Antago-miR-155 for treatment of v-src, c-src -tyrosine kinase-induced cancers

FIELD OF THE INVENTION

This invention relates to a therapeutic use of antago-miR-155 molecule an equivalent such as a source of an antago- miR with capability to inhibit/suppress/block of miR-155, as active compound for preventing, treating, reverting, curing and/or delaying v-src, c-src -tyrosine kinase-induced cancers (breast cancer, lung cancer, prostate cancer, colorectal cancer) or a disease or a condition, associated with v-src, c-src -tyrosine kinase pathway and induces transformation of cancer cells into non-cancerous cell form.

STATEMENT REGARDING FUNDINGS

This invention was supported, in whole or in part, by an funds of SID ALEX GROUP, s.r.o.

BACKGROUND

[0002] MicroRNAs are a class of small, non-coding RNAs with lengths 30-31 bps, which control gene expression by hybridizing to and triggering either translational repression. Small non-coding RNAs (sncRNAs) play an important role in many intracellular processes such as regulation of gene transcription, protein translation, epigenetic modification, genomic stability, and chromatin organization (Lytle et al, 2007; Varambally et al, 2008).

The majority of the primary genetic transformations occurred due to action of miRNAs that modify the cellular genome and structure. In tumor cells, genes can be regulated by epigenetic modifications and underlying genomic mutations that due to reversible and irreversible changes of cellular genome, which can result in the activation of oncogenes and the inhibition of tumor-suppressor genes. Tumorigenesis is a complex process that is driven by active and passive mutations. The resultant tumors generally comprise heterogeneous tissues with tumor cell characteristics (Nishikawa S. et al., 2012).

[0003] Previously miR-155 was identified as metastatic sncRNA in lung cancer. Increased levels of miR-155 correlated with increased cancer invasion and migration and, correlated with poor prognosis in patients with lung cancer. Moreover, it was shown that miR-155 promoted tumor formation in the lung when cells were injected directly in the bloodstream (Volinia et al, 2006; Wang et al, 2015). MiR-155 also inhibits apoptosis in lung cancer cells by inhibiting Apaf-1 expression (Zang et al, 2012). Among the microRNAs that have been linked to cancer, it is the most commonly overexpressed miRNA in malignancies of the breast, lung, liver, colon, and rectum, and prostate. It down- regulates BCL6, which modulates the STAT-dependent IL-4 responses of B-cells and increases their function. The reduction of BCL6 is due to the up-regulation of inhibitors of differentiation such as IL -6, cMYC, Cyclin DI, and Mipla/Ccl3, all of which promote cell survival and proliferation. MiR-155 also upregulates the Mxdl/Madl transcription factors that mediate cellular proliferation, differentiation, and apoptosis through the regulation BCL6. Thus, miR-155 leads to the resistance of cell death and enables replicative immortality. HDAC4 is also a target for BCL6. HDAC alters chromosome structure and affects the access of transcription factors to DNA. BCL6 acts with MEF2C and MEF2D. MiR-155 activates metastatic processes in breast cancer cells by activating Rho. It also represses SHIP, which is a negative regulator of myeloid cell proliferation and survival. MiR-155 suppresses BACH1 and SOCS-1 and induces G-2 arrest through the CD40 ligand (CD 154) and further represses of BCL2. MiR 155 also targets casein kinase (CKla) which enhances beta-catenin signaling and cyclin DI expression, thereby promoting tumor growth (Due et al, 2016; Wan et al, 2016; Huffaker. and O’Connell, 2015). All of these genetic and morphological changes are due to cell death or a cell’s irreversible transformation. As the result, structural modification of cancer tissues occurs.

[0004] Viruses are one of the main causes for induction, development and progression of majority of cancers. Such as colorectal cancer, prostate cancer, lung cancer, skin and liver cancers, etc. These cancers are first-placed causes of cancer-related deaths in United States and throughout the world, affecting more than 163.5 per 100,000 men and women per year (based on 2011-2015 causes of deaths).

[0005] Rous sarcoma virus (RSV) is carcinogenic retrovirus. The tumor -inducing gene of RSV and RSV-induced tumors is the viral src oncogene (v-src) (Sudol, 2011). The v-src oncogene is a truncated and active form of the wildtype proto-oncogene c-src. The src gene codes a non-receptor protein tyrosine kinase that is a member of the Src family kinases (SFKs). SFKs are involved in processes of tumor progression, invasion, and metastasis. Src kinase activity and protein levels are elevated in several cancers, including those of the colon, prostate, lung and breast (Rothschild and Gautschi, 2012; Gargalionis et al, 2014; Varkaris et al, 2014; Zhang et al, 2013). Oncogenic activity of the Src gene is connected with the activation of the src protein kinase after interactions with other receptor tyrosine kinases: epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), colony-stimulating factor-1 receptor (CSF-1R), HER2/neu, HER1, hepatocyte growth factor receptor (c-Met), and vascular epithelial growth factor (VEGF). These relationships eventually result in cancer development, progression, and metastasis. Activated src-tyrosine kinase interacts with ras/MAPK pathways, it activates statl and stat3, which promote cell transformation and tumor progression. Src-tyrosine kinase is associated with the regulation of adhesion factors such as, a-, β -. and y-catenins; cadherin; and plakoglobin (JUP). Src-tyrosine kinase activates the anti-apoptotic factor Bcl-xL by inducing Stat3 expression. Stat3 activation leads to the transcriptional regulation of cyclin DI and c-Myc. The expression of these genes results in the stimulation of proliferative processes (Irby and Yeatman, 2000; Giglione et al, 2001). Although the discovery of Src tyrosine kinase was first and significant step in identifying key mechanisms, involved in induction and progression of virus -induced cancers, it has become clear that activation and/or mutation of Src susceptibility to virus-induced cancers. In spite of considerable research into therapies for virus-induced cancers, lung, colorectal, prostate and breast cancers remain difficult to diagnose and treat effectively, and the high mortality observed in patients with indicates that improvements are needed in the diagnosis, treatment and prevention of the diseases.

[0006] No universally successful method for the treatment or prevention of v-src, c-src-tyrosine kinase-induced cancers is currently available. Management of cancer depends on a combination of early diagnosis (e.g., through routine physical screening procedures) and aggressive treatment, which may include one or more of a variety of treatments, such as surgery, radiotherapy, chemotherapy and immunotherapy. Virus-induced cancers have very quickly progression, hard to treat. Patients with these cancers have poor prognosis in late stages of diseases.

