PL236028B1 - Application of oligonucleotide molecule for modulation of inflammatory processes and/or processes depending on FAN protein - Google Patents

Description

Description of the invention

The invention relates to an oligonucleotide molecule for use in reducing an inflammatory response or preventing an increase in an inflammatory response in an organism. More specifically, the solution relates to the use of an oligonucleotide molecule to modulate inflammatory processes, and in particular to providing a new therapeutic method based on the action of an oligonucleotide molecule in the treatment of diseases, disorders or clinical conditions in the pathogenesis of which the FAN protein is involved and / or which are associated with inflammatory processes, malfunctioning of the immune system or an autoimmune response. The solution concerns the use of a plant-derived oligonucleotide molecule or a synthetic variant thereof, which by negatively regulating the expression of the FAN protein (factor associated with the activation of neutral sphingomyelinase) reduces or prevents an increase in the inflammatory response of the body.

The human body has many defense mechanisms against threats from the environment - inflammatory processes are one of them. Inflammation is defined as a complex, albeit dynamic sequence of interactions occurring in living, vascularized tissues between various biological factors (e.g. cytokines, chemokines, interferon or complement) and cells (e.g. macrophages, NK cells and leukocytes) following the action of a damaging stimulus. [1-3]. The aforementioned stimulus may be of a non-specific nature, including external (biological, physical, chemical) and internal factors (emboli, infarctions or neoplasms), and specific - related to an immune response [4]. Tumor necrosis factor alpha - TNF-α is a key player in regulating specific and non-specific responses and modulating inflammatory processes in the body. This protein, which is an inflammatory cytokine, is produced by activated cells of the immune system and its controlled synthesis is necessary to fight infection and promote the repair of damaged tissues [5-8]. Deregulation of TNF-α expression and the participation of T and B lymphocytes is a very important, and even critical factor causing the development of immune diseases such as rheumatoid arthritis, psoriasis, multiple sclerosis, atherosclerosis or Crohn's disease [9-14]. Currently, there are several effective methods of treating the aforementioned diseases, including anti-TNF-α therapies with the use of antibodies, synthetic or fusion proteins [15-17]. Among the approved drugs, the mechanism of which is based on anti-TNF-α activity, we can find, among others: Infliximab, Adalimumab, Certolizumab, Golimumab and Etanercept [18-22]. Despite the proven effectiveness, these therapies have many undesirable side effects - from increased susceptibility to infections (mainly), through an increased risk of lymphoma, reactivation of tuberculosis, or even in extreme cases, an increased likelihood of developing another type of autoimmune disease [23-27 ]. For this reason, further work is underway on new therapies to avoid or even reduce these side effects, and at the same time give a chance to cure patients who do not respond in any way to the available treatments. One such recently proposed potential therapeutic target for the treatment of immune diseases is the FAN protein (neutral sphingomyelinase activation factor), encoded by the NSMAF gene. The FAN protein is an adapter molecule that, by interacting with the NSD domain of the TNF-R1 receptor, modulates the expression of genes induced by TNF-α, and more specifically genes encoding inflammatory proteins such as interleukin 6 (IL-6) or chemokines - e.g. CCL5, CCL9 , CCL20 and CXCL-2 [28-30]. Studies in mice have shown that in individuals with a knockout NSMAF gene, the TNF-α-induced expression of these pro-inflammatory molecules was selectively altered. This resulted in a disturbance of lymphocyte population recruitment in secondary lymphoid organs, which was observed by reducing the number of macrophages, neutrophils, dendrites and lymphoid cells in the spleen [28, 29]. Apart from the important role of the FAN protein in inflammatory processes, it has been shown that it may also be responsible for the invasiveness and spread of melanoma cells [31].

As mentioned in the previous paragraph, the therapies available for the treatment of autoimmune diseases can have many adverse effects, and the effectiveness of these agents is often impaired due to the short half-life of the target molecules. An alternative to this type of method can be RNA-based therapies, which are divided into 3 main types: RNA interference (RNAi, including siRNA and miRNA), antisense oligonucleotides and steric blocking oligonucleotides. All these approaches are based on the antisense mechanism, namely the attachment of a complementary oligonucleotide to the target RNA and the formation of base pairs with it [32-35]. The exact difference between these technologies, however, is related to a further one

They are part of the way they work and as a result of their final functionalities. RNAi and antisense oligonucleotides regulate gene expression by inducing enzymatic degradation of the target mRNA, thereby reducing the amount of the gene product causing the disease [32, 34, 36]. In the case of prostate-blocking oligonucleotides (and in some situations also RNAi), the modulation of the target mRNA's function is not associated with its degradation, but with blocking its access to the translational machinery, which in the end also prevents the formation of the disease protein. An alternative product of the action of this type of molecules is the repair of abnormal RNA or, by modulation of the pre-mRNA assembly process, the synthesis of a new protein [35, 37].

