CN1705740B - Adenovirus with reverse gene expression and its application - Google Patents

Abstract

The present invention relates to an adenovirus expressing a first protein prior to expressing a second protein, said first protein being selected from the group comprising the E1B protein and the E4 protein, said second protein being selected from the group comprising the E1A-protein.

Description

Adenovirus with reverse gene expression and its application

The present invention relates to adenoviruses, nucleic acids encoding them and their applications, especially in the preparation of medicaments for treating tumors. the

A number of therapeutic principles are currently utilized in the treatment of tumors. In addition to the use of surgery, chemotherapy and radiotherapy are prevalent. All of these techniques, however, are associated with considerable side effects. The application of replication-selective oncolytic viruses provides a new platform for the treatment of tumors. In turn, selective intratumoral replication of the viral agent is initiated, resulting in viral replication, lysis of infected tumor cells and spread of the virus to adjacent tumor cells. Because viral replication capacity is limited to tumor cells, normal tissues are protected from viral replication and thus viral lysis. the

Currently, several viral systems are in clinical trials aimed at tumor lysis. An example of this adenovirus is dl1520 (Onyx-015), which has been successfully used in Phase I and Phase II clinical trials (Khuri, F. et al. Nature Medicine 6, 879-885, 2000). Onyx-015 is an adenovirus with a complete deletion of the E1B-55kDa gene. The complete deletion of the E1B-55 kDa protein of adenoviruses is based on the finding that adenoviral vectors with deletion of p53 may lead to replication and thus lysis of cells (Kirn, D. et al., Proc. Am. Soc. Clin. Oncol. 17 , 391a, 1998), wherein normal cells are not harmed. More specifically, the E1B-55kDa gene product is involved in the repression of p53, the translocation of viral mRNA and the shutting down of host cell protein synthesis. Inhibition of p53 occurs through the formation of a complex between p53 and the E1B-55kDa protein encoded by the adenovirus and/or a complex between E1B-55kDa and E4orf6. p53, encoded by TP53, is the starting point of the regulatory machinery of the complex (Zambetti, G.P. et al., FASEB J. 7, 855-865, 1993), which is particularly potent in inhibiting cellular replication of viruses such as adenoviruses. The gene TP53 is deleted or mutated in approximately 50% of all human tumors, leading to a lack of desired apoptosis by chemotherapy or radiation therapy, resulting in often unsuccessful tumor treatments. the

Another rationale for lysing tumorlytic adenoviruses is based on the discovery that Rb/E2F and/or p107/E2F and/or p130 are not affected if the E1A protein is present in a specific deleted form or contains one or more mutations /E2F, the adenovirus will not induce S phase in infected cells and will be able to replicate in tumor cells that do not contain a functional Rb protein. Additionally, the E1A protein may be N-terminally deleted and contain one or more mutations in the amino acid region 1-76 of the E1A protein, respectively, in order to inhibit the binding of E1A to p300 and thus provide for selective replication in tumor cells. These methods are described by way of example in European patent EP 0 931 830. Examples of these viruses are AdΔ24, dl922-947, E1Ad/01/07 and CB016 (Howe, J.A. et al., Molecular Therapy 2, 485-495, 2000; Fueyo, J. et al., Oncogene 19, 2-12, 2000; Heise , C. et al., Nature Medicine 6, 11341139, 2001; Balague, C. et al., J.Virol.75, 7602-7611, 2001). These adenoviral systems known in the prior art for oncolysis therefore contain various deletions in the E1A protein, which have been made under the assumption that a functional Rb protein and a combination of an inactive Rb protein and The complex composed of E2F blocks efficient in vivo replication, respectively, so that the adenovirus replicates in vivo only in Rb-negative/mutant cells. These adenoviral systems according to the prior art are based on E1A in order to control replication in vivo by the early E2 promoter (E2 early promoter in English) and free E2F (Dyson, N. Genes & Development, 12, 2245-2262, 1998). the

Another version of the tumor-lytic adenoviral system is based on the use of a selective promoter specifically expressing the viral oncogene E1A, which provides selective replication in tumor cells (Rodriguez, R. et al., Cancer Res. 57, 2559-2563, 1997). the

As mentioned above, selection of the cellular context appropriate to the principle of each mode of action is important for the various principles of adenoviral oncolytic viruses. In other words, the various currently known adenoviral systems can only be used if the different molecular biological prerequisites are recognized. This limits the application of these systems to different patient populations. the

A particular problem in the treatment of oncological diseases arises once a patient develops so-called multidrug resistance (MDR in English), which represents a particularly well-studied tumor effect on cytostatics. ) resistance (Gottesman and Pastan, Annu. Rev. Biochem. 62, 385-427, 1993). It is based on overexpression of the membrane-bound transporter P-glycoprotein, which belongs to the so-called ABC transporters (Stein, U. et al., JBC 276, 28562-69, 2001, J. Wijnholds, Novartis Found Symp., 243, 69-79, 2002). Bargou, R.C. et al. and Oda, Y. et al. (Bargou, R.C. et al., Nature Medicine 3, 447-450, 1997; Clin. Cancer Res. 4, 2273-2277, 1998) were able to demonstrate that the nuclear localization of the human transcription factor YB-1 Directly involved in the activation of P-glycoprotein expression. Further studies confirmed that YB-1 was induced by various stress conditions such as UV irradiation, cytostatic administration (Koike, K. et al., FEBS Lett 17, 390-394, 1997) and overheating (Stein, U. et al., JBC 276, 28562-69, 2001) and transported into the nucleus. Further studies confirmed that the nuclear localization of YB-1 has an effect on another ABC transporter. The ABC transporter is called MRP (English is multidrug resistance-related protein, multidrug resistance-related protein), and participates in the formation of so-called atypical non-P-glycoprotein-dependent multidrug resistance (Stein, U. et al., JBC 276, 28562-69, 2001). the

The problem solved by the present invention is that of providing a technical teaching and in particular a method which allows in particular the treatment of an organism, more particularly a human organism and a group of patients, respectively, with a tumor-lytic agent. Another problem addressed by the present invention is to provide methods suitable for causing tumor lysis in patients suffering from cytostatically resistant neoplastic diseases, especially those with multidrug resistance. Finally, a problem solved by the present invention is to provide an adenovirus suitable for cell lysis. the

The first aspect of the problem to be solved by the present invention is solved by an adenovirus capable of expressing a first protein selected from the group comprising E1B protein and E4 protein before expressing a second protein selected from the group comprising E1A-protein. the

In one embodiment, the first protein is an E1B protein, preferably an E1B55kd protein. the

In another embodiment, the first protein is an E4 protein, preferably an E4orf6 protein. the

In a preferred embodiment, the first protein is a combination of E1B protein and E4 protein, preferably a combination of E1B55kD protein and E4orf6 protein. the

In a preferred embodiment, the E1A protein is an E1A12S protein. the

The second aspect of the problem to be solved by the present invention is solved by adenovirus, wherein said adenovirus comprises at least one nucleic acid encoding a protein selected from the group consisting of E1B protein, E4 protein and E1A protein, wherein at least one protein It is under the control of a promoter different from that which controls protein expression in wild-type adenovirus. the

In one embodiment of the second aspect, the adenovirus is an adenovirus according to the first aspect of the invention. the

In one embodiment of the second aspect, at least one protein is an E1B protein, preferably an E1B55kD protein. the

In one embodiment of the second aspect, at least one protein is an E4 protein, preferably an E4orf6 protein. the

In one embodiment of the second aspect, at least one protein is an E1A protein, preferably an E1A12S protein. the

In a preferred embodiment of the second aspect, at least one protein is a combination of E1B protein and E4 protein, preferably a combination of E1B55kD protein and E4orf6 protein. the

In one embodiment of the second aspect, at least one protein is a combination of an E1B protein and an E1A protein, preferably a combination of an E1B55kD protein and an E1A12S protein. the

In a preferred embodiment of the second aspect, at least one protein is a combination of E4 protein and E1A protein, preferably a combination of E4orf6 protein and E1A12S protein. the

In one embodiment of the second aspect, at least one protein is a combination of E1B protein, E4 protein and E1A protein, preferably a combination of E1B55kD protein, E4orf6 protein and E1A12S protein. the

In one embodiment of the second aspect, the expression of the E1B protein is controlled by a promoter, wherein the promoter is selected from the group consisting of tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterogeneous A set of a source promoter and an adenoviral promoter, wherein the adenoviral promoter is different from the E1B promoter. the

In one embodiment of the second aspect, the expression of the E4 protein is controlled by a promoter, wherein the promoter is selected from the group consisting of tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterogeneous A set of a source promoter and an adenoviral promoter, wherein said adenoviral promoter is different from the E4 promoter. the

In a preferred embodiment of the second aspect, the promoter of the adenovirus is the E1A promoter. the

In one embodiment of the second aspect, the expression of the E1A protein is controlled by a promoter, wherein said promoter is selected from the group comprising: tumor-specific promoters, organ-specific promoters, tissue-specific promoters Promoters, heterologous promoters, and adenoviral promoters, wherein the adenoviral promoter is different from the E1A promoter. the

In a preferred embodiment of the second aspect, the promoter controlling the expression of the E1A protein is controlled by YB-1, or may be regulated by YB-1. the

In a preferred embodiment of the second aspect, the promoter controlling the expression of the E1A protein is the adenovirus 2 late promoter. the

In one embodiment of the first and second aspects, the E4 protein, preferably the E4orf6 protein and the E1B protein, preferably the E1B55kd protein are under the control of the same or a common promoter. the

The third aspect of the problem solved by the present invention is solved by adenovirus, wherein said adenovirus provides YB-1 in the nucleus through at least one adenovirus protein, or YB-1 supply in the nucleus is provided by at least one mediated by an adenoviral protein, wherein said adenoviral protein is preferably different from E1A. the

In one embodiment of the third aspect, the adenovirus is an adenovirus according to the first and/or second aspect of the invention. the

The fourth aspect of the problem solved by the present invention is solved by adenovirus, wherein said adenovirus provides YB-1 for the replication of adenovirus through at least one adenovirus protein, or mediated by at least one adenovirus protein The provision of YB-1 for the replication of adenovirus, wherein said adenovirus protein is preferably different from E1A. the

In one embodiment of the fourth aspect, the adenovirus is an adenovirus according to the first and/or second and/or third aspect of the invention. the

In one embodiment of the third and fourth aspects, the adenoviral protein is a complex of E4orf6 and E1B55kd. the

The fifth aspect of the problem solved by the present invention is solved by adenovirus, wherein the nucleic acid of the adenovirus contains at least one functionally inactive adenovirus region, wherein the region is selected from the group consisting of E1 region, E3 region, Groups of E4 regions and combinations thereof. the

In one embodiment of the fifth aspect, said adenovirus is an adenovirus according to the first and/or second and/or third and/or fourth aspects of the invention. the

In one embodiment of the fifth aspect, the region is the El region. the

In one embodiment of the fifth aspect, the region is the E3 region. the

In one embodiment of the fifth aspect, the region is the E4 region. the

In one embodiment of the fifth aspect, the region comprises an El region, an E3 region and an E4 region. the

The sixth aspect of the problem to be solved by the present invention is solved by an adenovirus, wherein said adenovirus comprises at least one expression cassette, wherein said expression cassette comprises at least one promoter and a nucleic acid encoding an adenoviral protein, wherein said The adenovirus protein is E1B protein, preferably E1B55kD protein. the

In one embodiment of the sixth aspect, the adenovirus is an adenovirus according to the first and/or second and/or third and/or fourth and/or fifth aspects of the invention. the

In one embodiment of the sixth aspect, the promoter is different from the E1B promoter. the

In one embodiment of the sixth aspect, the promoter is selected from the group comprising tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenoviral promoters, The promoter described therein is different from the E1B promoter. the

The seventh aspect of the problem to be solved by the present invention is solved by an adenovirus, wherein said adenovirus comprises at least one expression cassette, wherein said expression cassette comprises at least one promoter and a nucleic acid encoding an adenoviral protein, wherein said The adenovirus protein is E4 protein, preferably E4orf6 protein. the

In one embodiment of the seventh aspect, said adenovirus is the first and/or second and/or third and/or fourth and/or fifth and/or Sixth aspect of adenovirus. the

In one embodiment of the seventh aspect, the promoter is different from the E4 promoter. the

In one embodiment of the seventh aspect, the promoter is selected from the group comprising tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenoviral promoters, wherein The adenovirus promoter is different from the E4 promoter. the

In one embodiment of the seventh aspect, the promoter is an E1A promoter. the

The eighth aspect of the problem to be solved by the present invention is solved by an adenovirus, wherein said adenovirus comprises at least one expression cassette, wherein said expression cassette comprises at least one promoter and a nucleic acid encoding an adenoviral protein, wherein said The adenovirus protein is E1A protein, preferably E1A12S protein. the

In one embodiment of the eighth aspect, the adenovirus is according to the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh aspects of the present invention of adenovirus. the

In one embodiment of the eighth aspect, the promoter is different from the E1A promoter. the

In one embodiment of the eighth aspect, the promoter is selected from the group comprising tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenoviral promoters. the

In one embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus comprises nucleic acid , wherein said nucleic acid encodes YB-1. the

In a preferred embodiment of the eighth aspect, the nucleic acid encoding YB-1 is under the control of a promoter, wherein said promoter is preferably an E2 late promoter. the

In one embodiment of the eighth aspect, the nucleic acid encoding YB-1 is under the control of a promoter, wherein said promoters are YB-1 dependent and YB-1 controlled, respectively. the

In one embodiment of the eighth aspect, the nucleic acid encoding YB-1 is part of an expression cassette comprising a nucleic acid encoding an E1A protein, preferably a nucleic acid encoding an E1A12S protein. the

In one embodiment of the eighth aspect, the nucleic acid encoding the E1A protein is separated from the nucleic acid encoding YB-1 by an IRES sequence. the

In one embodiment of the sixth and/or seventh and/or eighth aspects, the nucleic acid encoding an E4 protein, preferably an E4orf6 protein, and the nucleic acid encoding an E1B protein, preferably an E1B55kD protein, are contained in an expression cassette, wherein two encoding The sequences are preferably separated by IRES sequences. the

In a preferred embodiment of the eighth aspect, the promoter of the expression cassette is selected from the group consisting of tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenovirus promoters The group wherein the adenovirus promoter is different from the E4 promoter and also different from the E1B promoter, preferably different from the wild type E4 promoter and also different from the wild type E1B promoter. the

In an embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus comprises a The expression cassette comprises a promoter and a nucleic acid sequence, wherein the nucleic acid sequence is selected from the group comprising aptamer, ribozyme, aptazyme, antisense molecule and siRNA. the

In an embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus comprises a An expression cassette comprising a promoter and a nucleic acid sequence, wherein said nucleic acid sequence is an encoding nucleic acid, wherein said nucleic acid encodes a molecule selected from the group consisting of peptides, polypeptides, proteins, anti-enzymes, antibodies and antibody fragments . the

In an embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus comprises a Expression cassette, wherein said expression cassette comprises a promoter and a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of apoptosis-inducing gene, prodrug gene, protease inhibitor, tumor suppressor gene, cytokine and blood vessel Group to generate inhibitors. the

In one embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus is a recombinant Adenovirus. the

In an embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus is an adenovirus Virus mutant. the

In an embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus is replicating defective type. the

In one embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus is capable of Replicates in cells that contain deregulated YB-1 or contain YB-1 in the nucleus. the

In one embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the cell is independently The nucleus of the cell cycle contains YB-1. the

In one embodiment of the first and/or second and/or third and/or fourth and/or fifth and/or sixth and/or seventh and/or eighth aspects, the adenovirus does not comprise Any E1A13S protein and/or the adenovirus does not contain any nucleic acid encoding an E1A13S protein. the

The ninth aspect of the problems to be solved by the present invention is solved by a nucleic acid encoding the adenovirus of any one of the first to eighth aspects. the

The tenth aspect of the problem to be solved by the present invention is solved by a replication system comprising the nucleic acid according to the ninth aspect and the nucleic acid of the helper virus, wherein the nucleic acid of the helper virus comprises one or more The adenovirus expression cassette according to any one of aspects 1 to 8. the

In one embodiment of the tenth aspect, the adenovirus or nucleic acid encoding it lacks the expression cassette comprised in the helper virus. the

The eleventh aspect of the problems to be solved by the present invention is solved by a vector comprising the nucleic acid according to the ninth aspect and/or the replication system according to the tenth aspect. the

In one embodiment of the eleventh aspect, the vector is an expression vector. the

The twelfth aspect of the problem to be solved by the present invention is solved by an adenovirus cell comprising the adenovirus according to any one of the first to eighth aspects and/or the nucleic acid according to the ninth aspect and/or Or the replication system according to the tenth aspect and/or the carrier according to the eleventh aspect. the

In one embodiment of the twelfth aspect, the cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell. the

In a preferred embodiment of the twelfth aspect, the mammalian cell is a cell selected from the group comprising cells of mouse, rat, guinea pig, pig, sheep, goat, cow, horse, dog, cat and human. the

A thirteenth aspect of the problems to be solved by the present invention is solved by an organism, preferably a mammalian organism, comprising an adenovirus according to the first to eighth aspects, a nucleic acid according to the ninth aspect, a nucleic acid according to the tenth aspect The replication system according to the eleventh aspect, the vector according to the eleventh aspect or the cell according to the twelfth aspect, wherein said organism is preferably selected from the group consisting of mice, rats, guinea pigs, pigs, sheep, goats, cows, horses, Group of dogs and cats. the

The fourteenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the Use of the vector of the eleventh aspect or the cell according to the twelfth aspect for replicating an adenovirus, preferably in vitro. the

The fifteenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the Use of the vector of the eleventh aspect or the cell according to the twelfth aspect for the production of adenovirus, preferably in vitro. the

The sixteenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the Use of the vector of the eleventh aspect or the cell according to the twelfth aspect for expressing a gene, preferably said gene promotes cell lysis, preferably during adenovirus replication, and/or promotes adenovirus-mediated cell lysis crack. the

In one embodiment of the sixteenth aspect, the expressed gene is a transgene disclosed herein. the

The seventeenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the Use of the carrier according to the eleventh aspect or the cell according to the twelfth aspect in the preparation of a medicament. the

In one embodiment of the fourteenth to seventeenth aspects, the cell in which the adenovirus replicates contains YB-1 in its nucleus, preferably YB-1 in its cell cycle independent nucleus. the

In one embodiment of the fourteenth to seventeenth aspects, the cell in which the adenovirus replicates comprises deregulated YB-1. the

In one embodiment of the use of the seventeenth aspect, the medicament is for the treatment of neoplastic diseases. the

In a preferred embodiment of the use of the seventeenth aspect, the neoplastic disease is selected from the group comprising malignant disease, cancer, cancerous venereal disease and tumor. the

In one embodiment of the use of the seventeenth aspect, the neoplastic disease is selected from the group comprising solid tumors, non-solid tumors, malignant tumors and benign tumors. the

In one embodiment of the use of the seventeenth aspect, at least a part of the tumor-forming cells has YB-1 in the nucleus, preferably YB-1 in the nucleus independent of the cell cycle. the

In one embodiment of the use of the seventeenth aspect, at least a portion of the tumor-forming cells comprise deregulated YB-1. the

In one embodiment of the use of the seventeenth aspect, at least a portion of the tumor-forming cells are Rb positive or Rb negative. the

In one embodiment of the use of the seventeenth aspect, at least a portion of the tumor-forming cells are resistant, preferably resistant, to a pharmaceutically active agent. the

In one embodiment of the use of the seventeenth aspect, the resistance is multiple resistance. the

In one embodiment of the use of the seventeenth aspect, the resistance is to an antineoplastic agent, preferably a cytostatic agent, and/or the resistance is caused by irradiation. the

In one embodiment of the use of the seventeenth aspect, the target patient of the medicament comprises a plurality of cells, wherein said cells are the cells described in each embodiment of the use according to the seventeenth aspect of the present invention. the

In one embodiment of the use of the seventeenth aspect, the medicament comprises at least one other pharmaceutically active agent. the

In one embodiment of the use of the seventeenth aspect, the medicament is or should be administered together with an other pharmaceutically active agent. the

In one embodiment of the use of the seventeenth aspect, the other pharmaceutically active agent is selected from the group consisting of cytokines, metalloproteinase inhibitors, angiogenesis inhibitors, cytostatics, tyrosine kinase inhibitors and cell cycle inhibitors Group. the

In one embodiment of the use of the seventeenth aspect, the medicament is administered before, during or after irradiation. the

In one embodiment of the use of the seventeenth aspect, the radiation is administered for the purpose of treating a tumor. the

In one embodiment of the use of the seventeenth aspect, a measure is taken on the cell or organism to be treated, wherein said measure is selected from the group comprising irradiation, administration of cytostatic agents and hyperthermia. the

In one embodiment of the use of the seventeenth aspect, the measure is a local or systemic application. the

In one embodiment of the use of the seventeenth aspect, the irradiation uses high energy irradiation, preferably any irradiation used in the treatment of neoplastic diseases. the

The eighteenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the The application of the carrier according to the eleventh aspect or the cell according to the twelfth aspect in the preparation of a drug for treating tumor diseases, characterized in that the tumor diseases are selected from the group consisting of breast tumors, bone tumors, gastric tumors, intestinal tumors, and gallbladder tumors. , tumors of the pancreas, tumors of the liver, tumors of the kidney, brain tumors, ovarian tumors, skin tumors, tumors of the skin appendages, head and neck cancers, uterine tumors, joint tumors, laryngeal tumors, esophageal tumors, tongue tumors, prostate tumors, Preferably one of the aforementioned neoplastic diseases has the features of any of the preceding claims. the

The nineteenth aspect of the problems to be solved by the present invention is solved by the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replication system according to the tenth aspect, the The use of the carrier according to the eleventh aspect or the cell according to the twelfth aspect in the preparation of a drug for treating tumor diseases, wherein the tumor-specific promoter is a tumor-specific promoter to which the drug is to be administered. the

The twentieth aspect of the problems to be solved by the present invention is solved by a pharmaceutical composition comprising the adenovirus according to any one of the first to eighth aspects, the nucleic acid according to the ninth aspect, the replicator according to the tenth aspect A system, a vector according to the eleventh aspect or a cell according to the twelfth aspect, and optionally a pharmaceutically acceptable carrier. the

