Gene delivery vector capable to induce cell apoptosis

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

 

The invention relates to gene delivery vectors containing the nucleic acid molecule coding for inducing apoptosis proteins VP2 and/or apoptin (VP3) with similar activity.

The invention also relates to cancer therapy and diagnosis of cancer. Infection of cells of various human cancers by gene delivery vectors according to the invention will, in consequence, to the induction of apoptosis of tumor cells and significantly reduced apoptosis, if not complete absence, of normal diploid normal/non-cancerous cells.

Expression of protein apoptin (VP3)derived from chicken anemia virus (CAV), in vitro transformed chicken cells induces apoptosis (Noteborn et al., 1994, Noteborn and Koch, 1995). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into fragments, approximately corresponding to the size of the domain, followed, in most cells, mineclearance degradation. Finally, apoptotic cells disintegrate into enclosed in a shell apoptotic cells that are rapidly phagocytized by neighboring cells. Therefore, apoptosis causes significantly less tissue destruction than necrosis, representing the non-physiological type of cell death (Wyllie et al., 1980, Arenas and Wyllie, 1991, and White, 1996).

Apoptin is a short protein length is only in the 121 amino acid, which is more of a base, with a high content of Proline, serine and threonine (Noteborn et al., 1991). In the analyzed transformed chicken cells, which all undergo induced apoptin apoptosis, apoptin strictly localized within the nucleus of the cell, the processing of the C-terminal of the main site apoptin leads to reduced localization in the nucleus and significantly reduced apoptotic activity (Noteborn et al., 1994).

Apoptin and other proteins with adapteroptions activity can also induce apoptosis in the lines of human malignant and transformed cells, but not in nontransgenic lines of human cells. The authors found that induced apoptin apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995 a) and cannot be blocked by Bcl-2, BCR-ABL (Zhuang et al., 1995)that binds Bcl-2 protein BAG-1 and protein cowpox virus CrmA (Noteborn, 1996). In vitro apoptin is not able to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial and smooth muscle cells. However, when normal cells become transformed, they become susceptible to apoptosis involving apoptin or other proteins with adapteroptions activity. Long expression apoptin in normal human fibroblasts shows that APO is tin does not have toxic or transforming activity in these cells, in normal cells, apoptin is found mainly in the cytoplasm, whereas in transformed or malignant cells, i.e. characterized by hyperplasia, metaplasia, or dysplasia, it is localized in the nucleus, suggesting that localization apoptin related to its activity (Danen-Van Oorschot et al., 1997, Noteborn, 1996).

Apoptosis is a programmed physiological process for the elimination of redundant, changed or malignant cells (Earnshaw, 1995). The process of apoptosis can be initiated by various regulatory incentives (Wyllie, 1995, and White, 1996). Changes in the level of survival of cells play an important role in the pathogenesis of a person, for example, in the development of cancer caused by increased cell proliferation, but also reduced cell death (Kegg et al., 1994). It is shown that various chemotherapeutic compounds and radiation induce apoptosis in tumor cells in many cases through the wild type p53 (Thompson, 1995, Bellamy et al., 1995, Steller, 1995).

However, many tumors acquire a mutation in the p53 during its development, often correlated with poor response to anti-cancer therapy (Hooper, 1994). In the case of some (leukemia), tumors of the high level of expression of proto-oncogene l-2 is associated with strong resistance from various inducing apoptosis chemotherapeutic agents (Hockenberry, 1994, Kerr et al.,1994, and Sachs and Lotem, 1993).

Therefore, apoptin can be a potential candidate for the role of a tool for the destruction of tumor cells or other cells, characterized by hyperplasia, metaplasia or dysplasia, which have become resistant to (chemo)therapeutic induction of apoptosis due to loss of functional p53 and increased expression l-2 and other infringing apoptosis factors. The fact that apoptin does not induce apoptosis in normal untransformed human cells, at least in vitro, suggesting that the toxic effect of the treatment apoptin in vivo must be very weak.

However, until now the expression apoptin in tumor cells is accomplished through the use of temporary transfection of cells in tissue cultures. The disadvantage of this method of expression is a very low percentage of cells that can Express apoptin in vitro. Techniques currently used for transfection in vivo will be cumbersome and inefficient, if any, will be possible, and will not make any contribution to the effective treatment of cancer.

You can get an adenovirus of human adenoviruses (Ads), which are bezobolochnye dvadtsatikartnym DNA viruses. The genome consists of a linear double-stranded DNA molecule of approximately 36 KB containing inverted terminal repeats (Horvitz, 1990). The serotypes used in the e for the development of the vector (Ad2 and Ad5), not associated with severe human pathology (Horvitz, 1990). The virus is extremely effective when introducing its DNA into the cell host. Ads can infect a variety of dividing and non-dividing cells a wide range of species, and the virus can be obtained in large quantities with relative ease. In contrast to retroviruses, Ads does not integrate into the genome of the host cell. All currently used Ads have a deletion in region E1 where you can enter a new DNA. As a result of deletions of E1 recombinant virus becomes defective for replication (Stratford-Perricaudet and Perricaudet, 1991). On the one hand, this provides a significant increase security: rAdV can not replicate in human cells in the absence of proteins EA. Thus, rAdV can deliver their genetic information in the cell in the absence or productive lytic infection. On the other hand, the problem arises of obtaining these vectors. However, for functions E1 there is no need to encode the vector itself. They can be provided in trans special helper cells that Express genes E1. After infection or transfection of these helper cells Ad-vector with a deletion of the E1 cellular proteins E1 will complementarity replication rAdV that will lead to the production of offspring rAdV. Ad-helper cells must be of human origin which I and they must contain and Express the area of AdE1, ie, Ad-transformed human cells, such as cell line 293 (Graham and Prevec, 1991), cell line 911 (Fallaux et al., 1996) and cell line PER.C6 (Fallaux, 1996).

