Method and compositions containing the dna-damage agents and p53

 

(57) Abstract:

The invention relates to medicine, in particular to the use of suppressor genes in malignant tumors, in combination with a DNA damaging agent or factor, with the aim of killing cells, in particular cells of malignant tumors. Suppressor gene in malignant tumors, p53, was delivered in vivo carrier of the gene with an intermediate adenovirus is a recombinant, in combination with a chemotherapeutic agent. Treated cells showed a state of apoptosis in specific DNA-fragmentation. Direct subcutaneous injection of p53 adenovirus composition in malignant tumors with subsequent interperitoneal the introduction of a DNA damaging agent, cisplatin, caused massive apoptotic destruction of tumors. The invention also provides a method that combines the movement of a gene, through the use of replication-defective adenovirus p53 wild-type (natural adenovirus p53) in combination with drugs that affect DNA, for the treatment of malignant tumors. The technical result of the invention is the expansion of the means to fight with malignant neoplasms. 4 C. and 111 C.p. f-NII methods chemotherapeutic intervention. In addition, the present invention provides new methods and compositions that combine the potential DNA-damaging agents with combined shipping gene suppressor of malignancy. The Association of DNA-damaging factors with xenogenic gene expression of suppressor of tumors leads to tangible synergies, suppressive activity of individual components.

Description of the prior art

Modern methods of cancer treatment, including radiation therapy, surgery and chemical treatment, have a limited effectiveness. Only lung cancer kills in the United States, more than 140,000 people annually. Recently installed age group mortality from lung cancer was higher than the mortality from breast cancer in women. Although the introduction of the program of fight against Smoking has reduced the incidence of diseases from Smoking, the mortality rate from lung cancer will remain high even in the 21st century. The development of new therapeutic methods for the treatment of lung cancer will depend on the penetration mechanism of the biology of lung cancer at the molecular level.

It is now established that cancers are caused, at least justicexxii rejected from normal or mutant gene or genes. For example, in many cases, as it is known, expression of oncogenes cause cancer. "Oncogenes" are genetically modified genes whose mutant the expression product somehow disrupts the normal functioning of cells, or the management of this process. (Spendidos et al., 1989).

The most studied to date oncogenes got the definition of "activated" by mutation, often point mutations in the coding region of a normal cellular gene, i.e., "proto-oncogene", resulting in the substitution of amino acids in a protein product whose expression is changed. This product with altered expression manifests rejected from the norm of biological functions that affect the flow of the process of tumor (Travali et al., 1990). Not manifesting itself mutations may develop under different circumstances, for example by chemical mutagenesis or ionizing radiation. A number of oncogenes or oncogenic families, including ras, myc, neu, raf, erb, src, fims, jun, abl, currently already identified and in varying degrees described (Travali et al., 1990, Bishop, 1987).

As installed, under normal cell growth, proto-oncogenes can cause an imbalance in the interaction of these two forces, leading to a condition involving the formation of tumors. One such factor is the mutation in the genes suppressors of tumors (Weinberg, 1991).

One of the important suppressor genes neoplasms is a gene that encodes a cellular protein, p53, which is a nuclear phosphoprotein 53 kD (substituted in cycle kD phosphoprotein 53), which controls cell proliferative (proliferation of cells). Mutations occurring with p53, and loss of allelic gene on chromosome 17p, where this gene is located, is one of the most common mutations identified in malignant neoplasms. The p53 protein has a high degree of safety in the process of evolution and occurs in most normal tissues. It was demonstrated how natural p53 in p53 wild-type) should be involved in the control of cell cycle (Mercer, 1992), in the process of transcriptional regulation (Fields et al., Mietz et al., 1992), DNA replication (Wilcock and Lane, 1991, and Bargonetti et al. 1991) and in the initiation of apoptosis (Yonish - Rouach et al., 1991, Shaw et al., 1992).

There are various mutant allelic genes p53, in which a single base substitution leads to protein synthesis, which is the malignant tumors (Hollstein et al., 1991). Indeed, it was found that the p53 gene is the most frequently mutated gene in cancer in humans (Hollstein et al., 1991) and, in particular, is associated with those cancers, the occurrence of which is associated with cigarette smoke (Hollstein et al., Zakut - Houri et al., 1985). Over-expression of p53 in breast cancer has also been documented (Casey et al., 1991).

One aspect that attracts the most attention of cancer gene therapy is the use of suppressor genes such as p53 for the treatment of tumors. It was found that transfection (infection of the cells of alien nucleic acid, e.g. viral) natural p53 gene in certain types of tumor cells of breast and lung cancer can recover control of the process of inhibition of growth of cell lines (Casey et al., 1991, Takahasi et al. , 1992). Although transfection of DNA is not a viable means of introducing DNA into the cells of patients, these results serve as a demonstration that the introduction of natural p53 gene in cancer cells containing mutant p53, can be an effective method of treatment if they developed improved ways of implementation of the p53 gene.

Currently researching the problem and created systemowe virus for gene transfection are of particular interest, due to the ability of viruses to infect really living cells. There is a way in which moves himself viral genetic material. In this regard, there were several successful solutions, such as playing retroviral vectors designed to deliver a wide range of genes. However, there are serious problems associated with the use of retroviral vectors in gene therapy. Since their effectiveness depends on the availability of retroviral receptors on target cells, it is difficult to concentrate and to disinfect, and in addition, they can be effectively implemented only in proliferating cells (replicating cells).

The main problem of clinical Oncology is the resistance of cells of malignant tumors to the effects of chemotherapeutic drugs. Almost 80% of lung cancer is lung cancer with involvement of a significant number of cells (NSCLC). However, patients with this form of cancer, mostly immune to chemotherapeutic treatment (Doyle, 1993). One of the tasks of modern research in the field of treatment of malignant tumors is the search for ways to improve the effectiveness terpadu product of the gene and chemotherapeutic drugs. The gene of herpes simplex-thymidine kinase (HS-tk), being embedded in the brain tumor using a retroviral vector system, successfully raised susceptibility to gancilclovir anti-virus agent (Culver et al. , 1992). The gene product HS-tK is an exogenous viral enzyme, and because the expression of protein wt-p53 appears in normal tissues, there is the assumption that the modulation of resistance to chemotherapeutic agents-induced alteration in the expression of wt-p53 may represent an alternative approach, which uses a path that is indirectly determined endogenous genetic program.

Adenovirus system has potential advantages when moving the gene in vivo, such as the ease of obtaining high titer virus, a high degree of infectious activity and immunity to many types of cells. However, the stability and duration of expression of the introduced gene is still problematic. The increased levels of p53 in cells, which differ in sensitivity to chemotherapeutic drugs, may occur within 6 hours after exposure to DNA-damaging stimulants (Fritche et al., 1993, Zhan et al., 1993), although the value is in, if the stimulator removed (Tishler et al., 1993). Hence the high level of expression of p53 can be maintained even after termination of exposure on the body of a medicinal product. Ensuring the expression of the protein wt-p53 with Ad-p53 culminates on the third day after infection (14 - fold greater than endogenous wild-type) and decreases to a low level for the ninth day (Zhang et al. , 1993). This suggests that alternating high level of expression of wt-p53 enough to initiate cytotoxic program in a cancer cell.

p53 plays an important role in determining the sensitivity of cancer cells of the human lung to chemotherapeutic drugs. A variety of treatment protocols (histories), including surgery, chemotherapy and radiotherapy, has been studied in connection with the treatment of cancers of the human lung NSCLS, however, the survival rate in the long term remains unsatisfactory. What need is in the combined therapeutic modality, which could be used alone or as adjunctive treatment to prevent local recurrence following predvaritelnogo was initially immune to the effects of drugs and in which there are metastases, or when local recurrence of lung cancer. Compositions and methods require further research, development and improvement of methods of clinical application of new tools for cancer treatment. In addition, these methods and compositions must confirm their right to use in vivo.

The invention

The invention is aimed at improving therapeutic agents and methods of their use in the process of destroying cells by combining the effects of the gene suppressor of malignant neoplasms or of protein and DNA-damaging agent or factor. The present invention also provides compositions and methods, including those that use viral gene movement intermediary, to ensure the expression of the natural gene suppressor of malignant neoplasms, such as p53, in target cells and to direct the agent or factor that causes damage to the DNA. The inventors unexpectedly discovered that, by using the compositions disclosed in this invention, they were able to call the programmed destruction of cells, a phenomenon known as "apoptosis" in a large number of cells targets.

Using the infusion of the new growths. The formation and growth of tumor cells is also known as "transfection", which describes the education and proliferate cells that have lost the ability to control the process of cell division, i.e. turned into cancer cells. Obviously, there are a number of different types of transformed cells can be considered as potential targets for the use of the methods and compositions according to this invention, for example, sarcoma, melanoma, lymphoma, with a wide range of solid tumors, etc., Although any tissue in which the growth of malignant cells, can serve as the object of use of the invention, the preferred area of use constitute the tissue of the lungs and chest. The inventors have found that the vector migrating recombinant, causing the expression of p53,was able to significantly reduce the degree of cell growth in the case when it is used in combination with a DNA damaging agent.

The invention provides in certain embodiments methods and compositions for the destruction of cells or cells such as cancer cells or cells, by providing a contact or exposure of the cells or population of cells by a p53 protein or gene and one ilnuw cell (cells). The list of cells that can be destroyed by use of the present invention, includes for example, unwanted, but benign tumor cells, such as cell hyperplasia, benign prostate tumors, the cells of the thyroid gland, characterized by increased activity, cells related to autoimmune diseases, such as B cells, which produce antibodies present in arthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasia, etc., Although the invention is applicable for disposal, basically, all unwanted cells, the specific problem that it solves, is the destruction of malignant cells. The term "cancer cells "refers to cells that have lost the ability to control the cycle of cell division that leads to the emergence of a "transformed" or "cancer" phenotype.

To kill cells, such as malignant or metastasis, using the methods and compositions of the present invention, it should be ensured that contact the target cells with a p53 protein or gene and at least one DNA damaging agent, the total amount which effectively enough to destroy this cell-Misha and factor(s) at the same time. This can be achieved by ensuring contact of the cell with a single composition or pharmacological unit, which includes both components, or by providing contact of the cell with two distinct compositions or pharmaceutical units at the same time, provided that one composition includes the p53 protein or gene, and the other includes affecting DNA agent.

It is obvious that the cell target may be initially irradiated damaging (and) DNA agent(s), and then brought into contact with the p53 protein or gene, or Vice versa. However, for embodiments of the invention, where striking DNA factor and p53 use to influence cell separately, it should be clear that between each of the inputs of these components should not be a significant period of time that affects DNA agent and p53 would be able to fully combined efforts to have a positive impact on the cell. It is established that in such cases, contact with both components should be implemented at intervals within 12-24 hours, and more preferably, at intervals within 6-12 hours, with the most preferred delay time (opendiv the", applied to the cell used in the present description in order to describe the process by which a gene suppressor of malignant tumors or protein, such as p53, and affects DNA agent or factor is delivered to the target cell or are placed in direct contact with the cell target. To ensure the killing of cells, both components are delivered into the cell in the total number sufficient to ensure the destruction process of this cell, i.e., programmed cell death, or apoptosis. The terms "killing", "programmed cell death" and "apoptosis" in the present context are interchangeable and are used to describe a series of intracellular reactions, which lead to the destruction of target cells. The destruction of target cells results in the active state of intracellular proteases and nucleases, which provides nuclear involution cells and fragmentation of nuclear DNA. Understanding the exact mechanisms of intracellular molecular interactions aimed at the realization of killing cells, not necessarily when using the present invention.

Amazing DNA factors or agents in this context are defined as any and factors include radiation and waves, which stimulate DNA damage, such as radiation, X-radiation, ultraviolet (UV) radiation, microwaves, electronic emissions, etc., a Variety of chemical compounds present in the description of the term "chemotherapeutic agents" that act as a means for causing the defeat of DNA, all of which can be used in the combined treatment methods disclosed in the present invention. The list is intended for the use of chemotherapeutic agents include, for example: adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The present invention also encompasses the use of combinations of one or more DNA damaging agents, which are based on the principle of radiation, or represent active compounds, for example, the use of radiation in conjunction with cisplatin or cisplatin with etoposide. In some cases, the use of cisplatin in combination with p53 protein or gene is a far better choice in comparison with the mentioned connection.

Can be used any way ensure contact of the cells with the p53 protein in techenclave as direct delivery of the protein p53, and delivery of a gene or DNA segment that encodes p53, with this gene will have to initiate the expression and formation of p53 within cells. With this method of delivery of the protein lacking, which is the degradation of protein and low absorptive capacity of the cell. It is concluded that the method of using recombinant vector that causes the expression of p53 protein has significant advantages.

Can be designed in a wide variety of recombinant plasmids and vectors capable of inducing the expression of p53 protein and, being so used to deliver p53 in the cell. For example, it is known the use of "naked" DNA and p53 plasmid for direct delivery of genetic material into the cell (Wolfe et al., 1990), the compounds of the p53 - coding DNA that are trapped in liposomes (Ledley et al., 1987) or by proteoliposome that contain receptor proteins in the viral membrane (Nicolau et al., 1983); and p53-encoding DNA that is attached to the complex-vector of policysimulation. Was studied using virus recombinants developed to facilitate the expression of p53 expression.

A set of viral vectors, such as retroviral weak), the cytomegaly virus, etc. can be used, as described by Miller (Miller, 1992); can be used recombinant adeno-associated viruses (AAV vectors), described in U.S. patent N 5139941 and included in the list of references, and, in particular, vectors adenoviruses-recombinants. Cooking techniques replication-defective infecting viruses are well known in the prior art and examples are described in the works of authors: Ghosh-Choudhury & Graham (1987); McGrory et al., (1988); and Gluzman et al. (1982), each of which is listed in the list of references.

