AU2006201883B2 - Tumour-Specific Vector for Gene Therapy - Google Patents

Description

Po01 Section 29 Regulation 13.2(2) AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Tumour-Specific Vector for Gene Therapy The following statement is a full description of this invention, including the best method of performing it known to us: Tumour-specific vector for gene therapy The present invention relates to a vector for gene therapeuti cal treatment of tumours, in particular in connection with a radiotherapy, whose DNA sequence has at least one tissue specific promoter and at least one therapeutic gene whose ex 5 pressing is controlled by the promoter. DE 44 44 949 C1 discloses a vector of this type. 10 2 For the purposes of the present invention, "tumours" means both malignant and benign tumours. Malignant neoplastic disorders account for approx. 30 % of deaths in the civilized world, and there is at present no safe therapy for any tumour yet, in spite of worldwide efforts over the last decades. Many tumours can be treated only with diffi-1 culties, if at all.. Examples of benign tumours include tumours of the vascular wall for which partly the same therapies are used as for the treat ment of malignant tumours, i.e. cancer. Such tumours of the vascular wall form as recurring stenoses essentially due to in duction of smooth muscle-cell proliferation in the vascular wall caused by so-called "balloon dilatations" (PTCA) for the therapy of local stenoses of the vascular wall which may limit an organ's blood supply. The treatment of such arteriosclerotic disorders includes in addition to balloon dilatation also by pass surgery, stents and other alternative therapeutic methods 3 which, however, likewise have the problem of recurring steno sis, i.e. constriction of the lumen of the treated vessel due to a benign tumour. Besides surgical removal of both malignant and benign tumours 25 and the treatment thereof with cytostatics, radiotherapy repre sents from the present point of view one of the most important pillars of tumour therapy. In this connection, the probability of destruction of the tumour depends on the dose administered, with the dose to be administered being limited by the radio 30 sensitivity of normal tissue which inevitably is also irradi ated. Therapeutic successes thus depend on the relative radio- 3 sensitivity of the tumor cells compared with the cells of the neighboring tissue. Consequently, an increase in the therapeutic range due to se 5 lective radiosensitization of tumour cells would mean a sig nificant step forward in the treatment of tumours and improve the rates of cure. For this reason, pharmacological radiosensi tizers which ought to make tumour cells more sensitive to ra diation are already in use clinically. 10 Gene therapy, for the first time, offers the opportunity of achieving significant progress in controlling cancer by making it possible to use therapeutic genes for enhancing radiotherapy or therapy with cytostatics. 15 In this connection, viral vector systems play a great part in transducing therapeutic genes. Besides retroviral vector sys tems which, to a certain extent, prefer proliferating cells and very often integrate into the cellular genome, especially ade 20 noviral vector systems which make it possible to attain a high titre of virus particles and which have good transduction effi ciency and a very low rate of integration are in discussion; see Ali et al., Gene Ther. (1991), Volume 1, 367-384. 25 It is crucial for a safe gene therapy that the therapeutic gene is used only in the desired target cells. It would therefore be desirable to use in the gene therapy of tumours a tumour cell specific promoter which is active in various tumour species and not active in all types of normal tissue. Such a promoter, how 30 ever, has not yet been found.

4 In this connection, DE 44 44 949 Cl, mentioned at the outset, describes a vector for the gene therapy of patients who after surgical removal of a tumour, undergo an aftertreatment using conventional radiation and/or cytostatic methods. The vector 5 contains an expressible Insert-DNA which is located behind a promoter active in tumour cells and codes for the DNA-binding domain (DBD) of a poly(ADP-ribose) polymerase (PARP). The idea on which that publication is based is to inhibit the activity of the enzyme PARP which is required for repairing DNA-damages 10 by adding DBD molecules so that repair of damaged DNA is pre vented. As example of a tissue-specific promoter the MVM P4 promoter is mentioned. The vector may be a viral vector, and the viruses 15 are replication-incompetent and can be complemented in trans in order to obtain viruses which code for DBD but are unable to propagate in patients. It has turned out to be a disadvantage of the known vector that 20 the P4 promoter does not yet have the tissue specificity re quired for a safe gene therapy so that DNA repair is also in hibited in normal tissue, and this is, for reasons that need no further explanation, undesirable. 25 Most gene therapeutic approaches therefore include an enhance ment of the tumour-specific immune response, i.e. they are a priori systemic, since the local increase in the immune re sponse is not limited to the tumour. At the same time, this is also a desired advantage, because in principle it is also pos 30 sible to destroy metastases using this therapy.