The antisense effect of the synthetic oligonucleotide molecule was first presented in the late 1970s by Zamecnik and Stephenson [38]. Since then, there has been a huge interest and dynamic development of this method, focusing its activities on the newer and newer "improvement" of oligonucleotides by various types of chemical modifications (e.g. the backbone or sugar ring) of native RNA or packing the molecule into appropriate transporters that will enable the use of these oligonucleotides for therapeutic purposes [39-42]. Currently, more and more therapies based on RNA technology are entering the phase of clinical trials, and several of them have already achieved considerable success in this field, such as Fomivirsen or Mipomersen [34, 36, 43, 44]. The reports of Zhang et al. And Wang et al., Who presented evidence for the presence and stability of plant miRNAs in human blood, are also extremely interesting from the point of view of the regulation of gene expression using oligonucleotide molecules of natural origin [45, 46]. Moreover, Zhang et al. Showed that one of the molecules detected by them, MIR168a, may negatively regulate the expression of the gene encoding the low density lipoprotein receptor 1 protein (LDLRAP1) [45]. These reports open up new perspectives for natural medicine and treatments for many known diseases.

Diseases related to inflammatory processes, improper functioning of the immune system or autoimmune response are a serious problem all over the world - it is estimated that autoimmune diseases affect up to 7% of the population [47]. Undoubtedly, therefore, new measures / methods for the treatment of this type of disease are necessary, and therapeutic targets such as the FAN protein in combination with the use of oligonucleotide molecules (especially of natural origin) may be an ideal alternative.

Patent application WO2012153854 (published 2012-11-15) describes novel agents for modulating the expression of cytokine-chemokine genes related to inflammation and infection. In particular, the invention provides an antisense nucleotide which comprises a sequence complementary to the 3'UTR of the CCL2, CCL20, CX3CL1, IL-23A, CD69, NF-kB p65, TNF-α gene. Fam89a, Grk5. Phospholipid scramblase 1, Runx1, semaphorin 4A. Steap4, Psmb10 β lymphotoxin or TLR2 induced by IL-13, and which is capable of modulating gene expression; sense oligonucleotide containing a sequence complementary to an endogenous antisense transcription product containing a sequence complementary to the 3'UTR of the gene and which is capable of modulating expression of the gene; an agent that prevents and / or treats inflammation or infection and contains a sense or antisense nucleotide.

Patent application AU2012201409 (published 2012-03-29) relates to inhibition of tumor necrosis factor alpha (TNF-α) by RNAi by silencing TNF-α mRNA expression of TNF receptor I cell surface receptors (TNFRI), or by silencing mRNA expression of the enzyme TNF-α converter (TACE / ADAM17). Silencing such TNF-α targets is primarily useful for treating patients with TNF-α-dependent disorders or at risk of developing TNF-α-dependent disorders, such as ocular disorders such as dry eye, allergic conjunctivitis, and ocular inflammation, as well as such like dermatitis, rhinitis, or asthma.

Patent application WO2012115469 (published 2012-08-30) relates to a functional anti-inflammatory food composition for oral use, and in particular relates to a method of isolating active ingredients effective in preventing inflammation and relieving symptoms from grain and fruit. The anti-inflammatory food composition contains an extract for oral use. To this end, a composition according to the invention is prepared by obtaining an extract of Morus alba L. (mulberry), black turtle beans, onion, and mixing the said extracts in an appropriate ratio. The composition prepared in this way can be used as an anti-inflammatory component in painkillers, and is effective in preventing and relieving the symptoms of arthritis, as it has (to an appropriate extent) greater antioxidant and anti-inflammatory effects, reduces the effects of edema, has an inhibitory effect on acute and chronic conditions. inflammatory, inhibits the expression of lipoxygenase, cyclooxygenase-2, induced nitric oxide synthase (iNOS)

PL 236 028 B1 and collagenase type I, which are pro-inflammatory enzymes, and inhibits the production of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-Ιβ), interleukin 6 (IL-6) and prostaglandin, which are pro-inflammatory factors.

Patent application KR101090177 (published 2011-12-06) relates to an RNA aptamer antagonistic to IL-32 to inhibit TNF-α expression and to develop a therapeutic agent for the treatment of inflammatory diseases. The RNA aptamer has the sequence SEQ. ID No. 7 and binds to IL-32 as an antagonist. The RNA aptamer blocks the function of the internal IL-32. The RNA aptamer has a high affinity for IL-32. The RNA aptamer is used to develop a therapeutic agent for the treatment of chronic inflammation such as rheumatoid arthritis.