In the twenty-first aspect, the problems of the present invention are solved by using a virus, preferably an adenovirus, in the preparation of a medicament, wherein the virus is in a normal cell that does not contain YB-1 in the nucleus, in an independent Replication-deficient in cells that do not contain YB-1 in the nucleus of the cell cycle, and in cells that do not contain deregulated YB-1, and the virus encodes an oncogene or oncogene product, particularly in YB-1 1 An oncogene protein that transactivates at least one viral gene, preferably an adenoviral gene, in a nucleus-positive cell, wherein said gene is selected from the group comprising E1B55kDa, E4orf6, E4orf3 and E3ADP. Preferably, the virus expresses the viral proteins E1B55kD, also denoted herein as E1B55kDa and E4orf6. the

In a twenty-second aspect, the problems of the present invention are solved by the use of a virus, preferably an adenovirus, for replication in a cell comprising YB-1 in the nucleus, wherein the virus does not Replication-deficient in cells comprising YB-1, or in cells that do not contain YB-1 in the cell cycle-independent nucleus, or in cells that do not contain any deregulated YB-1, wherein the virus encodes An oncogene or an oncogene product, in particular an oncogene protein that transactivates at least one viral gene, preferably an adenoviral gene, wherein said gene is selected from the group comprising E1B55kDa, E4orf6, E4orf3 and E3ADP. the

In one embodiment of use according to the twenty-first and twenty-second aspects of the invention, the virus, in particular an adenovirus, replicates in a cell that contains YB-1 in the nucleus or in a cell cycle-independent The nuclei of cells did not contain YB-1, or did not include any deregulated YB-1. the

In another embodiment applied according to the twenty-first and twenty-second aspects of the present invention, the viral oncogene protein is E1A and/or the oncogene is a gene encoding E1A and/or the oncogene protein is E1A. the

In a preferred embodiment, the viral oncogene protein E1A is capable of binding a functional Rb tumor suppressor gene product. the

In an alternative embodiment, the viral oncogene protein E1A is unable to bind a functional Rb tumor suppressor gene product. the

In another embodiment applied in accordance with the twenty-first and twenty-second aspects of the present invention, the viral oncogene protein E1A does not induce the nuclear localization of YB-1. the

In another embodiment for use in accordance with the twenty-first and twenty-second aspects of the invention, the medicament is administered to a patient whose cells are Rb positive or Rb negative. the

In a preferred embodiment, said cells are those cells involved in the development of the condition affected by said drug. the

In another embodiment for use according to the twenty-first and twenty-second aspects of the invention, said cells are Rb-negative or YB-1-positive in the nucleus, especially in the nucleus independent of the cell cycle Is YB-1 positive. the

In another embodiment of use according to the twenty-first and twenty-second aspects of the invention, the medicament is used to treat tumors. the

In another embodiment of use according to the twenty-first and twenty-second aspects of the present invention, said cells, in particular cells forming tumors or parts thereof, are resistant, in particular to drugs with multiple Resistance, the drug is preferably an antineoplastic agent, more preferably a cytostatic agent. the

In a preferred embodiment for use according to the twenty-first and twenty-second aspects of the invention, the cell expresses, preferably overexpresses, the membrane bound transport proteins P-glycoprotein and/or MRP. the

In another embodiment for use in accordance with the twenty-first and twenty-second aspects of the invention, the cells are p53 positive or p53 negative. the

In one embodiment of use according to the twenty-first and twenty-second aspects of the invention, the oncogene protein comprises one or several mutations or deletions compared to the wild-type oncogene protein E1A, wherein said deletion is preferably are those selected from the group comprising deletions of the CR3 region and N-terminal deletions and C-terminal deletions. It is intended that the E1A oncogene protein can bind Rb. the

In another embodiment applied according to the twenty-first and twenty-second aspects of the invention, the oncogene protein comprises one or more mutations or deletions compared to the wild-type oncogene protein, wherein said deletion is The preferred one in the CR1 region and/or the CR2 region. It is intended that the oncogene protein E1A cannot bind Rb. the

In one embodiment applied according to the twenty-first and twenty-second aspects of the invention, the viral oncogene protein, in particular ElA, is under the control of a tissue-specific and/or tumor-specific promoter. the

In another embodiment applied according to the twenty-first and twenty-second aspects of the invention, the virus, in particular the adenovirus, encodes YB-1. the

In another embodiment for use in accordance with the twenty-first and twenty-second aspects of the invention, YB-1 is under the control of a tissue-specific and/or tumor-specific promoter. the

In a preferred embodiment of the application according to the twenty-first and twenty-second aspects of the present invention, the virus, particularly the adenovirus, encodes a protein selected from the group consisting of E4orf6, E4orf3, E1B55k and the E3ADP protein of adenovirus at least one protein. the

In an alternative embodiment for use in accordance with the twenty-first and twenty-second aspects of the invention, the cells comprise YB-1 in the nucleus, in particular cells forming a tumor or part thereof comprise YB-1 in the nucleus . the

In another embodiment for use in accordance with the twenty-first and twenty-second aspects of the invention, the tumor comprises YB-1 in the nucleus after inducing translocation of YB-1 into the nucleus. the

In a preferred embodiment for use in accordance with the twenty-first and twenty-second aspects of the present invention, the translocation of YB-1 to the nucleus is caused by at least one method selected from the group consisting of radiation, administration of cells Group of inhibitors and hyperthermia. the

In a particularly preferred embodiment of use according to the twenty-first and twenty-second aspects of the invention, the method is applied to a cell, organ or organism. the

In a preferred embodiment for use according to the twenty-first and twenty-second aspects of the invention, the virus, in particular an adenovirus, is selected from the group comprising: AdΔ24, d1922-947, E1Ad/01/07, dl1119/1131, CB 016, dl520, and viruses lacking an expressed viral E1A oncogene that binds a functional Rb tumor suppressor gene product. the

In a twenty-third aspect, the problem is solved by the use of a virus, preferably an adenovirus, for the preparation of a medicament, wherein said virus, in particular an adenovirus, is adapted such that replication is via or mediated by YB-1 Activation of the E2-late promoter is controlled, preferably primarily by activation of the E2-late promoter. In one embodiment, YB-1 is transgenic YB-1 or cellular YB-1, in particular cellular deregulated YB-1 or deregulated YB-1. The transgenic YB-1 is preferably YB-1 expressed in cells by a vector, especially by one or said adenovirus. The E2-late promoter is preferably an adenovirus E2-late promoter as contained in a wild-type adenovirus or as used in connection with transgene expression as described herein. the

In a twenty-fourth aspect, the problem is solved by the use of viruses, especially adenoviruses, for replication in cells comprising YB-1 in the nucleus, wherein said viruses, especially adenoviruses are suitable Replication is allowed to be controlled by YB-1 through activation of the E2 late promoter, preferably primarily through activation of the E2 late promoter. In one embodiment, YB-1 is transgenic YB-1 or cellular YB-1, in particular cellular deregulated or deregulated YB-1. The transgenic YB-1 is preferably YB-1 expressed in cells by a vector, in particular one or said adenovirus. The E2-late promoter is preferably the adenovirus E2-late promoter as present in wild-type adenovirus, or the E2-late promoter as used in connection with the expression of the transgenes described herein. the

In a preferred embodiment of the twenty-third and/or twenty-fourth aspect of the invention, said adenovirus as disclosed herein is suitable, particularly suitable, such that it can be used according to the invention. the

In a twenty-fifth aspect, the problem is solved by a viral oncoprotein, in particular an isolated viral oncoprotein, wherein said viral oncoprotein is characterized by:

a) transactivation of at least one viral gene selected from the group comprising: E1B55k, E3ADP and E4orf6 and E4orf4 in YB-1 nucleus-positive cells;

and

b) In the nucleus. In particular, there was no induction of YB-1 in the nuclei of cells where the viral oncogene protein was present. the

The solution is the use of the nucleic acid of an adenovirus for the preparation of a medicament, wherein said virus is suitable such that replication is controlled by YB-1 via activation of the E2-late promoter, preferably mainly via the activation of the E2-late promoter . In one embodiment, YB-1 is transgenic YB-1 or a cell, in particular a deregulated cell, or a deregulated YB-1. The transgenic YB-1 is preferably YB-1 expressed in cells by a vector, especially by one or said adenovirus. The E2-late promoter is preferably an adenoviral E2-late promoter such as that contained in wild-type adenovirus, or an E2-late promoter as used in connection with transgene expression as described herein. the

In a thirtieth aspect, the problem is solved by the use of a nucleic acid encoding a virus as used according to the invention, in particular an adenovirus, for replication in a cell, wherein said virus is adapted such that replication is initiated via E2-late Activation of promoters, preferably mainly through activation of E2-late promoters, is controlled by YB-1. In one embodiment, YB-1 is transgenic YB-1 or cellular, especially cellular, deregulated YB-1. The transgenic YB-1 is preferably YB-1 expressed in cells by a vector, preferably by one or said adenovirus. The E2-late promoter is preferably an adenovirus E2-late promoter as present in wild-type adenovirus, or an E2-late promoter as used in connection with transgene expression as described herein. the

In a thirty-first aspect, the problem is solved by using a vector comprising one of the aforementioned nucleic acids according to the twenty-first or twenty-second aspect of the invention. the

In a thirty-second aspect, the present invention relates to the use of agents that interact with YB-1 for characterizing cells, cells of tumor tissue or patients, in order to determine whether they will be used as in accordance with the present invention exposure to and/or handling of viruses, especially adenoviruses. the

In one embodiment, the reagent is selected from the group comprising antibodies, anticalines, aptamers, aptazymes and spiegelmers. the

In a thirty-second aspect, the problem is in the preparation of viruses, in particular The application of adenovirus was solved. the

In one embodiment, the virus includes a nucleic acid encoding a transgene. the

In another embodiment, the virus includes translation products and/or transcripts of transgenes. the

In a preferred embodiment, the nucleic acid of the adenoviral replication system and/or the nucleic acid of the helper virus comprises a transgene or a nucleic acid encoding a transgene. the

In another embodiment, the nucleic acid comprises a transgene or a nucleic acid encoding a transgene. the

In an alternative embodiment, the transgene is selected from the group comprising prodrugs, cytokines, apoptosis-inducing genes, tumor suppressor genes, metalloproteinase inhibitor genes and angiogenesis inhibitor genes and tyrosine kinases Inhibitor gene. the

In one embodiment, the transgene is selected from the group of nucleic acids comprising siRNAs, aptamers, antisense molecules and ribozymes, wherein said siRNAs, aptamers, antisense molecules and/or ribozymes are directed against a target molecule. the

In another embodiment, the target molecule is selected from the group comprising resistance-associated factors, anti-apoptotic factors, oncogenes, angiogenic factors, DNA synthetases, DNA repair enzymes, growth factors and their receptors, Transcription factors, metalloproteinases, especially matrix metalloproteinases, and plasminogen activators of the urokinase type. In one embodiment, the resistance-associated factor is preferably selected from the group comprising P-glycoprotein, MRP and GST, and also comprises nucleic acids encoding them. In one embodiment, the anti-apoptotic factors are selected from the group comprising BCL2, and also nucleic acids encoding them. In one embodiment, the oncogene is selected from the group comprising Ras, in particular mutated Ras, Rb and MYC, and also nucleic acids encoding them. In one embodiment, angiogenic factors are selected from the group comprising VEGF and HMG proteins, and also nucleic acids encoding them. In one embodiment, the DNA synthetases are selected from the group comprising telomerases, and also nucleic acids encoding them. In one embodiment, the DNA repair enzymes are selected from the group comprising Ku-80, and also nucleic acids encoding them. In one embodiment, the growth factor is selected from the group comprising PDGF, EGF and M-CSF, and also comprises nucleic acids encoding them. In another embodiment, the receptor is preferably a receptor for a growth factor, wherein preferably the growth factor is selected from the group comprising PDGF, EGF and M-CSF, and also includes nucleic acids encoding them. In one embodiment, the transcription factors are selected from the group comprising YB-1, and also nucleic acids encoding them. In one embodiment, the metalloprotease is in particular a matrix metalloprotease. In a preferred embodiment, the matrix metalloprotease is selected from the group comprising MMP-1 and MMP-2, and also comprises nucleic acids encoding them. In one embodiment, plasminogen activators of the urokinase type are selected from the group comprising uPa-R, and also nucleic acids encoding them. the

In another embodiment, the medicament further comprises at least one pharmaceutically active compound. the

In a preferred embodiment of any aspect of the present invention, the pharmaceutically active compound is selected from the group comprising: cytokines, metalloproteinase inhibitors, angiogenesis inhibitors, cytostatic and cell cycle inhibitors and tyrosine kinase inhibitors agent. the

The above-disclosed adenoviruses according to the present invention, especially those described in relation to the first to eighth aspects of the present invention, are also referred to herein as group I adenoviruses; and have transactivated oncogene proteins (such as , E1A and/or here and in particular those mentioned above to be used according to the invention) are also referred to herein as group II adenoviruses. Group I adenoviruses and group II adenoviruses are also collectively referred to herein as adenoviruses or adenoviruses according to the invention or viruses according to the invention. the

The present invention is based on the unexpected discovery that inversion of the expressed sequence of an adenovirus gene results in efficient replication and optionally lysis of adenovirus-infected cells. With regard to the temporally variable expression of the adenoviral genes, the emphasis is on the E1B and E4 proteins, which are also referred to here individually or collectively as the first protein, which are expressed before the second protein. The second protein is selected from E1A proteins. This order of expression is reversed compared to wild-type adenovirus, in which the E1A protein is expressed first, followed by the E1B and E4 proteins, to ensure the activation of transcription factors such as the in the nucleus and influence or control further replicative activity. The dynamics of adenoviral transcripts in wild-type adenoviruses is described, for example, by Glenn G.M. and Ricciardi R.P. Virus Research 1988, 9, 73-91, which reported that, in wild-type, there is usually a difference between transcripts and translation products (respectively E1A transcripts (ie, E1A12S transcripts and E1A13S transcripts) could be detected before E4orf6 and E1B55k). Herein, unless stated to the contrary, the E1B protein is generally preferably the E1B-55kD protein herein. Herein, if there is no indication to the contrary, the E4 protein is generally preferably the E4orf6 protein. Herein, if not stated to the contrary, the E1A protein is generally preferably the E1A12S protein, or the E1A protein described herein in relation to an E1A-modified adenovirus. the

The present invention includes that in principle the E1A protein (and more specifically also the E1A12S protein) may be substituted. If not stated to the contrary, such substituted E1A protein and E1A12S protein are also referred to herein as E1A protein and E1A12S protein, respectively, or are deemed to be encompassed by that term. Not only E1A12S protein, but also E1A protein with tumor suppressor function, such as Dickopp A, Esche H, Swart G, Seeber S, Kirch HC, Opalka B. CancerGene Ther.2000, Jul;7(7):1043-50 recorded in. Other derivatives of the E1A protein (in particular the E1A12S protein), as used and/or referred to herein, are also proteins capable of releasing the factor E2F from the Rb/E2F complex. They are especially simian virus 40 tumor antigen (SV40 large T antigen), papillomavirus E7 protein (HPV E7) as described in Chellappan S. et al., Proc. . the

The present invention also includes that derivatives of E4orf6 and E1B55k may be used, as used herein, wherein the terms E4orf6 and E1B55k include such derivatives. Derivatives are described, for example, in Shen Y et al., J. of Virology 2001, 75, 4297-4307; Querido E. et al., J. of Virology 2001, 75, 699-709. the

The present invention includes that the E1B protein is expressed before the E1A protein, or the E4 protein is expressed before the E1A protein, or both the E1B protein and the E4 protein are expressed before the E1A protein, each as described above. the

Adenoviruses designed in this manner are capable of replicating at exceptionally high levels after infecting cells expressing YB-1 in the nucleus, preferably in the nucleus independent of the cell cycle, or which contain Regulated YB-1, preferably in the cytoplasm. Without wishing to be limited thereto, the inventors of the present invention hypothesize below that either a complex of E1B protein and/or E4 protein and either protein alone can transport deregulated YB-1 into the nucleus, or Replication of the adenovirus can be initiated there under the influence of the E1B protein and/or the E4 protein expressed prior to the E1A protein. Once in the nucleus or present there in an activated form, YB-1 replicates efficiently, as described here, more specifically using the E2-late promoter. Thus, temporally earlier expression of E1B protein and/or E4 protein avoids the cascade observed in wild type with initial expression of E1A protein. In a preferred embodiment, the E1A protein is an E1A protein which in particular no longer transactivates or which is only able to transactivate the E1B protein and/or the E4 protein to a very limited extent. Preferably, the transactivation is neither sufficient to ensure efficient replication nor sufficient to ensure replication in cells whose nuclei do not contain YB-1. Preferably, transactivation does not occur in cells that do not contain YB-1 in the cell cycle independent nucleus, or do not have deregulated YB-1. the

Furthermore, the present invention is based on the unexpected discovery that an adenovirus can replicate in a particularly efficient manner if it comprises at least one nucleic acid encoding a protein selected from the group consisting of E1B, E4 and E1A proteins The group wherein at least one protein is under the control of a promoter different from the promoter controlling the expression of each protein in wild-type adenovirus. Such replication is particularly efficient, and often results in tumor lysis, if the cell contains YB-1 in the nucleus, especially in a cell cycle-independent nucleus, or if the cell contains deregulated YB-1 1, specifically containing deregulated YB-1 in the cytoplasm. What has been said above with respect to the E1B protein, the E4 protein and the E1A protein also applies here. In wild-type adenovirus, the E1B protein is controlled by the E1B promoter, the E4 protein is controlled by the E4 promoter, and the E1A protein is controlled by the E1A promoter. By selecting a different promoter than that controlling the expression of the aforementioned proteins in wild-type adenoviruses, the expression of the aforementioned proteins and the regulatory interactions between the respective adenoviral nucleic acids and proteins are altered. Through the choice of promoters, temporally distinct expression profiles can be established, hopefully without limitation below, leading to the observed replication in cells, the mechanisms of which may be those already described above for the adenoviral proteins E1B, E4 and E1A temporally Different expressions are described. Examples of specific designs in which the proteins are controlled by promoters (different from those controlling the expression of the individual proteins in wild-type adenoviruses) can be obtained from the claims and Examples section below, where more specifically, The viruses XVirPSJL1 and XVirPSJL2 are representative of them. Preferably, the E1B protein is an E1B55kD protein, the E4 protein is an E4orf6 protein, and the E1A protein is an E1A12S protein. the

Preferably, the promoters controlling the E1B protein and the E4 protein are selected from the group comprising tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenovirus promoters, provided that , when adenovirus promoters are used, they are different from the E1B promoter, which controls the expression of the E1B protein, and different from the E4 promoter, which controls the expression of the E4 protein. It is particularly preferred that the E1A promoter is used to control the expression of the E1B protein and/or the E4 protein. The E1A promoter is described, for example, in Boulanger P.A. and Blair, G.E. Biochem. J. 1991, 275, 281-299. In addition, each and any other heterologous promoters, ie promoters different from those controlling the expression of the respective proteins in the wild-type adenovirus, may also be used. A representative example is the CMV promoter, and others will be apparent to those skilled in the art. the

The promoter used to control the E1A protein can also be selected from the group comprising tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and adenovirus promoters, provided that the adenovirus Viral promoters are different from E1A promoters. The present invention includes that one or more of the aforementioned proteins (i.e. E1B protein, E4 protein or E1A protein) are under the control of the same promoter, however, it is preferred, in particular, that the E1B protein and the E4 protein are under the control of the same promoter. under the control of the promoter. Particularly preferably, the expression of the E1A protein is controlled by a YB-1-controlled promoter or a promoter which can be regulated by YB-1. Such promoters are disclosed herein in relation to other aspects of the invention. It is particularly preferred to use the adenoviral 2-late promoter to control the expression of the E1A promoter, since firstly it can be regulated by YB-1 and secondly also exhibits only slight, practically negligible transcription in the absence of YB-1 , thus ensuring very good control of the expression of nucleic acids controlled by the E2-late promoter. This significantly increases biosafety, especially when used in the field of medicine. the

Furthermore, the inventors of the present invention have found that adenoviruses replicate particularly well in cells containing YB-1 in the nucleus, more specifically in the nucleus independent of the cell cycle, and / or contain deregulated YB-1, preferably in the cytoplasm, if YB-1 is provided for replication directly or indirectly, especially in the nucleus, or if the provision of YB-1 is direct mediated directly or indirectly by an adenoviral protein that differs from E1A. This aspect of the invention differs from that already disclosed herein in that the use of transactivating E1A-modified adenoviruses, preferably group II adenoviruses, enables the replication of these viruses in YB-1 nuclear-positive tumor cells, in particular YB-1-positive, cell-cycle-independent YB-1 nuclear-positive cells, as well as those cells containing deregulated YB-1, especially YB-1 in the cytoplasm, do not utilize E1A proteins here (especially E1A13S protein), i.e. with respect to group I adenoviruses, but in a preferred embodiment, the E1A13S protein is functionally inactive, so that it can no longer transactivate E4orf6 and E1B55k, respectively, in the nuclear Directly or indirectly involved in the transport and provision of YB-1. As a result, according to this aspect of the invention, adenovirus cannot replicate efficiently. In this context, provision of YB-1 in the nucleus and for adenovirus replication is now no longer controlled by direct or indirect involvement of the E1A protein, but by the E1B protein ( Especially the expression of E1B55kD protein) and/or E4 protein (especially E4orf6 protein). the

This embodiment of the adenovirus can also be achieved by one of the above-mentioned measures, for example by expressing the E1B protein and/or the E4 protein earlier than the expression of the E1A protein, or by combining the E1B protein, the E4 protein and the One or more of the E1A proteins are placed under the control of a different promoter than that controlling the expression of the individual proteins in the wild-type adenovirus. the

Finally, the inventors of the present invention started from the unexpected discovery that efficient adenovirus replication can also occur especially in cells containing YB-1 in the nucleus, and more specifically in cells that contain YB-1 in the nucleus independent of the cell cycle or in cells containing deregulated YB-1, preferably in the cytoplasm, if at least one of the E1B protein, the E4 protein and the E1A protein, particularly their preferred form, is Expression is in an expression cassette under the control of a promoter. In one embodiment of the invention, essentially 3 expression cassettes are provided, each containing a single said protein. In an alternative embodiment, the expression cassette may also contain respectively two or more proteins E1B, E4 and E1A and their derivatives and possible substitutes, especially in the case of E1A12S. What has been said previously regarding the adenovirus containing nucleic acids associated with the proteins E1B, E4 and E1A also applies to the design of the individual proteins and the respective promoters used. When using such an expression cassette, it is preferred that the proteins corresponding to the individual proteins of the expression cassette are completely or partially deleted from the genome of the wild-type adenovirus, to ensure that the virus is stable. , and prevent recombination, at least to a large extent. the

