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    • 专利摘要:
    Procedure for the effective control of cocoid insect pests. The present invention relates to a method for determining the effective flow rate of at least one semiochemical for the control of at least one pest of cocoid insects by means of an artificial semiochemical matrix, and the use of effective flow rate for the effective control of at least one pest. of cocoid insects, through the diffusion of at least one semiochemical. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2718121A1
    申请号:ES201731480
    申请日:2017-12-27
    公开日:2019-06-27
    发明作者:Fuertes Ismael Navarro；González Sandra Vacas；Llopis Vicente Navarro；Bargués Javier Marzo；Millo Jaime Primo；Garcia Alejandro Carbonell
    申请人:Ecologia Y Proteccion Agricola S L；
    IPC主号:
    专利说明:

    [0001] VARIANTS OF THE NON-VIRIONIC PROTEIN OF THE VIRUS OF HEMORAGIA
    [0002]
    [0003]
    [0004]
    [0005] The present invention relates to a set of polypeptides comprising an amino acid sequence having a sequence identity of at least 99% with the non-virionic protein (NV) of the Oncorhynchus 2 novirhabdovirus virus (known as septicemic hemorrhage virus viral, HSVV) comprising SEQ ID NO: 2, and said polypeptides further comprise at least one substitution of the natural amino acid present in any of positions 33 to 41 of said SEQ ID NO: 2, by the amino acid alanine (A ). Such polypeptides are useful for the treatment of diseases that occur with cell proliferation, in particular, for the treatment of cancer and metastasis. Therefore, the present invention is included within the field of biology, molecular biology and medicine, in particular, in the field of cancer treatment, as well as any proliferative disorder.
    [0006]
    [0007] STATE OF THE TECHNIQUE
    [0008]
    [0009] Tumors appear due to deregulation in cell proliferation, differentiation and death programs, the immune system and chronic inflammation processes having a determining role. MAPK signaling cascades (Mitogen-Activated Protein Kinase) are involved in cell proliferation, differentiation and survival or cell death. In mammals, four MAPK pathways whose terminal kinases are ERK1 / 2, JNK1 / 2/3, p38 kinases and ERK5 have been characterized. These MAPKs activate substrates that exert various functions such as gene transcription regulation and cell cycle regulation resulting in increased proliferation. In the study of tumor progression, the determining role of the pathway in which ERK participates, and the previous markers in cancer progression have been established (Shields, JM, et al., Trends Cell Biol, 2000. 10 ( 4): 147-54) denominating the RAS-RAF-MEK-ERK route. It has been estimated that ERKs can regulate the activities of 160 proteins (Yoon, S. and R. Seger. Growth Factors, 2006.
    [0010] 24 (1): 21-44) that are located in organelles, cytoplasm and mostly in the nucleus. When ERK is activated, it translocates to the nucleus where it regulates several factors of transcription resulting in gene expression changes (Zuber, J., et al., Nat Genet, 2000. 24 (2): 144-52; Schulze, A., et al., Mol Biol Cell, 2004. 15 ( 7): 3450-63).
    [0011]
    [0012] In many types of cancer, the RAS-RAF-MEK-ERK pathway is deregulated with excessive activity due to the mutation of some of its components, such as, for example, the EGFR, RAS and RAF proteins, which leads to uncontrolled proliferation. Mutations in the gene coding for B-RAF (BRAF) cause 70% of the melanoma tumors studied and to a lesser extent other tumors (Davies, H., et al., Nature, 2002. 417 (6892): 949 -54).
    [0013]
    [0014] One of the main objectives in the treatment of cancer is to decrease the deregulation of proliferation by inhibiting the initial, middle and final markers, and thus promoting apoptosis for the elimination of tumor cells. Different inhibitors have been developed for different signaling agents such as A-RAF, BRAFV600E or MEK kinases (Table 1), but over time the tumor cells generate resistance to such treatments, thus ceasing to be useful. In melanoma, resistance to treatment appears one year, while in colon carcinomas this resistance appears from the beginning of treatment because there is a feedback due to the activation of the epidermal growth factor receptor (Sun, C., et al. , Nature, 2014. 508 (7494): 118-22). More recently, compounds that inhibit ERK (Table 1) and that are functional in cells with BRAF mutations, resistant to other inhibitors (Morris, EJ, et al., Cancer Discov, 2013. 3 (7): 742-50 have been obtained ). However, the study with the largest number of cases has shown that they also show resistance to inhibition due to mutations in ERK (Jha, S., et al., Mol Cancer Ther, 2016. 15 (4): p. 548-59 ).
    [0015]
    [0016] Table 1. Inhibitors used in the RAS-RAF-MEK-ERK pathway.
    [0017]
    [0018]
    [0019]
    [0020]
    [0021] AML: Acute Myelogenous Leukemia ; CRC: Colorectal cancer, MEK: mitogen-activated protein kinase / extracellular signal-regulated kinase kinase; NSCLC: non-small-cell lung cancer, RCC: renal cell cancer.
    [0022]
    [0023] As mentioned above, in addition to the existing imbalance in MAPK pathways and their effector kinases, in cell proliferation and tumor growth there is also an imbalance between the different phases of the cell cycle, its promoter proteins (cyclines A, B, D and E), and cyclin kinase inhibitors (CDKs), promoting uncontrolled cell division. In this sense, overexpression of cyclin B1 has been determined in breast, cervical, gastric, colorectal and lung tumors (Banerjee, SK, et al., Am J Pathol, 2000.
    [0024] 156 (1): 217-25; Wang, A., et al., J Cancer Res Clin Oncol, 1997. 123 (2): 124-7; Zhao, M., et al., Exp Oncol, 2006. 28 (1): 44-8), this increase in cyclin B1 being a poor prognosis factor in several types of cancer, including breast cancer (Soria, JC, et al., Cancer Res, 2000. 60 (15): 4000-4; Suzuki, T., et al., Cancer Sci, 2007. 98 (5): 644 51; Nozoe, T., et al., Clin Cancer Res, 2002. 8 (3): 817-22). In addition, overexpression of cyclin B1 is involved in radiotherapy resistance in head and neck carcinoma (Hassan, KA, et al., Cancer Res, 2002. 62 (22): 6414-7). Studies in breast and cervical cancer cells showed that the decrease in cyclin B1 Transcriptional inhibition (inhibition by siRNA) led to lower cell proliferation and increased sensitization of cells to proapoptotic compounds (Androic, I., et al., BMC Cancer, 2008. 8: 391). Alternatively to the decrease in cell proliferation, it was observed that inhibition of cyclin B1 also induces an increase in the number of cells in the G2 / M phase, suggesting an arrest at this point in the cell cycle (Androic, I., et al. , BMC Cancer, 2008. 8: 391).
    [0025]
    [0026] The role of cyclin B1 as a promoter of cell proliferation in cancer has been revealed in other studies. Thus, an inverse relationship has been established between the concentration of the miR-379 microRNA, the concentration of cyclin B1 and the prognosis of breast cancer. Patients with breast cancer showed less concentration of miR-379 than controls, and this concentration decreased further in advanced stages of the disease. In addition, it was observed that the lower concentration of miR-379 increased the concentration of cyclin B1 (Khan, S., et al., PLoS One, 2013. 8 (7): e68753).
    [0027]
    [0028] In view of the foregoing, in the prior art there is a need to provide new alternative compounds to those described in the prior art, capable of inhibiting cell proliferation, acting both on the proteins of the pathways of MAPKs, such as cell cycle proteins, that are capable of reducing resistance to treatments already used in proliferative disorders, such as cancer, and improving the efficacy of classic treatments such as radiotherapy.
    [0029]
    [0030] DESCRIPTION OF THE INVENTION
    [0031]
    [0032] The inventors have discovered that variants (also called mutants) of the non-virionic protein (NV) of the Oncorhynchus 2 novirhabdovirus virus (known as viral septicemic hemorrhage virus, HSVV) comprising SEQ ID NO: 2, and which is encoded by the nucleotide sequence comprising SEQ ID NO: 1, it inhibits the gene and protein expression of genes and proteins involved in cell proliferation and in the cell cycle, such as MAPK pathway proteins, inflammatory proteins and cyclin, respectively. Additionally, the variants of the NV protein of the HSVV virus described in the present invention induce an increase in cell death of the cells that express them and therefore, such variants may be useful in the treatment of proliferative disorders, such as cancer. , being used in antitumor therapies either alone and / or in combination with other antitumor therapies. The variants described in the present invention are capable of improving the effectiveness of antitumor treatments, including those resistant to radiotherapy.
    [0033]
    [0034] To demonstrate the effectiveness of the polypeptide variants described in the present invention, the inventors transfected different tumor and notumoral cell lines with a vector containing the different polypeptides described herein, and observed that the expression of said variants inhibited the protein activity of the MAPK pathway. , inflammatory proteins and cyclin, and as a consequence of such inhibition, a lower cell proliferation occurred and an increase in cell death is also induced.
    [0035]
    [0036] The use of the polypeptide variants described in the present invention have the added advantage that it is possible to link them with cell internalization sequences so that, when administered to an individual, they can be internalized in the cell to exert their effect without the need to use gene therapy.
    [0037]
    [0038] Based on this discovery, a series of inventive aspects have been developed which are described below.
    [0039]
    [0040] Polypeptides of the invention
    [0041]
    [0042] In a first aspect, the present invention relates to polypeptide variants of the NV protein of the HSVV virus comprising SEQ ID NO: 2, and which is encoded by the nucleotide sequence comprising SEQ ID NO: 1, wherein said polypeptide variants they comprise at least 99% identity with the sequence SEQ ID NO: 2, or a functionally equivalent variant thereof.
    [0043]
    [0044] In the context of the present invention, "polypeptide" means a molecule formed by the binding, in a defined order, of alpha-amino acids by a peptide bond, and includes modifications or derivatives thereof, for example, glycosylation, phosphorylation, acetylation, amidation, etc. The amino acids of the polypeptide of the invention, depending on the orientation of the amino group carrying the alpha carbon atom, may belong to the L series or the D series.
    [0045] The polypeptide of the invention can be obtained by techniques widely known in the state of the art, such as chemical synthesis, genetic recombination, expression of the polynucleotide encoding the polypeptide of the invention, etc. All these techniques are routine practice for the person skilled in the art.
    [0046]
    [0047] Additionally, the carboxyl and amino terminal ends of the polypeptide of the invention may be protected against proteolysis. For example, the amino terminal end may be in the form of an acetyl group and / or the carboxyl end may be in the form of an amide group. It is also possible to carry out internal modifications of the peptides so that they are resistant to proteolysis. Examples of these internal modifications include, but are not limited to, modifications in which at least one peptide bridge -CONH- is modified and replaced by a reduced bond (CH2NH), a backlink (NHCO), an oxymethylene bond (CH2-O) , a thiomethylene bond (CH2-S), a ketomethylene bond (CO-CH2), a hydroxyethylene bond (CHOH-CH2), a bond (NN), an E-alcene bond or a -CH = CH- bond. The peptides can also be stabilized by intramolecular crossing, for example, by modifying at least two amino acid residues with oleophin side chains, preferably, C3-C8 alkenyl chains, preferably, pentel-2-yl chains, followed by crosslinking of the chains as described in the technology called "staple" (Walensky et al., 2004, Science 205: 1466-1470). All of these chemically modified peptides to resist proteolysis are also contemplated within the present invention.
    [0048]
    [0049] Additional modifications to the polypeptide of the invention comprise covalent binding to a polyethylene glycol (PEG) molecule by its terminal carboxyl end or to a lysine residue, in order to decrease its urinary elimination and therapeutic dose, and increase the half-life of the blood plasma polypeptide. The half-life of the polypeptide can also be increased by including the polypeptide in a biodegradable and biocompatible polymeric material to form microspheres that are employed as a drug delivery system. Polymers and copolymers include, but are not limited to, poly (D, L-lactide-co-glycolic) or PLGA. The techniques and procedures of how to manufacture lipid microspheres or nanocapsules for use in the administration of drugs are widely known to those skilled in the art. Any method of administration of drugs selectively targeting a tumor population can be employed in the context of the present invention.
    [0050] For the purposes of the present invention, the term "variant" or "mutant" used interchangeably throughout the present invention and referred to NV proteins of HSVV viruses and having at least one mutation, as described in the present invention and which result in increased activity in the inhibition of cell proliferation, modifying the expression of MAPK pathway and cell cycle proteins, as well as an increase in cell death, relative to wild-type NV protein (wild- type) of the VHSV virus.
    [0051]
    [0052] The variants or polypeptide mutants of the NV protein of the HSVV virus described in the present invention comprise the substitution of the natural amino acid present in any of positions 33 to 41 of SEQ ID NO: 2, by the amino acid alanine (A), giving place to the variants described in Table 2.
    [0053]
    [0054] Table 2.
    [0055]
    [0056]
    [0057]
    [0058]
    [0059]
    [0060]
    [0061]
    [0062]
    [0063]
    [0064]
    [0065] Therefore, the invention relates to a polypeptide, hence the polypeptide of the invention, comprising an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO: 2, and further comprising at least one substitution of the natural amino acid between positions 33 to 41 of SEQ ID NO: 2 with the amino acid alanine (A), or a functionally equivalent variant thereof.
    [0066]
    [0067] In a particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a replacement of the original aspartic acid (D) amino acid with alanine (A) at position 41 (D41A). In a more preferred embodiment The polypeptide of the invention is characterized in that it comprises SEQ ID NO: 20, or a functionally equivalent variant thereof.
    [0068]
    [0069] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a substitution of the original leucine (L) amino acid for alanine ( A) at position 33 (L33A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 4, or a functionally equivalent variant thereof.
    [0070]
    [0071] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a replacement of the original asparagine (N) amino acid with alanine ( A) at position 34 (N34A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 6, or a functionally equivalent variant thereof.
    [0072]
    [0073] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a substitution of the original amino acid cysteine (C) for alanine ( A) in position 35 (C35A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 8, or a functionally equivalent variant thereof.
    [0074]
    [0075] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a replacement of the original aspartic acid (D) amino acid with alanine (A) at position 36 (D36A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 10, or a functionally equivalent variant thereof.
    [0076]
    [0077] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a substitution of the original amino acid leucine (L) for phenylalanine ( F) at position 37 (F37A). In a more preferred embodiment The polypeptide of the invention is characterized in that it comprises SEQ ID NO: 12, or a functionally equivalent variant thereof.
    [0078]
    [0079] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a replacement of the original aspartic acid (D) amino acid with alanine (A) at position 38 (D38A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 14, or a functionally equivalent variant thereof.
    [0080]
    [0081] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a substitution of the original amino acid arginine (R) for alanine ( A) at position 39 (R39A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 16., or a functionally equivalent variant thereof
    [0082]
    [0083] In another particular embodiment, the polypeptide of the invention comprises an amino acid sequence that has a sequence identity of at least 99% with SEQ ID NO: 2, and further comprises a substitution of the original serine amino acid (S) for alanine ( A) at position 40 (S40A). In a more preferred embodiment, the polypeptide of the invention is characterized in that it comprises SEQ ID NO: 18, or a functionally equivalent variant thereof.
    [0084]
    [0085] In yet another more preferred embodiment, the polypeptide of the invention is selected from the list consisting of: SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 20, or a functionally equivalent variant thereof. More preferably, the polypeptide of the invention comprises SEQ ID NO: 20, or a functionally equivalent variant thereof.
    [0086]
    [0087] The family of Rhabdoviridae virus, particularly the genus Novirhabdovirus, groups four species called Oncorhynchus 1 novirhabdovirus (known as infectious hematopoietic necrosis virus, IHNV), Oncorhynchus 2 novirhabdovirus (known as viral septicemic hemorrhage virus, VHSirhabdovirus) (HIRV) and Snakehead Novirhabdovirus (SHRV). Virus infections of this genus affect more than 50 species of wild fish (Brudeseth, BE and O. Evensen, Dis Aquat Organ, 2002. 52 (1): 21-28) and farmed (Skall, HF, et al. J Fish Dis, 2005. 28 (9): 509-529) , producing large economic losses in commercial species. Novirhabdoviruses are negative polar RNA viruses whose genome codes for five virion proteins (N, P, M, G and L) and for the NV protein. The presence of the gene that codes for the NV protein is characteristic common to all viruses belonging to this genus, and therefore they have been classified as novirhabdovirus, unlike other rhabdovirus, such as SVCV virus (spring virus of the carp) that lacks the NV protein. The NV protein is necessary for efficient replication of the IHNV and HSVV novirhabdoviruses in trout and Japanese flounder (Paralichthys olivaceus), respectively. On the other hand, SHRV NV protein is not necessary for efficient replication of this virus in hot water fish.
    [0088]
    [0089] In the present invention, "identity" or "sequence identity" is understood as the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST (Altschul SF et al. J Mol Biol. 1990 Oct 5 ; 215 (3): 403-10). The BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI).
    [0090]
    [0091] The introduction of the polypeptide into the cell can be done by any of the procedures known in the state of the art, such as direct injection, electroporation, transfection, transformation, etc. but these are relatively complex techniques and with limitations when they have to be applied in vivo and access the entire population of tumor cells or that have uncontrolled cell proliferation. In case the polypeptide is to be administered to an individual, the polypeptide can be introduced into the cell by means of gene therapy techniques thanks to the use of viral vectors, or by means of cellular internalization sequences that allow the polypeptide to cross the plasma membrane.
    [0092] Thus, in a particular embodiment, the polypeptide of the invention is covalently bound to an amino acid sequence of cellular internalization.
    [0093]
    [0094] In the present invention, "cell internalization amino acid sequence" or "cell internalization sequence" or "cell penetration peptides" (CPPs) is understood as the amino acid sequences that possess the ability to transport molecules across the plasma membrane , without loss of integrity, among the most commonly used sequences are TAT, Antennapedia (Antp) and oligo-arginines, whose common characteristic is the presence of cationic amino acid groups.These internalization sequences allow internalization of the polypeptide directly to the cell As the person skilled in the art understands, the cell internalization polypeptide can come from a natural source, or it can be derived from chemical synthesis and be an artificial sequence that does not exist in nature. Also, the cell internalization polypeptide can be attached to markers (for example, without limiting to fluorophores) that you facilitate n its location.
    [0095]
    [0096] The cell internalization sequence may be linked to the polypeptide of the invention to both the amino terminal and the carboxyl terminal of the polypeptide.
    [0097]
    [0098] As previously indicated, the polypeptide of the invention can be obtained by techniques widely known in the state of the art, such as expression in a cell of the polynucleotide encoding the polypeptide of the invention, and its subsequent isolation. Therefore, in another aspect, the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", which encodes the polypeptide of the invention.
    [0099]
    [0100] The term "polynucleotide", as used in the present invention, refers to a polymeric form of nucleotides of any length and formed by ribonucleotides and / or deoxyribonucleotides. The term includes both single chain and double chain polynucleotides, as well as modified polynucleotides, that is, methylated, protected polynucleotides and the like. The polynucleotide of the invention can be DNA, RNA or cDNA.
    [0101]
    [0102] In a second aspect the present invention therefore relates to the polynucleotide encoding the polypeptide of the invention. In a preferred embodiment, the The polynucleotide of the invention is selected from the list consisting of: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 and / or any combination thereof. In yet another more preferred embodiment, the polynucleotide of the invention is selected from the list consisting of SEQ ID NO: 9, SEQ ID NO: 15 and SEQ ID NO: 19. In another even more preferred embodiment, the polynucleotide of the invention is the polynucleotide of SEQ ID NO: 19.
    [0103]
    [0104] In another aspect, the invention relates to a gene construct, hereinafter "gene construct of the invention", comprising the polynucleotide of the invention.
    [0105]
    [0106] Preferably, the gene construct of the invention comprises the polynucleotide of the invention operably linked to expression regulatory sequences of the polynucleotide of the invention. In principle, any promoter can be used in the gene constructs of the present invention, provided that said promoter is compatible with the cells in which it is desired to express the polynucleotide.
    [0107]
    [0108] A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a particular gene (nucleotide sequences). In the present invention it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention. Promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one gene or more genes that are placed in tandem with the first. "In tandem" means that the 3 'end of the first gene is followed, either consecutively or separated by a particular nucleotide sequence, by the 5' end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription in two opposite directions, that is, that a bidirectional promoter directs the transcription of two genes located divergently, that is in the opposite direction, the 5 'end of both sequences being nucleotide closer to each other than the 3 'end. In the present invention the terms "promoter" and "promoter region" are used interchangeably. In addition, the promoters in the present invention can be constitutive or inducible. The term "inducible", as used in the present description, refers to the possibility that the promoter has a control element that allows activating or deactivating (repress) the transcription of the gene that regulates, in the presence of a factor external to the promoter.
    [0109]
    [0110] Additionally, the gene construct of the invention may contain markers or tags that allow isolation of the polypeptide of the invention once it is synthesized in the cell.
    [0111]
    [0112] On the other hand, the polynucleotide or gene construct of the invention may be part of a vector. Thus, in another aspect, the invention relates to a vector, hereinafter "vector of the invention", which comprises the polynucleotide or gene construct of the invention.
    [0113]
    [0114] The person skilled in the art will appreciate that there is no limitation as to the type of vector that can be used, since said vector may be a cloning vector suitable for propagation or an expression vector. Thus, suitable vectors according to the present invention include, but are not limited to, (i) prokaryotic expression vectors such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, ColEl, pCRl, RP4, phages and "shuttle" vectors such as pSA3 and pAT28, (ii) yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids and the like, (iii) cell expression vectors of insects such as pAC series and pVL series vectors, (iv) plant expression vectors such as pIBI series, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like and (v) expression vectors in upper eukaryotic cells well based on viral vectors, including, but not limited to, adenoviruses, adenovirus-associated viruses as well as retroviruses and lentiviruses, as well as non-viral vectors, including, without limit ra, pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFVHis, pIND / GS, pRc / HCMV2, pSV40 / Ze02, pTRACER HCMV, pUB6N5-His, pVAXl, pZeoSV2 pCl, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.
    [0115]
    [0116] The vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector.
    [0117] Said cells can be prokaryotic or eukaryotic. By way of example, the vector where said DNA sequence is introduced can be a plasmid or a vector that, when introduced into a host cell, is integrated into the genome of said cell and replicated together with the chromosome / s in the / those that have been integrated. The obtaining of said vector can be carried out by conventional methods known to those skilled in the art.
    [0118]
    [0119] Therefore, in another aspect, the invention relates to a cell, hereinafter "cell of the invention", which comprises a polypeptide, a polynucleotide, a gene construct or a vector according to the present invention, for which said cell has been able to be transformed, transfected or infected with the construct or vector provided by this invention Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art.In a particular embodiment, said host cell is a animal cell, preferably a mammalian cell and more preferably a human cell, transfected or infected with an appropriate vector.
    [0120]
    [0121] Suitable host cells for expression of the polypeptide of the invention include, without limitation, mammalian cells, plant cells, insect cells, fungal cells and bacterial cells. Mammalian cells suitable for the present invention include epithelial cell lines (swine, human, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.). .), glial cells (murine, human, etc.), liver cell lines (mono, etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER.C6, human ECCs 5 NTERA-2 cells, D3 cells of the mESCs line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells and hMSCs cells ( human mesenchymal stem cells), and GliNS2 cells (glioma stem cells).
    [0122]
    [0123] Uses of the polypeptide of the invention
    [0124]
    [0125] The inventors have discovered that the NV protein of the VHSV virus (SEQ ID NO: 2), as well as the variants or mutants that are derived from said protein and that are described in the present invention, and more particularly the variants of SEQ ID NO: 10, 16 and 20, more particularly the variant of SEQ ID NO: 20, are capable of inhibit cell proliferation, by cell arrest in the G2 / M phase, in particular, to inhibit the proliferation of tumor cells, preferably HeLa cells, and also to promote cell death of cells expressing said variants, which allows the use of the polypeptides of the invention in the treatment of proliferative disorders, such as tumors or cancer, present in a subject.
    [0126]
    [0127] Thus, in another aspect, the invention relates to the use of a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, in preparation of a medicine. Alternatively, the present invention relates to a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2 for use as a medicine. The techniques and procedures for developing medications are described below.
    [0128]
    [0129] Thus, in another aspect, the invention relates to the use of a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, in preparation of a medicament for the prevention and / or treatment of tumors, cell proliferation and / or metastasis. Alternatively, the present invention relates to a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2 for use as a medicine for the prevention and / or treatment of tumors, cell proliferation and / or metastasis. The techniques and procedures for developing medications are described below.
    [0130]
    [0131] In a preferred embodiment of these aspects of the invention, the polypeptide comprises SEQ ID NO: 2. In another preferred embodiment, the polypeptide is a polypeptide comprising an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO: 2, and also a substitution of the original aspartic acid (D) amino acid with alanine (A) at position 41 (D41A), or a substitution of the original aspartic acid (D) amino acid with alanine (A) in the position 36 (D36A), or a substitution of the original amino acid arginine (R) with alanine (A) at position 39 (R39A).
    [0132] In another preferred embodiment of the uses of the polypeptides of the invention, these are characterized in that the polypeptide is selected from the list consisting of SEQ ID NO: 10, SEQ ID NO: 16 or SEQ ID NO: 20. In another embodiment Even more preferred, the polypeptide is the polypeptide comprising SEQ ID NO: 20.
    [0133]
    [0134] In another preferred embodiment of the uses of the polypeptides of the invention, these are characterized in that the tumors are benign or malignant and / or to prevent cell proliferation and / or prevent metastasis.
    [0135]
    [0136] In the present invention, "treatment" is understood as the set of means that are used to treat, alleviate or cure a disease, in particular, diseases that occur with proliferative disorders, such as tumors or cancer.
    [0137]
    [0138] In this report, "proliferative disorders" means any growth of a tissue by uncontrolled cell proliferation, such as, for example, benign prostatic hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, Pulmonary fibrosis, arthritis, glomerulonephritis and post-surgical stenosis and restenosis. Proliferative disorders, also sometimes called tumors, can be benign or malignant. A tumor is considered benign when the cells that make up the tumor do not invade other tissues or cause metastases in other parts of the body. Normally, the benign tumor is well encapsulated and the cells have no structure changes. On the contrary, a tumor is considered to be malignant when the cells that form the tumor invade adjacent tissues, which is known as metastasis, and its cells have anaplasia. In general, malignant tumors are known as cancer. Thus, in a particular embodiment, "malignant proliferative disorders" or "malignant tumors" comprise, but is not limited to: carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodking lymphoma, hair cell lymphoma, and Burkett lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannommas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, pigmentary xeroderma, keratoacantoma, thyroid follicular cancer and Kaposi's sarcoma.
    [0139]
    [0140] It is known in the state of the art of the existence of tumor or cancerous stem cells that, in addition to possessing the typical properties of a stem cell, that is, self-renewal and the ability to differentiate into multiple types of cells, are resistant to Conventional treatments and persist in tumors as a distinct population, causing relapse and metastasis of the tumor by giving rise or origin to new tumors. Thus, in a particular embodiment, the tumor comprises stem cells, in particular tumor stem cells.
    [0141]
    [0142] In addition to the therapeutic applications that the peptides of the invention may have and the inventive aspects derived therefrom, their application in experimental in vitro tests is also possible . Therefore, in another aspect, the invention relates to the use of the polypeptides described in this section as a reagent to inhibit cell proliferation in vitro .
    [0143]
    [0144] Thus another aspect of the invention relates to the use of a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2, as a reagent to inhibit cell proliferation in vitro . In a more preferred embodiment, the polypeptide comprises SEQ ID NO: 2.
    [0145]
    [0146] In another more preferred embodiment of this aspect of the invention the polypeptide comprises the polypeptides, polynucleotides, the gene construct, the vector or the cell of the invention. In another more preferred embodiment, the polypeptide comprises the sequences from the list consisting of: SEQ ID NO: 10, 16 or 20. In yet another more preferred embodiment, the polypeptide comprises SEQ ID NO: 20.
    [0147]
    [0148] In the present invention, "inhibition of cell proliferation" is understood as the reduction, decrease, attenuation or blockage of cell division or cycle.
    [0149] In the present invention, "subject" is understood as any animal, preferably a mammal, more preferably a primate, in particular, a human being, of any race, sex or age.
    [0150]
    [0151] Another aspect of the present invention relates to the use of a polypeptide comprising an amino acid sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2, as a reagent to inhibit in vitro the expression of inflammatory proteins selected from the list consisting of: mx, ERK-1 and IL-8; or the activity of regulatory proteins of the cell cycle selected from the list consisting of cyclin B and A. In a preferred embodiment of the present aspect of the invention, it is characterized in that the polypeptide comprises SEQ ID NO: 2. In one embodiment more preferred, the polypeptide of the invention is characterized in that it inhibits the expression of ERK-1 and cyclines A and B.
    [0152]
    [0153] In another preferred embodiment, this aspect of the invention is characterized in that the polypeptide comprises at least one of the polypeptides of the present invention, more preferably it comprises a polypeptide comprising an amino acid sequence having a sequence identity of at least 99 % with SEQ ID NO: 2 and a substitution of the original aspartic acid (D) amino acid with alanine (A) at position 41 (D41A), or a substitution of the original aspartic acid (D) amino acid with alanine (A) in the position 36 (D36A), or a substitution of the original amino acid arginine (R) with alanine (A) at position 39 (R39A). In another more preferred embodiment, the polypeptide comprises SEQ ID NO: 10, 16, 20 and / or any combination thereof.
    [0154]
    [0155] Pharmaceutical Composition of the Invention
    [0156]
    [0157] As explained in the previous inventive aspect, the polypeptide, polynucleotide, gene construct, vector or cell of the invention can be used in the preparation of a pharmaceutical composition.
    [0158]
    [0159] Therefore, in another aspect, the invention relates to a pharmaceutical composition, hereinafter, pharmaceutical composition of the invention, comprising a polypeptide having a sequence identity of at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2, as describes in the present invention, in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
    [0160]
    [0161] In the present invention, "pharmaceutical composition" or "medicament" is understood as any pharmaceutical preparation or form, whose composition formula expressed in units of the international system, is constituted by a substance or mixture of substances, with constant weight, volume and percentages , prepared in legally established pharmaceutical laboratories, packaged or labeled to be distributed and marketed as effective for diagnosis, treatment, mitigation and prophylaxis of a disease, physical anomaly or symptom, or the restoration, correction or modification of the balance of the organic functions of the human beings and animals. The preparation of the pharmaceutical composition can be carried out by any of the methods described in the state of the art.
    [0162]
    [0163] The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the minimum amount of the compound or pharmaceutical composition of the invention necessary to produce the desired effect and, in general, will be determined, among other factors, by the characteristics of said compound or said pharmaceutical composition and the therapeutic effect to be achieved. "Adjuvants" and / or "pharmaceutically acceptable carriers" that can be used in the compositions of the invention are widely known in the state of the art.
    [0164]
    [0165] In the present description, the term "vehicle" refers to a diluent or excipient with which the active ingredient is administered. Such pharmaceutical vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, or may come from a natural source or be a diluent or excipient that does not exist naturally. Preferably water or aqueous solutions of saline solution and aqueous solutions of dextrose and glycerol are used as vehicles, particularly for injectable solutions. Preferably, the vehicles of the invention are approved by the regulatory agency of a state or federal government or are listed in the US Pharmacopoeia or other pharmacopoeia generally recognized for use. in animals, and more particularly in humans. The vehicles and auxiliary substances necessary to manufacture the desired pharmaceutical form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical form of administration chosen. Said pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.
    [0166]
    [0167] In a preferred embodiment of the pharmaceutical composition of the invention, this is characterized in that the polypeptide comprises SEQ ID NO: 2.
    [0168]
    [0169] In another preferred embodiment of the composition of the invention, it is characterized in that the polypeptide has a sequence identity of at least 99% with SEQ ID NO: 2, and also a replacement of the original aspartic acid amino acid (D) by alanine (A) at position 41 (D41A), or a substitution of the original aspartic acid (D) amino acid with alanine (A) at position 36 (D36A), or a substitution of the original arginine amino acid (R) by alanine (A ) in position 39 (R39A). Preferably, the polypeptide comprises SEQ ID NO: 20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof. In a more preferred embodiment, the pharmaceutical composition of the invention may further comprise at least one of the polypeptides that are selected from the list consisting of: SEQ ID NO: 22, SEQ ID NO: 26 and / or any combination thereof.
    [0170]
    [0171] The compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, they can be, without being limited to, in aqueous or non-aqueous solutions, in emulsions or in suspensions. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with various inert carriers or excipients, including but not limited to: binders, excipients, dispersing agents, lubricants, glidants, sweetening agents, or flavoring agents. Additionally, the composition of the invention may comprise an adjuvant. By "adjuvant" is meant any substance that enhances the effectiveness of the pharmaceutical composition of the invention.