Patent application WO03070897 (published 2003-08-28) relates to methods and reagents useful in modulating the expression of genes of the TNF superfamily and the TNF superfamily receptor, in a variety of applications, including therapeutic and diagnostic application. In particular, the invention relates to small nucleic acid molecules such as short interfering RNA (siRNA), dsRNA, miRNA, shRNA, siNA, RNAi, capable of carrying out RNA interference (RNAi) against the expression of TNF superfamily genes and TNF superfamily receptor and / or his activities. Small nucleic acid molecules are useful in the treatment of septic shock, rheumatoid arthritis, HIV and AIDS, psoriasis, inflammatory and autoimmune diseases, and many other diseases or disorders in response to modulation of TNF and / or TNF receptor expression or activity.

The patent application WO2015026249 (published 2015-02-26) describes the use of a plant-derived miR172 molecule or its synthetic equivalent, selected from miR172a or miR172b for reducing inflammatory processes in the body, a method for reducing B and T lymphocyte proliferation, as well as methods for lowering protein levels FAN (a factor associated with the activation of neutral sphingomyelinase). The aim of the present invention is to provide a new therapeutic method based on miRNA molecules which, by interacting with the mRNA encoding the FAN protein, negatively regulate its expression and thus reduce the inflammatory response of the organism.

Patent application WO2014003742 (published 2014-01-03) describes methods of preventing or inhibiting inflammation in a patient. In one aspect, the method comprises administering to a patient diagnosed with an inflammatory disease an effective amount of an anti-inflammatory agent that (1) inhibits the expression of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3, and / or CXCR5, or (2) inhibits the interaction between CXCR3 and CXCL9, CXCL10 or CXCL11 or between CXCR5 and CXCL1 3, or (3) inhibits the biological activity of CXCL9, CXCL10, CXCL11, CXCL13, CXCR3 and / or CXCR5, which agent includes an antibody, antibody fragment, short interference RNA (siRNA), aptamer , synbody, binding agent, peptide, aptamer-siRNA chimera, single-stranded antisense oligonucleotide, triplex forming oligonucleotide, ribozyme, EGS sequences or an expression vector encoding a factor.

Patent application US2003166159 (published 2003-09-04) discloses an isolated polypeptide that binds to a DNA binding domain located from -550 to -487 in the promoter of the human TNF-alpha gene. This isolated polypeptide is referred to herein as a LITAF protein. Also disclosed is a nucleic acid sequence that encodes the LITAF protein. Such nucleic acid molecules can be inserted into an expression vector that can be introduced into a cell. LITAF-dependent induction of TNF-alpha gene expression in cells can be inhibited by delivering an inhibitor of LITAF gene expression to the cell. Such an inhibitor can be, for example, a construct that encodes an anti-sense RNA molecule that is complementary to a LITAF mRNA fragment and that is greater than 200 nucleotides in length. Preferably, the antisense RNA molecule is complementary to the translation initiation site, upstream of the contiguous 5'UTR sequence and downstream of the contiguous LITAF coding sequence. Optimal lengths and specific nucleotides for complementarity are also discussed in the application. Inhibition of the induction of LITAF-dependent expression of the TNF-alpha gene in cells can also be achieved by attaching an inhibitor of LITAF protein function to the LITAF protein in the cell. Such an inhibitor may be an antibody that binds to the LITAF protein or alternatively a small molecule that inhibits the function of the LITAF protein. An example of such an inhibitor is a recombinant mutant of the LITAF protein. Administration of a LITAF inhibitor can be a therapeutic method for treating patients with diseases related to chronic inflammation - e.g., rheumatoid arthritis, gum disease, Crohn's disease, and graft versus host disease. Therapeutic methods for treating patients with diseases in which TNF-alpha plays a role in pathology are also presented. Examples of such diseases are diabetes, cancer, cachexia, breast cancer, HIV, sepsis, malaria, trypanosomy, and asthma. Other methods of the invention include methods of identifying genes that are regulated by proteins

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LITAF, a method for identifying a molecule that inhibits the binding of LITAF to the TNF-alpha promoter, and a method for identifying proteins, molecules that bind LITAF from the matrix.