In principle, the expression cassettes can be cloned separately into each region and each site of the adenovirus, wherein preferably one or several expression cassettes are inserted individually or in combination with each other into the E1 region, E3 region and/or E4 area. The nucleic acids in the E1, E3 and E4 regions may be completely, partially deleted, or not deleted at all, however it is preferred for adenoviruses according to the present invention that the nucleic acid encoding the E1A13S gene is inactivated or deleted, so that the virus Does not provide any transactivation of the E1A protein. The extent of this deletion of one or more regions E1, E3 and E4 depends on the expression cassette used and optionally further introduced foreign genes or transgenes or other expression cassettes containing them, i.e. genes different from the adenoviral genes, They differ at least to the extent that they are not provided by the regulatory content of the adenoviral nucleic acid that predominates in the wild-type adenovirus, nor by the adenoviral nucleic acid sequence of the wild-type adenovirus at that site. The present invention includes partial or complete deletion of nucleic acids contained in one or more expression cassettes encoding E1B protein, E4 protein and/or E1A protein in the adenovirus genome. In one embodiment, for example in the adenovirus XvirPSJL1 or 2 according to the invention, the adenoviral nucleic acid encoding E4orf6 is partially deleted, but the expression cassette contains the complete nucleic acid encoding it. Preferably, this is also achieved in the E1B55k (also called E155Kd) protein and/or the E1A12S protein. The extent of the deletion will in preferred embodiments be selected so that the maximum packaging size reaches about 103% of that of wild-type adenovirus, although this limit is only a preferred limit. Possible deletions made in the adenoviral genome are only limited in a preferred embodiment to ensure that infectious and packaging particles can also be produced. One skilled in the art will be able to determine the precise extent of the deletion based on the disclosure herein and standard experimentation. the

As a starting point for the construction of the adenoviruses described herein, any wild-type adenovirus can be used, but other adenoviruses can also be used, as long as they are constructed according to the technical teachings of the present invention. Particular preference is given to using subgroup C adenoviruses, within which group adenovirus 2 and adenovirus 5 are also preferably used. the

If not stated to the contrary, the singular and plural forms of the terms E1B protein, E4 protein and E1A protein are used synonymously herein. the

The term "de-regulated" YB-1 as used herein refers to a YB-1 molecule or YB-1 protein as used herein in a form similar to the YB normally present in cells, preferably in non-neoplastic cells -1 Quantitatively and/or qualitatively different. Deregulated YB-1 can be characterized and identified by specific viruses as being able to replicate in the presence of deregulated YB-1 in the context of cells containing such deregulated YB-1. A particular virus related to it is one in which the E1A protein is mutated and exhibits a transactivation function. Examples of these special viruses are: ADδ24, dl 922-947, E1 Ad/01/07 and CB 016 and/or [Howe, J.A et al., Molecular Therapy 2, 485-495, 2000; Fueyo J. et al., Oncogene 19, 2 -12, 2000; Heise C. et al., Nature Medicine 6, 1134-1139, 2001; Balague, C. et al., J.Virol.75, 7602-7611, 2001; Bautista, D.S. et al., Virology 1991, 182, 578-596 ; Jelsma T.N. et al., Virology 1988, 163, 494-502; Wong, H.K. and Ziff E.B., J. of Virology 1994, 68, 4910-4920]. Such cells and cells with such a background can be used for the replication of group I adenoviruses and/or group II adenoviruses, respectively. In addition, the adenovirus according to the invention can lyse tumors containing such cells. the

Furthermore, the present invention is based on the unexpected discovery that DNA replication of E1A-modified adenoviruses in YB-1 nuclear positive tumor cells is based on activation of the E2-late promoter. An E1A-modified adenovirus is understood to be one that (a) replicates in YB-1 nuclear-negative cells with reduced or no replication at all, (b) for at least one The viral gene has transactivation activity, wherein the gene is specifically selected from the group comprising E1B-55kDa, E4orf6, E4orf3 and E3ADP, and/or (c) cannot transfer the cellular YB-1 into the nucleus by the adenovirus. Optionally, the adenovirus used according to the invention has the additional characteristic that the binding of the E1A protein encoded by the adenovirus interferes with the binding of E2F to Rb and is able to dissociate the respective complexes composed of E2F and Rb. Adenoviruses having one or several of the aforementioned features a) to c), preferably all of the features a) to c), are replication deficient in cells that do not contain YB-1 in the nucleus. the

In one embodiment, significantly reduced replication as used herein specifically refers to a 2-fold, preferably 5-fold, more preferably 10-fold and most preferably 100-fold reduction in replication compared to wild type. In a preferred embodiment, the same or similar cell line, the same or similar infected virus titer (multiplicity of infection, MOI, or plaque forming unit, pfu) and/or the same or similar routine experimental conditions are used to Compare copy. Replication as used herein refers in particular to the formation of particles. In another embodiment, the measure of replication may be the extent of viral nucleic acid synthesis. Methods of determining the extent of viral nucleic acid synthesis as well as methods of measuring particle formation are known to those skilled in the art. the

The discoveries, methods, applications or nucleic acids, proteins, replication systems, etc. described herein are not necessarily limited to adenoviruses. In principle, these systems are also present in other viruses, which are also included here. the

With the virus according to the invention or with the application of the virus according to the invention a comparable wild type can be achieved when using an infection rate of 1-10 pfu/cell compared to 10-100 pfu/cell according to the prior art copy. the

YB-1 of a cell shall mean any YB-1 encoded by and preferably also expressed by a cell, wherein the YB-1 is present in the cell, in particular prior to infection of each cell with an adenovirus, preferably as described herein adenovirus and/or helper virus as described above. However, the invention also includes that the YB-1 of the cells is YB-1 introduced into the cells or produced by these cells only when external measures are applied (eg infection with a virus, preferably adenovirus). the

Without wishing to be bound thereby, the inventors of the present invention postulate that the E2-early promoter (i.e. the early E2 promoter) is not turned on by the human cell E2F transcription factor which is associated with the replication of the virus used according to the invention , is also relevant for the use according to the invention of the adenoviruses of the invention. In such circumstances, initiation of replication is independent of the Rb status of the cells, ie, tumor cells infected with the viruses disclosed herein and preferably subsequently lysed may contain functional as well as inactive Rb proteins. Additionally, replication of adenoviruses using the adenoviruses disclosed herein or using the conditions disclosed herein does not require any functional p53 protein, but is not negatively affected by its presence. Thus, this technical teaching departs from the principle of using adenoviruses of the type AdΔ24, dl922-947, E1Ad/01/07, CB016, or e.g. Those adenoviruses described in 830, have introduced one or several deletions in the E1A protein under the assumption that an intact and functional Rb protein would prevent efficient replication in vivo, and thus only in Rb-negative and Rb-mutant cells Adenovirus replication is provided in vivo. These prior art adenoviral systems are based on E1A in order to control the in vivo replication of the adenovirus by the early E2 promoter (E2 early promoter) and "free E2F". However, these viruses known from the prior art can be used according to the invention for replication in cells containing YB-1 in the nucleus independent of the cell cycle, or deregulated YB-1. the

The viruses described in said European patent EP 0 931 830, in particular adenoviruses, can be used according to the invention. More specifically, the viruses described in said patent are replication deficient and incapable of expressing a viral oncoprotein capable of binding a functional Rb tumor suppressor gene product. The adenovirus may specifically be any adenovirus incapable of expressing a viral E1A oncoprotein capable of binding a functional tumor suppressor gene product, more specifically Rb. The viral E1A oncoprotein may exhibit inactivating mutations, for example in the CR1 domain at amino acid positions 30-85 of adenovirus Ad5 (also referred to herein as Ad5), at nucleotide positions 697-790 and/or Ad5 The amino acid positions 120-130 of the CR2 domain, and the nucleotide positions 920-967, are involved in the binding of the p105 Rb protein, p130 and p107 proteins. However, the invention also includes that the adenovirus is type 2 dl 312 or type 5 NT dl 1010. the

Regarding the preparation of medicines according to the adenovirus of the present invention, especially the preparation of medicines for the treatment of tumor diseases and other diseases disclosed herein, the application of the adenovirus of the present invention and the application of the adenovirus of the present invention to replicate in cells, the The cells contain YB-1 in the nucleus, preferably in the nucleus independent of the cell cycle, or contain deregulated YB-1, preferably in the cytoplasm, and replication ultimately occurs in such cells that Contain YB-1 in the nucleus, preferably cell cycle independent, in other words, they are YB-1 nuclear-positive, or occur in cells containing deregulated YB-1. It is particularly noteworthy that adenoviruses do not replicate or replicate at significantly reduced levels in cells that do not contain YB-1 in the nucleus but only in the cytoplasm, or that do not contain any deregulated YB-1. Successful replication of these viruses therefore requires the presence of YB-1 in the nucleus, preferably cell cycle independent, or the presence of deregulated YB-1. As will be described in more detail below, this can be achieved, for example, by applying to the cell a measure that results in the expression or presence of YB-1, preferably independent of the cell cycle, or deregulated YB-1 in the nucleus , or to deregulate the expression of YB-1. Various measures can be made, for example, by using according to the invention or undergoing the coding and expression of the adenovirus YB-1 according to the invention, which, in addition to the adenovirus gene, also carries a gene coding for YB-1 and in particular coding for the expression of YB-1. Genetic information. Other means of causing translocation, induction or expression of YB-1 in the nucleus are the use of stress, such as application of cytostatics, irradiation, overheating, etc. to the cell and organism containing the cell. In a preferred embodiment, the radiation may be any radiation, which is used, for example, in the treatment of neoplastic diseases. the

In a preferred embodiment, the adenoviruses used according to the invention, in particular for tumor lysis, and the adenoviruses according to the invention are characterized in that they do not replicate in cells that are independent of the nuclear either do not contain YB-1 and are thus YB-1 nuclear-negative, or do not contain any deregulated YB-1. the

A further feature of some of the adenoviruses to be used according to the invention (which differ from the adenoviruses of the invention) is that they encode a viral oncogene, also referred to herein as an oncogene protein, wherein the oncogene protein is preferably E1A, wherein the oncogene The protein is capable of activating at least one viral gene that has an effect on viral replication and/or cell lysis of virus-infected cells. Preferably, the effect on replication is such that the virus replicates better in the presence of the oncogene protein than in a situation where the respective virus' oncogene protein is absent. This process is also referred to herein as transactivation, specifically E1A transactivation when the transactivation is mediated by E1A. The term "transactivation" preferably describes the process by which various viral oncoproteins have an effect on the expression and/or transcription of one or more genes other than the genes encoding the viral oncoprotein genes themselves, i.e. control their expression and/or or translations, especially activations. These viral genes are preferably E1B55kDa, E4orf6, E4orf3 and E3ADP, and any combination of each of the foregoing genes and gene products. the

Another, albeit optional, feature of the adenoviruses used according to the invention and of the adenoviruses of the invention are their binding characteristics to the tumor suppressor Rb, and the binding properties of specific ones of the proteins they encode to the tumor suppressor Rb, respectively. Combine features. In principle, the invention includes that the adenovirus used according to the invention is capable or incapable of binding Rb. The use of either of the two adenovirus alternative embodiments is independent of the Rb status of the treated or treated cells. the

To confer on E1A the ability not to bind Rb, the following deletions can be made to the E1A oncoprotein: deletion of the CR1 region (amino acid positions 30-85 in Ad5) and deletion of the CR2 region (amino acid positions 120-139 in AD5). During the process, the CR3 region is preserved and may have transactivation functions for other early viral genes. the

However, in order to confer the ability of E1A to bind Rb, the following deletions to the E1A oncoprotein are in principle possible: deletion of the CR3 region (amino acid positions 140-185); deletion of the N-terminus (amino acid positions 1-29); Deletion of amino acid positions 85-119; and deletion of the C-terminus (amino acid positions 186-289). The regions listed above do not affect the binding of E2F to Rb. Transactivation function remains intact but is reduced compared to wild-type Ad5. the

The invention also includes, in particular with respect to the adenoviruses of the invention, that the E1A protein, especially the E1A12S protein, is designed such that in one embodiment it binds to Rb and in a different embodiment it cannot bind to Rb , wherein such an E1A12S protein is an E1A protein, more specifically an E1A12S protein in the sense of the present invention, which however is sometimes referred to as a modified E1A12S in the prior art. Various designs of the E1A12S protein, more specifically with regard to the aforementioned deletion of the E1A protein (which is also referred to herein simply as E1A), are known to those skilled in the art. the

These adenoviruses, which are basically known in the prior art and do not show any transactivation, are generally considered to be replication deficient. However, it is a contribution of the inventors of the present invention that they have been found to still be able to replicate in an appropriate context, especially a cellular context. The presence of YB-1 in the nucleus, preferably a cell cycle-independent presence of YB-1 in the nucleus, or deregulated YB-1, can create or provide this appropriate cellular context. The term cell or cell system as used in one and any other aspect of the invention encompasses fragments or fractions of cell extracts, as well as cells in vitro, in vivo or in situ. To this extent, the term cellular system or cell also includes cells present in cell culture, tissue culture, organ culture or any in vivo and in situ tissue or organ, respectively, isolated, in clusters or as tissue , an organ or a part of an organism, but as such also exists preferably in a living body. The organism is preferably any vertebrate, more preferably a mammal. More preferably, the organism is a human organism. Other preferred organisms are those disclosed according to the various aspects of the present invention. the

In addition, the present invention includes, based on the technical teachings provided herein, the production of novel viruses in cells which exhibit the replication behavior of the adenoviruses described herein as well as those described in the prior art, said cells Is YB-1 nuclear-positive, preferably cell cycle-independent YB-1 nuclear-positive, or comprises deregulated YB-1. In other words, preferably starting especially from known adenoviruses, other viruses can be designed which have the characteristics defined herein which are relevant for use according to the invention. the

In connection with the present invention, in contrast to the viruses of the present invention, the modified E1A oncoproteins of the various adenoviruses to be used according to the present invention are capable of expressing in YB-1 nuclear-positive cells or in cells comprising deregulated YB-1 Transactivates early viral genes such as E1B55K, E4orf3, E4orf6, E3ADP. Preferably, there are no other changes to the viral genome, to the extent that each adenovirus may otherwise correspond to a wild-type adenovirus or a derivative thereof. the

The viruses disclosed herein encoding or comprising transactivating oncogene proteins in the sense of the present invention comprise for example adenoviruses AdΔ24, dl922-947, E1Ad/01/07, CB106 and/or in European patent EP 0 931 830 Said adenoviruses are each capable of transactivating early genes, such as E1B, E2, E3 and/or E4, and are comparable to wild-type adenoviruses, especially wild-type Ad5. In these cases, a specific region of the E1A protein is responsible for transactivation. Among various adenovirus serotypes, there are three highly conserved regions in the E1A protein. The CR1 region at amino acid positions 41-80, the CR2 region at amino acid positions 120-139 and the CR3 region at amino acid positions 140-188. The transactivation function is mainly based on the presence of the CR3 domain in the E1A protein. The amino acid sequence of CR3 exists unchanged in the aforementioned adenoviruses. This results in transactivation of the early genes E1B, E2, E3 and E4 independent of whether YB-1 is present in the nucleus or cytoplasm. the

In contrast, in the recombinant adenovirus dl520, the CR3 region has been deleted. Thus, dl520 expresses the so-called E1A12S protein, which does not contain the amino acid sequence of the CR3 region. As a result, dl520 can only exert a very weak transactivation function, especially in the E2 region, and thus does not replicate in YB-1 nuclear-negative cells. In YB-1 nuclear-positive cells, YB-1 transactivates the E2 region and thus allows efficient replication of dl520. Applications of systems like dl520 and systems derived from them for the purposes described herein are based on this. Another important difference between the two aforementioned groups of adenoviruses (e.g. delta24 (also referred to herein as AdΔ24) and e.g. dl520) is that in contrast to cells that are nuclear-negative for YB-1 or cells that do not contain deregulated YB-1 Than, the early genes E1B, E3 and E4 were more fully transactivated in YB-1 nuclear-positive cell cycle-independent cells or cells containing deregulated YB-1. In contrast, there are no or only minor differences in δ24. However, the transactivation of dl520 and more specifically the E1A12S protein was significantly reduced compared to wild-type adenovirus. However, this transactivation was sufficient to provide efficient replication in YB-1 nuclear-positive cells, as also shown in Example 10. The design of the E1A proteins described herein, especially in this regard, and the nucleic acids encoding them, so that the E1A protein has one or several deletions and/or mutations compared to the wild-type oncogene protein E1A, including And particularly preferred are those designs of the E1A protein as described in relation to dl520 or AdΔ24, dl922-947, E1Ad/01/07, CB106 and/or adenoviruses described in European Patent EP 0 931 830, viruses especially An embodiment of the adenovirus, the replication of which is controlled, preferably predominantly by activation of the E2-late promoter. Preferably, the deletion is selected from the group comprising a deletion of the CR3 region and a deletion of the N-terminus and a deletion of the C-terminus. Based on the disclosure provided herein, one of skill in the art can generate other embodiments of the E1A protein that permit replication of this form of adenovirus. Embodiments of the E1A protein as previously described are embodiments that can also be used in accordance with the adenoviruses of the invention, also referred to herein as adenoviruses of the invention or group I adenoviruses. ``

The adenoviruses of the invention, in particular group I adenoviruses, are also referred to herein as derivatives and can be used according to the invention, they typically comprise an E1 deletion, an E1/E3 deletion and/or an E4 deletion, i.e. the corresponding adenovirus Functionally active E1 and/or E3 and/or E4 expression products and corresponding products cannot be produced, respectively. Or in other words, these adenoviruses are only capable of producing functionally inactive E1, E3 and/or E4 expression products, wherein the functionally inactive E1, E3 and/or E4 expression products are expression products which either It does not exist as an expression product at all, either at the level of transcription and/or translation, or it exists in a form lacking at least one of the functions attributed to it in the wild-type adenovirus. This/these functions inherent in the expression product of wild-type adenovirus are known to those skilled in the art, for example described in Russell, W.C., Journal of Virology, 81, 2573-2604, 2000. Russell (supra) also describes the principles of adenovirus and adenoviral vector design, which is incorporated herein by reference. Also included in the invention are modified E1A oncoproteins, i.e. E1A proteins that are no longer transactivated and other proteins such as E1A12S, E1B-55K, E4orf6 and/or E3ADP (adenovirus death protein (ADP)) (Tollefson, A etc., J. Virology, 70, 2296-2306, 1996) are expressed in such vectors alone or in any combination. The genes mentioned individually as well as the transgenes disclosed herein can be cloned independently of one another into the E1 and/or E3 and/or E4 region and can be expressed using or under the control of a suitable promoter. Essentially, each of the regions El, E3 and E4 are suitable as cloning sites within the adenoviral nucleic acid. In some embodiments, suitable promoters are those disclosed herein involved in the control and expression of E1A, preferably modified E1A, respectively. the

Finally, in one embodiment, the group II adenovirus used according to the invention is E1B deficient, in particular E1B 19 kDa deficient. As generally used herein, the term defective generally refers to a condition in which an E1B is unable to exhibit all the characteristics of a wild-type E1B, and lacks at least one of these characteristics. the

At least some embodiments of group II adenoviruses for use in accordance with the invention disclosed herein are as such known in the art. The adenovirus used according to the invention is preferably a recombinant adenovirus, more particularly if it has been altered in the sense of the technical teaching provided herein compared to the wild type. Deletion or mutagenesis, respectively, of adenoviral nucleic acid sequences not relevant to the present invention is known to those skilled in the art. As described herein, these deletions may, for example, be associated with a portion of the nucleic acid encoding E3 and E4. If these deletions do not extend to the protein E4orf6, in other words, the adenovirus to be used according to the invention encodes E4orf6, an E4 deletion is particularly preferred. In a preferred embodiment, these adenoviral nucleic acids may also be packaged in viral capsids and thus form infectious particles. The same applies to the use of the nucleic acids according to the invention. It should generally be noted that adenoviral systems may lack single or several expression products. In connection with this, which is associated with group I adenoviruses and group II adenoviruses, it should be considered that this may be caused by a mutation or deletion of the nucleic acid encoding the expression product, wherein such mutations and deletions, respectively, are complete, or to the extent that no The degree to which the expression product is re-formed, or is caused by regulatory elements and elements controlling expression, such as promoters and transcription factors that are deleted or activated in a manner different from wild-type, respectively, at the nucleic acid level (absent promoter; cis -acting elements) or at the level of translation and transcription systems (trans-acting elements). Especially the latter aspect may depend on the context of each cell. the

In addition to the use of adenoviruses known as such, it is also possible according to the invention to use novel adenoviruses, for example group II adenoviruses, which can be used for the purposes already disclosed and described herein for other adenoviruses. The novel adenoviruses of the present invention are derived from the technical teachings provided herein. Particularly preferred representatives are, for example, the viruses Xvir03 and Xvir03/01 described in FIGS. 16 and 17 , the design principles of which are also further illustrated in Examples 11 and 12. the

In the case of vector Xvir03, the CMV promoter was cloned into the E1 region controlling the nucleic acids of E1B 55K and E4orf6 separated by an IRES sequence. Due to the cloning of these two genes into the virus and due to the gene products produced by them, respectively, maintenance of replication efficacy results such that replication occurs specifically in YB-1 nuclear-positive cells, more specifically inclusion in the sense disclosed in the present invention In those cells with deregulated YB-1, the result actually corresponds to a wild-type virus, which replicates selectively in cells, especially tumor cells. In one embodiment, cells in which deregulated YB-1 is present exhibit increased expression of YB-1 compared to normal or non-neoplastic cells, preferably compartment-independent expression of YB-1. the

A further developed virus Xvir03 is the virus Xvir03/01 in which, in a preferred embodiment, a therapeutic gene or transgene has been cloned under the control of a specific promoter, especially a tumor-specific or tissue-specific promoter. In relation to this virus, the E4 region is also functionally inactive, preferably deleted. The transgenes described herein can also be cloned into the E4 region, where this can occur as an alternative or in addition to the transgene cloning into the E3 region. the