[0007] Different classes of Src-tyrosine kinase inhibitors have been synthesized. Of these, the most heavily clinically investigated are the dual inhibitors of Src and Bcr-Abl protein kinases (Fernandez et al, 2019). In 2006, 100 years after Rous’s discovery, one such inhibitor, SPRYCEL (dasatinib), gained U.S. Food and Drug Administration (FDA) approval for the complex therapy of patients with chronic myelogenous leukemia (W02017144109A1). Other Src inhibitors with initial US approval are ICLUSIG (ponatinib) (US8114874), CAPRELSA (vandetanib) (US7173038), BOSULIF (bosutinib) (US7417148). Other inhibitors of src-tyrosine kinases are still in different phases of preclinical and clinical investigations. However, none of the investigated src-tyrosine kinase inhibitors has shown appreciable activity in the monotherapy of patients with solid tumors (Puls et al, 2011). All proposed drugs are very toxic and have hepatotoxicity, cardiotoxicity, misbalance of blood cells count, changes of the hemostasis and immune activity.

Table 1. Comparative analysis of v-src, c-src-tyrosine kinase inhibitors.

0008] The main hallmarks of cancer are: genetic and epigenetic modifications, changes in the cell cycle with inhibition of apoptosis, metastatic activity of cancer cells, unlimited proliferation and division, and regulation of the tumor microenvironment. In tumor cells, genes can be regulated by epigenetic modifications and underlying genomic mutations that due to reversible and irreversible changes of cellular genome, which can result in the activation of oncogenes and the inhibition of tumor-suppressor genes. Tumorigenesis is a complex process that is driven by active and passive mutations. (Nishikawa et al, 2012). Genetic and epigenetic modifications are enabling characteristics that generates random mutations including chromosomal rearrangements. Rare genetic changes can occur that orchestrate hallmark capabilities of cancer growth and progression. Because cancer initiation and development are based on genetic information, the uncontrolled behavior of cancer cells is probably due to the combined deregulation of genetic and epigenetic programs. Artificial epigenetic reprogramming, or remodeling of the tumor cells and microenvironment, can modify the functional capability, tumor growth and clinical relapse. Given that the number of parallel signaling pathways supporting a given hallmark must be limited, it may become possible to target all of these supporting pathways therapeutically, thereby preventing the development of adaptive resistance (Hanahan and Weinberg, 2011; Kaczkowski et al, 2016; Nath et al, 2015).

[0009] Eventually, almost all cells could be reprogrammed to pluripotency, although with different latency periods. Induction of the apoptosis pathway and overexpression of pluripotency factors accelerate the kinetics of reprogramming by increasing the cell division rate, which may facilitate the acquisition of DNA and/or histone modifications (Hanna et al, 2009; Cox et al, 2008). The reprogrammed cells had morphological, genetic and protein marker changes compared to control lung cancer cells. A two-step process was used to fully reprogram cells. First, sncRNAs reprogrammed genetic program of lung cancer cells and transformed them into intermediate, ready-to- differentiate form. The transformation of cancer cells is possible by the combination of genetic modifications and full reprogramming of the intracellular genome. Therefore, only epigenetic regulators could be effective drugs for tumor treatment. SncRNAs are possible candidates for this role (Lam et al, 2015; Naidu and Garofalo, 2015).

DESCRIPTION OF INVENTION

[0010] The disclosure is use of antago-miR-155 molecule or source thereof as identified herein. The invention is provided an antago-miR-155 molecule, an equivalent or a source thereof or a composition comprising said miRNA molecule antago-miR-155, said equivalent or said source thereof, preferably for use as a medicament for preventing, treating, reverting, curing and/or delaying v-src, c-src-tyrosine kinase-induced cancers (breast cancer, lung cancer, prostate cancer, colorectal cancer) or a disease or a condition, associated with v-src, c-src-tyrosine kinase pathway. Antago-miR-155 or any molecule, an equivalent or a source thereof or a composition comprising said antago-miR- 155 molecule, equivalent or source thereof for use as a medicament for induction/initiation/stimulation/activation v- src, c-src-tyrosine kinase-induced cancer cell transformation into non-cancerous cell form. The term “antago-miR- 155 (a-miR-155, antago-miRNA-155, a-miRNA-155) of present invention is molecule or an equivalent, or a mimic, or an isomiR with capability to inhibit/suppress/block of miR-155. The present invention disclosure nucleic acid sequence to target the miR-155 to inhibit v-src, c-src-tyrosine kinase pathways in virus-induced cancers.

[0011] In the context of the invention, an antago-miR-155 molecule or an equivalent, or a mimic, or an isomiR thereof may be a synthetic or natural, or recombinant. Antagonism for miR-155 may be for immature, or part of a mature, or mature miR-155. An antago-miR-155 molecule or an equivalent, or a mimic thereof may be a single stranded or double stranded RNA molecule.

Preferably an antago-miR-155 of a miR-155 molecule is from 8 to 30 nucleotides in length, preferably 10 to 31 nucleotides in length, preferably 12 to 28 nucleotides in length, more preferably said molecule has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more. A source of an antago-miR-155 of a miR-155 molecule or a source of an equivalent of an antago-miR with capability to inhibit/suppress/block of a miRNA molecule may be any molecule, which is able to induce the production of said antago-miR-155. It is specifically related to deliver the nucleic acid molecule antago-miR-155 molecule or source thereof as active component with the carrier and use it as a medicament for suppression of miR-155, result in preventing, treating, reverting, curing and/or delaying v-src, c-src-tyrosine kinase or a disease or a condition associated with v-src, c-src- tyrosine kinase pathway, thereby realize the treatment of v-src, c-src-tyrosine kinase-induced cancers. An antago- miRNA molecule or a source of an equivalent of an antago-miR with capability to inhibit/suppress/block of a miR- 155 may be made by any known technique, such as for example, enzymatic or chemical synthesis, or biological production. Though antago-miR according to the invention could be produced using recombinant methods. Nucleic acid synthesis is performed according to standard methods. (U.S. Patent Nos 4704362A, 5221619A).

[0012] The v-src, c-src-tyrosine kinase-induced anti-cancer activity definition

An activity of a given antago-miR-155 molecule or an equivalent or a source thereof or a corresponding source thereof all as defined herein is preferably the ability to exhibit a detectable anti-v-src, c-src-tyrosine kinase activity and/or induce a decrease of v-src, c-src- tyrosine kinase pathways. Within the context of the invention, an anti-v-src, c-src- tyrosine kinase activity may comprise or comprises at least one of the following: reduction or decrease of v-src or c- src-tyrosine kinase pathways protein activity, induction of apoptosis, decrease of proliferation, decrease of metastatic activity, death of cancer cells or theirs transformation into non-cancerous form.