Patent application US2004009503 (published 2004-01-15) describes the immunomodulatory activity of human ribonuclease. Human extracellular ribonuclease (RNase) is widely distributed in various organs and body fluids along with other members of the mammalian RNase A superfamily. In addition to their ribonuclease activity, some RNases have specific biological activities, for example angiogenic, antitumor and antiviral properties. However, the molecular mechanisms of these properties are not fully understood. Using RCA protein microarrays, the influence of EDN (RNase 2), ECP (RNase 3) and RNase 1 on the production of cytokines by leukocytes was investigated. The levels of 78 different cytokines and growth factors present in the culture supernatants were measured to determine the cytokine profile of cells exposed to various combinations of RNases and RNase inhibitors. Members of the human ribonuclease family (such as RNase 1, hEDN (RNase 2) and RNase 3) induced the expression of certain sets of cytokines in human leukocytes, including ENA-78, EOT2, BLC, GDNF, 1309, IFN-alpha, IFN-gamma, IL-10, IL-12p40, IL-12p70, IL-13, IL-16, IL-18, IL-1 beta, IL-1ra, IL-2Sra, IL-3, IL-6, IL-6SR, IL-7, IL-8, IP-10, MCP-1, MCP-2, MCP-3, MCSF, MIG, MDC, MIP-1 alpha, MIP-1 beta, MPIF-1, NAP-2, RANTES, sCD23, OSM, TARC, TNF-alpha, TNF-R1 and uPAR. Therefore, members of the RNase superfamily are therapeutic targets in the treatment of inflammatory diseases and clinical conditions. Inhibiting or increasing RNase expression is used to modulate the immune system and is beneficial in the host's defense against various diseases and is used as an adjuvant. Expression of RNases is a diagnostic marker for inflammation related disorders and serves to define the different stages of the disease. Moreover, the expression of cytokines, chemokines, and growth factors is used to monitor the effectiveness of RNase therapy.

Patent application CN102397269 (published 2012-04-04) describes the use of certain chalcone compounds in the preparation of medicaments for the treatment of diseases associated with inflammation. The use of chalcone compounds can inhibit the expression and release of various inflammatory factors such as TNF-alpha, IL-6, COX-2, IL-1 beta, and IL-12; nuclear translocation associated with the nuclear factor kappaB can be largely inhibited; Phosphorylation of ERK and p38 inflammatory signaling pathways may also be inhibited. As a result of in vivo experiments with these compounds, the survival rate of LPS-injected mice was significantly improved.

Patent application US2014193366 (published 2014-07-10) describes a miR-323-3p inhibitor for the prevention or treatment of rheumatoid arthritis, containing a sequence capable of hybridizing with miR-323-3p in the form of an oligonucleotide or vector from which such an oligonucleotide is transcribed, or in the form of a "trap" or "sponge" miRNA.

Patent application P-378879 (Application 2003-12-24) describes methods of treating inflammatory or immune conditions. Methods of treating inflammatory or immune conditions using inhibitors of IL-1 and inhibitors of B-cell or T-cell activation are provided. Methods of treating inflammatory or immune conditions using inhibitors of TNF and inhibitors of B-cell or T-cell activation are provided.

Patent application P-369739 (Application 2002-11-11) discloses a modified anti-TNF-alpha antibody. The invention relates to the modification of human tumor necrosis factor alpha (TNF-alpha) reactive antibodies to obtain anti-TNF-alpha antibodies that are substantially non-immunogenic or less immunogenic than any unmodified counterpart when used in vivo. The invention also relates to peptide molecules containing T cell epitopes of the V regions of the starting antibody that have been modified by amino acid change to reduce or eliminate said epitopes in a lymphocyte.

Patent application WO2008116267 (published 2008-10-02) discloses methods and compositions for treating or preventing inflammatory processes or conditions related to inflammation in a patient, comprising administering to the patient an effective amount of at least one antagonist for one or more miRNAs overexpressed in a patient. disease states and inflammatory responses to an allergen. The invention also provides methods for the diagnosis of inflammatory diseases depending on the miRNA expression profile.

Patent application WO2010118979 (published 2010-10-21) relates to methods and compositions for use in the treatment of specific conditions. Compositions of the invention include serum preparations such as activated serum preparations. The invention also relates to use

Blood serum preparations for the treatment of diseases and disorders related to inflammation and / or unwanted activation of the immune system, e.g. periodontitis, abortus habitualis, ulcerative colitis, polymuscular pain syndrome, disorders associated with cervical spine injuries, endometriosis, unexplained infertility and adenomyosis.

Despite its proven effectiveness, the solutions presented in the state of the art, or the currently available therapies in the treatment of diseases / conditions associated with inflammatory processes, may have many undesirable effects. Moreover, their effectiveness may be impaired due to the short half-life of the target molecule and the low stability of the "therapeutic" molecule itself. It is therefore necessary to continue searching for new drugs and treatments for these diseases. One of the recently proposed potential therapeutic targets, incl. in autoimmune diseases, there is the FAN protein which, through its interaction with the tumor necrosis factor alpha receptor 1 (TNF-R1), mediates inflammatory responses in the body [28-30]. On the other hand, an alternative to the methods that deal with the rapid degradation of the therapeutic molecules used may be oligonucleotide molecules in the form of duplex. The current scientific reports show that duplex RNA molecules, especially those containing chemical modifications of nucleotides, are more stable and more resistant to the action of nuclease [48-50]. Moreover, thanks to the currently available various synthetic techniques / laboratory methods, it is possible to obtain them easily and efficiently, both in a synthetic and natural form, isolated from plants [51-56].