To kill a cell in accordance with the present invention, it is necessary to ensure contact of the cells with a p53 protein or gene and DNA damaging agent when combined amount effective enough to kill the cell. The term "when combined number effective enough to kill this cell" means that the amount of p53 and DNA damaging agents must be sufficient so that, if they will be merged inside the cell, it will induce apoptosis in this cell. Although this observation and is not required for each of the embodiments of the invention, as the United effektivnoye killing a significantly larger number of cells, than each item individually. The most preferred can be considered such an effective combined amount that will be equal to the value that triggers a synergistic killing of cells in comparison with the results observed when using each of the elements separately.

A certain number of parameters in vitro can be used to determine the result, which is achieved by application of the compositions and methods proposed by the present invention. These parameters include, for example, the counting result of the number of cells before and after exposure to the compositions described in this invention, and measured the size of multicellular tumor spheroids formed in the same way that formed their colonies in tissue culture. The In vitro process of killing cells, in particular, as shown in example 7, the method of the present description. Additionally, you can measure the parameters that are characteristic of cells undergoing programmed death, such as fragmentation of cellular genomic DNA fragments with the size of the nucleosome, which are mainly identified by vyd the size of the nucleosome identified as ascending stairs (like a dropped stitch or chain monomers or polymers, having the main unit of about 200 pairs.

By analogy, the term "therapeutically effective amount" means the combined amount of p53 protein or gene DNA damaging agent that, when introduced into the animal organism able to kill cells within the body of the animal. This, in particular, by killing cancer cells, for example, lung cancer cells, breast or colon cancer cells in the body of an animal or a human affected by a malignant tumor. The term "therapeutically effective compounds" means, basically, so the combined amount of p53 and DNA damaging agents, which are aimed at killing a larger number of cells than each of the elements separately, and, preferably, such combined amount, which provide a synergistic reduction of the genetic load in tumor growth.

The study of in vivo and ex vivo specific killing of cells are also an effective means by which proves the effectiveness of the compositions and methods of the present invention. For example, the observation impact on the suppression of oncogenic potential, as measured by experto is not news to the pathologist. Of course, other methods of determining the mass of the tumor, its growth and viability can also be used to establish the fact of killing of target cells. In particular, you can define this behavior in vivo of various models of cancer, including those in which human cancer cells are localized within the body of the animal. It is known that models of cancer of the animal, in contrast to the model AIDS, are very similar (predictable) with treatment regimens developed for the human body (Roth et al., editors, 1989). There is one instance of sample forecast of the model animal in which the subcutaneously grown cancer cells of the human lung, the variation is small size cells (H358 cells). Using this system, the inventors have shown that adenovirus carrying p53 introduced in the form of drops inside the tumour region, together with the introduction of the chemotherapeutic agent leads to unexpected efficient reduction of tumor cells.

One of the preferred methods of delivery of the p53 protein in the cell is ensuring contact of the cells with the adenovirus virion-recombinant or particle, which includes vectorlogo to direct the expression of p53 in this cell type.

Region of p53 expression in the vector may contain a sequence of genomes, but for simplicity we can assume that it is preferable to use the sequence of the cDNA of p53, as it is easily available for analysis of the prior art in this field and is easier to manage the process. In addition to the expression of p53 and promoter vector should also include signal polyadenylation (polyadenilation), for example early gene, SV40 or Protamine gene, or something like that.

In preferred embodiments of the invention, it is assumed that you may need in the location of the zone of expression of p53 under the control of a strong, significant (structural) promoter such as the promoter of cytomegalovirus (CMV), a virus LTR promoter, RSV or SV40 or promoter associated with genes that show expression at high levels in mammalian cells (breast cancer), for example the promoter factor-1 elongation or actin. All of these options have the right to exist in connection with the use of the invention. Currently, the most preferred promoter is the promoter of IE cytomegaly virus (CMV).

The p53 gene or cDNA can be inserted in the adenovirus is a recombinant, in accordance with this is to as the preferred adenoviruses should be considered defective virus replication, in which viral gene essential for replication and/or packaging has been removed from the adenoviral vector patterns, allowing the area of expression of p53 to move into place. Any gene, or essential, for example E1, E2, E4, or immaterial, such as E3, for replication, can be removed and replaced by p53. In particular, preferred are those vectors and virions, in which the area E1A and E1B of adenovirus vectors deleted and the area of expression of p53 embedded in their place, as shown in the example on the genome structure of Fig. 1.

Technological processes of preparation replication defective adenoviruses are well known in the prior art, as evidenced described in this description analogues Ghoss-Choudhury and Graham (1987); McGrory et al. (1988); and Gluzman et al., (1982) listed in the references to the literature. In addition, it is well known that can be used in a variety of cell lines, with the aim of reproducing adenovirus-recombinants and their play can continue as long as there is a possibility to add replication defect, if present. A preferred cell line is the cell line 293 people, but there are also the AE expression of E1A and E1B. Moreover, cells can be played as plastic cups, and in the culture suspension to obtain a stock culture of viruses.

The invention is not limited to viruses with the missing link E1 and cells with the expression E1. Indeed, in connection with the present invention, can be used complemented by other combinations of viruses and host cells. Can be used in cells with expression of E2 and the virus loses functional element E2 is exactly the same as cells with expression of E4 and the virus loses functional element E4 and so on In the case, if not essential for the replication gene is deleted or substituted, as, for example, is the E3 gene, such defect there is no need separately to complement with the help of a host cell.

For successful use of the invention, the only requirement is the creation of vectors adenoviruses, which can stimulate the expression of p53. The nature of the original adenovirus critical to the use of the invention does not have. Can be used any of adenovirus 42 various known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material for obtaining a solid replication is virus type 5 is selected because that, in connection with this adenovirus person has accumulated a significant amount of biochemical and genetic information, and, in addition, it is traditionally used for most structures, using adenovirus as a vector.

Methods and compositions related to the present invention is equally suitable for killing cells or cells in vivo and in vitro. In the case when cells are designed for killing, focus inside the body of the animal, such as cancer cells, lung cancer, breast or rectum, or other cells bearing mutated p53, in this case, the p53 protein or gene, as well as affecting DNA agent should be introduced into the animal organism in a pharmacologically acceptable form. The term "pharmaceutically acceptable form" in this context refers both to the form of any of the compositions, which can be introduced into the body of the animal, and to a method of providing contact of a living organism to radiation radiation, i.e., the method by which irradiate any area of the body of the animal, for example by radiation, X-rays, ultraviolet (UV) radiation, microwaves, electronic emissions, etc. Use affecting DNA radiation and waves known to all who had the cancer, which mainly include the introduction into the organism of an animal or human, a cancer patient, a therapeutically effective combination of a p53 protein or gene and a DNA damaging agent. This can be achieved through the use of recombinant virus, in particular an adenovirus, which is a vector capable of inducing the expression of p53 in the cells of a tumor. Composition, carrying the p53 gene, in the main, should be introduced into the body of the animal often while ensuring close contact with the tumor, in the form of a pharmaceutically acceptable composition. The preferred method is direct injection of a therapeutically effective amount of p53 gene, which is located within the boundaries of the recombinant virus in the place in the body that is affected by the tumor. However, have a right to exist and other parenteral routes of administration, such as intravenous, through the skin, endoscopically or via subcutaneous injection.

In the treatment of cancer, in accordance with the present invention, it is possible to ensure the contact of tumor cells with a DNA damaging agent, in addition to the effects of p53 protein or gene. This method can be carried out by irradiation spot socredo the radiation, - rays or even microwaves. In addition, tumor cells can be brought into contact with the DNA damaging agent is injected into the body of the animal pharmaceutically effective amount of pharmaceutical products containing damaging DNA connection, for example adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, and even better - cisplatin. Affecting DNA agent may be prepared and used in the form of a complex therapeutic composition or pharmacological set, obtained by combining DNA damaging agent with the protein p53, a gene or gene delivery system, as described above.

Striking a positive effect arising from the use of the present invention, disclosed that the use of virus Ad5CMV-p53 in combination with cisplatin showed excellent results in studies using model hairless mice. The combined mode, the virus is affecting DNA therapy significantly reduced carcinogenesis H358 cells, cells that normally produce significant tumor mass. Carcinogenesis cancer lung cancer cells was suppressed by treatment with Ad5CMV-p53,but not due to the treatment of the control virus, causes the Shui therapeutic efficacy.

Professionals, competent in this field of knowledge, the known methods of delivery of chemotherapeutic agents, including patterns of expression of DNA in eukaryotically cells. In the light of the present invention an experienced specialist will be able to deliver as affecting DNA agents and p53 proteins or genes to cells using a variety of effective means.

For DNA delivery in vivo, the inventors suggest the use of various gene delivery systems, such as viral and with an intermediate liposome transfection. In the context of describing the present invention, the term "transfection" is used to describe the targeted delivery of DNA in eukaryotically cells by using such delivery systems such as adenoviral, AAV, retrovirus, or delivery system, gene using plasmid. The specificity of viral delivery can be selected for the preferred direct gene transfer in some specific the target cell. Using viruses that can infect these separate specific types of cells. Of course, the various features of the virus determine the specificity of the selection of the appropriate virus for gene transfer in prinzio malignant cells of the specified type.

It was found that the use of damaging DNA chemotherapeutic agent using a whole variety of means, for example, through the use of parenteral delivery methods, such as intravenous or subcutaneous injection, etc., Such methods known to experts in the field of ingestion of drugs and will be described in the section relating to pharmaceutical preparations and methods of treatment.

For gene delivery in vitro can be used in different ways, for example using calcium phosphate or transfection with an intermediate of dextrin sulfate, electrophoresis; positioning (designation) using glass microcard, etc., These methods are well known in the prior art, and the exact composition of the means and methods of their use are given in the description of this invention.

Other embodiments of the invention relate to compositions, including pharmaceutical agents, consisting of a p53 protein or gene in combination with a DNA damaging agent, such as cisplatin. In the compositions of p53 can be represented in the form of a segment of DNA recombinant vector or recombinant virus that is able to induce the expression of proteinenergy for gene delivery, for example, adenoviral particle, can be prepared for the introduction of in vitro by dispersion in a pharmacologically acceptable solution or buffer mixture. Preferred pharmacologically acceptable solutions include neutral saline solutions, protected phosphate, lactate, TRIZ (Tris), etc.

Of course, everyone who uses a viral delivery system, there is a desire to clear the virion to the extent that is substantially free from undesirable contaminants, such as defective interfering with adenoviral particles or endotoxins and other pyrogens that the virion did not cause adverse reactions in the body of an animal or person receiving a vector structure. The preferred means of purification of the vector is the creation of conditions for use of gradients floating density, for example, centrifugation of caesium chloride in the density gradient.

Preferred pharmaceuticals according to the invention are those that include, within a pharmacologically acceptable solution or buffer mixtures, protein p53, or preferably the p53 gene, in combination with chemotherapeutic DNA damaging agent. PR is xed hydrogen, mitomycin C, cisplatin (CDDP) and etoposide (VP-16), it is particularly preferred is the use of cisplatin.

Further embodiments of the present invention are kits designed for killing cells, namely cancer cells, which can be defined as a therapeutic kits for the treatment of cancer. Such kits according to the present invention, should mainly contain placed in the appropriate container shell (capsule) pharmaceutical means of a recombinant vector, which is able to induce the expression of p53 protein in a cell of the animal, and pharmaceutical remedy DNA damaging agent. Recombinant vectors and affecting DNA agents may be present in the same capsule or these components can be entered in different capsules. In the preferred embodiment, as a recombinant vector, a vector is used adenovirus is a recombinant, causing the expression of p53, this vector is present within adenoviral particles, and as a DNA damaging agent used cisplatin.

The components of the kit preferably introduced in the form of liquid solutions or dry powder. In that case, when the especially preferred is a sterile aqueous solution. In the case when the reagents or components are introduced in the form of a dry powder, the powder can change its state by adding a suitable solvent. It is obvious that the solvent may be present in another container means.

Brief description of drawings

Part of the present description are drawings, which are included for additional disclosure of certain aspects of the invention. The invention can be better understood when referring to one or a number of the following drawings in conjunction with a detailed description of the specific embodiments of the invention.

Fig. 1. Given a diagram of the formation of adenovirus-recombinant p53. Cassette expression of p53 was inserted between the Xba - and Cla plots pXCJL. 1. The p53 expression vector (pEC53) and recombinant plasmids (pJM17) were subjected to cotransfected in 293 cells. Infected cells were maintained in medium up until not manifested cytopathogenic effect. Identification of newly established adenovirus-recombinants p53 (Ad5CMV - p53) was carried out on samples of chain polymerisation reaction (PCR) DNA using DNA matrices prepared from the supernatant, treated with proteinase K and extract phenol.

In Fig. 2A p the cassettes in the expression of p53, primary materials chain polymerisation reaction (PCR) and limiting stations. The genome size of about of 35.4 kb, divided into 100 cells of the map (1 m.u. (cell card = 0,35 kb). Cassette expression took the place of zone E1 (1,3-9,2 m.u.) the Ad5 genome. The primary element 1 is concentrated in the first intron, located down the path of movement of the main promoter of the gene 1E cytomegalovirus (CMV) human. The primary element 2 is concentrated in the area of early signal polyadenylation SV40. Both of these elements, located in 15-20 bp from the input CDNA of p53 at the two ends, determine the product of 1.40 kb chain polymerisation reaction (PCR). Primary elements 3 and 4 are concentrated in 11 cell card (m.u.) and 13.4 cell card (m.u.) the Ad5 genome, respectively, and define the specific product 0,86 kb chain polymerisation reaction (PCR) of the genome of the virus.

