA), and (C) lung cancer (HCC95), 3 days after miRNA transfection. Data represent mean ± SEM (*p<0.05, **p<0.01, and ***p<0.001), ns = not significant. (n = 3–4).

[0016] Figure 6 shows the in vivo tumor growth of tumor cells implanted in the flank of immunodeficient (nude) mice. After implantation of tumor cells from (A and D) pancreatic cancer (MIA-PACA), (BC) lung cancer (HCC95), and (E) breast cancer (MDA-MB-231), tumor volume was measured weekly using calipers (n = 5–6 mice per group). The combination of miRNA mimetics miR-3161 and miR-323a is designated “Combo 3.” The combination of miRNA mimetics miR-3127 and miR-323a is designated “Combo 4.” The combination of miRNA mimetics miR-3161, miR-323a, and miR-3127 is designated “Combo.”

[0017] Figure 7 shows the in vivo tumor growth of tumor cells implanted in the flank of immunodeficient (nude) mice. After implantation of tumor cells from (A) lung cancer (HCC95), (B) pancreatic cancer (MIA-PACA), and (C) breast cancer (MDA-MB-231), tumor volume was measured weekly using calipers (n = 7–8 mice per group). The combination of miRNA mimetics miR-3161, miR-323a, and miR-3127 is designated as “Combo.”

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] Definitions

[0020] MicroRNAs are involved in the post-transcriptional regulatory pathways of gene expression. MiRNAs are encoded by the genome and then transcribed into a stem-loop precursor called pri-miRNA. RNase III enzymes, such as DROSHA and DICER, act successively to cleave the pri-miRNA and then the pre-miRNA into a small, single-stranded fragment approximately 22 nucleotides long, which corresponds to the mature and active form of the microRNA that regulates the expression of target genes.

[0021] The "microRNAs" (also referred to herein as "miRNA" or "miR" interchangeably) used in the context of the present invention are single-stranded non-coding RNAs (generally containing 18 to 25 nucleotides).

[0022] As used here, the term “miRNA mimetic” (also referred to interchangeably as “mimetic” and “miR mimetic”) designates an RNA, such as a single-stranded RNA, a double-stranded RNA, or a hairpin RNA, that mimics an endogenous miRNA and enables the upregulation of the miRNA’s activity. The miRNA mimetic is further capable of augmenting or replacing the intracellular function of the miRNA by counteracting the expression of gene products whose expression is downregulated by the mimetic miRNA. The terms “prevent” or “prevention” here refer to the prevention of the onset, recurrence, or spread of a disease or disorder, or one or more of its symptoms.

[0023] The terms "treat" or "treatment," as used here, refer to a therapeutic approach whose objective is to alleviate, reverse, or eliminate one or more symptoms of a disease or disorder. Beneficial or desired clinical outcomes include, but are not limited to, the elimination of symptoms, the alleviation of symptoms, the reduction in the extent of the disease or disorder, the stabilization (i.e., the absence of worsening) of the condition of the disease or disorder, and the delay or slowing of the progression of the disease or disorder. Preferably, this treatment leads to the complete elimination of cancer cells in the patient.

[0024] The terms "patient", "subject" or "individual" are used interchangeably to refer to a human or non-human animal, preferably a mammal, including male, female, adult and child, in need of treatment in which an anticancer effect is desired.

[0025] Cancers

[0026] As used here, the term "cancer" refers to the growth, division, or proliferation of abnormal cells in the body. It encompasses any type of malignant (i.e., non-benign) tumor. A malignant tumor can be a primary tumor or a secondary tumor (i.e., a metastasis).

[0027] In some embodiments, the cancer is chosen from digestive cancer, breast cancer, skin cancer, brain tumor, and lung cancer.

[0028] In one particular embodiment, the cancer is a digestive cancer selected from esophageal cancer, stomach cancer, colorectal cancer, liver cancer, gallbladder cancer, and pancreatic cancer, preferably liver or pancreatic cancer. In another particular embodiment, the cancer is a skin cancer, preferably melanoma. In yet another particular embodiment, the cancer is a brain tumor such as a glioma, selected from astrocytoma, oligodendroglioma, and ependymoma, preferably astrocytoma, and even more preferably glioblastoma.

