MXPA99009486A - A gene delivery vehicle expressing the apoptosis-inducing proteins vp2 and/or apoptin - Google Patents

Abstract

The invention relates to gene delivery vehicles which comprise nucleic acid molecules encoding apoptosis-inducing proteins VP2 and/or apoptin (VP3) like activity. VP2 and VP3 are viral proteins of the Chicken Anaemia Virus. Also, the invention relates to anti-tumor therapies. Infection of various human tumor cells with the gene delivery vehicles of the invention will result in the induction of apoptosis in tumor cells and much reduced apoptosis, if at all, in normal diploid, non-transformed/non-malignant cells. Also the invention relates to the diagnosis of cancer, and related forms of hyperplasia, metaplasia and dysplasia.

Description

VEHICLE OF SUPPLY OF A GENE EXPRESSING THE PROTEINS INDUCTOR OF APOPTOSIS VP2 AND / OR APOPTINE DESCRIPTION OF THE INVENTION The invention relates to gene delivery vehicles which comprise nucleic acid molecules that encode apoptosis-inducing proteins with activity similar to VP2 and / or apoptin (VP3). In addition, the invention relates to antitumor therapies and to the diagnosis of cancer. The infection of various human tumor cells with the delivery vehicles of the gene of the invention will result in the induction of apoptosis in tumor cells and a very reduced apoptosis, if present, in normal diploid cells, non-transformed / non-malignant. In vitro, expression of the protein derived from chicken anemia virus (CAV), apoptin (VP3) in transformed chicken cells induces apoptosis (Noteborn et al., 1994, Noteborn and Koch, 1995). Apoptosis is characterized by a shrinkage of cells, segmentation of the nucleus, condensation and separation of DNA into fragments of domain size, in most cells followed by degradation between nucleosomes. Finally, the fragment of apoptotic cells in the apoptotic bodies enclosed in REF .: 31489 membrane, which are rapidly phagocytosed by neighboring cells. Therefore, apoptosis causes much less tissue destruction compared to necrosis, the non-physiological type of cell death (Wyllie et al., 1980, Arends and Wyllie, 1991 and White, 1996). Apoptin is a small protein, only 121 amino acids long which is rather basic, and rich in proline, serine and threonine (Noteborn et al., 1991). In the transformed chicken cells analyzed and which in their entirety experienced apoptosis induced by apoptin, apoptin is located strictly within the cell nucleus. Truncation of the C-terminal base chain of apoptin results in a reduced nuclear localization and significantly reduced apoptotic activity (Noteborn et al., 1994). Apoptin and other apoptin-like proteins can also induce apoptosis in malignant and transformed human cell lines, but not in untransformed human cell lines. We have established that apoptosis induced by apoptin occurs in the absence of functional p53 (Zhuang et al., 1995a), and can not be blocked by Bcl-2, BCR-ABL (Zhuang et al., 1995), the protein that associates Bcl-2, BAG-1, and Cowpox protein crmA (Noteborn, 1996). In vitro, apoptin does not induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial and smooth muscle cells. However, when normal cells are transformed, they become susceptible to apoptosis by apoptin or other proteins with activity similar to apoptin. The long-term expression of apoptin in normal human fibroblasts reveals that apoptin has non-toxic or transforming activity in these cells. In normal cells, apoptin has been found predominantly in the cytoplasm, whereas in transformed or malignant cells, that is, characterized by hyperplasia, metaplasia or dysplasia, it is localized in the nucleus, suggesting that apoptin localization is related with its activity (Danen-Van Oorschot et al., 1997, Noteborn, 1996). Apoptosis is an active and programmed physiological process for the elimination of superfluous, altered or malignant cells (Earnshaw, 1995). The apoptotic process can be initiated by various regulatory stimuli (Wyllie, 1995 and White, 1996). Changes in the cell survival rate may play an important role in human pathogenesis, for example in the development of cancer, which is caused by increasing cell proliferation, but also by decreased cell death (Kerr et al., 1994). It has been shown that various chemotherapeutic compounds and radiation induce apoptosis in tumor cells, in many cases, by means of wild type p53 (Thompson, 1995, Bellamy et al., 1995, Steller, 1995).