This invention relates to a vector for gene delivery, which provides the ability to use features of antineoplastic agents apoptin or other proteins with adapteroptions activity for the treatment of cancer using gene therapy or for the treatment of malignant tumors, characterized by hyperplasia, metaplasia, or dysplasia. This vector gene delivery, which is independent of infectious vector may be, for example, a virus or liposome, or polymer, or something similar, which in itself can infect or any other way to deliver genetic information, for example, to tumor cells that can be processed. Genetic information includes a nucleic acid molecule encoding adapterordinal activity. The invention also relates to a vector for gene delivery, which significantly increased the ability to Express adapterordinal activity. It has been unexpectedly found that the change in non-coding nucleotide sequences in the reverse direction, located within the site of translation initiation, which precede posledovatelno the pits, encoding adapteroptions protein significantly increases the expression of the indicated protein in tumor cells. The invention also relates to a vector for gene delivery, containing nucleic acid encoding a VP-2-like activity. Unexpectedly, it was shown that VP-2-like activity acts in synergy with adapteroptions activity against induction of apoptosis in tumor cells, VP-2-like protein itself may also act in a synergistic or additive, for example, in the case of (chemo) therapeutic induction of apoptosis. The invention also relates to a vector for gene delivery, containing nucleic acid encoding a VP-2-like activity, in addition to the nucleic acid molecule that encodes adapterordinal activity. According to the invention, for example, vector gene delivery is a virus. In addition, the invention relates to a vector for gene delivery, which itself is replication defective virus, but which can be replicated in helper or packaging cells to obtain the vector gene delivery. Thus, the vector gene delivery according to the invention may constitute, for example, adenovirus or retrovirus, or any other DNA-type or RNA-type recombinant viruses, which can be used as a vector or plazmofereza. In addition, invented the e refers to the vector gene delivery, which is further provided with a specific ligand or molecule-target or by target molecules, by which the vector gene delivery may specifically be directed to deliver its genetic information to the selected the target cell. This molecule is a target may represent, for example, viral spike protein or receptor molecule or antibody, the reaction is capable surface receptor or protein tumor cells.

The invention also relates to a vector for gene delivery that can be used in diagnostics, for example, cancer. This vector gene delivery can be used for in vitro diagnostics when a person or animal is taken tissue samples or cells or biopsies. These samples will then be evaluated or tested by infection, culture or directly specified by the vector for the delivery of genes capable of expression, i.e. with adapteroptions activity. Only transformed cells or cells showing different stages of hyperplasia, dysplasia or metaplasia, or tumor or cancer cells Express a protein with adapteroptions activity within the nucleus. The presence of the indicated protein can be demonstrated classic (immuno) histochemistry methods, i.e. methods of light microscopy or methods AB is matsyagandha cell sorting. On the other hand, the above infected cells are characterized by apoptosis, and thus, they can be identified by well-known characteristics of apoptosis.

The invention also relates to or describes all the stages required to build a recombinant replication defective adenovirus, expressiruemogo inducing apoptosis factor apoptin. High titers of recombinant containing the apoptin, adenovirus can be obtained using lines to adenovirus packaging cells such as 293, 911 and PER.C6. Negative impacts apoptin at all necessary stages of replication of adenovirus and other events of the life cycle of adenovirus in the conditions of growing cells was not detected.

In addition, the invention describes the construction of a control recombinant adenovirus that contains all the sequences, and what the recombinant containing the apoptin, adenovirus, but, because of the orientation of the 3’-5’ encoding apoptin sequence, under the control of a regulatory promoter elements, incapable of expression apoptin. With this control adenoviral vector to explore a specific action expression apoptin recombinant adenovirus.

Recombinant replication defective adenovirus expresses apoptin in large quantities in various of Oholibah and/or transformed cells, the result is the induction of apoptosis. In contrast, expression apoptin using recombinant adenovirus in normal untransformed human cells does not result in the induction of induced apoptin apoptosis.

in particular, the invention relates to antitumor therapy. Treatment of tumor cells (cells) will occur through the expression of apoptin by infection (tumor) cells by gene delivery vectors, such as adenoviral vectors, which contain the coding sequence for a protein with adapteroptions activity. Consequently, the invention relates to gene delivery vectors, such as adenovirus, experienceui apoptin, which is a very powerful anti-tumor agent. Adenovirus regulation apoptin does not induce apoptosis, or at least not significantly induces apoptosis in normal cells, indicating that the toxicity of the treatment in vivo of recombinant containing the apoptin adenovirus will be low. Using infection with recombinant containing the apoptin adenovirus significantly more (tumor) cells will Express apoptin. This discovery is a major improvement in the expression of apoptin compared with transfertime DNA.

The invention that the same applies to the construction element of the expression of VP2 without synthesis apoptin and/or part apoptin. In addition, the authors proved that the expression of VP2 protein of chicken anemia virus (CAV) increases induced by apoptin apoptosis in cells of human cancers. More precisely, VP2 and apoptin act of synergy in relation to the induction of apoptosis of tumor cells. This discovery shows that coexpressed VP2 and apoptin will give, as a result, improvement in the treatment, based on apoptin.

The invention describes a significant improvement in the expression apoptin by changing the direction of its direct sequences in the opposite direction from the initiating codon ATG. Improved expression does not require changes of amino acids in apoptin, as it predicts a rule Kozak. Improving sequences before the initiating ATG-codon other proteins CAV will also result in the improvement of their synthesis.

The invention also relates to the construction of retroviral vectors that Express apoptin in cells of human cancers, resulting in the induction of apoptosis. Data on recombinant containing the apoptin retrovirus in combination with data on recombinant containing the apoptin adenovirus show that the expression of apoptin is not toxic factor for DNA replication-type and RNA-type viruses.

Expression apoptin in (tumor) cells can also occur Ave is infected cells other DNA-inhibiting and/or RNA-type viral vectors, in addition to the adenoviral and retroviral vectors containing the coding sequence for apoptin. In addition, for the induction of induced apoptin apoptosis of tumor cells can be used based on virus vector system, such as plasmaverse.

The invention will be explained more in detail with the following experimental part. This is done only for the purpose of explanation and should not be interpreted as limiting the scope of claims of the invention.

Experimental part

Cells and the conditions of their cultivation

Transformed by Ad5 E1 cell line and primary human kidney (SOME; 293) and primary human retina (HER; 911 and PER. C6) grown in modified according to the method of Dulbecco environment Needle (DMEM) supplemented with 10% fetal calf serum (FCS) in an atmosphere with 5% CO2at 37°C. Cell line 293 was obtained from the American type culture collection (ATSS CRL 1573). Cell lines 911 and PER. C6 obtained by Fallaux et al. (1996). Environment for growing cells, reagents and sera obtained from GIBCO Laboratories (Grand island, new York). Cultural tablets obtained from Greiner (nürtingen, Germany).