Exhibiting such a detectable anti-v-src, c-src-tyrosine kinase pathway proteins activity and/or inducing such a reduction or decrease of v-src, c-src-tyrosine kinase-induced cancerogenesis is crucial in the present invention in order to be able to prevent, delay, cure and/or treat v-src, c-src-tyrosine kinase-induced cancers and/or any disease or condition associated with v-src, c-src-tyrosine kinase pathways.

[0013] Therapeutic v-src, c-src-tyrosine kinase-induced anti-cancer effects

In the context of the invention, preventing, treating, reverting, curing and/or delaying v-src, c-src-tyrosine kinase- induced cancerogenesis or a disease, or condition associated with v-src, c-src-tyrosine kinase-induced cancerogenesis may be replaced by achieving an anti-tumor effect. Unless otherwise indicated, an anti-tumor effect is preferably assessed or detected after at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or more in a treated subject. An anti-tumor effect is preferably identified in a subject as: an inhibition of proliferation of tumor cells, a decrease of proliferation markers in cancer cells, and/or an induction or increased induction of tumor cells death, an increase of markers of apoptotic activity, and/or a delay in occurrence of metastases and/or of tumor cell migration and inhibition of markers of metastatic activity and/or an inhibition or prevention or delay of the increase of a tumor weight, and/or a prolongation of patient survival of at least one month, several months or more (compared to those not treated or treated with a control or compared with the subject at the onset of the treatment), and/or an improvement of the quality of life and observed pain relief.

A patient may survive and/or may be considered as being disease free. Alternatively, the disease or condition may have been stopped or delayed. An improvement of quality of life and observed pain relief may mean that a patient may need less pain relief drugs than at the onset of the treatment. Alternatively or in combination with the consumption of less pain relief drugs, a patient may be less constipated than at the onset of the treatment. "Less" in this context may mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less. A patient may no longer need any pain relief drug. This improvement of quality of life and observed pain relief may be seen, detected or assessed after at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or more of treatment in a patient and compared to the quality of life and observed pain relief at the onset of the treatment of said patient.

An inhibition of the proliferation of tumor cells may be at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Proliferation of cells may be assessed using known techniques.

An induction of tumor cell death may be at least 1%, 5%, 10%, 15%, 20%, 25%, or more. Tumor growth may be inhibited at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumor cell death may be assessed using techniques known to the skilled person. Tumor growth may be assessed using MRI or CT. In certain embodiments, tumor weight decrease or tumor growth may be inhibited at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Tumor weight or tumor growth may be assessed using techniques known to the skilled person.

To test the effect of antago-miR-155 molecule on tumour growth in an animal model in vivo, an experimental system as described in Example 5 may be used.

A delay in occurrence of metastases and/or of tumor cell migration may be a delay of at least one week, one month, several months, one year or longer. The presence of metastases may be assessed using MRI, CT or Echography or techniques allowing the detection of circulating tumour cells (CTC). In certain embodiments, tumor growth may be delayed at least one week, one month, two months or more. In a certain embodiment, an occurrence of metastases is delayed at least one week, two weeks, three weeks, four weeks, one months, two months, three months, four months, five months, six months or more.

[0014] Preferably, a decrease of the expression level of miR-155 molecule or equivalent or source thereof means a decrease of at least 10% of the expression level of the miRNA using qPCR, microarrays or Northern blot analysis. A decrease of the expression level of a miRNA molecule or equivalent or source thereof means a decrease of at least 15%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100 %. Preferably, an expression level is determined ex vivo in a sample obtained from a subject (sample is derived from a tumor biopsy, blood, sputum, stool or urine).

[0015] Effective amount and therapeutically effective amount

The molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the molecule of the invention or its pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent the symptom/s, or prolong the survival of the patient being treated. For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. Such information can be used to determine effective doses in humans.

Therapeutically effective serum levels may be achieved by administering multiple doses. In cases of local administration or selective uptake, the effective local concentration of the proteins may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. The amount of molecules administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the a fiction, the manner of administration and the judgment of the prescribing physician. The therapy may be repeated intermittently while symptoms detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs or treatment (including surgery). [0016] Toxicity

Preferably, a therapeutically effective dose of the molecules described herein will provide therapeutic benefit without causing substantial toxicity. Toxicity of the molecules described herein can be determined by standard pharmaceutical procedures in cell cultures and experimental animals (LD50, LD100). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition, such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. In therapeutic applications, an effective amount of the antago-miR-155 of the present invention is administered to a cell, which may or may not be an animal. In some embodiments, a therapeutically effective amount of the antago-miR-155 of the present invention is administered to an individual for the treatment of disease or condition. The term "effective amount" as used herein is defined as the amount of the antago-miR-155 molecule or any molecule, an equivalent such as a source of an antago-miR with capability to inhibit/suppress/block of miR-155 that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term "therapeutically effective amount" as used herein is defined as the amount of the molecule of antago-miR-155 that achieves a desired effect with respect to a disease or condition associated with v- src, c-src -tyrosine kinase-induced cancers and/or any disease or condition associated with v-src, c-src-tyrosine kinase pathways. Thus, an amount of molecule that provides a physiological changes is considered an "effective amount" or a "therapeutically effective amount."

[0017] In in vivo experiments, antago-miR-155 sequence may differ in the in vivo tests as compared to the human sequence. In that case, antago-miRNA that differs from the human sequence might be used to demonstrate therapeutic effect in the animal. Results obtained with this sequence tested in an animal may be extrapolated expected results in human with a corresponding antago-miRNA molecule.

[0018] Modes of Administration and Formulations

The nucleic acid molecule of the invention may be administered to a subject alone or in the form of a pharmaceutical composition for the treatment of a condition or disease. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the antago-miR-155 into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For topical administration the antago-miR-155 may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration by injection (subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal), as well as those designed for transdermal, transmucosal, inhalation, oral or pulmonary administration. For injection, antago-miR-155 may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the nucleic acid molecule may be in powder form for constitution with a sterile pyrogen-free water, before use. For oral administration, the nucleic acid can be readily formulated by combining the molecule with pharmaceutically acceptable carriers. Such carriers enable the nucleic acid of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. If desired, solid dosage forms may be sugar-coated or enteric- coated using standard techniques. For buccal administration, the molecules may take the form of tablets, lozenges, etc. formulated in conventional manner. For administration by inhalation, the molecule for use according to the present invention are conveniently delivered in the form of an aerosol spray. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. The RNA molecules may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas.

In addition to the formulations described previously, the molecule may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Alternatively, other pharmaceutical delivery systems may be employed.

[0019] Delivery systems

The present invention involves in some embodiments delivering a nucleic acid into a cell to be related to a therapeutic application.