However, many tumors acquire a mutation in p53 during their development, which often correlates with a poor response to cancer therapy (Hooper, 1994). For several tumors (leukemic), a high expression level of the Bcl-2 proto-oncogene is associated with a strong resistance to various chemotherapeutic agents inducing apoptosis (Hockenberry, 1994, Kerr et al., 1994, and Sachs and Lotem, 1993 ). Therefore, apoptin can become a potential candidate for the destruction of tumor cells, or other cells characterized by hyperplasia, metaplasia or dysplasia, which become resistant to the induction (chemo) therapeutic of apoptosis, due to the lack of p53 Functional and (on) -expression of Bcl-2 and other apoptosis inhibiting agents. The fact that apoptin does not induce apoptosis in normal non-transformed human cells, at least not in vitro, suggests that the toxic effect of apoptin treatment in vivo may be very low. However, up to now, the expression of apoptin in tumor cells is carried out by the use of transient transfection of tissue culture cells. The disadvantage of this expression method is the very low percentage of cells which can express apoptin under in vitro circumstances. In vivo, the transfection methods used will be problematic and not efficient, if they are possible, and not all contribute to an effective treatment against cancer. Human adenovirus adenovirus (Ads) can be derived, which are icosahedral DNA viruses, without wrapping. The genome consists of a double-stranded linear DNA molecule of approximately 36 kb that has inverted terminal repeats (Horvitz, 1990). Serotypes that have been used for vector development (Ad2 and Ad5) have not been associated with severe human pathology (Horvitz, 1990). The virus is extremely efficient at introducing its DNA into the host cell. Ads can infect a wide variety of dividing and non-dividing cells from a wide range of species, and the virus can reproduce in large numbers with relative ease. In contrast to retroviruses, Ads are not integrated into the genome of the host cell. All currently used rAdVs have a deletion in the El region, where novel DNA can be introduced. The deletion makes the recombinant virus defective in its replication (Stratford-Perricaudet and Perricaudet, 1991). On the other hand, this provides an essential security feature. RAdV can not be replicated in human cells in the absence of ElA proteins. Therefore, rAdV can supply its genetic information in a human cell, but this will not result in a lytic or productive infection. On the other hand, it presents a problem for the production of these vectors. However, the functions of El do not necessarily need to be encoded by the vector itself. They can also be provided in trans, especially in helper cells which express the genes for El. When an infection or transfection of these helper cells occurs with an Ad vector to which El was deleted, the cellular proteins will complement rAdV replication, which results in the production of rAdV progeny. The Ad helper cells must be of human origin, and must contain and express the AdEl region, ie, human cells transformed with Ad such as the 293 cell line (Graha and Prevec, 1991), the 911 cell line (Fallaux et al., 1996) and the PER.C6 cell line (Fallaux, 1996). The invention now provides a gene (or vector) delivery vehicle which allows traits of the antitumor agent apoptin, or other proteins with apoptin-like activity, to be used for the treatment of cancer through the use of gene therapy or for the treatment of cancer. treatment of malignant cancers characterized by hyperplasia, metaplasia or dysplasia. Such a delivery vehicle for a gene, which is an infectious vector independently, can be, for example, a virus or a liposome, or a polymer or the like, which itself can infect, or otherwise provide genetic information to, for example, tumor cells that can be treated. The genetic information comprises a nucleic acid molecule that codes for apoptin-like activity. The invention also provides a delivery vehicle for a gene that has greatly increased its ability to express activity similar to apoptin. Surprisingly, it has been found that changing the non-coding nucleic acid sequences towards the 5 'end, located within the translation start site preceding the coding sequences for the apoptin-like protein greatly improves the expression of such a protein. in tumor cells. The invention also provides a delivery vehicle for a gene comprising a nucleic acid encoding an activity similar to VP2. Surprisingly, activity similar to VP2 is shown to act synergistically with apoptin-like activity in relation to the induction of apoptosis in tumor cells, the VP2-like protein itself can also act synergistically or additively, for example, for induction (chemo) therapeutic apoptosis. The invention also provides a delivery vehicle for a gene comprising a nucleic acid encoding VP2-like activity additionally comprising a nucleic acid molecule encoding apoptin-like activity. The invention provides, for example, a delivery vehicle for a gene according to the invention which is a virus. Additionally, the invention provides a vehicle from delivery of a gene that itself is a virus defective in its replication but which can be replicated in helper or packaging cells to generate delivery vehicles for a progeny gene. The delivery vehicle for a gene provided by the invention in this manner can be, for example, an adenovirus, or a retrovirus or other recombinant DNA or RNA virus that can be used as a delivery vehicle or a pasmovirus. Additionally, the invention provides a delivery vehicle for a gene which has been additionally supplemented with a specific target or ligand molecule or target molecules, by which the delivery vehicle of a gene can be specifically engineered to deliver its genetic information into a target cell of choice. Such a target molecule may be, for example, a viral spike protein, or a receptor molecule, or antibody, reactive with a tumor cell surface protein or receptor. In addition, the invention provides a vehicle for the delivery of a gene which can be used in diagnosis, that is, cancer. Such a delivery vehicle for a gene can be used, for example, for in vitro diagnosis where tissue or cell samples or biopsies are taken from a human or an animal. Such samples can be evaluated or can be tested by infecting them, in culture or directly, with the delivery vehicle of a gene capable of expressing, for example, apoptin-like activity. Only transformed cells, or cells showing various stages of hyperplasia, dysplasia or metaplasia, or tumor or cancer cells that express the protein with apoptin-like activity within the nucleus. The presence of the protein can be demonstrated with classical (immuno) histochemical techniques, for example microscopically or with automated cell sorting techniques. Alternatively, the above infected cells are characterized by apoptosis and can therefore be diagnosed on the basis of known characteristics of apoptosis. Furthermore, the invention provides or describes all the steps necessary for the construction of a recombinant adenovirus defective in its replication that expresses people apoptosis-inducing apoptosis. High titers of recombinant adenovirus can be produced with apoptin by means of adenovirus packaging cell lines, such as 293, 911 and PER.C6. Apoptin does not show a detectable negative effect at all stages of replication necessary for adenovirus and other adenovirus life cycle events under cell culture conditions. Furthermore, the invention describes the construction of a recombinant control adenovirus, which contains all the sequences of the recombinant adenovirus with apoptin, but due to the 3'-5 'orientation of the coding sequence for apoptin under the control of the promoter elements regulators, is not able to express apoptin. By means of this adenovirus control vector, the specific effects of apoptin expression can be examined by a recombinant adenovirus. The recombinant adenovirus defective in its replication expresses apoptin in high amounts in various tumor cells and / or transformed which results in the induction of apoptosis. In contrast, the expression of apoptin in normal human cells not transformed by means of recombinant adenovirus does not result in the induction of apoptosis induced by apoptin. In particular, the invention relates to antitumor therapies. The treatment of tumors (tumor cells) takes place by expression of apoptin by means of infectious (tumor) cells with gene delivery vehicles such as adenovirus vectors containing a coding sequence for a protein with apoptin-like activity. Therefore, the invention provides delivery vehicles for a gene such as adepts that express apoptin which are potential oral antitumor agents. The regulation of apoptin by the adenovirus does not induce, or at least does not induce detectably apoptosis in normal cells, indicating that the toxicity of in vivo treatment with recombinant adenovirus with apoptin will be low. By means of a recombinant adenovirus infection with apoptin, a much higher amount of (tumor) cells expressing apoptin can be obtained. This finding is a remarkable improvement of apoptin expression compared to DNA transfections. The invention also relates to the construction of a VP2 expression unit without the synthesis of apoptin and / or a part of apoptin. In addition, we have provided evidence that the expression of chicken anemia virus (CAV) VP2 protein improves apoptosis induced by apoptin in human tumor cells. To be more precise, VP2 and apoptin act synergistically with respect to the induction of apoptosis in tumor cells. This finding indicates that the coexpression of VP2 and apoptin will result in an improvement of apoptin-based therapies. The invention describes a significant improvement of apoptin expression by changing its direct sequences towards the 5 'end of the ATG start codon. The improvement of expression does not need to be an amino acid change in the apoptin protein, as predicted by the KOZAK rule. An improvement in the sequences towards the 5 'end of the ATG start codon of the other CAV proteins will also result in an improvement in their synthesis. The invention also relates to the construction of retroviral vectors which express apoptin in human tumor cells which results in the induction of apoptosis. This result with recombinant retroviruses with apoptin in combination with recombinant adenovirus data with apoptin indicate that the expression of apoptin is not toxic to the replication of a DNA virus or RNA virus. The expression of apoptin in (tumor) cells can also be carried out by infecting cells with other viral vectors of DNA and / or RNA, in addition to the adenovirus or retrovirus vectors, which contain a coding sequence for apoptin. In addition, vector systems derived from viruses such as plasmoviruses can be used for the induction of apoptosis induced by apoptin in tumor cells. The invention will be explained in more detail based on the following experimental part. This is solely for the purpose of illustration and should not be construed as limiting the scope of protection.