Epidermal carcity person is extracted from the flesh and grow in the presence of a layer of mouse fibroblasts T irradiated with a lethal dose of 137-Cs. Primary culture keratinocyte is in (FSK-1) initiate the full environment as described previously (Rheinwald and Green, 1975), with minor changes.

Tumorigenic keratinocytes SCC-15 (Rheinwald and Beckett, 1981), derived from squamous cell carcinoma, cultivated in a mixed environment DMEM and F12 (3:1)containing 5% fetal calf serum, 0.4 µ g of hydrocortisone and 1 μm of isoproterenol. The HepG2 cells derived from human hepatoma (Aden et al., 1979), and U20S and Saos-2 derived from human osteosarcoma (Diller et al., 1990), grown in DMEM (GIBCO/BRL) supplemented with 10% fetal calf serum. Strain spontaneously transformed keratinocyte Nasat (Boukamp et al., 1988) presented to Dr. R. Fusenig, DKFZ, Heidelberg, Germany. Cells Nasal grown in DMEM with addition of 10% fetal calf serum.

Murinova cell lines grown in modified according to the method of Dulbecco environment Needle with high glucose (4.5 g / liter) and 10% fetal calf serum in an atmosphere with 5% CO2at 37°C. Line ecotropic packing cells Psi-2 (Mann et al., 1983) and the line amphotropic packing cells RA described previously (Miller, 1990 a, b).

Methods viruses

Analyses of belascoaran performed as described previously (Fallaux et al., 1996). Briefly, strains of adenoviruses serially diluted in 2 ml of DMEM containing 2% horse serum, and added to almost fused cells 911 6-hole tablets. After incubation for 2 h at 37°With media is replaced with minimum supporting environment F-15 (MEM), containing a 0.85% agarose (Sigma, USA), 20 mm HEPES (pH 7.4), 12.3 mm MgCl2, 0,0025% L-glutamine and 2% horse serum (teploizolirovannye at 56°C for 30 minutes).

Getting small amounts of adenovirus carried out as described previously (Fallaux et al., 1996). Briefly, monolayers almost fused cells 911 or PER. C6 infect approximately 5 plaque-forming units (PFU) per cell in PBS containing 1% horse serum. After incubation for 1 hour at room temperature, inoculum replaced with fresh medium (DMEM/2% horse serum). After 48 hours almost completely exfoliated cells collected and placed in 1 ml PBS containing 1% horse serum. The virus isolated from the cells-producers through 3 cycles of rapid freezing and thawing. The clear lysates by centrifugation at 3000 rpm for 10 minutes and stored at -20°C.

Strains rAdV produced 911 and PER. C6, sceneroot the presence of recombinant-competent adenovirus by PCR with primers obtained for the region of Ad5 itr (5_-GGGTGGAGTTTGTGACGTG-3_) AND region coding EA (5_-TCGTGAAGGGTAGGTGGTTC-3_), as described in Noteborn and De Boer (1995), using the apparatus for Perkin Elmer PCR. The presence of the amplified fragment of 600 BP indicates that the analyzed strain of the virus has replication-competent (containing region E1) adenovirus (Hoebon, unpublished the results), or (this is confirmed) by a series of experiments with infection of HepG2 cells rAdV. For at least 10 days cells are checked for possible cytopathogenic action and using the method of indirect immunofluorescence assay using specific monoclonal antisera to protein EA.

Plasmids and DNA transfection

Adaptera plasmid pMad5 was constructed from pMLP10 (Levrerno et al., 1991) as described below. Plasmid pMLP10-lin obtained from pMLP10 by cloning a synthetic DNA fragment with unique sites for restriction endonucleases MluI, SplI, SnaBI, SpI, AsuII and MunI in the HindIII site pMLP10. BglII fragment of adenovirus from nt 3328 up 8914 Ad5 genome embed in website MunI pMLP-lin. From the resulting plasmid remove the SalI-BamHI fragment for inactivation of the gene of resistance to tetracycline. The resulting plasmid was characterized by restriction analysis and named pMad5. The expression of genes that are built into the multiple cloning site will be monitored by the major late promoter of adenovirus, which in this configuration is associated with enhancer immediate-early gene 1 (E1) of adenovirus.

All DNA sequences CAV originally derived from the plasmid pIc-20H/CAV-EcoRI (Noteborn and Boer, 1990). Expressing plasmid pCMV-fs, previously named pCMV-VP3 (Noteborn et al., 1994), contains a DNA sequence CAV encoding eliminate the Ino apoptin (nt 427-868).

Plasmid pCMV-VP2mu (Noteborn, unpublished results) contains a DNA sequence CAV from 380 to 1512. This DNA fragment CAV contains the coding region for VP2, flanked 5’-noncoding region of the plot in 25 BP and 3’-noncoding region of the plot in a 484 BP DNA sequence CAV. 106 BP in the forward direction from the start codon for VP2 in another frame read is the start codon for apoptin. To prevent synthesis apoptin without changing the synthesis VP2 introduce a mutation in the initiator codon apoptin (ATG replaced with ACG) and, in addition, a point mutation at position 549 (T is replaced by (A), resulting in an additional stop codon within the gene encoding apoptin. Therefore, cloning sequence CAV will lead to the expression of only the full-size protein VP2. Using indirect immunofluorescence assay, it was shown that VP2 can be produced, but apoptin is not synthesized.

For cloning of PCR amplified DNA fragments, the authors used the plasmid pCR-3.1, which was obtained from InVitrogen (carlsbad, California). To create a recombinant replication defective, containing the apoptin retrovirus used the retroviral vector pLXSN (Miller, 1990 a, b).

All stages of cloning with plasmid DNA is carried out, in principle, the methods described in Maniatis et al. (1992).

Seismology enzymes obtained from Boehringer Mannheim, Germany, and/or BioLabs, USA.

Plasmid DNA purified by centrifugation in a gradient sl and column chromatography on sephacryl S500 (Pharmacia, Uppsala, Sweden). Cell line human Nasal, HepG2, SCC-15, 293, 911 and PER.C6 transferout plasmid DNA by precipitation with calcium phosphate as described in Graham and Van der Eb (1973). Normal diploid human keratinocyte (FSK-1; the second passage), U20S cells and Saos-2 transferout DOTAP (Fisher et al., 1996).