In Fig. 2B shows a sample of the gel agarose product chain polymerisation reaction (PCR). Two pairs of primary elements (parameters) that define the DNA fragments of 1.4 kb (p53) and 0,86 kb (Ad5) were used in each reaction. In each reaction were used matrix DNA: plasmids pEC53 (1st band), Ad5 DNA/RCV/GL2 (a) 1-I band, no DNA (3rd band) and DNA Ad5CMV-p53 (4th band). The band with the designation (M) corresponds to genes-molecular weight markers.

citizenbane on the basis of the absence of enzyme (U), Hind III (H) (rear, the hind leg of the carcass). Bam HI (B), Eco RI (E) and Cla I (C), respectively, and analyzed on 1% agarose gel. The track with the designation (M) corresponds to genes-molecular weight markers.

In Fig. 3A, 3B, 3C and 3D show the results of observation cytopathogenic effects on 293 cells, obtained by using adenovirus-recombinant.

In Fig. 3A, 3B, 3C and 3D are a series of phase contrast images (400) 293 cells.

In Fig. 3A, 3B, 3C and 3D are given four pictures one of the figures shown on the next page. Fig. 3 A - before transfection, Fig. 3B - negative control result on the 12th day after transfection, Fig. 3C - emergence cytopathogenic effect (CPE) on the 12th day after transfection, Fig. 3D full manifestation cytopathogenic effect (CPE) on the 14th day after transfection.

In Fig. 4A, 4B, 4C and 4D are given immunology cells infected with adenovirus-recombinants.

In Fig. 4A, 4B, 4C and 4D shows a series of immunological images of H358 cells.

In Fig. 4A, 4B, 4C and 4D are 4 pictures of the figures presented on the same page.

Manifestation abilities to infection in H358 cells. H358 cells were infected with Ad5CMV-p53 or Ad5/RCV/GL2 50 PFU/cell for 24 hours. Nutrient creditevery using immunostaining. In Fig. 4A presents the results of the tests simulated infection using antibodies anti-p53. Ha Fig. 4B shows cells infected with the control Ad5/RSV/GL2 and tested using antibodies anti-p53. Ha Fig. 4C are infected cells Ad5CMV-p53, tested using unbound antibody (MOPC21). In Fig. 4D shows cell infection Ad5CMV tested antibody anti-p53. As antibodies anti-p53 was used Pab 1801, and staining was used avidin-biotherapy method.

In Fig. 5A shows a comparison of the relative level of expression of exogenous p53 in H358 cells using gel SDS-PAGE, stained blue (Coomassie blue). Samples of H358 cells that were infected with Ad5CMV-p53 or Ad5/RSV/GL2 30 PFU/cell, preparirovali within 24 and 72 hours after infection. Tested samples samples SDS-PAGE, stained blue (Coomassie blue), to determine the relative content of protein. Lanes 1 and 4 contain samples of cells infected with Ad5/RSV/GL2. Lanes 2 and 3 contain samples of cells infected by two individual groups Ad5CMV-p53 in 24 hours after infection. Lanes 5 and 6 are samples of Ad5CMV-p53 - infected cells collected at 72 hours after infection. Lane 7 is a sample H358 with simulated in REGO weight in kDu (CIBCO - BRL).

In Fig. 5B Dan batrouny Western analysis of gel is placed on the strip, identical to the one that belongs to SDS-PAGE in Fig. 5A. Relative levels of expression of p53 were studied Western-blokirovaniem using anti-p53. Primary antibodies were monoclonal antibodies against protein p53 (Pab 1801, Oncogene Science Inc.) and - actin (Amersham Inc.) HRP-conjugated secondary antibody and ECL-the developer were obtained from virus-infected cells H358, past the Western blokirovanie (Amersham Inc. ). Imprint, received the Western blokirovaniem, Fig. 5B, has a equivalent methods of recovery and treatment, and that those shown in Fig. 5A.

In Fig. 6 shows the time course of expression of p53, a certain Western-blokirovaniem. Multiple cups of H358 cells were infected with Ad5CMV at 10 PFU/cell. Cell lysates were prepared at set time points after infection. Western blokirovanie was tested using antibodies anti-p53 and antiactin at the same time. Lanes marked 'C' represent negative results. The histogram shows the relative amount of p53 defined using a densitometer.

In Fig. 7A shows the growth curve of virus-infected cells cancer easy the process involved 6 cups per cell line. After 24 hours, cells were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (multiplicity of infection, i.e., PFU/cell). After infection cells were counted daily for 6 days. Growth curves represent data obtained during the observation of four separate studies.

In Fig. 7B shows the growth curve of the virus-infected cell lung cancer human cell lines H322. Cells were seeded in 105cells per Cup (60 mm), were involved in the process 6 cups per cell line. After 24 hours, cells were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (multiplicity of infection, i.e., PFU/cell). After infection cells were counted daily for 6 days. Growth curves represent data obtained during the observation of four separate studies.

In Fig. 7C shows the growth curve of the virus-infected cell lung cancer human cell lines H460. Cells were seeded in 105cells per Cup (60 mm), were involved in the process 6 cups per cell line. After 24 hours, cells were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.about.i. (multiplicity of infection, i.e., PFU/cell). After infection cells were counted, aged the work.

In Fig. 8 shows a diagram of the sequence of operations in the study of Ad5CMV-p53 in an orthotopic model of lung cancer. Dosage and method of treatment of hairless mice infected cells H226Br and viruses represented in the specified schema.

In Fig. 9A, 9B, 9C and 9D shows sample mediastinal, lung of mice treated and control mice. Fig. 9A, 9B, 9C and 9D are photographs of one shape. The specimen was sacrificed at the end of the 6-week period commencing after the end of treatment. The tissue of the lung and mediastinum were excised for studying tumor formation.

In Fig. 9A shows a sample mediastinal unit from the normal hairless mouse.

In Fig. 9B given sample mediastinal block of the mouse, which was subjected to treatment with a carrier of phosphate-saline buffer solution (PBS).

In Fig. 9C shows a sample mediastinal block from the mouse, which took place course of treatment Ad5CMV-p53.

In Fig. 9D presents the sample mediastinal unit derived from mouse, previous treatment Ad5/RSV/GL2, the Arrows point to the tumor mass.

In Fig. 10A shows the continuous effect of CDDP on the degree of growth of the parent Ad-Luc-infected and Ad-p53-INFI is rez 24 hours in 100 l of medium was added Ad-Luc viral strain (stock biomass) (108PFU/ml) or Ad-p53 virus strain (108PFU/ml). After an additional 24 hour incubation period, the medium containing the virus was replaced with fresh medium that contained 10 g/ml CDDP.

In Fig. 10B shows the effect of CDDP for 24 hours on the growth of parental Ad-Luc-infected and Ad-p53-infected cells H358. Cells were exposed to CDDP continuously (Fig. 10A) or within 24 hours (Fig. 10B), followed by recovery free from drugs environment. Cells that remained attached monolayer, were tested for viability for more than five days by determining the degree of absorption of blue Triana. Shown is SE. On the fifth day the cell number for Ad-p53:CDDP group differed significantly from all other groups for A and B (p<0,05 identified through research 'Student st-test' (student's t-test).

In Fig. 10C shows the effect of different concentrations of CDDP on the viability of Ad-53-infected cells H358. After 24-hour exposure to the virus, Ad-Luc or Ad-p53 in cells, they were treated with 0, 10, or 100 g/ml CDDP for 24 hours, and then measured their viability.

In Fig. 11A shows nucleosidase and CDDP treated within 24 hours by the method, which was described in the comments to Fig. 10.

In Fig. 11B, 11C, 11D, 11E, 11F, 11G shows H358 cells, which were grown on slides, infected with Ad-p53 within 24 hours, treated for an additional 24 hours with CDDP and recorded for marking the location of the DNA fragments. There are the images of the parent H358 cells (B) or without (C) with CDDP; Ad-Luc-infected cells (D) or without (E) with CDDP; and Ad-p53-infected cells (F) or without (G) with CDDP. Arrowhead shows an example on dark colored fragments of the nucleus. Bar = 100 m

In Fig. 12A shows the effect of the combination of Ad-p53 infection with CDDP treatment for malignant tumor spheroids H358 cells. Environment multicellular tumor with H358 cells were prepared according to the method described previously (Takahashi et al., 1989). In zero day spheroids with diameters from 150 to 200 m were placed into 24 wells of agar plates, cover plate, and exposed to Ad-p53 or Ad-Luc for 24 hours. In the first day, the medium containing 10 g/ml of CDDP was added after removal of the vaccinated environment. On the second day after 24-hour incubation period, the agar plate was replaced with 1 ml fresh environment free from drugs. Perpendicular diameters were Asciano by the formula: a2b/a12b1where a and b are the smallest and largest diameters of the spheroid, and a1and b1the diameters of the first day. Only the relative amount of Ad-p53/CDDP spheroids significantly less (p<0.05, the value obtained in the tests Student's t-test (student's t-test) than the control group (Ctl).

In Fig. 12B, 12C, 12D, 12E shows the marking of the place dUTP with TdT-determination of apoptosis. Spheroids H358 were recorded on the third day and painted according to the method described in materials and methods of example 7. Control untreated spheroid (B); spheroid (C) treated with CDDP; spheroid (D), infected with Ad-p53, and spheroid (E), infected with Ad-p53 and processed CDDP. Bar = 100 m

In Fig. 13A shows a diagram of apoptosis using CDDP after infection in vivo by Ad-p53 on the results of measurement of changes occurring in the changing volumes of the tumor. H358 cells (5106) in 0.1 ml of balanced salt solution Hank were injected subcutaneously into the right block BALB/c female nu/nu mice. Thirty days 200 of the medium containing Ad-Luc (108Make/ml) or Ad-p53 (108Make/ml) was introduced into the tumour with a diameter of 5 to 6 mm Were made intratumoral injection (100 l) and akolouthia injection in two PR is temperatureare. Tumors were measured in two perpendicular diameters without information on medical groups, and tumor volume was calculated by obtaining the spherical shape with an average diameter of the tumor was calculated as the square root of the product of diameters of cross-sections. It was used by five mice for each treatment group, at the same time manifests the value of SE. Data were analyzed using Student's t-test. The arrow indicates the day of treatment. Given the independent evidence p<0,05 in the fifth day of the experiment 1; p<0,05 on the seventh day of the experiment-2 (B-E).

In Fig. 13B, 13C, 13D, 13E shows a histological study using Biotin-dUTP technology labeling with TdT-intermediate. Tumors were analyzed within five days after the start of treatment and immediately placed in O. C. T. (prepared histological material. Frozen tissues were excised in the cryostat at a thickness of 5). Excision were treated with 1 g/ml proteinase and stained according to the method described in the comments to Fig. 12. Presents images H358 tumors treated with: (B) only CDDP; (C) only Ad-p53; (D, E) Ad-p53 in combination with CDDP. Bars = 5 mm. Animals were kept in compliance with UT MD UT MD Anderson Institutional Animal Care and Use Committee) organization assiento of carrying out the invention

A. the Phenomena occurring at the molecular level in the development of lung cancer.

Research conducted by the authors of the present invention, identified critical phenomena at the molecular level, leading to the emergence and progression of cancer. This has enabled the inventors to develop new methods to restore some of the normal functions of the protein to such an extent, when the phenotype that causes cancerous tumors, can be suppressed in vivo.

The most common histology of lung cancer (80%) are United under the name of "lung cancer with involvement of a significant number of cells (NSCLC) and include squamous cancer, adenocarcinoma and unidentified form of cancer with extensive involvement of the affected cells. Accumulated a large Bank of data on the molecular biology of lung cancer, which is based on the study not so common form of lung cancer with involvement of a small number of cells (SCLC). SCLC can be different from NSCLC by neuroendocrine characteristics of the cells; SCLC is very sensitive to chemotherapy, but quickly resumed after the completion of the patient treatment. NSCLC can also serve as a model for other forms of epithelial cancer, vyzvannomu to be used to treat other forms of epithelial cancer.

Accumulated rich experience, confirming the fact that the process of malignant transformation is preceded by a genetic paradigm. The main pathological changes that occur in cancer cells relate to dominant oncogenes and suppressor genes neoplasms. Dominant oncogenes have alternatives in the class of genes, called proto-oncogene involved in the most important functions of normal cells, including signal transduction and transcription. Of paramount importance modification of dominant oncogenes, which provide the possibility of transformation include point mutations, transformation, reclassification and amplification (expansion, amplification). Genes suppressors of tumors needed to implement homozygotes loss-of-function due to mutations and deletions or a combination of the latter, with the aim of obtaining transformations. Some genes suppressors of tumors are designed to perform process control growth of cells by regulating transplantation. Modification expressions of dominant oncogenes and suppressor genes, in all likelihood, affect certain characteristics of the cells that contribute to the emergence of evil is the process of transformation, experiencing indirect impact of oncogenes, underdevelopment received therapeutic treatment methods, which should be aimed specifically at oncogenes and their products, serve as the target for impact on them during the treatment process. Initially, research in this area have focused on dominant oncogenes, since they first demanded descriptions. Studies of the process of DNA transfer intermediate gene demonstrated the mechanism of acquisition of malignant phenotype of normal cells, resulting in the transfer of DNA from human malignant tumors.

B. p53 and mutant p53 in cancer malignancy.

p53 is known as suppressor gene tumor (Montenarh, 1992). High levels were detected in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and a variety of viruses, including SV40. The p53 gene is a common target mutating inactivation in a wide variety of tumor neoplasms, and already documented that it is the most frequently mutated gene in traditional cancer tumors (Mercer, 1992). He aceste other neoplasms.

The p53 gene encodes a phosphoprotein amino acid - 375, which may form complexes with proteins-owners, such as extensive T-antigen and E1B. The protein found in normal cells and tissues, but in concentrations that are insignificant in comparison with the transformed cells or tissues of a tumor. Interesting is the fact that the natural p53 has been found to play an important role in regulating cell growth and division. Excessive natural expression of p53, as it turned out, in some cases suppresses proliferation cell lines tumor neoplasms. Thus, p53 may act as a negative regulator of cell growth (Weinberg, 1991) and can directly inhibit the uncontrolled growth of cells or to perform this function indirectly by activating genes that inhibit this growth. The absence of natural p53 or deprivation of its activity may contribute to the transformation process.