[0029] In a particular embodiment, the cancer is lung cancer, preferably small cell lung cancer, even more preferably squamous cell carcinoma of the lung (SCLC).

[0030] MicroRNAs and microRNA mimetics

[0031] There is an international nomenclature for miRNAs where each endogenous miRNA is assigned a unique name with a predefined format, as follows:

[0032] For a mature miRNA: sss-miR-XY, where:

[0033] "sss" is a three-letter code indicating the species of the miRNA (e.g., "hsa" for the species Homo sapiens)

[0034] "X" is a unique alphabetic suffix assigned to the miRNA sequence in the species concerned, which may be followed by a letter if several highly homologous miRNAs are known. For example, "323a" and "323b" denote highly homologous mRNAs, also referred to as "sister miRNAs";

[0035] "Y" is a numerical suffix that indicates whether the mature miRNA, having been obtained by cleaving the pre-miRNA, corresponds to the 5' arm (Y is then "5p") or to the 3' arm (Y is then "3p") of said pre-miRNA.

[0036] The RNA sequences of the miRNAs described in the present invention are publicly available, for example on the miRBase database (https: / / www.mirbase.org / ) where each miRNA is assigned an access number to its sequence.

[0037] In some embodiments, the described miRNAs are human RNA sequences, i.e. hsa-miRs, as detailed in Table 1 below.

[0038]

[0039] Table 1: miRBase accession number and sequence of each miRNA

[0040] In some embodiments, each of the described miRNAs covers both the mature miRNAs corresponding to the 5' arm and the mature miRNAs corresponding to the 3' arm of the pre-miRNA, preferably the mature miRNAs corresponding to the 5' arm.

[0041] Preferably, the miRNAs used here are in the -5p form. In one particular embodiment, "miR-3127" refers to either miR-3127-5p or miR-3127-3p, preferably miR-3127-5p. The sequences are described below:

[0042] Table 2

[0043] In one particular embodiment, "miR-323a" refers to both miR-323a-5p and miR-323a-3p, preferably miR-323a-5p. The sequences are described below:

[0044] Table 3

[0045] In one particular embodiment, "miR-3161" refers to both miR-3161-5p and miR-3161-3p, preferably miR-3161-5p. The sequences are described below:

[0046] Table 4 In a particular embodiment, "miR-340" is understood to mean either miR-340-

[0047] 5p and miR-340-3p, preferably miR-340-5p. The sequences are described below:

[0048] Table 5. In a particular embodiment, "miR-708" refers to both miR-708-5p and miR-708-3p, preferably miR-708-5p. The sequences are described below:

[0049] Table 6

[0050] In one particular embodiment, "miR-660" refers to both miR-660-5p and miR-660-3p, preferably miR-660-5p. The sequences are described below:

[0051] Table 7

[0052] In one particular embodiment, "miR-30d" refers to both miR-30d-5p and miR-30d-3p, preferably miR-30d-5p. The sequences are described below:

[0053] Table 8

[0054] In the context of the present invention, the miRNA used in the prevention and / or treatment of cancer in a patient is a mimetic of said miRNA.

[0055] In a preferred embodiment, the mimetic is chosen from the group consisting of a miR-323a mimetic, a miR-3127 mimetic, a miR-3161 mimetic, a miR-340 mimetic, a miR-708 mimetic, a miR-660 mimetic, and a miR-30d mimetic. This means that the mimetic chosen from among a miR-323a mimetic, a miR-3127 mimetic, a miR-3161 mimetic, a miR-340 mimetic, a miR-708 mimetic, a miR-660 mimetic and a miR-30d mimetic will have the same cellular function as the mimetic miRNA, namely respectively miR-323a, miR-3127, miR-3161, miR-340, miR-708, miR-660 and miR-30d.