Delivery methods (Any):

Direct: by direct delivery of nucleic acid by injection (U.S. Patent Nos. 5,981,274, 5780448A, 5736524A and 5702932A), including microinjection (US5789215A); by electroporation (US5384253A); by direct sonic loading (Fechheimer et al, 1987); by micro projectile bombardment (U.S. Patent Nos. 5610042A, 5550318A, 5538877 A);

Indirect: Using a vector, that is a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted; Using avidin fusion proteins (US6287792B1); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Rippe et al, 1990); by using DEAE -dextran followed by polyethylene glycol (US7122384B2); by liposome mediated transfection (U.S. Patent Nos. 5049388A and US6120798A, JP6382380B2); by photochemical internalization (W02008007073A2); by agitation with silicon carbide fibers (US5302523A); by PEG-mediated transformation of protoplasts (US4684611A);

A variety of compounds have been developed that complex with nucleic acids, deliver them to surfaces of cells, and facilitate their uptake in and release from endosomes. Among these are: (1) a variety of lipids such as DOTAP (or other cationic lipid), DDAB, DHDEAB, and DOPE and (2) non-lipid-based polymers like polyethylenimine, polyamidoamine, and dendrimers of these and other polymers. In certain of these embodiments a combination of lipids is employed such as DOTAP and cholesterol or a cholesterol derivative (US6770291B2).

Carriers that stabilize and/or transport nucleic acids within cells enhance the stability and activity of nucleic acids because they should protect and guide the bound oligonucleotides once they are in cells. In this invention compare toxicity and transfection efficiency of poly(N-vinylpyrrolidone) (PNVP) (CA2547038) and cationic graft-copolymer DDMC® (2-diethylaminoethyl-dextran methyl methacrylate copolymer), a non-viral transfection reagent (DDMC) (JP2017149787A) polymer carriers, see Examples 1 and 2.

[0020] Pharmacologically acceptable carrier

Pharmaceutical compositions of the present invention comprise an effective amount of antago-miR-155 molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce or produce acceptable adverse, allergic or other undesired reactions when administered to an animal, such as, for example, a human, as appropriate. Whether certain adverse effects are acceptable is determined based on the severity of the disease. Moreover, for animal or human administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by U.S. FDA or EMA Offices of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof (Remington's Pharmaceutical Sciences).

The nucleic acid may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (Remington's Pharmaceutical Sciences or other Handbooks on Pharmacy).

[0021] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol, lipids and combinations. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0022] The developed treatment with any types of oligonucleotides as active medicine component (antago-miR in the present invention) is effective tool in anti-cancer therapy. The invention mentioned herein may be combined with standard treatments of disease or condition associated with v-src, c-src-tyrosine kinase cancers such as chemotherapy, radiotherapy or surgery.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0023] Description of the figures

Figure 1.

A. Graphical changes of Fisher’s index described cell toxicity of the DDMC and PNVP in MTT assay (two-way ANOVA (p < .05)). Increasing of the DDMC vector dose due to increasing of cellular toxicity. PNVP had not cell toxic effect in used range of doses (ANOVA (p < .05)). B. Graphical changes of Fisher’s index of Transfection Efficiency in different types of cells in dynamic of transfections using the DDMC vector/pmKate2 and PNVP/pmKate2 (p < .05).

Figure 2.

Microscopic photos of CaCo2 colorectal adenocarcinoma cells: A. Control cells stained using the Leishmann- Romanowsky method. (Magnification 60x). B. Transfected CaCo2 cells on the 14 ^th day after transfection with complex of PNVP and pmKate2-N vector (Magnification 40x). C. Transfected CaCo2 cells separated using CD117+ positive magnetic separation and labeled using CD117+/FITC reagent (Magnification 40x); D. Transfected cells selected using CD117+ positive magnetic separation (stained using the Leishmann-Romanowsky method). (Magnification 40x).

Figure 3.

A. A549 cells viability (ANOVA) detected by MTT-test on 7 days after treatment with naked antago-miR-155 10 pl/ml (1), DDMC 5 mg/ml (2), DDMC 7 mg/ml (3), DDMC 10 mg/ml (4), and complexes of DDMC 5 mg/ml with antago-miR-155 10 pl/ml (5), complexes of DDMC 7 mg/ml with antago-miR-155 10 pl/ml (6) and complexes of DDMC 10 mg/ml with antago-miR-155 10 pl/ml (7) (p < .05, $ - increase of statistically significant interactions F > Fcrit). B. Microscopic photos of A-549 lung adenocarcinoma cells on 21 ^st day of control group (a.) and group after adding of antago-miR-155 with DDMC carrier (b.). (Magnification x60). C. a. Heat maps of the 13 differentially expressed genes levels in two independent microarray studies in A-549 lung adenocarcinoma cells after treatment using complexes of antago-miR-155 with DDMC vector on 11 ^th , 21 ^st and 31 ^st days after transfection. The heat map was produced by hierarchical clustering of the probeset data. Probesets for genes are represented by rows with the gene dendrogram at left. Green color indicates decreased expression and red color indicates increased expression of genes (AltAnalyze Platform), b. Photo of agarose gel with separate genes expression in A-549 cells on the 31 ^st day after treatment using complexes of antago-miR-155 with DDMC: 1. beta-actin, 2. Caspase8, 3. mTOR, 4. SOX2, 5. VMAF, 6. PIWIE1, 7. SOX2, 8. EBB, 9. ICOS1B, 10. FET3, 11. GITR3A, 12. FOXP3, 13. GATA1, 14. HM0X1, 15. BACH2, 16. CKIT1B, 17. RUNX1, 18. KEF4, 19. NOTCH1, 20. KIR2DE1.

Figure 4.

Kaplan-Meier plots of the lifespan of animals from the 1 ^st (n=10) (A.) and 2 ^nd (n=10) (B.) groups compared to that of the controls (n=10) (p < .05).

Figure 5.

A. The dynamics of tumor growth in mice from all three groups (means ±SEMs (p < .05)). B. Photographs of mice from the control (a.) and 1 ^st (b.) groups on the 20 ^th day after tumor inoculation.

Figure 6.

A. Annexin A4 translocation to the nucleus in labeled tumor cells from animals in the 1 ^st group, a. Tumor cells from the control group (24 days after beginning the experiment); b. tumor cells from mice in the 1 ^st group (16 days after beginning the experiment); c. tumor cells from mice in the 1 ^st group (24 days after beginning the experiment). (Magnification 60x). B. Tumor cells from animals of the 1 ^st group. Immunofluorescence labeling of cells with CD4+ (a.), CD117+ (b.), and Oct4 (c.). (Magnification 40x). Cells from tumors from animals of the 1 ^st group were stained with the Leishman- Romanowsky method (d.). (Magnification 60x).