The aim of the present invention is to provide a new therapeutic method that, by using the oligonucleotide molecule in the form of a duplex, allows to reduce the level of the FAN protein in diseases dependent on the FAN protein and / or reduce the inflammatory reaction of the organism in various types of immune diseases. In the described invention, the oligonucleotide molecule in the form of a duplex is represented by a molecule consisting of a strand having the RNA sequence SEQ. ID No. 1 and its complementary strand having the RNA sequence of SEQ. ID No. 3. Both strands of the duplex are of natural origin and have the 2'-O-methylation of the ribose of the last nucleotide at the 3 'end.

The bioinformatic analysis performed for the purposes of the invention showed that the above-described oligonucleotide molecule in the form of a duplex can interact with the mRNA encoding the FAN protein, and thus negatively regulate its expression. In vitro experiments carried out on human fibroblasts with the use of ELISA test confirmed that the presence of the oligonucleotide molecule in the form of a duplex significantly reduces the level of the FAN protein.

The realization of such a defined goal and the presentation of evidence for the action of the described oligonucleotide molecule in the form of a duplex, resulting in the reduction of the FAN protein level in cells, has been achieved in the present invention.

The present invention relates to a plant derived oligonucleotide molecule or a synthetic variant thereof as defined by the RNA sequence SEQ. ID No. 1, having a sequence of 6 out of 7 uninterrupted nucleotides, defined by the RNA sequence of SEQ, in position 2-8 from the 5'-end of the molecule. ID No. 2 and having at least 80% sequence similarity to SEQ. ID No. 1, wherein the oligonucleotide molecule is an antisense (leader) strand forming a duplex with at least 80% complementary to it sense (passenger) strand, the resulting duplex having at most two free, unpaired nucleotides at the 3 'end of each of its components strand, and in addition to the two free, unpaired nucleotides at the 3 'end, additionally include up to four mismatches for use in reducing an inflammatory response or preventing an increase in the inflammatory response of the body.

Preferably, the antisense (leader) oligonucleotide molecule and / or the sense (passenger) duplex oligonucleotide molecule is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.

Preferably, the sense (passenger) strand is an oligonucleotide molecule of plant or synthetic origin consisting of ribonucleotides.

Preferably, the antisense (leader) oligonucleotide molecule and / or the sense (passenger) duplex oligonucleotide molecule consists of ribonucleotides, none or at least one of which contains a ribose modification selected from the group: 2'-O-methyl ribose, 3 '-O-methyl ribose, 2'-O-methoxyethyl ribose or 2'-fluororibose.

Preferably, the inflammatory response of the body is reduced by reducing the amount of FAN protein (factor associated with the activation of neutral sphingomyelinase).

Preferably, the amount of FAN protein is reduced.

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Preferably, expression of the FAN protein is negatively regulated and the amount of the FAN protein is reduced.

Preferably, the molecule described above is used in the treatment and / or prevention of the development of diseases, disorders or clinical conditions associated with inflammatory processes, a malfunction of the immune system or an autoimmune response.

Preferably the molecule is used in the treatment and / or prevention of the development of diseases, disorders or clinical conditions in the pathogenesis of which the FAN protein is involved.

The attached figures allow for a better explanation of the essence of the invention:

Figure 1 shows the binding site of the oligonucleotide molecule to the mRNA encoding the human FAN protein, determined by bioinformatic analysis using four different programs.

Figure 2 shows the results of the determination of the FAN protein concentration (ng / ml) in fibroblasts 48 hours after transfection with the duplex oligonucleotide molecule at the three different concentrations analyzed. The determination was performed using the ELISA test, and the presented results are the average of three measurements.

Figure 3 shows the results of the determination of the FAN protein concentration (ng / ml) in fibroblasts 72 hours after transfection with the duplex oligonucleotide molecule at the three different concentrations analyzed. The determination was performed using the ELISA test, and the presented results are the average of three measurements.

Exemplary embodiments of the above-defined invention are presented below.

EXAMPLES

Example 1

In silico analysis of the interaction of an oligonucleotide molecule with the mRNA encoding the FAN protein

In order to evaluate the potential interaction of the oligonucleotide molecule presented in the invention with the mRNA encoding the human FAN protein, four bioinformatics tools were used based on different algorithms and assessing various properties of small RNA interactions: mRNA. They were: PITA [57], RNAhybrid2.2 [58], miRanda [59] and RNA22v2 [60]. Appropriate parameters were set for each of the programs mentioned, and the following were used as input sequences: sequence of the oligonucleotide molecule in duplex form represented by SEQ. ID No. 1 and the mRNA sequence encoding the human FAN protein represented by SEQ. ID No. 4. The obtained results were analyzed in terms of the consistency of the place of interaction of the oligonucleotide molecule with the mRNA encoding the FAN protein determined by the tools used and the level of assessment of this interaction.