[0056] The mimetics that can be used here are known to those skilled in the art and are commercially available. They can also be produced by any technique known to those skilled in the art, for example by chemical synthesis.

[0057] The mimetics used here have the same nucleotide sequence, as shown in the tables above.

[0058] In the context of the invention, the miRNA mimetic can be chemically modified. Advantageously, the miRNA mimetic can thus carry chemical modifications that increase its stability, for example, against the action of nucleases, or enhance its cellular uptake. More specifically, modifications to the phosphate backbone, nitrogenous bases, or ribose sugar can mask the charge of the modified miRNA and promote its adhesion to the cell surface, thereby facilitating cellular uptake.

[0059] In a preferred embodiment, the miRNA mimetic comprises nucleotide analogs. Preferably, at least 20%, preferably at least 40%, at least 50%, at least 60%, at least 70% or at least 80%, for example at least 90% or 100%, of the nucleotides in the miRNA mimetic are nucleotide analogs.

[0060] As used here, the term "nucleotide analogs" refers to variants of natural nucleotides, such as DNA or RNA nucleotides, resulting from modifications in sugar and / or base groups. In principle, analogs could simply be "silent" or "equivalent" to the natural nucleotides in the context of the miRNA; that is, they have no functional effect on how the oligonucleotide hybridizes to the miRNA. These "equivalent" analogs can nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable under storage or manufacturing conditions, or serve as a tag / marker. Preferably, however, nucleotide analogs have a functional effect on how the miRNA increases resistance to nucleases and / or facilitates transport within the cell.For example, the introduced nucleotide analogue can be chosen from, but not limited to:

[0061] - A 2',4' methylene bridge in the ribose to form a bicyclic nucleotide designated as a "locked nucleic acid nucleotide" (LNA);

[0062] - A 2'-fluoro (2'-F) group in the 2' oxygen of ribose;

[0063] - A 2'-O-methoxy ethyl (2'-MOE) group.

[0064] In a particular embodiment, the nucleotide analogs are locked nucleic acid (LNA) nucleotides. In a preferred embodiment, at least 20%, preferably at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, for example at least 90% or 100%, of the miRNA nucleotides are LNA nucleotides.

[0065] In another particular embodiment, the nucleotide analogs are 2'-F nucleotides. In a preferred embodiment, at least 20%, preferably at least 40%, at least 50%, at least 60%, at least 70% or at least 80%, for example at least 90% or 100%, of the nucleotides of the miRNA are 2'-F nucleotides.

[0066] Also in a particular embodiment, the nucleotide analogs are 2'-MOE nucleotides. In a preferred embodiment, at least 20%, preferably at least 40%, at least 50%, at least 60%, at least 70% or at least 80%, for example at least 90% or 100%, of the nucleotides of the miRNA mimetic are 2'-MOE nucleotides.

[0067] Nucleotide analogs, such as 2'-F / MOE nucleotides, are particularly advantageous when they have a functional effect on increasing miRNA resistance to nucleases and / or on ease of absorption and transport into the cell.

[0068] To further protect the oligonucleotide from exonuclease-mediated degradation and facilitate delivery and / or localization within the cell, it is also possible to modify the miRNA with a cap. As used here, the term "cap" refers to a terminal modification that protects the modified oligonucleotide. The cap can be located at the 5'-terminus, the 3'-terminus, or both ends. The term "cap" includes, for example, inverted deoxyabasic caps. Suitable caps include, but are not limited to, 4',5'-methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L nucleotide, alpha nucleotide, modified base nucleotide, phosphorodithioate bond, threopentofuranosyl nucleotide, 3',4'-seco acyclic nucleotide, 3,4-dihydroxybutyl acyclic nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, an inverted 3'-3'-nucleotide group, an inverted 3'-3'-abasic group, an inverted 3'-T-nucleotide group, an inverted 3'-T-abasic group, an inverted 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate group, and a non-bridging methylphosphonate group, a 5'-aminoalkyl phosphate, an 1,3-diamino-2-propyl phosphate, a 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecyl phosphate, a phosphate hydroxypropyl, a 5'-5'-inverted nucleotide group, a 5'-5'-inverted abasic group, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and / or non-bridging 5'-phosphoramidate, a phosphorothioate and a 5'-mercapto group.

[0069] microRNA mimetics used alone or in combination with each other

[0070] The inventors identified miR-323a, miR-3127, and / or miR-3161 mimetics as microRNA mimetics useful in the prevention and / or treatment of cancers. Surprisingly, the inventors specifically discovered that miR-323a, miR-3127, and / or miR-3161 mimetics, used alone or in combination, led to inhibition of tumor cell survival.

[0071] As used here, the term "combination" refers to a composition comprising a plurality of components. The use of the term "combination" is not limiting with respect to the relative contents of the different components of said composition, which may be found in equimolar ratios, in identical mass ratios, or in different molar or mass ratios.

[0072] The present invention therefore relates to a microRNA mimetic or a combination of microRNA mimetics for use in the prevention and / or treatment of cancer in a patient, said microRNA mimetic being selected from a miR-323a, miR-3127, and / or miR-3161 mimetic, alone or in combination. In particular, said microRNA mimetic or said combination of microRNA mimetics may be used in combination with the administration of a miR-340 mimetic.

[0073] In one embodiment, said microRNA mimetic combination comprises the combination of miR-323a and miR-3127 mimetics.

[0074] In another embodiment, said microRNA mimetic combination comprises a combination of the following four miR mimetics: miR-323a, miR-3127, miR-3161, and miR-340. In another embodiment, said microRNA mimetic combination comprises a combination of miR-323a and miR-3161 mimetics or a combination of miR-3127 and miR-3161 mimetics.

[0075] In one embodiment, said microRNA mimetic combination comprises the combination of the following three miR mimetics: miR-323a, miR-3127, and miR-708. Advantageously, but not limitingly, this combination is particularly useful for the treatment of a cancer selected from pancreatic cancer, breast cancer, melanoma, and lung cancer.

[0076] In another embodiment, said microRNA mimetic combination comprises the following three miR mimetics: miR-3161, miR-660, and miR-30d. Advantageously, but not limitingly, this combination is particularly useful for the treatment of a cancer selected from breast cancer, pancreatic cancer, and melanoma.

[0077] The following combinations are described more specifically here, for use in the prevention or treatment of cancers.

[0078] The combination of miR-323a and miR-3127 mimetics;

[0079] The combination of miR-323a and miR-3161 mimetics;

[0080] The combination of miR-3127 and miR-3161 mimetics;

[0081] The combination of miR-323a, miR-3127 and miR-3161 mimetics;

[0082] The combination of miR-323a and miR-340 mimetics;

[0083] The combination of miR-3127 and miR-340 mimetics;

[0084] The combination of miR-3161 and miR-340 mimetics;

[0085] The combination of miR-323a, miR-3127 and miR-340 mimetics;

[0086] The combination of miR-323a, miR-3161 and miR-340 mimetics;

[0087] The combination of miR-3127, miR-3161 and miR-340 mimetics;

[0088] The combination of miR-323a, miR-3127, miR-3161 and miR-340 mimetics;

[0089] The combination of miR-323a, miR-3127 and miR-708 mimetics;

[0090] The combination of miR-3161, miR-660 and miR-30d mimetics.

[0091] Pharmaceutical Composition The microRNA mimetic or combination of microRNA mimetics provided herein may be formulated in a single pharmaceutical composition or in separate pharmaceutical compositions, optionally by mixing it or them with one or more pharmaceutically acceptable excipients.

[0092] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one active compound (e.g., a microRNA mimetic or a combination of microRNA mimetics) and at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those competent in the technical field and generally depend on the chosen route of administration. Pharmaceutical compositions according to the present invention can be supplied in any form or formulation suitable for the chosen route of administration, for example, a solution for intravenous administration, or capsules, pills, or tablets for oral administration, etc.