C. Cells from tumors of the 1 ^st group treated with the complex of antago-miR-155 with the DDMC carrier at the same concentration per milliliter of culture medium as to the tumor cells on the 11 ^th day (Leishman-Romanowsky method (a., b.). (Magnification 60x). Same cells labeled with anti-CD4+ antibodies and analyzed by immunofluorescent method (c.). (Magnification 40x).

Figure 7.

Heat map of the hierarchical clustering of gene expression profiles in different cells from mice in all investigated groups. B. Images of RT-PCR products obtained after electrophoresis on 2 % agarose gels, stained with ethidium bromide.

[0024] Examples

Materials and Methods Cell culture and Chemicals

Cells: Human pancreatic ductal adenocarcinoma cell line (ATCC® CRL-1420™), Human glioblastoma cell line (ATCC® CRL-1620™), Human neuroblastoma cell line (ATCC® CCL-127™), Human retinoblastoma cell line (ATCC® HTB-18™), Human colorectal adenocarcinoma cell line (ATCC HTB-37™), Human lung non-small adenocarcinoma cell line (ATCC® CCL-185™), RSV (Prague C strain)-induced (NCBI_TaxID: 11888) RVP3 cell line (RRID: CVCL_L978).

Chemicals and equipment: Dulbecco’s Modified Eagle Medium (DMEM) medium, with 10% Fetal Bovine Serum, 2 mM 1-glutamine, 100 mg/ml penicillin, and 100 ME/ml streptomycin (GE Healthcare); Cationic graft-copolymer DDMC Vector® (2-diethylaminoethyl-dextran methyl methacrylate copolymer), a non-viral transfection reagent developed by Ryujyu Science Corp. (Aichi, Japan); The pmKate2-Annexin vector (mouse) was purchased by SID ALEX GROUP, s.r.o. (US7638615B1), RNAeasy mini kit (Qiagen, US); Dynabeads ® CD4 Positive Isolation Kit (Invitrogen, US); DETACHaBEAD™ DYNAL™ Dynabeads™ (Invitrogen, Life Technologies, US); CD4+/FITC staining reagent (R&D, US), CD117 purified mouse anti -human antibodies (Caltag Laboratories by Invitrogen, US); Oct4+ purified mouse anti -human antibodies (Santa Cmz Biotechnology, Inc., US); Dynabeads® Pan Mouse IgG (Invitrogen, Life Technologies, US); Fluorescent microscope (AxioVertAl, Zeiss, Germany); Two-step reverse transcriptase-PCR reagents and ferments (Fermentas, Thermo Fisher, US); Automatic thermocycler (TProfessional, Biometra, Germany); Gel doc system (Syngene, India); MTT reagent (Life Technologies, US); Spectrophotometer (ELx808 - Biotek, Winooski, US).

[0025] Example 1. Toxicity of antago-miR-155 with DDMC carrier, toxicity of antago-miR-155 with PNVP delivery system

In the present invention, active compound antago-miR-155 was incorporated in two polymer carriers: in amphiphilic PNVP and in DDMC to choose the best polymer carrier with minimal toxicity and optimal transfection effectivity. These two complexes form the nanoparticle formulations of average diameter 100-500 nm. Administering naked nucleic acids is challenging due to their rapid degradation by RNases within and outside of cells. Method. MTT Assay. The MTT assay was used to evaluate all the cell lines described above and human monocytes and lymphocytes, and toxicity was evaluated at 8 days after transfection. The MTT assay method employed was that of da Silva Gasque, et al. (2014), with 200 pL of DMSO (dimethyl sulfoxide) used as the crystal solvent to determine the cell densities. The absorbance at 570 nm was read using a spectrophotometer 30 min after the solubilization of the crystals.

Results. The results of the two-way ANOVA revealed a strongly significant interactions (p < .05) between the doses of the DDMC vector and its toxic effect. Increasing the concentration of the DDMC vector increased its toxic effect on cells. However, the ANOVA results did not reveal such a tendency (p < .05) for the doses of PNVP and its toxic effect (Figure I .A.). In a series of experiments, the toxicities of the DDMC and PNVP as delivery systems for the antago-miR-155 were investigated. The ANOVA results revealed that the presence of antago-miR-155 significantly affected the toxicity of the DDMC. The optimal doses of the DDMC/antago-miR-155 and PNVP/antago-miR-155 complexes that were non-toxic to cells were calculated.

Different doses of the DDMC were used to investigate its toxicity. According to the manufacturer’s protocol, the recommended starting dose of the DDMC is 10 mg/ml. However, the results of the MTT assay showed that the DDMC was highly toxic at this dose. The optimal doses of this carrier for the different cell lines ranged from 5 to 7.5 mg/ml in the cell culture medium. In this concentration range, the level of cell viability was 85-90 %. At a dose of 10 mg/ml, the level of viability of the various cell lines ranged from 30 to 40 %. In the different cell lines, the level of toxicity slightly varied. The adhesive cell lines were the most resistant to DDMC-related toxicity. The cells that were the most highly resistant to DDMC -based toxicity were the cultured monocytes prepared from the peripheral blood cells of healthy men.

[0026] Example 2. Transfection efficiency of antago-miR-155 with DDMC carrier, transfection efficiency of antago-miR-155 with PNVP delivery system

Method. The pmKate2-N vector, vector, which is a mammalian expression vector encoding the far -red fluorescent protein mKate2 (Evrogene), was used as the nuclear transfection control. (Shcherbo et al, 2007). The cells were incubated for 40 days, with the transfection efficiency dynamics analyzed on days 7, 14, 30 and 40. Fluorescence microscopic images were used to quantitate the transfection efficiency levels before further processing of the cells. The controls were cells not given any treatment and cells treated with unloaded nanoparticles.

PNVP/pmKate2 and DDMC vector/pmKate2 complexes were used in this study. In three independent experiments, I calculated the percentage of cells expressing the mKate2 deep-red fluorescent protein (transfection efficiency) was determined as previously described (at 40x magnification).

Transfection efficiency, % =

C _FP - The number of cells expressing pmKate2 fluorescent protein (fluorescent microscopy).

C _v -The total number of viable cells (phase contrast microscopy).