EXPERIMENTAL PART Cells and cell culture conditions Human embryonic kidney cell lines transformed with El5 (HEK; 293) and human embryonic retina (HER; 911 and PER.C6) are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FCS). ) 10% in an atmosphere of C02 5% at 37 ° C. Cell line 293 is obtained from American Type Culture Collection (ATCC CRL 1573). Cell lines 911 and PER.C6 are obtained from Fallaux et al., (1996). The cell culture media, reagents and sera are purchased from GIBCO Laboratories (Grand Island, NY). Plastic culture vessels are purchased from Greiner (Nürtingen, Germany). Human epidermal keratinocytes are isolated from the forearm and grown in the presence of a layer of murine 3T3 mouse fibroblasts irradiated with 137-CS. The primary cultures of keratinocytes (FSK-1) are started in complete medium as described (Rheinwald and Green, 1975) with minor modifications. Tumorigenic keratinocytes, SCC-15 cells (Rgeinwald and Beckett, 1981), derived from squamous cell carcinoma, are cultured in DMEM / F12 medium (3.1) containing 5% fetal bovine serum, 0.4 μg hydrocortisone and 1 μM isoproterenol. HepG2 cells derived from human haptoma (Aden et al., 1979) and U20S and Saos-2 cells derived from human osteosarcoma (Diller et al., 1990) are grown in DMEM (GIBCO / BRL) supplemented with 10% fetal bovine serum. The keratinocyte strain spontaneously transformed HaCaT (Boukamp et al., 1988) is a donation from Professor Doctor R.

Duseign, DKFZ, Heidelberg, Germany. HaCaT cells are grown in DMEM supplemented with 10% fetal bovine serum.

Urine cell lines are grown in Dulbecco's modified Eagle's medium with an elevated glucose concentration (4.5 grams per liter) and 10% fetal bovine serum in a 5% C02 atmosphere at 37 ° C. The ecotropic packaging cell line Psi-2 (Mann et al., 1983) and the amphotropic packaging cell line PA317 have been described before (Miller, 1990a, b).

Viral Techniques Plaque assays are performed as previously described (Fallaux et al., 1996). In brief, adenoviconcentrates diluted in 2 ml of DMEM containing 2% horse serum are serially diluted and added to nearly confluent 911 cells in 6-well plates. After 2 hours of incubation at 37 ° C, the medium is replaced by minimal essential medium F-15 (MEM) containing 0.85% agarose (Sigma, USA), 20 mM HEPES (pH 7.4), 12.3 mM MgCl2, L- glutamine 0.0025% and horse serum 2% (inactivated by heat) at 56 ° C for 30 minutes). Small-scale production of batches of adenoviis carried out as previously described (Fallaux et al., 1996). In brief, almost confluent monolayers of 911 or PER.C6 cells are infected with approximately 5 plaque forming units (pfu) per cell, in phosphate buffered saline (PBS) containing 1% horse serum. After 1 hour at room temperature, the inoculum is replaced by fresh medium (DMEM / horse serum 2%). After 48 hours, the cells that have almost completely detached are harvested, and harvested in 1 ml of PBS / horse serum 1%. The viis isolated from producer cells by 3 freeze / fast reheat cycles. The used ones are clarified by centrifugation at 3,000 rpm for 10 minutes and stored at -20 ° C. Lines 911 and PER.C6 produce rAdV concentrates which are analyzed for the presence of recombinant competent adenoviby carrying out PCR analysis with primers derived from the Ad5 ITR region (5_-GGGTGGAGTTTGTGACGTG-3_) and the region coding for E1A (5_-TCGTGAAGGGTAGGTGGTTC-3_) as described by Notebor and De Boer (1995) using a Perkin Elmer PCR apparatus. The presence of an amplified fragment of 600 bp indicates that there is a competent replication (containing the El region) of adenoviin the viconcentrate analyzed (Hoeben, unpublished results) or when infecting HepG2 cells with the batch rAdV. Over a period of at least 10 days, the cells are monitored for potential cytopathogenic effects and by indirect immunofluorescence using a specific monoclonal antiserum directed against the E1A protein.

Plasmids and DNA Transfections The adapter plasmid pMad5 is constructed from pMLP10 (Levrerno et al., 1991) as described below. Plasmid pMLP-10-lin is derived from pMLPlO by insertion of a synthetic DNA fragment with unique sites for the restriction endonucleases Mlul, Spll, SnaBI, SpI, AsuII, and Muñí within the HindIII site of pMLPlO. The BglII adenovirus fragment spans nt 3328 to 8914 of the Ad5 genome and is inserted into the Muñi site of pMLP-lin. From the resulting plasmid, the SalI-BamHI fragment is deleted to inactivate the tetracycline resistance gene. The resulting plasmid is monitored by restriction enzyme analysis and is referred to as pMad5. The expression of genes inserted at the multiple cloning site will be driven by the adenovirus major late promoter, which in this configuration is linked to the adenovirus (El) immediate early gene extender. All CAV DNA sequences are originally derived from the pIc-20H / CAV-EcoRI plasmid (Noteborn and De Boer, 1990). The expression plasmid pCMV-fs, formerly called pCMV-VP3 (Noteborn et al., 1994), contains CAV DNA sequences encoding exclusively for apoptin (nt 427-868).

Plasmid pCMV-VP2mu (Noteborn, unpublished results) contains CAV DNA sequences from positions 380 to 1512. This CAV DNA fragment contains the VP2 coding region flanked by 25 bp of non-coding CAV DNA sequences. 'of 25 bp and non-coding 3' of 484 bp. At 106 bp towards the 3 'end of the start codon for VP2, the start codon for apoptin is located in another reading frame. To avoid synthesis of apoptin without interference from VP2 synthesis, a mutation is introduced into the start codon of apoptin (ATG is changed into ACG) and in addition, a point mutation at position 549 (T is changed to A), which results in an additional stop codon, within the gene that codes for apoptin. Therefore, inserted CAV sequences will only express full length VP2 proteins. By means of indirect immunofluorescence it is demonstrated that VP2 can be produced but that apoptin is not synthesized. For the cloning of DNA fragments amplified by PCR, the plasmid PCR-3.1 which is commercially purchased from InVitrogen (Carlsbad, CA) has been used. For the construction of a recombinant retrovirus with apoptin, defective in its replication, the retrovirus vector pLXSN is used (Miller, 1990a, b).

All the cloning steps with the plasmid DNAs are in principle carried out according to the methods of Maniatis et al. (1992). All the enzymes used are obtained commercially from Boehringer Mannheim Germany and / or BioLabs, United States. The plasmid DNA is purified by centrifugation in a CsCl gradient and column chromatography on Sephacryl S500 (Pharmacia, Uppsala, Sweden). The human cell lines HaCAT, HepG2, SCC-15, 293, 911 and PER.C6 are transfected with the plasmid DNA by calcium phosphate precipitation as described by Graham and Van der Eb (1973). Normal human diploid keratinocytes (FSK-1, second passage), U20S and Saos-2 cells are transfected with DOTAP (Fischer et al., 1996).