Example 2

Assessment of the effect of a duplex oligonucleotide molecule on the level of FAN protein in cells

K21 fibroblast culture line:

Fibroblasts were grown in MEM / EBSS medium (HyClone) supplemented with amino acids (non-essential aminoacids, Gibco) and 10% FBS (inactivated bovine serum) at 37 ° C in an atmosphere of 5% CO2.

Fibroblast Transfection:

The day before transfection, fibroblasts were passaged into a 6-well plate (2.8 × 10 5 cells / well). The next day, the medium was replaced with fresh (1.75 ml). Duplex oligonucleotide to be tested (final concentration in the well 60 pM, 1 nM or 10 nM) was added to 125 μl of medium without addition of FBS and mixed with lipofectamine RNAiMAX (7.5 μl; Invitrogen) diluted in 117.5 μl of medium without addition FBS. After 5 minutes incubation at room temperature, the mixture (250 μθ was added to the cells. Cells transfected with MISSION microRNA Mimics (Sigma) at a final concentration of 10 nM (Sigma) and untransfected cells (induced with LPS) were a negative control. From the moment of transfection, cells were incubated 48 or 72 hours at 37 ° C in an atmosphere with 5% CO2. 4 hours before the end of the experiment, fibroblasts were induced with LPS (1 μg / ml). After 4 hours from the addition of LPS, cells were detached with trypsin, and then washed 3 times in buffer PBS was resuspended in 400 µl PBS buffer and stored at -20 ° C until the ELISA assay The cell transfection efficiency was checked by BLOCK-iT Alexa Fluor Red Fluorescent Oligo (Invitrogen) at a final concentration of 10 nM according to the manufacturer's protocol.

Determination of the FAN protein concentration by ELISA test:

FAN protein concentration was determined using the NSMAF Human kit (Cloud-Clone Corp.) according to the manufacturer's protocol. Absorbance was measured with a plate reader at the wavelength

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450 nm. The FAN protein concentration was calculated as the mean of the measurements from three wells based on the plotted standard curve.

DESCRIPTION OF THE RESULTS

Determination of the potential interaction of an oligonucleotide molecule with mRNA encoding the human FAN protein

The bioinformatics analysis carried out with the use of four independent tools showed that the oligonucleotide molecule presented in the invention can interact with the mRNA encoding the FAN protein. The designated mRNA potential binding site of the oligonucleotide was consistent for each of the programs used and included the coding sequence (CDS) coding region of the mRNA molecule (Fig. 1). Assessments of this impact, calculated by individual tools, were high, which may indicate a strong bond between both molecules: 1) miRanda - the assessment of the complementarity of the sequences of both molecules was 177, while the free energy of the structure was -24.16 kcal / mol; 2) RNAhybrid2.2 - the determined free energy of the structure is -27.50 kcal / mol; 3) PITA - site accessibility represented by AAG was -15.94; while in 4) RNA22v2 - the free energy of binding was -21.20 kcal / mol with a p-value of 2.73e ^-4 .

Effect of a duplex oligonucleotide molecule on the level of FAN protein in cells

A critical element in the implementation of the described invention was to study the in vitro effect of the oligonucleotide molecule presented in the invention in the form of a duplex on the level of the FAN protein. The ELISA test carried out on the K21 fibroblasts showed that the said oligonucleotide molecule in the form of a duplex at a concentration of 1 nM and 10 nM significantly reduced the level of the FAN protein (both 48 and 72 hours after transfection - Fig. 2 and Fig. 3). The greatest reduction in the FAN protein level was observed 48 hours after the transfection of the oligonucleotide molecule in the form of a duplex at 10 nM (Fig. 2).

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Sequence List

SEQ. ID No. AND

Oligonucleotide molecule, RNA sequence, 21 nucleotides long

AGAAUCUUGAUGAUGCUGCAU

SEQ. ID No. 2

"Sicd" region of oligonucleotide molecule, RNA sequence, 7 nucleotides in length

GAAUCUU

SEQ. ID No. 3

Oligonucleotide molecule, RNA sequence, 21 nucleotides long

GCAGCACCAUUAAGAUUCACA

SEQ. ID No. 4

NM 003580, mRNA protein FAN, organism - Homo sapiens, variant 1 trans crypt, RNA sequence, 3582 nucleotides long