[0093] As used here, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse reaction, allergic reaction, or other adverse effect when administered appropriately to a mammal, particularly a human. A pharmaceutically acceptable vehicle or excipient means a nontoxic, solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type.

[0094] In some embodiments, the microRNA mimetic or combination of microRNA mimetics may be formulated in one or more pharmaceutical compositions suitable for administration by any suitable route, including intravenous, oral, transdermal, subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal, parenteral, rectal, vaginal and topical, preferably to intravenous administration.

[0095] In some cases, administration may be intratumoral or targeted systemically to the tumor. For oral administration, such a composition may be prepared, for example, as tablets or capsules, particularly orodispersible tablets, or for parenteral administration, particularly as liquid solutions, suspensions, or emulsions. It may be prepared by any of the methods well known in the art of pharmacy, for example, as described in Remington: The Science and Practice of Pharmacy, 20th edition; Gennaro, AR, Ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2000. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Oral compositions generally include an inert diluent vehicle or an edible vehicle. They may also be administered as unit doses.

[0096] As used here, the term "unit dose" refers to a single dose that can be administered to a patient and can be easily handled and packaged, remaining a physically and chemically stable unit dose that comprises either the active compound itself or a pharmaceutically acceptable composition.

[0097] Tablets, pills, powders, capsules, and other forms may contain, but are not limited to, one or more of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose or tragacanth gum; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a sliding agent such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint or methyl salicylate. Capsules may be in the form of a hard capsule or a soft capsule, which are generally made from mixtures of gelatin, possibly mixed with plasticizers, as well as in the form of a starch capsule.In addition, dosage unit forms may contain various other materials that alter the physical form of the dosage unit, such as coatings of sugar, shellac, or enteric agents. Other oral dosage forms, such as syrups or elixirs, may contain sweeteners, preservatives, colorings, and flavorings. Furthermore, the active compounds may be incorporated into rapid-dissolving, modified-release, or extended-release preparations and formulations, with extended-release formulations preferably being bimodal. Liquid preparations intended for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid compositions may also include binders, buffers, preservatives, chelating agents, sweeteners, flavorings, colorings, and so on.Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, acrylate copolymers, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous vehicles include mixtures of alcohols and water, hydrogels, buffered media, and saline solutions. In particular, biocompatible and biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be useful excipients for controlling the release of active compounds. Intravenous vehicles may include fluid and nutrient reconstituters, electrolyte reconstituters, such as those based on Ringer's dextrose, and others.

[0098] Administration diagrams

[0099] As previously described, the combination of microRNA mimetics according to the present invention can be formulated in a single pharmaceutical composition or in separate pharmaceutical compositions.

[0100] In certain embodiments, said separate pharmaceutical compositions are adapted for concomitant administration.

[0101] In alternative embodiments, said separate pharmaceutical compositions are adapted for sequential administration.

[0102] As used here, the term "concomitant" means that the pharmaceutical compositions are administered simultaneously or almost simultaneously. If this is not the case, then these compositions may be administered "sequentially," that is, within a time frame that allows the respective active compounds of said compositions to all be available to act therapeutically during the same period. Thus, "sequential" administration may allow one pharmaceutical composition to be administered within 5 minutes, 10 minutes, or a few hours following another pharmaceutical composition, provided that the half-life of the active compound(s) it comprises, in the bloodstream, is such that all the active compounds of each of the pharmaceutical compositions are present simultaneously in therapeutically effective quantities.The interval between administrations of the compositions will vary depending on the exact nature of the components, their interaction, and their respective half-lives. Those skilled in the art know how to adjust the interval between administrations based on these parameters.