Indirect Immunofluorescence Assay Cells are fixed with 80% acetone and used for immunofluorescence assays with monoclonal antibodies specific for CAV or specific for adenovirus ElA and goat anti-mouse and / or goat anti-rabbit fluorescein conjugate (Jackson Immunoresearch Laboratories Inc. , West Grove PA), as described by Noteborn et al. (1990) . The nuclear DNA is stained with 2,4-diamino-2-phenylindole (DAPI) or with propidium iodide (Pl).

RESULTS AND DISCUSSION Construction of the adapter vector pMab To introduce a BamHI restriction enzyme site into the adapter plasmid pMAd5, it is digested with the restriction enzyme Clal and treated with bovine intestinal alkaline phosphatase. A Clal-BamHI linker is treated with T4-kinase and ligated to itself by the use of T4 DNA ligase and subsequently by ingestion with Clal. The Clal / BamHI / Clal linker is isolated and ligated to the linearized pMad5 vector. The bacterial strain JM109 is transformed with the ligation products. By means of digestions with restriction enzyme, the final pMab vector is characterized. By means of the pMab vector, foreign genes can be ligated into unique BamHI site under regulation of the adenovirus major late promoter. In figure 1 a schematic representation of pMad5 and pMab is shown.

Construction of a recombinant apoptin and a control adapter vector To construct an adapter vector for introducing the apoptin gene into an adenovirus, pMab is treated with BamHI and a 0.45 kb DNA fragment containing the sequences encoding apoptin is subsequently isolated. The apoptin DNA fragment is ligated into the BamHI site of the linearized pMab adapter vector. The ligation products are cloned into bacterial strain JM109. The orientation of apoptin in pMab is determined by restriction enzyme analysis. The construction of pMab containing the apoptin gene in the 5 '-3' orientation under the regulation of the adenovirus major late promoter will express the gene for apoptin. This adapter vector is called pMab-VP3 and will be used to generate an adenovirus vector that expresses apoptin. The plasmid DNA of pMab containing the sequences coding for apoptin in the 3 '-5' orientation towards the 3 'end of the adenovirus major late promoter can not express apoptin and will be used to establish a control recombinant adenovirus vector. In Figure 2 a schematic representation of both recombinant adapter vectors is shown.

Induction of apoptosis by a CMV plasmid versus a recombinant apoptin adapter vector expressing apoptin First, we examined whether the pMab-VP3 DNA vector is actually capable of expressing apoptin in transfected cells, and whether pMab-con does not do so. For this purpose, human 293 and 911 cells transformed with adenovirus are transfected with pMab-VP3, pMab-con and with a positive control with pCMV-VP3. Approximately two days after transfection, the cells are mixed and examined for their apoptin expression by means of an indirect immunofluorescence assay. The cell cultures are transfected with pCMV-VP3 and pMab-VP3 containing about 1% of the cells that react with a monoclonal antibody specific for apoptin, while cultures of cells transfected with DNA for pMab-con do not. These results imply that pMab-VP3 expresses apoptin and, as expected, pMab-con does not do so. In another transfection experiment, we analyzed the induction of apoptosis in 911 cells after transfection with pMab-VP3 versus pCMV-VP3. Three days after trnasfection, 911 cells are harvested and examined by indirect immunofluorescence to determine apoptin expression. In addition, the cells are stained with DAPI or Pl, which strongly stains intact DNA, but weakly and / or irregularly apoptotic DNA (Telford, 1992). Approximately 60% of apoptin-positive 911 cells transfected with pCMV-VP3 are apoptotic, whereas approximately 40% of apoptin-positive cells transfected with pCMV-VP3 undergo apoptosis. These results indicate that the expression regulated by pMab-VP3 of apoptin results in a similar or slightly higher level of apoptosis induction, and that apoptin is expressed by pCMV-VP3. In Figure 3 these results are shown. In addition, apoptin is capable of inducing apoptosis in human cells transformed with adenovirus, E1B does not inhibit apoptosis induced by apoptin. In contrast, E1B is capable of blocking apoptosis induced by a wide variety of chemotherapeutic agents. These results indicate that apoptin is a very potent antitumor agent.

Construction of recombinant adenovirus with apoptin Recombinant adenovirus vectors are generated with apoptin by co-transfection with the 911 line of helper cells of the adapter plasmids pMab-VP3 which present the coding sequences for apoptin plus some adenovirus sequences, and the plasmid DNA JM17 which contains all of the adenovirus DNA minus the El region and E3 (McGrory et al., 1988). The cotransfections are transformed with DNA precipitated with calcium phosphate as described by Graha and Van der Eb (1973). The recombinant adenovirus DNA is formed by homologous recombination between the homologous viral sequences that are present in the plasmid pMab-VP3 and the adenovirus DNA of the JM17 DNA.

In a similar manner, cotransfections of 911 cells are carried out with pMab-con and pJM17 DNA to generate the control recombinant adenovirus that can not express apoptin, and which will be used as adenovirus control for the apoptotic effects induced by apoptin. Several hours after transfection, the monolayers of 911 cells are covered with an agarose overlay and incubated at 37 ° C until the plates induced by recombinant adenovirus become clearly visible. The virus is harvested from plates as PBS-horse serum concentrates, as described by Fallaux et al. (nineteen ninety six). Subsequently, a portion of the recombinant virus concentrates are added to 24 wells containing fresh 911 cells. Several days later, these infected 911 cells are lysed and the recombinant viruses are harvested. Next, the expression of apoptin relative to the potential of recombinant adenovirus with apoptin (rAd-VP3) or its absence of expression by the control recombinant adenoviruses (rAd-con) is examined. A portion of the recombinant virus concentrates derived from the 24-well infected plates are used to infect fresh 911 cells, which grow as monolayers on glass coverslips. One day later, the infected 911 cells are fixed with acetone and analyzed by immunofluorescence using the monoclonal antibody specific for apoptin 85.1. Five of the 5 analyzed 911 cell cultures infected with putative rAd-VP3 contain cells expressing apoptin. None of the 911 cells infected with Ad-con and uninfected 911 cells were positive for apoptin. These results imply that, in the case of cotransfection of cell lines that pack adenovirus, such as 911 cells, with the required adapter and adenovirus DNA, viable rAd-VP3 expressing apoptin can be generated. Two concentrates derived from rAd-VP3 or rAds-con plates were used for purification of the rAds when carrying out three subsequent plate purifications with 911 cells or in parallel with a limited dilution assay in PER.C6 cells, as described by Fallaux (1996). Based on the methods described above that result in the production of apoptin expressing rAd-VP3 under the regulation of the adenovirus major late promoter, we also succeeded in an adenovirus vector expressing apoptin under the control of a cytomegalovirus promoter ( CMV). These results show that various types of recombinant adenoviruses can be produced by one of their own or heterologous promoter elements.