Analysis by indirect immunofluorescence assay

The cells were fixed with 80% acetone and used for immunofluorescent analyses specific to CAV or specific to EA adenovirus monoclonal antibody and goat artemisinin and/or goat anti-rabbit IgC, conjugated with fluorescein (Jackson Immunoresearch Laboratories Inc., West grove, PA), as described in Noteborn et al. (1990). Nuclear DNA was stained with 2,4-diamino-2-phenylindole (DAPI) or propitiation (PI).

Results and discussion

Creating adaptorname vector pMab

In order to enter the site restrictase BamHI in adapting plasmid pMAd5, it splits ClaI and treated with alkaline phosphatase intestine of the calf. The linker ClaI-BamHI was treated with T4 kinase and ligated with itself using the T-4 DNA ligase, followed by ClaI cleavage. The linker ClaI/BamHI/ClaI was isolated and ligated with the linear vector pMAd5. Bacterial strain JM109 t who was informirovali products of ligation.

The resulting vector b characterized by cleavage with restriction enzymes. Using vector b foreign genes can be cloned into the unique BamHI site under the control of adenovirus late chief promoter. Schematic representation pMad5 and b is shown in figure 1.

The development of recombinant containing the apoptin, and control adaptorname vector

To create adaptery vector for introduction of a gene apoptin in adenovirus, pMab was treated with BamHI, and then the intestinal phosphatase calf. Then pCMV-fs was treated with BamHI and was isolated DNA fragment in the 0.45 KB containing encoding apoptin sequence. The DNA fragment apoptin ligated into the BamHI site of the linear adaptorname vector pMab. The ligation products were cloned into a bacterial strain JM109. Orientation apoptin in pMab was identified by restriction analysis.

Design pMab containing the apoptin gene orientation 5’-3’, under the control of adenovirus late chief promoter will Express apoptin gene. This adaptery vector outlined pMAb-VP3 and used to generate adenoviral vector expressing apoptin. Plasmid DNA pMab containing encoding apoptin sequence in the orientation of the 3’-5’ in the forward direction from the adenovirus late chief promoter, does not provide the expression of apoptin is, and was used to create the control recombinant adenoviral vector. The scheme adapting vectors shown in figure 2.

Induction of apoptosis by plasmid CMV compared to recombinant containing the apoptin adapternum vector expressing apoptin

First, the authors tested whether the fact DNA vector pMAb-VP3 able to Express apoptin in transfected cells, while b-SOP does not possess this ability. For this human 293 cells and 911, transformed with adenovirus, was transferrable pMAb-VP3, b-SOP and, as positive control, pCMV-VP3. Approximately two days after transfection the cells were fixed and examined for the expression of apoptin method of indirect immunofluorescence. Cell culture transfected with pCMV-VP3 and pMAb-VP3, contained approximately 1% of cells reacting with apophysomyces monoclonal antibodies, while cell cultures transfected b-SOP, they are not contained. These results confirm that pMAb-VP3 expresses apoptin, and b-SOP, as expected, it expresses not.

In another experiment, transfection, the authors analyzed the induction of apoptosis in cells 911 after transfection pMAb-VP3 compared with pCMV-VP3. Three days after transfection cells 911 collected and determined the expression of apoptin method nepr is my immunofluorescence assay. In addition, cells were stained with DAPI or PI; these dyes are intensely stained intact DNA and apoptotic DNA stained weakly and/or unevenly (Telford, 1992).

Approximately 60% of apoptin-positive cells 911, transfected pMAb-VP3 were apoptotic, while of apoptin-positive cells 911, transfected with pCMV-VP3, apoptosis has been about 40%. These results indicate that the expression of apoptin, adjustable pMAb-VP3, leads to the same, and sometimes to a higher level of apoptosis induction, compared with the expression apoptin, adjustable pCMV-VP3. The results are shown in figure 3.

Moreover, apoptin can induce apoptosis in human cells transformed by adenovirus, while EU did not inhibit apoptosis induced by apoptin. In contrast, EW able to block apoptosis induced by many chemotherapeutic agents. These results indicate that apoptin is a very strong anti-tumor agent.

Creation of recombinant adenovirus containing the apoptin

Recombinant adenoviral vectors containing the apoptin get through cotransfection in a helper cell line 911 adapting plasmids b-V3 carrying coding sequences for apoptin and also some adenovirus sequence, the plasmid DNA JM17, contains the complete DNA of adenovirus with the exception of areas E1 and E3 (McGrory et al., 1988). Cotransfection spent the calcium-phosphate method according to Graham and Van der Eb (19735. DNA recombinant adenovirus is generated by homologous recombination between homologous viral sequences present in the plasmid pAMb-VP3, and adenovirus DNA from DNA J17.

Likewise conducted cotransfection cells 911 with b-SOP and DNA pJM17 for receiving a control recombinant adenovirus, which may not Express apoptin and which was used as a control in the case of the apoptotic effects induced by apoptin.

A few hours after transfection monolayers of cells 911 covered the top agarose layer, and incubated at 37°until a clearly visible plaques formed by recombinant adenovirus. The virus was collected from the plaques in the form of culture, preserved in PBS with horse serum as described in Fallaux et al., (1996). Then part of the biomass of recombinant virus was added in 24-hole tablets containing fresh cells 911. After a few days these infected cells 911 was literally and collected recombinant viruses.

Then investigated the expression of apoptin recombinant containing the apoptin adenovirus (rAd-VP3), or the absence of such expression in the case of control is lnyh recombinant adenovirus (rAd-con). Part of the biomass recombinant virus obtained from infected 24-well plate, was used to infect fresh cells 911, which were grown as monolayers on cover glasses. The next day infected 911 cells were fixed with acetone and analyzed using immunofluorescence assay using apophysomyces monoclonal antibodies 85.1. Five of the 5 analyzed cell cultures infected with the alleged rAd-VP3, contained cells expressing apoptin. None of the cultures of 911 cells infected with Ad-con, and uninfected cells 911 is not apoptin-positive.

These results show that after cotransfection lines adenovirus packaging cells such as cells of the 911, the required adapter and adenoviral DNA can be obtained viable rAd-VP3 expressing apoptin.

Two isolates obtained from plaques rAd-VP3 or rAd-con, used for cleaning the rAd through the implementation of three consecutive passages plaques on 911 cells or in parallel, in the system of limiting dilutions on PER.C6 cells, as described in Fallaux (1996).