However, some studies indicate that the presence of mutant p53 gene may be necessary for the full expression of the transforming potential of the gene. Although it is recognized that natural gene p53 is extremely important resolutely are normal p53 gene and is essential for the transforming ability of the oncogene. The only genetic change, prompted by point mutations, can create kancerogeny p53. However, it is known that, unlike other oncogenes, point mutations of p53 occur at least 30 individual codons, often creating a dominant allelic genes that contribute to the formation of offsets in the phenotype of the cells without reposition to homozygote. In addition, many of these dominant negative alleles of genes is sometimes tolerated in the body and passed in the line of origin of the infection. Various mutant allelic genes can move from minimally dysfunctional state is extremely penetrating, dominant negative alleles of genes (Weinberg, 1991).

Casey and his colleagues showed that the transformation of the DNA encoding the natural p53 in two cell lines of breast cancer person, restores the function of the control growth inhibition in these cells (Casey et al., 1991). A similar effect was also demonstrated by transfection of natural, but not mutant, p53 cell lines human lung cancer (Takahasi et al., 1992). P53 exerts the dominant characteristics in comparison with the mutant gene and may be selected against proliferation after transfection in cells with mutant GE So such patterns may be perceived by normal cells without side effects.

Proceeding from the above, it is possible to conclude about the possibility of treatment of cancer related to p53, a natural p53 by reducing the number of cancer cells. However, the studies above, is far from practical application, though, because transfection of DNA cannot be used to introduce DNA into cancer cells in the body of the patient.

C. Methods of gene therapy

Developed several experimental methods for gene therapy, but they all have significant drawbacks (Mulligan, 1993). As mentioned above, there are a number of basic methods of transfection, in which DNA containing the gene of interest to us, is introduced into cells not by biology, but, for example, by physical or chemical penetration through cellular membranes. Naturally, such methods are limited to cells that can be temporarily removed from the body and able to move the cytotoxicity of the treatment, i.e., lymphocytes. Liposomes or protein conjugates formed by certain lipids, infofilename peptides, can be used for transfection, but the effectiveness of integrated the frame of the gene is often limited days in proliferating cells and weeks in nerazrushaushsii cells. Therefore, it is obvious that the method of transfection may not be considered appropriate for the treatment of cancer.

The second method is based on the natural ability of viruses to enter cells by introducing into their own genetic material. Retroviruses have an advantage as vectors for gene transfer, due to its ability to inject their genes into the genome of the host, transferring a large amount of foreign genetic material, infecting a broad spectrum of specific groups of cells that are arranged in specific cell lines. However, there are three main problems that hinder the practical use of retroviral vectors. First, retroviral infection depends on the availability of viral receptors on the surface of the target. The second retroviruses effectively integrate only in replicating cells. Finally, the last retroviruses difficult to concentrate and to disinfect.

D. Adenovirus structure intended for use in gene therapy

Adenoviruses person is tumor viruses DNA double with a genome size of about 36 kb (TOOZA, 1981). Adenoviruses have been widely studied and described as a model system for acne adenoviruses as a system for gene transfer. This group of viruses is easy to grow and use, while they exhibit a broad host range in vitro and in vivo. In lytic infected cells adenoviruses are able to trim protein synthesis - master, directing cellular mechanisms in the synthesis of a large number of viral proteins and receiving copious quantities of viruses.

Area E1 of the genome includes both E1A and E1B, which encode proteins responsible for the regulation of transcription of the viral genome and some cellular genes. Expression of E2, including E2A and E2B, provides a synthesis of viral replication functions, such as DNA-associated protein, DNA polymerase and the final protein that creates the ground for replication. The products of the gene E3 prevent cytolysis caused by cytotoxic T cells, and tumor necrosis factor and plays an important role for viral reproduction. Functions associated with the E4 proteins include DNA replication, the expression of the latter gene and clipping of the host cell. The products of the latter gene include most of the protein capsid of the virion, while they appear only after most of the production is the only main transcript of the last main promoter will be performed. Lastly the et and Perricaudet, 1991a).

Since only a small portion of the viral genome required in cis (Tooza, 1981), adenomyosarcoma vectors offer a great potential to replace large DNA fragments using such cell lines, as for 293 cells. Ad5-transformed cell line mesonephros (primary kidney) (Graham et al., 1977) was established to provide basic viral proteins in trans. The inventors, therefore, have shown that these features of adenoviruses showed good preconditions for their use, with the purpose of sighting ingress into cancer cells in vitro (Grunhaus &Horwitz, 1992).

Special advantages adenovirus system for transferring a foreign protein into the cell include:

(i) the ability to substitute relatively large fragments of viral DNA with alien DNA;

(ii) structural stability of adenovirus-recombinants;

(iii) the safety of adenoviral purpose for man;

(iv) the lack of any known Association of adenoviral infection with cancer or malignancy;

(v) the ability to obtain high titers using recombinant virus in the treatment;

(vi) the high is overuse, are high levels of gene expression. In addition, replication of the adenovirus does not depend on the replication of a gene-master, which distinguishes his use of the application of the retrovirus. Because adenovirus transforming genes in zone E1 can be freely removed and to ensure the existence of effective vectors of expression, there is an opinion that the carcinogenic risk from adenoviral vectors is minimized and is not taken into account (Grunhaus &Horwitz, 1992).

Basically, adenoviral gene transfer systems are based on the constructed recombinant adenovirus, which is a replication, not completed due to lack of its genome, such as E1, but still preserving the ability to infection. A relatively large amount of foreign protein can be taken if the adenoviral genome to perform additional removal. For example, adenoviruses, not stocked in both zones E1 and E3, capable of carrying up to 10 kb of foreign DNA and can be grown to high titers in 293 cells (Strattford - Perricaudet & Perricaudet, 1991a). In addition, there was also a surprisingly prolonged transgene expression accompanying adenoviral perenosa gene intermediary in eukaryotically cells of living organisms. For example, in the treatment of mice with a rare recessive genetic disorder - chlamydia psittaci transcarbamylase-deficiency (Ornithine transcarbumylase (OTC) - it was found that adenoviral patterns can be used to supply normal (OTC)-enzymes. Unfortunately, expression of normal levels (OTC) was achieved in 4 of the 17 examples (Strattford - Perricaudet et al., 1991b). Therefore, the defect was only partially fixed most of the mice that did not lead to physiological or to phenotypic changes, and therefore, these results leave little hope for the possibility of using adenoviral vectors in cancer therapy.

Attempts to use adenovirus during transfer of the gene for transmembrane conductance regulator with fibrous-cystic fibrosis (CFTR) in the lung epithelium of cotton rats also had only partial success without the possibility of obtaining the biological activity of the transferred gene in the epithelium of animals (Resenfeld et al., 1992). And again, these studies have demonstrated gene transfer and expression of CFTR protein in the cells of the pulmonary Airways (air ducts), but not confirmed any physiological effect. In 1991 science article authors Resenfeld et al. showed pulmonary expr and came to the conclusion, what levels of expression, which they observed, make up only 2 percent from the level that you want to protect the lung of a person, i.e., these levels are well below those that are able to provide a physiological effect.

The gene for human1-antitrypsin was introduced into the liver of normal rats by intraportal injection, which saw its expression, resulting in the discovery of secretion entered the human protein in the plasma of these rats (Jaffe et al., 1992).

However, these levels were not high enough to have any therapeutic effect.

Thus, these results do not confirm that the adenovirus sufficiently capable of directing the expression of sufficient protein in recombinant cells to obtain relevant physiological effect, and therefore they do not make predictions about the use of potential adenovirus system for cancer treatment. In addition, existing up to the present time the prior art it has been suggested that p53 may not be incorporated into the Packed cell such that they used to obtain adenovirus, as it could be toxic. That cannot be combined.

E. Patterns of adenovirus p53 and suppression of tumor

The present invention provides gene therapy of cancer with a new and more efficient vector suppress tumor. This recombinant virus takes advantage of adenoviral vectors, such as high titers, a wide range of lesions, efficient transduction and inability to integrate into the target cell. In one of the embodiments of the invention receive replication-defective, does not require a helper-independent adenovirus, which helps to bring out the natural expression of p53 (Ad5CMV-p53) under the control of the human cytomegalovirus promoter.

Control functions on the expression vectors are often provided with viruses when you want to achieve expression in mammalian cells. For example, commonly used promoters are derived from polyoma, adenovirus 2, and simian vacuolating virus 40 (SV40). Early and late promoters of SV40 virus are particularly often used, since they are both easily obtained from the virus as a fragment, which contains the SV40 viral source replication. Smaller or larger SV40 fragments can also be used the structure of a section of Bg II, self-contained viral replication source. In addition, there is also the possibility, and often desirable, promoter or control sequences normally associated with on the gene sequence, provided such control sequences are compatible with the systems of the host cell.

The replication source can be provided by the structure of the vector to include an exogenous source, such, for example, which may be derived from SV40 or other (for example, polyoma, adeno, VSV, BPV) viral sources, or can be equipped with a mechanism of chromosomal replication in the host cell. If the vector integrates into the chromosome of the host cell, this is enough.

The structure and reproduction of the preferred adenovirus p53 presented in a graphical chart of Fig. 1. In this regard, has been developed an improved Protocol for playback and identification of adenovirus-recombinant (to be described below). Once identified, the adenovirus is a recombinant p53 was structurally confirmed by analyses of chain polymerisation reaction (PCR), as shown in Fig. 2. After isolation and confirmation of the structure of adenovirus p53 was used for epitiro) Westrn showed that exogenous p53 protein showed expression at a high level (Fig. 4 and Fig. 5), and the peak of its expression peaked at day 3 after infection (Fig. 6).

It was also shown in the cell line H322 point mutations of p53 that mutant p53 was adjusted by lowering the levels of expression of exogenous p53. As an experimental control element was used virion Ad5/RSV/GL2, which had a structure similar to the structure of Ad5CMV-p53. This virus contained with DNA-luciferase produced LTR-promoter of the virus Rous-sarcoma in the expression cassette of the virion. Neither the expression of p53, no changes in the expression of actin was not detected in cells infected with Ad5/RSV/GL2 of the virion. The growth of H358 cells infected with Ad5CMV-p53, was largely suppressed, as compared with uninfected cells or cells infected with a control virion (Fig. 7A). The growth of H322 cells was also largely suppressed by the virion p53 (Fig. 7B), while the growth of H460 cells cancerous human tumors containing natural p53, had a smaller effect (Fig. 7C).

Ad5CMV-p53 had a strong overwhelming impact on the growth of lung cancer cells in vitro. Growth suppression was not as evident when cells were infected with Ad5CMV-p53 at MOI (the centre of the control virus Ad5/RSV/GL2. In our studies, the optimal dose for the study of the age composition of the population was set to 10 to 50 PFU/cell. In this dose range the suppression of cell growth was indicative of the p53 protein with the manifested expression of p53.

Tests conducted on hairless mice, demonstrated that the ability to form tumors in H358 cells treated Ad5CMV-p53 was significantly reduced. In experimental models in mice orthotopic human lung cancer tumorigenic cells H226Br with point mutations were introduced vnutritrahealno three days prior to treatment with the virus. Intratracheal instillation Ad5CMV-p53 warned the formation of tumors in these model systems, suggesting that the modified adenovirus is an efficient vector for the intermediate transfer and gene expression of tumor suppressor level cancerous human cells and that the virus Ad5CMV-p53 can be improved to obtain a therapeutic agent, with the aim of its use in gene therapy of cancer.

Ad5CMV-p53 mediates when receiving a high level of expression of the p53 gene in cancer cells of the human lung, as demonstrated by Western-blokirovanie analyses. The ind is for the more abundant, than active internal control in H358 cells. High levels of expression can be explained by the presence of highly efficient carrier of the gene, a strong promoter of the cytomegalovirus (CMV) driving p53 CDNA, and adenovirus E1-amplifier, activating the transcription of CDNA-p53. The duration of expression of p53 after infection was more than 15 days in H358 cells. However, there was a rapid attenuation of the expression after 5 days after infection. Analyses of a chain reaction of polymerization on DNA samples on infected H358 cells showed a low level of viral DNA with reduced protein levels, indicating a loss of viral DNA in the process of continuous growth of cancer cells in vitro.

The decrease in the expression of p53 can also be the result of cell attenuatio promoter of cytomegalovirus, which controls the level of expression of p53, since the phenomenon of cut-off promoter of cytomegalovirus intermediate host cell is already known (Dai et al., 1992). Adenoviral vectors are reintegrirovat vectors-vectors of genes, and therefore the duration of gene expression depends on a number of factors, including cells-the owners of transferred genes and special relevant conductance in cystic degenerations in epithelial cells of the cotton rat, that manifested itself in 6 weeks after infection (Rosenfeld et al., 1992). Laboratory Perricaudet showed minimal expression of a gene-minidystrophin in the mdx mouse muscle, which lasted more than three months after infection. Transient expression of high levels of natural protein p53 observed in this study may have promising results, which results in reduction of possible side effects on normal cells, accompanying in vivo treatment with Ad5CMV-p53.

Disclosed in this specification, the results indicate that adenovirus is a recombinant p53 has the ability to suppress tumors, however, he performs these actions by restoring the function of the p53 protein in cancer cells. These results provide an opportunity to make a statement about the use of the virion Ad5CMV-p53 as a therapeutic agent for cancer treatment.

F. Striking DNA agents

In connection with the present invention can be used with a wide variety of DNA damaging agents, such as agents that structure the DNA (form cross-links), agents that are embedded in DNA, and agents that lead to chromosomal emitations.com distortions by creating conditions for the synthesis of kleinhovia acid, specifically DNA, are used in this situation to defeat the DNA that leads to a synergistic antineoplastic (antitumor) connection. Can be used cisplatin and other DNA alkylating agents. Cisplatin is widely used for the treatment of cancer. The effective dose used in clinical assignments 20 mg/m2within every three weeks to complete a course of treatment consisting of three cycles. Cisplatin is not absorbed orally and must be administered intravenously, subcutaneously, directly in tumor tissue or interperitoneal.