[0103] In some embodiments, the microRNA mimetic or combination of microRNA mimetics provided herein may be used in combination therapy with at least one additional therapy useful in oncology. The microRNA mimetic or combination of microRNA mimetics and the second therapy may be administered in a combined amount effective in achieving the desired effect, such as inhibiting the proliferation and / or viability of a cancer cell. This may be achieved by administering the microRNA mimetic or combination of microRNA mimetics and the additional therapy concurrently or sequentially. More specifically, the patient may also be receiving chemotherapy or immunotherapy, or radiotherapy.

[0104] Doses

[0105] The dose to be administered is determined by a person skilled in the art in order to obtain the desired anticancer effect, and may depend on the active compound used (i.e. miRNA mimetic alone or a combination of miRNA mimetics), the route of administration as well as the pharmaceutical form used.

[0106] The dose administered to a subject, in one or more doses, may be, for example, in the range of 5pg to 100mg / kg of body mass, preferably from 100pg to 50mg / kg of body mass, preferably still from 1 to 20mg / kg of body mass.

[0107] Dosage unit compositions may contain these quantities or submultiples to achieve the required daily dose. However, it is understood that the specific dose level for a given patient will depend on various factors, including body weight, general health, sex, diet, timing and route of administration, absorption and excretion rates, combination with other medications, and the severity of the condition being treated.

[0108] The following examples illustrate the invention without limiting its scope.

[0109] EXAMPLES

[0110] Materials and methods

[0111] The tested miRNA mimetics were obtained from Thermo Fisher Scientific.

[0112] Cell culture

[0113] Pancreatic cancer (MIA PACA-2 and / or PANC-1), lung cancer (HCC95), and breast cancer (MDA-MB-231 and / or MCF-7) cell lines were used. A human fibroblast (HFF) cell line was used as a non-tumor control line.

[0114] The HCC95 cell line was cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin streptomycin (PS), 1% sodium pyruvate and 1% HEPES.

[0115] The MDA-MB-231 and MCF-7 cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin streptomycin (PS) and 1% NEA.

[0116] The MIA PACA-2 and PANC-1 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS).

[0117] Cell transfection The tested miRNA mimetics were transfected using lipofectamine RNAiMax (Life Technologies) according to the protocol provided by the supplier, i.e. 5pL of lipofectamine RNAiMax and 20pL of miRNA mimetic (52nM).

[0118] Cell viability assessment

[0119] To assess cell viability, for each cell line tested, 150,000 cells were seeded in 96-well plates and transfected for 72 hours according to the method described previously (Bassot et al., 2023, Cell Death Dis. 2023 Sep 25;14(9):630). After 72 hours, corresponding to day 0 post-transfection, a cell count and a Cell TiterFluor viability assay (CTF, Promega) were performed. The cells were then reseeded in 96-well plates for further cell counting and CTF (corresponding to days 3, 7, and 10 post-transfection). Luminescence quantification was performed using a Spectramax plate reader, and the viability of the treated groups was assessed by normalizing the values ​​obtained with those of the control group.

[0120] Evaluation of cellular apoptosis

[0121] To assess cell apoptosis, 150,000 cells were transfected for 72 hours according to the method described previously, and then maintained in culture for an additional 24 hours or 7 days. Apoptosis analysis was performed by flow cytometry using the Annexin V / PI kit (Life Technologies), according to the manufacturer's recommendations.

[0122] Evaluation of cell migration

[0123] To assess cell migration, 150,000 cells were transfected for 72 hours using the method described previously. The cells were then reseeded into 96-well plates at a density of 20,000 cells per well for 24 hours. The resulting cell mat exhibited 95% to 100% confluence. A gap (scratch) was created in this cell mat using a woundmaker. The 96-well plates were then placed in an IncuCyte incubator for continuous observation of cell migration into the created gap over a period of four days. For each well, images were automatically captured every two hours by the IncuCyte incubator, providing a precise time series of gap closure. The images were analyzed to measure the reduction of this gap, which corresponds to the progression of cell migration.The data obtained were then processed using image analysis software, allowing the percentage of empty space created to be closed at different time intervals. This percentage was used to determine the cell migration rate.

[0124] Quantitative real-time PCR (qPCR)

[0125] Following 72 hours of cell processing, total TRNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's instructions. This step includes cell lysis, contaminant removal, and RNA purification. The purity of the extracted RNA is then verified using a Nanodrop to ensure the necessary quality for subsequent steps.

[0126] Once the RNA was purified, 500 ng of total RNA was used for each reverse transcription reaction. Conversion to complementary DNA (cDNA) was performed using a reverse transcriptase and specific primers (Takara kit) for the target genes Bcl2111, ARF6, RICTOR, AXL, JAK1, FZD6, RAP1A, FZD4, CDK16, FOXO1, RAP2A, HIFI A, FOXO3, RAB5B, XIAP, SCN9A, BCL11A, CDK6, and G0S2, following an incubation protocol at different temperatures in a Ristretto thermocycler (VWR) to optimize complete transcription of RNA to cDNA. The resulting cDNAs were then diluted 1:10 to adjust concentrations and ensure the accuracy and sensitivity of qPCR measurements.

[0127] Finally, the expression of the target genes was quantified using the SYBR Green dye for detection in quantitative PCR (qPCR), performed with the Bio-Rad CFX96 system. qPCR accurately measured the expression levels of the target genes, providing a quantitative overview of the impact of the tested miRNAs on the regulation of these genes. Data analysis was then performed using a heatmap, which allows for easy visualization of the expression variations of the different target genes. The qPCR results, expressed as AACt values, were transformed into color scales to represent the relative expression levels.

[0128] In vivo models tested: The mice tested were immunodeficient (nude), female, 4 weeks old and weighing 25g. The mice had a pre-acclimation period of 1 week in the animal facility.

[0129] Injection of tumor cells in vivo

[0130] Four to five million cells were injected into the right flank of nude mice. Tumor size was measured using calipers until it reached 50 mm 3 Once this tumor size was reached, the mice were divided into treatment groups: three groups of 5 to 6 mice for MIA PACA-2 and four groups of 3 to 4 mice for HCC95.

[0131] Intraperitoneal injection of miRNA mimetics

[0132] Treatments were administered three times a week, with an intraperitoneal (IP) injection of 100 pL of a complexed miRNA mimetic using the Invivofectamine kit (Thermo Fisher). For complex preparation, the miRNA mimetic (6000 nM) was mixed with Invivofectamine according to the manufacturer's instructions to achieve a stable and efficient bond that facilitates in vivo transfection.

[0133] Monitoring of tumor volume evolution in vivo

[0134] Tumor volume was monitored twice a week using calipers, allowing for regular assessment of treatment effectiveness.

[0135] Quantification and statistical analysis

[0136] The results were analyzed using Graph Pad software, Prism and t-tests or one-way ANOVA and correspond to the mean ± standard deviation from the mean and (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0137] Results

[0138] In this study, the inventors tested the efficacy of several microRNA mimetics, alone or in combination, on tumor aggressiveness markers in vitro (on viability tests (FIGURES 1 and 2), migration (FIGURE 3), apoptosis (FIGURE 4) and expression of target genes involved in their aggressive phenotype (FIGURE 5) and in vivo, on tumor growth in a subcutaneous graft model (FIGURES 6 and 7).

[0139] The results showed an inhibitory effect of all miRNA mimetics, tested alone or in combination, on cancer cell lines 7 days post-transfection (FIGURES 1 and 2). In particular, the combination including the miRNA mimetics miR-3127, miR-323a, and miR-708 (also referred to as "Combo 2") showed the strongest inhibition (60–80%) of the viability of the tested cell models (FIGURE 2).

[0140] To assess the inhibitory potential of miRNA mimetics on several tumor cell aggressiveness criteria, migration (scratch assay) and apoptosis (Annexin V / PI) were measured following mimetic treatment. The results show inhibition of cancer cell migration following treatment with miRNA mimetics, alone or in combination (FIGURE 3). Similarly, induction of cell death (necrosis and apoptosis) is observed following treatment with miRNA mimetics, alone or in combination (FIGURE 4). Thus, the inhibition of tumor cell viability is explained, in particular, by the induction of cell death following treatment with the tested miRNA mimetics, alone or in combination. This inhibition is also coupled with inhibition of tumor cell migration.