Production of rAd-VP3 and rAd-con by using PER.C6 cells Small-scale production of batches of rAd-VP3 and rAd-con using PER.C6 cells is done as described (Fallaux, 1996). Briefly, the procedure is described in the experimental section. By means of the plate assay, titers were determined, which are approximately 1011"12 per ml of clarified lysate for both rAd-VP3 and rAd-con.The titres obtained are not lower than those observed in our laboratory for other rAd By means of PCR analysis and HepG2 infection with rAd-VP3 and rAd-con it was examined whether the batches of virus produced contain adenovirus competent for replication (see also the experimental section). Both batches of rAd-VP3 and rAd-con were free of RCA, as demonstrated by both methods. We conclude that the expression of apoptin does not interfere negatively with all the necessary stages of the adenovirus life cycle under cell culture conditions. Therefore, gene therapy based on an adenovirus vector expressing apoptin is feasible. Due to the expression of Ad5 proteins The antiapoptotic (White, 1996), apoptin optimally induces apoptosis after recombinant adenovirus with apoptin has been produced in large quantities. The fact that the adenovirus vector expressing apoptin can be produced in human cells transformed with adenovirus type 5 (Ad 5) El proteins, such as 293, 911 and PERC6 cells, indicates that the El protein allows this DNA virus is replicated with high titers in the presence of the apoptosis-inducing protein apoptin. These results indicate that it is also possible to generate other recombinant DNA virus vectors expressing apoptin in cell lines transformed by the El protein 5 adenovirus. For example, recombinant parvovirus vectors based on Hl or parvovirus MVM can be propagated in 293T cells, which are transformed by the Ad5 El protein (Dinsart et al., 1996). Parvovirus H-1 and MVM specifically induce cell death in transformed cells, but not in all (López-Guerrero, 1997). Parvovirus vectors expressing apoptin will be more potent in inducing tumor-specific apoptosis compared to parvoviruses as such, due to the additional tumor-specific induction of apoptosis by apoptin (Dinsart et al., 1996, Danen-Van Oorschot, 1997 ). A protocol for the production of recombinant virus vectors with apoptin based on cells transformed with Ad5 protein also remains valid for RNA virus species such as retroviruses.

Induction of apoptosis in transformed and / or malignant human cell lines We have examined whether infection of human tumor cells with rAd-VP3 will result in apoptosis induced by apoptin. For this purpose, human hepatoma cells HepG2, osteosarcoma U20S, SCC-15 cells derived from squamous cell carcinoma and cells from a line of spontaneously transformed keratinocyte cells HaCaT were infected with rAd-VP3. One day after transfection, the cells are fixed by means of immunofluorescence and stained with DAPI, the cells are examined for apoptin synthesis, and if they are under developing apoptosis. One day after infection, almost all apoptin-positive human tumor cells analyzed were apoptotic. In uninfected cultures, only a small percentage of human tumor cells were apoptotic. The results for HepG2 and U20S cells are shown in the figure. These results indicate that apoptin expressed by rAd-VP3 can induce apoptosis in tumorigenic and / or transformed mammalian cell lines.

Apoptine expression in normal cells infected with rAd-VP3 To analyze the effect of apoptin expressed by rAd-VP3 on normal non-transformed infected cells, FSK-1 cells are infected with rAd-VP3. Four days after transfection, the cells are analyzed by indirect unofluorescence using monoclonal antibody 85.1 and DAPI staining. A maximum of 8% of apoptin-positive cells show an abnormal DAPI staining, indicating that they may be experiencing induced apoptosis (by apoptin). However, 7% of the cells that are not infected also have an aberrant DNA pattern stained with DAPI. The results are shown in Figure 4. Given the hepatotropic nature of human Ad5 after systemic delivery, it is also important to investigate the effect of apoptin on normal diploid hepatocytes. For this purpose, isolated rat hepatocytes were cultured in Williams E medium (Gibco / Life Technologies, Grand Island, NY, USA) supplemented with insulin (2 mU / ml) and dexamethasone (1 nM). The cells are grown on culture plates coated with collagen (Micronic). Primary rat hepatocytes are infected by the Ad-VP3 adenoviral vector which expresses apoptin, a control adenovirus that expresses LacZ or is infected in false. After two days, the cells are fixed and, by means of immunofluorescence and staining with DAPI, the percentage of apoptotic cells is examined. No differences were observed in the percentage of dead cells expressing apoptin, LacZ or false infected cells. These observations indicate that hepatocytes (from rat) do not undergo apoptosis induced by apoptin, in addition when apoptin is synthesized by means of an adenovirus vector. These results indicate that the expression directed by rAd-VP3 of apoptin does not result in apoptosis induced by apoptin in normal non-transformed human cells, in contrast to transformed / tumorigenic human cells.

Increase of apoptin synthesis To examine the effect of direct sequences against the ATG start codon of apoptin, two constructs of pCR-3.1-apoptin were performed. pCR-VP3ori contains the sequences towards the original direct 5 'end (5_-TTTCAA-3_) of the ATG codon, while the other construction, pCR-Vp3mu, contains the sequence to the direct 5 'end 5_-GCCAAC-3_. By means of in vitro transcription / translation in a wheat germ assay, it was determined that the apoptin expression of pCR-VP3mu is at least five times higher than that observed for pCR-VP3ori. These data indicate that the nature of the direct sequences towards the 5 'end of apoptin-ATG influences apoptin synthesis. The construction of (viral) vectors with direct sequence towards the 5 '5_-GCCAAC-3_ terminus versus the ATG codon of apoptin will result in increased apoptin production and indirectly in increased apoptosis, induced by apoptin. It is also important to mention that the amino acid sequence of apoptin is not altered as predicted to be necessary for increased translation efficiency, according to the "Kozak rule" (Caventer and Stuart, 1991). According to this rule, we have changed the nucleotides in the +4 position from an A to a G, which results in a second amino acid different from apoptin which would change its activity.

Identification of an essential fragment of apoptin that contains apoptotic activity To examine whether a part of the apoptin protein is essential for its apoptotic activity, a plasmid is constructed that codes for chimeric or recombinant proteins, consisting of the green fluorescence protein (GFP; Rizzuto, 1995) and the 71 amino acids of the part N terminal apoptin (N-apoptin) or its 50 amino acids from the C-terminal part (C-apoptin). Human transformed cells, such as Saos-2 cells (Zhuang, 1995) are transiently transfected with the plasmids expressing chimeric GFP / N-apoptin or GFP / C-apoptin. Only Saos-2 cells expressing GFP / C apoptin undergo apoptosis specific for apoptin. This coincides with the fact that C-apoptin (bound to GFP) can enter the nucleus. These results indicate that a part of apoptin may also be sufficient to induce apoptosis in (original) human / transformed cells. Therefore, an effective therapy (of genes) based on vectors (viral) expressing only that part of apoptin can also be developed. In addition, these data indicate that a part of apoptin contains its apoptotic activity when it is covalently bound to a foreign protein.