Agents that affect DNA, also include components that interfere with DNA replication, mitosis and chromosome segregation. Examples of such compounds can serve as adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, etc. widely used in clinical assignments for the treatment of malignant tumors, these compounds are administered through injection loading dose of the substance intravenously with doses of 25 - 75 mg/m2at intervals of 21 days for adriamycin, to 35-50 mg/m2for etoposide intravenously or intravenously twice the subgroups, also lead to the destruction of DNA. There was obtained a number of precursors of nucleic acids. In particular, deserve the attention of the agents that have been subjected to extensive testing and are quite amenable to evaluation. In particular, can be useful such agents as 5-fluorouracil (5-FU), which are preferably used neoplastic tissues, making this agent is particularly useful when transferring cells of a tumor. Despite the extreme toxicity of 5-FU can be used for a wide range of conductors (vectors), including local; commonly used intravenous doses of 3 to 15 mg/kg/day.

There are other factors causing the defeat of DNA, which are widely used and are well known as rays, X-rays and/or controlled delivery of radioisotopes to tumor cells. Can be applied to other types of DNA damaging factors, such as microwave and ultraviolet radiation. Most probably assume that all of these

factors have extensive damaging impact on a large range of precursors of DNA, replication, DNA repair, as well as on the Association and maintaining the viability of the chromosome. Dosage ur the and of time (three to four weeks) before a single dose value from 2000 to 6000 x-ray. Dosage for radioisotopes varies within very wide limits and depends on the half life of the isotope, the strength and type of the emitted radiation, and the degree of absorptivity cells with malignant neoplasm.

Cm. "Remington''s Pharmaceutical Sciences, 15th edition, Chapter 33, page 624-652. May be some variations in the dosage, depending on the conditions associated with the characteristics of patients. The person responsible for the introduction of drugs, in any case individually determines the appropriate dose for the individual patient. In addition, a medicine intended for the reception of patients, must be sterile, protected from progenote, to meet the security requirements and disinfected in accordance with the standards of the FDA Office of Biologics.

G. treatment with the use of p53 and cisplatin

To determine the effectiveness of combined therapies, including the movement of a gene and the influence of chemotherapeutic agents for the treatment of human cancer, the inventors investigated the question of whether to initiate apoptosis in vivo continuous Ad-p53 and CDDP. During the following three days after direct intratumoral injection of Ad-p53 or interperitoneal introduction CDDP tumor cells H35 the ATA slowdown. If Ad-p53 and CDDP were introduced at the same time, the tumor regressed partially, and the size of the investigated tumors remained statistically significantly less than those who participated in any of the other treatment groups. Increase the absorption effect became even more noticeable after two cycles of treatment (Fig. 13A). Histological studies were found massive destruction of cancer cells in the zone, which was introduced Ad-p53 in the body of a mouse exposed to CDDP treatment. Staining of the place showed a large number of dead cells around the intercellular spaces (Fig. 13B-E). In contrast, tumors treated only CDDP or only Ad-p53, did not show any zone of saturation or zones of apoptosis.

The present invention demonstrates a new direction in human gene therapy in combination with conventional chemotherapy using DNA structure agent. Resistance of tumor cells to chemotherapeutic drugs is a major problem in clinical Oncology. A type of cancer NSCLC is at least 80% of lung cancer cases; patients with NSCLC, but mostly immune to chemotherapeutic drugs (Doyle, 1993). One yoke of cancer treatment, based on the principle of moving a gene, a study of the mechanisms of interaction between the gene product and chemotherapeutic drug. The gene of herpes simplex-thymidine-kinase (HS-tK) being transferred retroviral vector system in the tumor tissue of brain, successfully demonstrated sensitivity to antiviral agent gancilclovir (Culver et al., 1992). The gene product HS-tK is an exogenous viral enzyme, and because the expression of protein wt-p53 appears in normal tissues, there is the assumption that the modulation of resistance to chemotherapeutic agents-induced alteration in the expression of wt-p53 may represent an alternative approach, which uses a path that is indirectly determined endogenous genetic program.

Adenovirus system has potential advantages when moving the gene in vivo, such as the ease of obtaining high titer virus, a high degree of infectious activity and immunity to many types of cells. However, the stability and duration of expression of the introduced gene is still problematic. The increased levels of p53 in cells, which differ in sensitivity is oragami stimulants (Fritche et al. , 1993, Zhan et al., 1993), although increased activity caused by the action of DNA, may be returned in original condition within four hours, if the stimulator removed (Tishler et al., 1993). Hence the high level of expression of p53 can be maintained even after termination of exposure on the body of a medicinal product. Ensuring the expression of the protein wt-p53 with Ad-p53 culminates on the third day after infection (14 - fold greater than endogenous wild-type) and decreases to a low level for the ninth day (Zhang et al. , 1993). This suggests that alternating high level of expression of wt-p53 enough to initiate cytotoxic program in a cancer cell.

H. Patients and treatment protocols (medical history)

The inventors suggest that the regional delivery of adenoviral p53 gene structures, such as inoperable obstructive endobronchial forms of cancer, will be an effective way of delivering a therapeutically effective gene to withstand disease clinic. Carrier p53 gene must act in combination with agents or factors that lead to the destruction of DNA. This combined approach represents a significant improvement in suprasec efforts on killing or removing the last cancer cell with an effective destruction of the DNA. But as "dormant" tumor cells is an established fact, it makes the possibility of such killing in the highest degree unlikely.

There is an opinion that the uptake of adenoviral cell structures NSCL should reduce the degree of proliferation of these cells, however, the existing models show that the combined use of DNA damaging agents or factors, together with the adenovirus leads to profound suppression of cell growth and tumor size, which does not detect none of the components separately. The compositions and methods disclosed herein, to a large extent determine the increase over time of the probability that a sick light may remain in the expanded condition, preventing the resumption of growth and division of tumor cells that can extend the life of the patient.

Patients with recurrent inoperable endobronchial tumors, which partially or completely covers the respiratory tract and which unsuccessfully undergone or may not be subjected to external radiotherapy, were selected for treatment Protocol proposed combined method. Existing therapeutic methods of treatment in these conditions suggest that is terapia. There is the possibility of introducing brachytherapies catheter and the appointment of additional radiotherapy, intravenous DNA damaging agents. Patients who received such treatment could live on average 6 months. Patients for whom brachytherapies treatment proved ineffective, were also selected to receive gene therapy. The tumor may be removed from the breathing passages by using a laser or biopsy forceps. This may be done in conjunction with input adenoviral structures, thus reducing the quantity that should be entered. The purpose of viral structures should not prevent the appointment of patients other palliative means, if the tumor progresses.

I. Other means of gene transfer

The successful application of gene therapy depends mainly on the integration of the gene is able to correct genetic disorders in the genome of the host, where he could live and play in DNA-owner, and to show their expression level, can compensate for missing components of the defective gene. Ideally, the disease would be cured after one or more courses of treatment with no serious adverse phenomenon is applied using the present invention.

The first approach is to transfection DNA containing the desired gene into the cells, for example, by penetration through the cell membrane by chemical or physical means. This approach is applicable mainly in relation to the cells, which can be temporarily removed from the body and which bear the cytotoxicity of treatment (namely, lymphocytes). Liposomes or protein conjugates formed by certain lipids and infofilename peptides, can be used for transfection in vivo (Stewart et al., 1992; Torchilin et al., 1992; Zhu et al., 1993), however, the actual effectiveness of the integration of the gene is very low. It is established that the desired gene integrates into the genome of only one cell in 1,000 to 100,000. In the absence of integration, the expression of the introduced gene is limited to a few days in growing cells or a few weeks in nerazrushaushsii cells, due to the non-integrated DNA.

The second approach is based on the natural ability of viruses to enter cells, bringing in their own genetic material.

Retroviruses showed himself as vectors carrying genes, because of their ability to integrate their genes into the genome of the host, transferring a large number turkleton lines (Miller, 1992).

In the basis of the third method is the use of other viruses, such as adenoviruses, herpes simplex viruses (HSV), cytomegaly virus (CMV) and adenoassociated virus (AAV), which are designed to act as vectors carrying genes. Although some viruses are able to accept foreign genetic material is limited in the number of nucleotides, they can accommodate, and within the boundaries of the cells that they infect, the virus was successfully demonstrated the effect of gene expression. However, adenoviruses do not integrate their genetic material into the genome of the host and therefore do not require replication master for the occurrence of gene expression, which makes them ideal for excitation of fast and efficient heterogeneous gene expression.

Although the invention has been described with a certain degree of private manifestations, it is evident that many alternatives, modifications and variations may occur from experts in the study of this specification, revealing the essence of the invention. It is important that all these alternatives, modifications and variations were within the range of characteristics defined by the claims.

Example 1. Structure of the p53 expression vector.

This example describes the structure of a p53 expression vector. This vector is designed and used for replacement of zone E1 (1,3 - 9,2 m.u.) the genome of the Ad5 adenovirus strain and obtain patterns adenoviral virion described in example 2.

Cassette expression of p53, as shown in Fig. 1, containing the promoter of cytomegalovirus (CMV) human (Boshart et al., 1985), p53 cDNA and early signal polyadenylation SV40, was installed between the sites Xba I & ClaI pXCJLI (described: Dr. Frank L. Graham, Mc Master University, Canada).

The size of the AI p53 have been replaced by a zone of E1 (1,3 - 9,2 m.u.) the Ad5 genome.

The primary element 1 has the sequence 5'-GGCCCACCCCCTTGGCTTC-3' (SEQ ID No. 1) and is concentrated in the first intron down movement of the main promoter of the gene 1E human cytomegalovirus (Boshart et al., 1985). The primary element 2 has the sequence 5'-TTGTAACCATTATAAGCTGC-3' (SEQ ID No. 2) and concentrated in early signal polyadenylation SV40. Both the primary element, 15-20 bp from including cDNA of p53 at both ends determine the product of the chain reaction of polymerizatio of 1.40 kb. The primary element 3 has the sequence 5'-TCGTTTCTCAGCAGCTGTTG-3' (SEQ ID No. 3) and the primary element 4 has the sequence 5'-CATCTGAACTCAAAGCGTGG-3 (SEQ ID No. 4) and are located in 11 m.u. and 13.4 m. u. the Ad5 genome, respectively, which define the product 0,86 kb-viral-genome-specific chain polymerization reactions.

Example 2. Education and the reproduction of adenovirus-recombinant p53.

This example describes the method used for the formation of a helper-independent recombinant adenovirus expressing the expression of p53. Molecular algorithm is applied to obtain the adenovirus is a recombinant, based on the fact that, due to the limit of packaging (dosage) of adenovirus, pJM17 cannot form the virus yourself. Therefore, homologous recombinational virus which can be Packed only in cells in which expression of the protein. In this example, 293 cells are used as host cells for reproduction of viruses that contain replacement cassettes for the expression of heterologous DNA in zones E1 and E3. This process requires cotransfection DNA in 293 cells. Transfection widely determines the efficiency of viral reproduction.

The method used for DNA transfection in 293 cells to generate the present invention, generally included the use of calcium phosphate/coprecipitation DNA (Graham &van der Eb, 1973). However, this method together with rosethorne relatively difficult to implement and usually ends with the low efficiency of reproduction of viruses. As shown in this example, transfection and subsequent identification of the infected cells were significantly improved through the use of the method of transfection with the intermediate moving element of the liposomes in the identification of infected cells using cytopathogenic effect (CPE).

Cell line 293 was maintained in a slightly modified nutrient medium Dulbecco with 10% inactivated by heating horse serum. Vecto ledah 293 using a transfection method with an intermediate DOTAP, according to the Protocol execution (Boehringer Mannheim Biochemicals, 1992). Schematically this is shown in Fig. 1.

The 293 cells (passage 30, 60% intersection) were infected for 24 hours before transfection or 60 mm cups, or in 24-hole Petri dishes, Cells in each well were subjected to transfection with 30 l of DOTAP. 2 g of p53 expression vector and 3 g of plasmid pJM17. After transfection the cells were given food in the form of MEM medium every 2-3 days until, until you appear cytopathogenic effect.

Example 3. Confirmation of the identity of adenovirus-recombinant.

This example illustrates a new way to explore the chain polymerisation reaction (PCR) to confirm the identity of recombinant virions that accompany the process of cotransfection appropriate cell line.

Layers supernatant of cell culture (from 50 to 370 liters ) were collected from participating in the research Petri dishes treated with proteinase K ( 50 g/l with 0.5% SDS and 20 mM EDTA) at 56oC for 1 hour using phenolchloroform, and nucleic acids were removed precipitated using ethanol. The precipitated DNA was re-suspended in 20 l dH2O and used as template for amplification chain reaction polymerizes the tee shown in Fig. 1 and marked SEQ ID No. 1, 2, 3, and 4, respectively. The primary elements of specific inclusions DNA determine the product of 1.4 kb chain polymerisation reaction (PCR), and viral genome-specific primary elements that define a product 0,86 kb chain polymerisation reaction (PCR). Chain reaction polymerization was carried out in a volume of 50 l containing 4 mM MgCl2, 50 mM KCl, 1.1% of Triton X-100, 200 M of each dNTP, 10 mM Tris-CI (pH 9,0), 2 M of each of the primary element and 1.0 unit of Tag polymerase (Promega). The reaction was carried out at 94oC for 0.5 min, 56oC for 0.5 min, 72oC for 1 min for 30 cycles.