Results. The levels of transfection of the carriers and complexes were investigated. An initial sign of cell transfection, the expression of pmKate2, was observed after adding the DDMC/pmKate2 and PNVP/pmKate2 complexes to the cells. The DDMC/pmKate2 complex produced better transfection indices, and a sign of cell transfection by these complexes was observed earlier than that following cell treatment with the PNVP/pmKate2 complex. The expression of the mKate2 deep-red protein was detected 5-7 days cell treatment with the DDMC/pmKate2 complex. The maximum rate of transfection of 90-96 % was observed on the lOth-l lth day after treatment with DDMC/pmKate2 (Figure I.B.). The expression of mKate2 after treatment with the PNVP/pmKate2 complex was detected on day 11 -14. The maximum cell transfection rate observed after treatment with PNVP/pmKate2 complex was 98-100 % on the days 21 ^st -24 ^th days. The duration of transfection after treatment with the DDMC/pmKate2 complex was longer than that following treatment with the PNVP/pmKate2 complex. Upon cell treatment with the DDMC/pmKate2 complex, the expression of Kate2 protein continued for 35-40 days and then decreased, whereas the treatment of cells with the PNVP/pmKate2 complex prolonged transfection for 17-21 days. Supplementation of the medium with different doses of serum, ranging from 1 to 20 %, did not affect the transfection efficiency levels of the different cell lines.

Comparative characteristics of DDMC and PNVP delivery systems are presented in table.

Table 1. Summary characteristics of the DDMC and PNVP as delivery systems for oligonucleotides.

*DDMC solution can be autoclaved and treated using 0.05 % of DEPC, and incubated overnight. The trace of the DEPC could be removed from DDMC solutions by autoclaving again. [0027] Example 3. Transformation of colorectal cancer cells after treatment with antago-miR-155 and PNVP carrier

Methods. On the 31 ^st day of incubation, a portion of treated with antago-miR-155/PNVP cells was treated using the CD117 purified mouse anti-human antibodies, treated with Dynabeads® Pan Mouse IgG and stained using the Leishman-Romanowsky method. The last part of the cells, which were negatively separated, were treated using the Dynal ® Monocyte Negative Isolation Kit and stained using the Leishman-Romanowsky method.

Leishman-Romanowsky staining of the CaCo2 cells was made in accordance with the Blood safety and clinical technology guidelines on standard operating procedures for hematology.

Results. CaCo2 colorectal adenocarcinoma cells were transformed into CD117+ cells after treatment with antago- miR-155 and PNVP carrier. The morphology of the adenocarcinoma cells changed during the dynamic transformation. Cellular and nuclear forms, sizes and number were different when compared with control cells (Figure 2). Cells had apoptotic changes such as: pyknosis of nuclei, apoptotic vesicles in the cytoplasm and in the extracellular medium, and increased cellular size.

An absence of adhesive properties and changes in the sizes and morphology of cells and nuclei were obtained following treatment with complexes of antago-miR-155 and PNVP. Cells were separated with magnetic nanoparticles in order to isolate CD117 cells (Figure 3) and CD-I 17+ cells were obtained, which indicate the pro-T stage and the subsequent pre-T stage, respectively, of af> T cell development (Abbas et al, 2007).

[0028] Example 4. Transformation of lung cancer cells after treatment with antago-miR-155 and DDMC carrier

Methods. Toxicity. The A549 (ATCC® CCL-185™) human lung adenocarcinoma cell line was used for the MTT test and toxicity was detected 7 days after treatments. In MTT experiments on toxicity investigation were used DDMC, and complex of DDMC with antago-miR-155 (Figure 3). The concentration of DDMC was 5, 7 and 10 mg/ml of medium. The concentration of naked RNA oligonucleotide was 10 pl/ml of medium. The concentration of the DDMC complexes was 7 mg/ml of medium plus 10 pl/ml of medium RNA oligonucleotide.

Gene expression analysis: Total RNA was extracted from cell culture using the RNAeasy Mini kit according to the manufacturer’s protocol. In these series of experiments standard two-step reverse transcriptase-PCR procedure was used. Amplification of Caspase8, mTOR, PIWI1L1, EBB, ICOS1B, FLT3, GITR3A, HM0X1, BACH2, CKIT1B, NOTCH1, KIR2DL1, DICER1 and beta-actin cDNA (as an internal control) was performed with an automatic thermocycler. The primers sequences 5 ’ -3 ’ are indicated below. The PCR protocol was made as described previously. I added 6.25pM primer (sense and antisense) for reaction mix. Amplification proceeded according standard protocol. PCR products were loaded on 1.5 % agarose gel and electrophoresed then stained with Ethidium Bromide, exposed to a gel doc system and quantified with Quantity One Software (Bio-Rad). For comparison inner control - expression of beta-actin gene, and external control for which were used A549 cells without any treatments.

Staining of the A549 cells was made using the Leishman-Romanowsky method (Lewis and Kumari, 2000).

Statistics. Data are presented as the mean ± SEM (p < .05). MTT assay data were analyzed using two-way analysis of variance (ANOVA) based on the concentrations of DDMC and complex of DDMC and antago-miR-155 (p < .05). All gene expression data were normalized to internal control gene expression levels of beta-actin. For external control were used culture of cells without any treatment in the same moment of time as experimental cells. All samples were prepared in triplets. Experiments were repeated three times (N=9). For gene expression analysis, I used AltAnalyze software.

Results. The DDMC was toxic to cells at a concentration more than 10 pg/ ml (Figure 3). In my experiments, I used concentration of DDMC 7 mg/ml. The toxicity was decreased after treatment with a complex of the DDMC and oligonucleotide (F observed < F crit). The transfection efficiency of the DDMC was lower than that particles of PNVP, which were used in previous studies; however, the transfection maximum was reached 11-14 days faster than that of the PNVP complexes (21 days). DDMC based particles in non-toxic concentrations had greater stability and increased cell transfection with low nucleic acid and DDMC concentrations.

Three weeks after a complex of antago-miR-155 plus DDMC was added, A-549 cells were morphologically changed compared to non-treated control cells. In the microscopic photos was observed an increase in number of sharp large cells (approximately 50 pm in diameter) with single nuclei, displaying different morphological forms. These cells had vesicles of varying sizes on the periphery of the cytoplasm with properties of suspension cells. Un -stained cells and cells full of DDMC crystals were microscopically detected. These cells were large (approximately 60 pm in diameter) and sharp with polymorphic nuclei that took 1/3 - 2/3 of the cell volume (Figure 5). Giant dendritic-like cells with multiple pseudopodia approximately 90 pm in diameter were also detected.

Gene expression in A-549 cells was changed after addition of DDMC with antago-miR-155 compared to the control group (Figure 6). Differences were observed in separate genes’ expression levels. The expression of some genes was increased on different days, whereas that of other genes was fully inhibited after the transfection procedure on other to compared to the control cells. In the control A-549 cells, the expression of PIWIL1L1, ICOS1B, GITR3A, cKITIB and KIR2DL1 genes was not detected. However, the expression of these genes was obtained in transfected cells. The expression levels of HM0X1 and EBI3 were increased more than two-fold compared controls. The BACH2 gene expression levels were found to be variable in the control cells. cKITIB gene expression was detected only after transfection with antago-miR-155 on day 21. Other genes were also investigated, but did not exhibit a response.