The transgenes described herein and in particular below may also be expressed by or in connection with the adenoviruses of the invention (i.e. group I adenoviruses and their nucleic acids) or the replication system of the invention, respectively, and thus comprise In an expression cassette comprising a promoter and a nucleic acid sequence encoding one or more of said transgenes. The El, E3 and/or E4 regions are particularly suitable cloning sites in the adenovirus genome, however, the cloning sites are not limited thereto. the

The therapeutic gene may be a prodrug gene, a cytokine gene, an apoptosis-inducing gene, a tumor suppressor gene, a metalloproteinase inhibitor and/or an angiogenesis inhibitor and a tyrosine kinase inhibitor gene. In addition, siRNAs, aptamers, antisense molecules and ribozymes can be expressed, preferably directed against cancer-associated target molecules. Preferably, the single or multiple target molecules are selected from the group consisting of resistance-related factors, anti-apoptotic factors, oncogenes, angiogenic factors, DNA synthetases, DNA repair enzymes, growth factors and their receptors, transcription factors, metal Group of proteases, in particular matrix metalloproteases and plasminogen activators of the urokinase type. Preferred embodiments thereof are those already disclosed herein in relation to other aspects of the invention. the

Possible prodrug genes that can be used in preferred embodiments are, for example, cytosine deaminase, thymidine kinase, carboxypeptidase, uracil phosphoribosyltransferase; or purine nucleoside phosphorylase (PNP) [Kirn et al., Trends in Molecular Medicine, Vol. 8, No.4 (Suppl.), 2002; Wybranietz W.A. et al., Gene Therapy, 8, 1654-1664, 2001; Niculescu-Duvaz et al., Curr.Opin.Mol.Therapy, 1, 480.486, 1999; Koyama et al., Cancer Gene Therapy, 7, 1015-1022, 2000; Rogers et al., Human Gene Therapy, 7, 2235-2245, 1996; Lockett et al., Clinical Cancer Res., 3, 2075-2080, 1997 ; Vijayakrishna et al., J. Pharmacol. and Exp. Therapeutics, 304, 1280-1284, 2003]. the

Possible cytokines that may be used in a preferred embodiment are, for example, GM-CSF, TNF-α, Il-12, Il-2, Il-6, CSF, Interferon-γ; [Gene Therapy, Advances inPharmacology, Vol. 40, editors: J.Thomas August, Academic Press; Zhang and Degroot, Endocrinology, 144, 1393-1398, 2003; Descamps et al., J.Mol.Med., 74, 183-189, 1996; Majumdar et al., Cancer Gene Therapy, 7, 1086-1099, 2000]. the

Possible apoptosis-inducing genes that can be used in preferred embodiments are, for example, decorin: [Tralhao et al., FASEB J, 17, 464-466, 2003]; Retinoblastoma 94: [Zhang et al. , Cancer Res., 63,760-765, 2003]; Bax and Bad [Zhang et al., Hum.GeneTher., 20, 205 1-2064, 2002]; Apoptin (apoptin) [Noteborn and Pietersen, Adv. Exp.Med.Biol., 465, 153-161, 2000]]; ADP [Toth et al., Cancer GeneTherapy, 10, 193-200, 2003]; bcl-xs [Sumantran et al., Cancer Res, 55, 2507-25 12 , 1995]; E4orf4 [Braithwaite and Russell, Apoptosis, 6, 359-370, 2001]; FasL, Apo-1 and Trail [Boehringer Manheim, Guide to Apoptotic Pathways, Arai et al., PNAC, 94, 13862-13867, 1997] ; Bims [Yamaguchi et al., Gene Therapy, 10, 375-385, 2003; GNR163: Oncology News, 17 June, 2000]. the

Possible tumor suppressor genes that may be used in preferred embodiments are, for example, ElA, p53, p16, p21, p27, or MDA-7. [Opalka et al., Cell Tissues Organs, 172, 126-132, 2002, Ji et al., Cancer Res., 59, 3333-3339, 1999, Su et al., Oncogene, 22, 1164-1180, 2003]. the

Possible angiogenesis inhibitors that can be used in a preferred embodiment are, for example, endostatin or angiostatin [Hajitou et al., FASEB J., 16, 1802-1804, 2002] , and antibodies against VEGF (Ferrara, N., Semin Oncol 2002 Dec; 29(6 Suppl 16): 10-4). the

Possible metalloproteinase inhibitors that can be used in preferred embodiments are, for example, Timp-3 [Ahonen et al., Mol Therapy, 5, 705-715, 2002]; PAI-1; [Soff et al., J.Clin. Invest., 96, 2593-2600, 1995]; Timp-1, [Brandt K. Curr. Gene Therapy, 2, 255-271, 2002]. the

Other transgenes within the meaning of the invention which can be expressed by group I and group II adenoviruses are also tyrosine kinase inhibitors. An exemplary tyrosine kinase is EGFR (epidermal growth factor receptor) [Onkologie, Entstehung und Progression maligner Tumoren; author: Christoph Wagner, Georg Thieme Verlag, Stuttgart, 1999]. A preferred tyrosine kinase inhibitor is trastuzumab (herceptin) [Zhang H et al., Cancer Biol Ther. 2003, Jul-Aug; 2(4 suppl 1):S122-6]. the

siRNA (short interfering RNA) consists of 2, preferably 2 separate RNA strands, which hybridize to each other due to base complementarity, which means that they exist essentially in base pairing, and preferably with up to 50 nucleotides, preferably 18-30 nucleotides, more preferably less than 25 nucleotides and most preferably 21, 22 or 23 nucleotides in length, where these numbers refer to a single strand of siRNA, in particular a stretch The length of a single-stranded sequence that hybridizes or base-pairs with one, more specifically, a second single strand. SiRNA specifically induces or mediates the degradation of mRNA. Its desired specificity is mediated by the sequence of the siRNA and thus its binding site. The target sequence to be degraded is substantially complementary to either the first or second strand of the siRNA forming strand. Although the exact mode of action is unclear, it is speculated that siRNAs represent a biological strategy by which cells suppress distinct alleles during development and protect themselves against viruses. siRNA-mediated RNA interference can be used as a method to specifically inhibit or completely knock out protein expression by introducing gene-specific double-stranded RNA. For higher organisms, an siRNA comprising 19-23 nucleotides is particularly suitable as such, since it does not activate non-specific defense responses, such as interleukin responses. Direct transfection of 21 nucleotide double-stranded RNA with symmetrical 2 nucleotide long 3' overhangs is suitable for mediating RNA interference in mammalian cells and is comparable to other techniques such as ribozymes and antisense molecules than is efficient (Elbashir, S.Harborth J.Lendeckel W. Yalvcin, A.WeberK Tuschl T: Duplexes of 21-nucleotide RNAs mediate RNA interference incultured mammalian cells. Nature 2001, 411: 494-498). Only very few siRNA molecules are required to inhibit the expression of target genes. To avoid the limitations of exogenously administered siRNAs, particularly in the interference phenomenon and the transient nature of the specific delivery of siRNA molecules, the prior art uses vectors that allow the expression of endogenous siRNAs. For this purpose, for example, oligonucleotides with a length of 64 nucleotides are introduced in sense and antisense orientation into a vector containing a target sequence of 19 nucleotides long, which is replaced by, for example, a 9 nucleotide long Spacer sequences are separated. The resulting transcript folds into a hairpin structure with a stem structure of, for example, 19 base pairs. The loops are rapidly degraded in cells in order to generate functional siRNAs (Brummelkamp et al., Science, 296, 550-553, 2002). the

The nucleic acid encoding YB-1 may contain a nucleic acid sequence that mediates the transport of YB-1 into the nucleus, and the YB-1 may be an adenovirus used according to the present invention, especially a group II adenovirus, or the adenovirus of the present invention The virus, ie the group I adenovirus, is part of an adenovirus in one embodiment. Nucleic acids according to the invention, adenoviruses and adenovirus systems and adenoviruses known from the prior art, such as Onyx-015, AdΔ24, dl922-947, E1Ad/01/07, CB016, dl 520 and in patent EP 0 931 830 The described adenoviruses can be used as adenoviruses and adenovirus systems, respectively, and the corresponding nucleic acids, in combination with these nucleic acids according to the invention. Suitable nucleic acid sequences for mediating nuclear transport are known to those skilled in the art, for example in (Whittaker, G.R. et al., Virology, 246, 1-23, 1998; Friedberg, E.C., TIBS 17, 347, 1992; Jans, D.A. et al., Bioassays 2000 Jun; 22(6):532-44; Yoneda, Y., J. Biocehm. (Tokyo) 1997 May; 121(5):811-7; Boulikas, T., Crit. Rev. Eukaryot. Gene Expr. 1993; 3(3): 193-227; Lyons RH, Mol. Cell Biol., 7, 2451-2456, 1987). Nucleic acid sequences that mediate nuclear transport can use different principles. One such principle is that YB-1 forms a fusion protein with a signal peptide, or provides it with such a signal peptide, by means of which it is introduced into the nucleus, whereby the replication of the adenovirus according to the invention takes place. the

Another principle that can be used in the design of the adenoviruses used according to the invention, more specifically group II adenoviruses, but also the adenoviruses according to the invention, i.e. group I adenoviruses, is that YB-1 is provided with a transit sequence , which preferably starts with synthesis in the cytoplasm, transfers or translocates YB-1 into the nucleus and promotes viral replication there. An example of a particularly efficient nucleic acid sequence that mediates translocation to the nucleus is the TAT sequence of HIV, which is described, for example, together with other such suitable nucleic acid sequences in Efthymiadis, A., Briggs, LJ, Jans, DA., JBC 273, 1623 -1628, 1998. The present invention includes that the adenoviruses used according to the invention, in particular group II adenoviruses, but also the adenoviruses according to the invention, ie group I adenoviruses, comprise a nucleic acid sequence encoding a peptide mediating nuclear transport. the

The present invention includes that YB-1 exists in its full length, especially in a form corresponding to wild-type YB-1. Furthermore, the invention includes the use or presence of YB-1 as a derivative, for example in a shortened or truncated form. YB-1 derivatives which can be used or which can be present in connection with the present invention are YB-1 which are preferably able to bind the E2-late promoter and thus activate gene expression in the E2 region of the adenovirus. The derivatives specifically comprise the YB-1 derivatives disclosed herein. Other derivatives can be produced by deletion of single or several amino acids at the N-terminus, at the C-terminus or within the amino acid sequence. The invention encompasses that YB-1 fragments are also used as YB-1 proteins in the sense of the invention. In the paper by Jürchott K et al. [JBC2003, 278, 27988-27996] various YB-1 fragments are disclosed which are characterized by deletions at the C- and N-terminus. The distribution of various YB-1 fragments has demonstrated that both the cold shock domain (CSD) and the C-terminus are involved in cell cycle-regulated transport of YB-1 into the nucleus. The invention therefore includes that the truncated YB-1 (also referred to herein as the YB-1 protein) associated with creative expression of E1B55k and E4orf6 better migrates into the nucleus and thereby mediates Stronger CPE without better binding to the E2-late promoter, where it cannot be ruled out that the truncated YB-1 also migrates better into the nucleus and causes both effects, induction of CPE and binding to E2 - late promoter. Finally, this truncated YB-1 fragment could also migrate better into the nucleus and bind more efficiently to the E2-late promoter without inducing better CPE. The present invention also includes that the truncated YB-1 protein and fragment respectively contain other sequences related to the full-length YB-1 disclosed here, especially the cell localization signal sequence (NLS) and the like. the

Regarding the aforementioned various other genes and gene products respectively encoded and expressed by adenoviruses, they may also be encoded and expressed in any combination, respectively, in principle. the

The present invention encompasses that the terms adenovirus and adenovirus system should be understood to have substantially the same meaning. The term adenovirus is to be understood in particular in relation to whole virus particles comprising capsid and nucleic acid. The term adenoviral system focuses specifically on the fact that the nucleic acid is altered compared to the wild type. Preferably, these changes comprise changes in the genome structure of the adenovirus, eg resulting from deletions and/or additions and/or mutations of promoters, regulatory sequences and/or coding sequences such as open reading frames. In addition, the term adenoviral system is more preferably used as a vector, which can be used, for example, in gene therapy. the

The foregoing description, including any use and any design of adenoviruses and adenovirus systems, also applies to nucleic acids encoding them, and vice versa. the

It is possible according to the invention that the adenoviruses used according to the invention, more particularly group II adenoviruses, but also group I adenoviruses and the nucleic acids encoding them, respectively, can be any respective adenovirus nucleic acid according to This or combination with other nucleic acid sequences results in a replication event. As explained herein, the sequences and/or gene products required for replication may be provided by a helper virus. With regard to the reference to a coding nucleic acid sequence and to the extent that the nucleic acid sequence is a known nucleic acid sequence, the invention encompasses the use not only of identical sequences but also of sequences derived therefrom. The term derived sequence shall in particular refer to any sequence which still produces a gene product (nucleic acid or polypeptide), which has a function corresponding to or a function of the non-derived sequence. This can be determined by routine experiments known to those skilled in the art. Examples of such derived nucleic acid sequences are those encoding the same gene product, especially the same amino acid sequence, however, having different base sequences due to the degeneracy of the genetic code. the

In one embodiment, with regard to the II adenovirus according to the invention and/or the corresponding adenovirus replication system according to the invention and their use according to the invention, respectively, the adenoviral nucleic acid is deficient for the expression of oncogene proteins, in particular The E1A protein is absent, i.e., neither encodes the 12S E1A protein (also referred to herein as the E1A12S protein) nor the 13S E1A protein (also referred to herein as the E1A13S protein), or it neither encodes the 12S E1A protein nor the 13S E1A protein, or is Modified, as defined herein, if not stated to the contrary, the adenoviral replication system additionally comprises a nucleic acid of a helper virus, wherein the nucleic acid of the helper virus comprises a nucleic acid sequence encoding an oncogene protein, in particular an E1A protein, which respectively has the following characteristics and endow the adenovirus with the following characteristics: it preferably does not replicate in YB-1 nuclear-negative cells, but replicates in cells cycle-independent of YB-1 nuclear-positive cells or cells expressing deregulated YB-1, Transactivation of at least one viral gene, particularly E1B55kDa, E4orf6, E4orf3 and/or E3ADP, in YB-1 nuclear-positive cells, and/or non-translocation of cellular YB-1 into the nucleus. The invention encompasses that the transgenes described herein are encoded and/or expressed individually or collectively by a helper virus. Helper virus was administered for both Group I and Group II adenoviruses. the

Furthermore, in one embodiment of this adenoviral replication system according to the invention, the nucleic acid of the adenovirus and/or the nucleic acid of the helper virus is present as a replicable vector. the

The invention also encompasses that the nucleic acid encoding the group I adenovirus and/or the group II adenovirus is preferably present in an expression vector which is used according to the invention. the

In another aspect, the present invention also relates to a vector set comprising at least two vectors, wherein the vector set comprises in total an adenoviral replication system for group I adenoviruses and/or group II adenoviruses as described herein, the set of vectors according to The present invention uses. In one embodiment, each element of the adenoviral replication system is arranged on a single vector, preferably an expression vector. the

Finally, another aspect of the invention also relates to the use of cells containing one or several nucleic acids encoding a group I adenovirus and/or a group II adenovirus preferably used according to the invention and to be used according to the The invention uses, and/or the corresponding adenovirus replication system and/or the corresponding vectors and/or the set of vectors according to the invention, for the very same purpose as described herein for the various adenoviruses. the

The aforesaid adenoviral constructs and in particular their nucleic acids and nucleic acids encoding them can also be introduced in multiple copies into cells, preferably tumor cells, so that due to the presence of the various individual elements they can thus act together as if a single element Derived from a single nucleic acid and a single or several adenoviruses, respectively. the

Nucleic acids encoding group I adenoviruses and/or group II adenoviruses, corresponding adenovirus systems or parts thereof for use according to the invention may also be present as vectors. Preferably, these vectors are viral vectors. When the nucleic acid comprises adenoviral nucleic acid, preferably the viral particle is the vector. However, the invention also includes that said nucleic acid is present as a plasmid vector. In each case, the vector comprises elements that permit and control the propagation (ie, replication) and, optionally, expression of the inserted nucleic acid. Suitable vectors, preferably expression vectors and the respective elements are known to those skilled in the art, for example in Grunhaus, A., Horwitz, M.S., 1994, Adenovirus as cloning vectors. In Rice, C., editor., Seminars in Virology , London: described in Saunders Scientific Publications. the

Aspects relating to sets of vectors contemplate the foregoing embodiments, ie the various elements of the nucleic acid are not necessarily contained in only one vector. Thus, a vector set consists of at least two vectors. Additionally, any discussion made with respect to vectors also applies to vectors and vector groups, respectively. the

Group I adenoviruses and/or group II adenoviruses are characterized by the various nucleic acids and gene products disclosed herein, respectively, and may additionally contain all those elements known to those skilled in the art and inherent to wild-type adenoviruses (Shenk, T.: Adenoviridae: The virus and their replication. Fields Virology, Volume III, eds. Fields, B.N., Knipe, D.M., Howley, P.M. et al., Lippincott-Raven Publishers, Philadelphia, 1996, Chapter 67). the

In order to illustrate and not limit the present invention, adenovirus replication will be briefly discussed below. the

Adenovirus replication is a very complex process, usually based on the human transcription factor E2F. During viral infection, the "early genes" El, E2, E3 and E4 are first expressed. The group of "late genes" is responsible for the synthesis of the structural proteins of the virus. For the activation of early genes as well as late genes, the E1 region, composed of two transcriptional units E1A and E1B encoding distinct E1A and E1B proteins, plays a key role because they induce the transcription of E2, E3 and E4 genes (Nevins, J.R. , Cell 26, 213-220, 1981). In addition, E1A proteins can initiate DNA synthesis in quiescent cells and thus drive them into S phase (see Boulanger and Blair, 1991). In addition, they interact with tumor suppressors of the Rb class (Whyte, P. et al., Nature 334, 124-127, 1988). In this case, the cellular transcription factor E2F is released. E2F factors can then bind to the corresponding promoter regions of cellular genes as well as viral genes (in particular the adenovirus E2 early promoter) and initiate transcription and thus replication (Nevins, J.R., Science 258, 424-429, 1992). The activities of pRb and E2F are regulated by phosphorylation. The hypophosphorylated form of pRb is mainly present in G1 and M phases. In contrast, hyperphosphorylated forms of pRb are present in S and G2 phases. Upon phosphorylation of pRb, E2F is released from the complex consisting of E2F and hypophosphorylated pRb. Release of E2F from the complex formed by E2F and hypophosphorylated pRb results in the transcription of E2F-dependent genes. The E1A protein only binds to the low phosphorylated form of pRb, and most of the binding of E1A to pRb occurs through the CR2 region of the E1A protein. In addition, it also binds to the CR1 region, albeit with lower affinity (Ben-Israel and Kleiberger, Frontiers in Bioscience, 7, 1369-1395, 2002; Helt and Galloway, Carcinogenesis, 24, 159-169, 2003). the

Gene products in the E2 region are specifically required for initiation and completion of replication, as they encode three key proteins. Transcription of the E2 protein is controlled by two promoters, the "E2-early E2F-dependent" promoter, which is also referred to herein as the E2-early promoter or the early E2 promoter, and the "E2-late" promoter ( Swaminathan and Thimmapaya, The Molecular Repertoire of Adenoviruses III: Current Topics in Microbiology and Immunology, Vol. 199, 177-194, Springer Verlag 1995). In addition, the product of the E4 region plays an important role in the activity of E2F and the stability of p53 together with the E1A and E1B-55kDa proteins. For example, the E2 promoter is more transactivated by direct interaction of E4orf6/7 encoded by the E4 region with a heterodimer composed of E2F and DP1 (Swaminathan and Thimmapaya, JBC 258, 736-746, 1996). In addition, p53 inactivates a complex consisting of E1B-55kDa and E4orf6 (Steegenga, W.T. et al., Oncogene 16, 349-357, 1998) in order to successfully complete the lytic infection cycle. In addition, the E1B-55kDa protein has another important function, that is, when interacting with the E4orf6 protein, it promotes the export of viral RNA from the nucleus, where the cellular RNA remains (Bridge and Ketner, Virology 174, 345-353, 1990). Another important finding is that the protein complex consisting of E1B-55kDa/E4orf6 localizes to so-called "viral inclusion bodies". These structures are presumed to be sites of replication and transcription (Ornelles and Shenk, J. Virology 65, 424-429, 1991). the

Another important region for replication, especially for the release of adenoviruses, is the E3 region. The E3 region more specifically contains the genetic information for a number of relatively small proteins that are not required for the adenovirus infection cycle in vitro (ie in cell culture). However, they play an important role in the survival of the virus during acute and/or latent infection in vivo, as they have, inter alia, immunomodulatory and apoptotic functions (Marshall S. Horwitz, Virololgie, 279, 1-8, 2001; Russell, supra arts). It could be demonstrated that a protein with a size of about 11.6 kDa induces cell death. Due to its function, this protein is called ADP - the English term adenovirus death protein - (Tollefson, J. Virology, 70, 2296-2306, 1996). This protein is mainly formed late in the infection cycle. Furthermore, overexpression of the protein leads to better lysis of infected cells (Doronin et al., J. Virology, 74, 6147-6155, 2000). the

Furthermore, the inventors of the present invention have known that E1A-deficient viruses, specifically those that do not express any 12S E1A protein and also do not express any 13S E1A protein, can replicate very efficiently at higher MOIs (Nevins J.R., Cell 26, 213-220, 1981), however it cannot be realized in clinical application. This phenomenon is referred to in the literature as "E1A-like activity". It is also known that from the 5 proteins encoded by E1A, two proteins, namely 12S and 13S proteins, control and induce the expression of other adenovirus genes respectively (Nevins, J.R., Cell 26, 213-220, 1981; Boulanger, P. and Blair, E.; Biochem. J. 275, 281-299, 1991). This confirmed that the CR3 region of the 13S protein specifically displays a transactivation function (Wong HK and Ziff EB., J Virol., 68, 4910-20, 1994). However, adenoviruses with specific deletions in the CR1 and/or CR2 regions and/or CR3 regions of the 13S protein are essentially replication-defective and still transactivate viral genes and promoters, especially the E2 region ( Wong HK, Ziff EB., J Virol. 68, 4910-20, 1994; Mymryk, J.S. and Bayley, S.T., Virus Research 33, 89-97, 1994). the