[0141] Furthermore, the inventors also performed an in silico analysis to identify the mRNAs targeted by the miRNA mimetics (FIGURE 5). These targets were validated by quantitative RT-PCR, demonstrating the modulation of their expression following miRNA transfection.

[0142] Finally, in vivo experiments showed significant inhibition of tumor growth by the tested miRNA combinations (FIGURES 6 and 7).

Claims

25 DEMANDS 1. MicroRNA mimetic or combination of microRNA mimetics, for use in the prevention and / or treatment of cancer in a patient, said microRNA mimetic being selected from a miR3161, miR323a, and / or miR3127 mimetic, alone or in combination.

2. MicroRNA mimetic or combination of microRNA mimetics, for use according to claim 1, said microRNA mimetic being miR3161, alone or in combination.

3. Combination of microRNA mimetics, for its use according to claim 1, the combination of mimetics comprising miR323a, in combination with miR3127 and / or miR3161.

4. Combination of microRNA mimetics, for its use according to claim 1, the combination of mimetics comprising miR3127, in combination with miR323a and / or miR3161.

5. MicroRNA mimetic or combination of microRNA mimetics, for use according to any one of claims 1 to 4, in combination with administration of a miR340 mimetic.

6. Combination of microRNA mimetics, for use according to claim 1 or 3-5, comprising a combination of a miR323a mimetic and a miR3127 mimetic.

7. Combination of microRNA mimetics, for use according to claim 1 or 3-6, comprising a combination of a miR-3161 mimetic, a miR323a mimetic and a miR3127 mimetic.

8. Combination of microRNA mimetics, for use according to claim 7, comprising a combination of miR323a, miR3127, miR3161 and miR340 mimetics.

9. Combination of microRNA mimetics, for use according to any one of claims 1 to 8, comprising the combination of miR323a and miR3161 mimetics or the combination of miR3127 and miR3161 mimetics.

10. MicroRNA mimetic or combination of microRNA mimetics, for use according to any one of claims 1 to 9, said miR323a, miR3127 and / or miR3161 mimetics being in -5p form.

11. MicroRNA mimetic or combination of microRNA mimetics for use according to any one of claims 1 to 10, said cancer is selected from pancreatic cancer, breast cancer, melanoma, liver cancer, brain tumor, preferably glioblastoma, lung cancer, preferably small cell lung cancer, even more preferably squamous cell carcinoma of the lung (SCC).

12. Combination of microRNA mimetics, for use according to claim 11, comprising the combination of miR323a, miR3127 and miR708 mimetics, cancer being a cancer selected from pancreatic cancer, breast cancer, melanoma and lung cancer.

13. MicroRNA mimetic, for use according to claim 11, said microRNA mimetic being miR323a, and the cancer being selected from pancreatic cancer, breast cancer, melanoma, liver cancer, brain tumor and lung cancer.

14. MicroRNA mimetic, for use according to claim 11, said microRNA mimetic being miR3127, and the cancer being selected from pancreatic cancer, breast cancer, melanoma, liver cancer and brain tumor.

15. Combination of microRNA mimetics, for use according to claim 11, comprising the combination of miR3161, miR660 and miR30d mimetics, cancer being a cancer selected from breast cancer, pancreatic cancer and melanoma.

16. Combination of microRNA mimetics for use according to any one of claims 1 to 15, wherein the microRNA mimetics of said combination are formulated within a single pharmaceutical composition or are formulated in separate pharmaceutical compositions suitable for sequential or concomitant administration.

17. MicroRNA mimetic or combination of microRNA mimetics, for use according to any one of claims 1 to 16, the microRNA mimetic or combination of microRNA mimetics being formulated in one or more pharmaceutical compositions suitable for administration by intravenous, oral or subcutaneous route.

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