GCCGGAGTCCCCACGCGAGGATGCTGCGGTGAGCGCGGCCGGGACGCCGCGTCGGGCTCTCGGCCGCCCA

GCGGCGCCGCAGGGGAAGCCAGGCCGGCAGCCGCGCGCTCCCCAGCCGGCCACCAATCCCGCCCTCCCCG GCTCCTCCGGACCTCCTCGGCAGGCGGCGGCGGGGCCTGCCTGTGCGCTCTGCGCCCGCCCGCGCCTACC CTCCATGGCGTTTATCCGGAAGAAGCAGCAGGAGCAGCAGCTGCAGCTCTACTCCAAGGAGAGATTTTCC TTGCTGCTGCTTAACTTGGAGGAGTACTACTTTGAACAGCATAGAGCCAATCACATTTTGCACAAGGGCA GTCACCATGAAAGGAAAATCAGAGGCTCCTTAAAAATATGTTCAAAATCGGTGATTTTTGAACCAGATTC AATATCCCAGCCCATCATCAAGATTCCTTTGAGAGACTGTATAAAAATAGGAAAGCATGGAGAAAATGGA GCCAATAGACACTTCACAAAGGCAAAATCTGGGGGTATTTCACTCATTTTCAGTCAGGTATATTTCATTA AAGAACATAATGTTGTTGCACCATATAAAATAGAAAGGGGCAAAATGGAATATGTTTTTGAATTGGATGT TCCCGGGAAAGTGGAAGATGTTGTGGAGACGTTGCTTCAGCTTCACAGAGCATCCTGCCTTGACAAATTG GGTGACCAAACCGCCATGATAACAGCTATTTTGCAGTCTCGTTTAGCTAGAACATCATTTGACAAAAACA GGTTCCAAAACATTTCTGAAAAGCTGCACATGGAATGCAAAGCAGAAATGGTGACGCCTCTGGTGACTAA TCCTGGACACGTGTGCATCACGGACACAAACCTGTATTTTCAGCCCCTCAACGGCTACCCGAAACCTGTG GTCCAGATAACACTCCAAGATGTCCGCCGCATCTACAAAAGGAGGCACGGCCTCATGCCTCTGGGCTTGG aagtattttgcacagaagatgaictgtgttccgacatctacgtaaagttctatgaacctcaagatagaga TGATCT CTATTTTTACATTGCCACATACCTAGAGCACCATGTGGCGGAGCACACTGCTGAGAGCTACATG ctgcagtggcagcgtggacacctttccaactatcagtacctccttcacctcaacaacctggccgaccgca GCTGCAACGACCTCTCCCAGTACCCTGTGTTTCCATGGATAATACATGATTATTCCAGCTCAGAACTAGA TTTGTCAAATCCAGGAACCTTCCGGGATCTCAGTAAGCCAGTAGGGGCCCTAAATAAGGAACGGCTGGAG AGACTACTGACACGCTACCAGGAAATGCCTGAACCAAAGTTCATGTATGGGAGTCACTACTCTTCCCCGG