To simplify the identification process again reproduced recombinant virus was developed way directly chain polymerization reactions (PCR) on DNA samples taken from the supernatant of cell culture. Layers (50 or 370 l ) of the supernatant liquid cell environment with cytopathogenic effect were treated with proteinase K and phenol/chloroform/extract. After ethanol precipitation, the DNA samples were analyzed using a chain polymerisation reaction (PCR), which was attended by two pairs of primary cells, to enhance specific sequence VK is polarization and their sequence shown in Fig. 1. Primary elements 1, 2, 3 and 4 represented by SEQ ID No. 1, 2, 3, 4, respectively.

As a result, the inclusion of the cDNA of 1.4 kb and a fragment of the viral genome 0,86 kb were amplified from the expression vector of (positive) and DNA samples positive cell cultures (Fig. 2B, lane 1 and 4, respectively). Only a fragment 0,86 kb was amplified from the sample DNA virus Ad5/RSV/GL2 (a negative result, lane 2). No amplified bands, bundles of fibers was not found at chain polymerization reactions (PCR), who used a raw positive supernatant layer cell culture (3rd band)

These results indicate that adenoviruses, secreted in the cell culture medium, can be defined chain polymerization reaction (PCR), using only 50 l of the supernatant of the cell culture environment for the preparation of matrix DNA. The results should allow to develop a quantitative method when using this method, to determine adenoviral titers that are usually obtained by using the method of sockets.

The natural sequence of the cDNA of p53 in the virus Ad5CMV-p53 was confirmed by dideoxy DNA sequence CsCl-gradient - purified winiam virus promoter, Rous sarcoma, and luciferase cDNA, which were used in the expression cassette. Adenovirus is a recombinant that carries the gene for E. coli - galactosidase (LacZ), Ad5CMV-LacZ, has a structure similar to the Ad5CMV-p53, and was obtained based on the research of Dr. Frank L Graham (see Graham et al., 1991).

Viral biomass, titer and infection. Individual clones of Ad5CMV-p53, Ad5/RSV/Gl2 and Ad5CMV-LacZ viruses were obtained by disinfection plaques (sockets) according to the method of Graham & Prevec (1991). Single viral clones were reproduced in 293 cells. The cultural environment of 293 cells that detect finished cytopathogenic effect, was collected and centrifuged at 1000 g for 10 min supernatant Collected masses were stratified and placed for storage at -20oC as strain (biomass). Viral titers were determined in the reaction of rosethorne (Graham and Prevec, 1991). Infection of cell lines were produced by adding viral solution (0.5 ml per 60-mm Cup) to the cell monolayers and the creation of conditions of incubation period at room temperature for 30 minutes with a short shaking every 5 minutes. This process was accompanied by the addition of the cultural environment and the return of infected cells in Inca use Ad5CMV-LacZ in a number of cell lines, for example H226Br, H322, HeLa, HepG2, LM2 and Vero. Using color X-gel all cell lines were painted. 97-100% in blue color after infection with Ad5CMV-LacZ at MOI 30 PFU/cell.

Example 4. The expression of p53 gene directed Ad5CMV, in cells of human lung cancer.

This example discloses the use of adenovirus-recombinant p53, with the aim of infection of lung cancer cells with homozygotes removal of the p53 gene. The results show that the growth of these cells and expression of mutant p53 was suppressed by detecting the potential of the virion Ad5CMV-p53, showing the signs of an agent capable of controlling the status of the metastatic cells.

The infected cell monolayers were subjected to immunohistochemical processing, which consisted in the fact that these layers were fixed with 3.8% formalin and processed 3% H2O2in methanol for 5 minutes. Immunohistochemical analyses were performed using the kit Vectastain Elite (Vector, Burlingame, CA). As primary antibodies used antibody PAb 1801 anti-p53 (Oncogene Science, Manhasset, N 4); MOPC-21 (Organon Tekhica Corp., West Chester, PA) was used as a negative control group. As the second antibody ispolzovalsya ABC reagent batrouney peroxidase horseradish) was used as the determinant of the antigen-antibody-complex. Finally, the cells after washout of the drug were zakrasheny Harris with hematoxylin (Sigma) and mounted with sealing material - Cytosine-60 (Stephens Scientific, Riverdale, NJ).

Immunohistochemical analysis of infected cell lines were performed to study the expression of in situ expression of p53, excited by the promoter of cytomegalovirus (CMV) virus Ad5CMV-p53. In cell line H358, which had homozygote destruction of p53, the p53 gene was transferred with the efficiency of 97-100%, as shown by immunohistochemical analyses, when cells were infected with Ad5CMV-p53 at a multiplicity of infection - 30-50 rosette-forming units (PFU/cell) (Fig. 4).

High transfer efficiency of adenovirus-recombinant was confirmed by Ad5CMV-LacZ virus, which carries the LacZ gene, transformed 1E promoter of CMV (cytomegalovirus) human. When a plurality of human infection 30-50 PFU/cell, all cells were subjected to the study, including HeLa, G2, LM2, and cell lines cancer NSCLC man showed 97-100% positive result for b-galactosidase activity using X-gel staining. These results indicate that adenoviral vectors are an effective tool for the delivery of g is Oh, prepared by dissolving cell monolayers in the cups, using a sample buffer for SDS-PAGE (0.5 ml for 60-mm Cup) after washing the cells with phosphate-saline buffer solution (PBS). For analyses SDS-PAGE lines were loaded with lysates equivalent to 5104cells (10-15 ml). Proteins were transferred to Hybond TM-ECL-membrane (Amerrsham, Arlington Heights, IL). Membranes were blocked with 0.5% milk powder in phosphate-saline buffer solution and conducted research using primary antibodies: mouse anti-human p53 monoclonal antibody PAb 1801 and mouse anti-human active monoclonal antibodies (Amersham), washed and analyzed using secondary antibodies, namely rabbit antimisting gamma-globulin G, linked to horseradish peroxidase (horseradish buffer-conjuated rabbit antimouse IgG) (Pierce Chemical Co. Rocliford, lL). Membranes were developed in accordance with the enhanced chemiluminescent Protocol. The relative amount of exogenous p53 expressed by expression were determined by densitometry (Molecular Dynamics Inc., Sunnyvale, CA).

Western-blots showed that exogenous p53 protein showed its expression at high levels (Fig. 5A, lanes 2, 3 and 5, 6). Peak protein was observed on day 3 after infection (Fig. 6, vedet recombinant Ad5CMV-p53 in example 1. The virion contains cDNA-luciferase contained in the active state of the LTR promoter of the virus Rous-sarcoma in the expression cassette of the virion. Neither the expression of p53, no changes in the expression of actin was not detected in cells infected with virion Ad5/RSV/GL2.

Adenovirus is a recombinant was used to infect three lines of lung cancer NSCLC:

cell line H358, which has homozygote deletion of p53 gene,

cell line H322, which has a point mutation of p53 gene at codon 248 (G to T), and

cell lines H460, which has a natural gene p53.

The degree of growth of NSCLC cells of a person was determined based on inoculation of H322 and H460 (1105or H358 (2105in 60-mm dishes 24 hours prior to viral infection. Cells were infected with virus at a multiplicity of infection of 10 PFU/cell. The cultural environment was used to control the simulated infection. Matrix cultures of each cell line with various types of treatment were counted daily from the 1st to the 6th day after infection.

The growth of H358 cells infected with Ad5CMV-p53 was significantly suppressed, compared with uninfected cells or cells infected with the control wiryono person, containing natural p53, were subjected to a lesser extent (Fig. 7C). The growth of H358 cells infected with Ad5CMV-p53 was suppressed by 79%, whereas uninfected cells or cells infected with the control virus, were not suppressed. The growth of the cell line H322, which had a point mutation was suppressed by 72% by Ad5CMV-p53, whereas the growth of cell lines H460, containing natural p53 was suppressed less (suppression of growth was 28%). The results indicate that adenovirus is a recombinant has properties inhibit the growth of malignant tumors, due to the restoration of function of the p53 protein in the cells of a tumor.

Example 5. The use of Ad5CMV-p53 in the treatment of cells with p53-deficiency.

This example relates to the use of adenovirus-recombinant p53 in restoring the function of suppressing the growth of tumor cells in vitro and implementation thus treatment of diseases associated with malignant or metastatic cell growth. He describes some of the methods according to this invention, intended for use in the treatment of cancer using adenoviral gene therapy.

H358 cells were inficon is operated by environment, modeled as infection. 24 hours after infection treated cells were collected and washed twice with phosphate-saline buffer solution (PBS). For each of the types of processing three million (3106) cells in a volume of 0.1 ml were subcutaneously injected in each of hairless mice (Harlan Co. Houston TX). For each of the treatments was used by 5 individuals. Mice were irradiated (300 cGy,60Co) before injection and were examined weekly after injection. Neoplastic lesions were detected at the end of the six-week period, and tumor volume was determined on the basis of the model of spheres with an average diameter of the tumor, obtained as the square root of the magnitude of the product of diameters of the cross-section.

To determine the effect of suppressing the oncogenic potential with Ad5CMV-p53, hairless mice were infected subcutaneously H358 cells (cell cancer type NSCLC person) to cause malignancy. Each mouse received a single injection of the cells, which were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 10 PFU/cell for 24 hours. H358 cells treated only with medium were used as control with false infection. Tumors that initially PR is zirovanii cells, as shown in the table. 1.

As shown in the table. 1, the mice that received cells treated with Ad5CMV-p53, tumor lesions did not develop. Tumors at the end of the 6-week period was 4-5 mm in diameter. This study began with 5 animals per group. One mouse in each group research with Ad5CMV-p53 or Ad5/RSV/GL2 did not participate and were not able to complete the cycle of research. Early death occurred, presumably from nosocomial infections.

Example 6. Ad5CMV-p53 in the treatment of lung cancer.

This example relates to the use of recombinant adenovirus p53 to restore the function of the suppression of cell growth of tumors in vivo and treatment thus malignant tumors in animals. In this example, there are several ways of carrying out the invention, with a view to its use for the treatment of cancer by the method of gene therapy using adenovirus as an intermediary for the proxy.

The effectiveness of Ad5CMV-p53 to suppress oncogenic potential has been repeatedly proven in a murine model of orthotopic human lung cancer. Because cells H358 and H322 in this model do not cause a tumor, the study used cell line is and. H226Br has a point mutation (ATC to GTC) in exon 7, codon 254, p53 gene is oncogenic in the mouse body.

Testing procedure in a murine model of orthotopic human lung cancer has been previously described (Gorges et al., 1993). In a nutshell, hairless mice were irradiated (300 c Gy 60 Co), then inoculated intratracheal installation of cells H226Br. Each mouse received 2106cells in a volume of 0.1 ml PBS (phosphate-saline buffer solution). Three days after inoculation 10 mice per group were treated with 0.1 ml of the virus or of a carrier medium (PBS) by intratracheal instillation of 1 times a day for two days. Used dosage of the virus Ad5CMV-p53 or Ad5/RSV/GL2 - 5107one individual. Mice were euthanized at the end of the 6-week period. Neoplastic lesions were studied as follows. The tissue of the lung and mediastinum were excised and identified the size of the tumor. Lesions were confirmed by histological analysis of the excision of the tumor mass.

Irradiated hairless mice were inoculated cells H226Br - 2106at one individual by intratracheal instillation. Three days after vaccination from each of the mice (8-10 mice per group) were instilled with 0.1 ml or Ad5CMV-p5, or a day for two days. The dosage of the virus was 5107(PFU)/mouse. Neoplastic lesions were investigated at the end of the 6-week period by excision of the tissues of the lung and mediastinum and defined the size of the tumors. The procedure of the study is shown in Fig. 7 submission of samples excision of Fig. 8. Detected tumors were confirmed by histological analysis. Measurements of the tumors are shown in table. 2.

Only 25% of mice treated with Ad5CMV-p53, has been formed tumors, whereas in the group of mice treated with media or Ad5/RSV/GL2 (control group) tumors received 70 to 80% of infected mice. The average tumor size for a group of Ad5CMV-p53 was significantly lower than for control groups. These results indicate that Ad5CMV-p53 can protect H226Br from the formation of a tumor in a murine model of orthotopic human lung cancer.

Example 7. Synergies between p53 DNA damaging effect.

Biochemical characterization of programmed killing (apoptosis) is the appearance of the characteristic image of the DNA fragments resulting from the fragmentation of nuclear DNA. Recent studies have demonstrated that the occurrence of t to be relevant to the status of the p53 gene and to the fact that what amazing DNA stimulants can increase intracellular levels of p53 protein in cells that occurs during apoptosis (Lowe et al., 1993; Clarke et al., 1993; Fritsche et al., 1993; Harper et al., 1993; El-Deiry et al., 1993). Suppression of the cell cycle in the G1 phase by increasing the levels of natural protein wt-p53 provides increased time for DNA repair; optimal if it cannot be repaired, p53 can initiate a program of killing cells. Thus, p53 may contribute to apoptotic cell death caused by chemotherapeutic agents.

Inactivation of p53 gene due to missense mutations or delizie is the most common genetic alteration in cancer diseases (Levine et al., 1991; Hollstein et al., 1991). Loss of p53 function was the result of increased resistance of cells to the effects of a variety of chemotherapeutic agents (Lowe et al., 1993). Studies of the inventors have shown that in NSCLC, a type of lung cancer person H358 cells that are erased both alleles of the gene p53, were resistant to chemotherapeutic drugs, whereas cell line WTH 226b with endogenous wt-p53, showed apoptotic death really is). Therefore, the inventors have set the task to determine whether the introduction of the gene wt-p53 in the H358 cell, using adenoviral vector, to increase the sensitivity of cells to agents CDDP, forming cross-links with DNA, in vivo and in vitro.

Materials and methods

H358 cells were set A. Gazdar & J. Minna (Takahashi et al., 1989).