The coexpression of VP2 and apoptin in human tumor cells synergistically increases the induction of apoptosis To examine the effect of co-expression of VP2 and apoptin on the induction of apoptosis, we (co) -transfect Saos-2 cells with pCMV-fs, which expresses apoptin and / or with pCMV-VP2mu, which expresses VP2. The cells are fixed with acetone at various time intervals after transfection. By means of indirect immunofluorescence, VP2 positive cells are determined with monoclonal antibody (Noteborn and Koch, 1996) and apoptin positive cells with monoclonal antibody CVI-CAV-85.1. On day 3 after transfection, only 3% of the cells expressing VP2 experienced apoptosis, and only about 10% of the cells expressing apoptin. In contrast, approximately 40% of the Saos-2 cells expressing both VP2 and apoptin were already apoptotic. In addition, at four days after transfection, the percentage of VP2 / apoptin positive cells that experienced apoptosis is significantly higher compared to cells expressing only apoptin or VP2. These results show that VP2 increases the apoptosis induced by apoptin and this is shown in figure 5.

Construction and production of rAD-VP2 To construct a recombinant adenovirus expressing viral protein 2 (VP2) of chicken anemia virus, the adapter plasmid pMAb-VP2 is made. A 1.1 kb BamHI fragment is isolated from the pCMV-VP2mu plasmid which contains all the sequences coding for VP2, but with two point mutations within the apoptin coding region (see experimental section) and which binds to an adapter pMAb vector treated with bovine intestinal alkaline phosphatase linearized with BamHi. The final construction pMab-VP2 is characterized by restriction enzymes and sequence analysis as shown in Figure 3. By cotransfection of 911 cells with DNA for pMab-VP2 and DNA for pJMl7, rAD-VP2 is made. Cotransfection and all additional necessary steps for the characterization, purification and production of rAd-VP2, were carried out as described for rAd-VP3 and for rAd-con. By indirect immunofluorescence using monoclonal antibody CVl-CAV-111.1, it is shown that 911 and PER.C6 cells are infected with rAd-VP2, and can actually express the VP2 protein.

Construction of a retrovirus vector that expresses apoptin To generate plasmid pL-VP3-SN (see Figure 7), a BamHI fragment presenting the sequences coding for apoptin is inserted into the unique BamHI site of pLXSN. Appropriate orientation of the insert is confirmed by restriction enzyme analysis. To test the integrity of the insertion, the plasmid pL-VP3-SN is transfected with the calcium phosphate coprecipitation technique in COS-7 and HepG2 cells. Four days after transfection, the cells are fixed and analyzed with monoclonal antibody 85.1 to determine the expression of the apoptin protein. In about 1-2% of the cells, the expression of apoptin can be detected. Most of the cells underwent apoptosis, determined by staining with DAPI. These data show that the proviral LTR promoter is capable of activating apoptin protein expression, that its gene is intact in the DNA construct and that, in transfected HepG2 and COS-7 cells, the expression of apoptin induces apoptosis. To generate the viruses, plasmid pL-VP3-SN is transfected into Psi-2 cells and into PA 317 cells with the calcium phosphate coprecipitation technique. At 48 hours after transfection, the supernatant of the cell is harvested and dilutions are used to infect HepG2 cells (the PA317 supernatant) and NIH3T3 cells (the Psi-2 supernatant) in the presence of 4 μg / ml polybrene. Four days after infection, the cells are fixed and analyzed for apoptin expression by staining with monoclonal antibody 85.1. It is found that approximately 1% of the cells express apoptin. The majority of HepG2 cells positive for apoptin are apoptotic. These data demonstrate that the cells have undergone transduction with the retrovirus L-apoptin-SN. In addition, it is demonstrated that a single copy of the provirus is sufficient to express sufficient amounts of the apoptin protein which can be detected by immunofluorescence and that this amount is sufficient to induce apoptosis in a human tumor cell line, specifically, the cell line of hepatoma HepG2. Taken together, these data demonstrate that retroviral vectors presenting the gene for apoptin can be generated and that they can be used to induce apoptosis in human tumor cells. It is formally demonstrated that neither the apoptin gene nor its expression interfere with the essential stages in the life cycle of the retrovirus. It also demonstrates that retroviruses containing apoptin can be produced as batches in amounts sufficient to be used to transduce human tumor cells in tissue culture. These results further imply that the expression of apoptin, and consequently the apoptosis induced by apoptin in tumor cells (human), is also possible by means of vector systems derived from (retro) viruses, such as plasmoviruses (Noguiez-Hellin, 1996 ). The critical step for such a recombinant plasmovirus system with apoptin is if the retrovirus replication is not blocked by the expression of apoptin. We have provided evidence that this is not really the case for the recombinant retrovirus with apoptin described above that involves a successful production of recombinant plasmovirus with apoptin.

Diagnostic assay for cancer cells based on rAD-VP3 The apoptin cell position is different in human tumorigenic / transformed cells compared to normal untransformed cells. In addition, another marker is the specific ability of apoptin to induce apoptosis in tumorigenic / transformed cells and not in normal cells. By infecting cells with rAd-VP3 and analyzing the position of apoptin and / or the induction of apoptosis within these cells, one is able to demonstrate whether a cell is malignant or not. Primary cells of tissue are isolated (suspect) and cultured in the required medium. The cells are infected with rAd-VP3 and, in parallel, with rAd-con, and subsequently analyzed. For example, by using an immunofluorescence assay based on monoclonal antibodies specific for apoptin, 85.1. Cells will be checked for apoptin in the cytoplasm (normal cells) or in the nucleus (transformed cells). In addition, or alternatively, the percentage of apoptotic cells will be estimated. If the percentage of apoptotic cells is significantly higher for rAd-Vp3 in comaprtion with cells infected with rAd-con, these cells will become malignant.

