Methods and compositions for the diagnosis and treatment of cancer

 

(57) Abstract:

The invention relates to medicine, in particular to the biology of the tumor, and in particular to methods and compositions for the treatment of squamous (squamous cell carcinoma. Also provides an animal model for the study of microscopic residual tumors and tumor contamination in the body cavities, as well as methods of their treatment. The inventive developed expression construct that contains a promoter functional in eukaryotic cells, and polynucleotide encoding p53, and polynucleotide is in the sense orientation to a promoter. The technical result of the invention is the expansion of the Arsenal against cancer. 2 C. and 41 C.p. f-crystals, 7 tab., 28 Il.

The invention relates, generally, to the field of tumor biology. More specifically, the invention relates to compositions and methods for treating squamous (squamous cell carcinoma. Also provides an animal model for the study of microscopic residual tumors and tumor contamination in the body cavities, as well as methods of their treatment.

Balancing the speed of cell proliferation and programmed cell death is important to support the process of carcinogenesis, and inhibition of apoptosis, or programmed death of cells, is one of the reasons for this violation. The influence of such defects is catastrophic, causing about half a million deaths annually in the United States alone.

There is substantial evidence of the involvement of mutations of the p53 gene, a tumor suppressor, the etiology of many human cancer. The message showed that the growth of various cancer cell lines person, including representatives of colon cancer, glioblastoma, breast cancer, osteosarcoma and lung cancer functionally suppressed by using viral-mediated transfer of the p53 gene of the wild type. It was shown that the induction of exogenous p53 expression p53 wild-type induces apoptosis in cancer cell lines, colon cancer, and lung cancer spheroids person, suggesting about the role of the p53 gene in programmed cell death.

Patients with squamous cell carcinoma of the head and neck (SCCHN) are affected by the disease, which often has a strong influence on speech, swallowing and surgery, affecting the appearance of the patient. In addition, overall survival among these patients, will bring iacona therapy. Relapses are mainly local and regional as opposed to systematic, indicating that microscopic residual carcinoma in the primary tumor site is the main cause of death. Due to these facts, the ability to effectively work on microscopic residual disease in SCCHN is an attempt, which may improve therapeutic efficacy of cancer treatment.

Therefore, the present invention is the creation of improved methods of treatment of squamous cell carcinoma in vivo. Another objective of the present invention is a method of development evaluation, and treatment of microscopic residual carcinoma and microscopic tumor seeding of body cavities.

To solve these problems we propose a method of treatment of a subject with squamous cell carcinoma, including stage (a) ensure expression constructs comprising the promoter function in eukaryotic cells and polynucleotide encoding p53, where polynucleotide is located in sense orientation to a promoter and is under the control of the promoter; (b) contacting expression constructs with squamous kletochnom squamous cell carcinoma may be, or may not be mutated. Expression design is preferably a viral vector such as a retroviral vector, an adenoviral vector, and adeno-associated viral vector, with replication deficient adenoviral vector, which is the most preferred. In a specific embodiment, the p53 gene is directed in such a way that can be detected expression of p53 expression vector. The preferred target is immune target, such as a sequential epitope antibodies.

The method may further include surgical resection of the tumor with additional contacting the tumor bed or artificial body cavity with expression design after resection. The volume used for contact with the tumor bed is from about 3 ml to about 10 ml in areas where an adenovirus vector, the number of adenovirus entered in each process of the probe is 107, 108, 109, 1010, 1011or 1012pfu.

Also considered continuous perfusion expression constructs. A number of designs, delivered during continuous perfusion will be determined on the basis of the first time period, although there may be achieved a few large total dose using continuous perfusion.

In another embodiment, the expression construct is introduced in the form of injection into the natural body cavity such as the mouth, throat, trachea, pleural cavity, peritoneal cavity, or cavities of hollow organs, including the bladder, the colon, or other visceral organs.

Also claimed is the method of determining the effectiveness of therapy for microscopic residual cancer, comprising (a) providing a rodent with dissection in the subcutaneous tissue; (b) seeding dissection of tumor cells; (C) treatment of rodent therapeutic method; (d) assessment of the impact of treatment on tumor development. Dissection can be isolated after stage (b) and before stage (C). In addition, the mode may include the introduction of a therapeutic composition in dissection, which re-opens after isolation and re-sealed after the introduction of the specified therapeutic composition.

Other objectives, features and advantages of the present invention will be apparent from the following detailed description. Must be, however, it is clear that the detailed description and specific examples, while they indicated the changes and modifications within the essence and scope of the invention will become obvious to a person skilled in this field from this detailed description.

The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these drawings together with the detailed description of specific options presented here.

Fig. 1 - efficiency transduction of SCCHN cell lines Tu-138 (solid triangles) and Tu-177 (solid squares). Was used recombinant-Gal adenovirus to infect cells at different MOI areas from 10 to 100. Percentage-Gal-positive cells was obtained from counting 500 cells located on each Cup for replication.

Fig. 2A and 2B inhibition of growth of SCCHN cells in vitro. Fig. 2A - growth curve mock-infected Tu-138 cells (solid circles), d-1312-infected cells (solid triangles) and Ad5CMV-p53-infected cells (solid squares). Fig. 2B - growth curve mock-infected Tu-177 cells (open circles), d1312-infected cells (open triangles), Ad5CMV-p53-infected cells (open squares). At any given time three cups cells were subjected to trypsinization and counted. The SEM value of counted cells in triple wells after and then the - Astana growth curve four SCCHN cell lines. Fig. 3A - Tu-138. Fig. 3B - Tu-177. Fig. 3C - MDA 686-LN. Fig. 3D MDA 886. Mock-infected cells (solid circles), d1312-infected cells (solid triangles) and Ad5CMV-p53 infected cells (solid squares). The value of counted cells in triple wells after infection were represented as depending on the number of days from the moment of infection; bars, SEM (scanning electron microscopy).

Fig. 4 - growth curve of cell lines of normal fibroblasts. Mock-infected cells (solid circles), d1312-infected cells (solid triangles) and Ad5CMV-p53 infected cells (solid squares).

Fig. 5A and 5B is an integral curve of growth of SCCHN cell lines. Fig. 5A - Tu-138. Fig. 5B - MDA 686LN. Mock-infected cells (open squares), d1312-infected cells (open triangles) and Ad5CMV-p53 infected cells (open circles). At any given time three cups cells were subjected to trypsinization and counted. The value of counted cells in triple wells represented in the form depending on the number of hours after infection; bars, SEM.

Fig. 6A and 6B - labeling DNA breaks in apoptotic cells biotinylated dUTP using the TUNEL method. After infection, the flowing 1312, replication-deficient adenovirus (panel 1 panel 4), Tu-138 cells infected with p53 adenovirus wild-type (panel 5 - panel 8). Fig 6B - MDA 686LN cells that were infected d1312, replication-deficient adenovirus (A-D), MDA 686LN cells infected with p53 adenovirus wild-type (E-H). Ap stands (Ap stands for apoptosis.

Fig. 7A and 7B is an integral curve of growth of SCCHN cell lines. Fig. 7A - Tu-138. Fig. 7B - MDA 686LN. Mock-infected cells (open circles), d1312-infected cells (closed triangles) and Ad5CMV-p53 infected cells (closed squares). At any given time three cups cells were subjected to trypsinization and counted. The value of counted cells in triple wells represented in the form depending on the time (number of hours) after infection; bars, SEM.

Information is available data suggests that one of the primary disadvantages of the treatment of SCCHN is the inability to complete elimination of the disease in the primary tumor site or in the immediate local or regional tissues, therefore the present invention aims at providing methodologies for gene therapy, which allow a more complete and effective treatment of SCCHN, especially due to the impact on microscopies togami treatments, such as chemo - and radiotherapy or surgery. In addition, the use of animal models specifically aimed at the treatment of microscopic residual carcinoma, as well as microscopic tumor seeding of body cavities, the present invention demonstrates the effectiveness of these methods. Details of the invention are described more fully below.

Usually observed that p53 gene therapy of cancer may be effective regardless of the p53 status of tumor cells. Unexpectedly therapeutic effects were observed when the viral vector carrying the p53 gene of the wild type, was used for the treatment of neoplastic diseases, cells that expressed p53 molecule. This result was not predictable on the basis of the current understanding function as tumor suppressors. Unexpected is the fact that normal cells that also Express functional p53 molecule, clearly not affected by the expression of high levels of p53 viral constructs. This increases the possibility that p53 gene therapy may be more broadly applicable to the treatment of cancer than originally expected.

A. P53 proteins and polynucleotides.

In the sun the other species. "Wild-type" and "mutant" p53 refers respectively to the p53 gene expressing a normal tumor suppressor activity, and p53 gene lacking or having reduced suppressor activity and/or with transforming activity. Thus, the "mutant" p53's are just variants of the sequence, and not variants showing altered functional profiles.

p53 is now recognized as a tumor suppressor gene (Montenarh, 1992). Were found high levels of the gene in many cells, transformed with the help of chemical carcinogenesis, ultraviolet radiation and certain viruses, including SV40. p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented as being the most frequently mutated gene in normal human tumor cancer (Mercer, 1992). Gene motirola in about 50% of human NSCLC (Hollestein et. al., 1991) and a wide range of other tumors.

While tumors contain mutated p53 gene are the preferred target according to the present invention, the suitability of the stated p53 expression vectors the awn clear in the present invention found that p53 expression may limit the growth of tumors expressing functional p53 product and even to induce apoptosis in these cells. Thus, the p53 status of the tumor, although potentially useful for diagnostic purposes, it is not necessary to practice the present invention. This phenomenon is not limited to SCCHN tumors, but may be applicable to a wide variety of malignant diseases, including glioma, sarcoma, carcinoma, leukemia, lymphoma, and melanoma, including tumors of the skin, liver, testes, bone, brain, pancreas, head, neck, stomach, liver, lung, ovary, breast, blind gut, prostate, and bladder.

p53 Polypeptides.

the p53 gene encodes a 375-amino acid phosphoprotein that can form complexes with viral proteins such as the large T antigen E1B. Protein is found in normal tissues and cells, but at concentrations that are minimum when compared with many transformed cells or tumor tissue. Interestingly, appears p53 wild-type, which is important in the regulation of cell growth and division. In some cases, it was shown surexpreso, p53 can act as a negative regulator of cell growth (Weinberg, 1991) and can directly cause suppression of uncontrolled cell growth or indirectly activate genes that suppress the growth. Thus, the absence or inactivation of p53 wild-type may contribute to transformation. However, some studies indicate that the presence of mutant p53 may be required for full expression, transforming the potential of the gene.

Although p53 wild-type is recognized as centrally important regulator of growth in many cell types, its genetic and biochemical traits also seem to have value. MIS-sense mutations are normal p53 gene and are essential for the transforming ability of the oncogene. A single genetic change, caused by point mutations can create carcinogenic p53. It is known that in contrast to other oncogenes, however, p53 point mutations occur at least 30 distinct codons, often creating a dominant alleles that produce changes in cellular phenotype without recovery of homozygosity. In addition, many of these dominant negative alleles okazyvaets and from minimally dysfunctional to highly penetrating, dominant negative alleles (Weinberg, 1991).

Casey colleagues reported that transfection of DNA that encodes a p53 wild-type, in two cancer cell lines human breast restores the control of suppressing the growth of such cells (Casey et al., 1991). A similar effect was also demonstrated by transfection of wild-type but not mutant p53 in cancer cell lines of human lung (Takahasi et al. , 1992). p53 wild type is dominant within the mutant gene and is selective with respect to proliferation, when transfercases in cells with a mutant gene. Expression transfectional p53 does not affect the growth of normal cells with endogenous p53. Thus, such designs can be absorbed by normal cells without the negative effects.

Thus, it becomes possible to make the treatment of p53-associated cancers with p53 wild-type could reduce the number of malignant cells. However, studies such as those described above, are far from achieving such a goal at least, but not because DNA transfection may not be used for introducing DNA into cancer cells inside the patient's body.

p53-kodiruyushchuyu p53 protein domain or any p53 polypeptide. "Complementary" polynucleotide are those which are capable of base pairing according to the standard rules of complementarity Watson-Crick. Thus, base pair with a large number of purine is a pair of bases with fewer pyrimidines to form combinations of guanine, paired with cytosine (G:C) and adenine, paired with simple ether thymine (A:T) in the case of DNA, or adenine, paired with uracil (A: U) in the case of RNA. The inclusion of fewer bases such as inosine, 5-methylcytosine, 6-methyladenine, gipoksantin and others, hybridize sequence is not a hindrance mating.

The term "complementary sequences", as used here, refers to polynucleotide sequences that are essentially complementary throughout their length and have a very small number of erroneous mating grounds. For example, the sequence of fifteen bases in length can be called complementary when they have complementary nucleotide in the thirteen or fourteen positions. Naturally, sequences that are fully complementary", will be the succession is of rivani grounds.

Also considered other sequences with lower degrees of homology. For example, can be constructed antisense construct that contains limited areas of high homology, but also contains non-homologous region (e.g., a ribozyme). These molecules, albeit with less than 50% homology, will contact the sequences of targets under appropriate conditions.

Polynucleotide can be obtained from genomic DNA, i.e., cloned directly from the genome or specific organism. In other embodiments, however, polynucleotide can be complementary DNA (cDNA). cDNA is DNA, obtained using information (matrix) messenger RNA (mRNA) as a matrix. Thus, the cDNA does not contain any broken coding sequences and usually contains almost exclusively the coding region(t) for the corresponding protein. In other embodiments, polynucleotide can be obtained synthetically.

It may be convenient to combine proteins genomic DNA with cDNA or synthetic sequences to generate specific structures. For example, where it is desirable intron in the past to niteline to p53. cDNA, or synthesized polynucleotide can provide a more convincing restriction enzymes cut sites for the remaining parts of the structure and therefore can be used for the remainder of the sequence.

Human or mouse DNA sequence for p53 is provided in SEQ ID NO:1 and SEQ ID NO:3, respectively, with the corresponding amino acids, which are provided in SEQ ID NO:2 and SEQ ID NO:4, respectively.

Also considered that there are natural (wild) variants of p53, which contain sequences that differ from those disclosed here. Thus, the present invention is not limited to the use of the provided polynucleotide sequences of p53, but rather includes the use of any options of natural origin. The present invention also includes chemically derived mutants of these sequences.

Another type of variant sequences arises from radonaway variations. Because there are several codons for most of the 20 normal amino acids, many different DNA can encode p53. Table. 1 allows to identify such options.

Given the degeneration of the genetic code, posledovatelnoy nucleotides, disclosed here will be preferred. Sequences that are in range "p53 coding polynucleotide" are those which are capable of base pairing with the segment polynucleotide above under intracellular conditions.

As stated above, although p53 coding sequences can be copies of the full length cDNA or copies, or large fragments, the present invention can also be applied to shorter oligonucleotides p53. Sequence length 17 bases can appear only once in the human genome, and therefore are sufficient to identify a unique target sequence. Although shorter oligomers should be more easy to get and increase in vivo accessibility, many other factors are involved in determining specificity mating grounds. And binding affinity and sequence specificity of the oligonucleotide to its complementary target increase with length. It is assumed that the oligonucleotides 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs will be used, for example, in the preparation of p53 mutants and PCR reactions.

Any sequence 17 osnovatelnee target sequence. Although shorter oligomers easier to get and increase in vivo accessibility, many other factors are involved in determining the specificity of hybridization. Both the binding affinity and sequence specificity of the oligonucleotide to its complementary target, increase with the length.

In certain embodiments each may apply designs that include other elements, such as those that include the C-5 propilovyi pyrimidines. Oligonucleotides containing C-5 propilovyi analogues of uridine and cytidine, bind RNA with higher affinity (Wagner et al., 1993).

The expert in this field also it is clear that the integral in the definition of a biologically functional equivalent protein or peptide is a concept that imposes a limitation on the number of changes that can be made within a certain part of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are defined here, such as peptides, in which some, but the greatest part of them or all amino acids can be substituted. In particular, ocelot can be changed within a given peptide. Of course, many distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

Amino acid substitutions are typically based on the relative similarity of the amino acid side substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and similar properties. Analysis of size, shape and type of amino acid side substituents shows that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of a similar size; and phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based on these reviews, arginine, lysine and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine; defined here as biologically functional equivalents.

When changes can be considered the degree of hydropathicity (hydropathic) amino acids. Each amino acid is assigned a degree of hydropathicity on the basis of its hydrophobicity and charge characteristics, which are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); Seri (-3.5); lysine (-3.9); and arginine (-4.5).

The role of the degree of hydropathicity amino acids in the discussion of the biological function of interaction on protein usually understood by a person skilled (Kyte and Doolittle, 1982, which is introduced here by reference). It is known that certain amino acids may be substituted for other amino acids having similar degree of hydropathicity or label and still retain a similar biological activity. When changes on the basis of the degree of hydropathicity substitution of amino acids whose hydropathicity is within +/-2, is preferred amino acids that have a degree of hydropathicity within +/-1, are particularly preferred, and amino acids that have a degree of hydropathicity within +/-0.5, are even more preferred.

It is clear that the amino acid may be substituted for another having a similar hydrophilicity value is obtained biologically equivalent protein. As described in detail in U.S. patent 4554101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +/-1); glutamate (+3.0 +/-1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); Strait is phenylalanine (-2.5); tryptophan (-3.4).

When changes on the basis of similarity of hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +/-2, are preferred, and amino acids, which have hydrophilicity values are within +/-1, are particularly preferred, and amino acids, which have hydrophilicity values are within +/-0.5, are even more preferred.

B. Expression vectors.

Within this application, the term "expression design" refers to the inclusion of any type of genetic constructs containing nucleic acid encoding a gene product in which part or all of the coding sequence of the nucleic acid is able to be transcribed. The transcript can be translated into protein, but this is not necessary. Thus, in certain embodiments, expression includes both transcription of the p53 gene and translation of p53 mRNA in p53 gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding p53 or its complement.

In order to design influenced the expression of at least p53 transcript, polynucleotide, coders DNA sequences recognizable synthetic mechanism of the host cell, or introduced synthetic mechanism, which is required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct position in relation to polynucleotide to control initiation of RNA polymerase and expression of polynucleotide.

The term "promoter" can be used here to apply to the group of modules transcriptional control, which are clustered around the initiation site for RNA polymerase II. A large part of thinking about how promoters organized, comes from analyses of several viral promoters, including HSV Trimilin kinase (tk) and SV40 units early transcription. These studies, most recently updated work has shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognized sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The most famous example of this assetsliabilities transportnogo gene of the mammal, and promoter, SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements to regulate the frequency transcriptional initiation. They are usually located in the area of 30-110 bp before starting a website, although it was recently shown that a number of promoters also contains functional elements after the launch of the website. The space between promoter elements is often flexible, so that the function of the promoter is saved when the elements are inverted or moved relative to each other. In the tk promoter, the space between promoter elements can be increased up to 50 bp regardless of what activity before beginning to decrease. Depending on the promoter becomes obvious that the individual elements can work collaboratively or independently to activate transcription.

The particular promoter used to control expression of polynucleotide p53, is not considered as important because he is able to Express polynucleotide in the targeted cell (the target cell) with sufficient levels. Thus, when a human cell is targeted, atory able to be expressed in the human cell. In General, such a promoter might include either human or viral promoter.

In various embodiments, the promoter direct early genes of human cytomegalovirus (CMV) early promoter of SV40 and the long terminal repeat of Rous sarcoma virus can be used to obtain high-level expression of p53 polynucleotide. We also consider the use of other viral promoters or cellular promoters mammal or bakteryjnych phage promoters which are well known in the field to achieve the expression of polynucleotides, provided that the levels of expression are sufficient for producing inhibitory growth effect.

By applying a promoter with well-known properties, the level and structure of expression of p53 polynucleotide can be optimized after transfection. For example, selection of a promoter that is active in specific cells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin (liver tumor), CC10 (lung tumor) and prostate-specific antigen (prostate cancer), will contribute to tissue-specific

expression of p53 polynucleotides. Table. 2 gives the list several elements/promoters, to the t list does not claim to be exhaustive of all the possible elements involved in the acceleration of p53 expression, but only contains examples.

The enhancers were originally discovered in the form of genetic elements that increase transcription from a promoter that is located at some distance on the same DNA molecule. This ability to act over large distances has few precedents in classical studies prokaryotic transcriptional regulations. Subsequent work has shown that DNA activity enhancers arranged substantially like the promoters. That is, they consist of many individual elements, each of which is associated with one or more transcriptionally proteins.

The main difference between enhancers and promoters is in the way. The enhancer region as a whole may be able to stimulate transcription at a distance; not necessarily that it exactly matches the region of the promoter or of its constituent elements. On the other hand, the promoter must have one or more elements that directly initiate RNA synthesis at a specific site in a specific orientation is minimi, often creating the appearance that they have a similar modular organization.

In addition, any combination of the promoter/enhancer (according to data Bank eukaryotic promoter EPDB) could also be used to trigger the expression of p53 constructs. The use of T3, T7 or SP6 cytoplasmic expression system is another possible option. Eukaryotic cells can maintain cytoplasmic transcription from certain bacterial promoters, if there is a corresponding bacterial polymerase, or as part of exempted complex or as additional genetic expression vector.

In addition, the selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of p53 constructs. For example, polynucleotide under the control of the human PAI-1 promoter: expression is induced factor tumor necrosis. Table. 3 illustrates some combinations of promoter/inducer.

In certain embodiments of the invention the delivery of the expression vector into the cell may be identified in vitro or in vivo introduction of a marker in the expression vector. The submission of the expression. Usually the introduction of selective drug marker AIDS in cloning and in the selection of transformants. An alternative can be used enzymes, such as (tk) thymidine kinase herpes simple virus (eukaryotic) or chloramphenicole acetyltransferase (CAT) (prokaryotic). Can also be used immunological markers. Applied breeding marker, is not considered as important because it is able to be expressed together with polynucleotide encoding p53. Other examples are selectively capable of markers known to experts in this field.

Typically, the sample will include a polyadenylation signal to implement the appropriate polyadenylation of the transcript. There is no reason to believe that the nature of the polyadenylation signal is crucial for the successful application of the invention, and can be used any such sequence. In the invention was used, the SV40 polyadenylation signal in the sense that it was standard and well-known for successful functioning in the target cells that were used. Considered also as an element of expression design is a term is of the construction in other sequences.

In preferred embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and, in some cases, to integrate into the chromosomes of host cells makes them attractive candidates for gene transfer in mammalian cells. However, due to the fact that it has been demonstrated that direct uptake of "naked" DNA as well as receptor-mediated uptake of DNA is complicated (discussed below), expression vectors should not be viral, but instead they can be any plasmid, kosmidou or ragovoy design, which is capable of supporting expressvu encoded genes in mammalian cells, such as pUC or BluescriptT plasmid series.

Retroviruses

Retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by the method of reverse transcription (Coffin, 1990). The resulting DNA is then stably integrated into the cell chromosome as a provirus and directs the synthetic and its derivatives. The retroviral genome contains three genes, gag, pol and env, which encode capsid proteins, polymerase enzyme and shell elements, respectively. The sequence, which is detected ahead of the gag gene, designated functions as a signal for packaging of the genome into virions. Two sequences of the long terminal repeat (LTR) are present at the 5' and 3' ends of the viral genome. They contain a strong sequence of the promoter and enhancer, and also required for integration into the genome of the host cell (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a p53 incertitude in the viral genome in the place of certain viral sequences to produce a virus that is replication-deficient. To create virions, we construct a packaging cell line containing the genes gag, pol and env, but without the LTR and components (Mann et al. , 1983). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and sequences is introduced into this cell line (by calcium phosphate precipitation for example), the sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which is rovirosa, then is collected, optionally concentrated and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression require separation of host cells (Paskind et al., 1975).

A new approach designed to facilitate specific targeting of retroviral vectors has been developed recently based on the chemical modification of retroviruses by chemical additives leftovers lactose to the viral membrane. This modification may allow specific infection of hepatocytes through sialoglycoprotein receptors.

Was developed another approach for targeting of recombinant retroviruses were used biotinylated antibodies against retroviral protein shell and in relation to specific cellular receptor. Antibodies combined through bitenova components through the use of streptavidin (Roux et al., 1989). Using antibodies against major histochemistry complex antigens class I and class II, they demonstrated the infection of a variety of human cells that bore those surface antigens with IVF the DNA tumor viruses with a genome size of approximately 36 base pairs (Tooze, 1981). As a model system for eukaryotic gene expression have been extensively studied and well characterized adenoviruses, which make them an attractive system for the development of adenovirus as a gene transfer. This group of viruses is easy to grow and manipulate, and they exhibit a wide area master in vitro and in vivo. In lytic infected cells, adenoviruses are able to close the protein synthesis of the host, directing cellular mechanisms in the synthesis of large quantities of viral proteins and induce distributed groups number of viruses.

E1 region of the genome includes both E1A and E1B, which encode proteins responsible for regulation of transcription of the viral genome, as well as a small number of cellular genes. E2 expression, including E2A and E2B region, contributes to the synthesis of viral replicative functions, such as DNA-binding protein, DNA polymerase and terminal protein, which serve as seed replication. E3 gene products prevent cytolysis due to cytotoxic T cells and tumor necrosis, and obviously are important for viral growth. Functions associated with the E4 proteins include DNA replication, the expression on the Cove, and these proteins are only expressed after reprocessing the most part, a single primary transcript of mostly late promoter. A large part of the late promoter (MLP) shows high efficiency during the late phase of infection (Stratford-Perricaudet and Perricaudet, 1991 a).

As only a small part of the viral genome, obviously, is required in cis (Tooze, 1981), vectors derived from adenovirus represent excellent potential for substitution of large DNA fragments, when used in conjunction with cell lines such as 293 cells. Cell lines Ad5 transformed human embryonic kidney (Graham, et al., 1977) were developed to provide basic viral proteins in trans. In the invention, therefore, argues that the characteristics of adenovirus transformed them good candidates for use in targeting cancer cells (cancer cells as target cells) in vivo (Grunhaus and Horwitz, 1992).

Specific advantages of adenoviral systems for the delivery of foreign proteins in the cell are (i) the ability to substitute relatively large areas of viral DNA alien DNA; (ii) structural stability of recombinant adenoviruses; (iii) the security is om or malignancy; (v) the ability to obtain high titers of recombinant virus; and (vi) the high infectivity of adenovirus.

A further advantage of adenoviral vectors compared with retroviruses includes higher levels of gene expression. In addition, adenoviral replication is independent of gene replication master, unlike retroviral sequences. Due to the fact that adenovirus transforming genes in the E1 region can be easily dellarovere and still provide efficient expression vectors, it is assumed that the carcinogenic risk from adenoviral vectors is small (Grunhaus and Horwitz, 1992).

In General, systems of adenoviral gene transfer based on the constructed recombinant adenovirus, which is replication-incompetent due to the deletion of part of its genome, such as E1, and still retains its competence to infection. Sequence encoding relatively large foreign proteins can be expressed, when produced additional deletions in the adenoviral genome. For example, adenoviruses, deleteregvalue in the E1 and E2 regions, are able to carry up to 10Kb of foreign DNA and can grow danchenkov after adenoviral infection.

Gene transfer mediated by adenovirus, has recently been investigated as a means of mediating gene transfer in eukaryotic cells and in all animals. For example, when processing mouse with a rare recessive genetic disorder ornithine transcarbamylase (OTC) deficiency, it was found that adenoviral constructs can be used to supply the normal OTC enzyme. Unfortunately, expression of normal levels of OTC achieved only in 4 of the 17 examples (Stratford-Perricaudet et al., 1991b). Therefore, the defect is only partially corrected in most mice did not lead to physiological or phenotypic change. Therefore, these results are not attractive for the application of adenoviral vectors for cancer therapy.

Attempts to use of adenovirus in gene transfer for citiescape fibrosis transmembrane conductive regulator (CFTR) in the lung epithelium cotton hamster also been partially successful, although it was not possible to assess the biological activity of the transferred gene in the epithelium of animals (Rosenfeld et al., 1992). Again, these studies have demonstrated that gene transfer and expression of the CFTR protein in cells of the pulmonary route does not show physiological eff is biologicheskogo effect. In fact, they found that the levels of expression, which they observed, was only about 2% of the levels required to protect the lungs in humans, i.e., considerably lower than what is required for physiological effect.

The gene for human 1-antitrypsin was introduced into the lungs of normal rats due to vnutriaortalina injection, where it was expressed and resulted in secretion entered the human protein in the plasma of these rats (Jaffe et al., 1992). However, disappointingly, the levels that were obtained were not sufficiently high to provide therapeutic value.

These results demonstrated that the adenovirus is capable of directing the expression of a sufficient amount of protein in recombinant cells to achieve a physiologically relevant effect, and they do not involve the utility of adenoviral system for use in conjunction with cancer therapy. In addition, prior to the present invention it was believed that p53 may not enter into upravyauschaya cell, how those genes are used for the preparation of adenovirus, as it can be toxic. As E1B of adenovirus binds to p53, it is believed that this is another reason why technology is ion designs

As expression constructs in the present invention can use other viral vectors. Can be used with vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses. They present several attractive features for various mammalian cells (Friedmann, 1986; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

With the recent detection of defective hepatitis B virus was committed new penetration into structural-functional relationships of different viral sequences. In vitro studies have shown that the virus can maintain the ability to helperservice packaging and reverse transcription, despite the deletion of up to 80% of its genome (Horwich et al., 1990). This implies that a significant part of the genome can be replaced with alien genetic material. Hepatotropic and persistence (integration) were especially attractive qualities for liver-directed gene transfer. Chang et al. recently introduced chloramphenicol acetyltransferase (CAT) gene in the viral genome of hepatitis B ducks instead polymerase, surface, and Yu line hepatoma birds. For infection of primary duck hepatocytes were used culture medium containing high titers of recombinant virus. Was detected robust expression of the CAT gene by at least 24 days after transfection (Chang et al. 1991).

Alternative methods of gene delivery

In order to induce the expression of p53 constructs, the expression vector must be delivered into the cell. As described above, the preferred mechanism for delivery is via viral infection where the expression vector is encapsulated in infectious adenoviral particle.

The present invention also covers some non-viral transfer methods expression vectors in cultured mammalian cells. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al. 1990), DEAE-dextranase way (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), the way DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and the way lipofectamine-DNA complexes, cell treatment with ultrasound (Fechheimer et al., 1987), gene bombardment using high-speed microbreccia (Yang et al. , 1990), polycation (Boussif et. al., 1995) is hastily adapted for use in vivo or ex vivo.

In one embodiment of the invention adenoviral expression vector may consist simply of naked recombinant vector. The transfer of the construction can be carried out using any of the methods mentioned above, which provides a physically or chemically permeability of the cell membrane. For example, Dubensky et al. (1984), successfully completed the injection polyomavirus DNA in the form of precipitation CaPO4in the liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection CaPO4precipitated plasmids results in expression of transfection genes. You can anticipate that DNA encoding a p53 design can also be transferred in a similar manner in vivo.

Another variant of the invention for transferring the expression vector naked DNA into cells may involve particle bombardment. This method depends on the ability to accelerate microparticles coated with DNA to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Have created several devices for acceleration of small particles. One such device is based on the Yu force (Yang et al., 1990). Used microparticles consisted of biologically inert substances, such as tungsten or gold beads.

Selected organs, including the liver, skin, and muscle tissue of rats and mice, bombardopolis in vivo (Yang et al., 1990; Zelenin et al., 1991). These authorities may require surgical dissection of tissues or cells in order to remove any interfering fabric between the gun and target organ. DNA encoding a p53 design, can be delivered in this way.

In a further embodiment of the invention the expression vector can be captured in a liposome. Liposomes are cellular structures, characterized by a phospholipid bilayer membrane and internal watery environment. Multilayer liposomes have multiple lipid layers separated aqueous environment. They are formed spontaneously when phospholipids suspendered in excess of an aqueous solution. The lipid components undergo SOMO-rearrangement before the formation of closed structures and capture of water and solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). It is also expected to consider lipofectamine-DNA complexes.

Shipping liposome-mediated nucleic acid and expression of alien Dnevni and expression of foreign DNA in cultured cells HeLa and hepatoma embryo chick. Nicolau et al., (1987) have successfully completed liposome-mediated gene transfer in rats after intravenous injection.

In some embodiments of the invention, the liposome may be complexometry with hemagglutinin virus (HVJ). This, as shown, facilitates fusion with the cellular membrane and accelerates cellular entry liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may complexometry or be used in conjunction with nuclear megatonnage chromosomal proteins (HGM-1) (Kato et al., 1991). In other embodiments, the liposome may complexometry or to be used in conjunction with both HVJ and HMG-1. The fact that such expression vectors have been successfully employed in transfer and expression of polynucleotide in vitro and in vivo, they are applicable to the present invention. Where used bacteriotherapy promoter in the design of DNA would also be desirable to include inside liposomes suitable bacteriophagous polymerase.

Another mechanism for transferring the expression vectors into the cells, is a receptor-mediated delivery. This approach has the advantage of selective absorption macromolecule through receptor-mediated endocytosis in almost all eukaryotically (Wu and Wu, 1993).

Guiding the conductors receptor-mediated genes usually consist of two components: a cell receptor-specific ligand and DNA binding agent. Several ligands have been used for receptor-mediated gene transfer. The most widely characterized ligands are asialoorozomukoidom (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1993). Recently synthetic neoglycoproteins, which recognizes the same receptor as ASOR, has been used as a conduit for delivery of a gene (Ferkol et al. , 1993; Perales et al., 1994) and was also used epidermal growth factor (EGF) to deliver genes to squamous cell carcinoma (Myers, EPO 0273085).

In other embodiments, the conductor of delivery may include a ligand and a liposome. For example, Nicolae et al. (1987) used lactose-ceramide (ceramid), galactose-terminal asianangels entered into liposomes and observed an increase in the absorption of the insulin gene by hepatocytes. Thus, it is possible that adenoviral expression vector, can also be delivered to specific type of cells, such as lung cells, epithelial or tumor cells, by any number receptore-ligand systems with liposomes or without liposomes. Such is instrukcii in many tumor cells, show sverrehelena EGF receptor. Can be used to target mannose mannose receptor on liver cells. In addition, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T cell leukemia) and MAA (melanoma) can likewise be used as a target half.

In certain embodiments, gene transfer can more easily be performed in ex vivo conditions. Ex vivo gene therapy refers to the separation of cells from the animal, delivery polynucleotide in cells in vitro and then return the modified cells back into an animal. This may include surgical removal of tissue/organs from an animal or a primary culture of cells and tissues. Anderson et al., U.S. patent 5399346, disclose ex vivo therapeutic methods. During ex vivo culture expression vector can Express the p53 design. Finally, the cells can be re-introduced in the original animal or entered into an individual animal in a pharmaceutically acceptable form using any of the techniques described below.

D. Pharmaceutical compositions and routes of administration.

When considering the clinical application of adenoviral expression vector according to the present invention,consider the application. Usually this will lead to the preparation of pharmaceutical compositions, which, essentially, is free of pyrogens, as well as any other impurities that can be harmful to people or animals. Moreover, it is often desirable to employ appropriate salts and buffers to make complex sustainable, and in order to allow the complex to be absorbed by the target cells.

Aqueous compositions of the present invention include an effective amount of the expression vector, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be attributed to the so-called inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular compounds or compositions that do not produce harmful allergic or other adverse reactions when introduced to an animal or person. The term "pharmaceutical carrier", as used here, includes any and all solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents and slowing down the absorption of the agents and the like. The use of such media and agents for ICNA medium or agent is incompatible with the active ingredient, discusses its use in therapeutic compositions. Can also enter additional ingredients in the composition.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water, respectively, mixed with a surface-active compound, such as hydroxypropylcellulose. Can also be prepared dispersions in glycerol, liquid polyethylene glycols and their mixtures in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Expresiones vectors and guides the delivery of the present invention may include classic pharmaceutical preparations. Introduction therapeutic compositions according to the present invention can occur in any usual way of introduction, while the target tissue is available for this route of administration. This path includes oral, nasal, introduction to cheek, rectal, vaginal or point introduction. Or introduction can be carried out ostatochnoi, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions will generally be administered is whether other fillers.

Therapeutic compositions of the present invention mainly introduced in the form of compositions for injection or in the form of liquid solutions or suspensions; can be prepared solid forms suitable for dissolution in the liquid or suspension in liquid prior to injection. These preparations can also be emulsified. A typical composition for such a purpose includes a pharmaceutically acceptable carrier. For example, the composition may contain 10 mg, 25 mg, 50 mg or up to 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and organic esters for injection, such as etiloleat. Aqueous carriers include water, water-alcohol solutions, saline solutions, parenteral fillers such as sodium chloride, dextrose and ringer, etc. Intravenous fillers include liquid and nutritious fillers. Preservatives include antimicrobial agents, antioxidants, chelating agelena well-known parameters.

Additional formulations are suitable for oral administration. Oral compositions include typical fillers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like reagents pharmaceutical quality. The composition is chosen in the form of solutions, suspensions, tablets, pills, capsules, and compositions with controlled release or powders. If the way the point is, the shape may be a cream, ointment, liquid or spray.

An effective amount of therapeutic agent is determined on the basis of the intended purpose, for example, (1) inhibition of proliferation of tumor cells or (11) removal of tumor cells. The term "standard dose" refers to physically discrete units suitable for use with the subject, this unit contains a predetermined quantity of therapeutic composition calculated to produce the desired responses, discussed above, together with its introduction, i.e. the correct path and processing mode. The number that is entered in accordance with the number of treatments and the standard dose, depends on the subject being treated, sostoyalo doctor and are specific for each individual.

In certain embodiments it may be desirable to provide continuous supply of therapeutic compositions to a patient. When intravenous or intraarterial administration ways this submission may be accompanied by drip systems. For local applications, you should use re-use. Slow release compositions can be

used for a variety of approaches, which provides a limited but continuous amount of therapeutic agent within a certain extended period of time. For internal use, it may be preferable to continuous perfusion of the interested area. This can be accompanied by the introduction of the catheter, in some cases posioperating, followed by continuous introduction of therapeutic agent. The duration of perfusion will be selected by the attending physician for a particular patient and situation, but the time may be in the range from about 1-2 hours to 2 to 6 hours, up to about 6-10 hours to about 10-24 hours, until about 1-2 weeks or more. The usual dose of therapeutic composition during continuous perfusion will be equivalent to the dose that is given to using one or multiple injections, adjusted for a period in the dose of perfusion.

Clinical Protocol for SCCHN

Was developed clinical Protocol to facilitate the treatment of SCCHN disease using adenoviral constructs discussed below in the examples. In accordance with this Protocol will be selected patients have histological evidence of squamous cell carcinoma of the head and neck. Patients could, but not necessarily received prior chemo-, radio - or gene therapy. Optimally, patients should have adequate bone marrow function (defined as the number of peripheral absolute granulocyte >2000/mm3and platelet count of 100,000/mm3), adequate liver function (bilirubin 1.5 mg/DL) and adequate renal function (creatinine < 1.5 mg/DL).

The Protocol requires a single dose at intratumoral (intratumoral injections of pharmaceutical compositions containing between 106and 109infected particles p53 adenoviral expression constructs. For tumors 4 cm injected volume will be 4-10 ml, preferably 10 ml), while for tumors < 4 cm is used, the volume of 1-3 ml (preferably 3 ml). Multiple injections will be delivered for a single dose, entered in two weeks. When choosing a clinical test mode can be continued, six doses every two weeks or less frequently (monthly, within two months, four months, and so on).

When patients are suitable for surgical resection, the tumor will be treated as described above for at least two consecutive two-week courses of treatment. Within one week of the end of the second year (or more, for example, the third, fourth, fifth, sixth, seventh, eighth, and so on) the patient will be undergoing surgical resection. Before closing the excision of 10 ml of pharmaceutical composition containing p53 adenoviral expression design (106-109infected particles), will be delivered to the surgical site (operational bed) and left in contact for at least 60 minutes. The wound is closed and in it is placed a drain or catheter. On the third postoperative day, which introduces an additional 10 ml of the pharmaceutical composition through the drain and left in contact with the surgical bed for at least two hours. Then remove song suction and removal of drainage in the clinically primary sources recurrent SCCHN is residual microscopic disease, which remains in the primary tumor site, as well as locally or regionally after tumor excision. In addition, there is a similar situation when the natural body cavity obamanauts using microscopic tumor cells. Effective treatment of such microscopic disease will create a significant advantage in therapeutic regimes.

Thus, in certain embodiments the cancer can be removed by surgical excision, creating a "cavity". During and after surgery and (periodically or continuously) in the body cavity is entered therapeutic composition of the present invention. This is, in essence, the "local" processing cavity surface. The amount of the composition should be sufficient to ensure that the entire surface of the cavity has been contacting through expression construct.

In one embodiment, the introduction is just going to lead to the injection of therapeutic composition into the cavity formed by the excision of the tumor. In another embodiment, it may be desirable mechanical application through a siphon, a pad or other device. Can be used any of these approaches, once removed the described operational site. The cavity can then be subjected to continuous perfusion within the desired period of time.

In another form of this treatment local application therapeutic compositions of the targets in a natural cavity such as the mouth, throat, esophagus, larynx, trachea, pleural cavity, peritoneal cavity or cavities of hollow organs, including the bladder, the colon, or other visceral organs. In this situation, may or may not be significant primary tumor in the cavity. Treatment targets the microscopic disease in the cavity, but accidentally may also affect primary tumor mass, if it was not previously removed, or pre-neoplastic damage which may be present inside the cavity. Again can be applied in various ways in order to influence local application in these visceral organs or surfaces of the cavity. For example, oral cavity in the throat can be affected simple oral dissection and rinsing solutions. However, local processing within the pharynx and trachea may require endoscopic visualization and local delivery of therapeutic composition. VI what's catheter with infusion or re-direct visualization by cytoscope or other endoscopic instrument. Cavity such as the pleural and abdominal cavities, can be accessed using indwelling catheters or surgical approaches that provide access to these areas.

Observation of p53 expression after injection

Another aspect of the present invention includes the observation of p53 expression after administration of therapeutic composition. Due to the destruction of microscopic tumor cells, they may not be observed, so it is important to determine effectively whether the contact area target with expression design. This phenomenon may be accompanied by the identification of cells in which expression design actively produces p53 product. However, it is important to be able to distinguish between exogenous p53 and the fact that is present in the tumor and healthy cells in the treated area. Tagging of exogenous p53 indicator element will provide definite evidence of the expression of such molecules, and not its endogenous version.

One such indicator element is provided FLAC Biosystems (Hopp et al., 1988). FLAC polypeptide is oktapeptid (AspTyrLysAspAspAspAspLys) and its small size does not destroy the expression of the delivered gene therapeutic protein. Coexp the A.

Can also be used other immune system markers, such as 6XHis system (Qiagen). For this purpose, can be used any linear epitope to generate a composite protein p53, because (i) the immunological integrity of the epitope is not comprometida composite protein and (ii) the functional integrity of the p53 not comprometida composite protein.

E. combined therapeutic protocols.

The resistance of tumor cells to agents that damage DNA is a major problem in clinical Oncology. One goal of current cancer research is to find ways to improve the effectiveness of chemo - and radiotherapy by combining it with gene therapy. For example, the simplex-Trimilin kinase (HS-tK) gene of herpes when it is delivered to brain tumors through the retroviral vector, successfully induces sensitivity to ganciclovir antiviral agent (Culver, et al., 1992). In the context of the present invention, it is assumed that the p53 therapy could be used in conjunction with chemo - or radiotherapeutic intervention.

To kill cells, such as malignant or metastatic cells, using the methods and compositions Nikodym DNA damaging agent. These compositions can be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with an expression vector with a DNA damaging agent(s) or factor(s) at the same time. This can be achieved by contacting the cell with a single composition or pharmacological composition that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, where one composition includes p53 expression vector, and the other includes a DNA damaging agent.

Alternatively, p53 treatment may precede or follow the treatment with DNA damaging agent by intervals varying from minutes to weeks. In embodiments where the DNA damaging factor and p53 expression vector are applied separately to the cell, usually take care that was not significant period of time between the time of each delivery, so that the DNA damaging agent and expression vector were even able to provide mainly the combined effect on the cell. In such examples, it is assumed that there will be a place contact the cell with both agents in what aderemi time only about 48 hours, which is the most preferred. However, in some situations, it may be desirable to significantly prolong the time period of processing, in which between the respective applications goes from a few days(2, 3, 4, 5, 6 or 7) to several weeks(1, 2, 3, 4, 6, 7 or 8).

It is also expected that more than one introduction of either p53 constructs, or DNA damaging agent is desirable. Can be used in various combinations, where p53 is "A", and the DNA damaging agent is "B":

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/ A/B/B/B

B/B/B/A B/B/A/B A/A/B/B A/B A/B A/B A/B A/A B/B/A/A B/A/B/B

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A B/A/A/B

To achieve killing cells both agents are delivered into the cell in a combined amount effective to kill cells.

DNA damaging agents or factors are defined here as any chemical compound or a method of treatment, which induces DNA damage when delivered into the cell. Such agents and factors include radiation and waves that induce DNA damage as well as-irradiation, x-irradiation, UV-irradiation, microwave irradiation, electron radiation and the like. A variety of chemical compounds, also described ka is obtained for use in the methods of the combined treatment disclosed here. Chemotherapeutic agents, which are assumed to apply, include, for example, adriamycin, 5-florouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also includes the use of a combination of one or more DNA damaging agents, or based on radiation or real connections, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide (etoposide). In specific embodiments, the use of cisplatin in combination with p53 expression vector is particularly preferred.

When treating cancer in accordance with the invention will contact the tumor cells with a DNA damaging agent in addition to the expression vector. This can be achieved by irradiating the localized tumor site DNA damaging radiation, such as X-rays, UV-light-rays or even microwaves. Alternatively, tumor cells can be subjected to contact with a DNA damaging agent by introducing to the subject a therapeutically effective amount of a pharmaceutical composition containing the DNA damaging compound, such as adriamycin, 5-ferragens can be prepared and used as a combined therapeutic composition, or kit, by combining it with p53 expression vector, as described above.

Agents that directly sew polynucleotide, specifically DNA, are considered and are shown here in order to cause DNA damage leading to a synergistic antineoplastic combination. Can be applied agents, such as cisplatin and other DNA alkylating agents. Cisplatin is widely used for cancer treatment, with effective doses used in clinical applications of 20 mg/m2for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed for oral use and should therefore be delivered by injection intravenous, subcutaneous, intratumoral injection, or intraperitoneally.

Agents that damage DNA, also include compounds that hinder DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin and the like. Widely used in clinical setting for the treatment of neoplasm, these compounds are injected by bolus injections intravenously at doses varies from 25-75 mg/m2

Agents that disrupt the synthesis and attachment of polynucleotide precursors and subunits, also lead to DNA damage. Essentially, has developed a number of polynucleotide precursors. Especially useful are agents that have been widely tested and is readily available. Essentially, agents such as 5-fluorouracil (5-FU), are preferably used neoplastic tissue, making this agent is particularly useful for targeting neoplastic cells. Although moderately toxic 5-FU is applicable in a wide range of media, including local, but usually applied dose intravenous varies from 3 to 15 mg/kg/day.

Other factors that cause DNA damage and have been intensively used include those that are commonly known as rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Also considered other forms of DNA damaging factors, such as microwaves and UV radiation. In all likelihood, all of these factors create a wide range of DNA damage, or precursors of DNA replication and DNA repair, and Assembly and maintenance of chromosomes. The dosing intervals for X-rays from 0 to 6000 x-ray. The dosing intervals for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation and absorption of neoplastic cells.

Specialist in this field, refer to "Remington''s Pharmaceutical Sciences" 15th edition, Chapter 33, in particular pages 624-652. Some changes in doses will inevitably occur depending on the condition of the subject being treated. Specialist responsible for the administration of the drug, will, in any case, to determine the appropriate dose for the individual subject. In addition, for introducing human preparations should meet sterility, progenote, General safety and purity standards as required by FDA Office of Biologics standards.

The inventors believe that the regional delivery of p53 expression vectors patients suffering from cancer associated with p53, is a very effective way of delivering a therapeutically effective gene to counteract the clinical disease. Similarly, chemotherapy and radiotherapy can be directed in specific, the affected area of the body of the subject. Alternatively, systemic delivery of gene-expression vector or DNA damaging agent may be appropriate is but also, what cytokine therapy is an effective partner for combined therapeutic regimens. In such combined approaches can be applied to various cytokines. Examples of cytokines include IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-, GM-CSF, M-CSF, G-CSF, TNF-, TNF-, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-, IFN-, IFN-. Cytokines are entered according to the standard modes, as described below, consistent with clinical guidelines, such as the patient's condition and the relative toxicity of the cytokine.

In addition to the combination of p53-targeted therapies with chemo - and radio - and cytokine therapies it is also expected that combination with other gene therapies will be predominant. For example, targeting K-ras and p53 mutations at the same time may produce an improved anti-cancer treatment. Any other associated with tumor gene can be targeted in this way, such as p21, p16, p27, E2F, Dp family of genes, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-1, MEN-II, BRCA1, VHL, FCC, MCC, other ras molecules, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. It may also be desirable to combine the p53 gene therapy therapeutic treatment based on the antibody, including the use of design of single-chain antibodies, in Coti more cytokines, listed above. Also, it may be preferable to combine p53 with other genes that were involved in the processes of apoptosis, such as adenovirus E1A, Bax, BclXsand so on

Sets.

All the necessary materials and reagents required for inhibition of proliferation of tumor cells can be connected together in a set. This set can typically include the selected adenovirus expression vectors. Also included can be a different environment for replication of expression vectors and cells of the hosts for such replication. Such kits may include separate containers for each individual reagent.

If the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred. For use in vivo expression vector may comprise a pharmaceutically acceptable composition, is able to be introduced with a syringe. In this case, the container means may be in itself a container for inhalation, syringe, pipette, eye dropper, or any other similar means, which is in the form of injections, or it can even be added to and mixed with the other components of the set.

The components of the kit may also be provided in dry or liofilizovannyh forms. If the reagents or components are provided in a dry form, re-preparation is carried out usually by adding an appropriate solvent. It is obvious that the solvent may also be provided in another container means.

The kits of the present invention may also typically include a means for storing tubes in a closed form for commercial sale, such as, for example, containers for injection or molded plastic containers that hold the desired tube.

Regardless of the number and type of containers, the kits of the invention may also include or be packaged with the tool to facilitate injection/injection, or placement of a homogeneous complex of the composition into the body of the animal. Such a tool can be a pump, syringe, pipette, forceplay (forcepts), measuring spoon, eye dropper or any such accepted medical releasing a guide.

Animal model for microscopic tumor obsemeniteli for the analysis of microscopic residual carcinoma and microscopic seeding of body cavities. The term "cancer" as used herein may refer to a single cell or multicellular tumor mass. Microscopic disease "tumor" will consist of one or more carcinoma cells, which may not be observed with the naked eye.

The animal model described here is particularly useful to simulate (i) the post-operative environment for patients with head and neck cancer, especially in the development stages of the disease and (ii) the body cavity of the infected subject, which was established microscopic carcinoma. The model, similar to other animal models for cancer, comes from the inoculation of tumor cells in the animal. The difference, however, lies in the creation, subcutaneously, pockets (cavities), which is physiologically equivalent to the natural body cavity or post-surgical cavity created by excision of the tumor mass.

The present invention as example uses the Nude mouse as a model organism. In fact, however, there may be used any animal for use according to the present invention. Particularly preferred animals will be small mammals, which are routinely used in LSI, rats, Guinea pigs and hamsters. Rabbits are the preferred species. The criterion of choice of the animal will largely depend on the specific preferences of the researcher.

The first stage is the creation of a tissue flap in the experimental animal. The term "tissue graft" refers to any excision in the body of the animal, which withstands the target tissue. Usually it is preferable excision, performed at the rear side of the animal, as it is easily accessible area. However, it should be clear that excision can be performed in other areas on the animal, and the selection of the tissue may depend on various factors, such as a particular type of therapies, which will be investigated.

Once the site of the target tissue is exposed to, carcinoma cells, either individually or in the form of microscopic tumors in contact with the tissue. The most convincing way contamination of the cancer cells in the tissue is the filing of a suspension of the cultural environment of tissue containing cells for effects on the fabric. The use of cancer cells can be achieved simply by using a sterile pipette or any other the bottom version 2.5 106SCCHN cells inoculated in the affected tissue flap naked mouse. Specialist in this field will be able to easily determine for this purpose, what the appropriate number of cells will be required. The number of cells will depend on various factors such as the size of the animal, the site of excision, replicative ability of the tumor cells themselves, the time estimated for tumor growth, a potential anticancer therapy, which will be tested, and similar factors. Although the establishment of an optimal model system for any particular tumor type may require a certain throttling input cells, it does not create an excessive number of experiments. Specialist in the field of test animals will evaluate what you want this optimization.

This may be accompanied, for example, preliminary studies in animal delivered to different numbers of cells and growth of cells after release of the tissue flap. Naturally, the introduction of a larger number of cells will lead to greater populations of microscopic residual tumor cells.

At present chevigny, the experts in this field can use any variety of ways, which are normally used for sealing excision, such as the use of adhesives, brackets, stitches, seams, etc. specific to the considered application.

The following examples are provided more to illustrate certain specific options and should not, in any case, be considered as limiting the scope of invention.

EXAMPLE 1

Growth inhibition of cancer cells of the head and neck of a person by the introduction of the gene p53 wild type by recombinant adenovirus

Materials and methods

Cell lines and culture conditions. Cell lines human SCCHN, Tu-138, Tu-177, both were installed at the Department of head and neck surgery, Cancer center M. D. Anderson. Tu-138, Tu-177 were isolated from Desna-mouth moderately differentiated squamous carcinoma and poorly differentiated squamous carcinoma larynx, respectively. Both cell lines were constructed using the basic techniques of explantation, and are cytokeratin positive and oncogenic have etimicin Nude and SCID mice. These cells were grown in DMEM/F12 supplemented with 10% heat inactivated fetal b is the infection. Recombinant adenovirus p53 (Ad5CMV-p53) (Zhang et al., 1994) contains a cytomegalovirus (CMV) promoter, the cDNA of p53 wild-type, and the SV40 polyadenylation signal in the cassette minigene, insertional in E1-depletirovannoi region of the modified type 5 adenovirus (Ad5). Viral original cultures were grown in 293 cells. Cells were collected after 36-40 hours after infection, alloy preformed, resuspendable in phosphate-buffered saline, literally, and cellular debris was removed by gradient purification due to the impact of CsCl. Concentrated virus deliberately, were divided into aliquots and stored at -80oC. Infection was performed by adding virus in DMEM/F12 and 2% FBS with cell monolayers and the cells were incubated at 37oC for 60 min with constant stirring, then add complete medium (DMEM/F12/10% FBS) and the cells were incubated at 37oC for the desired period of time.

Analysis of the Northern blot. Total RNA was isolated using the acid-guanidine-thiocyanato method (Chomczynsky and Sacchi, 1987). Northern-blot analyses were performed on 20 μg total RNA. The membrane hybridized probe p53 cDNA, labeled using the method of random primers in 5 x SSC/Denhardt''s solution/0.5% SDS/DNA denaturised control. The relative amount downregulation of p53 was determined using a densitometer (Molecular Dynamics Inc., Sunnyvale, CA).

Analysis of the Western blot. Prepared full cell lysates by ultrasonic treatment of the cells after 24 hours of infection in RIPA buffer (150 mm NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mm Tris, pH 8.0) for 5 seconds. Fifty micrograms of protein samples were subjected to 10% SDS-PAGE and transferred to Hybond-ECL membrane (Amersham). The membrane was blocked Blotto/Tween (5% nonfat dry milk, 0.2% Tween 20, 0.02% sodium azide in phosphate-buffered saline and probed with primary antibodies, mouse monoclonal antibody Pab1801 anti-human p53 and murine monoclonal antibody (Amersham) anti-human-actin, and the secondary antibody horseradish peroxidase, conjugated sheep artemisinin IgG (Boehringer Mannheim, Indianapolis, IN). The membrane was treated and showed, as suggested by the manufacturer.

Immunohistochemical analysis. Infected cell monolayers were fixed with 3.8% formalin and treated with 3% H2O2in methanol for 5 minutes Immunohistochemical staining was performed by using a set Vectastain Elite (Vector, Burlingame, CA). The primary antibody used was Pab1801 the antibodies is complex ABC reagent was used for detection of complex antigen-antibody. Used pre-adsorption controls in each experiment immunostaining. The cells are then protivorechivii by Harris hematoxylin (Sigma Chemical Co., St. Louis, MO).

Analysis of cell growth. Cells were sown at a density of 2104cells/ml in 6-well plates thrice repeated versions. Cells were infected either with wild-type adenovirus (Ad5CMV-p53) or replication-deficient adenovirus as a control. Cells were collected every 2 days, counted and their survival was determined with the exception trepanovy blue.

Inhibition of tumor growth in vivo. The effect of Ad5CMV-p53 on education subcutaneous tumor nodules was determined in naked mice in certain non-pathogenic environment. The experiment was re-examined and confirmed by both committees for the protection and utilization of animals for research with recombinant DNA. After induction acepromazine/ketamine anesthesia were raised three separate subcutaneous flap on each animal and 5 106cells in 150 ml of complete medium were injected subcutaneously into each rag, using a blunt needle; the cells were kept in the pocket with a horizontal mattress suture. For each cell line used four animals. Posy front flap; 2) replication-defective virus (50 MOI) in the right rear flap; and 3) a single transport medium in the left rear side. All injection sites were developed subcutaneous visible and palpable (tangible) nodes before processing was carried out. Animals were examined daily and were killed on day 20. Tumor volume in vivo was calculated by the assumption of a spherical shape with an average diameter of the tumor, defined as the square root of the work transversely intersecting diameters. Following the killing was carried out three-dimensional measurement of the tumor using microstresses to determine the extent of the tumor. Applied nonparametric Friedman's 2-way ANOVA test to determine the magnitude of differences between sample values; used SPSS/PC + software (SPSS Inc., Chicago, II).

Results

Adenoviral infection of SCCHN cells. Conditions for optimal adenoviral transduction Tu-138, Tu-177 cells were determined by infection of these cells with adenovirus expressing E. Colip-gal gene. The transduction efficiency was assessed by counting the number of blue cells after X-gal staining. It turns out that there is a linear relationship between the number of infected cells and the number of Ali 60% blue cells (Fig. 1), and this was demonstrated in 100% of cases by a variety of infections. The transduction efficiency of the vector in SCCHN cells is quite different from the efficiency of transduction of different cell line, was investigated earlier: HeLa, HepG2, LM2 and human cell lines considerable cell lung cancer showed 97-100% efficiency of infection after incubation 30-50 MOI-gal adenovirus (Zhang et al., 1994).

Expression of exogenous p53 mRNA in SCCHN cells infected with adenovirus. Two human SCCHN cell lines were selected for this study: both cell lines Tu-138, Tu-177 had a mutated p53 gene. The recently established recombinant p53 adenovirus wild-type Ad5CMV-p53 was used to infect Tu-138, Tu-177 cells. 24 hours after infection was isolated total RNA and the analysis was performed Northern blotting. Transformed primary cell line 293 human embryonic kidney was used as a positive control because of its high level of expression of the p53 gene product, whereas t cell line lymphoblastoma with homozygous deletion of the p53 gene served as a negative control. Levels of 2.8-kb endogenous p53 mRNA detected in samples isolated from mock-sarado 10-fold higher levels of exogenous 1.9-Kb p53 mRNA was present in the cells, infected with Ad5CMV-p53, indicating that exogenous p53 cDNA was subjected successful transduction in these cells and effectively transcribable. Interestingly, the level of endogenous p53 mRNA in these cells was 5-fold higher than in the experimental control samples. Analysis Northern blotting was not confirmed Ad5CMV-p53 (DNA) RNA contamination.

Expression of p53 protein in SCCHN cells infected with adenovirus

To compare the levels of p53 mRNA relative to the quantity of biogas produced p53 protein was analyzed by Western blotting. p53 strip defined using monospecific anti-p53 antibody PAb1801, was observed in cell extracts isolated from all samples with the exception of K562 cells. Line 293 cells showed high levels of p53 protein. Samples isolated from mock-infected Tu-138, Tu-177 cells showed low levels of p53 protein. The level of p53 expression remained similar to the level in cells infected with adenovirus d1312. The levels of p53 antigen detected in Ad5CMV-p-53-infected cells, were significantly higher than the levels of endogenous mutated proteins in both cell lines. This result demonstrates that exogenous p53 mRNA produced from cells infected with Ad5CMV-p-53, effectively translates into immunore the nuclear staining of p53 protein, while mock-infected cells are not able to show similar staining, despite the presence of p53 protein in these cells. This inability to detect protein can be attributed to the insensitivity of the analysis.

The influence of exogenous p53 in SCCHN cell growth in Vitro. Cells infected with the control virus d1312, had a growth rate similar to the growth rate of mock-infected cells (Fig. 2A and 2B), whereas the growth of Ad5CMV-p53 cells infected Tu-138 (Fig. 2A) and Tu-177 (Fig. 2B) cells was strongly suppressed. 24 hours after infection was apparent morphological changes with areas of cell populations that were rounded, and their outer membranes that formed blisters. It was part of a series of histologically predictable events that are programmed death of cells. The effect was more pronounced for Tu-138, than for Tu-177 cells. Cells infected with adenovirus defective replication, d1312 showed normal growth patterns with no histological abnormalities. Analyses of growth were reproduced in four repeated experiments.

Inhibition of tumor growth in Vivo. For each cell line were tested four animals. Odontella, mainly due to deep anesthesia and subsequent injury due to the cell mating. The autopsy showed no evidence of metastases or systemic effects. Measurable tumor was observed on both rear flaps animals (i.e., areas that have not received Ad5CMV-p53). The absence of tumor progression is significant in the right front flaps of the animals that received Ad5CMV-p53 (p<0.4). Such Tu-177 cells have a slower rate of growth than was previously installed in these animals. Two animals in Tu-138 group were euthanized earlier due to the fact that they had undergone rapid growth and ulceration of the control tumor sites. All surgical sites were developed damage at least 9 mm3before the intervention. The volume of tumors at autopsy is shown in table. 4. Differences in volume were not statistically significant in Tu-177 group, which could reflect the limited sample size.

Example 2

Molecular therapy in vivo by adenovirus p53 for microscopic residual squamous carcinoma of the head and neck.

Materials and methods

Cell line and condition of culture. Human SCCHN cell line Tu-138, Tu-177, MDA 686-LN and MDA 886 were installed and PR is th Eagle's (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin.

Preparation of recombinant adenovirus and infection. Analysis of cell growth. Western blotting analyses. All procedures were previously described in Example 1. Analyses of cell growth were all carried out three times.

Transduction in vivo by adenovirus-galactosidase. X-gal staining of tissue samples was conducted on O. C. T. frozen tissue sections to determine the transduction efficiency. Eight micrometer thickness, the samples were washed in cold PBS and fixed in 0.5% glutaraldehyde the aldehyde at room temperature for 5 minutes. Then the slides were washed twice 4oC PBS and preincubator for 4 hours in X-gal solution (1.3 mM MgCl2, 15 mM NaCl, 44 mm Hepes buffer, pH 7.4, and 3 mM potassium ferricyanide; 3 mM potassium ferrocyanide, and 2% X-gal in DMF). Slides were protivootechny with hematoxylin and eosin.

Immunohistochemical analysis. Experimental animal tissue in vivo, fixed with formalin and enclosed in paraffin, cut into pieces 4-5 μm, dried at 60oC, were subjected to dewaxing and hydrational distilled water. The sections were treated with 0.5% solution of saponin in distilled water and washed several times, replacing the distilled water; endogenous peroxidase AK water. The medium was subjected to microwave irradiation in distilled water for 3 minutes using a microwave oven Sharp Model R9H81 operating at a frequency of 2450 MHz at 700 watts. After cooling, the sections were washed, replacing several times in distilled water, and placed in PBS; immunohistochemical study was performed using the method of Hsu et al., (1981), using the avidin-Biotin-peroxidase complex (ABC) as follows: the sections were blocked with normal horse serum, and incubated overnight at 4oC rabbit anti-human p53 polyclonal antibody, clone OM-1, 1:80 (Signet Laboratories, Denham, MA). Was then used anti-rabbit IgG-Elite kit (Vector Laboratories, Burlingame, CA) for use biotinylated anti-rabbit IgG and ABC complexes, which were preincubator for 45 minutes each. Reactions were subjected to immunostaining visualization through the use of 0.5% DAB in PBS containing 0.01% hydrogen peroxide (pH 7.6), protivorechivii 0.01% toluidine blue, were dehydrational, prosvetlili and was introduced in Permount. To confirm the specificity of the immunostaining reaction was performed immunoperoxidase staining using the same method as in test samples, nm control rabbit monoclonal antibodies.

Inhibition of tumor growth in vivo. This procedure was performed as described in Example 1. All surgical sites were assessed for pathology, as well as through analysis of autopsy on a systematic toxicity.

Results

The influence of exogenous p53 in SCCHN cell growth in vitro. Example 1 described the inhibition of cell growth in vitro with Ad5CMV-p53 in SCCHN cell lines, endogenous mutant p53. This is a real example is given to determine whether SCCHN cell lines similarly be affected by endogenous p53 wild-type. Also studied the effect of Ad5CMV-p53 in non-malignant fibroblasts.

Four human SCCHN cell lines were selected for this study: Tu-138, Tu-177 had a mutated p53 gene, whereas MDA 686-LN and 886 are both homozygous for p53 wild-type gene. Cell line of fibroblasts derived from normal growing fibroblast, which is kriticheskie normal and non-carcinogenic, was used as a non-cancerous control cell lines. Cells infected with the control virus d1312, had a growth rate similar to the growth rate of mock-infected cells, whereas the growth of tumor cells, infected with the ü hours after infection was apparent morphological change with portions of the cell population, which were rounded, and their outer membranes that formed blisters. It was part of a series of histologically predictable events that are programmed death of cells. The effect of previously occurred rather in cells with endogenous mutant p53 than in cells with p53 wild-type. Cells infected with adenovirus defective replication d1312, showed normal growth patterns with no histomorphologically anomalies. Analyses of growth were reproduced in four repeated experiments.

Expression of exogenous p53 protein in normal fibroblasts infected with adenovirus, and its influence on the growth rate. In addition it was also investigated the effect of Ad5CMV-p53 on karyotyping normal and not opuholevidnoe cell line of fibroblasts. These cells were selected in the process of establishing a primary tumor cell lines. Twenty-four hours after infection were analyzed by Western blotting for the comparison of protein levels produced different infected cell types. The band p53, the focus of monospecific anti-p53 antibody PAb1801, was observed in cell extracts isolated from all samples infected with Ad5CMV-p53. As can be seen from P and served as control. The level of p53 expression remained similar in both types of cells in mock-infected and d1312 infected cells. Ad5CMV-p53 infected fibroblasts showed higher levels of p53 protein levels than control cells. This result indicates that the p53 gene is efficiently transmitted in normal fibroblasts infected with Ad5CMV-p53, as proved by production of immunoreactive p53 protein. The protein expression and transduction efficiency of cytospins Ad5CMV-p53 infected fibroblasts were confirmed using immunohistochemical analyses. This fibroblastoma cell line showed a normal growth rate and morphology regardless of the intervention (mock, replication-defective virus, or Ad5CMV-p53) (Fig. 4). These experiments were repeated twice and also confirmed in other normal human fibroblastoid cell lines.

The effectiveness of tranzactii in vivo. To measure the efficiency of gene transfer in vivo, the site of subcutaneous flap was cut through 72 hours after molecular or control intervention. Experiments dose-response vector marker of adenovirus-galactosidase demonstrated the transduction efficiency of the dose-response in this model. This was confirmed by visimosti in vivo dose-response which was previously described in vitro (Example 1). None of the examples cited, the virus dose in excess of 1010PFU, did not cause the expression of p53 in other organ systems, including brain, liver, lung, heart, abdominalgia visceral organs and the skin. These experiments illustrated the ratio of the dose-response between viral titer and transduction efficiency, as well as the possibility of achieving extensive temporal expression of the gene subjected transduction within the desired surgical model area.

Suppression of tumor growth in Vivo. Were planned research to determine whether Ad5CMV-p53 mediated gene transfer in vivo to influence the establishment or growth of SCCHN cells were implanted in the subcutaneous flap. To achieve this purpose, we created a model of microscopic residual disease. In this model, three subcutaneous flap was raised on atomiclog naked female mice and were sowing 2.5 106tumor cells with a pipette. Instead of allowing tumor cells to form nodes (usually taking place over 4 days), single dose molecular intervention was carried out 48 hours after seeding of tumor cle surgical site, simulating clinical dilemma surgical excision of the entire large tumors. The development of tumors was directly related to the number of tumor cells, the time allocated for implantation and the dose of Ad5CMV-p53. Of the mice that received microscopically implanted tumor cells (2.5 106) and were treated with Ad5CMV-p53 at a concentration of 108plasmopara units (PFU) or more, only two developed tumors in both mice implanted cell line p53 wild-type (MDA 886-LN). All other cell lines showed no tumor development (table. 5). These experiments clearly demonstrate that the growth of microscopic tumor cells can be effectively suppressed in vivo when exposed to Ad5CMV-p53. The tumors was assessed at the end of the 12 week period (before the animal was slaughtered under circumstances of excessive tumor burden) with total and histological analysis of the surgical sites. The data establish tumors are summarized in table. 5.

Immunohistochemical analysis was performed on tumor sections of experimental animals. This cell line had the endogenous p53 gene of the wild type. Was V-p53 showed peripheral tumor necrosis with immunoablative in a more Central part of the tumor. 108PFU Ad5CMV-p53 was found complete necrosis of the tumor immunoablative found around the surgical pocket with multiple layers, expressing the protein, including stroma and superficial muscle layers. 109PFU Ad5CMV-p53 showed similar results with the results of the 108PFU Ad5CMV-p53, but noticeable was the increased exogenous p53 expression around the surgical site and swelling.

Using animals served as their own internal control, implants 4.0 106or more cells significantly increased the establishment of subcutaneous implants compared with the tumor implantation 2.5 106cells (P < 0.01), even after being processed in the surgical site Ad5CMV-p53 at 48 hours after inoculation. Permission implantirovaniem cells to take root within 72 or 96 hours before Ad5CMV-p53 intervention similarly increased tumor outcome (infection). Experiments dose-response was found that 108and 109PFU Ad5CMV-p53 were equally effective in the inhibition of tumor-loaded 2.5 106cells implanted within 48 hours (Fig. 6). Endogenous p53 status implanted tumor cell lines (whether the reduction of tumor development.

Example 3

The induction of apoptosis mediated gene transfer of adenovirus p53 wild type in squamous cell carcinoma of the head and neck.

Materials and methods

Cell lines and culture conditions. Preparation and infection with recombinant adenovirus. All procedures were performed and cell lines were maintained as described previously in Examples 1 and 2.

Analysis of DNA fragmentation. After incubation with adenovirus p53 wild-type and replication defective adenovirus controls with different time intervals, cells were collected, resuspendable in 300 µl PBS with the addition of 3 ml of extraction buffer (10 mM Tris, pH 8.0, 0.1 M EDTA, 20 μg/ml RNA, 0.5% SDS) and preincubator at 37oC for 1-2 hours. By the end of the incubation was added proteinase K to a final concentration of 100 μg/ml and the solution placed in a water bath at 50oC for at least 3 hours. DNA was Proektirovanie equal volume of 0.5 M Tris (pH 8.0) saturated phenol, then the extraction is repeated with a mixture of phenol/chloroform. Precipitated DNA was analyzed in 1% agarose gel.

Cell fixation. For the method TUNEL cells were fixed in 1% formaldehyde solution in PBS (pH 7.4) for 30 minutes -20oC before use. For cell cycle analysis the cells were fixed in 70% ethanol, cooled in ice.

Analysis of terminal deoxynucleotidyl transferase. The analysis was performed according Gorczyca et al. procedures (Gorczyca et al., 1993). After fixation and washing, cells were resuspendable in 50 μl of TdT buffer containing 0.2 M cacodylate sodium (pH 7.0), 2.5 mM Tris-HCl CoCl2(Sigma Chemical Company, St. Louis, MO), 0.1 mM DTT (Sigma Chemical Company), 0.25 mg/ml BSA (Sigma Chemical Company), 5 units of terminal transferase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.5 nmole Biotin-16dUTP together with dATP, dGTP and dCTP at a concentration of 20 μm. Controls were prepared by incubating separate aliquot of each test sample without dUTP. Cells were preincubator in solution at 37oC for 30 minutes, washed in PBS and resuspendable in 100 μl FITC, coloring solution containing 4X SSC, 0.1% Triton X-100 and 2.5 µg/ml fluoresceine avidin (Vector Labs. Inc. , Burlingame, CA). The tubes were preincubator for 30 minutes in the dark at room temperature. Cells were washed in PBS with 0.1% Triton X-100 and resuspendable in 0.5 ml PBS containing providine iodide (5 μg/ml) and 70 μl (1 mg/ml) RNase. The tubes were preincubator in the dark in ice for 30 minutes prior to flowing the use of flow cytometrical analysis EPICS Profile II (Coulter Corp. , Healeah, FL) with a standard optical configuration. For each sample were collected at least 5,000 events. Positive for TdT end-labeling was determined by subtracting the reference histogram from the histogram of the test sample using the working program immuno-4 Elite (Coulter Corp., Healeah, FL).

Analysis of cell growth. Cells were sown and growth were recorded as described in Example 1.

Analysis of apoptosis in Vivo. Gene therapy in a model of microscopic residual disease SCCHN was described above in Example 2.

End-labeling in Situ. The procedure was performed as previously described (Wijsman et al., 1993). Paraffin sections were deparaffinization dissolution in xylene for 5 minutes, three times each and have successfully hydrational by immersing the slides for 3 minutes each in 100%, 90%, 70% and 30% solutions of ethanol. Endogenous peroxidase was inactivated by immersing the slides for 20 minutes at 0.75% H2O2about./about. in 100% methanol. After that, the slides were washed in PBS, sections hydrolyzed in 0.1% pepsin (Fisher Scientific, Houston, TX) weight./about. in 0.1 N HCl for 5 minutes at 37oC and intensively washed in PBS. Then the slices were preincubator in a humid chamber at 37oC for 1 hour cocktail is/µl; biotinylated dUTP, 0.06 mM; 5X tdt buffer, 10 μl; bidistilled up to 50 ál. The reaction was interrupted by immersing the slides in a buffer containing 300 mM NaCl and 30 mM Na-citrate in redistillate. After washing the slides in PBS, the sections were preincubator in avidyne, kongugirovannom horseradish peroxidase for 1 hour at 37oC in a humid chamber. Staining showed using 3,3'-diaminobenzidine and slicers protivorechivii methyl green.

Results

Suppression of growth of SCCHN cell lines adenovirus p53. The above Examples demonstrate that the gene p53 wild type can be effectively transmoldovan in SCCHN cell lines using recombinant adenoviral vector. Therefore, the damaged tumor cells lose their ability to proliferation in vitro, as well as proliferation in vivo. The effect of suppression is independent of endogenous p53 status of the cell lines. Analyses preliminary growth rate were performed over a one-week period of time. This example explores the early effects of p53 wild-type on SCCHN cell growth (i.e., earlier time intervals in hours).

In this research, we used two representative cell lines. Kletochnaia-defective virus d1312, had a growth rate similar to the growth rate of mock-infected cells (Fig. 5A and Fig. 6B). On the other hand, the growth of Ad5CMV-p53 infected Tu-138 (Fig. 5A) and MDA 686LN (Fig. 5B), was significantly depressed. Obviously, exogenous p53 protein had earlier and more profound suppression of growth, Tu-138, compared to MDA 686-LN. Observed apparent morphological changes with areas of cell populations, the rounding top, and with their outer membranes, forming blisters resembling apoptosis, which is accompanied by initiation of suppression of growth. Cells infected replication-defective adenovirus d1312, showed normal growth patterns with no histomorphologically anomalies. Importantly, these effects were not observed after infection of p53 by adenovirus karyotyping normal fibroblasts, as described in detail in Example 2 above, as well as in human oral keratinocytes (dead, but not oncogenic).

Analysis of DNA fragmentation. One of the characteristic markers of apoptosis, which distinguishes apoptosis from necrosis, is biochemically observed manifestation of aliasing DNA fragments. To confirm the idea that cells are exposed to up the NC, extracted from viable cells after infection replication-defective adenovirus or adenovirus p53 wild-type, was subjected to agarose gel electrophoresis. View DNA fragments equivalent to approximately 200 bp (base pairs) and their multiplicity was observed in both cell lines. Fragmented DNA appeared in 22 hours after infection, p53 adenovirus in cell lines Tu-138, whereas in cell line MDA 686 LN, fragmented DNA was visible after 30 hours and more evident after 48 hours after infection of p53-wild-type adenovirus. Did not find fragmented DNA from mock-infected cells and d1312-infected cells.

Analysis of terminal deoxynucleotidyl transferase in Vitro. Another characteristic marker of apoptosis is the morphological alteration and destruction of the structural organization of the nucleus, which leads to condensation of chromatin. Electron microscopy has been widely used to detect such ultrastructural changes. However, recently running cytometrics ways to identify apoptotic cells has achieved great success owing to the ability of scanning and analysis of cell populations on sravnenitel transferase-mediated-dUTP-Biotin nick end-labeling) (Gorczyca et al., 1993), which is based on the discovery of vast gap DNA to identify apoptotic cells. Fifteen hours after infection, p53 adenovirus 4.4% viable Tu-138 cell population was in apoptotic stages compared with the absence of such state for MDA 686LN (Fig. 6A and Fig. 6B). The number of apoptotic cells increased in proportion to the increase in the duration of observation after incubation of p53 by adenovirus. About 31% of Tu-138 cells underwent apoptosis after 22 hours. Although there has been a slowdown in the initial induction of apoptosis, approximately 60% of MDA 686 LN cells were apoptotic stages after 48 hours of infection p53 adenovirus. Noteworthy that the percentage of apoptotic cells, as determined using the TUNEL method, may be largely unknown, since only viable cells were subjected to analysis. These data correlate with data analysis growth rate and DNA fragmentation. Did not observe detectable in the cell population undergoing apoptosis in the control experiments using mock-infected, and replication defective viral controls (100 M. O. I). Therefore, apoptosis is not , in Vivo. The examples above show that p53 adenovirus inhibits the formation of tumors in vivo. This example is designed to show whether the suppression of tumor growth in vivo after apoptosis. The analysis was performed end-labeling in situ detection of apoptotic cells in sections enclosed in paraffin, which were obtained from the Example 2. It is clear that the staining is not observed in tissue sections isolated from animals bearing MDA 686LN, which as a control was carried out processing of PBS. On the other hand, tissue slices, isolated from mice bearing MDA 686LN treated with adenovirus p53 wild type, showed vysokopoligonalnaya staining, demonstrating that apoptosis was indeed the event is included in the suppression of tumor growth in vivo.

In addition, these studies came to the determination of whether the suppression of growth, partly due to arrest of the cell cycle by inducing p21 protein, or mainly, is the result of apoptosis. Analysis of Western blotting showed that p21 protein was induced infected with adenovirus p53 wild-type SCCHN cells. However, analyses of the cell cycle indicated that despite the increased level of p21 protein in the Yes was compared with the S phase.

Example 4

Suppression of growth of squamous cell head and neck using p53-FLAG: effective marker for testing gene therapy.

Materials and methods

Cell line and condition of culture. Preparation and infection with recombinant adenovirus. Analysis Northern blotting. Analysis by Western blotting. Analysis of cell growth. Immunohistochemical staining of cell layers in vitro. All procedures were performed and cell lines were maintained as described in Example 1.

The generation of p53-FLAG adenovirus. p53 cDNA sequence was cut from pC53-SN by hydrolysis BamHI and cloned into the BamHI site pGEM7Z. The recombinant plasmid with the appropriate orientation of the inserts was then hydrolyzed with AccI and kpni restriction sites for deletion of 22 amino acids from the 3' end of the p53 cDNA. The linker with AccI-kpni restriction sites compatible ends, containing a sequence of FLAG peptide, including the stop codon, was then Legerova in gidralizovanny plasmid to generate p53-FLAG compound of the gene. The resulting p53-FLAG composite gene was then cloned in the expression vector of the human CMV promoter and SV the best of polyadenylation. The final design was subsequently insertion in the Shuttle vector pXCJL.1 (Zhang et al., 1994) to generate rekomendowane were conducted in a free environment specific pathogen using a model system atomiclog naked mouse, described in Example 1. Were performed two different sets of duplicate experiments. The first experiment was a dose-response using AdCMV-p53-FLAG virus in three pieces with decreasing concentration (1010pfu, 109pfu, 108pfu). The fourth flap served as control and was subjected to the method of blind selection with either PBS or with replication-defective adenovirus (DL312). A second study was performed using 1010pfu AdCMV-p53-FLAG, AdCMV-p53 and replication-defective adenovirus in three separate pieces. The fourth flap was inoculated with the same volume (100 μl) of sterile PBS. Forty-eight hours after treatment, two of these animals were euthanized and collected scraps for immunohistochemical analysis. The remaining animals were observed for 21 days and then killed. The tumor volumes were measured for comparison with the use of compasses.

Results

Expression of mRNA after infection with AdCMV-p53 and AdCMV-p53-FLAG virus. Both Tu-138 and MDA 686LN were investigated on the expression of p53 mRNA. Total RNA was isolated after infection with adenovirus. The analysis was performed Northern blotting. Were detected similar levels of exogenous AdCMV-p53 mRNA between AdCMV-p53 and AdCMV-p53-FLAG of sarajen the change of the intensity is palpable, so it can be associated with a loading dose. Endogenous expression of p53 mRNA is seen in lines 2 and 3 in mutated cell lines Tu-138. There was no significant endogenous p53 mRNA expression in MDA 686LN cell line, which is wild type for p53 gene. These data confirm that AdCMV-p53-FLAG virus similar AdCMV-p53 virus successfully transductions and efficiently transcribed. Analysis Northern blotting confirms the contamination of AdCMV-p53 DNA.

Expression of exogenous p53 protein in infected with AdCMV-p53 and AdCMV-p53-FLAG SCCHN cell lines. To compare the amount of protein expressed by cells infected with AdCMV-p53 and infected with AdCMV-p53-FLAG, was analyzed by Western blotting. The protein bands were identified using monospecific p53 antibodies (Ab1801) and anti-FLAG M2 antibodies (IB13025) on two simultaneously tested gels. Using p53 antibody (pAB1801) obtained similar high levels of expression of p53 protein in both cell lines, which were infected AdCMV-p53 and AdCMV-p53-FLAG. Also been tested Tu-138 and MDA 686LN cells infected with AdCMV-p53. No marked changes in the expression of p53 protein or in cells infected replication-defective adenovirus, nor in the group of mock-infected cells, when similarly about the Yu, downregulation after tagging p53 antibody, but was not observed detectable bands in these cells infected with AdCMV-p53 virus. Mock - and DL312 infected cells did not show detectable levels of immunoreactive p53 or FLAG protein in any cell line.

The influence of AdCMV-p53 and AdCMV-p53-FLAG on SCCHN cell growth in Vitro. The cytotoxic effect of therapy p53 wild-type Tu-138 and MDA 686LN cell lines was presented in detail above. Tu-138 cell line contains endogenous mutant p53 gene and MDA 686LN cell line possesses the p53 gene of the wild type. This study looks to determine whether any difference in performance observed after recombination of the AdCMV-p53 virus by interturbine FLAG sequence. Cells infected replication-defective adenovirus, have a similar rate of growth in mock-infected cells. Moderate cytotoxic effect can be seen with replication-defective adenovirus (Fig. 7A). In contrast, those cells that are infected with either AdCMV-p53 or AdCMV-p53-FLAG was actually experienced the total destruction of tumor cells on the third day. Histological examination confirmed the formation of the blister through the plasma membrane, which is characteristic APE 1). As noted above, the effect was more pronounced for Tu-138 cell line (mutated p53), than he was noticeable for MDA 686LN cell lines (p53 wild type). The growth curve analyses were reproducible in three repeated trials without significant differences, which would be noticeable between the effect AdCMV-53 and AdCMV-53-FLAG viruses, suggesting that the addition of FLAG peptide does not affect the ability of p53 in suppressing cell growth.

Immunohistochemical staining of SCCHN cell lines infected with adenovirus. Infected cell monolayers were compared to the expression of p53 and p53-FLAG protein using standard immunohistochemical method. Neither p53 nor FLAG protein could not be clearly identified in the mock infection DL312 infected cells in MDA 686LN cell lines. However, in Tu-138, which contains a mutated p53 gene, endogenous staining for p53 was positive. When cells were infected with AdCMV-p53 virus, it was observed a strong staining in both cell lines. Visual observation of these cells infected with AdCMV-p53-FLAG virus, showed identical staining intensity and number of positive cells with PAb1801 for antibodies compared with cells infected with AdCMV-p53 virus. Cells is by antibodies. Although the staining differed both within the nucleus and, to a lesser extent, in the cytoplasm.

Suppression of growth in Vivo. Study of dose-response with 108, 109and 1010plasmopara units (pfu) of AdCMV-p53-FLAG virus compared with the control flap, which has been treated with either PBS or DL312, were performed using the microscopic model of the method described in Example 1, Tu-138 cell line. The average tumor size for mock infection was 1205 +/- 205 mm3. Size linearly decreased with increasing concentration of virus used in molecular intervention. The average tumor size was 637 +/- 113 mm3, 392 +/- 109 mm3and 193 +/- 74 mm3for flaps, which were treated with a 108, 109and 1010pfu AdCMV-p53-FLAG, respectively. A comparison was made between each animal relative to itself using paired t test and noted the importance of the dose-response at p<0.05 in all comparisons, except for the comparison between flap, treated with 109and 1010pfu. It is clear that the greater the amount of virus in more visually inhibits tumor growth. In an additional study, the effects of AdCMV-p53 were compared with effects AdCMV suppression of exogenous tumor in an animal model of microscopic residual disease. After ensuring comparable in vivo and in vitro activity of AdCMV-p53 and AdCMV-p53-FLAG in the invention were used immunohistochemical methods to demonstrate p53-FLAG compound protein product in vivo. Using Tu-138 and MDA 686LN cell lines, pieces of microscopic residual disease were collected at 48 hours after treatment, fixed in formalin and embedded in a paraffin. On adjacent sections of tumor cells treated with AdCMV-p53-FLAG virus was applied staining for p53 and FLAG protein. The intensity of staining and the number of cells increased positively, was directly proportional to the number used in the infection of the virus. The controls were negative for staining with p53 and FLAG antibodies to MDA 686LN cells. Endogenous staining for p53 was observed in Tu-138 tumor cells. Histological specimens were stained with Hematoxylin and Eosin p53 antibody and FLAG antibody. Typical cytoplasmically staining FLAG M2 antibody contrasted with intranuclear staining of p53 antibodies. This, firstly, is that FLAG M2 antibody, as has been proven an effective enclosed in paraffin fixed tissue. Staining demonstrates that the tumor suppressive effect of napravle is whether the FLAG system.

In conclusion, it is clear that the joint delivery of FLAG protein with the desired gene therapy is a potential suitability as a marker for gene therapy. The invention clearly shows that he was simultaneously promotional together with p53 gene and the expression of messenger RNA and protein was not decreased. More importantly, the biological activity of released tumor suppressor gene has not been changed. For the first time it was proved that FLAG antibody effectively when immunohistochemical analysis performed on formalin fixed enclosed in paraffin tissue. These factors suggest the suitability of this new protein as a marker in future studies of gene therapy.

Example 5

Treatment of squamous cell carcinoma of the head and neck using p53 adenovirus.

The patient is A

Presents the 53-year-old man with unresectable SCCHN tumor of the head. The tumor weight was approximately 6.5 cm in diameter. After study the function of the bone marrow, platelet count and renal function, the patient received the first processing 108infected particles expression constructs of p53 adenovirus diluted in sterile phosphate buffered saline is UNT received identical treatment, until there have been six full treatment.

Three days after six treatments were investigated tumor and found that it is more than 4.0 cm in diameter. Histological examination shows a significant fragmentation of the cells in the tumor region. We had a second course of six treatments, after which the tumor was greater than 2.0 cm in diameter and was necrotic. The patient continued to receive weekly treatment within three months, the time after which the tumor was no longer found.

Patient B

Presents 44-year-old woman with resectable SCCHN tumor of the neck. Tumor size is approximately 2.5 cm in diameter. After study the function of the bone marrow, platelet count and renal function, the patient receives the first processing 5 107infected particles expression constructs of p53 adenovirus diluted in sterile phosphate buffered saline solution, through three separate intratumoral injection (total volume 3 ml). Every three days the patient receives identical treatment until there have been six full treatment.

Three days after six treatments, performed excision puheliasta, the wound closed and in the tumor bed leave drainage. 4, 7, 10 and 14 days after surgery are infusion through the drainage 5 107infected particles expression constructs of p53 adenovirus diluted in sterile buffered saline solution (total volume 3 ml). After contact with the tumor bed within two hours of the AMF inoculum is removed by suction. Six months after the end of treatment is not observed primary local or regional tumors.

Example 6

Gene transfer of wild-type via an adenoviral vector in A phase I trial of patients with progressirovanii recurrent squamous carcinoma of the head and neck.

An adenoviral vector containing the p53 gene "wild type", was taken in logarithmically increasing doses to patients with recurrent squamous carcinoma of the head and neck, confirmed by biopsy. Were conducted by direct tumor injections three times weekly for two current weeks. The patients were divided into two groups: 1) patients with recurrent disease, which can undergo resection, 2) patients with recurrent disease, which cannot undergo resection.

Patients separated into a group with tableware neoplasma 72 hours after the sixth event of gene transfer during the 2-week period. An adenoviral vector was also delivered vnutrikorporativnoj and 72 hours after the surgical procedure through regressive catheter infusion. Patients who could not undergo resection, were subjected to a repeated attempt of gene transfer by direct tumor injections monthly for a two-week cycles until then, haven't seen the progression of the disease or the deterioration of the activity status of the patient. The reliability of this processing is controlled by a closed hospital surveillance biopsies to assess the efficiency of gene transfer, analysis of water content in the body for trombotsitnoy (Shed) vectors and analysis of necropsy.

Ways

The research subjects. Twenty-one patients with progressirovanii recurrent squamous carcinomas of the upper europamusicale tract (aerodigestive) with Eastern Cooperative Oncology Group activity status 2 patients were enrolled into one of two study groups (arms), consisting of patients who were able to undergo resection (Group 1) or who could not undergo resection (Group 2) with recurrent malignant disease. Characteristics of the study subjects and doses of adenoviral vector are shown in the table. 6 and 7, respectively the person. This information was obtained from all patients before the study began.

Vector gene transfer. The present study used the vector replication-defective adenovirus serotype 5 with enhancer (CMV)-promoter, designated Ad5CMVp53. Were produced three series of adenoviral vector with plasmoptysis units (PFU) in the range of 109up to 1011due to good manufacturing practice Magenta, Inc and Introgen Therapeutics, Inc., and were stored frozen (-70oC) University of Texas, M. D. Anderson Cancer Centre. Each series was effective for transduction using Western blotting, as well as for analyses of suppressing the growth of tumor cells in vitro. The vector was thawed and diluted in phosphate buffered saline solution (binder) immediately prior to gene transfer and sent to the rooms of patients in the 4oC.

The dosage. An adenoviral vector was administered to each of five groups of patients in logarithmically increasing doses. Dosage increase was installed after a two-week observations of the last patient treated by the previous dose. After recording the first six patients in the study: three patients were recorded at each dose level regardless of javljaetsja in terms of full dose (plasmopara units). Set the number of vectors introduced in malignant epithelial cells, was not fitted. The total amount of injection is shown in table. 7. The amount of adenoviral vector, introduced in solid malignant tumor, was determined by clinical and radiographic method for defining the extent of the tumor. The vector was directly injected into recurrent squamous carcinoma by direct observation and by manual palpation. The injection was performed over the entire space by 1 cm along the tumor masses. After gene transfer, the subject remained under constant surveillance for at least 1-1/2 hours. Respiratory secretions and secretion excess body supported within 72 hours after gene transfer vector.

Detection of the vector. Samples of urine and serum was registered for inseminated adenoviral vector using viral culture of 293 cells, as well as the polymerase chain reaction (PCR) using primers that amplified E1b region of adenovirus 5' end of the p53 gene in wild-type, which were specific to the vector. PCR products were then moved through southern to improve detection of the virus in 1-5 viral particles and verify the response.

Security. Were carried out registration of symptoms, vital signs and the amount of blood, and patients were examined physically and daily photographically documented. Radiography of the chest, chemical analysis of blood and analysis of health status were performed at the start of each processing cycle. Serum titers of adenovirus antibodies were

measured before and after each cycle gene transfer. Three days after the sixth gene transfer of the first cycle were held tumor biopsy (or surgical resection). Samples of each example were stored in the form of frozen-snap, pathologically included samples, and in samples fixed in formalin.

Extraction of nucleic acids from serum and urine. Ad5CMV-p53 adenoviral DNA was extracted from 0.5 ml aliquot of serum or urine using a modified method of Cunningham et al., (1995). Distilled water was added to obtain 1 ml, and aliquots were besieged by 30% polyethylene glycol (PEG). As one SDS was not sufficient to release the viral DNA of its particles, proteinase K was added to the SDS at 50oC for 2-16 hours after precipitation with polyethylene glycol (Norder et al., 1990). The samples were proektirovanii phenol-Finance at 1400 g for 10 minutes at 4oC, resuspendable 0.3 ml of distilled water and re-precipitated with ethanol. The DNA pellets were washed in 70% ethanol, dried in vacuum and dissolved in 10 μl of distilled water. The samples were analyzed either immediately or stored at -20oto use. Extraction of nucleic acids was performed in biological safety cameras to prevent possible cross-contamination of samples.

PCR reactions on DNA extracted from serum samples. Were designed primers for specific amplification of p53 gene of the adenovirus vector. The upper primer (5'-CACTGCCCAACAACACCA-3', SEQ ID NO:5) corresponds to the 3' end of the p53 gene and the lower primer (5'-GCCACGCCCACACATTT-3', SEQ ID NO: 6) corresponds to the E1B region of adenovirus type 5 (nucleotides 3517-3533 sequence of the wild type). Each swab PCR reaction contained 0.2 mM of each oligonucleotide, 0.4 mM dNTPs, 1X TaqPlus Long weakly saline buffer (from Stratagene), 0.6 ál of TaqPlus Long (5U/ml) (from Stratagene), and 5 µl of the test DNA. The samples were placed in a MJ Research Pelter Thermal Cycle (PTC-200), programmed for 93oC for 3 minutes followed a three-stage profile: 93oC for 30 seconds, 65oC for 45 seconds, and 72oC for 45 seconds for a total of 30 or 35 cycles. 5 UNL 6X nagruzochnogo the CGS by the end of the PCR experience and were loaded in 1% agarose, 1X TBE gel containing acidini bromide (0.6 mg/ml). The samples were subjected to electrophoresis at 100 V for 1-1.5 hours and then photographed under UV light.

The polymerase chain reaction (PCR). Only 5 ál of the prepared DNA can be used in a single polymerase chain reaction. For serum PCR was performed in 20-µl volume containing 2 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μm each of deoxyribonucleotide of triphosphates (dNTP), 10 mM Tris-HCl (pH 9.0), 5 μm of each primer, and 1.7 units of Taq DNA polymerase (Promega). The reaction was carried out at 94oC for 30 seconds, 58oC for 30 seconds and 72oC for 60 seconds for 35 cycles, followed by 10-minute period at 72oC. PCR primers were selected from a sequence of Ad5CMV-p53 with sense primer located at the 3' end of the p53 cDNA (5'-GCCTGTCCTGGGAGAGACCG-3', SEQ ID NO:7) and antisense primer was selected from the E1B region of adenovirus type 5 (5'-CCCTTAAGCCACGCCCACAC-3', SEQ ID NO: 8). PCR product (fragment 838 base pairs) was separated in 1% agarose gel. The same PCR product was subcloned into pCR-Script vector (Stratagene), sequenced and the insert purified gel, was used as a probe for detection of the PCR product. Urine PCR was performed in 20-µl volume containing 2 mM MgSO Lumina, 200 μm each of deoxyribonucleotide of triphosphates (dNTP), 5 μm of each primer and 2.5 units of Taq Plus long DNA polymerase (Stratagen). The reaction was carried out at 93oC for 60 seconds and then at 93oC for 30 seconds, 65oC for 45 seconds and 72oC for 45 seconds for 35 cycles followed by a 10-minute period at 72oC. PCR primers were selected from a sequence of Ad5CMV-p53 with sense primer located at the 3' end of the p53 cDNA (5'-CACTGCCCAACAACACCA-3', SEQ ID NO:9) and antisense primer was selected from the E1B region of adenovirus type 5 (5'-GCCACGCCCACACATTT-3', SEQ ID NO:10). PCR product (fragment 724 base pairs) was separated in 1% agarose gel.

Southern blotting analysis. In the analysis of the southern blot used to verify the specificity of PCR products, the DNA in the gel was denaturiruet and neutralized prior to blotting on a nylon membrane (Hybond-N+, Amersham) by capillary absorption. The membrane is pre-hybridized for 15 min at 65oC in Rapid-hyb buffer (Amersham) and hybridized in the same buffer containing32P-labeled probe, within 1-2 hours. The membrane was washed in 0.1 x SSC and 0.1% SDS at room temperature twice, and twice again at 65oC (15 min per wash). The washed membrane waterlance test specimens. The following types of controls are included with each series of samples. At the stage of DNA extraction were used two negative control serum (funded and brought to aliquot the serum sample from the owners Introgen) and two positive control containing negative serum, increased to a peak of 10 or 100 pfu pfu of virus AdCMV-p53, which has been applied. This was done to get a window of sensitivity where 10 (100) pfu controls was positive, but the negative controls were negative. If negative controls were positive, PCR was repeated only 30 cycles. If 10 pfu was negative, DNA was further cleaned by additional deposition of ethanol, and repeated PCR. The above two stages are always placed the experimental parameters in the corresponding window of sensitivity.

At the stage of PCR were used a positive control 1ng AdCMV-p53 DNA (isolated from clinical batches) and negative (H2O) control. PCR series was repeated if any of the controls was wrong. Failures with negative controls was not. To confirm the alleged positives DNA was re-isolated from serum (along with a few temporarily adjacent samples) and repeated this PCR DNA. The samples were evaluated as positive, terrazzo, it was considered negative for reporting purposes. Putative positives that cannot be repeated (due to the lack of additional untreated sample), were not included in the database.

Measuring the efficiency of gene transfer. Surgically removed tissue samples were placed in a cryostat, immediately instantly froze and then kept in storage with liquid nitrogen until use. Frozen samples decantation in the hole spray for fabric Bessman stainless steel (Spectrum, Houston, TX), which was pre-cooled by plunging into liquid nitrogen. The tissue was ground to a fine powder, hitting pestle Bessman with a steel cylinder, five or ten times. Polarisavenue tissue was transferred to a glass tissue homogenizer (Fisher Scientific, Pittsburg, PA), containing 1 ml of TRI reagent (Molecular Research, Cincinnati, OH), 50 mg of tissue, and homogenized by five to ten strokes at the top and bottom Teflon pestle.

After homogenization RNA was isolated according to the instructions attached to the TRI reagent. The homogenates was transferred into a polypropylene centrifuge tubes (Molecular Research) and kept for 5 min at room temperature before adding chloroform (0.2 ml per 1 ml TRI reagent). Samples C at 12000 g for 15 min at 4oC to separate the RNA containing aqueous layer from the phenol-CHLOROFORMATES phase. Added isopropanol to the aqueous phase and the RNA precipitated with incubation at room temperature for 15 minutes to Pellet the RNA was isolated by centrifugation at 12000 g for 15 min at 4oC, washed once with 75% ethanol, air dried, dissolved in diethyl pyrocarbonate (DEPC)-treated water and quantitatively determined by measuring absorption at 260 nm. Contaminating DNA was removed by incubation of up to 50 µg RNA with 60 U DNase has I (Pharmacia, Piscataway, NJ) for 25 min at 37oC in a total reaction volume of 260 μl. RNA was then extracted with a mixture of phenological, precipitated with ethanol, washed once with 75% ethanol, was grained by centrifugation at maximum speed in microcentrifuge for 15 min at 4oC, dried air, resuspendable in DEPC-water and stored at -80oC. the Quality of RNA was evaluated by testing samples on a regular Undenatured 0.8% agarose gel and the manifestation of 28S and 18S ribosomal bands colouring ethidium bromide. To exclude cross-contamination between samples and to minimize RNase activity all reusable instruments that are used is of n from debris, were transferred to 10% bleaching solution for 3 min, were intensively rinsed with deionized water, was sprayed 100% ethanol, dried, was immersed in chloroform and dried again before using.

Was carried out reverse transcription using 1.5 µg full cellular RNA in 23.5 μl reaction mixture containing 111 ng statistical hexamers (Gibco BRL, Grand Island, NY). 40 units of RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), 0.4 mm of each dNTP (Perkin Elmer, Foster City, CA), and 300 units of Superscript II Rnase-reverse transcriptase (Gibro BRL) in 1 x RT buffer (50 mm Tris, pH 8.3, 75 mm KCl, 3 mm magnesium chloride and 20 mm dithiothreitol). RNA and statistical hexamers were heated to 70oC for 10 min and cooled in ice before adding the remainder of the reaction mixture. The reaction was incubated at 25oC for 5 min 200 units of reverse transcriptase and then an additional 10 min at 25oC following the addition of the following 100 units of reverse transcriptase to facilitate annealing of primer before incubation at 42oC for 50 min, the RT-reaction was interrupted by heat inactivation of the reverse transcriptase inhibitor for 15 min at 70oC. RNA complementary to the cDNA was removed by hydrolysis of one IU is you and neck, infected with recombinant adenovirus Ad5CMV-p53 (multiplicity of infection of 100:1), was used as a positive control for the detection of viral transcribed p53 and TU167 cells, living with variant adenovirus vector d1312 (1), which does not contain p53 transcriptionally unit, which was used as a negative control.

For detection of Ad5CMV-p53 transcript PCR was performed in a reaction volume of 30 μl containing 0.2 mm of each dNTP, 1.5 mm magnesium chloride, 1 unit taq polymerase (Promega, Madison, WI) and 0.5 mm of each primer CMV2 (5'-GGTGGATTGGAACGCGGATT, SEQ ID NO:11) and P53EX3 (5'-GGGGACAGAACGTTGTTTTC, SEQ ID NO: 12) in 1 x PCR buffer (50 mm KCl, 10 mm Tris pH 9.0, 0.1% Triton X-100). Primers CMV2 and P53EX3 amplified 295-grounds fragment specific to adenovirus, created p53 transcript. PCR conditions for detection of Ad5CMV-p53 transcripts were as follows: 94oC for 1 min, followed 94oC for 30 sec, 58oC for 40 sec, 70oC for 1 min for 35 cycles and 70oC during the 10-minute period of time.

To ensure that the product amplificatory during PCR detected mRNA and do not contaminate the DNA in the RNA preparation, PCR was performed using RT productliability-3-phosphate dehydrogenase (GAPDH), conducted in order to monitor the integrity of the RT reactions. 3 μl volume of the RT reaction was diluted in 30 μl of PCR mixture containing 0.2 mm each DNTR, 2 mm magnesium chloride, 1 unit taq polymerase, and 0.5 mm of each primer GAPDH1 (5'-ACGGATTTGGTCGTATTGGG, SEQ ID NO:13) and GAPDH2 (5'-TGATTTTGGAGGGATCTCGC, SEQ ID NO:14) in 1 x PCR buffer. GAPDH primers cover 3 axona in GAPDH gene of the person and amplified product 231-base-specific mRNA. The PCR conditions for detection of GAPDH were as follows: 94oC for 1 min, followed 94oC for 30 sec, 60oC for 12 sec, 72oC for 1 min for 35 cycles and 72oC during the 7-minute period of time.

PCR was performed using Perkin Elmer Gene Amp 9600 thermal cycler, and all primers were commercially synthesized (Genosys, the Woodlands, TX).

Immunohistochemical determination of intratumoral gene. Immunoperoxidase studies were performed on formalin-fixed, included in paraffin tissue sections using the method avidin-Biotin-peroxidase complex (ABC) (1). The specimens were cut to a thickness of 3-4 μm, was deparaffinization in xylene and re-hydrational in decreasing concentrations (100-70%) of ethanol. The activity of endogenous peroxidase were blocked with 3% piroxicam is aStore (PBC), the sections were incubated with a 1:10 dilution of normal horse serum, to minimize background staining. That was followed by incubation overnight at 4oC monoclonal antibodies to p53 (DO-1, Oncogene Science, Inc., Uniondale, NY; 1:80 dilution) and p21 (Oncogene Science, Inc., 1:100). Procedure peroxidase staining was performed using sets of ABC Elite (Vector Laboratories, Burlingame, CA). The immunostaining reaction was visualizability using 0: 05% 3.3'-diaminobenzidine in Tris-HCl buffer containing 0.01% peroxide hydrogen, pH 7.6. Slices protivorechivii 0.01% toluidine blue and were placed in permount. The evaluation was carried out by counting positive staining nuclei 200 cells 10 consecutive high-power fields by two independent observers.

TUNEL analysis for DNA fragmentation. TUNEL analysis was performed using the ApoptagTMPLUS kit (Oncor, Gaithersburg, M. D.) according to the instructions provided by the manufacturer. Slides protivorechivii 4% methylene green. Accordingly stained with hematoxylin and eosin slides were evaluated for the presence of infiltrates of inflammatory cells and were scored on a scale of 1-4.

The analysis procedure for cytopathic effect. Urine samples of the patient were also examined for the presence of Ad5p53 analysis, in which the love of cytopathic effect (CPE): the cells are rounded at the top and detach from the surface. Urine samples of the patient, which were analyzed for CPE were from samples of the first morning urine collected during the second week of the first treatment cycle and on day 0 or 1 (preprocessing). Samples, approximately 15 ml each, were stored in sterile 15-ml conical tubes at -80oC before use. IT293 cells, which form a cell monolayer recipient in this analysis are contained in DMEM plus 10% FBS in an incubator when wet 10% CO2at a temperature of 37oC. For 2 days before testing a patient sample cells were inoculated with 2 105per well in 12-well plates.

At the time of analysis, urine samples were thawed in an ice bath, and aliquot the sample was mixed 1:1 with DMEM and sterile filtered using a 0.22 μm syringe filter. 350 ál aliquot of sample this 1:1 mixture was slowly added into each well after removal of the growth environment. The tablet was carefully shaken after 15 minutes. After 30 minutes was added 2.0 ml of DMEM plus 10% FBS in each well to dilute the sample. On day 3 (72 hours later) and 6-day analysis was added 0.5 ml of aliquot samples of fresh medium to each well to maintain the cell monolayer for a maximum of 6-7 days.

Patient samples were subjected to the tests three times. The UP>5pfu Ad5p53 on the hole for the registration of any component of urine, which could interfere with detection of the virus using this procedure. Control wells were incubated with only one DMEM with decreasing concentration of 105, 104, 103, 102or 101pfu per well, each twice. Peak 105pfu causes CPE in these circumstances, on the 2nd day of analysis; in each subsequent day following descending control will show CPE. Therefore, the time at which the CPE is detected in each patient sample indicates the level Ad5p53 in this sample.

Recombinant competent adenovirus. Adenoviral p53 used in a clinical study, was tested for the presence of RCA using A549 cells, using Biotechnology Services Division of Microbiological Associates, Inc., (Rockville, Maryland).

The statistical analysis. Was applied odnoseriynoy research project. To prevent the involvement of a larger number of patients than is necessary in the study of excessive toxicity, which was detected, has applied a rule early stop spam. Marked as WILCOXON airman test and the post test was used for comparison between before and after treatment, the percentage of cells showing TUNEL and them the and Survpac SPSS-Statistical.

Response and toxicity. Survival and response were evaluated in the analysis in the interval between them (interim), but was not considered the purpose of the analysis. The purpose of this analysis was to determine the potential for transduction of this strategy of gene transfer. Patients were valuable for response and toxicity following the 30-day observation after one cycle gene transfer. The effects of toxicity of the treatment was evaluated according to the criterion of the overall toxicity of the National cancer Institute. (Xref.) Response to therapy was assessed by CT scan or ultrasound of the neck before each treatment course. Patients were evaluated for response if they received at least one course of therapy, followed by appropriate documentation of response. Patients undergo surgical resection ozretich tumors could not be evaluated for response, as the surgery was carried out before the 30-day follow-up period. All CT scans were evaluated by a single radiologist, and ultrasounds. Partial response was defined as a 50% or greater decrease in the sum of diameters of measurable tumors; minor response was estimated as 25% to less than 50% reduction of the product of diameters of measurable pathological changed the ditch.

Duration of survival was measured from time of entrance of the Protocol prior to expiry. Each response of the patient was re-examined using the data governance Committee, including oncologist surgery head and neck radiologist and oncologist on drugs.

Results

Detection of the vector. Adenoviral vector DNA was detected using PCR in serum and in urine samples of patients up to 48 hours after gene transfer. The detection limit for vector is 1-5 viral particles. Viral DNA was isolated in the urine of the patients that were subjected to gene transfer with each dose of the virus, but were not detected after 48 hours after gene transfer. Detection of viral DNA in serum were increased with increasing virus dose in excess of 107PFU, but was undetectable after 48 hours after gene transfer.

Urine samples were first analyzed for infectious virus, measured CPE on cell monolayers to PCR nucleotide analysis. The presence of virus in the urine was attributed to 293 cells, as described above, in order to observe the CPE, which is observed at the time, as the cells are rounded at the top and appear from the Petri dishes. CPE rarely identified in the mod who were as amenable to expression. Not random CPE was confirmed using PCR and southern blot testing on the same sample of urine for registration adenoviral nucleotide.

Analysis of viral DNA of other organ systems. PCR analysis showed the viral DNA more than two months after the 109Ad5CMVp53 gene was transferred into the skin, cardiac muscle, lung and testicular tissue in frozen autopsies samples, carefully analyzed to prevent cross-contamination between tissues. Renal parenchyma, adrenal gland and pancreatic tissue showed no viral DNA sequences that are specific to this vector. Immunohistochemical analysis for these autopsychic samples showed no confirmation of overexpression of the protein product of p53 wild-type.

Evaluation of gene transfer. All analyses were performed after at least 1 hour after incubation of the tissue with the vector. mRNA product was detected at 4 h and 48 h after RT-PCR. In contrast, biopsy samples, frozen after 1 hour after exposure to the impact or less, showed no p53 mRNA. In addition, samples negative control obtained with an operational site, which was not subjected to gene transfer, were also negativnyi by RT-PCR for the presence of Ad5CMVp53 transcribed mRNA. Transduced samples from two patients were positive on EtBr stained gels, while no Ad5CMVp53 product was not detected in any of nitrosulfonic samples, except for the fact that GAPDH may be amplified from all samples. Specificity 295 br PCR product of two positive patients was confirmed by southern-blotting. As the PCR product was not observed when reverse transcriptase was excluded from the RT reactions, 295 br product is detected in the picture was most likely generated from mRNA than from contaminating DNA.

Immunohistochemical analysis. All patient samples with pre - and postgene transfer simultaneously analyzed positive and negative controls in each experiment. Many sections of each sample were analyzed and compared with hematoxyline and eosin-stained, and TDT concetatenii samples. Biopsy samples subjected postvictorian the injecting confirmed gene transfer in three patients whose samples with pre-migrated biopsy showed no endogenous sverhagressivnomu p53 protein. In 5 patients out of 21 patients (27%) p21 (CIP/WAFI) was not significantly downregulation of endogenous; this was proved by informativna also observed in tumour-related lymphocytes as well as in the cells of the tumor stroma, thus also pointing to the transduction of non-neoplastic cells.

Serological antibody response. Adenovirus serotype 5 antibodies induced in all patients followed by re-injection of the vector. However, the transduction efficiency was found not to have changed significantly in comparison with the initial cycle gene transfer and subsequent cycles. Weak tumor erythema at the injection site was found starting with 3109PFU, however, these local reactions are not restricted vector tolerance. No evidence of systemic hypersensitivity was found in each patient, in spite of repeated viral dosing in continuation of the longer period, such as six months, patients treated with 109PFU.

Pathological observations. Traces of needles were identified in most of the biopsy samples, and expression of the gene product was shown in the deeper tissues outside the sites of injection. Similarly, the presence of cells with magnetic tagging in transduced areas suggested the induction of apoptosis in tumor cells, but the absence of apoptosis in stromal or vospalitelnye among patients who received a dose of 109PFU or more. Interestingly, the patient number 7, from which the sample was collected downregulation of endogenous p53 wild type, showed necrosis bleeding with no evidence of viable tumor in serialno chopped surgical samples in the mass of his recurrent 2-cm sample neck after one cycle gene transfer. Pathological findings of necrosis as well as induction of apoptosis, were often observed in the samples.

Clinical observations. Patients transferred direct tumor injections of the vector with the most frequent side effect associated with anxiety in the injection site. Sign local erythema at the injection site is observed at 109PFU of vector and above, although more significant systemic sign of hypersensitivity was not observed, despite the evidence of high titers of systemic antibodies. Patients under the numbers 5, 10, and 16, there has been no sign of disease on average over the next seven months. Patients 7 and 13 showed stable disease for 3 and 5 months respectively indicative of the damage zone. Patient number 20 had a partial response, when he was checked using a CAT Scan, ultraslut disease.

In conclusion, patients with recurrent squamous carcinomas of the head and neck, promoting using adenoviral gene transfer of p53 wild-type, are safe and can effectively transducible cancer and normal cells by direct tumor injections or intraoperative injection of the vector. None of the patients showed no toxicity, which could limit the introduction of the vector to increase the dose. Expression of the transgenic product has not changed, despite the improvement in the response of the system antibodies. Answers local inflammation detected pathologically within samples esecanna tumors may actually be valuable.

1. The method of treatment of a subject with a malignant tumor, characterized in that it comprises the following stages a) providing expression constructs containing the promoter, functional in eukaryotics cells, and polynucleotide encoding p53, where the specified polynucleotide is in the sense orientation to a specific promoter and is under control of the specified promoter, and b) the introduction of the indicated expression constructs in vivo in a tumor cell expressing endogenous molecule p53 wild-type.

3. The method according to p. 1, wherein the tumor cell is a glioma, sarcoma, carcinoma, lymphoma, or melanoma.

4. The method according to p. 3, wherein the tumor cell is a squamous cell carcinoma of the head and neck.

5. The method according to p. 1, characterized in that the specified expression design is a viral vector.

6. The method according to p. 5, characterized in that the viral vector is selected from the group consisting of a retroviral vector, adenoviral vector, and adeno-associated viral vector.

7. The method according to p. 6, characterized in that the viral vector is replication-deficient adenoviral vector.

8. The method according to p. 7, characterized in that the specified replication-deficient adenoviral vector is a vector that lacks at least in part of the E1 region.

9. The method according to p. 1, characterized in that the promoter ptx2">

11. The method according to p. 1, wherein stage (b) is repeated at least once.

12. The method according to p. 11, characterized in that the specified tumor is subjected to resection after re-introduction.

13. The method according to p. 12, characterized in that the expression vector comes into contact in the amount of from about 3 ml to about 10 ml

14. The method according to p. 11, characterized in that the adenovirus entered each time the contact is about 107- 1012PFU (plaque-forming units).

15. The method according to p. 1, characterized in that the introduction is an intratumoral injection.

16. The method according to p. 1, characterized in that the introduction is carried out in a natural or artificial body cavity.

17. The method according to p. 16, characterized in that the introduction includes a continuous perfusion of the location of the tumor.

18. The method according to p. 16, characterized in that the said introduction is carried out in an artificially created body cavity resulting from the excision of the tumor.

19. The method according to p. 1, characterized in that the p53 coding polynucleotide targets with the ability to detect expression of p53 decree continuous epitope antibodies.

21. The method of determining the effectiveness of therapy for microscopic residual cancer comprising performing a dissection in the subcutaneous tissue of rodents, characterized in that conduct seeding specified excision of tumor cells, treatment specified rodent in therapeutic mode and the assessment of the impact of the regime on the development of tumors.

22. The method according to p. 21, wherein the specified excision close after a specified contamination and to specified treatment of the rodent therapeutic regime.

23. The method according to p. 22, characterized in that therapeutic regimen includes the introduction of a therapeutic composition in the specified excision, which re-opened after closing and re-close after injection of the indicated therapeutic composition.

24. The method according to p. 23, characterized in that therapeutic composition includes an expression construct containing a promoter functional in eukaryotic cells, and polynucleotide encoding p53, where the specified polynucleotide is in the sense orientation to a specific promoter and under the control of a specified promoter.

25. The method according to p. 24, characterized those who motor CMV IE promoter.

26. The method according to p. 11, wherein the tumor is in contact with the specified expressional design at least three times.

27. The method according to p. 11, characterized in that the said multiple injections contain amounts of about 0.1 - 0.5 ml at a distance of approximately 1 cm from each other.

28. The method according to p. 1, characterized in that it further comprises contacting the specified tumor destructive DNA agent.

29. The method according to p. 28, characterized in that the specified destroying the DNA of the agent is the agent of radiation therapy.

30. The method according to p. 29, characterized in that the specified agent is radiation therapy selected from the group consisting of gamma-irradiation, x-ray irradiation, ultraviolet irradiation, and microwave irradiation.

31. The method according to p. 28, characterized in that the destructive DNA agent is a chemotherapeutic agent.

32. The method according to p. 31, characterized in that the said chemotherapeutic agent is chosen from the group consisting of adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, verapamil, doxorubicin, podofillotoksina and cisplatin.

33. The method according to p. 1, wherein otlichayushiesya fact, he further comprises contacting the specified tumor with a second therapeutic gene other than the gene encoding the p53 polypeptide.

35. The method according to p. 34, characterized in that the second therapeutic gene is selected from the group consisting of Dp gene, p21, p16, p27, E2F, Rb, APC, DC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, mys, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl, abl, Bax, Bcl-Xsand E1A.

36. The method according to p. 1, characterized in that said tumor is in the body cavity selected from the group consisting of oral cavity, pharynx, esophagus, larynx, trachea, pleural cavity, abdominal cavity, the inner part of the bladder and the cavity of the large intestine.

37. The method according to p. 11, wherein the tumor is in contact with the specified expression design at least six times during a two-week treatment regimen.

38. The method according to p. 1, characterized in that it further comprises a surgical detection of the indicated tumor cells before the introduction of the indicated expression constructs.

39. The method according to p. 17, characterized in that the specified location of the tumor represents a natural or artificial body cavity.

42. The method according to p. 1, characterized in that the expression vector is administered intravenously.

43. The method according to p. 1, characterized in that the expression vector is administered orally.

 

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The invention relates to medicine, in particular to biotechnology, and can be used in biological studies of a wide profile for diagnostics of the functional state of a biological object and optimum nutrition

The invention relates to biotechnology and can be used for the regulation of cell proliferation

The invention relates to biotechnology and related analogue of erythropoietin

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets

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The invention relates to the field of molecular biology and concerns a new factor in the growth/differentiation of ТGЕ-collection and coding of its DNA sequences

Therapeutic protein // 2155810
The invention relates to a new protein - human stem cell factor (FGC, SCP), DNA sequences coding for this protein, its use in therapy, in particular for in vitro fertilization, as well as to pharmaceutical compositions containing such protein

The invention relates to the field of medicine

The invention relates to medicine, in particular to method-specific "targeting" and transfer DNA repair in mammalian cells in vivo

The invention relates to the field of medical biotechnology

The invention relates to medicine, namely to somatic mutations in the gene multifunctional tumor suppressor (MTS) in the case of neoplasms of human rights and the use of these mutations for diagnosis and prognosis of cancer

The invention relates to a recombinant adenovirus expression vectors, characterized by partial or complete deletion of the DNA fragment of adenovirus encoding the protein IX, and containing the gene of a foreign protein, or a functional fragment or mutant form

The invention relates to biotechnology, immunology and medicine and can be used to direct cellular immune response to an infectious agent

The invention relates to biotechnology and can be used for the regulation of cell proliferation

The invention relates to biotechnology and can be used for selective destruction of cells infected with hepatitis C virus (HCV) or infectious RNA

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

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