Adenoviral vectors

Structure and method for the identification of adenovirus vector is a recombinant containing cDNA encoding human wt-p53 (Ad-p53) or luciferase (Ad-Luc), were described previously (Zhang et al., 1993). Briefly characterize the way to obtain. Cassette expression of p53 containing the promoter of the human cytomegaly virus wt-p53 cDNA and early signal polyadenylation SV40, was inserted between the Xba1 and Cla1 provisions pXCJL1. The Shuttle vector and recombinant p53 plasmid pJM17 were transferred into 293 cells (Ad5-transformed cell line, derived from human embryo kidney) using a method involving the use of liposomes as an intermediate link. The culture supernatant of 293 cells, demonstrating a complete cytopathic effect was collected and used in the following as the infective funds. The control virus Ad-Luc was obtained in the same way. VIR is the query result of experiments on Hela cells. Viral titers were determined in the reaction of rosethorne (Graham et al., 1991).

Detection nucleosomal destruction of DNA

DNA was isolated from the parent, Ad-Luc-infected and Ad-p53-infected cells that were exposed or not exposed to CDDP treatment, by keeping cells at 55oC for 6 hours in Lisina buffer solution (50 mm TRIZ-HCl, pH 8.0; 100 mm EDTA, 100 mm NaCl; 1% SDS and 50 l/ml proteinase K). DNA was extracted twice with equal volumes of phenol and once with chloroform isoamyl alcohol (24:1) and then precipitated in adinole. The samples were processed by electrophoresis on a 1.5% agarose gel, and then shown by staining with ethyl bromide compounds (ethidium bromide).

Marking and labelling dUTP with an intermediate TdT were carried out in accordance with the process described previously (Gavrielli et al., 1992). Monomolecular cells were treated with 0.01% NP-40. Slides were immersed in TdT buffer solution (30 mm TRIZ-HCl, pH 7,2; 140 mm coodinate sodium; 1 mm cobalt chloride) and placed in the incubator with the processed dUTP vitamin H (Biotin) (Boehringer Mannheim, Indianapolis, IN) and TdT at 37oC for 45 minutes. Glass slides were coated with 2% bullish, cyberotic the CA) for 30 minutes. Colorimetric determination was performed using diamino-benzidine.

Results

H358 cells were transformed in vitro using cDNA of human wt-p53 by exposing them to Ad-p53. Analyses Western-blokirovaniya showed a high level of protein expression of wt-p53 immediately after 24 hours after infection with Ad-p53. However, wt-p53 was not detected neither in the parent cells (untreated) or in control cells infected with Ad-Luc (data not shown). Related research immunohistological examination showed detectable protein wt-53 more than 80% of infected cells, proving that the transfer and expression of p53 expression with Ad-p53 highly effective (data not shown).

Continuous treatment of H358 cells infected with Ad-p53, using CDDP quickly reduced their viability, while the death of a significant number of parental cells and cells infected with Ad-Luc, occurred after 72 hours of treatment with CDDP (Fig. 10A). Loss of viability was greatly increased in cells transformed with Ad-p53. Moreover, the decreased viability was also observed even when cells were in an environment that is free is the effect lasted for 24 hours (Fig. 10B). The sensitivity of H358 cells, transformed wt-p53, to the effects of CDDP depended on the dosage (Fig. 10C).

Internucleosomal ladder (like descending loop) DNA, indicating the destruction of the DNA was visible in cells that detect the manifestation of the expression of wt-p53 after 24 hours exposure to CDDP, however, the parent and Ad-Luc-infected cells destruction DNA showed (Fig. 11A). End tags nick (break one internal phosphodiester bond in DNA double) Biotin-triphosphate (average salt of orthophosphoric acid) (dUTP)-2'-deoxyuridine-5' with an intermediate limit deoxynucleotidyl-transferase (TdT), which (label) specifies the destruction of DNA in situ as a result of apoptosis, revealed many apoptotic cells among Ad-p53-infected cells treated with CDDP for 24 hours, as shown in Fig. G, which shows a dark nuclei and nuclear fragments that are missing in Fig. 11B-F.

Known methods of introduction of wt-p53 in order to initiate apoptosis in some types of tumor cell lines with erased or mutated p53 (Yonish-Rouach et al., 1991; Shaw et al., 1992; Ramqwist et al., 1993). However, over-expression of wt-p53 in itself cannot lead to the destruction of DNA in p53-negative letocnala, showing that stable clones H358 can be obtained after transfer of wt-p53 with an intermediate retrovirus, and that these clones grow much more slowly than the parental cells (Cai et al., 1993).

The potential therapeutic effectiveness of the combination of Ad-p53 with CDDP was found in terms of the relative changes in the volume of spheroids H358. The model of multicellular spheroid tumor shows in vitro histological structure similar to the one that has the tumor at the initial stage and at the stage of metastases. Treatment with CDDP caused a decline in the relative volume number of Sviridov infected with Ad-p53, but had no significant influence on the parent or Ad-Luc-infected spheroids (Fig. 12A). In situ labeling dUTP with an intermediate TdT showed many cells are in a state of apoptosis on the surface of Ad-p53-infected Sviridov, while apoptotic cells were absent on the spheroids not infected with Ad-p53 (Fig. 12B-E). The inventors previously reported that the expression of wt-p53 with an intermediate retrovirus inhibited the growth of spheroids H322a, thanks to the transforming growth factor (TGF- ) (Fujiwara et al., 1993). However, the retroviral vector may not infect the sphere, the effect of CDDP reduces the size of the spheroids H358 infected with Ad-p53, due to the initiation of apoptosis, suggesting that Ad-p53 infects indestructible cells and that CDDP initiates the process of apoptosis in asymptomatic (latent) cells.

Example 8. The use of p53 and DNA damaging agents in medical schemes.

Naturally, the model of treatment developed for animals, shall be used as part of research prior to clinical assignments, as shown in examples 5, 6, 7. So sick, you have an indication for treatment with gene transfer by adenovirus, can be tested for the presence of antibodies which are directed against adenovirus. If the antibodies are present and established allergic patient response to any pharmacological or natural resources that can be assigned using experimental doses of the order of 103up to 106adenovirus is a recombinant under close clinical supervision.

To assign a person in the treatment of cancer with the use of Ad5CMV-p53 adenovirus is a recombinant, showing the expression of p53 under the control of appropriate promoters/reinforcing elements, such as CR is the organization for supervision of food and drugs (FDA). Such a method includes, but is not limited to, centrifugation in density gradient of cesium chloride and subsequent tests on the efficiency and cleanliness.

There is an opinion that there are two methods of treatment using adenovirus p53, immediate or local function or a more General treatment. These methods give results only for the treatment of a variety of malignant tumors that are known to be related to p53 mutations. With regard to the General treatment, as was proved by the experiments, quite ordinary intravenous injection to infect entered virus tissue located at a distance from the site of drug administration (Stratford-Perricaudet et al., 1991b). Thus, viral infection through intravenous injections can be considered as a suitable method for the treatment of all malignant tumors related to p53. The virus can be assigned to patients as intravenously in the form of any pharmaceutically acceptable solution and in the form of infusions over a period of time. Frankly, there is a deep conviction that in order to obtain the expected effect of the number of appointed acting viral particles should be parasportas method is more direct physical effects of adenovirus-recombinant on target cells, similar intratracheal purpose of transmembrane conductance regulator when fibropenobetona degeneration (Rosenfeld et al., 1992). The result of this method can be delivery of adenovirus-recombinant p53 closer to the place where the target cells.

Methods

Marking dUTP in situ with an intermediate TdT to determine apoptosis. Spheroids H358 were recorded on the third day and dyed as described in example 7. Labeled samples TdT was put in contact with the slide, immersed in TdT buffer solution and was maintained in an incubator with dUTP and TdT treated with Biotin at 37oC for 45 minutes. Slides were covered with 2% bovine serum albumin for 10 minutes and placed in an incubator (cultivated) with avidin-biotinyl solution for 30 minutes. Colorimetric determination was performed using diamino-benzidine.

Initiation of apoptosis using CDDP after Ad-p53 infection in vivo.

H358 cells (5106) in 0.1 ml of balanced salt solution Hank was injected in the right flank of BALB/c female mice nu/nu hairless mice. Thirty days 200 l homogeneous medium or medium containing Ad-Luc (108PFU/ml) or Ad-p53 (108injection in two opposite zones ( 50 l each). CDDP (3 mg/kg) or control saline were introduced interperitoneal. Tumor volume (A) changes. Tumors were measured with a compass on two perpendicular diameters without regard to relationship to treatment group, and tumor volume was calculated by the model with a spherical shape with an average diameter of the tumor was calculated as the square root of the product of the diameters of cross-sections. Five species of mice were used for the formation of each treatment group, and shows the average value of SF. Data were analyzed using Student's t-test. The arrow indicates the day of treatment. Presents two independent notation: p<0.05 on day 5 in experiment 1; p<0.05 on day 7 in experiment 2. Histological examination was carried out using the technology of labeling with Biotin-dUTP with an intermediate TdT. The tumor was removed after 5 days from the start of treatment and was immediately placed in the composition O. C. T. Samples with a thickness of 5 m was secalis in frozen tissues in the cryostat. Excision processed 1 g/l proteinase K and stained according to the method described above. The animals were kept in accordance with the requirements of the UT M. D. Anderson Institutional Animal Care &Use Committee.

Results

To demonstrate in vivo eff what kimioterapia in the treatment of human cancer, the inventors investigated the question of whether to be induced apoptosis in vivo sequential introduction of Ad-p53 and CDDP. Three days after direct intratumoral injection of Ad-p53 or interperitoneal introduction CDDP tumor cells were implanted subcutaneously in the body of nu/nu mice and the results showed modest results slowdown. However, in cases where Ad-p53 and CDDP were introduced at the same time, cancer partially regressed, and the tumor size remained statistically significantly smaller than for any other treatment groups. The effect of inhibition of growth was even more pronounced after two treatment cycles (Fig. 13A). Histological examination revealed massive destruction of cancer cells in the zone of the body of the mouse treated with CDDP, which was introduced Ad-p53. Staining in situ showed a lot of dead cells around the intercellular spaces (Fig. 13A-E). In contrast to the above, cancer cells treated only CDDP or only Ad-p53, showed neither apoptotic zones or zones of intercellular spaces, surrounded by dead cells.

In more detail the preferred treatment protocols can be developed in accordance with the following algorithm. Initially, the patient can can much of the tumor. Pre-bronchoscopy should be performed under local or General anesthesia. Transbronchial aspiration needle (21 g) Stifcortm(hard core) must be conducted in a biopsy channel of the bronchoscope. The preserved parts of the tumor can then be fed a small amount of adenovirus p53 in the amount of 10 ml or less.

In any case, even if the applied adenovirus is not sufcient for the emergence of replication effect, by itself, the virus on the human body no harmful effects will not have. However patients during treatment should be in the hospital for at least 48 hours to monitor the possible emergence of acute or remote side reactions. Although the effective and safe use of adenovirus deficit replication as a carrier during the transfer of a gene in the human body have been paid attention to in the past (Rosenfeld et al., 1992, Jaffe et al., 1992), the dosage of adenovirus appointed to enter into the body, should be seriously explored in order to further reduce the potential for any adverse effects.

There are various criteria that should be taken in vnimanie determine toxicity, before you begin the course of therapeutic treatment, it is necessary to photograph the tumor bed, measuring its greatest diameter and the perpendicular. Tumor size is defined as the product of the diameters. With these data, it is possible to calculate the degree of tumor recurrence.

Time to progression in the development of the disease can also be measured from the first survey with the discovery of reducing tumor mass before the appearance of visible signs of progression. The progression in the disease development is defined as an increase of 25% of the sum of the products of diameters measured neoplasms. The patient must pass at least two courses of treatment before you receive testimony on the progression of the disease. Observations on the patients should be evaluated since the record (history).

Subsequent studies should include all actions (appointments) therapeutic treatment of cancer, including the determination of clinical tests and the taking of biopsies for standard and molecular biological analyses, which can be investigated the structure of the expression of different genes p53. These analyses can also provide data on the number leakproofing data can be made the necessary adjustments in the course of treatment. These adjustments may include patterns of adenovirus, which use different promoters, or changes in the number of PFU (multiplicity of infection), introduced to ensure that infected all or most of the tumour cells without excessive non-physiological expression of recombinant genes.

It is established that the expression of exogenous genes transferred in vivo adenovirus, can exist for a long time. Therapeutically effective, long time, the expression of the exogenous gene carried by the virus, should be directed to the patient's body (nature) of the patient. Marker genes are limited in their influence determination of therapeutically relevant duration of gene expression, because the expression levels necessary to correct any specific genetic disorders are significantly different from the level required for the integrated treatment of another disease.

Although the compositions and methods according to this invention have been described referring to the preferred embodiments, specialists with experience in this obrazovatelnoi techniques, methods, as described above, without derogating from the entity set forth in the claims. More specifically, this description implies that certain agents that can be both chemical and physiological nature, are replaced by agents described in this invention and showing identical or similar effect. All such replacements shall be signs in the claims. All protected by a formula of the invention compositions and methods may be implemented without additional experimental studies.

Sequence listing

1. General information:

(i) Applicant:

A. Name: "BO S REGENT, JO UNIVERSITY OV TEKSAS SYSTEMS

B. Street (Address): 201 West Sevens (7th) Street

C. city (City) Austin

D. state (State) Texas

E. Country (Country) USA

F. Postal code: (ZIP) 78701

G. Phone: (512) 499-4462

H. TELEFAX: (512) 499-4523

(ii) Inventors: Roth, Jack A., Fujiwara, Toshiaki, Grimm, Elizabeth Hey, Mukhopadhyay, Tapas Zhang, Wei - Wei and Owen Staples, Lori B.