Adenovirus toxicity experiments with recombinant apoptin in healthy rats Under tissue culture conditions, apoptin expressed by rAd-VP3 of recombinant adenovirus in normal cells, for example derived from human or rodent origin, do not induce apoptosis. In the experiment described below, we have examined whether the expression of apoptin by means of the rAd-Vp3 vector of recombinant adenovirus in healthy rats does not result in acute toxicity. The vector used rAd-VP3 and the control vector rAd are grown on PER.c6 cells and, by means of PCR analysis, they are shown to be negative for the adenovirus replication component (RCA-free). RAd are purified by centrifugation in a CsCl gradient. Male Wag / Rij rats (Harían, The Netherlands) with a body weight of approximately 200 grams are injected with recombinant adenovirus expressing apoptin (rAd-VP3; 2.5xl09 pfu plate-forming units), with recombinant control adenovirus expressing the product of luciferase gene (rAd-luc, -2.5xl09). Both adenovirus vectors are resuspended in phosphate buffered saline, containing 0.1% bovine serum albumin and 10% glycerol (PBS +). This solution without the adenovirus vector is also injected into rats and serves as an additional negative control. Two rats are injected intravenously, intraperitoneally or subcutaneously, either with rAd-VP3, rAd-luc or PBS + suspension.

Macroscopic pathological analysis of rats treated with Ad-VP3 The first method to examine a possible toxic effect of apoptin expressed by Ad-VP3 is to determine the general health condition and in particular the body weight of the treated rats, which is done every day after the injections. All rats are in good health during the experiment. Body weight is not significantly different in the various groups. After 1 week, all the rats examined, including those injected with rAd-VP3, have gained body weight which indicates that none of the animals suffers from acute toxicity due to one of the rAd-VP3 treatments. To further establish the absence of acute toxicity, the following determinations were made. Two hours before sacrifice, all the rats were injected with BrdU. After sacrifice, various tissues (liver, kidney, intestines, heart, lung, spleen, adrenal glands and penis) were examined pathologically and / or collected for later histopathological analysis (see below).

The macroscopic analysis showed that none of the rats treated with Ad-VP3 presents organs with significant pathological effects.

Determination of Ad-VP3 DNA in the liver The main target of the (vector) adenovirus (recombinant) injected intravenously is the liver and to some extent the spleen. Therefore, any toxic effect of apoptin will be observed in the liver. A panel of experiments was carried out to examine the presence of DNA for Ad-VP3, the expression of apoptin by Ad-VP3 and some possible cytopathological effect on the liver. First, we examined by means of Southern blot analysis whether the DNA isolated from the livers of rats treated with Ad-VP3 contains the DNA for apoptin on the day of sacrifice, which means 8 days after the injection . As negative controls, DNA from rat livers treated with Ad-luc was examined in parallel. Before loading the DNA isolated on agarose gel, the DNA is digested with BamHI, which results in a DNA fragment for apoptin of approximately 0.5 kbp. The Southern blot hybridizes with a 32P-labeled apoptin DNA probe.

The DNA fragment BamHl and apoptin is clearly visible in the Southern blot in the case of DNA derived from animals treated with Ad-VP3, and as expected, is absent in the rails containing the DNA isolated from the livers of rats treated with rAd -luc control. To examine the number of copies of Ad-VP3 in the liver, various amounts of DNA for apoptin were loaded in parallel in the Southern blot and hybridized with the labeled DNA probe for apoptin. Even eight days after the intravenous infection, 0.25 copies of Ad-VP3 per cell could be determined, which indicates a very significant transduction of Ad-VP3 in the liver.

Expression of apoptin and its toxic effect in liver cells By means of immunostaining of paraffin sections of livers treated with Ad-VP3 or control livers using monoclonal antibodies specific for apoptin CVI-CAV-85.1 or CVl-CAV-111.3. , we have shown that approximately 20-30% of the liver cells of animals treated with Ad-VP3 have expressed apoptin. The liver sections of the control rats were negative for apoptin. To examine the possible cytotoxic effects on apoptin expressed by Ad-VP3 in liver cells, two different methods were carried out. First, liver sections from all rats treated with Ad-VP3 and those from both types of control animals were stained with hematoxylin-eosin (HE). For all liver sections examined no morphological pathological changes could be observed, indicating that apoptin expression is not toxic to rat liver cells. Damage effects can be observed by means of BrdU labeling which detects newly divided liver cells. In the case of livers containing Ad-VP3, the amount of liver cells labeled with BrdU is of a similar degree (approximately 2%) compared to the livers of control rats treated with Ad-luc. Therefore, the expression of apoptin, as such, does not have a significant toxic effect in vivo.

Apoptin does not have an acute toxic effect in an in vivo model Both macroscopic and histological analyzes, in combination with biochemical and immunological data, reveal that apoptin expression does not have a toxic (acute) effect in an in vivo model. These results indicate that therapy based on the expression of apoptin by the use of a vehicle that delivers the gene or by other methods will have limited negative side effects.

Antitumor studies in a human hepatoma model The regulated expression of Ad-VP3 of apoptin results in the induction of apoptosis in human transformed cells under tissue culture conditions. For example, the expression of apoptin activated by Ad-VP3 results in the induction of apoptosis in HepG2 cells derived from human hepatoma. Hitherto, in vivo anti-umoral activity of apoptin has not been examined (e.g., expressed by Ad-VP3). Therefore, we have determined whether the expression of apoptin regulated by Ad-VP3 will result in antitumor activity in an in vivo model. For this purpose Balb / C / nu / nu nude mice were injected subcutaneously with IxlO6 of human HepG2 cells per side. At least two to three positions were injected per mouse of human hepatoma cells. Three weeks after the injection, clear hepatoma tumors had developed which were visible subcutaneously and had an average size of at least 5 x 5 mm. Vectors rAd-VP3 and conrol rAd-conl, suspended in phosphate buffered saline, 5% sucrose and 0.1% bovine serum albumin, were injected intratu orally. The rAd-VP3 vector used expressing apoptin and the rAd-conl control vector containing the apoptin gene in the 3'-5 'orientation (inverse or antisense) as opposed to the Ad MLP promoter, both grew in PER.C6 cells. Both batches of recombinant adenovirus were shown to be free of RCA by PCR analysis. The rAds were purified by the use of a CsCl gradient centrifugation. For tumor, 7xl09 pfu of rAd particles in a suspension of 40 microliters were injected. By type of vector rAd, 6 mice were treated with 2 to 3 HepG2 tumors. As an additional control, a group of 4 nude mice containing HepG2 tumors were injected intratumorally with phosphate buffered saline containing 5% sucrose and 0.1% bovine serum albumin (PBS + - group).

Apoptin has an antitumor effect in an in vivo model To examine the possible antitumor effect of apoptin expressed by Ad-VP3 in human HepG2 tumors, the size of the subcutaneous tumors was measured during the experiment which continued until 7 days after the injection of rAd-VP3 suspensions and control . Both PBS + groups and the group treated with rAdconl control showed a mean progressive increase in HepG2 tumor size. In contrast to the group that was treated intratumorally with rAdVP3, which shows a reduced tumor size. Seven days after the injection, athymic mice were sacrificed. The tumors were isolated and examined macroscopically. It is evident that hepatoma tumors treated with the vector rAd-conl control or with PBS + show to be highly vascularized HepG2 tumor tissues and have become larger after treatment. A completely different pattern is observed with HepG2 tumors treated with rAd-VP3. The residual tumor mass has a pale appearance, due to a reduced vascularization of the tumor. The tumors present a reduced size after treatment with rAd-VP3. The tumors also contain white structures similar to bubbles, which is indicative of dead cells. In addition to apoptin-induced tumor regression, a negative effect of apoptin expression on organs could not be observed (particularly the liver and spleen were examined).