Based on the above-described methods, the result of which is the production of rAd-VP3 expressing apoptin under control adenoviruses main late promoter, the authors also received an adenoviral vector, the expression is yuushi apoptin under the control of the cytomegalovirus (CMV) promoter. These results show that it is possible to obtain different types of recombinant adenoviruses, which are controlled by one of its own or a heterologous promoter elements.

Getting rAd-VP3 and rAd-con using PER.C6 cells

Getting on a small scale parties rAd-VP3 and rAd-con using PER.C6 cells was performed as described previously (Fallaux, 1996). Briefly the procedure described in the experimental section.

By analyzing belascoaran defined titles, amounting to approximately 1011-12per ml of clarified lysate for rAd-VP3 and rAd-con. Received titles not inferior to those authors observed for other rAd.

Using PCR analysis and infected HepG2 rAd-VP3 and rAd-con investigated included whether the obtained preparations of viruses complementary replication of the adenovirus (see also experimental section). As the party rAd-VP3 and party rAd-con did not contain RCA, which was confirmed by two methods.

The authors come to the conclusion that the expression of apoptin no negative impact on all the necessary stages of the life cycle of adenovirus in conditions of cell cultivation. Therefore, gene therapy based on adenoviral vectors expressing apoptin possible.

Due to the expression of anti-apoptotic proteins of Ad5 El (White, 1996) apoptin optimally induces apoptosis in the Le of producing recombinant, containing the apoptin adenovirus in large quantities. The fact that an adenoviral vector expressing apoptin, you can get in human cells, transformed proteins E1 adenovirus type 5 (Ad5), such as 293 cells, 911 and PER.C6 shows that the E1 protein allows this DNA virus replicated to high titers in the presence of inducing apoptosis protein apoptin.

These results show that it is also possible to construct other recombinant DNA viral vectors expressing apoptin in cell lines transformed with the E1 protein of adenovirus 5. For example, you can grow a recombinant parvovirus vectors on the basis of parvovirus H-1 or MVM in cells T transformed protein Ad5 E1 (Dinsart et al., 1996).

Parvovirus H-1 or MVM specifically induce cell death in transformed cells, but not in all (Lopez-Guerrero, 1997). Parvovirus vectors expressing apoptin will be stronger when the induction of tumor-specific apoptosis than parvovirus as such, because of the additional tumor-specific induction of apoptosis by apoptin (Dinsart et al., 1996, Danen-Van Oorschot, 1997).

The scheme of preparation of recombinant containing the apoptin viral vectors based on the transformed protein Ad5 E1 cells also true for species of RNA viruses such as retroviruses.

Induction of AP is ptosis in the lines of human transformed and/or malignant cells

The authors investigated whether infection of human tumor cells rAd-VP3 in apoptosis induced by apoptin. for this, cells human hepatoma HepG2, osteosarcoma U20S cells, SCC-15, derived from squamous cell carcinoma, and cells from spontaneously transformed lines of the keratinocytes Nasat were infected with rAd-VP3. One day after transfection the cells were fixed and, by using the immunofluorescence assay and DAPI staining, cells were investigated for the synthesis apoptin and the emergence of apotosis. Already the next day after infection almost all analyzed apoptin-positive cells in human cancers were apoptotic. In uninfected cultures, only a small percentage of cells in human cancers appeared to be apoptotic. Results for HepG2 cells and U20S shown in figure 4.

These results demonstrate that apoptin, downregulation of rAd-VP3, can induce apoptosis in various oncogenic and/or transformed cell lines mammal.

Expression apoptin in normal cells, inficirovannyh rAd-VP3

In order to analyze the effect apoptin, downregulation of rAd-VP3, uninfected normal untransformed cells, rAd-VP3 infected cells FSK-1. Four days after transfection, the cells are analyzed using an indirect immunofluorescence assay used is eat monoclonal antibodies 85.1 and DAPI staining. At most 8% of apoptin-positive cells show staining with DAPI deviation from the norm, which indicates that they could endure induced (apoptin) apoptosis. However, 7% of the cells that were not infected, also have a pattern of aberrant staining of DNA DAPI. The results are shown in figure 4.

Data hepatotropic nature of human Ad5 after systemic delivery are also important for studies on the action of apoptin in normal diploid hepatocytes. To obtain the selected rat hepatocytes were grown in medium E. Williams (Gibco/Life Technologies, Grand island, new York, USA) supplemented with insulin (2 IU/ml) and dexamethasone (1 nm). Cells were grown on sensitised collagen slides (Micronic).

Primary rat hepatocytes were infected with vector-based adenovirus Ad-VP3 expressing apoptin, a control adenovirus expressing LacZ, or pseudoinfection. After two days the cells were fixed and, by using the immunofluorescence assay and DAPI staining, examined the percentage of apoptotic cells. Did not observe differences in the percentage of dead cells between cells, positive for expression apoptin, lacZ, or pseudoisocyanine. These observations suggest that (rat) hepatocytes do not undergo induced apoptin apoptosis, also in the case when the APOP is synthesized in an adenoviral vector.

These results show that guided rAd-vp3 expression apoptin does not lead to induced apoptin apoptosis in normal untransformed human cells, in contrast to transformed or tumorigenic human cells.

The increased synthesis apoptin

In order to investigate the effect of direct sequence before initiating ATG-codon apoptin, the authors have created two designs pCR 3.1-apoptin. pCR VP3ori contains the original direct sequence in the opposite direction (5_-TTCAA-3_) from the ATG-codon, while the other design pCR VP3mu includes a direct sequence in the opposite direction 5_-GCCAAC-3_. Using a cell-free system (in vitro transcription/translation from wheat germ was shown that the expression of apoptin pCR VP3mu at least five times the expression observed in the case of pCR-Vp3ori. These data show that the character sequences directly in front of the ATG-codon apoptin affects the synthesis apoptin. Create (viral) vectors with a straight line, in the opposite direction, the sequence 5_-GCCAAC-3_ before ATG-codon apoptin will lead to higher production apoptin and, indirectly, to increased induced apoptin apoptosis.

It is also important to mention that the amino acid sequence apoptin does not change as it clean the needs for increased effectiveness broadcast according to the "rule Kozak" (Caventer and Stuart, 1991). In accordance with this rule, the authors would have to replace the nucleotide at position +4 And G, the result of which was to change the second amino acid apoptin, which may change its activity.