In vitro experiments with lung adenocarcinoma, cervical adenocarcinoma, skin adenocarcinoma, and colorectal adenocarcinoma cells, treated with antago-miR-155 result in transformation of cancer cells into non-cancerous cell forms. Treated cells had morphological and genetic changes to compare with non-treated cells. They had induced apoptotic genes, inhibited genes activating proliferation and had down-regulated genes controlling metastatic activity. Treated cells expressed markers, which did not expressed control cells without the treatment with antago-miR-155. Using of DDMC or PNVP polymers as delivery system prolong the effect of active component (antago-miR-155) until 40 ^th day providing irreversible transformation of cancerous cells into non-cancerous cell form. These carriers prevent biodegradation of antago-miR-155 (Figure 3).

[0029] Example 5. Changes of lifespan and tumor growth in treated with antago-miR-155 with DDMC carrier and non-treated mice

Methods. Cells: In our experiments, we used the RSV (Prague C strain) -induced (NCBI_TaxID: 11888) RVP3 cell line (RRID: CVCL_L978) to produce primary sarcoma in mice (NCIt: C21603). The nucleotide sequence of RSV (Prague C strain) see in NCBI nucleotide sequences base ID: J02342.1. These cells have the possibility to produce primary mouse sarcoma in 100 % of cases, and the number of transformation -defective cells is minimal compared with that of other RSV cell line strains (Rynditch et al, 1985). The Prague strain of RSV is competent for viral replication and transforms mammalian cells but does not produce virus (Sainerova and Svoboda, 1981). Animals and Passage of Tumors. Inbreed 4-week-old female C57BL/6 (n=15) mice were used for RVP3 cell inoculation to investigate the tumor-inducing activity of the cells, select the optimal concentration of cells for inoculation and the cell concentration/tumor growth rate for further therapy, determine the lifespan of the mice after tumor inoculation, and measure the changes in tumor size. Animals were purchased by SID ALEX GROUP, s.r.o. Animals were injected subcutaneously in the interscapular region with an optimal concentration of 5xl0 ⁴ of cells in 1 ml of culture medium (DMEM, 10 % FCS, and an antibiotic mixture of 100 pg/ml penicillin-streptomycin and 32 pg/ ml gentamicin). The tumors were removed aseptically, freed of necrotic material, minced with scissors and frozen in culture medium with 20 % DMSO. Some samples were very finely minced in PBS for the staining of obtained cells with Leishman-Romanowski dye. All mice were kept in a temperature-controlled environment with a 12-hour light/dark cycle and free access to food and water. Animal care and experimental procedures were conducted in compliance with the Declaration of Helsinki and were approved by an institutional animal care committee.

Design of in vivo experiments. For the main experiments, 3 groups of mice (n=10 in each group) were used. All three groups were inoculated with RVP3 cells. The first group consisted of untreated animals (control). The second group was treated with one injection of complex 24 hours after tumor inoculation (1 ^st group). The third group was treated with one injection of complex 24 hours after tumor inoculation and with the second injection one week after the first injection (2 ^nd group). The complexes of antago-miR-155 with the DDMC were injected intravenously in the tail vein of the mice.

Statistics. Kaplan-Meier plots were generated using InVivoStat software (ver. 3.7.0.0), a freely available statistical analysis system for the analysis of in vivo data (developed by Bate S. and Clark R.). The lifespan of the controls was compared with the lifespan of the mice from the 1 ^st and the 2 ^nd groups (p < .05).

The differences in the treatments performed in the 1 ^st and 2 ^nd groups and in tumor progression compared to the treatments and tumor progression in the control group were calculated as the means ±SEMs (p < .05).

Results. In in vivo experiments is the mice virus-induced model of human v-src, c-src -tyrosine kinase-dependent cancers was used (Bister, 2015; Rubin, 2011). In the series of experiments, was observed a two-fold increase in the lifespan of mice with Rous sarcoma after one injection of the complex of the antago-miR-155 with DDMC (the 1 ^st group). After two intravenous injections of the antago-miR-155 plus DDMC, the full recovery of mice inoculated with RVP3 virus-induced sarcoma cells was attained (the 2 ^nd group). Animals from the 2 ^nd group were killed with the decapitation (AVMA Guidelines for the Euthanasia of Animals: 2013 Ed.). The Kaplan-Meier plots indicated a significant difference between the lifespan of the controls and the mice from the 1 ^st and 2 ^nd groups. (Figure 4).

A significant difference was found in the tumor growth in the animals from the 1 ^st group compared with that in the control animals (Figure 5). Tumor growth in animals from the 1 ^st group was significantly decreased compared with that in animals from the control group. Were no any visual or palpable signs of tumors in animals from the 2 ^nd group till theirs deaths. The animals looked physically healthy until they were euthanized.

In the current experiments, was used the antago-miR-155 with DDMC as a treatment in a virus-induced mouse model of human cancer. In preliminary experiments, were established the toxic doses for the delivery system but not for the antago-miR-155. All treatments were administered at the optimal doses of the complex. All animals were divided into three groups: the 1 ^st group was the untreated control group, the 2 ^nd group was composed of animals with 1 intravenous treatment, and the 3rd group was composed of animals with two intravenous treatments. A significant decrease in tumor progression was observed in animals after one injection; however, all animals from this group died. However, the animals with two injections had no visible tumors and were in good physiological condition. These animals were killed on the 83 ^rd day after the start of the experiments.

[0030] Example 6. In vitro experiments with tumor cells from non-treated mice and mice with one treatment with antago-miR-155 with DDMC (1 ^st group)

Methods. Design of in vivo experiments. For the experiments, 2 groups of mice (n=10 in each group) were used. All groups were inoculated with RVP3 cells. The first group consisted of untreated animals (control). The second group was treated with one injection of complex 24 hours after tumor inoculation ( 1 ^st group). The complexes of antago-miR- 155 with the DDMC were injected intravenously in the tail vein of the mice.

In vitro experiments. Treatment of tumor cells from non-surviving animals from the 1 ^st group and the control group. Transfection solutions for tumor cells were prepared per 1 milliliter of culture medium: 10 pg of each antago-miR- 155, and 4 pl of the DDMC were mixed in 60 pl water for injection. The solution of the DDMC in water was preliminarily boiled at 120°C for 15 minutes.

Immunofluorescence labeling of tumor cells from the control group and 1 ^st group with pmKate2- Annexin4 A.