After infection of cells, typically tumor cells, with wild-type adenovirus, YB-1 is introduced into the nucleus by E1A, E1B-55K, and E4orf6, where it colocalizes with E1B-55K in viral inclusion bodies, which allows the virus to and replicate efficiently in the nucleus in vivo. It has been previously found that E4orf6 can also bind E1B-55K (Weigel, S. and Dobbelstein, M.J. Virology, 74, 764-772, 2000; Keith N. Leppard, Seminars in Virology, 8, 301-307, 1998), and thus Mediates the translocation and distribution of E1B-55K into the nucleus, respectively, which ensures optimal virus production and adenovirus replication. Co-localization of YB-1 and E1B-55K in the so-called viral inclusion bodies in the nucleus through the synergy of E1A, E1B-55K and YB-1, respectively, and through a complex composed of E1B-55K/E4orf6 and YB-1 , efficient replication of the virus according to the invention is possible, thus the virus described herein is intended for replication in cells which are YB-1 nuclear-positive, preferably containing YB-1 in the nucleus independent of the cell cycle cells, and/or cells containing or expressing deregulated YB-1, and/or for the preparation of a medicament for the treatment of diseases in which YB-1 nuclear-positive cells are involved, preferably containing in a nucleus independent of the cell cycle YB-1 cells, and/or cells containing or expressing deregulated YB-1. Replication is therefore possible in this cellular context, leading to lysis of the cell, release of the virus and infection and lysis of adjacent cells, thus in the case of infection of tumor cells and tumors respectively, the eventual lysis of the tumor, i.e. oncolysis . the

YB-1 belongs to a class of highly conserved factors that bind an inverted CAAT sequence, which is called the Y-box. They can act in a regulated manner at the level of transcription as well as translation (Wolffe, A.P. Trends in Cell Biology 8, 318-323, 1998). An increasing number of Y-box-dependent regulatory pathways have been found in the activation and repression of growth and apoptosis-related genes (Swamynathan, S.K. et al., FASEB J. 12, 515-522, 1998). For example, YB-1 directly interacts with p53 (Okamoto, T. et al., Oncogene 19, 6194-6202, 2000), in Fas gene expression (Lasham, A. et al., Gene 252, 1-13, 2000), in MDR and MRP gene expression (Stein, U. et al., JBC276, 28562-69, 2001; Bargou, R.C. et al., Nature Medicine 3, 447-450, 1997) and activation of topoisomerases and metalloproteinases (Mertens.P.R. etc., JBC 272, 22905-22912, 1997; Shibao, K. et al., Int. J. Cancer 83, 732-737, 1999) play a substantial role. In addition, YB-1 is also involved in the regulation of mRNA stability (Chen, C-Y. et al., Genes & Development 14, 1236-1248, 2000) and repair process (Ohga, T. et al., Cancer Res.56, 4224-4228, 1996; Izumi H etc., Nucleic Acid Research 2001, 29, 1200-1 207; IseT. et al., Cancer Res., 1999, 59, 342-346). the

By YB-1 present in the nucleus independent of the cell cycle, or by deregulated YB-1 present in the cytoplasm that has been translocated into the nucleus by group I adenoviruses and/or group II adenoviruses, YB-1 Localizes in the nucleus of tumor cells, leading to E1A-independent viral replication in the absence and use of any 12S E1A protein or any 13S E1A protein (Holm, P.S. et al. JBC 277, 10427-10434, 2002), and in protein YB -1 overexpression results in multidrug resistance. In addition, adenoviral proteins such as E4orf6 and E1B-55K are known to have positive effects on viral replication (Goodrum, F.D. and Ornelles, D.A, J. Virology 73, 7474-7488, 1999), where the functional E1A protein is responsible for the activation of other viral gene products (such as E4orf6, E3ADP and E1B-55K) (Nevins J.R., Cell 26, 213-220, 1981). However, this does not occur with E1A-negative adenoviruses known in the art, in which the 13S E1A protein is absent. Nuclear localization of YB-1 in multidrug-resistant cells containing YB-1 in the nucleus allows replication and particle formation, respectively, of the E1A-negative virus. In this case, however, the efficiency of viral replication and particle formation was reduced several-fold compared to wild-type Ad5. In contrast, the combination of YB-1 allows YB-1-mediated extremely efficient viral replication and particle formation, and thus provides oncolysis, where YB-1 is already present in the nucleus of tumor cells, probably from Cell cycle-independent YB-1 in the nucleus, or where deregulated YB-1 present in the cytoplasm is transferred into the nucleus by group I adenoviruses and/or group II adenoviruses, or by external factors such as administration of Cytostatics or irradiation or overheating) into the nucleus, i.e. induced to exist in the nucleus, especially independent of the cell cycle, or as a transgene introduced by a vector with a system, preferably an adenovirus system, which can activate the adenovirus gene But does not show viral replication. This also applies to adenoviruses according to the invention, i.e. group I adenoviruses capable of replicating efficiently due to their specific design, and using effects, i.e. E1B protein, preferably E1B55K protein and/or E4 protein, preferably E4orf6 The protein provides efficient mobilization of YB-1, preferably in the nucleus. Suitable cytostatic agents that may be used with the adenoviruses disclosed herein according to various aspects of the invention are, for example, those belonging to the following group: anthracyclines, such as daunomycin and doxorubicin; Alkaloids such as cyclophosphamide; alkaloids such as etoposide; vinca-alkaloids such as vincristine and vinblastine; antimetabolites such as 5-fluorouracil and methotrexate; platinum derivatives substances, such as cisplatin; topoisomerase inhibitors, such as camphothecine, CPT-11; taxanes, such as taxol (taxole), paclitaxel (paclitaxel); histone-deacetylase inhibitors, such as FR901228, MS-27-275, trichostatine A; MDR modulators, such as MS-209, VX-710, and geldanamycin derivatives, such as 17-AAG. The adenoviruses disclosed herein, particularly recombinant adenoviruses, are capable of replicating only in YB-1 nuclear-positive cells and in cells containing, preferably deregulated, YB-1 in the cytoplasm, unlike wild-type adenoviruses. , especially wild-type Ad5, are limited in their ability to transactivate the viral genes E1B-55K, E4orf6, E4orf3 and E3ADP. The inventors of the present invention have now surprisingly found that these limited transactivation capacities can be overcome by expressing the corresponding genes, in particular E1B-55K and E4orf6, combined with the nuclear localization of YB-1. As shown in the Examples herein, virus replication and particle formation under these conditions increased to levels comparable to the replication activity and particle formation activity of wild-type adenovirus, respectively. the

Use according to the present invention of medicaments related to or prepared from the adenoviruses described herein is intended to be generally administered systemically, although topical administration or delivery of such medicaments is also within the scope of the invention. The use is intended in particular to infect those cells with adenoviruses, and in particular in which the replication of adenoviruses takes place, which preferably participates in a causal manner in the formation of disorders, typically diseases, for the diagnosis and/or prophylaxis and/or treatment of them , using a medicament according to the invention. the

Such medicaments are preferably used in the treatment of: malignancies, neoplastic diseases, cancerous venereal diseases, cancer and tumors, wherein these terms are used herein in a substantially synonymous manner if not stated to the contrary. The neoplastic disease is preferably: wherein the mechanism of the neoplastic disease, in particular due to a pathological mechanism, has resulted in YB-1 being localized in the nucleus, preferably independently of the cell cycle, or wherein YB-1 is present in the nucleus due to exogenous measures, The exogenous means described therein are suitable for transferring YB-1 into the nucleus, or inducing or expressing YB-1 there. The term tumor or neoplastic disease as used herein shall encompass malignant as well as benign tumors, solid and diffuse tumors, and various diseases. In one embodiment, the medicament comprises at least one other pharmaceutically active compound. The nature and amount of such other pharmaceutically active compounds will depend on the type of indication for which the drug is used. In the case of drugs for the treatment and/or prophylaxis of neoplastic diseases, cytostatic agents such as cisplatin and paclitaxel, daunoblastin, daunorubicin, doxorubicin and/or mitol are typically used Anthraquinones or other cytostatics or groups of cytostatics as described herein, preferably as described for cytostatic-mediated nuclear localization of YB-1. the

The medicament according to the invention may be present in various formulations, preferably in liquid form. Furthermore, the medicament will contain adjuvants such as stabilizers, buffers, preservatives and those known to those skilled in the art of formulation. the

The inventors of the present invention have surprisingly found that the use of the viruses described herein, preferably group I adenoviruses and/or group II adenoviruses according to the invention can be applied to tumors with a very high success rate, they can be used to produce therapeutic Drugs for tumors that contain YB-1 in the nucleus independent of the cell cycle. Usually, YB-1 is located in the cytoplasm, especially in the perinuclear cytoplasm. In the G1/S-phase of the cell cycle, YB-1 can be found in the nucleus of normal cells and tumor cells, and part of YB-1 remains in the cytoplasm [Jürchott K et al., JBC 2003, 278, 27988-27996]. However, the use of such modified adenoviruses is not sufficient to provide viral oncolysis. The relatively low efficacy of such attenuated adenoviruses described in the prior art is ultimately due to their misapplication. In other words, when these attenuated or modified viruses are used as described herein, preferably using group I adenoviruses and/or group II adenoviruses, to provide the molecular biological prerequisites for viral oncolysis, they can be treated with higher efficacy. These adenoviral systems are used in particular. When the adenoviruses described herein are to be used according to the present invention, such as AdΔ24, dl922-947, E1Ad/01/07, CB016, dl520 and recombinant adenoviruses described in European patent EP 0 931 830, in the following tumors Provides a prerequisite: its cells display a cell cycle-independent nuclear localization of YB-1. This form of nuclear localization may be caused by the nature of the tumor itself, or may be caused by the measures according to the invention disclosed herein or by the agents of the invention. The present invention thus defines a new group of neoplasms and neoplastic diseases, and thus also a group of patients, which can be treated with viruses according to the invention, such as preferably group I adenoviruses and/or group II adenoviruses, but also with existing Successful treatment with attenuated or modified adenoviruses has been described in the art. the

Another group of patients are those in whom YB-1 is ensured to migrate into the nucleus or to be introduced or transferred into it by imposing or achieving specific conditions, or where deregulated YB-1 is present, for which group I adenoviruses can be used according to the invention and/or adenoviruses of group II, or adenoviruses known for example from the prior art and to be used according to the invention, or adenoviruses described for the first time in this text, preferably with mutations and deletions respectively in the E1A protein Those adenoviruses which do not interfere with the binding of Rb/E2f or which do not replicate in YB-1 nuclear-negative cells, or exhibit significantly reduced replication as defined herein, and/or have and/or exhibit a deletion Oncoproteins, especially E1A, for example in the case of the viruses AdΔ24, d1922-947, E1Ad/01/07, CB106 and the adenoviruses described in European patent EP 0 931 830. The use of group I and/or group II adenoviruses in relation to this group of patients is based on the discovery that the induction of viral replication is based on the nuclear localization of YB-1 and subsequent binding of YB-1 to the E2-late promoter. Thanks to the findings disclosed herein, adenoviruses such as AdΔ24, dl922-947, E1Ad/01/07, CB106 and/or the adenoviruses described in European patent EP 0 931 830 can also replicate in the following cells: YB-1 nuclear-positive , and/or a cell in which YB-1 is present in a deregulated manner as defined in the present invention. These adenoviruses can therefore be used in the treatment of diseases and groups of patients according to the invention comprising cells with these characteristics, especially when these cells are involved in the development of the various diseases to be treated. This is the basis for the successful treatment of these tumors according to the invention of AdΔ24, dl922-947, E1Ad/01/07, CB016 and the adenoviruses described in patent EP0 931 830, as well as group I adenoviruses and/or group II adenoviruses, which The tumor contains YB-1 in the nucleus independent of the cell cycle, or deregulated YB-1 in the sense of the present disclosure. Another group of patients treated according to the invention can be treated according to the invention with the viruses described herein which can be used according to the invention and with the viruses described for the first time herein, in particular group I adenoviruses and/or group II adenoviruses Patients who are YB-1 nuclear-positive, and/or who become YB-1 nuclear-positive due to the treatment described below, and/or will undergo one of the following measures, preferably in a therapeutic sense, prior to the administration of adenovirus , patients following the use of each adenovirus or after administration of the adenovirus. Included in the present invention is a YB-1 nuclear-positive patient who has YB-1 in the nucleus of many tumor-forming cells independent of the cell cycle, and/or has deregulated YB-1 in such cells. One of these measures is the administration of a cytostatic agent as described herein as a whole and/or as used in tumor therapy. In addition, irradiation, especially that used in the treatment of tumors, falls under this category of measures. Irradiation refers in particular to irradiation with high-energy radiation, preferably radioactive radiation, preferably as used in tumor therapy. Further measures are respectively overheating and applied overheating, preferably overheating used in tumor therapy. In a particularly preferred embodiment, superheat is applied locally. Finally, a further measure is hormone therapy, in particular as used in tumor therapy. During such hormone therapy, anti-estrogens and anti-androgens are used. In relation thereto, antiestrogens such as tamoxifen are used in particular in the treatment of breast cancer, and antiandrogens such as flutamide or cyproterone acetate are used in particular in the treatment of prostate cancer. the

The adenoviruses described herein can also be used to treat tumors selected from the group comprising primary tumors, secondary tumors, tertiary tumors and metastatic tumors. In this connection, tumors preferably exhibit at least one feature that they have YB-1 in their cell cycle-independent nuclei, whatever the cause, and/or that they contain deregulated YB-1. the

The present invention includes that cells and tumors respectively comprise such cells in which the adenovirus according to the present invention replicates, or are capable of replicating, said cells or tumors have one or more of the characteristics described herein, more specifically , which contain YB-1 in the nucleus independent of the cell cycle, regardless of the cause, and/or which feature deregulated YB-1, and these cells and tumors, respectively, can be treated with adenovirus group I according to the invention and/or group II adenovirus for treatment, and said adenovirus can be used to produce medicines for treating them, wherein said adenovirus can express nucleic acid encoding YB-1. Thus, preferably there are 3 types of cells and thus tumors in which the adenoviruses of group I and/or group II according to the invention can replicate and with which they can be treated, preferably lysed, respectively:

Panel A: cells containing YB-1 in the nucleus independent of the cell cycle;

Panel B: cells that do not contain YB-1 in nuclei, especially those that are not cell cycle independent, but contain deregulated YB-1; and

Panel C: cells that do not contain YB-1 in nuclei, especially cell cycle-independent nuclei, and do not contain deregulated YB-1. the

For group A cells, adenoviruses according to the invention, in particular group I adenoviruses expressing no other YB-1, can be used for replication or lysis. However, such adenoviruses according to the invention expressing other YB-1, in particular group I adenoviruses, can also be used for replication or lysis. This also applies to Group B. Without wishing to be limited thereto, the reason seems to be that, through the localization of YB-1 in the nucleus and the transfer of YB- 1 respectively ensures efficient replication. This process is supported by the additional expression of YB-1 by adenovirus. the

In the case of group C, preferably those adenoviruses according to the invention, in particular group I adenoviruses will be used for replication or lysis, which additionally express YB-1. The reason for this seems to be, still wishing not to be limited, that the above-mentioned viral replication process is not activated in a specific cellular context so that efficient replication can take place. The mechanism by which efficient replication occurs simply by providing YB-1 and expressing YB-1, respectively, appears to be such that overexpression of YB-1 leads to nuclear localization of YB-1, as also described in Bargou [Bargout R.C. et al., Nature Medicine 1997, 3, 447-450] and Jürchott [Jürchott K. et al., JBC 2003, 278, 27988-27966]. the

The invention includes that some tumor-forming cells themselves contain YB-1 in the nucleus or do so or after induction and activation have been introduced into the nucleus, or contain deregulated YB-1 in the sense of this disclosure. Preferably, about 5% or any greater percentage (i.e. 6%, 7%, 8%, etc.) of tumor-forming cells are such YB-1 nuclear-positive cells, or in which deregulated YB-1 is present. 1 cell. For other tumors, such as breast tumors, osteosarcomas, ovarian cancers, joint cancers, or lung cancers, the percentage of tumor cells that contain deregulated YB-1 or exhibit cell cycle-independent YB-1 nuclear localization can be about 30-50 % [Kohno K. et al., BioEssays 2003, 25, 691-698]. Such tumors can preferably be treated using the adenovirus according to the invention. The nuclear localization of YB-1 can be induced by external stress and by locally applied stress, respectively. This induction can take place, for example, by irradiation, especially UV irradiation, application of cytostatic agents as already disclosed herein, and overheating. The important thing about hyperthermia is that it can now be achieved in a very specific way, more specifically in a localized way, and thus also cause a specific nuclear translocation of YB-1 in the nucleus, thus, providing adenoviral replication and thus cells and tumors A prerequisite for cleavage, which is preferably locally restricted (Stein U, Jurchott K, Walther W, Bergmann S, Schlag PM, Royer HD. J Biol Chem. 2001, 276(30): 28562-9; Hu Z, Jin S, Scotto KW. J Biol Chem. 2000 Jan 28; 275(4): 2979-85; Ohga T, Uchiumi T, Makino Y, Koike K, Wada M, Kuwano M, Kohno K. J Biol Chem. 1998, 273(11) : 5997-6000). the

The medicaments according to the invention can therefore also be administered to patients and groups of patients, or can be designed for them, in which the transport of YB-1, in particular in various tumor cells, is achieved by appropriate pre- or post-treatment or concomitant treatment, respectively , and produce deregulated YB-1 in cells. the

Based on the technical teaching provided herein, those skilled in the art are within the scope of their skill to make appropriate modifications, in particular to E1A, for example, which may include various embodiments for the generation of deletions or point mutations to thereby generate adenoviruses, which may be found in used in applications according to the invention. the

As already explained above, group I adenoviruses and/or group II adenoviruses are able to replicate in these cells and cell systems containing YB-1 in the nucleus. With regard to the question of whether the adenoviruses used according to the invention are capable of replicating and thus lysing tumors, the state of the cells with or without Rb (ie the retinoblastoma inhibitor product) is irrelevant. In addition, regarding the application of the adenovirus according to the present invention, when using the adenovirus system disclosed herein related to YB-1 nucleus-positive cells (i.e., cells containing YB-1 in the nucleus independent of the cell cycle) , need not consider the p53 status of the infected cells, cells to be infected or cells to be treated, the p53 status and the Rb status have no effect on the replication of the adenovirus implementing the technical teachings disclosed herein. the

Transactivated oncogenes and oncogene proteins, in particular E1A, preferably group II adenoviruses, respectively, can be under the control of all native adenovirus promoters and/or via tumor-specific or tissue-specific promoters. Suitable non-adenoviral promoters may be selected from the group comprising the cytomegalovirus promoter, the RSV (Rous sarcoma virus) promoter, the adenovirus-based promoter Va I and the non-viral YB-1 promoter (Makino Y. et al., Nucleic Acids Res. 1996, 15, 1 873-1878). Other promoters that may be used in any aspect of the invention disclosed herein are the telomerase promoter, the alpha-fetoprotein (AFP) promoter, the caecinoembryonic antigen promoter (CEA) (Cao, G., Kuriyama, S., Gao, J., Mitoro, A., Cui, L., Nakatani, T., Zhang, X., Kikukawa, M., Pan, X., Fukui, H., Qi, Z. Int. J. Cancer, 78, 242-247,1998), L-plastin promoter (Chung, I., Schwartz, PE., Crystal, RC., Pizzorno, G, Leavitt, J., Deisseroth, AB. Cancer Gene Therapy, 6,99 -106, 1999), arginine vasopressin promoter (Coulson, JM, Staley, J., Woll, PJ.British J.Cancer, 80, 1935-1944, 1999), E2f promoter (Tsukada et al. Cancer Res., 62, 3428-3477), uroplakin II promoter (Zhang et al., Cancer Res., 62, 3743-3750, 2002) and PSA promoter (Hallenbeck PL, Chang, YN, Hay, C, Golightly, D. , Stewart, D., Lin, J., Phipps, S., Chiang, YL.HumanGene Therapy, 10, 1721-1733, 1999), tyrosinase promoter (Nettelbeck, DM.Anti-Cancer Drugs, 14, 577-584, 2003), cyclooxygenase 2 promoter (Nettelbeck, DM., Rivera, AA, Davydova, J., Dieckmann, D., Yamamoto, M., Curiel, DT. Melanoma Res., 13, 287 -292, 2003), and induction systems such as tetracycline (Xu, XL., Mizuguchi, H., Mayumi, T., Hayakawa, T. Gene, 309, 145-151, 2003). In addition, the YB-1-dependent E2-late promoter of adenovirus as described in German patent application DE 101 50 984.7 is a promoter that can be used in the present invention. the

The telomerase promoter is known to be extremely important in human cells. Thus, telomerase activity is regulated by the transcriptional control of the telomerase reverse transcriptase gene (hTERT), the catalytic subunit of the enzyme. Expression of telomerase is active in 85% of human tumor cells. In contrast, it is inactive in most normal cells. Germ cells and embryonic tissues do not contain it (Braunstein, I. et al., Cancer Research, 61, 5529-5536, 2001; Majumdar, A.S. et al., Gene Therapy 8, 568-578, 2001). More detailed studies of the hTERT promoter have demonstrated that fragments of the promoter 283 bp and 82 bp away from the start codon, respectively, are sufficient for specific expression in tumor cells (Braunstein I. et al.; Majumdar AS et al., supra). Accordingly, the promoter and the specific fragment are respectively suitable for the specific expression of a gene and in particular a transgene, preferably one of the transgenes disclosed herein, in tumor cells. The promoter should allow expression of the modified oncogene, preferably the E1A oncogene protein, only in tumor cells. Furthermore, in one embodiment, the expression of a transgene in the adenoviral vector is under the control of these promoters, said transgene preferably being selected from the group comprising E4orf6, E1B55kD, ADP and YB-1. The invention also includes that the open reading frame of the transactivating oncogene protein, especially the E1A protein, is within the frame of the gene product of one or several adenoviral systems. However, the open reading frame of the transactivating E1A protein can also be independent from there. the

The present invention encompasses that the various promoters described above are also used in various embodiments of the adenovirus according to the present invention, preferably group I adenovirus, in particular when the promoter to be used is related to the control of the expression of the respective proteins in the wild-type adenovirus Or when the promoters of the expression products are different. The aforementioned promoters are thus suitable heterologous promoters in the sense of the present invention. In a preferred embodiment of the adenovirus according to the present invention, especially the group I adenovirus, when the adenovirus is applied to the above-mentioned group A and B cells, it is considered that the expression of the E1B protein and/or the E4 protein is derived from such heterogeneous A source promoter wherein preferably, but not exclusively, the expression of the E1A protein is controlled by YB-1. In this and other embodiments, expression of the E1A protein is under the control of a YB-1 controllable promoter, such as the adenovirus 2-late promoter. This also applies when the E1B protein and/or the E4 protein is expressed in one expression cassette. the

In a preferred embodiment of the adenovirus according to the present invention, especially the group I adenovirus, each promoter is independently tumor-specific, organ-specific or Tissue-specific promoters. In connection with this, it is sufficient when at least one promoter controlling the expression of the E1B protein, the E4 protein and/or the E1A protein is such a specific promoter. Through such tumor, organ and tissue specificity, it is ensured that the replication of the adenovirus according to the invention takes place only in the cells of the corresponding tumor, organ and tissue, and that, apart from this, no other tissues are subjected to the replication of the adenovirus damage, such as being cleaved. Preferably, also a second and more preferably, all 3 are controlled by such tumor-specific, organ-specific or tissue-specific promoters. Using such adenoviruses, it is also possible to lyse cells which are unable to form tumors or which cannot develop into such tumors, but for other reasons, such as medical reasons, they are to be destroyed or removed from an organism, preferably a mammalian organism, more Human organisms are preferred, for example because they produce undesired factors or produce such factors at excessively high levels. the

In one embodiment, cells lysed using said adenovirus according to the invention are considered resistant, preferably exhibiting multiple resistances. the

Resistance as mentioned here is characteristic of the tumor and of the patient to be treated, they are mediated by, but not limited to, the following genes: MDR, MRP, topoisomerase, BCL2, glutathione-2-transferase (GST), protein kinase C (PKC). Since cytostatics are especially based on the induction of apoptosis, the expression of genes associated with apoptosis plays a key role in the development of any resistance, so that the following factors are also associated with it, namely Fas, BCL2 family, HSP 70 and EGFR [Kim et al., Cancer Chemther. Pharmacol. 2002, 50, 343-352]. the

Levenson et al. [Levenson, V.V. et al., Cancer Res., 2000, 60, 5027-5030] have documented that the expression of YB-1 is significantly higher in resistant tumor cells than in non-resistant tumor cells. the

Resistance as used herein preferably refers to resistance to the cytostatic agents described herein. This multidrug resistance resistance is preferably consistent with expression, preferably overexpression, of the membrane-bound transporter P-glycoprotein, which can be used as a marker to determine various cells and thus also tumors and various types of patients with this marker Set of tags. The term resistance as used herein also encompasses resistance mediated by P-glycoprotein, also known as typical resistance, and resistance known as atypical resistance, which is MRP-mediated, or otherwise, non-P- Glycoprotein-mediated resistance. Another marker associated with YB-1 expression is topoisomerase IIα. Therefore, to determine whether a patient can be treated with adenovirus according to the invention with the expected success, topoisomerase IIα can be used in a screening method instead of or in addition to identifying YB-1 in the nucleus. In principle, a marker that can be used analogously for P-glycoprotein is MRP. Another marker, at least in the case of colorectal cancer cells or patients with colorectal cancer, is PCNA (proliferating cell nuclear antigen) (Hasan S. et al., Nature, 15, 387-391, 2001), as described e.g. by Shibao K. et al. (Shibao K et al., Int. Cancer, 83, 732-737, 1999). Finally, at least in the field of breast cancer cells and osteosarcoma cells, the expression of MDR (multidrug resistance) is a marker in the above sense (Oda Y et al., Clin. Cancer Res., 4, 2273-2277, 1998). Another feasible marker that can be used according to the present invention is p73 (Kamiya, M., Nakazatp, Y., J Neurooncology 59, 143-149 (2002); Stiewe et al., J. Biol. Chem., 278, 14230-14236 , 2003). the

Finally, YB-1 should also be considered a precursor marker for breast cancer, which can be used in the present invention. Only in patients with primary tumors with elevated YB-1 expression, recurrence occurs after surgery and chemotherapy [Janz M. et al. Int. J. Cancer 2002, 97, 278-282]. the

A particular advantage of the present invention is also that, using the adenoviruses according to the invention described herein, it is also possible to treat those patients who would otherwise be considered to be no longer treatable in a clinical sense, and therefore to treat tumors using the methods of the prior art The disease can no longer be expected to be successful, especially when the use of cytostatics and radiation is no longer reasonably possible and can no longer be done successfully in the sense of affecting or reducing the tumor. The term tumor generally refers herein to any tumor or cancerous disease which contains YB-1 intrinsically in the nucleus, preferably cell cycle-independent nucleus, or through the application of extrinsic measures as disclosed herein, and /or they contain deregulated YB-1. the

Furthermore, the viruses described herein could in principle be used in the treatment of tumors. Preferably, these tumors are selected from the group comprising breast cancer, ovarian cancer, prostate cancer, osteosarcoma, glioblastoma, melanoma, small cell lung cancer and colorectal cancer. Other tumors are those which are resistant as described herein, preferably those which are multi-resistant, in particular also those tumors of the above mentioned groups. Particularly preferred tumors are selected from the group consisting of breast tumors, bone tumors, stomach tumors, intestinal tumors, gallbladder tumors, pancreatic cancer, liver tumors, kidney tumors, brain tumors, ovarian tumors, skin and skin appendage tumors, head/neck tumors, Group of tumors of the uterus, joints, larynx, salivary glands, esophagus, tongue and prostate. In this connection, preferably these tumors are represented in their entirety as described herein. the

Adenoviruses according to the invention, preferably group I adenoviruses and adenoviruses to be used according to the invention, preferably group II adenoviruses. the

The use of the adenoviruses described herein, in particular group I adenoviruses and/or group II adenoviruses, as pharmaceuticals, in particular in relation to systemic administration, can be improved by appropriate targeting of the adenoviruses. Infection of tumor cells by adenoviruses in particular is somewhat dependent on the presence of the coxackievirus-adenovirus receptor CAR and unique integrins. Once they are strongly expressed in tumor cells, infection at very low titers (pfu/cell) is possible. To achieve the so-called retargeting of recombinant adenoviruses, various strategies have been tried so far, such as inserting heterologous sequences into the fibrous nodule region, using bispecific antibodies, coating adenoviruses with polymers, and Ligands were introduced into Ad fibers, serotype 5 nodules (knobs) and serotype 5 fiber shafts (shafts) and nodules were replaced by serotype 3 nodules and Ad 35 fiber shafts and nodules, and penton substrates (penton base) (Nicklin S.A. et al., Molecular Therapy 2001, 4, 534-542; Magnusson, M.K. et al., J. of Virology 200 1, 75, 7280-7289; Barnett B.G. et al., Biochimica et Biophysica Acta 2002, 1575, 1-14). The present invention includes aspects of the invention implementing such further embodiments and features in the adenoviruses according to the invention and in the adenoviruses used according to the invention, in particular group I adenoviruses and group II adenoviruses, respectively. the

In another aspect, the invention relates to a method of screening patients for treatment with a modified adenovirus, i.e. an adenovirus used according to the invention such as AdΔ24, dl922-947, E1Ad/01/07, CB016 or European patent EP A virus as described in 0 931 830, and/or adenovirus group I and/or adenovirus group II, wherein said method comprises the following steps:

- analyze samples of tumor tissue, and

- Determine whether YB-1 is localized in the nucleus independent of the cell cycle, or whether the cells contain deregulated YB-1. the

Instead of or in addition to YB-1, the presence of the above markers can be detected. the

In cases where tumor tissue or a part thereof contains YB-1 in the nucleus, in particular cell cycle independent, or deregulated YB-1, the adenoviruses disclosed herein, in particular group I adenoviruses, can be used according to the invention and group II adenoviruses. the

In one embodiment of the method according to the present invention, the analysis of tumor tissue is accomplished by using a reagent selected from the group consisting of antibodies against YB-1, aptamers against YB-1, and spiegelmers against YB-1 and against Group of anticalines of YB-1. In principle, reagents of the same kind can also be prepared and used separately for the respective labels. The preparation of antibodies, particularly monoclonal antibodies, is known to those skilled in the art. Another reagent that specifically detects YB-1 or the marker is a peptide that binds with high affinity to their target structure, which in the case of the present invention is YB-1 or said marker. In order to produce such peptides, methods are known in the prior art, such as phage-display. For this purpose, starting from a peptide library, where individual peptides have a length of about 8-20 amino acids, the size of the library is about 10 ² -10 ¹⁸ , preferably 10 ⁸ -10 ¹⁵ different peptides. A special form of target molecule capable of binding polypeptides is the so-called anti-enableins, which are described, for example, in German patent application DE 197 42 706 .

Another reagent for specifically binding YB-1 or the corresponding markers disclosed herein, and thus detecting the cell cycle-independent localization of YB-1 in the nucleus, is the so-called aptamer, i.e. a D-nucleic acid, which takes the form RNA or DNA based, exist as single or double strands, and bind specifically to target molecules. The generation of aptamers is described, for example, in European patent EP 0 533 838. A specific embodiment of an aptamer is the so-called aptazyme, which is described, for example, in Piganeau, N. et al. (2000), Angew. Chem. Int. Ed., 39, no. 29, pp. 4369-4373. They are specific embodiments of aptamers because they contain, in addition to the aptamer part, a ribozyme part which, after binding or releasing the target molecule bound to the aptamer part, acquires catalytic activity and cleaves the nucleic acid substrate, which Accompanied by the signal generation. the

Another form of aptamer is the so-called spiegelmer, ie a target molecule consisting of L-nucleic acid binding nucleic acid. Methods for preparing such spiegelmers are described, for example, in WO 98/08856. the

Samples of tumor tissue can be obtained by puncture or by surgery. Assessment of whether YB-1 is localized in the nucleus independent of the cell cycle is often performed using microscopy techniques and/or immunohistological analysis, typically using antibodies or any other of the aforementioned reagents. Other methods are known to those skilled in the art to detect the presence of YB-1 in the nucleus and its localization therein is independent of the cell cycle. For example, the localization of YB-1 can be readily detected when scanning tissue sections stained for YB-1. The frequency of YB-1 in the nucleus has been indicative of a cell cycle-independent localization in the nucleus. Another possibility to detect YB-1 in the nucleus independently of the cell cycle is to stain for YB-1 and detect whether YB-1 is localized in the nucleus and determine the stage of the cell. However, this and the detection of YB-1 can also be achieved by using the aforementioned reagents for YB-1. Detection of reagents is performed by methods known to those skilled in the art. Because the reagents are specifically directed to YB-1 and therefore do not bind to other structures in the sample to be analyzed, in particular other structures of cells, by means of appropriate labeling of the reagents and due to their specific binding to YB-1 , can detect and analyze the localization of the reagent, as well as the localization of YB-1. Methods of labeling reagents are known to those skilled in the art. The same technique can also be used to detect how much deregulated YB-1 is contained in the cells of a sample. Since deregulated YB-1 also exhibits overexpression compared to non-deregulated YB-1, the relative expression of YB-1 relative to a reference sample can also be used in order to test whether YB-1 is deregulated in the analyzed cells. regulated. the

The invention will now be further illustrated by means of figures and examples, from which new features, embodiments and advantages will be obtained. related to it,

Figure 1 shows the structural design of the adenovirus vector, which is referred to herein as an AdE1/E3-negative adenovirus vector, and is an E1/E3-deleted adenovirus of wild-type adenovirus and adenovirus dl520;

Fig. 2 shows the binding domain of E1A protein about binding p300, p107 and p105;

Figure 3 shows U2OS cells, which do not contain YB-1 in their nuclei after infection with E1/E3-deleted adenovirus Ad5 (referred to herein as E1/E3-negative Ad5) and dl520;

Figure 4 shows 257RDB cells containing YB-1 in their nuclei after infection with E1/E3-deleted adenovirus Ad5 (referred to herein as E1/E3-negative Ad5) and adenovirus dl520;

Figure 5 shows 257RDB cells and U2OS cells after infection with adenovirus dl1119/1131;

Figure 6 shows the results of EMSA analysis, which confirmed that YB-1 exists in the nuclei of multi-resistant cells and cell lines 257RDB, 181 RDB, MCF-7Ad, while YB-1 does not exist in the nuclei of U2OS and HeLa cells Inside;

Figure 7 shows the structural design of the E1A protein of wild-type adenovirus, adenovirus dl520 and adenovirus dl1119/1131;

Figure 8 is a histogram showing the replication efficiency of adenoviruses in the presence of additionally expressed viral proteins, as absolute values;

Figure 9 is a histogram showing the increase in adenovirus replication efficiency in the presence of additionally expressed viral proteins;

Figure 10 shows wells grown with U2OS cells after crystal violet staining and infection with dl520 at 10 and 30 pfu/cell and control (K), without and with 40 ng daunorubicin/ml, respectively;

Figure 11 shows wells grown with HeLa cells after crystal violet staining and infection with dl520 at 10 and 30 pfu/cell and control (K), without and with 40 ng daunorubicin/ml, respectively;

Figure 12 is a graph of tumor volume as a function of time for tumors of different origin (RDB257 and HeLa), after treatment with PBS and dl520, respectively;

Figure 13 shows photographs of killed mice that developed tumors based on RDB257 cells after treatment with PBS and 5 x ¹⁰⁸ pfu dl520, respectively;

Figure 14 is the result of the DNA blot analysis of the cell extract (subcutaneous growth tumor) of RDB257 cells and HeLa cells after being infected with dl520;

Figure 15 is a histogram showing the replication efficiency and particle formation of dl520 and wild-type adenoviruses in YB-1 nuclear-positive tumor cells (257RDB and 181RDB) and YB-1 nuclear-negative tumor cells (HeLa, U2OS) picture;

Figure 16 shows the structural design of wild-type adenovirus and adenovirus vector AdXVir03;

Figure 17 shows the structural design of the adenoviral vector AdXVir03/01; and

Figure 18A/B shows 18RDB cells (Figure 18A) and 272RDB cells (Figure 18B) after crystal violet staining and infection with Ad3 12 (20 pfu/cell), Xvir03 (5 pfu/cell) and control (uninfected) Outgrown wells in which crystal violet staining was performed 5 days post-infection;

Figure 19 has shown the result of the Northern blot analysis of the expression in A549 cells and U2OS cells of E2 gene after being infected with wild-type adenovirus Ad5 and adenovirus Ad3 12;

Figure 20 shows the results of Northern blot analysis of the expression of the E2 gene in U2OS cells 12 and 24 hours after infection with wild-type adenovirus and adenovirus δ24;

Figure 21 shows the structural design of the adenoviral vector XvirPSJL1;

Figure 22 shows the structural design of the adenoviral vector XvirPSJL2;

Figure 23 shows the wells of HeLa cell growth after crystal violet staining and infection with adenovirus dl520 of different pfu/cell;

Figure 24 shows a histogram representing the activity of luciferase in U2OS cells, HeLa cells and 257RDB cells using different promoter fragments of the adenovirus 2-late promoter;

Figure 25 shows a histogram representing the number of virus particles after 2 days and 5 days of infection of U2OS cells with adenovirus expressing YB-1 and virus Ad312, wherein virus particles remaining in the cells (indicated by black) and released A distinction is made between extracellular viral particles (horizontal bars). Example 1: Types of E1A modifications that adenoviruses used according to the invention may contain

Figure 1 shows the structural design of the adenoviral vector AdE1/E3-negative (i.e. E1/E3-deleted adenovirus), wild-type adenovirus and adenovirus dl520. the

Adenovirus AdE1/E3-negative, which does not contain the region encoding functional E1A or functional E1B or E3, was used as a toxicity control in this experiment. the

The wild-type E1A gene encodes a total of 5 proteins that are produced by alternative splicing of the E1A RNA. Among them, 2 different proteins were produced, namely a protein of 289 amino acids and a protein of 243 amino acids. dl520 does not encode a 289 amino acid protein because it has a deletion in the CR3 region of the E1A gene, resulting in a lack of 13S gene product. The adenovirus d1520 that can be used according to the invention is known by those skilled in the art as 12S-E1A virus. The adenovirus dl347 known in the prior art (Wong und Ziff, J. Virol., 68, 4910-4920, 1994) is also a 12S-E1A virus that can be used according to the invention. the

In the 289 amino acid protein encoded by 13S-E1A mRNA, there are 3 regions that are conserved among various adenovirus subclasses. These are called CR1, CR2 and CR3. Although CR1 and CR2 are present in two E1A proteins (E1A 12S and E1A 13S), ie in proteins of 289 amino acids and 243 amino acids, the CR3 region is only present in the larger of the two aforementioned proteins. the

The CR3 region is required for activation of viral genes, especially E1B, E2, E3 and E4. Viruses containing only a small (ie, 243 amino acid) protein were only very weak in transactivating viral genes and did not promote replication of adenovirus in those cells that did not contain YB-1 in the nucleus. Since YB-1 is only present in the nucleus of tumor cells and can only be detected there, this vector is suitable for inducing tumor-specific replication. the

Due to the absence of CR3 in dl520, the adenovirus is unable to translocate cellular YB-1 into the nucleus of the cell, which is also referred to herein as a translocation, and thus is not a site for replication in YB-1 nuclear-negative cells, Thus are viruses which can be used according to the invention, wherein said virus comprises the transactivation required according to the invention. the

Example 2: The mode of action of adenovirus depends on the Rb state of the cell

Figure 2 shows the binding domains of the E1A protein involved in the binding of p300, p107 and p105. P300 and p107 are cell-associated proteins. Retinoblastoma protein (pRb) is a tumor suppressor protein whose binding is mediated through CR1 and CR2. Studies have shown that pRb and p107/p300 bind the cellular transcription factor E2F, which is effective in regulating transcription. Wild-type E1A protein can interfere with the binding of E2F to Rb. The E2F thus released can bind to the E2 early promoter and thereby induce the replication of the adenovirus. the

It is known from the prior art that certain deletions in the E1A oncoprotein may result in recombinant adenoviral vectors such as those mentioned below, which are able to replicate predominantly in Rb-negative cells and which can be used according to the invention. For example, adenoviral vector dl922-947 contains a deletion in the CR2 region (amino acid positions 122-129), vector CB016 has deletions in the CR1 region (amino acid positions 27-80) and CR2 region (amino acid positions 122-129) . Vector E1Ad101/07 contains a deletion in the CR2 region (amino acid positions 111-123). Also, because of other deletions at the N-terminus (amino acid positions 4-25), otherwise, no binding to protein p300. The adenoviral vector Ad[Delta]24 contains a deletion in the CR2 region (amino acid positions 120-127). The adenoviral vector described in patent EP 0 931 830 contains deletions in the CR1 and CR2 regions. the

The mechanism of E2F/RB binding and E1A-mediated release of E2F is fundamentally different from the mechanism underlying the present invention. Contrary to what was assumed in the prior art, it was not the release of E2F from the Rb protein, which is essential if not critical for viral replication, but the nuclear localization of the human transcription factor YB-1. This transcription factor is present only in the cytoplasm during most of the cell cycle in normal cells. After infection with adenovirus, in some cases it is induced into the nucleus or already present in the nucleus in different cell systems, such as different neoplastic diseases, which include for example but not limited to breast cancer, ovarian cancer, prostate cancer , osteosarcoma carcinoma, glioblastoma, melanoma, small cell lung cancer and colorectal cancer. the

Example 3: Infection of U2OS cells

100,000 U2OS cells were plated per well. The next day the cells were infected with different adenoviruses as shown in FIG. 3 . Infect in 500 μl serum-free DMEM medium for 1 hour at 37°C. Subsequently, the infection medium was removed and replaced with 2 ml of complete medium (10% FCS/DMEM). Analysis was performed after 3 days using crystal violet staining. the

As can be obtained from Figure 3, U2OS cells that do not contain YB-1 in the nucleus show no lysis as indicated by crystal violet staining after infection with two different adenoviruses, namely E1/E3- Negative E1/E3-deleted adenoviruses, and adenovirus dl520, can be used according to the invention. In relation thereto, the culture medium is removed first. Subsequently, cells were covered with crystal violet (50% ETOH, 3% formaldehyde, 5% acetic acid, 1% crystal violet) and incubated at room temperature for 5-10 minutes. Subsequently, the plate with 6 wells was rinsed well with water and dried at room temperature. the

This confirms the discovery underlying the present invention that the presence of YB-1 is required in order to induce the virus used according to the invention to lyse infected cells. the

Example 4: Infection of 257RDB cells

100,000 257RDB cells were plated per well. The next day the cells were infected with different adenoviruses as shown in FIG. 4 . Infect in 500 μl serum-free DMEM medium for 1 hour at 37°C. Subsequently, the infection medium was removed and replaced with 2 ml of complete medium (10% FCS/DMEM). Analysis was performed after 3 days using crystal violet staining. the

The results of this experiment are depicted in FIG. 4 . The E1/E3-deleted adenovirus called E1/E3-negative Ad5 did not show any lysis at low MOI (pfu/cell) after infection of 257RDB cells containing YB-1 in the nucleus. In contrast, dl520, as shown in Example 3, does not replicate in YB-1 nuclear-negative cells, while encoding E1A for transactivating oncogene proteins according to the present invention, at an MOI of 40 pfu/cell (infection plural) resulted in virtually complete lysis, and still resulted in significant lysis at an MOI of 10 pfu/cell. From this it can be concluded that dl520 and similar viruses as described herein by dl1119/1131 or AdXvir 03 require about an order of magnitude (ten-fold) reduction compared to E1-deleted or E1/E3-deleted adenoviruses MOI, which justifies their clinical utility. the

As shown in Figure 7, protein E1A of dl520 is characterized by its deletion of the CR3 region, leading to transactivation and replication in YB-1 nuclear-positive cells required for the application according to the invention. the

Example 5: Infection of 257RDB and U2OS cells with dl1119/1131

As shown in Figure 5, there was no lysis at an MOI of 20 pfu/cell after infection of YB-1 nuclear-negative U2OS cells with adenovirus dl1119/1131, which lacks amino acids 4-138 of the E1A protein and encodes their nucleic acids, and additionally comprise a stop codon after amino acid 218, wherein the truncated E1A protein expressed comprises the CR3 region of the complete E1A protein. As a negative control, an uninfected cell layer was used. the

In contrast, in a cell system such as a cell containing YB-1 in the nucleus, ie a YB-1 nuclear-positive 257RDB, under the influence of adenovirus dl1119/1131, the cell layer was virtually completely lysed at an MOI of 20 pfu/cell. This example is therefore further evidence that a modified E1A oncogene protein as shown in Figure 7, comprising only, for example, the CR3 region and lacking the CR1 and CR2 regions, can provide the desired effect in YB-1 nuclear-positive cells. The required transactivation, which is required for the replication of the adenovirus according to the invention, leads to viral replication. Adenovirus dl1119/1131 is thus another adenovirus that can be used according to the invention. The present invention encompasses that viruses having a design similar to dl1119/1131 in the CR3 region but having the CR1 region and/or the CR2 region instead can also be used. the

Example 6: Detection of nuclear YB-1 in multidrug resistant cells

This example is based on the following considerations, that is, nuclear YB-1 should be used as a transcription factor to bind to the Y-box (CAAT sequence) in the mdr1 promoter (multiple drug resistance promoter in English). For detection, a so-called EMSA analysis (Electrophoretic Mobility Shift Assay) is carried out. In relation thereto, nucleoproteins are isolated and 1-10 μg of protein are subsequently incubated with short DNA fragments (oligos) at 37°C. For the determination of nuclear YB-1, the following oligonucleotides were used: mdr1 promoter (positions -86 to -67) opposite U2O3: TGAGGCTGATTGGCTGGGCA (X-box underlined). the

Prior to this, the DNA fragment was radiolabeled with ³² P at the 5' end. Subsequently, separation was carried out in native polyacrylamide gels. In the case of protein YB-1 binding to sequences in oligonucleotides, it can be detected because any unbound oligonucleotides migrate faster in the gel than bound oligonucleotides (Holm, P. S. et al., JBC 277, 10427-10434, 2002; Bargou, RC et al., Nature Medicine 3, 447-450, 1997).

As shown in Figure 6, EMSA analysis could confirm the presence of YB-1 in the nuclei of the multidrug resistant cells 257RDB, 181RDB and MCF-7Ad cells in contrast to the cell lines U2OS and HeLa cells. the

The results of Examples 4 and 5 demonstrate that, in contrast to U205, adenoviruses dl520 and dl1119/1131 replicate in YB-1 nuclear-positive cells such as 257RDB and induce their lysis. This confirms the findings regarding the use of the adenovirus according to the invention. In addition, the results have demonstrated weak transactivation of viral genes by modified or deleted E1A gene products in YB-1 nuclear-positive cells in the presence of nuclear YB-1 compared to wild-type adenovirus. Leading to successful replication and lysis of these cells, including, for example, multidrug resistant cells, the adenoviruses described herein can thus be used to lyse these tumors. the

Embodiment 7: Improve the replication efficiency of E1-negative adenovirus

This example demonstrates that early viral genes E1B-55K and E4orf6 can be replaced by transfection with plasmid pE4orf6 and infection with E1/E3-deleted adenovirus Ad-55K. Ad-55K is an E1/E3 deleted virus whereby E1B-55K was cloned into El and under the control of CMV. This substitution was required in light of the fact that AdYB-1 (adenovirus expressing YB-1) does not express these early genes, and the inventors of the present invention have realized that in a replication system containing YB-1 in the nucleus, these Replacement of early genes was able to improve replication efficiency and particle formation efficiency, respectively, to a degree comparable to wild-type adenovirus of one type Ad5. the

Do the following:

Each ¹⁰⁵ U2OS cells were transfected with plasmid pE4orf6 using lipofectamine. Plasmid pE4orf6 carries the DNA sequence encoding the early viral gene E4orf6 under the control of CMV.

24 hours after transfection with plasmid pE4orf6, E1/E3-deleted adenovirus AdYB-1 (50 pfu/cell) expressing YB-1 and E1/E3-deleted E1B-55K adenovirus Ad-55K (50 pfu/cell) cells) infected cells. Ad-55K is an E1/E3-deleted virus carrying the viral gene E1B-55K as a transgene under the control of CMV. the

Subsequently, cells were removed from the culture medium (2 ml) 5 days after infection (=post infection). Virus particles were released from isolated cells by alternating freezing and thawing 3 times (thaw/freeze). Subsequently, 293 cells were subjected to a plaque assay to measure the infectious particles produced (plaque forming units/ml (pfu/ml)). The results are shown in Figures 8 and 9. Figure 8 shows the results of the plaque assay, expressed as absolute values. The most striking difference compared to infection with AdYB-1 alone was shown by transfection with plasmid pE4orf6 and co-infection with both viruses, AdYB-1 and Ad-55K. Figure 9 shows the results of Figure 8, where the increase in replication efficiency is expressed as the replication factor determined for AdYB-1. Cells infected with plasmid pE4orf6 and subsequently with AdYB-1 and E1B-55K (Ad-55K) produced up to 25-fold more pfu/ml. the

Based on these results it can be concluded that substitution of E1B-55K and E4orf6 increased the amount of virus formed (pfu/ml) up to 25-fold upon infection with the E1/E3-deleted adenovirus AdYB-1. The additive effects of E1B-55K and E4orf6 on the generation of plaque forming units (pfu) were significantly higher compared to the individual effects of the two gene products. the

Control experiments using a plasmid expressing EGFP clearly demonstrated that only 10% of cells were successfully transfected with plasmid pE4orf6 in the chosen experimental method. The number of particles formed in cells expressing E1B-55K and E4orf6 was comparable to that of a human adenovirus type 5 (wild type). This confirms the discovery that forms the basis of the present invention that the expression of E4orf6 and E1B-55K, combined with the nuclear localization of YB-1, can provide adenoviral replication and particle formation, especially for E1A-deleted adenoviruses, which are comparable to A wild-type Ad5. the

Example 8: Elevated adenosis after administration of cytostatics in YB-1 nuclear-positive cells

Virus replication, the adenovirus does not replicate in YB-1 nuclear negative cells

It is known in the prior art that the addition of different cytostatic agents induces the nuclear localization of the human transcription factor YB-1. As the inventors of the present invention have found, YB-1 localized in the nucleus controls adenovirus replication by activating the adenovirus 2-late promoter. To provide specific tumor lysis, both actions can be used in combination. the

In the implementation of the tumor digestion assay, the following steps were followed: 200,000 cells (HeLa and U2OS, respectively) were seeded into each well of a 6-well plate. The next day, add 40 ng/ml (final concentration) of daunorubicin. After 3 h of incubation, cells were infected with 10 and 30 pfu dl520/cell, respectively. Subsequently, the cells were incubated in medium without cytostatics. After 3-5 days, cells were stained with crystal violet. the

As can be seen from Figures 10 and 11, addition of daunorubicin induces replication of dl520 through nuclear localization of YB-1. Thus, dl520 produced a greater tumor lytic effect in combination with the cytostatic growth inhibitor daunorubicin compared to daunorubicin alone. the

Example 9: In vivo tumor lysis of dl520

HeLa (YB-1 nuclear-negative) and 257RDB (YB-1 nuclear-positive) cells for in vivo studies were propagated under sterile cell culture conditions. Before injecting cells into mice (CD1NuNu strain) to generate subcutaneous tumors, cells were harvested by trypsinization, placed in DMEM medium (10% FCS), counted and washed once with PBS. Subsequently, the cells were centrifuged, the PBS was aspirated, and the cells were aliquoted in fresh PBS at the desired cell number. In this study, the number of cells injected subcutaneously was 5 x ¹⁰⁶ cells for each of the two cell lines. Subcutaneous injections were made to one side of the animal, where HeLa cells were injected on the right side and 257RDB cells on the left side for better differentiation. Tumor growth was compared twice a week, where the length and width of the tumor was measured using a vernier caliper. Based on this, the tumor volume was calculated based on the following mathematical formula:

3/4π*a/2*(b/2) ² a=length, b=width

Once the tumors had reached a volume of 200-520 ^mm3 , the virus and PBS as a negative control were administered intratumorally, respectively. Injection volumes were equal, 50 μl each time. Injections were repeated for 3 consecutive days. The total dose of virus administered was 5 x 10 ⁸ pfu. Subsequently, the tumor growth was recorded twice a week, and the volume was calculated. Mice were sacrificed at the end of the study and tumors were removed for further analysis.

The results are shown in Figures 12 and 13. the

Figure 12 shows a graph representing tumor volume as a function of time and different treatment regimens. In the case of tumor formation by RDB257, the tumor grew significantly to about 438 mm ³ -1466 mm ³ after PBS injection. Under the influence of the vector dl520 used according to the invention, the growth of tumors was significantly reduced. Starting from a mean tumor size of 344 mm ³ , tumor size increased by only 21% to a total of 543 mm ³ .

In this example, a tumor consisting of HeLa cells was used as a control, which behaved similarly to RDB257-based tumors after PBS administration. However, tumors based on HeLa cells and treated with dl520 still showed a significant increase in tumor growth, starting from 311 mm ³ to 1954 mm ³ .

Figure 13 shows photographs of killed nude mice with tumors grown using RDB257. It can clearly be seen that following administration of the adenovirus dl520 according to the invention, a significant reduction in tumors occurs. In this case there was even a reduction in tumor volume (day 1 after administration of virus dl520: 515 mm ³ ; day 30 after administration of virus dl520: 350 mm ³ ).

Example 10: Southern Blotting of Tumor DNA

DNA was extracted from tumor samples taken from the middle of the tumor propagated in Example 9. For isolation, Qiagen's Dneasy Tissue Kit was used. Complete DNA isolation according to the manufacturer's instructions. According to it, DNA is released from cells by alkaline lysis. Subsequently, the isolated DNA was column purified. Subsequently, the concentration of isolated DNA was determined by photometry at 260 nm. For the analysis, 2 μg of DNA samples treated with 10 units of restriction enzyme KpnI were used for analysis. Subsequently, electrophoretic separation of the samples was performed on a 0.8% agarose gel. Subsequently, the DNA was blotted on a nylon membrane (performed according to the Schleicher & Schuell system). The DNA blotted on the membrane was hybridized to a specific 1501 bp DNA probe. The 1501bp DNA probe specifically binds to the 3369bp Kpn I fragment within the Ad5 sequence encoding E2A. Probes were previously prepared by PCR (primers: 5'-GTC GGA GAT CAG ATC CGC GT (SEQ.ID.No.2), 5'-GAT CCT CGT CGT CTT CGC TT (SEQ.ID.No.3)), and radioactively labeled with ³² P. Subsequently, the membrane is washed and exposed to film.

The results of Southern blot of tumor DNA are shown in FIG. 14 . Analysis confirmed that only dl520 replicated in vitro in resistant cells RDB257, as shown in lanes 3, 4 and 5. Lane 1 shows the positive control Ad-5d, and lanes 6, 7 and 8 show DNA from HeLa cells infected with dl520. Since HeLa cells are not YB-1 nuclear positive, the virus dl520 does not replicate and thus the E2A sequence cannot be detected. the

Additional results for dl520 are shown in FIG. 15 . Particle formation (pfu/ml) was studied after infection with dl520 and wild type adenovirus based on plaque assay. Various YB-1 nuclear-positive (257RDB and 181RDB) tumor cells and YB-1 nuclear-negative tumor cells were infected with dl520 and wild-type adenovirus. the

Carry out the following steps:

100,000-200,000 cells were each seeded into so-called 6-well plates (ie, 6-well plates) in L15 medium (resistant cells) and DMEM containing 10% FCS (non-resistant cells). After 24 hours, infection was performed with dl520 and wild-type adenovirus (10 pfu/cell). Three days post-infection (after infection), virus particles were released from the cell suspension (3 ml) by alternating freezing and thawing three times. Subsequently, a plaque assay was performed on 293 cells to measure the infectious particles formed (plaque forming units/ml (pfu/ml)). The results are shown in Figure 15. The results of the plaque assay showed that dl520 replicated in YB-1 nuclear-positive cells (257RDB and 181RDB) similarly to wild-type adenovirus. Thus, when using the adenoviruses described herein according to the present invention, a replication efficiency similar to that of a wild-type adenovirus can be observed. the

Example 11: Structural Design of Adenoviral Vector Xvir03

Figure 16 shows the structural design of the adenoviral vector Xvir03. Adenovirus Xvir03 is a so-called E1/E3-deleted adenovirus. This means that the E1A, E1B and E3 proteins that function in adenovirus replication cannot be produced. The deletion of the El region extended from 342 to 3528; the E3 region was deleted at amino acid positions 27865-30995. As used herein, the term "E1-deleted virus" refers to a virus in which El is no longer functionally active. This can be achieved by inactivating an otherwise largely intact nucleic acid and amino acid sequence, however this can also mean deletions of proteins encoding the E1 region with different sizes. Because of the absence of the E1A and E1B proteins and the nucleic acids encoding them, the E4 region (such as E4orf6) is only weakly expressed (approximately 1-5% compared to wild-type adenovirus) or not expressed at all. Viral genes E1B55k and E4orf6 were expressed in the El region by introducing a heterologous CMV promoter (Clontech: PlasmidpShuttle) into Xvir03. Instead of the CMV promoter, various and any promoters disclosed herein that are relevant to ElA expression can be used. The open reading frames of the two genes are linked to each other by a so-called IRES sequence (internal ribosomal entry site in English) (Pelletier, J. and Sonenberg, N. Nature, 1 988, 334, 320-325) . This element (Novagen: pCITE) provides expression of 2 proteins from 1 mRNA. the

Prepare the vector as follows:

Plasmid E1B55k-pShuttle was generated by cloning the E1B55k open reading frame of pCGNE1B from M. Dobelstein (University of Marburg) with XbaI and BfrI into Clontech's pShuttle vector. Subsequently, E1B55k in pShuttle was linearized with Apal, blunt-ended and cut with NheI. the

In the second vector, pcDNA3.1(+) (Invitrogen), consecutive to each other, the IRES element as a PCR product was cloned into the EcoRV cleavage site by TA cloning using pCITE-4a(+) from Novagen as a template , E4orf6=IRES-E4orf6-pcDNA3.1(+) from plasmid pCMV-E4orf6 (M. Dobelstein, University of Marburg) was cloned by means of BamHI. IRES-E4orf6 in pcDNA3.1(+) was linearized with NotI, blunted at the ends, and then the fragment IRES-E4orf6 was excised with NheI. The fragment IRES-E4orf6 was ligated with the open vector E1B55k-pShuttle (blunt end, NheI). Cassettes were subsequently cloned into ΔE1, ΔE3 Adeno-X-plasmids (Clontech) from E1B55k-IRES-E4orf6-pShuttle and CMV promoters and bovine growth hormone (BGH)-PolyA using I-Ceu I and PI-SceI, It is called AdcmvE1B/IRES/E4orf6. Subsequently, adenovirus was prepared according to the manufacturer's instructions (Clontech). An adenoplasmic plasmid containing the expression element CMV-E1B55k-IRES-E4orf6-BGH polyA linearized with PacI was transfected into HEK293 cells, and 11 days after transfection, the detached cells were removed together with the culture medium to allow for repeated freeze-thaw cycles Release the adenovirus. the

The vectors described above are in principle suitable for the other viruses described herein for use according to the invention. In particular the above-mentioned vectors are suitable for replicating and inducing lysis in cells which are YB-1 nuclear-positive cells and cells in which YB-1 is deregulated (i.e. overexpressed compared to normal cells and non-tumor cells), respectively . The use of this vector is particularly applicable to those diseases and patient groups or patient populations disclosed together with other adenoviruses described herein for use according to the invention and other adenoviruses disclosed herein. the

Example 12: Structural Design of Adenovirus Vector Xvir03/01

As can be obtained from Figure 17, Xvir03/01 is a further development of Xvir03. Therapeutic genes (such as those described herein) and transgenes can be cloned into the E3 region. Additionally, a deletion was introduced in the E4 region in order to avoid homologous recombination with E4orf6 from the Xvir03 expression cassette. This allows larger transgenes to be cloned into the construct. The deleted E3 region contains SacI, NdeI and NheI restriction sites for the introduction of cassettes, such as where therapeutic transgenes can be cloned. the

Preparation of plasmids for cloning therapeutic genes into the E3 region and for making deletions in the E4 region:

Clontech's pAdenoX-Plasmid has a restriction site for SfuI after the 3'ITR region that is absent in wild-type adenovirus. The E3-E4 region was obtained from pAdenoX (Clontech) using SpeI (site 23644) and SfuI and transferred to pcDNA3.1(+) (Invitrogen) = pcDNA3.1-E3Δ27865-30995-E4. Most of E4ORF6 was removed by PstI, ie 33241-33875=pcDNA3.1-E3Δ27865-30995, E4Δ33241-33875. For further development of Xvir03, the deleted E3/E4 region was cloned from pcDNA3.1-E3Δ27865-30995, E4Δ33241-33875 into plasmid pAdenoX with SfuI and SpeI=pAdenoX E3Δ27865-30995, E4Δ33241-33875. the

The expression cassette was subsequently cloned from E1B55k-IRES-E4orf6-pShuttle with CMV promoter and bovine growth hormone (BGH)-PolyA with I-Ceu I and PI-SceI into pAdenoX E3Δ27865-30995, E4Δ33241-33875 as described for Xvir03 In , it is called AdcmvE1B/IRES/E4orf6-ΔE4. Subsequently, adenovirus was prepared according to the manufacturer's instructions (Clontech). the

The vectors described above are in principle as useful as the other viruses described herein for use according to the invention. In particular the vectors described above are suitable for replicating and causing lysis in cells that are YB-1 nuclear-positive as well as cells in which YB-1 is deregulated (ie overexpressed compared to normal cells and non-neoplastic cells). This vector can also be used in those diseases and patient groups and patient collectives which are disclosed herein with respect to other adenoviruses used according to the invention and adenoviruses according to the invention. the

Example 13: The effect of Xvir03 on tumor digestion in 257RDB and 181RDB cells

100,000 cells (257RDB and 181RDB) were seeded in each well of a plate with 6 wells (6-well plate). The next day, as shown in Figure 18, cells were infected with Ad312 (20 pfu/cell) and Xvir03 (5 pfu/cell). Infect in 500 μl serum-free DMEM medium for 1 h at 37 °C. Subsequently, the infection medium was removed and replaced with 2 ml of complete medium (10% FCS/DMEM). Analysis was completed after 5 days by crystal violet staining. The results are shown in Figures 18A and 18B. the

As can be obtained from Figures 18A and 18B, after infection with Ad312 and Xvir03, and only in the case of Xvir03, multidrug-resistant cells containing YB-1 in the nucleus showed lysis, as indicated by crystal violet staining of the cells . In relation thereto, the culture medium is removed first. Subsequently, cells were covered with crystal violet (50% ETOH, 3% formaldehyde, 5% acetic acid, 1% crystal violet) and incubated at room temperature for 5-10 minutes. Subsequently, the 6-well plate was rinsed well with water and dried at room temperature. the

The inventors of the present invention have known E1A-deleted viruses (such as Ad312), which however are not transactivating adenoviruses in the sense of the present invention, may replicate very efficiently at higher MOIs (Nevins J.R., Cell 26, 213 -220, 1981), but cannot be implemented in clinical applications. This phenomenon is referred to in the literature as "E1A-like activity". The adenovirus Ad312 used herein is an E1A-deleted virus. At the titer used (20 pfu/cell), the titer is still higher than the clinical ideal titer, and early adenovirus genes such as E1B55k and E4orf6 are not expressed or can only be expressed in a very small amount (Nevins J.R., Cell 26, 213-220 , 1981). As already described herein, these genes and proteins play an important role in viral replication. In contrast, these genes and proteins were expressed by adenovirus Xvir03, respectively (Fig. 16). As can be obtained from Figures 18A and 18B, expression of the genes E1B55k and E4orf6 will result in efficient viral replication and cell lysis with concomitantly required lower infection titers (expressed as pfu/cell). This confirms the discovery underlying the present invention that expression of E4orf6 and E1B-55K (and lack of E1A) combined with nuclear localization of YB-1 can induce very efficient adenovirus replication. Its required titer of only 1-5 pfu/cell is now clinically applicable. the

This confirms the finding that forms the basis of the present invention that the presence of YB-1 in the nucleus, in particular independent of the cell cycle, is necessary for the viruses used according to the invention to lyse infected cells. the

Example 14: Northern blot analysis of E2 gene expression of adenovirus Ad312

In each case, 1 million A549 and U2OS cells were seeded into 10 cm Petri dishes. The next day, cells were infected with Ad312 (50 pfu/cell) and Adwt (as a control, 5 pfu/cell). The high viral titers of Ad312 used resulted in El-independent replication in tumor cells. Infect in 1-2 ml serum-free DMEM medium for 1 hour at 37°C. Subsequently, the infection medium was removed and replaced with 10 ml of complete medium (10% FCS/DMEM). After 3 days, RNA was isolated. Subsequently, the concentration of isolated RNA was detected in a luminometer at 260 nm. Then, the RNA samples were separated by electrophoresis in a 0.8% formaldehyde agarose gel. Subsequently, the RNA was blotted on a nylon membrane (performed according to the system of Schleicher & Schuell). The RNA blotted on the membrane was hybridized to "early probe" E2 and "late probe" E2. The 1501bp "late probe" can specifically bind to the E2 late promoter. Probes were previously prepared by PCR (primers: 5'-GTC GGA GAT CAG ATC CGC GT (SEQ.ID.NO.4), 5'-GAT CCT CGT CGT CTT CGC TT (SEQ.ID.NO.5)), and radioactively labeled with ³² P. On the contrary, the early probe can bind between the E2-early promoter and the E2-late promoter (position: 226791-227002), and also by means of PCR (primer: 5'-AGCTGATCTTCGCTTTTG (SEQ.ID.NO.6 ), 5'-GGATAGCAAGACTCTGAC AAAG (SEQ.ID.NO.7)) produced. Subsequently, the membrane is washed and exposed to film.