Identification of significant fragment apoptin containing apoptotic activity

In order to investigate whether the protein apoptin essential for its apoptotic activity, was constructed a plasmid encoding a chimeric protein consisting of protein green florescence (GFP; Rizzuto, 1995) and N-terminal 71 amino acids apoptin (N-apoptin) or its C-terminal 50 amino acids (apoptin). Human transformed cells, such as cells Saos-2 (Zhuang, 1995), was transferrable plasmids expressing chimeric GFP/N-apoptin or GFP/C-apoptin. Apoptosis undergo only cells Saos-2 expressing GFP/C-apoptin. This coincides with the fact that-apoptin (associated with GFP), you can log in to the kernel.

These results indicate that induction of apoptosis in human oncogenic or transformed cells can also be quite part apoptin. Consequently, it is possible to develop an effective (gene) - based therapy (viral) vectors expressing only part apoptin. In addition, these data suggest that part apoptin contains its apoptotic act shall want to make, when it is covalently linked to foreign protein.

Coexpressed VP2 and apoptin in cells of human cancers synergy increases the induction of apoptosis

In order to investigate the effect of co-expression of VP2 and apoptin on the induction of apoptosis, cells Saos-2 (Ko)transferout pCMV-fs expressing apoptin, and/or pCMV-VP2mu expressing VP2. The cells were fixed with acetone after different time intervals after transfection. By indirect immunofluorescence assay with monoclonal antibody CVI-CAV-111.1 (Noteborn and Koch, 1996) was determined AR-positive cells, and monoclonal antibody CVI-CAV-85.1 - apoptin-positive cells. On day 3 after transfection underwent apoptosis only about 3% expressing VP2 cells and only about 10% of cells expressing apoptin. In contrast, about 40% of the cells Saos-2, as expressing VP2 and apoptin were apoptotic. Also 4 days after transfection, the percentage OR/apoptin-positive cells that have undergone apoptosis, significantly higher than for cells expressing one apoptin one or VP2.

These results, which are shown in figure 5, show that VP2 increases induced by apoptin apoptosis.

Designing and getting rAD-VP2

In order to construct a recombinant adenovirus expressing the viral protein 2 (VP2) of the virus Anh and chickens, was obtained adaptera plasmid pMAb-VP2. The BamHI fragment of 1.1 KB was isolated from the plasmid pCMV-VP2mu containing all coding sequences VP2, but with 2 point mutations within the region, encoding apoptin (see experimental section), and ligated with adapternum vector b, split BamHI and treated with alkaline phosphatase intestine of the calf. The resulting structure pMAb-VP2 shown in Fig.6, characterized using restriction analysis and sequence analysis.

rAD-VP2 create through cotransfection cells 911 DNA pMAb-VP2 and DNA pJM17. Cotransfection and all other required stages required for characterization, cleanup, and get rAD-VP2, was carried out as described in the case of rAd-VP3 and rAd-con. By indirect immunofluorescence assay using a monoclonal antibody CVI-CAV-111.1 showed that cells 911 and PER. C6, inifitsirovannye rAd-VP2, can really Express the protein VP2.

The creation of a retroviral vector expressing apoptin

In order to obtain the plasmid pL-VP3-SN (see Fig.7), the BamHi fragment carrying the encoding apoptin sequence, embedded in the unique BamHI site pLXSN. The correct orientation of the insert was confirmed by restriction analysis. To verify the integrity of the insert, cells COS-7 and HepG2 were transferrable the plasmid pL-VP3-SN method of co-precipitation of phosphate, calc who I am. Four days after transfection the cells were fixed and analyzed using monoclonal antibodies 85.1 on the expression of protein apoptin. Expression apoptin was detected in approximately 1-2% of the cells. Most of the cells underwent apoptosis, which was determined by DAPI staining. These data demonstrated that the proviral LTR-promoter capable of controlling expression of the protein apoptin that his gene remains intact in DNA constructs, and that in transfected cells HepG2 and COS-7 expression apoptin induces apoptosis.

In order to get viruses, plasmid pL-VP3-SN was transferrable cells Psi-2 in cells of RA 317 by the method of coprecipitation with calcium phosphate. Supernatant cells were harvested forty-eight hours after transfection, and their breeding was used to infitsirovanija HepG2 cells (supernatant RA 317) and NIH3T3 cells (supernatant Psi-2) in the presence of 4 μg/ml polybrene. Four days after infitsirovanija the cells were fixed and analyzed for the expression of apoptin using monoclonal antibodies 85.1. Found that approximately 1% of cells expressed apoptin. Most of the apoptin-positive HepG2 cells were apoptotic. These data confirm that the cells were transpulmonary retroviruses containing L-apoptin-SN. In addition, the results obtained demonstrate that one is the opium of provirus sufficient for the expression of sufficient amounts of protein apoptin, which can be detected by immunofluorescence, and this amount is sufficient for the induction of apoptosis in cell lines of human tumor, namely cell lines hepatoma HepG2.

Collectively, these data show that retroviral vectors carrying apoptin gene, can be obtained and can be used for the induction of apoptosis in cells of human cancers. This formally proves that neither the apoptin gene or its expression is not affected in a significant stage of the life cycle of the retrovirus. They also show that containing the apoptin retroviruses can be obtained in quantities sufficient for transduction of cells of human tumors in tissue culture.

These results also indicate that the expression of apoptin and, therefore, induced apoptin apoptosis of human tumor cells will also be possible using vector systems derived from (retro)virus, such as plasmaverse (Noguiez-Hellin, 1996). The critical moment for this system based recombinant containing the apoptin of plazmofereza is that not blocked replication of retrovirus expression apoptin. The authors obtained evidence that this actually happens in the case of the above-described recombinant containing the apoptin retrovirus, which means the successful receipt of recombinant containing and the optin of plazmofereza.

Diagnostic analysis on cancer cells on the basis of rAD-VPS

Cellular localization apoptin in oncogenic or transformed cells other than localization in normal untransformed cells. In addition, another indicator is a specific ability apoptin to induce apoptosis in tumorigenic or transformed cells and do not induce apoptosis in normal cells.