A portion of the tumor cells from the animals in the control group and the 1 ^st group was washed with PBS 3 times and put in culture medium. Twenty-four hours later, 10 pl/ml of pmKate2 -Annexin vector (mouse) and 3 pl/ml DDMC were added to the culture medium. Fluorescence was visualized every 72 hours, and images were acquired using fluorescence microscopy for the quantification of labeled cells. The percentage of cells expressing pmKate2 -Annexin deep-red fluorescent protein was calculated in magnification 40x and 60x.

Immunofluorescence labeling of tumor cells from animals in the 1 ^st group with CD4+, CD117+ and Oct4+. One portion of the tumor cells from the animals in the control group and the 1 ^st group was washed with PBS 3 times and air-dried in the air. Then, immunofluorescence labeling was performed. Separated cells were stained with a CD4+/FITC staining reagent, CD117 purified mouse anti-human antibodies, and Oct4+, and were observed and imaged with fluorescence microscopy.

Results. The changes in the tumor cells from the mice with one injection of the antago-miR-155 with DDMC carrier were observed. These cells were labeled with Annexin A4 for the detection of changes in the tumor cells.

In a series of studies, a portion of the tumor cells from non-surviving animals in the 1 ^st group and the control group were labeled with pmKate2- Annexin A4. In tumor cells from animals in the 1 ^st group, significant nuclear translocation of Annexin A4 was detected on the 24 ^th day after the start of the in vitro experiments (Figure 6. A.). In the control group, Annexin A4 was diffusely distributed in the cells.

To investigate the morphologic and phenotypic changes in the tumor cells from non-surviving animals from the 1 ^st group, the cells were stained with the Leishman-Romanowsky method. These cells were labeled with antibodies against CD4+, CD117+, and Oct4. Cells with morphologies corresponding to tumor cells, tumor stem cells, and apoptotic tumor cells were obtained. Immunofluorescence showed the expression of Oct4 and CD117+; however, CD4+ was not expressed in tumor cells from animals in the 1 ^st group (Figure 6.B.).

Next, were used scissors to mince tumors from the 1 ^st group and put the tumor cells in culture medium. Then, was added the complex of antago-miR-155 with the DDMC delivery system at the same concentration per milliliter of culture medium to the tumor cells. On the 11 ^th day, were assessed morphological changes in these cells. Were obtained ubiquitous small oval cells with round nuclei and dark, blue, small, round cells. Immunofluorescence labeling showed the expression of CD4+, CD117+ and Oct4 in the cells (Figure 6.C.).

This invention is disclosure that antago-miR-155 treatment result in transformation of cancer cells into non-cancerous cell form. Cells taken from tumors of mice treated with under -optimal dozes of medicine with subsequent treatment in vitro with optimal doses of antago-miR-155 result in morphological changes and due to full transformation of cancerous cells into non-cancerous cell form.

[0031] Example 7. Genetic changes of cells from treated with one (1 ^st group), two (2 ^nd group) injections of antago-miR-155 and non-treated (control) mice

Methods. Samples preparations. The tumors were removed aseptically, freed of necrotic material, minced with scissors and frozen in culture medium with 20 % DMSO. Spleen and lungs were washed with sterile PBS, cut into small pieces and frozen in culture medium with 20 % DMSO for subsequent RT-PCR. One portion of the cells obtained from different organs after mouse dissections was treated with lysis buffer from the RNeasy mini kit for the further isolation of total RNA, reverse transcription reaction and specific cDNA product amplification. Tumor samples obtained from the animals in the control group and the 1 ^st group, as well as spleens and lungs from all mice, were used for the RT- PCR analyses.

Gene expression analysis: In these series of experiments, a standard two-step reverse transcription-PCR standard procedure was used. The amplification of casp8, casp3, piwill, v-src, oct4, c-myc, tgfbrl, lin28a, sca-1, dicer, cdh2, nanog, c-src, and actb cDNA (as an internal control) was performed with an automatic thermocycler. The primer sequences from 5 ’-3’ are presented below.

PCR products were loaded on a 2 % agarose gel, electrophoresed, stained with ethidium bromide, exposed in a gel doc system and quantified using Quantity One software (Bio -Rad, Germany). For expression comparison, the betaactin gene was used as the internal control.

Statistics. All gene expression data were normalized to the expression of the beta-actin internal control. Cells from the control group were used as an external control. All samples were prepared in triplicate. Quantification of the gene expression intensity was performed using Quantity One software (Bio-Rad, Germany). AltAnalyze software, used for gene expression analysis (ver. 2.1.0), is an open-source, freely available application covered under the Apache open- source license (developed by the research group of Dr. N. Salomonis, Cincinnati Children Hospital Medical Center). The data are presented as the means ± SEMs (the observed differences between the study control and experimental animals were considered statistically significant for P= .05).

Results. In addition, the gene expression profiles of the treated and untreated mice showed differences. The expressions of v-src, c-src, lin-28, nanog, c-myc and stat3 were decreased in animals after treatment, and the expression of casp8 was increased (Figure 7). c-Src gene activity is associated with processes of cellular proliferation, motility, differentiation, and survival, which are four main “whales” mediating cancer development. Was observed significant down-regulation of src genes and src -dependent pathway genes (v-myc and stat3) after the treatment of animals. Moreover, the expression of caspases was significantly increased in the cells from the animals after treatment.

Thus, in this series of experiments, was obtained the full recovery of mice from Rous sarcoma after two intravenous treatments with antago-miR-155 plus DDMC. The epigenetic modifications in tumor cells after antago-miR-155 with DDMC treatment resulted in the full transformation of sarcoma cells into other types of cells. The sarcoma cells lost their tumorigenic functions; this change was observed at the morphological and genetic levels.

The present disclosure provides that, the complex of antago-miR-155 with the DDMC delivery system induced a full recovery from v-src, c-src -tyrosine kinase-induced cancer in mice. Processes of tumor regression were accompanied by the activation/induction of apoptosis and the inhibition of v-src, c-src-tyrosine kinase-associated pathways.

This invention as proposed, to be adopt the antago-miR-155 as a new medicine for the treatment of v-src, c-src- tyrosine kinase-induced cancers, such as colon, prostate, lung and breast cancers.

The pharmaceutical compositions of the present disclosure may comprise antago-miR-155 with pharmaceutically- acceptable carrier as treatment for v-src, c-src-tyrosine kinase-dependent cancers.

The present disclosure provides that treatment of cancer cells with antago-miR-155 due to transformation of cancer cells into non-cancerous cells. This transformation based on gene expression changes result in cellular morphological transformations.

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Antago-miR-155 sequence

The primer sequences from 5’-3’were as follows: