Poxviral oncolytic vectors. RU patent 2508401.

FIELD: biotechnologies.

SUBSTANCE: pox virus of variolovaccine is proposed, which includes a defect F2L gene and a suicide gene. Pox virus has oncolytic activity. Besides, a reproduction method of such pox virus and its use for treatment of proliferative diseases or diseases with increased activity of osteoclasts is proposed.

EFFECT: improving compound application efficiency.

31 cl, 12 dwg, 3 tbl

The technical field

viruses are a new class of therapeutic substances used for the treatment of cancer with the unique ability to tumor-dependent self-sufficiency (HERMISTON. A demand for next-generation oncolytic adeno viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.322-30.). viruses are capable of selective replication in malignant cells and therefore offer the levels of strength and specificity, which is potentially much bigger than standard antineoplastic therapy (FISHER. Striking out at disseminated metastases: the systemic delivery of oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.301-13.). The advantage of using these viruses is that when its replication they are lysed its host cells. Cancer cells are the perfect hosts for many viruses, and therefore, they are inactivated virus interferon way or genes-suppressors of tumors that allow unrestricted flow of viral replication (CHERNAJOVSKY, et al. Fighting cancer with oncolytic viruses. The British medical journal. 2006, vol.332, no.7534, p.170-2.).

Some viruses in nature can selectively replicate in tumor cells, but viruses can also be obtained by modification of natural viruses. With this purpose, the main strategies currently used for modifying viruses include: functional deletions in significant viral genes; tumor - or made of two fabrics-specific used to control the expression of these viral genes; and modification of the tropism to redirect adenovirus on the surface of a cancer cell. In the near future it is necessary to optimize adenoviruses, to fully realize their potential as critical anticancer instruments and, thus, improve the prognosis for patients with malignant gliomas (JIANG et al. Oncolytic adenoviruses as antiglioma agents. Expert review of anticancer therapy. 2006, vol.6, no.5, p.697-708.).

For example, ONYX-015, selectively modified adenovirus for replication and cell destruction, which have p53 mutation is designed by the company «became all swollen and Pharmaceuticals for the potential treatment of various solid tumors, including head and neck tumors, gastrointestinal and pancreatic tumors. He is a recombinant adenovirus, which carries the mutation of «loss of function» in the locus 1, the product of which is 55 kDa protein that binds to p53 tumor protein-suppressor and inactivate it. Thus, adenovirus ONYX-015 is assumed affects normal cells. Mutations in the p53 gene- tumors are the most common type of genetic pathology of cancer, which occurs in more than half of all the major types of cancer. Thus, these cells are susceptible to the virus that can easily be replicated and cause the death of cells. Research is ongoing phase III of the application of ONYX-015 regarding the treatment of relapsing cancer of the head and neck, a study of the phase II regarding the treatment of colorectal tumors, tumors of the ovary, pancreas and oral cavity, and the study of the phase I - relatively digestive diseases, tumors of the esophagus and liver (COHEN, et al. ONYX-015. Onyx Pharmaceuticals. Current opinion in investigational drugs. 2001, vol.2, no.12, p.1770-5.).

Natural viruses are a replication-competent viruses have the innate ability to selectively infect and destroy tumor cells. Despite the fact that they were used in the original efforts to treat cancer live viruses five decades ago, interest in the natural viruses behind support created by genetic engineering of adenoviruses and herpes viruses as cancer therapy. However, has recently been renewed interest in high potency and selectivity of these natural agents (ROBERTS, et al. Naturally oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.314-21.).

Among the natural virus, vaccinia viruses (from the poxviridae) have many of the key features required for an ideal viral frame for use in . They include a short half-life period, with rapid spread, strong political ability, greater ability to cloning and clear molecular biology. In addition, although they are capable of replication in human cells, they are not considered natural health problem and is especially well are administered to millions of people during the campaign for the destruction of smallpox. Early clinical results, use or strains of the vaccine or genetically modified vaccinia strains demonstrated antitumor effects (THORNE, et al. Vaccinia virus and oncolytic virotherapy of cancer. Current opinion in molecular therapeutics. 2005, vol.7, no.4, p.359-65.).

On the contrary, poxvirus is a new candidate who has no history of direct use in people, since it has a distinctive and absolute tropism for mind-owner in relation to (rabbits). Virus , as recently shown, can also selectively infect and destroy human tumor cells, unique tropism associated with intracellular signalling pathways, found in most human cancers. This review outlines the existing data on tropism to human cancer cells, as well as preclinical data demonstrating its ability to infect and destroy tumors in animal models of cancer (STANFORD, et al. Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer. Expert opinion on biological therapy. 2007, vol.7, no.9, p.1415-25.).

Technical problem

Injection of high doses of poxviruses, necessary for achievement of the antitumor effect, caused the problems associated with toxicity. The majority of adverse events represent minor adverse reactions that are typically associated with vaccinia virus are limited and include fever, headache, fatigue, , chills, local skin reaction, a nonspecific rash, multiformnuu erythema, swollen lymph nodes, and pain in the area of vaccinations. Other reactions could require additional treatment methods (e.g. VIG, first-line therapy and cidofovir, second-line therapy). Adverse reactions that might require further evaluation or treatment include unintentional inoculation, vaccinia (smallpox) (GV), eczema after vaccination (EV), progressive vaccinia (smallpox (PV), a disease of the Central nervous system, and fetal vaccinia (smallpox) (CONO, et al. Smallpox vaccination and adverse reactions. Guidance for clinicians. MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control. 2003, vol.52, no.RR-4, p.1-28.).

Thus, there is a need for safer with the same activity as their natural copies.

Preconditions of creation of the invention

In the US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 describe a modified recombinant poxvirus, especially vaccinia virus, which has inactivated minor encoded virus genetic functions so that the recombinant poxvirus had reduced toxicity and enhanced security. In particular, genetic functions inactivated by deletions open reading frame, the coding is a virulence factor, or inactivation of open reading frame, the coding is a virulence factor. In more detail, this patent describes a vaccinia virus, which open reading frame for J2R, B13R+B14R, A26L, A56R, C7L - K1L, and I4L was . This virus (NYVAC) can be designed as a vector for alien nucleic acid and used as a vaccine to induce immunological response in the animal host. However, N YVAC incapable of effectively replicated in most mammalian cells and cannot be used as virus (XIANGZHI, et al. Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2. The Journal of virology. 2006, vol.353, no.1, p.220-233.).

WO 2004/014314 (KIRN DAVID (US), 19/02/2004 describes the modified vaccinia virus, which includes one or more mutations in his viral genome. Described mutations are located in one or more of the following classes polyp : 1) interferon-modulating polypeptide; 2) complement-control polypeptide; 3) TNF or - modulating polypeptide; 4) serine protease inhibitor; 5) IL-lp-modulating polypeptide; 6) non-communicable EEV form polypeptides; and 7) viral polypeptide, which acts to inhibit the release of infectious virus from cells (anti-infective viral form of polypeptide). In addition, it reveals the mutation in the virus and vaccinia A41L or C11R.

Portions of the genome vaccinia, such as A34R, A41L, A53R, B5R, B7R, B8R, B13R, B15R, B18R, B22R, B28R, B29R, CUR, E3L, K2L, NIL, vC12L, vCKBP described in more detail in this proposal. Methods of an invention involving the use of any of poxviruses discussed by the authors. The inventors also reveal the methods of cancer treatment through the introduction of a cancer cell or the patient effective number of modified vaccinia virus.

Disclosure of the invention

Inventors to the surprise found that poxviruses, including defective F2L gene, have a superior safety profile, but retain the equivalent activity (compared to their natural copy).

The present invention relates to poxvirus, including defective F2L gene.

As used throughout the proposal, the singular shall be used in the sense that they mean "at least one", "at least one", "one or more" or "a lot" of components or steps that are referenced, unless the context clearly indicates otherwise. For example, the term "cell" includes a variety of cells, including their mixtures.

The term "and/or"used by the authors, includes the meaning of "and", "or" and "all-or any other combination of elements related to the specified term.

Used by the authors of the terms "including" and "include" means that the products, compositions and methods include components or stages, referenced, but exclude others. "Consisting essentially of"used to identify the products, compositions and methods, should mean the exclusion of other components or stages of any significant value. Thus, the composition consisting essentially of these components, does not exclude contaminants in trace quantities, and a pharmaceutically acceptable carrier. "Consisting of" shall mean the exclusion of more than elements in trace quantities of other components or stages.

Used by the authors of the term "poxvirus, including the defective gene" refers to , including the deletion, substitution or addition of one or more of the nucleic acid of the defective gene, or any combination of these opportunities, and these modifications lead to inability for the virus to produce a protein with the activity of the protein produced unmodified gene. In a preferred embodiment of the invention poxvirus, including the defective gene, concerns poxvirus, which was removed a whole gene sequence. A mutation can be implemented in many ways, known qualified in the field of technology, using recombinant methods. Methods for modifications of the genome poxvirus available in this area. For example, methods disclosed in MCCART, et al. Systemic cancer therapy with a tumor selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.. Cancer res.. 2001, no.61, p.8751-57., KIM, et al. Systemic armed oncolytic ans immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF. Molecular Therapeutic. 2006, no.14, p.361-70., WO 2004/014314 (KIRN DAVID (US), 19/02/2004 and US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 can be used to produce a poxvirus invention. The ways disclosed in this application, especially concerning the acquisition of poxvirus according to the invention. Genome sequence of the various poxviruses are available in this area, for example, the genomes of vaccinia virus, vaccinia virus, the virus , Ectromelia virus, the virus , available in Genbank (inventory number NC_006998, NC_003663, NC_005309, NC_004105, NC_001132, respectively).

Used by the authors of the term "poxvirus" refers to the virus belonging to the family from the poxviridae. According to their preferred option for implementation, poxvirus according to the invention belongs to Chordopoxvirinae, more preferably genus Orthopoxvirus and even more preferably to mind Vaccinia virus.

For example, can be used vaccinia virus strains Dairen I, IHD-J, L-IPV, LC16M8, LC16MO, Lister, LIVP, Tashkent, WR 65-16, Wyeth, Ankara, Copenhagen, Tian Tan and WR. According especially preferred option for implementation, poxvirus according to the invention represents the strains of Copenhagen from vaccinia virus,

Poxvirus vaccinia contains a large double genome DNA (187 pairs ) and is a member of the only known of a family of DNA viruses that replicate in the cytoplasm of infected cells. Because the infected cell must put the precursors of the DNA in the cytoplasmic sites of replication, the virus encodes and expresses many enzymatic activities required for the metabolism and synthesis of DNA, including 5'-triphosphate- (dUTPase).

5'- (dUTPase, EU 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate in the presence of ions (Mg 2+). dUTPaa, when you delete dUTP from the pool of dNTP and production dUMP, is involved in maintaining the loyalty of DNA replication and the provision of the predecessor for the production of TSR . dUTPaa vaccinia is a 15-kDa protein encoded by the genome F2L (MCGEOGH.. Nucleic Acids Research. 1990, no.18, p.4105-10; BROYLES.. Virology. 1993, no.195, p.863-5.). The sequence of the gene F2L vaccinia virus is available in the gene Bank by stock number 25392, sequence and location of the gene F2L in different genomes poxviruses are also available in the gene Bank, for example, by stock number NC_006998, DQ121394, NC_001611, AY689436, AY689437, NC_008291, DQ437594, DQ437593, AY313847, AY313848, NC_006966, NC_005309, NC_003391, NC_003389, NC_001132, NC_003310, NC_002188, M35027, AY243312, AF170726, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, X94355, Y16780, AY318871, U94848, AF198100 and M34368.

Gene nomenclature used in this description is nomenclature strain Copenhagen vaccinia, and is also used for homologous genes from the poxviridae other, if not indicated otherwise. However, gene nomenclature may differ depending on the strain of smallpox. For information, the correspondence between genes Copenhagen and MVA, see Table I ANTOINE. Virology. 1998, no.244, p.365-396.

Poxviruses, defective on a plot J2R, and their production methods are available in this technical field. For example, the management MCCART, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes, of cancer research. 2001, vol.61, no.24, p.8751-7., PUHLMANN, et al. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer gene therapy. 2000, vol.7, no.1, p.66-73., GNANT, et al. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene, and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice. Cancer Research. 1999, vol.59, no.14, p.3396-403 can be used to get poxviruses, with the deletion of the plot J2R.

According to the preferred option of the incarnation, poxvirus according to the invention further includes a target acid.

In a preferred embodiment, the incarnation of the target nucleic acid contains at least one target sequence encoding gene product, which is therapeutic molecule (i.e. therapeutic gene). "Therapeutic molecule is a molecule that has a pharmaceutical or protective activity with proper introduction to the patient, especially the patient suffering from morbid condition or illness or who should be protect from the disease or condition. This pharmaceutical or protective activity is an activity that is expected to be associated with a beneficial impact on the move, or a symptom of the disease or the specified state. When a qualified specialist chooses during the existing invention gene therapeutic molecule, he links his own choice with the earlier obtained results, and can reasonably be expected to have, without excessive experiment, in addition to carrying out the invention, according to the formula, to get this pharmacological property. According to the invention, the task sequence can be homologous or cells, in which it is entered. Preferably specified target sequence encodes all or part of the polypeptide, especially therapeutic or prophylactic polypeptide, giving therapeutic or prophylactic property. Polypeptide know, is any translational product regardless of size, and regardless of glycosylation, and includes peptides and proteins. Therapeutic polypeptides include as a primary example of the polypeptides, which can compensate for the defective or incomplete proteins in animal or human body, or those who act through toxic effects to limit or remove harmful cells from the body. They can also be giving immunity , which act as the endogenous antigen to induce humoral or cell-mediated response, or both.

Examples of proteins encoded by therapeutic gene, include the genes encoding the cytokine (alpha, beta or gamma interferon, interleukin, features of IL-2, IL-6, IL-10, and IL-12, tumor necrosis factor (TNF), -stimulating factor (GM-CSF, C-CSF, M-CSF...), polypeptide (7.1, 7.2 etc), coagulation factor (FVIII, FIX...), growth factor (transforming growth factor TGF, fibroblast growth factor FGF etc), enzyme (urease, renin, thrombin, metalloproteinase, nitric oxide synthase, NOS, SOD, catalase...), an inhibitor of the enzyme (alpha 1-antitripsin, antithrombin III, viral protease inhibitor, plasminogen activator inhibitor PAI-1), CFTR (regulator transmembrane conductivity cystic fibrosis) protein, insulin, dystrophy, antigen-MHC class I or II, polypeptide, which can modulate/regulate the expression of cellular genes, polypeptide, capable to inhibition of bacterial, parasitic or viral infections or her development (antigenic polypeptides, antigenic epitopes options, inhibiting the action of the native protein by competition...), an inductor or inhibitor of apoptosis (Bax, BCL-2, BclX...), cytotoxic agent (P21, P16, Rb...), apolipoprotein (ApoAI, ApoAIV, ApoE...), angiogenesis inhibitor (angiostatin, endostatin...), angiogenic polypeptide (family of vascular endothelial growth factors VEGF, FGF family, family CCN, including CTGF, Cyr61 and Nov), scavenger of oxygen radicals, polypeptide having antitumor effects by antibody-toxin, immunotoxin and marker (beta-galactosidase, luciferase system...) or any other target genes recognized in a given field of technology as useful for the treatment or prevention of clinical condition.

Suitable anticancer genes include, among others, the genes that encode genes-suppressors of the tumor (for example, Rb, p53, DCC, NF-1, tumor Wilma NM23, BRUSH-1, p16, P21, 56, 73, and their respective mutants), products of suicidal gene, antibodies, , inhibiting cell division or signal transduction.

According especially preferred option of the incarnation, poxvirus present invention further includes the suicide gene.

The suicide gene concerns the gene encoding the protein can convert predecessor of the medicinal product in connection.

The suicide gene

Thymidine kinase will

Ganciclovir;

ether ganciclovir acid;

penciclovir;

The suicide gene

acyclovir;

valacyclovir;

(E)-5-(2-)-2'-;

zidovudine;

2'-Exo-

5-

6-; fludarabine

5-; 5-fluorouracil inside the body

According to their preferred option for carrying out the invention, the suicide gene encodes a protein that has at as activity D. D involved in metabolic pathway, by which the exogenous cytosine is converted into uracil by hydrolytic deamination. While activity D were demonstrated in and lower (JUND, et al.). Journal of Bacteriology. 1970, no.102, p.607-15.; BECK, et al.). Journal of Bacteriology. 1972, no.110, p.219-28.; , et al.). Journal of Infectious Diseases. 1974, no.130, p.112-18.; ESDERS, et al.). J. biol. chem.. 1985, no.260, p.3915-22.), they are not present in mammals (Koechlin's, et al.. Biochemical pharmacology. 1966, no.15, p.435-46.; POLAK, et al.. Chemotherapy. 1976, no.22, p.137-53.),

CDaa also similar cytosine, i.e. a 5- (5-FC), thus forming a 5-fluorouracil inside the body (5-FU), which represents a connection, which is very when converting to 5-fluoro-UMP (5-FUMP). Cells which do not have activity D, or due to a mutation that inactivates the gene encoding the enzyme, or because they have no natural enzyme, as in mammalian cells, are resistant to 5-FC (JUND, et al. Journal of Bacteriology. 1970, no.102, p.607-15.; KILLSTRUP, et al.). Journal of Bacteriology. 1989, no.171, p.2124-2127.). In contrast, mammalian cells, which were transferred sequences encoding activity D, become sensitive to 5-FC (HUBER et al. Cancer Research. 1993, no.53, p.4619-4626.; MULLEN, et al.. the Proceedings of the National Academy of Sciences of the United States of America. 1992, no.89, p.33-37.; WO 93/01281 (US HEALTH)). Moreover, neighboring, untransformed cells also become sensitive to 5-FC (HUBER et al. Proceedings of the National Academy of Sciences of the United States of America. 1994, no.91, p.8302-6.). This is a phenomenon that is called effect of «witness», comes from the cells that Express the activity CD, 5-FU, which neighboring cells through direct diffusion through the plasma membrane. This property is 5-FU relatively passive diffusion is an advantage over tk/GCV of the reference system in which the effect of the «witness» requires contact with the cells that Express tk (MESNIL, et al. Proceedings of the National Academy of Sciences of the United States of America. 1996, no.93, p.1831-35.). All of the benefits that D offers within the context of gene therapy, particularly important for cancer gene therapy, can therefore be easily understood.

Genes codA Saccharomyces cerevisiae (S. cerevisiae) FCY1, Candida Albicans FCA1 and E. coli, which respectively encode D these two organisms are known, and their sequences were published (in SEQ ID N o :4; SEQ ID N o :5; SEQ ID no:6 respectively).

In this respect, according to the more preferred option embodiment of the present invention, a gene that encodes a protein with activity CD is FCY1, FCA1 or CodA or their equivalent. Analogues of these genes related gene having a nucleic acid sequence, which has a degree of identity, at least more than 70%, mostly more than 80%, preferably more than 90%, and it is most preferable to more than 95% with a sequence of nucleic acid parent gene.

Patent WO 2005/007857 describes the gene that encodes a protein with improved activity CD. These polypeptides obtained from native CD through the addition of the amino acid sequence. According to another preferred option embodiment of the present invention, a protein with activity CD, is a polypeptide, opened in WO 2005/007857, and more preferably polypeptide FCU1-8 presented by the ID sequence SEQ ID N o :2 and their analogues.

In and lower eukaryotes, such uracil is converted in UMP under the influence of (UR). This enzyme converts 5-FU to 5-FUMP. According to another preferred option embodiment of the present invention, the suicide gene encodes a protein with activity UPR-Tazy.

Considered UPR-Pelvis may be of any origin, especially origin, fungal or yeast origin. By way of illustration, nucleic acid sequence coding UR of E. coli (ANDERSEN, et al. Characterization of the upp gene encoding uracil phosphoribosyltransferase ofEscherichia coli K12. European Journal of Biochemistry. 1992, no.204, p.51-56.), from Lactococcus laclis (MARTINUSSEN, et al. Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis. Journal of Bacteriology. 1994, vol.176, no.21, p.6457-63.), from Mycobacterium bovis (KIM, et al. The Complete sequence of the UPP gene encoding uracil phosphoribosyltransferase from Mycobacterium bovis BCG. Biochemistry and molecular biology international. 1997, vol.41, no.6, p.1117-24.) and from Bacillus subtilis (MARTINUSSEN, et al. Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis. Journal of Bacteriology. 1995, vol.177, no.1, p.271-4.) can be used in the context of the invention. However, most especially it is preferable to use yeast UPR-Basin and in particular the coded S. cerevisiae FUR1 genome sequence that is disclosed in KERN, et al. The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene. 1990, vol.88, no.2, p.149-57, which is introduced by the authors by reference. As a guide gene sequences and sequence relevant UPRTa can be found in the literature and in the databases of specialists (SWISSPROT, EMBL, Genbank, Medline etc).

Application ER 0998568 AND describes the gene FUR1, not having 105 nucleotides in the 5' coding parts, creating a synthesis of UPRT, of which 35 were deleted first residues in the N-terminal position and starting from methionine in the position 36 in the native protein. The product of gene mutant called FUR1Δ105, capable of fur1 mutant S. cerevisiae. In addition, the truncated mutant shows higher activity UR than that of the native enzyme. Thus, according especially preferred option embodiment of the present invention, the suicide gene is mutated deletions native . Deletion preferably located in the N-terminal region of the original UPR-Tazy. It may be full (affecting all remnants of the specified N-terminal region) or partial (affecting one or more of continuous or intermittent residues in the primary structure). In General, polypeptide consists of the N-terminal, Central and C-terminal parts, each of which is approximately one-third of the molecule. For example, since the UPR-Pelvis S. cerevisiae has 251 amino acid, its N-terminal part consists of the first 83 balances, beginning with the so-called initiator methionine, located in the first position native form. With regard to UR E. coli, its N-terminal part includes provisions 1-69.

Preferred protein with activity UPR-Tazy, includes the amino acid sequence almost like presents in an identifier sequence SEQ ID N o :1 from UR 0998568 AND starting with the remainder Met in position 1 and ends with the remnant of Val in the position 216. The term "almost" refers to the degree of identity with the specified sequence SEQ ID N o :1 EP 0998568 more than 70%, mostly more than 80%, preferably more than 90%, and it is most preferable to more than 95%. Still more preferable it includes the amino acid sequence provided in the ID sequence SEQ ID N o : 1 EP 0998568 A. As mentioned above, it may include additional mutations. In particular, may be referred to the replacement of serine residue at position 2 (position 37 in the native UR) alanine residue.

According to another preferred option embodiment of the present invention, the suicide gene encodes a protein with at least one activity CD and one activity UR. Patent applications WO 96/16183 and the EP 0998568 AND describe the use of drained protein encoding the enzyme with two domains with activity CD and UR, and demonstrate that the transfer of hybrid gene codA::upp or FCY1::FUR1 or FCY1::FUR1A105 (i.e. FCU1), which bears a plasmid expression, increases the sensitivity 16 cells to 5-FC. According to the more preferred option embodiment of the present invention, the suicide gene is a polypeptide, including the amino acid sequence, almost like presents in an identifier sequence SEQ ID N o :3 (coda:: upp), SEQ ID N o :1 (FCU1) or FCY1:: FURL the Term "almost" refers to the degree of identity with the specified sequence, more than 70%, mostly more than 80%, preferably more than 90%, and it is most preferable to more than 95%. Still more preferable, it includes the amino acid sequence, almost like presents in an identifier sequence SEQ ID N o :3 (coda::upp), SEQ ID N o :1 (FCU1) or FCY1::FUR1. As mentioned above, it may include additional mutations.

sequence can be easily obtained by cloning, PCR or chemical synthesis according to the usual methods used. They can be native genes or genes derived from them by mutations, deletions, substitutions and/or additions to one or more nucleotides, in addition, their sequence to be widely described in the literature, which can be accessed by professionals skilled in the art.

includes, but the limitations listed, , and conveyors of the nucleoside. According to their preferred option embodiment of the present invention, is a purine or S. Cerevisiae. Conveyors S. cerevisiae consist of purine-cytosine known as FCY2, and known as FUR4. Purine - cytosine , FCY2 mediates protons and adenine, guanine, gipoksantina and cytosine through the plasma membrane of yeast (Grenson 1969, Jund and Lacroute 1970, Polak and Grenson 1973, Chcvallier et al. 1975, Hopkins et al. 1988). Protein FCY2 mediates transport 5-, analogue cytosine (Grenson 1969, Jund and Lacroute 1970). FCY2 gene encodes a protein of 533 amino acids (58 kDa), as originally intended, has 10-12 transmembrane rotating domains (Weber et al. 1990), nine of which today approved (Ferreira ct al. 1999). FCY2 shows such affinity for purine and cytosine (Brethes et al. 1992). Capture u in S. cerevisiae is mediated , FUR4 (Jund and Lacroute 1970, Jund et al. 1977). FUR4 is a uracil-proton (Hopkins et al. 1988), which apparently is a protein of 633 amino acids (71,7 kDa) with 10 domains and long cytoplasmic hydrophilic N - and C-terminal tails (Jund et al. 1988, Gamier et al. 1996). Protein FUR4 can also mediate transport 5-, analogue uracil (Jund and Lacroute 1970).

The amino acid sequences of FCY2 and Fur4 especially available in the swissprot database (inventory number R 17064 and 05316, respectively). Preferably, has the amino acid sequence selected from the group consisting of the amino acid sequence SEQ ID N0:1 and SEQ ID N0:2, as disclosed in the patent application WO 2006/048768.

In this respect, according to their preferred option embodiment of the present invention, is selected from the group consisting of FCY2 and Fur4 and their analogues. Analogues Fur4 and FCY2 concern polypeptide having the amino acid sequence, which has a degree of identity, at least more than 70%, mostly more than 80%, preferably more than 90%, and it is most preferable to more than 95% of the amino acid sequence of the parent protein as described by the authors above that retain the ability to transport medicine, including one balance through the cell membrane.

Specialist, skilled in the art can choose that will be associated with the drug or precursor of the drug, including one balance . For example, FCY2 and Fur4 preferably associated with 5- (5-FC).

In accordance with preferred embodiment, poxvirus invention may also include elements necessary for the expression of target nucleic acid.

In accordance with preferred embodiment, poxvirus invention may also include elements necessary for the expression of sequence, including the gene encoding . These elements needed for the expression of target nucleic acid and/or sequence, including the gene encoding included elements required for transcription of the indicated DNA into mRNA and, if necessary, to broadcast mRNA in the polypeptide. Transcription suitable for use in different systems of vertebrates, widely described in the literature. For example, among the appropriate viral promoters , such as RSV, Ministry, SV40, CMV or 7,5k, the promoter of the vaccinia, , etc. Preferred isolated from poxviruses, for example, 7.5, H5R, TC, P28, P11 or K1L vaccinia virus. Alternatively, you can use synthetic promoter, such as described in CHAKRABARTI.. Biotechniques. 1997, no.23, p.1094-97., HAMMOND, et al.. Journal of Virological Methods. 1997, no.66, p.135-38. and KUMAR.. Virology. 1990, no.179, p.151-8, as well as chimeric between early and late .

Target sequence and sequence, including the gene encoding may also include additional functional elements such as intron sequence, sequence, transport sequence, secretion signal, nuclear localization signal, IRES, poly And sequence termination of the transcription, tripartite leader sequences, sequences involved in replication or integration. About these sequences reported in the literature, and they can be easily obtained by the qualified specialists in the field of technology.

The invention also relates to a method for getting poxvirus in accordance with the invention, in which way:

(i) poxvirus according to the invention injected into the cell,

(ii) the specified cell cultured under conditions which are relevant in order to allow the products specified poxvirus, and

(iii) the specified poxvirus isolated from the cell culture.

The present invention also relates to a composition that includes the poxvirus in accordance with the invention, in combination with a pharmaceutically acceptable auxiliary substance.

Composition in accordance with the invention of more definitely is intended for preventive or healing therapy of diseases through gene therapy and more specifically focuses on proliferative diseases (cancer, tumor, restenosis, etc) or focus on diseases associated with increased activity of osteoclasts (e.g., rheumatoid arthritis, osteo porous).

Composition according to the invention can be made traditionally for its introduction locally parenteral or digestive path. In particular, a therapeutically effective amount of recombinant poxvirus vectors, or the present invention combine with pharmaceutically acceptable auxiliary substance. Possible to provide a large number of pathways of introduction. Examples that can be mentioned include the intragastric, subcutaneous, , intramuscular, intravenous, , intratumoral, , and way. In the case of these last three variants of the incarnation, it is preferable that the introduction happened spray or by instillation. Introduction may occur as a single dose or dose, which is repeated in one or more of the cases after a certain time interval. The appropriate route of administration and dosage varies depending on many parameters, such as the patient, the disease, which will be treated, or the target gene(s) (s) will be transferred(s). Drugs based on viral particles according to the invention, can be formulated in the form of doses of between 10 4 to 10 of 14 pfu ( unit), mainly from 10 5 10 13 pfu, preferably from 10 6 10 12 pfu, preferably from 10 6 10 7 .

The composition can also include solvent, adjuvant or auxiliary substance that is acceptable from a pharmaceutical point of view, and , stabilizing tool and a preservative. In the case of injecting preference is given to the composition in the water, or izotonicescom solution. It can be represented as a single dose or , in liquid or dry form (powder, freeze-dried etc), which can be cooked during use with a suitable solvent.

The present invention also relates to the use of poxvirus or composition according to the invention for the preparation of a medicinal product which is intended for treatment of the human or animal gene therapy. This medicine can be entered directly in vivo (for example, intravenous injection, the available tumor, in light spray, vascular system, using the appropriate catheter, etc). The preferred use is in the treatment or prevention of cancers, tumors and diseases that result from the undesirable proliferation of cells. Possible applications that can be mentioned include cancers of the breast, uterus (especially caused by papilloma viruses), prostate cancer, lung cancer, bladder, liver, intestines, pancreas, stomach, esophagus, larynx, Central nervous system (such as glioblastoma) and blood (lymphoma, leukemia, etc). Another preferred use is in the treatment or prevention of rheumatoid arthritis, osteoporosis and other diseases associated with increased activity of osteoclasts. It can also be used in the context of cardiovascular diseases, for example, for the inhibition or delay of the proliferation of the cells of smooth muscles of a wall of a blood vessel (restenosis). Finally, in the case of infectious diseases, it is possible to apply the drug in AIDS.

When poxvirus, the composition or the method of the invention is used for the treatment of cancer, the preferred route of administration is a systematic way so as poxvirus according to the invention able to specifically target the tumor cells.

The invention also covers a method for the treatment of diseases characterized by the fact that the poxvirus, composition according to the invention of the injected into the host organism or a cell that is in need of such treatment.

According to its favourable variant of the incarnation, therapeutic use, or a method of treatment also includes additional in which pharmaceutically acceptable number of prodrugs, mainly analogue cytosine, especially the 5-FC injected into the host organism or cell. By way of illustration possible to use a dose of 50 to 500 mg/kg/day, preferably at a dose of 200 mg/kg/ day or 100 mg/kg/day. Within the context of the present invention prodrug is administered in accordance with the standard practices (e.g., oral, systematically).

Preferably, introduction, what is happening after the introduction of the poxvirus or composition according to the invention, preferably at least 3 days later, more preferably at least 4 days and even more preferably at least 5 days after the introduction of the poxvirus or composition according to the invention. In accordance with the even preferred embodiment of the present invention, the introduction of prodrugs takes place after 7 days after administration of therapeutic agent. Preferred oral way. It is possible to single dose or prodrugs doses that are repeated for some time, which is enough to allow the production of a toxic metabolite within the host organism or cell.

In addition, the composition or method according to the invention can be combined with one or more substances that the cytotoxic effect of 5-FU. In particular, one can mention the medications that inhibit enzymes way for de novo pyrimidine biosynthesis (for example, mentioned below), medicines, such as leucovorin (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18, 685-692), which, in the presence of the product of the metabolism of 5-FU (5-FdUMP), increases the inhibition , reducing the pool of dTMP, which is required for replication, and finally, drugs, such as methotrexate (Cadman et al., 1979, Science 250, 1135-1137), which, inhibiting and increasing the pool of PRPP (), is the increase in the introduction of the 5-FU in cellular RNA.

According to the present invention, drugs that inhibit enzymes way for de novo pyrimidine biosynthesis, preferably selected from the group consisting of PALA (N-()-L-aspartate; Moore et al., 1982, Biochem. Pharmacol. 31, 3317-3321), Leflunomide, A771726 (the active metabolite of Leflunomide; Davis et al., 1996, Biochem. 35, 1270-1273) and (Chen et al., 1992, Cancer Res. 52, 3251-3257).

The composition or the way in accordance with the invention can be combined with one or more substances, effective in cancer treatment. Among pharmaceutical substances, effective in cancer treatment, which can be used in Association or in combination with compositions of the invention, mention can be made of alkylating agents, such as, for example, mitomycin C, cyclophosphamide, busulfan, ifosfamide, , melphalan, gexametilmelamin, , chlorambucil, or dacarbazine; antimetabolites such as, for example, gemcitabine, capecitabine, 5-fluorouracil inside the body, cytarabine, 2-, methotrexate, , or ; inhibitors topoisomerase II, such as, for example, doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone; inhibitors topoisomerase I, such as, for example, irinotecan (CPT-11), 7-ethyl-10-hydroxy- (SN-38) or topotecan; drugs, such as, for example, , , vinblastine, vincristine or vinorelbine; and derivatives of platinum, such as cisplatin, oxaliplatin, or carboplatin.

The composition or the ways in accordance with the invention can also be used in combination with radiation therapy.

The composition or the ways in accordance with the invention can also be used in combination with one or more other agents, including, among others, the immunomodulatory agents, such as alpha, beta or gamma interferon, interleukin (in particular, IL-2, IL-6, IL-10, and IL-12) or tumour necrosis factor; agents that affect the regulation of cell surface receptors, such as inhibitors of epidermal growth factor receptor (in particular, cetuximab, panitumumab, , , , , or ) or receptor inhibitors human epidermal growth factor 2 (particularly, ); and agents that affect angiogenesis, such as, for example, an inhibitor of vascular endothelial growth factor (in particular, bevacizumab or ).

Short description of the Figures in the drawings

Figure 1. Sensitivity in vitro to 5-FC from vaccinia virus infected human cells colorectal tumors (LoVo). Cells LoVo, infected with the MOI 0,0001 specified viruses (example (•) VVTK-/FCU1 (tiger) or VVTK-F2L-/FCU1 (◊), subjected to the action of different concentrations of 5-FC. Survival of cells define through 5 days after infection. The results expressed as a percentage of cell viability in the presence or in the absence of drugs. Values are presented as mean ±SD three individual determinations without mortality cells because the replication of viruses.

Figure 2. The effectiveness of viral replication in vitro in the LoVo, infected with the MOI 0,0001 specified viruses on day 5 after infection. Values are presented as mean ±SD three individual determinations.

Figure 4. The average volume of the tumor ±SEM p/to HepG2 tumors of the Swiss hairless mice following in/injections in the virus. After 14 days after inoculation tumors (palpable tumor), mice treated with 10 7 pfu buffer + water (◊), or buffer + 5-FC (diams), or 10 6 pfu VVTK-/FCU1 + water (about), or 10 6 pfu VVTK-/FCU1+5-FC (•) (A); or buffer + water (◊), or buffer + 5-FC (diams) or 10 6 pfu VVTK-F2L-/FCU1 + water (0), or 10 6 pfu VVTK-F2L-/FCU1+5-FC (diams) (A). Animals treated with 5-FC at 100 mg/kg twice a day feeding after 7 days of virus injection and within 3 weeks. Twice a week to determine the volume of the tumor.

Figure 5. The ratio of the output of the virus in dividing cells in comparison with merged cells. Cells PANC1 (cancer of the pancreas rights), H1299 (cancer of the lung) or U118MG (cancer of the human glioma) infect using 100 pfu (∎) VVTK-/FCU1 or (?) is VVTK-F2L-/PCU1. After 48 hours after infection depends on the viral titres. Values represent the ratio between the outputs of the virus in dividing cells in comparison with merged cells.

Figure 6. Viral titres (pfu/mg tissue) in the bodies or tumors in day 6 and day 21 after the on/in infection in Swiss hairless mice bearing human subcutaneous tumors with 1 x 10 6 PFU VVTK-/FCU1 (∎) or VVTK-F2L-/FCU1 (NASB).

Figure 7. Survival Swiss hairless mice after treatment with 1 x 10 8 pfu VVTK-/FCU1 (∎) or VVTK-F2L-/FCU1 (◊) in/in the injection.

Figure 8. Survival immunocompetent mice B6D2 after treatment with 1 x 10 7 pfu (A) or 1 x 10 8 pfu () VVTK-/FCU1 (tiger) or VVTK-F2L-/FCU 1 (◊) in/in the injection.

Figure 9. The average number of on the tails of the following in/injection in 1 x 10 6 pfu VVTK-/FCU1 or VVTK-F2L-/FCU 1 of the Swiss hairless mice day 13 after infection and on the day of 34 after infection.

Figure 10. The average number of on the tails of the following in/injection in 1 x 10 7 pfu VVTK-/FCU1 or VVTK-F2L-/FCU 1 of the Swiss hairless mice in day 15 after infection and the day 31 after infection.

Method(s) for the implementation of the invention

Design vector plasmids

Build the Shuttle plasmid to remove F2L using DNA strain Copenhagen vaccinia virus (inventory number 35027). Flanking DNA F2L using PCR. Primers rising plot F2L represent 5' - CGC GGA TCC GAA AGC GAT GAA HUNDRED AAT GTT C - 3' (SEQ ID no:7; section BamHI is underlined), and

5' - TCC CCC GGG GTT AGT TTC STT AAC AAA TST AAS - 3' (SEQ ID N o :8; plot. sv_-m {underlined), Primers downward site represent a 5' - GCC TGG SSA ASA AAT AGA GGA GAT CAA GGG T - 3' (SEQ ID N o :9; section MscI is underlined), and 5' - GCC CAG CTG ACC ACT ASA TCA ATT TTA CAA AAG - 3' (SEQ ID N o :10; plot PvuII is underlined). DNA fragment split restriction enzyme. sv_-m {/BamHI or MscI/PvuII and in the relevant areas of the plasmid PpolyIII. Re part of descending plot F2L by PCR using primers 5' - GCC GCA TGC TCC AGA ATT GAT CAT AGT GGA TA - 3' (SEQ ID N o :11; plot SphI is underlined), and 5' - GCT HUNDRED GAG TTA GTT TCC TTA ASA AAT HUNDRED AC - 3' (SEQ ID N o :12; plot XbaI is underlined) and inserted into a plasmid PpolyIII. Re plot is used for the removal of cassette breeding during production deleted viruses. Cartridge selection, corresponding merged gene GFP/GPT at the control pH5R promoter vaccinia, insert in plot. sv_-m {/SphI on a plasmid PpolyIII. Received a plasmid is a recombinant Shuttle plasmid called pAF2L to remove gene F2L.

Obtaining of recombinant vaccinia viruses.

Cells CEF infecting strain of Copenhagen VVTK-FCU1 (Vaccinia virus, a defective gene J2R, expressing the gene FCU1 under the control of synthetic promoter p11k7.5) at the MOI 0.1 and incubated at 37 OC for 2 hours, then with CaCl 2 recombinant Shuttle plasmids (0.2 mkg). Cells incubated for 48 hours at 37 deg C. Received cultivation of the virus uses to infect cells'KOV in the selective medium containing gipoksantin at a final concentration of 15 mg/ml, xanthine at a final concentration of 250 mg/ml and mikofenolovu acid final concentration of 250 mg/ml Fluorescent (GFP) and positive (GPT breeding) plaques isolates and filters a few rounds of selection in the cells of the CEF in the presence of selective environment GPT. The presence or absence of VVTk-FCU1 determine 40 cycles PCR with primers in the area of deletions. After elimination of the parent virus use the dual remote virus to infect the CEF without selective environment GPT to eliminate tape selection. plaques are isolated and are selected for 2 cycles in the CEF. Concluding recombinant viruses W in the CEF, clean and virus source solutions titrated on the CEF through a test to plaques.

Cell sensitivity in vitro to 5-FC.

Human tumor cells transform relevant recombinant W MOI 0,0001. A total of 3 x 10 5 cells/well placed on a 6-hole Cup for culture in 2 ml of medium containing different concentrations of 5-FC. Then the cells are cultured at 37 C for 5 days, and viable cell count with the exception of blue. The results, shown in the figures 1, 2, show that the activity FCU1 equivalent of viruses defective gene J2R than the virus, the defective gene J2R and F2L.

In vitro replication in cultured cells.

Dividing cells or merging cells to infect, 6- disks with 100 PFU viruses (almost MOI 0,0005), Add 2 ml of medium with 10% FCS for dividing cells and without additives for merging cells. The cells are collected after 48 hours after infection. Cells stored at -20 degrees and treated with ultrasound for the release of the virus, the virus is also to quantify the titration of the disks on the cells, SER. The ratio between replication in dividing cells and merging cells like in all cells. Both virus VVTK-/FCU1 and WTK-F2L-/FCU1 replicated more in dividing cells than in merged cells.

As an indirect value for the quantitative determination of specificity of viral replication, determine the yield of the virus, resulting in dividing cells compared to blend into tumor cells (human tumor of the pancreas PANC1; the human lung tumor 1299; human tumor glioma U118MG), Merging cells are placed on the tablets with 1 x 10 6 cells/well and cultivate in full environments within 7 days, then 1 day prior to infection of the cell washed and cultivated in the environment without serum. Dividing cells are placed on a plate at a 3 x 10 5 cells/well one day before the infection. To assess the level of cell division, the number of thymidine included in acid, determine after 5 hours, 24 hours and 48 hours after the cultivation of cells on the tablet. During this period, the inclusion of thymidine is relatively constant merging cells, whereas in dividing cells increase the involvement of notice over time. Then infect cells with the help of 100 pfu viruses, and after 48 hours after infection ratio between the output of the virus produced in dividing tumor cells and in merged tumor cells, can be determined drive to the CEF. The results depicted in figure 5, shows that both the virus VVTK-/FCU1 and WTK-F2L-/FCU1 more replicated in dividing cells than in merged cells.

Subcutaneous tumor model.

Females Swiss hairless mice derived from Charles River Laboratories. Animals used in research are uniform age (6 weeks), and body mass vary from 20 to 23 . naked mice injected subcutaneously (n/a) in the side 5 x 10 6 cells LoVo/100 ml. When the tumor reaches 50-70 mm diameter 3 , mice arbitrary manner and process the specified vectors for in vivo experiments.

Б virus.

The presence of VV-FCU1 and WTK-F2L-/FCU 1 assess virus titration in samples of organs and tumors of 1 x 10 6 viruses is administered by intravenous (IV) injection into the tail vein of naked mice with the set p/to tumors LoVo. Mice destroy the 14th day after infection and tumor and other organs harvested and weighed. Tumors and organs homogenized PBS, and credits are determined on the CEF, as described previously. Viral titres to standardise tissue. Viral titres to standardise tissue. The results, shown in Table 2 and 3 (range viral titers presented in pfu/mg tissue), show that after 14 days the virus, according to the invention mainly detected in the tumor. In the second set of experiments, in accordance with the same conditions as those described above, mice destroy the 6 day and 21 day after infection. The results depicted in figure 6 show that both the virus VVTK-/FCU1 and VVTK-F2L-/FCU1 target tumor approximately 1000-10000 times more virus in tumors than in other organs-reviewed except tails in the case of VVTK-/FCU1. A small number of VVTK-/FCU1 found in the lungs, spleen, kidneys, lymph nodes (less than 10 pfu/mg) and more in the skin, tail and bone marrow in day 6, and skin and tail day 21. On the contrary, VVTK-F2L-/FCU1 has a higher specificity to the tumor with only a small amount in the lungs, spleen, kidney, lymph nodes and skin in day 6, and in the skin and tail day 21.

Lymph nodes

VVTK-/FCU1

(0,2-3,3)x 10 5

VVTK-F2L/FCU1

0-8,1 x l0 4

Bone marrow

VVTK-/FCU1

13,5-7·10 4

VVTK-F2L/FCU1

Antitumor activity of poxvirus invention on the p/to the model of the tumor.

Naked mouse installed p/to tumors LoVo (50-70 mm) are treated once or twice intravenously (tail Vienna) specified vectors in a dose of 1.10 4 PFU, 1.10 6 PFU ABO 1.10 7 PFU, respectively. Starting from day 7 after a virus injection, 5-FC is administered orally through a tube at 100 mg/kg (0.5 ml of 5-FC 0.5% in water) twice a day for 3 weeks. The size of the tumor determine twice a week using . Tumor volume is calculated in mm 3 using the formula (p/6) (length x width 2 ). The results depicted in figure 3 show that the 2 viruses have this efficiency with activity (p<0.05), capable to monitor the growth of the tumor and the joint activity ( virus and therapeutic gene FCU1) with the introduction of the 5-FC, which could further improve the control of tumor growth (p<0.01).

Naked mouse installed p/to tumors HepG2 (cells hepatocellular liver carcinoma persons) are treated intravenously (tail Vienna) specified vectors in a dose of 1.10 6 PFU in accordance with the following scheme: after 14 days after inoculation tumors (palpable tumor) of the mice treated: buffer + water or buffer + 5-FC, or 10 6 pfu VVTK-/FCU1 + water, or 10 6 pfu VVTK-/FCU1+5-FC, or 10 6 pfu VVTK-F2L-/FCU1 + water, or 10 6 pfu VVTK-F2L-/FCU1+5-FC. Animals treated with 5-FC at 100 mg/kg twice daily orally through the probe, after 7 days of virus injection and within 3 weeks. The size of the tumor determine twice a week using . Tumor volume is calculated in mm 3 using the formula (p/6) (length x width 2 ). The results depicted in figure 4, show control over the development of a tumor after injection VVTK-/FCU1 and VVTK-F2L-/FCU1 (p<0.0001) relative to the buffer. Activity does not increase after the introduction of the 5-FC. Self activity of the virus is very strong already injections of viruses in a dose of 1 x 10 6 PFU.

Viral pathogenicity.

Viral pathogenicity evaluate research survival, which conduct and the Swiss hairless mice (figure 7) and in immunocompetent mice B6D2 ((6 weeks from Charles Rivers) (figure 8)). Mice in/in introducing 7 1.10 1.10 or 8 PFU all viruses in 100 MKL of the buffer on the mouse. The mice were observed daily during the experiment. The Swiss hairless mice (figure 7) injection of 1 x 10 8 PFU VVTK-/FCU1 leads to the death of 40% of the animals after 3 days after infection. The remaining mouse dying day between 50 and day 80 after infection. Introduction VVTK-F2L-/FCU1 is less pathogenic most animals dying day between 65 and 115 (p<0.05). There is no evidence of toxicity with both viruses at 10 7 pfu. All the mice die after in/in the injection 10 8 pfu VVTK-/FCU1. Group treatment VVTK-F2L-/FCU1 has a significantly longer survival up to 92% (p<0,00005) compared with VVTK-/FCU1 infected mice. Therefore, this result demonstrates the decrease of toxicity of the double-remote virus VVTK-F2L-/FCU1.

Model defeat tail .

Swiss naked mice in/in introducing 1.10 6 (figure 9) or 1.10 7 (figure 10) PFU each virus. The defeat of the tail is calculated once a week. In mice which impose 1.10 6 PFU VVTK-F2L-/FCU1, fewer than 1 Ospina/mouse compared with mice, which impose VVTK-/FCU1 with the average number equal to 8 the mouse day 13 after infection (p<0.001) as shown in figure 9 (A). The results are similar in the day 34 after infection with the average value of 4 with VVTK-/FCU1 compared with about 1 for VVTK-F2L-/FCU1 (p<0.0001) as shown in figure 9 (A). In mice which impose 1.10 7 PFU VVTK-F2L-/FCU1, notes, respectively, the average number equal to 3.5 /2 mouse and the pockmarks were/mouse compared with mice, which impose 1.10 7 PFU VVTK-/FCU1, with an average of 10 /mouse day 15 after infection (figure 10 (A)). Day 31 after infection in mice which impose VVTK-F2L-/FCU1, marked accordingly in average 3.5 /mouse compared with mice, which impose VVTK-/FCU1, with an average of 7 /mouse (figure 10). The difference in the number between VVTK-/FCU1 and VVTK-F2L-/FCU1 is statistically significant (p<0.05). Formation correlates with viral replication in the tail, as well as with virulence and toxicity. Injection, in/in VVTK-F2L-/FCU1 a less toxic than with a single remote virus TC.

The statistical analysis.

Conduct statistical studies using nonparametric U test Mann-Whitney and software STATISTICA 7.1 (StatSoft, Inc). P<0.05 is considered to be statistically significant.

- US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15.11.1994

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1. Poxvirus vaccinia, which has activity and includes defective F2L gene and target acid, including the suicide gene, and defective F2L gene leads to the inability of the virus to produce any protein, exhibiting activity of the protein produced unmodified gene.

2. Poxvirus according to claim 1, of the poxvirus additionally includes a defective gene J2R.

3. Poxvirus according to claim 1, of the poxvirus is a strain WR Vaccinia virus.

4. Poxvirus according to claim 1, of the poxvirus is a strain of Copenhagen Vaccinia virus.

5. Poxvirus according to claim 1, wherein said the suicide gene encodes a protein that has at least yeast cytosine deaminase activity.

6. Poxvirus of claim 5, wherein said the suicide gene is a FCY1, FCA1 or CodA, or the equivalent.

7. Poxvirus of claim 5, wherein said a protein that has at least yeast cytosine deaminase activity is kilodalton FCU1-8 presented in the identifier is a sequence SEQ ID no:2, and its analogs.

8. Poxvirus according to claim 1, wherein said the suicide gene encodes a protein that has at least one yeast cytosine deaminase activity and one .

9. Poxvirus on item 8, wherein said the suicide gene is a polypeptide, including the amino acid sequence almost the same as presented in the identifier is a sequence SEQ ID no:3 (coda::upp), SEQ ID no:1 (FCU1), or the amino acid sequence FCY1 ::FUR1.

10. Poxvirus according to claim 1, wherein said poxvirus additionally includes sequence, including the gene encoding .

11. Poxvirus to 10, where is a purine or S.Cerevisiae.

12. Poxvirus in paragraph 11, in which choose from the group consisting of FCY2 and Fur4 and their analogues.

13. Poxvirus according to claim 1, wherein said poxvirus additionally includes the elements needed for the expression of target nucleic acid.

14. Poxvirus to 10, in which a specified poxvirus additionally includes the elements needed for the expression of nucleic acid sequences, including the gene encoding .

15. Application of poxvirus any claims 1 to 14 for the treatment of proliferative diseases or diseases with increased activity of osteoclasts.

16. Application on item 15, which poxvirus additionally includes a target acid.

17. The application of the article 16, under which the target nucleic acid contains at least one target sequence that encodes therapeutic molecules.

18. How to play poxvirus according to claim 1, where in the way: i. poxvirus according to claim 1 enter in the cell; ii. the specified cell cultured under conditions which are relevant in order to allow to produce the specified poxvirus, and; iii. specified poxvirus isolated from the cell culture.

19. Composition, possessing activity, including poxvirus on any one of claims 1 to 14 in combination with a pharmaceutically acceptable auxiliary substance.

20. Application of poxvirus on any one of claims 1 to 14, to receive the medicinal product treatment of proliferative diseases or diseases with increased activity of osteoclasts.

21. Application for 20 to receive the medicinal product for the treatment of cancer.

22. The method of treatment of proliferative diseases or diseases with increased activity of osteoclasts, wherein the poxvirus on any one of claims 1 to 14 or arrangement .19 injected in the effective quantity in the master-organism or a cell that is in need of such treatment.

23. The method according to article 22 where the disease is cancer or restenosis.

24. The method according to article 22 where the disease is rheumatoid arthritis or osteoporosis.

25. The method according to article 22, in which a specified poxvirus or composition enter system path.

26. The method according to article 22, including an additional stage in which pharmaceutically acceptable number of prodrugs injected into the specified master-organism or cell.

27. The method according to .26, in which the introduction of the prodrug spend preferably after at least 3 days, more preferably at least 4 days and even more preferably at least 5 days after the introduction of the specified poxvirus or composition.

28. The method according to item 27, in which the introduction of the prodrug made after 7 days after a specified poxvirus or composition.

29. The method according to article 22, which poxvirus or composition is administered in combination with one or more substances that the cytotoxic effect of 5-.

30. The method according to clause 29, in which these substances, the cytotoxic effect of 5-, are medications that inhibit enzymes way for de novo pyrimidine biosynthesis, preferably selected from the group consisting of PALA, Leflunomide and A771726.

31. The method according to clause 30, in which the indicated substance the cytotoxic effect of 5-, represents methotrexate.