(iii) title of the invention:

Methods and compositions containing affecting DNA agents and p53

(iv) number of sequences: 4

(v) Corresponding address:

(F) Postal code: 77210

(vi) Form, readable on a computer:

A media Type: Floppy/ASCII

B Computer: IBM. PC-compatible

C Operating system: PC-DOS/MS-DOS

D Software: WORDPERFECT 5.1

Data on the application under consideration:

A requisition Number: unknown

B filing date: in accordance with attendant circumstances

C Classification: unknown

(viii) Previously filed application:

A requisition Number: USSEN 08/233,002

B filing date: April 25, 1994

C Classification: unknown

(ix) information about the patent attorney/agent

A Name: the Highlander, Steven L.

B Registration number: 37,642

C is the Link to the document on payment of fees: UTFC403PCE

(x) Information about communication channels:

A Phone: (512) 418-3000

B Fax: (713) 789-2679

C telex: 79-0924

(2) the Information about the sequence SEQ ID N 1

(i) sequence Characteristics:

A. Length: 20 base pairs (base pairs)

B. Type: Nucleic acid

C. the DNA Chain: Single

D. Topology: Linear

(ii) sequence Description: SEQ ID N 1:

GGCCCACCC CCTTGGCTTC 20

(2) Information for sequence SEQ ID N 2:

(iii) sequence Characteristics:

A. D. the I: Line

(iv) sequence Description: SEQ ID N 2:

TTGTAACCAT TATAAGCTGC 20

(2) Information for sequence SEQ ID No. 3:

(v) sequence Characteristics:

A. Length: 20 base pairs (base pairs)

B. Type: Nucleic acid

C. the DNA Chain: Single

D. Topology: Linear

(vi) sequence Description: SEQ ID N 3:

TCGTTTCTCA GCAGCTGTTG 20

(2) Information for sequence SEQ ID No. 4:

(vii) sequence Characteristics:

A. Length: 20 base pairs (base pairs)

B. Type: Nucleic acid

C. the DNA Chain: Single

D. Topology: Linear

(viii) sequence Description: SEQ ID N 4:

CATCTGAACT CAAAGCGTGG I

1. Method of destruction afflicted, the sick, or the like cells, including ensuring contact of the cells with a p53 protein or gene and a DNA damaging agent in a combined amount sufficient to destroy the cells.

2. The method according to p. 1, which provide the contact of cells with the protein p-53 or genome in combination with x-ray irradiation, ultraviolet irradiation, irradiation, microwave radiation, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C or cisplatin.

3.om and provide contact of cells with the protein p-53 or genome in combination with cisplatin.

5. The method according to p. 1, which provide the contact of cells with the recombinant vector, which stimulates the protein expression of p-53 in the cage, in combination with a DNA damaging agent.

6. The method according to p. 5, in which the recombinant vector, causing the expression of p53, is "naked" plasmid DNA, the plasmid within liposomes, retroviral vector, vector, adeno-associated virus (AAV) vector herpesvirus or adenovirus vector is recombinant.

7. The method according to p. 6, in which the recombinant vector, causing the expression of p53, is a vector, adenovirus-recombinant.

8. The method according to p. 7, in which the amount of adenoviral vector is from 1x105to 1x1012plaque-forming units (pfu).

9. The method according to p. 8, in which the amount of adenoviral vector is 5x107plaque-forming units.

10. The method according to p. 8, in which the amount of adenoviral vector is 2x107plaque-forming units.

11. The method according to p. 6, in which the viral vector is a herpes virus vector.

12. The method according to p. 6, in which the viral vector is delivered through the endoscope, intravenous, vnutritrahealno, intralesional through skin is eh tissue.

14. The method according to p. 6, where the viral vector is introduced in a quantity of approximately 0.1 ml.

15. The method according to p. 6, where the viral vector is introduced in an amount of about 10 ml.

16. The method according to p. 7, in which the recombinant vector, causing the expression of p53, is a vector, the adenovirus is a recombinant, including the area of expression of p53, located at controlling the impact of cytomegalovirus promoter IE.

17. The method according to p. 7, in which the use of the adenovirus vector is a recombinant includes the area of expression of p53, the promoter of the cytomegalovirus IE and early signal polyadenylation SV40.

18. The method according to p. 7, in which at least one gene responsible for replication of the adenovirus, is removed from the structure of the vector specified adenovirus, and the area of expression of p53 introduce in its place.

19. The method according to p. 18, in which the area of expression of p53 introduce in place of the remote areas of EIA and EIB adenoviral vector.

20. The method according to p. 7, in which the use of the adenovirus vector is a recombinant, which is present within the adenovirus is a recombinant.

21. The method according to p. 1, which provide first contact of the cells with a p53 protein or gene, and then provide it a contact of cells with a p53 protein or gene, and for the up contact of cells with a DNA damaging agent, and then provide its contact with p53 protein or gene.

23. The method according to p. 1, in which at the same time provide contact of the cells with a p53 protein or gene and DNA damaging agent.

24. The method according to p. 1, which ensures the contact of the cell with a first composition containing a p53 protein or gene, and the second composition comprising a DNA damaging agent.

25. The method according to p. 24, in which the first and second composition is dispersed in a pharmacologically acceptable form.

26. The method according to p. 1, which ensures the contact of the cell with a single composition containing a p53 protein or gene in combination with a DNA damaging agent.

27. The method according to p. 26, in which the composition is dispersed in a pharmacologically acceptable form.

28. The method according to p. 26, which provide contact of the cell with a single composition comprising a recombinant vector, which stimulates the expression of p53 in the cell in combination with a DNA damaging agent.

29. The method according to p. 28 which provide contact of the cell with a single composition containing the adenovirus is a recombinant comprising a recombinant vector, which stimulates the expression of p53 in the cell in combination with a DNA damaging agent.

32. The method according to p. 31 in which the specified cell is cell lung cancer.

33. The method according to p. 32, in which the cancer cell is a large cell lung carcinoma.

34. The method according to p. 33, which indicated a large cell lung carcinoma is a squamous cell cancer.

35. The method according to p. 33, in which a large cell lung carcinoma is a cell adenocarcinoma.

36. The method according to p. 33, in which a large cell lung carcinoma is a large undifferentiated cell carcinoma.

37. The method according to p. 32, in which cell lung cancer is small cell lung carcinoma.

38. The method according to p. 31 in which the specified cell is a cell of breast cancer.

39. The method according to p. 31 in which the specified cell is a cell having a mutation in a gene called p53.

40. The method according to p. 1, in which the specified cell is in an animal's body, and p53 protein or gene and affecting DNA agent is introduced into the animal organism in a pharmacologically acceptable form.

41. The method according to p. 40, including injection into the tumour place therapeutically effective amount is surrounding the expression of p53 in cancer cells, and ensuring contact of the tumor with DNA damaging agent.

42. The method according to p. 41, which provide the contact of the tumor with a DNA damaging agent by radioactive irradiation, ultraviolet irradiation, irradiation or microwave irradiation.

43. The method according to p. 42, which provide the contact of the tumor with a DNA damaging agent by radioactive irradiation of the tumour's location.

44. The method according to p. 42, in which the tumor cell is brought into contact with a DNA damaging agent by irradiating the tumor cells by x-irradiation.

45. The method according to p. 44, in which the dose of x-ray radiation is 2000 - 6000 x-ray.

46. The method according to p. 44, in which the dose of x-ray radiation is 50 - 200 x-ray.

47. The method according to p. 42, which provide the contact of the tumor with a DNA damaging agent by ultrafioletowego irradiation of the tumour's location.

48. The method according to p. 42, which provide the contact of the tumor with a DNA damaging agent by irradiating the location of the tumor.

49. The method according to p. 42, which provide the contact of the tumor with a DNA damaging agent by microwave irradiation of the tumour's location.

51. The method according to p. 50, which affects DNA agent is cisplatin.

52. The method according to p. 51, wherein said cisplatin is administered at 20 mg/m2.

53. The method according to p. 50, which affects DNA agent is doxorubicin.

54. The method according to p. 53, wherein said doxorubicin is administered at the level of 25 - 75 mg/m2.

55. The method according to p. 50, which affects DNA agent is etoposide.

56. The method according to p. 55, wherein said etoposide is administered at the level of 35 to 50 mg/m2.

57 the Method according to p. 50, which affects DNA agent is verapamil.

58. The method according to p. 50, which affects DNA agent is podophyllotoxin.

59. The method according to p. 50, which affects DNA agent is 5-fluorouracil.

60. The method according to p. 59, wherein said 5-fluorouracil is administered at the level of 3 to 15 mg/kg

61. The method according to p. 50, which affects DNA agent is camptothecin.

62. The method according to p. 50, which affects DNA agent is actinomycin D.

63. The method according to p. 50, which affects DNA agent is mitomycin C.

64. The method according to p. 40, which provide contact armaceuticals composition, containing affecting DNA connection.

65. The method according to p. 40, in which the p53 gene or protein is administered prior to DNA damaging agent.

66. The method according to p. 65, in which the period between the entry of the viral vector and the DNA damaging agent is 12 - 24 hours

67. The method according to p. 65, in which the period between the entry of the viral vector and the DNA damaging agent is 6 - 12 hours

68. The method according to p. 65, in which the period between the entry of the viral vector and the DNA damaging agent is about 12 o'clock

69. The method according to p. 40, in which the p53 gene or protein is administered after the DNA damaging agent.

70. The method according to p. 69, in which the period between input DNA damaging agent and viral vector is 12 - 24 hours

71. The method according to p. 69, in which the period between input DNA damaging agent and viral vector is 6 - 12 hours

72. The method according to p. 69, in which the period between input DNA damaging agent and viral vector is approximately 12 o'clock

73. The method according to p. 40, in which the p53 gene or protein is administered simultaneously with the DNA damaging agent.

74. A method of treating cancer, comprising introducing into the animal organism, a cancer patient, a therapeutically effective combination of a p53 protein or gene with powerstick effective amount of the pharmaceutical compounds containing the adenovirus is a recombinant comprising a recombinant vector, which causes the expression of p53 in tumour cell, and ensure contact of the tumor with DNA damaging agent.

76. The method according to p. 75, in which the viral vector is administered in amount of about 0.1 ml.

77. The method according to p. 75, in which the viral vector is administered in an amount of about 10 ml.

78. The method according to p. 75, which provide the contact of the tumor with a DNA damaging agent by irradiating the damaged tumor designated x-rays, ultraviolet rays, -rays, or by microwave irradiation.

79. The method according to p. 75, which provide the contact of the tumor with a DNA damaging agent by entering into the body of the animal a therapeutically effective amount of the pharmaceutical compounds containing affecting DNA connection.

80. The method according to p. 75, which affects DNA compound is cisplatin.

81. The method according to p. 78, which provide the contact of the tumor with a DNA damaging agent by radioactive irradiation of the tumour's location.

82. The method according to p. 78, which provide the contact of the tumor with a DNA damaging agent by ultraviolet irradiation mashantou by-irradiation of the tumour's location.

84. The method according to p. 78, which provide the contact of the tumor with a DNA damaging agent by microwave irradiation of the tumour's location.

85. The method according to p. 75, which affects DNA agent is doxorubicin.

86. The method according to p. 75, which affects DNA agent is etoposide.

87. The method according to p. 75, which affects DNA agent is verapamil.

88. The method according to p. 75, which affects DNA agent is podophyllotoxin.

89. The method according to p. 75, which affects DNA agent is 5-fluorouracil.

90. The method according to p. 75, which affects DNA agent is camptothecin.

91. The method according to p. 75, which affects DNA agent is actinomycin.

92. The method according to p. 75, which affects DNA agent is metalminer C.

93. The method according to p. 74, in which the p53 gene or protein is administered prior to DNA damaging agent.

94. The method according to p. 93, in which the period between the entry of the viral vector and the DNA damaging agent is from 12 to 24 hours.

95. The method according to p. 93, in which the period between the entry of the viral vector and the DNA damaging agent is 6 - 12 hours

96. The method according to p. 93, in which the period between the entry of the virus is theine injected after the DNA damaging agent.

98. The method according to p. 97, in which the period between input DNA damaging agent and viral vector is 12 - 24 hours

99. The method according to p. 97, in which the period between input DNA damaging agent and viral vector is 6 - 12 hours

100. The method according to p. 97, in which the period between input DNA damaging agent and viral vector is approximately 12 o'clock

101. The method according to p. 74, in which the p53 gene or protein is administered simultaneously with the DNA damaging agent.

102. Composition for the destruction of the afflicted, the sick and the like of cells containing p53 protein or gene in combination with a DNA damaging agent.

103. The composition according to p. 102 containing a p53 protein or gene in combination with adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C or cisplatin.

104. The composition according to p. 103 containing a p53 protein or gene in combination with cisplatin.

105. The composition according to p. 102 containing a recombinant vector, which provides the expression of p53 protein in the cell of an animal in combination with a DNA damaging agent.

106. The composition according to p. 105, in which the recombinant vector is "naked" plasmid DNA, plasmid within liposomes, the retroviral Vectora what I p. 106, in which the recombinant vector is an adenovirus vector is recombinant.

108. The composition according to p. 107, in which the recombinant vector is an adenovirus vector is a recombinant present within recombinant adenovirus particles.

109. The composition according to p. 102 containing the adenovirus vector is a recombinant present within recombinant adenoviral particles in combination with cisplatin.

110. The composition according to p. 102, which is dispersed in a pharmacologically acceptable form.

111 the Composition according to p. 110, suitable for introduction inside the affected places.

112. therapeutic set to destroy the afflicted, the sick and the like of cells containing in appropriate packaging (shell) cooked pharmacological means of a recombinant vector, which causes the expression of p53 protein in a cell of the animal, and cooked pharmaceutical remedy DNA damaging agent.

113. therapeutic set according to p. 112, in which the recombinant vector and affecting DNA agent are in the same package.

114. therapeutic set according to p. 112, in which the recombinant vector and affecting DNA agent are different is overuse-recombinant, includes a recombinant vector, which causes the expression of p53 protein in a cell of the animal, and the pharmaceutical agent is cisplatin.

 

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