The results are shown in FIG. 19 . Both early and late probes provided specific signals for wild-type adenovirus control infection, whereas Ad312-infected tumor cells only provided specific signals when late probes were used. This confirms the finding that underlies the present invention that expression of E4orf6 and E1B55K and deletion of E1A transports overexpressed and deregulated YB-1, respectively, into the nucleus and thereby induces a prerequisite for efficient adenoviral replication Expression of the E2 gene. Example 15: Northern blot analysis of E2 gene expression of adenovirus Adδ24

In each case, 1 million U2OS cells were seeded into 10 cm Petri dishes. The next day, cells were infected with adenovirus delta 24 (Adδ24) (10 pfu/cell) and wild-type adenovirus (Adwt) (as a control, 10 pfu/cell). The recombinant adenovirus Adδ24 used (Fueyo, J. et al., Oncogene 19, 2-12, 2000) has a specific deletion in the CR2 region of the E1A protein and thus can only replicate in Rb-negative tumors. In addition, the virus expresses genes E1B55k and E4orf6 comparable to wild-type adenovirus. Infect in 1-2 ml serum-free DMEM medium for 1 hour at 37°C. Subsequently, the infection medium was removed and replaced with 10 ml of complete medium (10% FCS/DMEM). After 12 and 24 hours, RNA was isolated. Subsequently, the concentration of isolated RNA was detected in a luminometer at 260 nm. Then, the RNA samples were separated by electrophoresis in a 0.8% formaldehyde agarose gel. Subsequently, the RNA was blotted on a nylon membrane (performed according to the system of Schleicher & Schuell). The RNA blotted on the membrane was hybridized to "early probe" and "late probe". The "late probe" comprising 1501 bp specifically binds to the E2 late promoter. Probes were prepared by PCR (primers: 5'-GTC GGA GAT CAGATC CGC GT (SEQ.ID.NO.4), 5'-GAT CCT CGT CGT CTT CGC TT (SEQ.ID.NO.5)) before, and Radioactively labeled with ³² P. However, the early probe can be combined between the E2-early promoter and the E2-late promoter, and also by means of PCR (primers: 5'-AGCTGATCTTCGCTTTTG (SEQ.ID.NO.6), 5'-GGATAGCAAGACTCTGAC AAAG( SEQ.ID.NO.7)) produced. Subsequently, the membrane is washed and exposed to film.

The results are shown in Figure 20. the

After 12 hours, only the late probe provided a specific signal. After 24 hours, only the early probe provided a specific signal in cells infected with Adδ24. However, the signal was significantly attenuated compared to wild-type adenovirus. This result also confirms the finding underlying the present invention that expression of E4orf6 and E1B-55K transports overexpressed and deregulated YB-1, respectively, into the nucleus where it subsequently binds to the E2-late promoter and induces the E2 gene Express. the

Example 16: Structural Design of Adenoviral Vectors XvirPSJL1 and XvirPSJL2

Vector Description: Vectors of the XvirPSJL group are embodiments of viruses referred to herein as group I adenoviruses, and are exemplified by the vectors and adenoviruses XvirPSJL1 and XvirPSJL2, respectively, which not only are capable of infecting YB-1 nucleus-positive cells like adenovirus dl520 , especially in tumor cells, and can replicate in tumor cells, and YB-1 in said tumor cells is overexpressed and deregulated respectively. Whereas the viral genes E1B55k and E4orf6 were only expressed under the influence of the E1B promoter and E4 promoter, respectively, in dl520-infected YB-1 nuclear-positive cells, E1B55k and E4orf6 in XvirPSJL were expressed via the cytomegalovirus (cmw) promoter. Express. However, not only the cmw promoter, but also other promoters, in particular tumor-specific, tissue-specific and organ-specific promoters, can be used. Due to the expression of E1B55k and E4orf6, overexpressed YB-1 and deregulated YB-1 were translocated into the nucleus, respectively, and initiated the replication of adenovirus. The adenoviral vectors of the XvirPSJL group as described herein thus combine the various elements and thus their functions of the adenoviral vectors dl520, Xvir03 and AdYB-1 in the same vector. Similar to the vector dl520, the XvirPSJL virus contains the E1A12S gene. This gene and the corresponding gene product are responsible for inducing the S phase of infected cells and promoting the replication of the virus, the effects of chemotherapy and irradiation, respectively. Like Xvir03, the XvirPSJL virus contains the expression cassette CMV-E 1B55k/IRES/E4orf6, which is required for efficient replication and indirectly or directly transfers deregulated YB-1 to the nucleus, which is preferably contained in in tumor cells. Thus, replication is only possible in cells, especially tumor cells, in which YB-1 is overexpressed or deregulated. Additionally, P53 is degraded by the E1B55k/E4orf6 complex. The sequence encoding human transcription factor YB-1 was taken from the virus AdYB-1. Endogenous, ie, YB-1 already present in the cell amplifies viral replication. Expression of E1A12S and YB-1 is controlled by the YB-1-dependent adenoviral E2-late promoter. Also related, specific promoters, especially tumor-specific, tissue-specific or organ-specific promoters may be used. Another characteristic of these viruses is the deletion of the E4 region. The vectors contain restriction sites through which, in the case of the adenoviral vectors XvirPSJL1 and XvirPSJL2, the various transgenes disclosed in the specification can be expressed, such as ribozymes, antisense molecules, siRNA, apoptosis-inducing genes, cytokines and prodrug genes. Their expression can also be controlled by the tumor-specific, tissue-specific or organ-specific promoters disclosed in the specification. The positioning of the expression cassettes is not fixed, in particular not about or within the El, E3 and E4 regions, but can be arranged in any way. Vector replication is independent of p53 or the Rb status of tumor cells. the

The structural designs of the recombinant adenoviruses XvirPSJL1 and XvirPSJL2 are shown in Figures 21 and 22. the

Generation of cassette E2-late-YB1IRES/12S:

The pAdenoX plasmid from Clontech/BD Biosciences, used as starting material herein, contains the genomic nucleic acid of the adenovirus Ad5 and has an SfuI restriction site behind the 3' ITR region (which wild-type adenovirus does not have). The E3-E4 region was transferred to pcDNA3.1(+) (Invitrogen) using SpeI (site 23644) and SfuI from pAdenoX (Clontech) and denoted as pcDNA3.1-E3Δ27865-30995-E4. Most of E4ORF6, bases 33241-33875, was removed by PstI. The fragments thus obtained are called pcDNA3.1-E3Δ27865-30995, E4Δ33241-33875. the

The E2-late promoter was isolated from pGL3-EGFP with SacI and NheI (Holm et al., JBC2002, 277, 10427-10434) and cloned into pcDNA3.1-E3Δ27865-30995, E4Δ33241-33875. During this process, the E3 region was further deleted in the Δ27593-31509 region of bases. The fragment thus obtained is called E2-late-pcDNA3.1-E3Δ27593-31509, E4Δ33241-33875. the

cDNA for the E1A-243AA product was generated by RT-PCR, isolated, sequence checked, and cloned into pcDNA3.1(+) vector (Invitrogen) using BamHI and EcoRI. E1A-12S-pcDNA3.1+ was linearized with NheI and BamHI, the ends were blunt-ended by T4 polymerase, and T-overhangs were provided by Taq polymerase and dTTP. The IRES element was cloned as a PCR product (template = pCITE, Novagen) into the E1A-12S-pcDNA 3.1(+) vector (TA cloning strategy). the

The YB-1-EcoRI fragment (Holm et al., JBC 2002, 277, 10427-10434) was isolated from the vector pHVad2c and blunt-ended. Vector pShuttle (commercially available from BD Biosciences) was linearized with XbaI, blunt-ended, dephosphorylated, and ligated to a previously generated nucleic acid encoding YB-1. The vector thus obtained was named YB-1-pShuttle. Cloning into the pShuttle vector will provide nucleic acid encoding the YB-1 fragment with an in-frame stop codon. The nucleic acid encoding YB-1 was cloned from YB-1-pShuttle into the vector IRES-E1A-12S in pcDNA3.1(+) with the aid of NheI and BfrI. The vector thus obtained was called YB-1 (EcoRI-EcoRI with stop codon)-IRES-E1A-12S-pcDNA3.1(+). the

Subsequently, the cassette YB-1-IRES-E1A12S was cut with PmeI and cloned into the NheI linearized, blunt-ended and dephosphorylated vector E2 late-pcDNA3.1E3Δ27593-31509, E4Δ33241-33875. Thus, the second cassette is in the deleted region of the E3 region. the

The transgene cassette comprises the nucleic acid construct E2 late stage-YB-1-IRES-E1A12S, which is cloned into Clontech's vector pAdenoX (=AdenoX/E2 late stage- YB-1-IRES-E1A12S/E3Δ27593-31509, E4Δ33241-33875). the

Cassette CMV-E1B55k/IRES/E4orf6 was excised from pShuttle as described above for Xvir03 via I-CeuI and PI-SceI and inserted into the vector AdenoX/E2 Late-YB-1-IRES-E1A12S/E3Δ27593-31509, E4Δ33241 -33875. the

Subsequently, the vector was linearized with Pac I and transfected into 293 cells. According to the manufacturer's instructions, the recombinant adenoviruses XvirPSJL1 and XvirPSJL2 that did not contain the transgene marked in the figure were isolated respectively. the

Example 17: Infection of HeLa cells with adenovirus dl520

100.000 HeLa cells were plated per plate. The next day, cells were infected with different titers (pfu/ml) of adenovirus dl520. Infect in 500 μl of serum-free DMEM medium for 1 hour at 37°C. Subsequently, the infection medium was removed and replaced with 2 ml of complete medium (10% FCS/DMEM). After 3-5 days, analysis was performed using crystal violet staining. the

The experimental results are shown in Figure 23. Adenovirus dl520 did not show any lysis at low MOI (5-10 pfu/cell) infecting HeLa cells without YB-1 in the nucleus, in contrast to dl520 at an MOI (multiplicity of infection) of 100-200 pfu/cell Practically complete lysis and still exhibited significant lysis at an MOI of 50 pfu/cell. From this it can be seen that dl520 and similar viruses that switch on the adenoviral genes E1B55k and E4orf6 are suitable at higher MOIs to transport overexpressed or deregulated YB-1 directly or indirectly into the nucleus and thus Induces cell lysis. Example 18: Luciferase assay for detecting E2-late promoter activity

YB-1 is known to bind to the adenovirus 2-late promoter in the nucleus (Holm et al., JBC 2002, 277, 10427-20434), and this promoter is also well suited for expression of nucleic acids. The use of the adenovirus 2-late promoter is particularly motivated by the fact that it can be controlled by YB-1, which acts as a positive effector, that is, the promoter is only present in the nucleus where YB-1 is present. case is active. The adenoviral E2-late promoter can thus be regulated in a highly selective manner when YB-1 is absent in the nucleus as an effector and regulator, respectively, and thus applies to systems where YB-1 is present in the nucleus In practically any expression of nucleic acids under the control of the adenoviral 2-late promoter is avoided. The E2-late promoter contains a 3Y-box (CCAAT), which is involved in the activation of the E2 gene. Different E2-late promoter constructs have been prepared and tested for their specificity and activity. Analysis is performed as follows. the

The cell lines EPG-257RDB (epithelial gastric carcinoma), HeLa (epithelial cervical carcinoma) and U2OS (osteosarcoma) containing YB-1 in nuclei were seeded in 6-well plates using 3 different cell concentrations. Wells showing 70% confluence the next day were used for transfection. For each well, 500 ng of Spin Miniprep (Qiagen) purified plasmid DNA of different E2-late promoters in a luciferase vector (commercially available from Promega, starting plasmid: pGL3-enhancer) was added to a 1.5 ml band immobilized 500 μl OptiMEM in the capped reaction tube, 5 μl DOTAP was added to 500 μl in another reaction tube with fixed cap. The 2 solutions were combined and mixed. The mixture was incubated at room temperature for 30 minutes to allow complex formation. Cells were washed 3 times with PBS and covered with a layer of transfection mixture. The plates were incubated at 37°C for 5 hours, after which the cells were washed 3 times with PBS and provided with complete medium. the

48 hours after infection, the cells were treated with Promega's luciferase assay system kit (Cat.No.E1500): provide each well with a layer of 500 μl lysis buffer, and after 10 minutes at room temperature, use a 1ml pipette to remove the cells from the Plate wells were rinsed out and transferred to 1,5 ml reaction tubes with fixed caps. The cell lysate was then centrifuged at 14.000 rpm for 15 minutes at 4°C. 100 μl of luciferase substrate was added per 50 μl of supernatant and detected at 945 nm in a 96-well black plate with a TopCount (Canberra-Packard GmbH, 63303 Dreieich) microplate scintillation & fluorescence counter. the

Molecular dynamics of proteins were measured at 570 nm in a bioluminometer (Biolumin ^™ 960) kinetic fluorescence/absorption plate reader using the BCA protein assay kit Cat. No. 23227 (PIERCE, Rockford, Illinois, USA). The relative light signal of the sample was converted to protein amount (RLU/μg protein).

The following plasmids were used: pGL3-enhancer (Promega) was used as blank read, from which the enhancer was removed with BamHI (2250 bp) and BsaBI (2003 bp). Each E2 promoter construct was cloned into the MCS of the enhancer-deleted pGL3 vector with the aid of the restriction sites Apa I and Sac I. The hCMV promoter was cloned into the pGL3 enhancer with Bgl II and Hind III and served as a positive control. This positive control allows assessment of transfection efficiency and also serves as a reference value for luciferase activity. For each cell line, the CMV control was set at 100%, and the enzymatic activity produced by the E2 promoter construct was correlated with it, and a histogram was made as shown in FIG. 24 . the

The constructs mentioned are as follows:

1. Contains Y-box I, II and III, corresponding to bases 25932-26179 bp (for the wild-type adenovirus sequence, also refer to the subsequent adenovirus E2 region part)

2. Contains Y-box II and III, corresponding to base 25932-26127bp (for the wild-type adenovirus sequence, also refer to the adenovirus E2 region part provided later)

3. Contains Y-box III, corresponding to base 25932-26004 bp (for the wild-type adenovirus sequence, also refer to the adenovirus E2 region provided later)

4. Does not contain Y-box, read as a blank

Adenovirus E2 region part (taken from Virology 1992, 186, 280-285)

(YB-1 binding sites are printed in bold)

25561 aggaactttatcctagagcgctcaggaatcttgcccgccacctgctgtgcacttcctagc

25621 gactttgtgcccattaagtaccgcgaatgccctccgccgctttggggccactgctacctt

25681 ctgcagctagccaactaccttgcctaccactctgacataatggaagacgtgagcggtgac

25741 ggtctactggagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtttgc

25801 aattcgcagctgcttaacgaaagtcaaattatcggtacctttgagctgcagggtccctcg

25861 cctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggct

25921 taccttcgcaaatttgtacctgaggactaccacgcccacgagattagggttctacgaaga

cccgcccgccaaatgcggagcttaccgcctgcgtcattacccagggccacattctt  25981

cccgcccgccaaatgcggagcttaccgcctgcgtcattacccagggccacattctt

26041 gg tgcaagccatcaacaaagcccgccaagagtttctgctacgaaagggacgggggg

26101 gtttacttggacccccagtccggcgaggagctcaac ccccccgccgccgcagccc

26161 tatcagcagcagccgcgggcccttgcttcccaggatggcacccaaaaagaagctgcagct

26221 gccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttgga

26281 cgaggaggaggaggacatgatggaagactgggagagcctagacgaggaagcttccgaggt

26341 cgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgcccca

26401 gaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcact

26461 gcccgttcgccgacccaaccgtagatgggacaccactggaaccagggccggtaagtccaa

26521 gcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcatggcgcgg

26581 gcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccg

26641 ccgctttcttctctaccatcacggcgtggccttcccccgtaacatcctgcattactaccg

26701 tcatctctacagcccatactgcaccggcggcagcggcagcggcagcaacagcagcggcca

26761 cacagaagcaaaggcgaccggatagcaagactctgacaaagcccaagaaatccacagcgg

(SEQ.ID.No.8) 

The results shown in Figure 24 strongly demonstrate that each promoter fragment containing the different E2-late/Y-boxes is suitable for expressing therapeutic transgenes in YB-1 nuclear-positive tumor cells and thus can be used as the present invention. A promoter in the sense of the invention. the

Example 19: Effect of YB-1 expressed by adenovirus on particle release

Human osteosarcoma cells (U2OS) were infected with adenoviral vector AdYB-1 with deletion of E1/E3 and Ad312 with deletion of E1A only, MOI was 50pfu/cell. AdYB-1 contains in its genome a sequence encoding the cellular transcription factor YB-1 and thus expresses the Y-box binding protein 1 (YB-1). To evaluate post-infection release of virus particles as "plaque forming units" (pfu), culture supernatants or residual cell layers were isolated 2 and 5 days post-infection, respectively. Through 3 thaw/freeze cycles, the intracellular particles were released. Particle numbers were analyzed using a plaque assay on 293 cells. the

The results are shown in Figure 25, where the solid bars represent the residual virus particles inside the cells, and the crossed lines represent the released extracellular virus particles. the

The results shown in Figure 25 confirm that AdYB-1 as a whole generates more pfu and releases more particles than Ad312. After 5 days, cells infected with AdYB-1 clearly exhibited the opposite cytopathic effect (CPE) to cells infected with Ad312. the

The features of the invention disclosed in the preceding description, the claims and the drawings are essential for the realization of the invention in its various embodiments both individually and in any combination. the

Claims (36)

1. A recombinant adenovirus expressing a first protein prior to expressing a second protein, said first protein being selected from the group consisting of E1B protein, E4 protein, and a combination of E1B protein and E4 protein, said second The protein is an E1A-protein, wherein the expression of said first protein is controlled by a promoter, wherein said promoter is selected from the group consisting of tumor-specific promoters, organ-specific promoters, tissue-specific promoters, heterologous promoters and The group consisting of adenoviral promoters, wherein if the promoter for the E1B protein is an adenoviral promoter, the adenoviral promoter is different from the E1B promoter, and if the promoter for the E4 protein is an adenoviral promoter, then The adenovirus promoter is different from the E4 promoter, and wherein the expression of the E1A protein is controlled by a promoter, wherein the promoter controlling the expression of the E1A protein is controlled by YB-1. 2. The recombinant adenovirus according to claim 1, characterized in that said first protein is E1B protein. 3. The recombinant adenovirus according to claim 2, characterized in that said E1B protein is E1B55kd protein. 4. The recombinant adenovirus according to claim 1, characterized in that said first protein is E4 protein. 5. The recombinant adenovirus according to claim 4, characterized in that the E4 protein is E4orf6 protein. 6. The recombinant adenovirus according to claim 1, wherein the first protein is a combination of E1B protein and E4 protein. 7. The recombinant adenovirus according to claim 6, characterized in that said E1B protein is E1B55kD protein and said E4 protein is E4orf6 protein. 8. The recombinant adenovirus according to claim 1, characterized in that the E1A protein is the E1A12S protein. 9. The recombinant adenovirus according to claim 1, characterized in that the promoter controlling the expression of the E1A protein is the adenovirus E2 late promoter. 10. The recombinant adenovirus according to claim 1, characterized in that the E4 protein, and the E1B protein are under the control of the same or a common promoter. 11. The recombinant adenovirus according to claim 1, characterized in that, the nucleic acid of the adenovirus comprises at least one functionally inactive adenovirus region, wherein the region is selected from the group consisting of E1 region, E3 region, E4 region and combinations thereof Group. 12. The recombinant adenovirus according to claim 11, characterized in that the region is the El region. 13. The recombinant adenovirus according to claim 11, characterized in that the region is the E3 region. 14. The recombinant adenovirus according to claim 11, characterized in that the region is the E4 region. 15. The recombinant adenovirus according to claim 11, characterized in that the region comprises an E1 region, an E3 region and an E4 region. 16. The recombinant adenovirus according to claim 1, characterized in that the adenovirus comprises nucleic acid, wherein said nucleic acid encodes YB-1. 17. The recombinant adenovirus according to claim 16, characterized in that the nucleic acid encoding YB-1 is under the control of a promoter. 18. The recombinant adenovirus according to claim 17, wherein said promoter is the E2 late promoter. 19. The recombinant adenovirus according to claim 17, characterized in that the nucleic acid encoding YB-1 is under the control of a promoter, wherein said promoter is controlled by YB-1. 20. The recombinant adenovirus according to claim 16, characterized in that the nucleic acid encoding YB-1 is a part of an expression cassette comprising a nucleic acid encoding E1A protein. 21. The recombinant adenovirus according to claim 20, characterized in that the E1A protein is the E1A12S protein. 22. The recombinant adenovirus according to claim 20, characterized in that, in the expression cassette, the nucleic acid encoding the E1A protein is separated from the nucleic acid encoding YB-1 by an IRES sequence. 23. The recombinant adenovirus according to claim 1, characterized in that, the adenovirus comprises an expression cassette comprising a promoter and a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of aptamers, ribozymes , suitable enzymes, antisense molecules and siRNA groups. 24. The recombinant adenovirus according to claim 1, characterized in that, the adenovirus comprises an expression cassette, the expression cassette comprises a promoter and a nucleic acid sequence, wherein the nucleic acid sequence is a coding nucleic acid, wherein the The molecule encoded by the nucleic acid is selected from the group comprising peptides, polypeptides, proteins, anti-enzymes, antibodies and antibody fragments. 25. The recombinant adenovirus according to claim 1, characterized in that, the adenovirus comprises an expression cassette, wherein said expression cassette comprises a promoter and a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group comprising cell apoptosis Panels of induced genes, prodrug genes, protease inhibitors, tumor suppressor genes, cytokines and angiogenesis inhibitors. 26. A nucleic acid encoding a recombinant adenovirus according to claim 1. 27. Vector comprising a nucleic acid according to claim 26. 28. The vector according to claim 27, characterized in that said vector is an expression vector. 29. The use of the recombinant adenovirus according to claim 1 in the production of drugs for tumor diseases. 30. The use of the nucleic acid according to claim 26 in the preparation of drugs for tumor diseases. 31. Use according to claim 29, characterized in that at least part of the tumor-forming cells have YB-1 in the nucleus independent of the cell cycle. 32. Use according to claim 30, characterized in that at least some of the tumor-forming cells have YB-1 in the nucleus independent of the cell cycle. 33. Use according to claim 29, characterized in that at least part of the tumor-forming cells contain deregulated YB-1. 34. Use according to claim 30, characterized in that at least part of the tumor-forming cells contain deregulated YB-1. 35. Use according to claim 29, characterized in that at least a part of the tumor-forming cells has resistance to pharmaceutically active agents, wherein said resistance is multi-resistance, the resistance is directed against antineoplastic agents, cytostatic agents resistance to the agent, and/or the resistance is caused by radiation. 36. Use according to claim 30, characterized in that at least a part of the tumor-forming cells has resistance to pharmaceutically active agents, wherein said resistance is multi-resistance, the resistance is directed against antineoplastic agents, cytostatic resistance to the agent, and/or the resistance is caused by radiation.

Images