PL 236 028 Β1

GTTATGTACTTTTTTATCTTGTTAGGATTGCACCAGAGTATATGCTGTGCCTGCAGAATGGAAGATTTGA TAATGCAGATAGAATGTTCAACAGTATTGCAGAAACTTGGAAAAACTGTCTGGATGGTGCAACGGATTTT AAAGAGTTAATTCCAGAATTCTATGGTGATGATGTGAGCTTTCTAGTCAATAGCCTGAAGTTGGATTTGG GAAAGAGACAAGGAGGACAGATGGTTGACGACGTGGAGCTTCCCCCTTGGGCTTCCAGTCCCGAGGACTT TCTCCAGAAGAGCAAAGATGCATTGGAAAGCAATTATGTGTCTGAACACCTTCACGAGTGGATTGATCTA ATATTTGGCTACAAACAAAAAGGGAGTGATGCAGTTGGGGCCCATAATGTATTTCATCCCCTGACCTATG AAGGAGGTGTAGACTTGAACAGCATCCAGGATCCTGATGAGAAGGTAGCCATGCTTACGCAAATCTTGGA atttgggcagacaccaaaacaactatttgtgacaccacatcctcgaaggatcaccccaaagtttaaaagt TTGTCCCAGACCTCCAGTTATAATGCTTCTATGGCAGATTCCCCAGGTGAAGAGTCTTTTGAAGACCTGA CCGAAGAAAGCAAAACACTGGCCTGGAATAACATCACCAAACTGCAGTTACACGAGCACTATAAAATCCA caaagaagcagttactggaatcacggtctctcgcaatggatcttcagtattcacaacatcccaagattcc ACCTTGAAGATGTTTTCTAAAGAATCAAAAATGCTACAAAGAAGTATATCATTTTCAAATATGGCTTTAT CGTCTTGTTTACTTTTACCAGGAGATGCCACTGTCATAACTTCTTCATGGGATAATAATGTCTATTTTTA ttccatagcatttggaagacgccaggacacgttaatgggacatgatgatgctgttagtmgatctgttgg CATGACA ACAGGCTATATTCTGCATCGTGGGACTCTACAGTGAAGGTGTGGTCTGGTGTTCCTGCAGAGA TGCCAGGCACCAAAAGACACCACTTTGACTTGCTGGCCGAGCTGGAACATGATGTCAGTGTAGATACAAT CAGTTTAAATGCTGCAAGCACACTGTTAGTTTCCGGCACCAAAGAAGGCACAGTGAATATTTGGGACCTC ACAACGGCCACCTTAATGCACCAGATTCCATGCCATTCAGGGATTGTATGTGACACTGCTTTTAGCCCAG ATAGTCGCCATGTCCTCAGCACAGGAACAGATGGCTGTCTTAATGTCATTGATGTGCAGACAGGAATGCT CATCTCCTCCATGACATCAGATGAGCCCCAGAGGTGCTTTGTCTGGGATGGAAATTCCGTTTTATCTGGC AGTCAGTCTGGTGAACTGCTCGTTTGGGACCTCCTrGGAGCAAAAATCAGTGAGAGAATACAGGGCCACA caggtgctgtgacatgtatatggatgaatgaacagtgtagcagtatcatcacaggaggggaagacagaca aattatattctggaaattgcagtattaagtgccttttcctctcctgaatattaaattgaactctatttaa TGCATTTTTAAACCAAACTTTTAAACGGACTGGTGAATGTGCAATGTTAGTAATTAGAAGTTTTACCACA TGGAAAATTTGTGGTTTTAAACTTTCTAAATCATGGTGACTTCATTGAAAGCCATTAGTTGCTATTCTCr TAGGGCAGATAAAATGCGGCTGTGTTAGGAAAAACATGTTACACTGTAAGGCAGATGATCGTCCCCGTAT GATGATTGTCAGAAGACAGGACTAAGTAGCAGAGAATAGCTAAGAGATAAATTGGGCTGGGGAAACTTGT CAGAAAGCACTGAACAATTAAGAAATTTTCCAAGAAAATGTGCAGTATTCTCTGCTACTTCTGAATCTGT TTTGTCTTCCTAA TCTATCACAATTGCCACCCATCGGGTTTTGGGTGTGTGTTTTCATAGCGTGGTTACT TTCTATAATGCTGTACCCAGATTCTAAGAACCTGGAGAAGGATTAGCAGTTCTTAGTAAGTTTACTGTGT ataggaacatttcctatacgagaacatttccta

Claims (9)

Patent claims 1. A plant derived oligonucleotide molecule or a synthetic variant thereof as defined by the RNA sequence SEQ. ID No. 1, having a sequence of 6 out of 7 uninterrupted nucleotides, defined by the RNA sequence of SEQ, in position 2-8 from the 5'-end of the molecule. ID No. 2 and having at least 80% sequence similarity to SEQ. ID No. 1, wherein the oligonucleotide molecule is an antisense (leader) strand forming a duplex with at least 80% complementary to it sense (passenger) strand, the resulting duplex having at most two free, unpaired nucleotides at the 3 'end of each of the two In addition to the two free, unpaired nucleotides at the 3 'end, the incinerating strands additionally contain up to four mismatches for use in reducing the inflammatory response or preventing the enhancement of the inflammatory response in the body. 2. The molecule for use according to claim 1 The method according to claim 1, characterized in that the antisense (leader) oligonucleotide molecule and / or the sense (passenger) duplex oligonucleotide molecule is of length 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides. 3. The molecule for use according to claim 1 The method of claim 1 or 2, characterized in that the sense (passenger) strand is an oligonucleotide molecule of plant or synthetic origin consisting of ribonucleotides. 4. The molecule for use according to any one of claims 1 to 3, characterized in that the antisense (leader) oligonucleotide molecule and / or the sense (passenger) duplex oligonucleotide molecule consists of ribonucleotides of which none or at least one it contains a ribose modification selected from the group: 2'-O-methyl ribose, 3'-O-methyl ribose, 2'-O-methoxyethyl ribose or 2'-fluororibose. 5. The molecule for use according to any one of claims 1 to 4, characterized in that the inflammatory response of the body is reduced by reducing the amount of FAN protein (factor associated with the activation of neutral sphingomyelinase). 6. A molecule for use according to any one of claims 1 to 5, characterized in that the amount of FAN protein is reduced. 7. The molecule for use according to any one of claims 1 to 6, characterized in that the expression of the FAN protein is negatively regulated and the amount of the FAN protein is reduced. 8. A molecule for use according to any one of claims 1 to 7 in the treatment and / or prevention of the development of diseases, disorders or clinical conditions associated with inflammatory processes, a malfunction of the immune system or an autoimmune response. 9. A molecule for use according to any one of claims 1 to 8 in the treatment and / or prevention of the development of diseases, disorders or clinical conditions in the pathogenesis of which the FAN protein is involved.