Apoptin has antitumor activity in an in vivo system In conclusion, tumors treated with rAd-VP3 show a reduced tumor size, whereas controls did not. This implies that the expression of apoptin has an antitumor activity in an in vivo model. The fact that apoptin expressed by Ad-VP3 can induce tumor regression in a rapidly growing tumor such as HepG2 demonstrates the strong antitumor potential of apoptin. The fact that apoptin reduces tumor growth in athymic mice shows that the expression of apoptin itself "destroys" tumor cells without an additional immune response. The toxicity described and the anti-tumor studies reveal that antitumor therapy based on the expression of apoptin is safe and feasible.

DESCRIPTION OF THE FIGURES Figure 1 shows the diagrammatic representation of the essential parts of the pMAdS vectors and pMab adenovirus adapters. Figure 2 shows the diagrammatic representation of the essential parts of the pMab-VP3 and pMab-vectors with recombinant adenovirus adapters. Figure 3 shows the activity of apoptosis induced by apoptin in 911 cells transfected with pMAb-VP3 or pCMV-VP3. Two batches of pMab-VP3 DNA were used, cloned and purified independently (pMab-VP3 / ml and pMab-VP3-m2) for the transfection of 911 cells. Cells are fixed 3 days after transfection and analyzed by immunofluorescence indirectly using the monoclonal antibody specific for apoptin CVI-CAV-85.1 (85.1; Noteborn et al., 1991). The percentage of cells that are normally stained with DAPI is given as the relative measure for apoptosis. Figure 4 shows the apoptin-induced activity (termed VP3) of HepG2 cells of human tumorigenic hepatoma, U20S osteosarcoma cells and untransformed normal diploid FSK-1 keratinocytes infected with the adenovirus Ad-VP3 defective in their recombinant replication with apoptin. Cells were analyzed by direct immunofluorescence using monoclonal antibody 85.1 and stained with DAPI. The HepG2 and U20S cells were fixed one day after transfection and the FSK-l cells were harvested and fixed 4 days after transfection. The percentage of apoptin-positive cells that were normally stained with DAPI is provided as a measure of apoptosis induced by apoptin (black squares). As a control, the percentage of uninfected cells that were abnormally stained with DAPI (white squares) is provided. Figure 5 shows apoptosis activity induced by apoptin and / or by VP2 in Saos-2 cells transfected with 2.5 μg of pCMV-fs DNA expressing apoptin (formerly called pCMV-VP3; apoptin is called VP3), and 2.5 μg of pCMV-neoBa DNA (Danen-Van Oorschot, 1997); or with 2.5 μg of pCMV-VP2 DNA expressing CAV protein 2 (VP2) and 2.5 μg of pCMV-neoBam DNA; or with 2.5 μg of pCMV-fs and 2.5 μg of pCMV-VP2 results in the expression of both apoptin (VP3) and VP2. The cells were fixed 3, 4 and 5 days after transfection and analyzed by indirect immunofluorescence using the monoclonal antibody specific for apoptin CVI-CAV-85.1 (85.1, Noteborn et al., 1991) or with monoclonal antibody CVI-CAV- 111.1 (Noteborn and Koch, 1996). The percentage of cells that were abnormally stained with DAPI is provided as a relative measure for apoptosis. Figure 6 shows the diagrammatic representation of the essential parts of the pMab-VP2 recombinant adenovirus adapter vectors. Figure 7 shows the diagrammatic representation of the essential parts of the pLS-VP3-N transfer vector of recombinant retrovirus.

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Claims (21)

1. A delivery vehicle for a gene, characterized in that it comprises a nucleic acid molecule that codes for apoptin-like activity.

The delivery vehicle of a gene, according to claim 1, characterized in that it additionally comprises a modified translation start site, directly towards the 5 'end of the ATG start codon in the nucleic acid molecule.

3. The delivery vehicle of a gene, according to claim 2, characterized in that the translation site comprises the GCCAAC nucleic acid sequence.

4. A delivery vehicle for a gene characterized in that it comprises a nucleic acid molecule that encodes activity similar to VP2.

The delivery vehicle of a gene, according to claim 4, characterized in that it additionally comprises a translation start site modified directly towards the 5 'end of the ATG start codon of the nucleic acid molecule.

6. The gene delivery vehicle according to any of claims 1 to 3, characterized in that it additionally comprises a nucleic acid molecule that encodes activity similar to VP2.

The delivery vehicle of a gene, according to claim 6, characterized in that it additionally comprises a translation start site modified directly towards the 5 'end of the ATG start codon of the nucleic acid molecule encoding similar activity to VP2.

8. The vehicle for delivery of a gene, according to any of claims 1 to 7, characterized in that it is a virus.

9. The vehicle for the supply of a gene, according to claim 8, characterized in that it is a virus defective in its replication.

10. The delivery vehicle of a gene, according to claim 8 or 9, characterized in that it is an adenovirus.

11. The delivery vehicle of a gene, according to claim 8 or 9, characterized in that it is a retrovirus.

12. The delivery vehicle of a gene, according to any of claims 1 to 11, characterized in that it additionally comprises at least one target molecule.

13. The delivery vehicle of a gene, according to claim 12, characterized in that the target molecule is reactive with a tumor cell surface receptor.

14. A host cell, characterized in that it comprises a delivery vehicle of a gene, according to any of claims 1 to 13.

The host cell according to claim 14, characterized in that it is an auxiliary or packaging cell.

16. The host cell according to claim 14, characterized in that it is selected from the group of HEK cells: 293, HER, 911, PER.C6, Psi-2 and PA317.

17. The use of a gene delivery vehicle, according to any of claims 1 to 13, characterized in that it is used in the treatment against cancer.

18. The use of a gene delivery vehicle, according to claim 17, characterized in that it is used in combination with conventional (chemo) therapy.

19. The use of a gene delivery vehicle, according to any of claims 1 to 13, characterized in that it is used for the treatment of hyperplasia, metaplasia and dysplasia.

20. The use of a gene delivery vehicle, according to any of claims 1 to 13, characterized in that it is used in diagnosis.

21. The use of a gene delivery vehicle, according to claim 20, characterized in that it is used in the in vitro detection of transformed or cancerous cells or of hyperplastic, metaplastic or dysplastic cells.