By infection of cells rAd-VP3 and analysis locations apoptin and/or induction of apoptosis in these cells it is possible to check whether the cell is cancerous or not. Primary cells isolated from the (suspicious) tissue and cultured in the desired environment. Cells infect rAd-VP3 and, in parallel, rAd-con, and then analyze. For example, use immunofluorescence assay based on monoclonal antibodies 85.1 specific apoptin. Cells control for the presence of apoptin in the cytoplasm (normal cells) or in the nucleus (transformed cells). In addition, or instead, we can estimate the percentage of apoptotic cells. If the percentage of apoptotic cells in the case inficirovannyh rAd-VP3 cells is significantly higher than in the case of cells infected with rAd-con, this means that the first cells to become malignant.

Experiments on the toxicity of recombinant containing the apoptin adenovirus for Zdor the o rats

In the conditions of culturing tissue apoptin, expressed by recombinant adenovirus rAd-VP3 in normal cells, for example, derived from human or rodent, does not induce apoptosis. In the following experiment, the authors investigated, and does the expression apoptin using recombinant viral vector rAd-VP3 in healthy rats to acute toxicity.

Both vector - used vector rAd-VP3 and the control vector rAd - grown cells R.C6, and by PCR analysis confirmed that the product of the virus does not contain adenovirus, complementiser replication (RCA). The rAd vectors were purified by centrifuging the gradient sl.

The male rats of the Wag/Rij (Harlan, the Netherlands) weighing approximately 200 grams were injected with recombinant adenovirus expressing apoptin (rAd-VP3; to 2.5×109plaque-forming units, PFU) and the control recombinant adenovirus expressing the gene product of the luciferase (rAd-luc; to 2.5×109). both adenoviral vector resuspendable in PBS containing 0.1% bovine serum albumin and 10% glycerol (PBS+). This solution is without adenoviral vector was injected with the rats, and he served as an additional negative control. rAd-VP3, rAd-luc or suspension PBS+ was injected intravenously administered intraperitoneally or subcutaneously two rats in every way.

Macroscopy the definition of pathological analysis of rats, processed rAd-VP3

The first way to study the possible toxic effects downregulation of rAd-VP3 apoptin is the definition of General health and, in particular, the body weight of treated rats, which is carried out after daily injections. In continuation of the experiment all rats were observed in good health. Body weight in different groups were not significantly different. After 1 week all tested rats, including injected rAd-VP3, gained weight, which indicates that no animal was suffering from acute toxicity due to one of the treatments rAd-VP3. For additional evidence of the lack of acute toxicity was carried out by the following definitions. Two hours before worldline all rats injected BrdU. After worldline some tissues (liver, kidney, intestine, heart, lung, spleen, gonads, and penis) investigated the pathology directly and/or collected for histological analysis (see below).

Macroscopic analysis shows that none of the treated rAd-VP3 rats no bodies with significant pathological changes.

Determination of DNA Ad-VP3 in the liver

The primary target of injected intravenously (recombinant) adenovirus (vector) is the liver and, to some extent, the spleen. Therefore, any toxic effect of apopt is to be observed in the liver.

Conduct a series of experiments to study the presence of DNA Ad-VP3, expression apoptin Ad-VP3 and possible cytopathological actions in the liver.

First, by the method of southern blotting, the authors investigated whether DNA extracted per day worldline, ie, 8 days after injection, the liver of rats treated with Ad-VP3, DNA apoptin. As a negative control in parallel examined DNA from the liver of rats treated with Ad-luc. Before applying the selected DNA on the agarose gel DNA was digested BamHI, resulting in the observed DNA fragment apoptin about 0.5 to.. southern blot was hybridisable labeled with32P probe DNA apoptin.

BamHI DNA fragment apoptin clearly visible on the southern band in the case of DNA, obtained from animals treated with Ad-VP3, and not, as expected, on the lanes containing DNA extracted from the liver of rats treated with control rAd-luc. To study the number of copies Ad-VP3 in the liver of different amounts of DNA apoptin was applied parallel to the southern blot and hybridisable with labeled probe DNA apoptin. Even eight days after intravenous infection can be defined 0,25 copies of Ad-VP3 on the cell, which indicates a very significant transduction of Ad-VP3 in the liver.

Expression apoptin and its toxic effect on liver cells

Through the immune okras is of paraffin sections of the liver, handled Ad-VP3, or control of the liver, using a monoclonal antibody CVI-CAV-85.1 or CVI-CAV-111.3, the authors showed that approximately 20-30% of the liver cells of animals treated with Ad-VP3, expressed apoptin. Sections of the liver of control rats were negative in relation to apoptin.

In order to explore the possible cytotoxic effect apoptin, expressed Ad-VP3, liver cells, was used by two different methods. In the first method, sections of the liver of all rats treated with Ad-VP3, and slices from control animals of both types were stained with hematoxylin and eosin (NOT). In all tested sections of the liver were not observed pathological morphological changes, indicating that the expression of apoptin is not toxic to the cells of rat liver.

Possible harmful effects could be detected with BrdU labeling, which identifies the newly divided cells. In the case of the liver, containing Ad-VP3, the number of BrdU labeled cells is about the same (about 2%)in the liver of control and treated with Ad-luc rats. Therefore, expression apoptin, as such, no significant toxic effects in vivo.

Apoptin has no toxic effect on the model in vivo

Both macroscopic and histological analysis in combination with biochemical and immunological data indicate that e is xpressia apoptin has no acute toxic effects on the model in vivo.

These results demonstrate that treatment based on the expression of apoptin through the application of vector gene delivery, or using other methods will have limited negative side effects.

Studies of antitumor activity in models of human hepatoma

Adjustable Ad-VP3 expression apoptin leads to the induction of apoptosis of transformed human cells in tissue culture conditions. For example, adjustable Ad-VP3 expression apoptin results in the induction of apoptosis of HepG2 cells derived from human hepatoma. Still antitumor activity apoptin (for example, downregulation of Ad-VP3) in vivo has not been studied.

Therefore, the authors determined on the model in vivo, whether the variable Ad-VP3 expression apoptin antitumor activity. This male Nude mice Balb/C/nu/nu were subcutaneously injected with 1×106human HepG2 cells per injection. Cells human hepatoma were injected with each mouse at least two or three places. Three weeks after injection develop distinct hepatoma tumors that are visible under the skin, having an average size of at least 5×5 mm

Tumors were injected with vectors rAd-VP3 and control rAd-conl suspended in PBS containing 5% sucrose and 0.1% bovine serum albumin.

Vecto the rAd-VP3, expressing apoptin, and the control vector rAd-conl containing the apoptin gene in the opposite orientation 3’-5’ (reverse or antisense) relative to the promoter Ad MLP, were grown on cells PER.6. By PCR analysis confirmed that both recombinant adenovirus is free from RCA. rAd was purified using centrifugation in CsCl gradient.

Were injected with 7×109the battle of particles rAd in 40 μl of the suspension on the tumor. Each type rAd-vector was treated with 6 mice with 2-3 HepG2 tumors. As an additional control group of 4 naked mice with HepG2 tumor, tumors were injected with PBS containing 5% sucrose and 0.1% bovine serum albumin (PBS group+-).

Apoptin has an antitumor effect on model in vivo

In order to check the possible antitumor effect of downregulation of Ad-VP3 apoptin on tumors from human HepG2, measured the size of subcutaneous tumors during the experiment continued for another 7 days after injection of rAd-VP3 and control suspensions.

As a group, PBS+, and the group treated control rAd-conl, show a gradual increase in the size of tumors. In contrast, the group, in which tumors were injected with rAd-VP3, shows the reduction in the size of tumors. On the seventh day after injection of naked mice omerville. The tumor was removed if and examined macroscopically. It is evident that hepatoma tumors treated with the control vector rAd-conl or PBS+, are strongly vascularizing the tumor tissue HepG2 and became larger after treatment. A completely different picture was observed in HG2-tumors treated with rAd-VP3. The mass of the remaining tumor pale in appearance due to reduced vascularization of the tumor. The tumor after treatment rAd-VP3 decreased in size. Tumors also contained white puzirkoviy patterns, which is an indicator of dead cells.

In addition to the induced apoptin regression of tumors of any other negative action expression apoptin on organs (tested, in particular, liver and spleen) was not registered.

Apoptin has antitumor activity in vivo

Thus, tumors treated with rAd-VP3, showed a decrease in size, while in the control treatments, it is not. This means that the expression apoptin has an antitumor effect on model in vivo.

The fact that downregulation of Ad-VP3 apoptin can induce regression of tumors in the fast-growing tumors, such as HepG2, proves a strong antitumor effect apoptin. The fact that apoptin slows the growth of tumors in Nude mice shows that the expression of apoptin kills tumor cells without addition the immune response.

Described studies of toxicity and antitumor actions show that the antitumor therapy based on apoptin, safe and feasible.

Description of figures

Figure 1 gives a schematic view of the essential areas of adenoviral adapting vectors pMAd5 and pMab.

Figure 2 gives a schematic view of the essential areas of recombinant adenovirus adapting vectors pMab-VP3 and b-SOP.

Figure 3 shows inducing apoptosis activity apoptin in 911 cells, transfected pMab-VP3 or pCMV-VP3. For transfection of cells 911 used two independently cloned and purified DNA preparation pMab-VP3 (b-V3/m1 and pMab-VP3/m2). The cells were fixed 3 days after transfection and analyzed by indirect immunofluorescence assay using apoptin-specific monoclonal antibody CVI-CAV-85.1 (85.1; Noteborn et al., 1991). The percentage of cells abnormally painted DAPI is given as a relative measure of apoptosis.

Figure 4 shows the activity apoptin (called VP3) at the induction of apoptosis in human cancer cells hepatoma HepG2, cells U20S osteosarcoma and normal normal diploid keratinocytes FSK-1 infected with recombinant containing the apoptin replication defective adenovirus Ad-VP3. Cells were analyzed by indirect immunofluorescence assay is the use of monoclonal antibodies 85.1 and DAPI staining. Cells HepG2 and U20S were fixed the next day after transfection, and cells FSK-1 was collected and recorded 4 days after transfection. The percentage of apoptin-positive cells that abnormally stained with DAPI, is given as the measure induced by apoptin apoptosis (black rectangles). As control is the percentage of uninfected cells, abnormally colored DAPI (where there's no shading rectangles).

Figure 5 shows the activity of apoptosis by induction of apoptin and/or VP2 in cells Saos-2, transfected with 2.5 µg DNA pCMV-fs expressing apoptin (previously named pCMV-VP3; apoptin is called VP3) and 2.5 µg DNA pCMV-neoBam (Danen-Van Oorschot, 1997); or 2.5 µg DNA pCMV-Vp2 expressing CAV protein 2 (VP2), and 2.5 µg DNA pCMV-neoBam; or 2.5 μg pCMV-fs and 2.5 μg pCMV-Vp2, resulting in expression as apoptin (VP3)and VP2. The cells were fixed in 3, 4 and 5 days after transfection and analyzed by indirect immunofluorescence assay using apoptin-specific monoclonal antibody CVI-CAV-85.1 (85.1; Noteborn et al., 1991) or monoclonal antibody CVI-CAV-111.1 (Noteborn and Koch., 1996). The percentage of cells that abnormally stained with DAPI, is given as a relative measure of apoptosis.

6 gives a schematic view of the essential areas of recombinant adenovirus adapting vectors pMab-VP2.

7 gives a schematic representation of su is the natural enemy areas recombinant retroviral vector vector PLS-VP3-N.

1. Vector gene delivery, is able to induce apoptosis in the cell, including the DNA fragment that encodes a protein having adapteroptions activity, while apoptosis is induced in cancer cells and to a lesser extent or not at all occur in normal diploid normal/non-cancerous cells, and the vector gene delivery is independent of the infective vector and represents the adenovirus.

2. Vector gene delivery according to claim 1, additionally containing modified the site of translation initiation immediately before the initiation codon ATG of the molecule of nucleic acid, and the site of translation initiation contains the nucleotide sequence GCCAAC.

3. Vector gene delivery according to claim 1 or 2, representing a replication defective virus.

4. Vector delivery of genes according to any one of claims 1 to 3 for the treatment of cancer.

5. Vector gene delivery according to claim 4 for the treatment of cancer in combination with conventional (chemo)therapy.

6. Vector delivery of genes according to any one of claims 1 to 3 for the treatment of hyperplasia, metaplasia and dysplasia.

7. Vector delivery of genes according to any one of claims 1 to 3 for the diagnosis.

8. Vector gene delivery according to claim 7 for detecting in vitro transformed, cancer or hiperplasia, metaplasia or displacing cells.



 

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