    [0172]
    [0173] In a particular embodiment, the pharmaceutical composition of the invention further comprises a chemotherapeutic agent.
    [0174] By "chemotherapeutic agent" is meant any substance that is capable of inhibiting cell proliferation without necessarily killing the cell, or that is capable of inducing cell death. Agents capable of inhibiting cell proliferation without causing cell death are generically called cytostatic agents, while those that are capable of inducing cell death normally by activating apoptosis are generically called cytotoxic agents. Non-limiting examples of chemotherapeutic agents suitable for use in the compositions of the invention include, but are not limited to, (i) microtubule stabilizing agents such as taxanes, paclitaxel, docetaxel, epothilones and laulimalides, (ii) kinase inhibitors such as Iressa (R), Gleevec, Tarceva ™, (Erlotinib HCl), BAY-43-9006, (iii) antibodies specific for receptors with kinase activity including, but not limited to, Trastuzumab (Herceptin (R)), Cetuximab (Erbitux (R) ), Bevacizumab (Avastin ™), Rituximab (ritusan (R)), Pertuzumab (Omnitarg ™); (iv) mTOR pathway inhibitors, such as rapamycin and CCI-778; (v) Apo2L1Trail, (vi) anti-angiogenic agents such as endostatin, combrestatin, angiostatin, thrombospondin and vascular endothelial growth inhibitor (VEGI); (vii) antineoplastic vaccines including activated T cells, nonspecific immunopotentiating agents (for example interferons, interleukins); (viii) antibiotic cytotoxic agents such as doxorubicin, bleomcin, dactinomycin, daunorubicin, epirubicin, mitomycin, mitozantrone, etc; (ix) alkylating agents such as Melphalan, Carmustine, Lomustine, cyclophosphamide, ifosfamide, Chlorambucil, Fotemustine, Busulfan, Temozolomide and thiotepa; (x) antineoplastic hormonal agents such as Nilutamide, Cyproterone Acetate, Anastrozole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin, Toremifene Citrate, Letrozole, Flutamide, Megestrol Acetate and Gelsrelin Acetate; (xi) gonadal hormones such as cyproterone acetate and medoxiprogesterone acetate; (xii) antimetabolites such as Cytarabine, Fluorouracil, Gemcitabine, Topotecan, Hydroxyurea, Thioguanine, Methotrexate, Colaspase, Raltitrexedo and Capicitabine; (xiii) anabolic agents such as nandrolone; (xiv) steroid adrenal hormones such as methylprednisolone acetate, dexamethasone, hydrocortisone, prednisolone and prednisone; (xv) antineoplastic agents such as Carboplatin, Cisplatin, Oxaliplatin, Etoposide and Dacarbazine and (xvi) topoisomerase inhibitors such as topotecan and irinotecan.
    [0175] Such compositions and / or their formulations may be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, intraarticular. , intratumoral, oral, enteral, parenteral, intranasal, ocular or topical. A preferred route of administration of the compositions and / or formulations of the compound of the invention for the prevention or treatment of a cancer is the intratumoral route. In a particular embodiment, the pharmaceutical composition of the invention is formulated for oral, parenteral, nasal or sublingual administration.
    [0176]
    [0177] Invention kit
    [0178]
    [0179] The administration of the polypeptide of the invention requires a series of components that can be arranged together in the form of a pack or kit.
    [0180]
    [0181] In another aspect, the invention relates to a kit, hereinafter "kit of the invention", comprising a polypeptide, a polynucleotide, a gene construct, a vector or a cell of the invention described in the above inventive aspects together to their corresponding particular embodiments.
    [0182]
    [0183] Particularly, the kit of the invention comprises a polypeptide having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 2. More preferably, the polypeptide comprises SEQ ID NO: 2.
    [0184]
    [0185] In another particular embodiment of the kit of the invention, this is characterized in that the polypeptide comprises an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO: 2 and, in addition, a substitution of the acidic amino acid original aspartic (D) by alanine (A) at position 41 (D41A), a substitution of the original aspartic acid (D) amino acid by alanine (A) at position 36 (D36A), or a substitution of the amino acid arginine (R) original by alanine (A) at position 39 (R39A). More preferably, the polypeptide is selected from the list consisting of SEQ ID NO: 20, SEQ ID NO: 10 or SEQ ID NO: 16. In a more preferred embodiment the kit of the invention comprises the peptides of SEQ ID NO: 2 , 20, 10 and 16. In another more preferred embodiment, the kit of the invention further comprises peptides comprising SEQ ID NO: 22 and / or SEQ ID NO: 26.
    [0186] Components useful for administration of the polypeptide of the invention and which may be included within the kit include, but are not limited to, buffer solution, lysis solution, sterile material (syringes, swabs, swabs, tweezers, etc.), distilled water , alcohols (ethanol), etc. Additionally, the kit may contain instructions or indications that guide the person skilled in the art in the administration of the polypeptide of the invention.
    [0187]
    [0188] In another aspect, the invention relates to the use of the kit of the invention for the determination of the effect of said polypeptide of the invention on the tumorigenicity of a cell line, to inhibit cell proliferation in vitro and / or to inhibit in vitro the inflammatory protein activity selected from the list consisting of: mx, ERK-1 and IL-8; preferably ERK-1, or the regulatory protein activity of the cell cycle selected from the list consisting of cyclin B and A.
    [0189]
    [0190] The terms and expressions used in the present inventive aspect have already been defined in previous inventive aspects.
    [0191]
    [0192] Method of treatment of the invention
    [0193]
    [0194] In another aspect, the invention relates to a method for the treatment and / or prevention of tumors in a subject, both benign and malignant (cancer or metastasis), which comprises the administration to said subject of a therapeutically effective amount of the polypeptide, of the polynucleotide, of the gene construct, of the vector, of the cell or of the pharmaceutical composition according to the invention.
    [0195]
    [0196] The invention also relates to a method for inhibiting (in vitro / in vivo) the activity of inflammatory proteins selected from the list consisting of: mx, ERK-1 and IL-8; preferably ERK-1, or the regulatory protein activity of the cell cycle selected from the list consisting of cyclin B and A.
    [0197]
    [0198] The terms and expressions used in the present inventive aspect have already been defined in previous inventive aspects. In turn, all the particular embodiments described above in the present invention are applicable to the methods of the invention.
    [0199] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0200]
    [0201] BRIEF DESCRIPTION OF THE FIGURES
    [0202]
    [0203] FIG. 1. Mortality analysis of EPC cells expressing the NVGFP (A) or GFP (B) protein. Quantification of the mortality of EPC cells expressing the NVGFP or GFP (C) protein, the difference between them being statistically significant (p <0.05).
    [0204] Fig. 2. Analysis of the expression of the mx gene in ZF4 cells transfected with the plasmids encoding the native NV (wild-type) VHSV protein and the different NV mutants described in the present invention. Asterisks (*) denote statistically significant differences ( p <0.05 ) with respect to the native NV HSV protein (wt-NV VHSV).
    [0205] Fig. 3. Analysis of the expression of the mx gene in ZF4 cells transfected with the plasmids encoding the native NV protein (wild-type), the different NV mutants described in the present invention and the NV proteins of IHNV, SHRV and HIRV Asterisks (*) denote statistically significant differences ( p <0.05) with respect to the native NV HSV protein (wt-NV VHSV).
    [0206] Fig. 4. Analysis of the expression of the IL8 gene in ZF4 cells transfected with plasmids encoding the native NV (wild-type) VHSV protein and the different NV mutants described in the present invention. Asterisks (*) denote statistically significant differences (p <0.05) with respect to the native NV HSV protein (wt-NV VHSV).
    [0207] Fig. 5. Analysis of ccnbl gene expression encoding cyclin B1 protein in ZF4 cells transfected with plasmids encoding VHSV native NV (wild-type) protein and for the different NV mutants of VHSV of the invention. The graph also shows the expression of the IL8 gene to see the correlation between both genes. Asterisks (*) denote statistically significant differences (p <0.05) with respect to the native NV HSV protein (wt-NV VHSV).
    [0208] Fig. 6. Analysis of the gene expression of ccnal, ccndl and ccnel in HeLa cells after transfection treatment with plasmids expressing NV41 protein (SEQ ID NO: 20), NV41GFP (SEQ ID NO: 64) at 96hpt or control at 48hpt (Control48).
    [0209] Fig. 7. Analysis of ccnbl gene expression in HeLa cells after transfection treatment with plasmids expressing at 48hpt and 96hpt the NV41 protein (SEQ ID NO: 20), NV41GFP (SEQ ID NO: 64), GFP (SEQ ID NO: 30) at 48hpt or control at 48hpt.
    [0210] Fig. 8. Analysis of erkl gene expression in HeLa cells after transfection treatment at 48 and 96hpt with plasmids expressing the NV41 protein (SEQ ID NO: 20), NV41GFP (SEQ ID NO: 64) or control (a 48hpt).
    [0211] Fig. 9. Ct values obtained in the qPCR for the gadph (gray bars) and erkl (black bars) genes in the analyzed samples of HeLa cells transfected with plasmid NV41 (SEQ ID NO: 21) at 48hpt, 96hpt and compared with a control at 48h. Fig. 10. Analysis of ERK1 kinase protein expression in HeLa cells transfected with plasmids expressing GFP proteins (SEQ ID NO: 30), NV41 (SEQ ID NO: 20) or control at 48hpt and 96hpt. The data is standardized by GFP at 48hpt for samples at this time, and by control at 96hpt for samples at this time.
    [0212] Fig. 11. Analysis of cyclin B protein expression in HeLa cells transfected with plasmids expressing GFP proteins (SEQ ID NO: 30), NV41 (SEQ ID NO: 20) at 48hpt and 96hpt or control at 48hpt and 96hpt. The data has been normalized with the expression of GFP at 48hpt.
    [0213] Fig. 12. Analysis of cyclin A protein expression in HeLa cells transfected with plasmids expressing GFP proteins (SEQ ID NO: 30), NV41 (SEQ ID NO: 20) or control at 48hpt and 96hpt. The data has been normalized by the expression of GFP at 48hpt.
    [0214]
    [0215] EXAMPLES
    [0216]
    [0217] The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    [0218]
    [0219] MATERIAL AND METHODS
    [0220]
    [0221] Cell lines
    [0222] The cell lines used were HeLa, from cervical carcinoma (ATCC® CCL-2 ™), the zebrafish cell line (Danio rerio) ZF4 (ATCC® CRL-2050 ™) and the EPC fish cell line (ATCC® CRL -2872 ™). All of them were grown in a 5% CO2 atmosphere, with DMEM medium: F12 (SIGMA, Spain), ZF4 and HeLa cells, and EPC cells with RPMI 1640 medium (SIGMA, Spain). Media were supplemented with 2 mM L-glutamine (Gibco, USA), 1mM sodium pyruvate (Gibco, USA), penicillin / streptomycin (Gibco, USA) and 10% fetal calf serum (FBS). ZF4 cells were maintained at 28 ° C and the rest of the lines at 37 ° C.
    [0223]
    [0224] Plasmids used
    [0225] By means of standard PCR techniques (Polymerase Chain Reaction) the primary sequence of the NV gene of the strain of the VHSV-07.71 virus (GenBank No. AJ233396: SEQ ID NO: 1) was obtained. This gene was cloned into an expression vector called pMCV 1.4 comprising the cytomegalovirus promoter. From the nucleotide sequence of the natural NV of HSV (SEQ ID NO: 1) nine mutated NV proteins were obtained where each of the mutated proteins comprised the change of the original amino acid to alanine (A) between positions 33 to 41, both inclusive, of SEQ ID NO: 2. The mutants obtained are: L33A (SEQ ID NO: 4), N34A (SEQ ID NO: 6), C35A (SEQ ID NO: 8), D36A (SEQ ID NO: 10 ), F37A (SEQ ID NO: 12), D38A (SEQ ID NO: 14), R39A (SEQ ID NO: 16), S40A (SEQ ID NO: 18), D41A (SEQ ID NO: 20).
    [0226]
    [0227] The mutant proteins L33A (SEQ ID NO: 4), D36A (SEQ ID NO: 10) and R39A (SEQ ID NO: 16), were generated by PCR using overlapping primers, and cloned into the same vector as previously used , the vector pMCV 1.4. On the other hand, mutants N34A (SEQ ID NO: 6), C35A (SEQ ID NO: 8), F37A (SEQ ID NO: 12), D38A (SEQ ID NO: 14), S40A (SEQ ID NO: 18) and D41A (SEQ ID NO: 20) by chemical synthesis (Invitrogen, Thermofisher, USA), and subsequently cloned into the expression vector mentioned above, the vector pMCV 1.4.
    [0228]
    [0229] For the comparative examples included herein, the above-mentioned pMCV 1.4 vector has been used in which the nucleotide sequences encoding the NV protein of the IHNV virus have been cloned (Oregon69 strain, UniProtKB: locus NV_IHNVO, no. Access Q08455 SEQ ID NO: 22), NV of SHRV virus (Accession No. NC_000903.1 SEQ ID NO: 24), NV of HIRV Virus (Accession No. U47847.1 SEQ ID NO: 26) or protein GFP fluorescent (SEQ ID NO: 30).
    [0230] Plasmid Transfection
    [0231] ZF4 or HeLa cells were distributed in 24-well culture plates (SPL Life Sciences Co., Ltd) at a concentration between 50-100 x103 cells / well, 24 hours before use. For the transfection of the different plasmids, indicated above, which code for the different natural and mutant NVs proteins described in the present invention, Lipofectamine 3000 was used following the manufacturer's instructions (Thermofisher, USA). ZF4 and EPC cells transfected with the different plasmids encoding the native and mutant NVs proteins of the present invention were collected at 48h post-transfection (hpt), while in the case of HeLa cells they were collected at 48hpt and 96hpt
    [0232]
    [0233] RNA extraction and retrotranscription
    [0234] After the estimated transfection time, the cells are collected from the culture plates by the use of trypsin (Gibco, Thermofisher). Subsequently, RNA extraction is carried out through the use of the E.Z.N.A.® HP Total RNA Kit (OMEGA bio-tek, USA). An additional step of DNAse incubation was performed to ensure that we completely removed the DNA from each sample. RNA quantification is carried out by Nanodrop ND1000 (Thermo Scientific, USA). To obtain the cDNA, an RNA retrotranscription was performed with the PrimeScript ™ RT reagent Kit (Takara, Japan) following the manufacturer's instructions.
    [0235]
    [0236] Quantitative PCR (qPCR)
    [0237] For the quantification of the different RNAs, these were re-transcribed to cDNAs and subsequently qPCRs were performed using different primer pairs (Table 3) and the SYBR green technology of the SYBR® Premix Ex Taq ™ commercial kit (Tli RNaseH Plus) (TAKARA, Japan ), following the manufacturer's instructions. To perform the qPCR, the ABI 7500 Fast real-time PCR equipment (ABI, Thermofisher, USA) was used. The reference genes used to normalize the expression of the genes of interest have been the RPLP0 gene for ZF4 cell cultures and the GADPH gene for HeLa cell cultures. The formula for obtaining the amount of expression is based on the Delta Ct method, with DeltaCt = CtRef-Ctgen and the reference genes RPLP0 (ZF4) and GADPH (HeLa), and subsequently calculating the power according to the Pot = 2 "Del * formula aCt
    [0238] Table 3. Sequences of the primers used in qPCR. n ni - Ani ni -
    [0239]
    [0240] n n i - An i n i -
    [0241]
    [0242]
    [0243]
    [0244] Hosa: HeLa human cell line. Dre: Danio rerio (Zebrafish).
    [0245]
    [0246] In the comparative study of the expression of transcripts in ZF4 with the different versions of the NV protein, both native and mutant, a normalization of each power value of mx was performed by its relative power of expression. For example, for the NV41 mutant, the power ratio of mx / power NV41 was made.
    [0247]
    [0248] Western blot
    [0249] In order to determine the relative amount of proteins involved in promoting tumor progression, HeLa cells transfected with plasmids expressing GFP and mutant NV41 (SEQ ID NO: 20) have been used, and compared with a control of untransfected cells The tests were performed at 48hpt and 96hpt. Western-blot (WB) determined by monoclonal antibodies (Santa Cruz Biotechnology, Quimigen) the amount of cyclin B1 proteins (sc-245 monoclonal antibody), cyclin A (sc-271682 monoclonal antibody) and ERK1 kinase (monoclonal antibody sc-271269). Briefly, the transfected cells were collected at the mentioned study times and the total soluble protein was extracted with PBS in the presence of protease inhibitors (cOmplete Protease Inhibitor Cocktail, Sigma-Aldrich), by 3 freeze-thaw cycles. Once quantified by the Nanodrop 1000 spectrophotometer (Thermo Scientific, USA), 10 micrograms of total soluble protein were subjected to 10% PAGE-SDS and subsequent transfer to nitrocellulose membrane. Next, WB was carried out as standard using the concentrations of the monoclonal antibodies suggested by the commercial house. WB image capture was performed with the Gel DocTM XR + documentation system (BioRad) and the relative quantification of the bands was performed with Image Lab software v5.2 (BioRad). The WB study was repeated in three independent trials.
    [0250]
    [0251] Flow cytometry
    [0252] The viability of EPC cells (Epithelioma Papulosum Cypríni) was analyzed by flow cytometry with the BD FACSCANTO II cytometer (BD Biosciences, Spain) at 48hpt with: (a) the plasmid expressing the GFP protein, and (b) the plasmid which expresses the NVGFP construct, with the NV protein being the native protein of SEQ ID NO: 1. The results were analyzed with the FACSDiva v6.1.3 program. The study was done in duplicate.
    [0253]
    [0254] Statistic analysis
    [0255] The results obtained are shown as mean ± the standard error of the mean (SEM). The significant differences between the means of two, three or more data sets were calculated using ANOVA (One-way ANOVA). The differences between 2 data sets and their level of significance were calculated using the Student's t-test. The criterion of statistical significance in all cases was established at p <0.05.
    [0256]
    [0257] Example 1. The HSV NV protein (SEQ ID NO: 2) increases the mortality of EPC cells.
    [0258]
    [0259] The cellular viability of the EPC (Epithelioma Papulosum Cypríni) cells was determined by flow cytometry at 48h post-transfection with: (a) the plasmid comprising the nucleotide sequence SEQ ID NO: 29 encoding the GFP protein of SEQ ID NO : 30, and (b) the plasmid comprising the nucleotide sequence SEQ ID NO: 27, which codes for the NVGFP protein of SEQ ID NO: 28. The results obtained demonstrate that there is an increase in mortality (1.65 times) of the EPC cells transfected with the plasmid encoding the NVGFP construct (SEQ ID NO: 28) with respect to EPC cells transfected with the plasmid encoding the GFP protein (SEQ ID NO: 30) (Figure 1).
    [0260]
    [0261] These results show that the NV protein (SEQ ID NO: 2) induces an increase in mortality in the cells in which it is expressed.
    [0262] Example 2. The mutants of the invention inhibit the gene expression of mx and IL-8 in zebrafish cells (ZF4).
    [0263]
    [0264] The analysis of the expression of the mx gene in ZF4 cells expressing the native NV protein of VHSV, wt-NV (SEQ ID NO: 2) or any of the mutants analyzed in the present invention: NV33 (SEQ ID NO: 4), NV34 (SEQ ID NO: 6), NV35 (SEQ ID NO: 8), NV36 (SEQ ID NO: 10), NV37 (SEQ ID NO: 12), NV38 (SEQ ID NO: 14), NV39 (SEQ ID NO : 16), NV40 (SEQ ID NO: 18) or NV41 (SEQ ID NO: 20), have shown that mutants NV36 (SEQ ID NO: 10), NV39 (SEQ ID NO: 16) and NV41 (SEQ ID NO: 20) decrease the gene expression of mx with respect to natural NV (wt) (Figure 2). Specifically, said mutants NV36 (SEQ ID NO: 10), NV39 (SEQ ID NO: 16) and NV41 (SEQ ID NO: 20) induce an inhibition of mx gene expression of the order of 3.3 times, 2 times and 5 times respectively, regarding the wt-NV protein.
    [0265]
    [0266] Additionally, expression of the mx gene in ZF4 cells expressing the NV protein of other different novirhabdoviruses such as: IHNV (SEQ ID NO: 22), snakehead rabdovirus (SHRV) (SEQ ID NO: 24) and hirame rabdovirus (also analyzed ) HIRV) (SEQ ID NO: 26). The results show that SHRV NV protein (SEQ ID NO: 24) induces an increase in the expression of the mx gene (Figure 3). These results are consistent with the information in the state of the art where it is shown that the NV protein of this virus does not behave like the rest of NV proteins of other novirhabdoviruses (Alonso M., et al. J Virol. 2004 Jun; 78 (11): 5875-82).
    [0267]
    [0268] On the other hand, the NV proteins of IHNV (SEQ ID NO: 22) and HIRV (SEQ ID NO: 26) inhibit mx expression , even more than the natural NV of VHSV (SEQ ID NO: 2), but less than the mutant NV41 (SEQ ID NO: 20) (Figure 3).
    [0269]
    [0270] In addition to the analysis of the expression of the mx gene , the expression of the IL8 gene in ZF4 cells transfected with the various NV plasmids described in the present invention and mentioned above was also analyzed. The results obtained have been similar to those shown for the mx gene , with the cells expressing the mutants NV36 (SEQ ID NO: 10), NV39 (SEQ ID NO: 16) and NV41 (SEQ ID NO: 20) the highest inhibitory capacity show the expression of the IL8 gene (Figure 4).
    [0271] Example 3. The mutants of the invention inhibit the gene expression of cyclin B1 in zebrafish cells (ZF4).
    [0272]
    [0273] In addition to the expression of the mx and IL8 genes , it was analyzed in ZF4 cells that express the different NV proteins (mutants and native) described in the invention, the expression of the ccnbl gene that codes for cyclin B1, a protein that regulates a point of control (checkpoint) G2 / M of the cell cycle and that allows mitosis of the cell.
    [0274]
    [0275] The results show that mutants NV35 (SEQ ID NO: 8), NV36 (SEQ ID NO: 10), NV37 (SEQ ID NO: 12), NV39 (SEQ ID NO: 16) and NV41 (SEQ ID NO: 20) they are those with the greatest inhibitory capacity of ccnbl gene expression (Figure 5). In addition, the correlation in the same graph of the expression levels of IL8 and ccnbl is analyzed to observe the behavior of each mutant, determining that they are the mutants NV36 (SEQ ID NO: 10), NV39 (SEQ ID NO: 16) and NV41 (SEQ ID NO: 20) are those that show a greater inhibition of the expression of both genes.
    [0276]
    [0277] Example 4. The mutant protein NV41 (SEQ ID NO: 20) inhibits the gene expression of ERK1 and cyclines A and B in cervical cancer cells (HeLa).
    [0278]
    [0279] Once the inhibitory capacity of the NV41 protein (SEQ ID NO: 20) on the transcription of the cyclin B1 gene ( ccnbl gene ) in zebrafish cells ZF4 was demonstrated, the effect that the mutant protein NV41 (SEQ ID NO) was analyzed : 20) can cause human HeLa tumor cells. To that end, said cells were transfected with the plasmids encoding the mutant protein NV41 (SEQ ID NO: 20), the GFP control protein (SEQ ID NO: 20) and its comparison with a control (untransfected cells analyzed thereto times) and were cultivated for 48h and 96h post-transfection. Subsequently, the expression levels of cyclins A, B, D, E, proteins that participate in the cell cycle, and ERK1 kinase, were determined as a signaling and effector molecule of the MAPK pathway, by quantitative PCR (qPCR).
    [0280]
    [0281] The HeLa cell line study showed that mutant protein NV41 (SEQ ID NO: 20) inhibits the expression of ccnal gene transcripts encoding for cyclin A1 and ccnbl gene transcripts encoding cyclin B1, with respect to control (untransfected cells) at 48hpt, and said inhibition is even greater than 96hpt (Figure 6 and 7, respectively). On the other hand, cyclin D1 (encoded by the ccndl gene ) increases its expression of transcripts in those HeLa cells that express the mutant protein NV41 (SEQ ID NO: 20), while the cyclin E1 (encoded by the ccnel gene ) remains unchanged in said cells whether or not they express the mutant protein NV41 (SEQ ID NO : 20) (Figure 6). Similarly, cyclin B1 transcript expression levels also decreased in HeLa cells expressing NV41 (SEQ ID NO: 20) relative to HeLa cells expressing GFP (SEQ ID NO: 30), and at those expressing NV41GFP construction (SEQ ID NO: 64) (Figure 7). As seen in said Figure 8, the expression of NV41GFP (SEQ ID NO: 64) in HeLa cells induced an increase in the concentration of ccnbl gene transcripts (cyclin B1) at both 48hpt and 96hpt.
    [0282]
    [0283] Additionally, erkl gene expression levels were analyzed for their relevance as a signaling protein in the MAPK pathway. The results showed that erkl gene expression decreases by action of NV41 (SEQ ID NO: 20). This decrease in erkl is time dependent since at 48hpt a very significant decrease in its expression is observed, but at 96hpt it was so low that it was not possible to detect the expression of said gene in HeLa cells expressing NV41 (SEQ ID NO: 20) (Figure 8). However, at this same time (96hpt) the transcripts of the gadph internal control gene are detected (Figure 9), indicating that said absence of expression is not due to cell culture degradation, but to the absence of gene expression induced by the NV41 mutant protein (SEQ ID NO: 20).
    [0284]
    [0285] Example 5. The mutant protein NV41 (SEQ ID NO: 20) inhibits the protein expression of ERK1 and cyclin B1, and increases the protein expression of cyclin A1 in HeLa cells.
    [0286]
    [0287] Once it has been demonstrated that the different mutant proteins described in the present invention, particularly the mutant NV41 (SEQ ID NO: 20), modulate the gene expression of erkl, mx, IL-8, cyclin b1 and cyclin a1, it is analyzed whether said modulation at the gene level it is also produced at the protein level.
    [0288]
    [0289] For this, protein expression was determined by Western-blot (WB) with the use of monoclonal antibodies (see materials and methods) that detect ERK1, cyclin B1 and cyclin A1 in cell extracts of HeLa cells treated at different times, as described previously. As seen in Figure 10, the cells HeLa transfected with the plasmid expressing the mutant protein NV41 (SEQ ID NO: 20) show a reduction in ERK1 expression by 32% compared to cells treated with GFP (SEQ ID NO: 30), and 26% compared to the control (untransfected cells), analyzed at 48hpt. On the other hand, ERK1 expression at 96hpt showed that GFP expression was reduced by 4% with respect to the control (non-transfected cells), while HeLa cells transfected with the plasmid expressing the mutant protein NV41 (SEQ ID NO : 20) showed a decrease in ERK1 expression by 45% compared to the control (non-transfected cells) (Figure 10).
    [0290]
    [0291] These data correlate with those shown for erkl gene expression (Figure 8) and demonstrate that the mutant protein NV41 inhibits ERK1 gene and protein expression by interfering with the MAPK pathway.
    [0292]
    [0293] Next, the protein expression of cyclines B and A, at 48 and 96hpt, was analyzed, as indicated above. Regarding cyclin B1, HeLa cells at 48 hpt show reduced expression of said protein when treated with the plasmid that expresses mutant protein NV41 (SEQ ID NO: 20) by 30% compared to cells treated with GFP ( SEQ ID NO: 30), and show a level similar to the control (untransfected cells) (Figure 11). Expression of cyclin B1 at 96hpt shows that HeLa treated with GFP (SEQ ID NO: 30) decreased cyclin B expression by 24% relative to the control (untransfected cells), while HeLa cells transfected with the plasmid that Expressing mutant protein NV41 (SEQ ID NO: 20) decreased cyclin B expression by 43% compared to the control (non-transfected cells) (Figure 11). In addition, 25% less cyclin B is observed in HeLa cells treated with NV41 compared to those treated with GFP. These data correlate with the data obtained for ccnbl gene expression , demonstrating that the mutant protein NV41 (SEQ ID NO: 20) lowers the protein and protein levels of cyclin B.
    [0294]
    [0295] When protein expression of cyclin A is analyzed by WB, HeLa cells treated with GFP (SEQ ID NO: 30) or NV41 (SE ID NO: 20) for 48 hpt maintain cyclin A expression with respect to control (untransfected cells ), although it decreases with respect to cells transfected with GFP (Figure 12). However, at 96hpt the expression of cyclin A increases in HeLa cells treated with NV41 (SEQ ID NO: 20) or GFP (SE ID NO: 30) with respect to the control (untransfected cells) and regarding their 48hpt counterparts (Figure 12). However, it is observed that at 96hpt the HeLa cells treated with NV41 (SEQ ID NO: 20) decrease the cyclin A with respect to the cells treated with GFP (SEQ ID NO: 30), indicating that NV41 inhibits the expression of cyclin A in 20% -30%. These results contrast in part with those shown in Example 4 for cyclin A, where ccnal gene expression at both 48hpt and 96hpt is decreased by treatment with the mutant protein NV41 (SEQ ID NO: 20). One possible explanation for this fact is that there is not always a real-time correlation between the amount of mRNA transcripts and the amount of protein. This could be due to the fact that cyclin A protein could have a longer half-life, that is, it degrades less and in order to detect a decrease thereof by inhibiting mRNA expression, such expression should be analyzed at times longer than those carried out in the examples shown here.
    权利要求:
    Claims (21)
    [1]
    1. Variation of the non-virionic protein of the Oncorhynchus 2 novirhabdovirus virus comprising an amino acid sequence having a sequence identity of at least 99% with SEQ ID NO: 2, and a replacement of the original aspartic acid (D) amino acid by alanine (A) at position 41 (D41A), or a substitution of the original aspartic acid (D) amino acid with alanine (A) at position 36 (D36A), or a substitution of the original arginine amino acid (R) by alanine ( A) at position 39 (R39A), and which also inhibits cell proliferation and / or metastasis.
    [2]
    2. Variant according to claim 1 characterized in that it comprises SEQ ID NO: 20, SEQ ID NO: 10 or SEQ ID NO: 16.
    [3]
    3. Polynucleotide encoding a variant according to any one of claims 1 to 2.
    [4]
    4. Gene construct comprising a polynucleotide according to claim 3.
    [5]
    5. Vector comprising a polynucleotide according to claim 3 or a gene construct according to claim 4.
    [6]
    6. A cell comprising a variant according to any one of claims 1 to 2, a polynucleotide according to claim 3, a gene construct according to claim 4 or a vector according to claim 5.
    [7]
    7. Use of a variant according to claim 1, in preparation of a medicament.
    [8]
    8. Use of a variant according to claim 1, in the preparation of a medicament for the treatment and / or prevention of tumors, cell proliferation and / or metastasis.
    [9]
    9. Use according to any of claims 7 to 8 wherein the variant comprises SEQ ID NO: 20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof.
    [10]
    10. Use according to claim 8, wherein the tumor is a malignant or benign tumor.
    [11]
    11. Use according to claim 10, wherein the malignant tumor is cancer.
    [12]
    12. Use of a variant according to claim 1, as a reagent to inhibit cell proliferation in vitro .
    [13]
    13. Use according to claim 12 wherein the variant comprises SEQ ID NO:
    20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof.
    [14]
    14. Use of a variant according to claim 1, as a reagent for inhibiting in vitro ERK-1 protein expression; or the activity of regulatory proteins of the cell cycle selected from the list consisting of cyclin B and A.
    [15]
    15. Use according to claim 14 wherein the polypeptide comprises SEQ ID NO:
    20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof.
    [16]
    16. Pharmaceutical composition comprising a variant according to claim 1, in a therapeutically effective amount, and a pharmaceutically acceptable carrier.
    [17]
    17. Pharmaceutical composition according to claim 16 characterized in that the variant comprises SEQ ID NO: 20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof.
    [18]
    18. Pharmaceutical composition according to any of claims 16 to 17, further comprising a chemotherapeutic agent.
    [19]
    19. Kit comprising a variant according to claim 1.
    [20]
    20. Kit according to claim 19, wherein the variant is selected from the list consisting of SEQ ID NO: 20, SEQ ID NO: 10, SEQ ID NO: 16 and / or any combination thereof.
    [21]
    21. Use of the kit according to any of claims 19 to 20, to inhibit cell proliferation in vitro and / or to inhibit ERK-1 protein activity in vitro ; or the activity of regulatory proteins of the cell cycle selected from the list consisting of cyclin B and A.
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    同族专利:
    公开号 | 公开日
    MA51427A|2021-04-07|
    PE20211309A1|2021-07-20|
    EP3732964A1|2020-11-04|
    BR112020012967A2|2020-12-01|
    US20210059246A1|2021-03-04|
    WO2019129908A1|2019-07-04|
    AU2018397349A1|2020-07-09|
    ES2718121B2|2020-05-21|
    CL2020001704A1|2020-12-18|
    引用文献:
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