5 In order to allow a more or less local gene therapy, the use of retroviruses which transduce genes only. into dividing cells, has already been discussed. Furthermore, adenovirus mutants were proposed, which ought to replicate only in p53-negative tumour 5 cells; Heise et al., Nat. Med. (1997), Volume 3, 639-645. It is assumed here that all p53-positive cells can prevent prolifera tion of the adenovirus. In addition, Joki et al., Hum. Gene Ther. (1995), Volume 6, 10 1507-1513 have already proposed using radiation-inducible pro moters and Nettelbeck et al., Adv. Exp. Med. Biol. (1998), Vol ume 451, 437-440 proposed using cell cycle-specific promoters. However, all of these known solutions have specific disadvan 15 tages. The concept of immunotherapy has the principal disadvan tage that immunosuppressed patients cannot respond to it, the neoplastic disorder itself often additionally adversely affect ing the immune system of the said patients. Furthermore, it has turned out that an immunotherapeutic gene or a "suicide gene" 20 which kills the target cell is on its own not sufficient for in vivo administrations; Uckert et al., HUM. GENE THER. (1958), Volume 9, 855-865. The combination of several immunostimulatory genes, where appropriate in connection with suicide genes, has also up to now not led to a breakthrough which would allow a 25 standard application for a given neoplastic disorder. The abovementioned retroviruses are furthermore not exclusively selective for tumours but infect all dividing cell types, as long as an appropriate receptor is present. Moreover, infection 30 of a healthy cell carries the risk of insertion mutagenesis, since retroviruses integrate into the cellular genome. Replica- 6 tion-competent adenoviruses which have been proposed for a specific tumor therapy are also not specific for tumor cells, as has been proved since then, the effect also being independent of the p53 state; Rothmann et al., J. Virol. (1998), Volume 72, 9470-9478. 5 The previously described use of suicide genes, too, has disadvantages, since the said suicide genes increase the side effects in healthy proliferating tissues. This problem could be avoided only if the suicide genes were expressed tumor-specifically. In view of the above, it is an object of the present invention to provide a 10 vector of the type mentioned at the outset, which acts tumor-specifically and has only slight side effects on normal tissue. According to the invention, this object is achieved with the vector mentioned at the outset by the promoter being selected from the group comprising the promoter for the catalytic subunit of telomerase and the promoter 15 for cyclin-A. The aim of the present invention is therefore to achieve/overcome at least one of the objects/prior art disadvantages referred to herein. The invention, the subject of this application is directed to a vector for treating tumors by gene therapy, the vector comprising a DNA sequence 20 comprising a) a first sequence encoding T7 RNA polymerase under control of a tissue-specific promoter that is a telomerase catalytic subunit promoter or a cyclin-A promoter; b) at least one therapeutic gene under control of a first T7 promoter, 25 wherein the therapeutic gene encodes a protein selected from the group consisting of: cytosine deaminase (CD), herpes simplex-virus thymidine kinase (HSV-TK), DNA-binding domain (DBD) of poly(ADP-ribose) polymerase (PARP), cytotoxic protease 2A and 3C; or a fusion protein comprising at least two proteins selected from the group of proteins; and 30 c) a second sequence encoding T7 RNA polymerase under control of second T7 promoter.

6a In fact, the inventors of the present application have recognized that the promoter for the catalytic subunit of telomerase is particularly well suited for a tumor-specific gene therapy. Namely, this promoter which is described, for example, by Horikawa et al., Cancer Res. (1999), Volume 59, 826-830, and 5 Takakura et al., Cancer Res. (1999), Volume 59, 551-557 is very 7 active in tumour cells and more than 90 % of all tumours are telomerase-positive. Furthermore, the said promoter is less ac tive in a few proliferative stem cells, for example germ line cells and blood stem cells, and not active at all in the vast 5 majority of proliferative cell types such. as, for example, en dothelial, fibroblasts, hepatocytes, etc. In this connection, WO 98/11207 discloses the use of a telom erase promoter in connection with the cell transfection, in, or 10 der to express products which inhibit cell growth with regard to a cancer treatment. However, the said publication does not mention application of a telomerase promoter in connection with a radiotherapeutic treatment. 15 The second promoter used according to the invention is the cy clin-A promoter which is activated in the S phase of the- cell cycle, while being repressed in resting cells, i.e. in the GO and early Gl phases; Henglein et al., PNAS (1994), Volume 91, 5490-5494. Cyclin-A is involved in several regulatory pathways 20 in cell division so that it is exceptionally well suited to ex pressing therapeutic genes in proliferating cells. Here, the inventors of the present application start from the finding that tumour cells are distinguished in particular by defects in cell cycle regulation and, as a result, enter the S phase in an 25 uncontrolled manner. Choosing the cyclin-A promoter thus would make the therapeutic gene express only in the tumour but not in the surrounding normal tissue, since this tissue in most cases has no or only little proliferative activity. The cyclin-A promoter in combination with a therapeutic gene is 30 particularly suitable for treating benign tumours, especially 8 in connection with the recurring stenoses mentioned at the be ginning, whose therapy is considerably improved by the vector of the invention. Against this background, the present inven tion also relates to the use of the cyclin-A promoter in con 5 nection with a therapy of benign tumours, in particular in con nection with recurring stenoses. Besides the safety provided by the tumour-specific promoters for the catalytic subunit of telomerase and for cyclin-A a 10 second point of safety results from applying these promoters, or the therapeutic genes controlled thereby in connection with a radiotherapy which, according to the current state of the art, can be directed very locally only towards the tumour tissue. 15 By combining various therapeutic genes which are expressed tis sue-specifically and the locally directed radiotherapy, it is possible to provide for the tumour cells to react more sensi tively than the neighbouring tissue, albeit perhaps only by a small factor, but as a result the tumour can then be destroyed 20 efficiently. Besides using the new vector in radiotherapy, it is also advan tageous to use it in connection with chemotherapy, because in this way it is possible to destroy non-localized metastases. 25 In this connection, preference is given to the therapeutic gene coding for a protein selected from the following group of pro teins: cytosine deaminase (CD), herpes simplex-virus thymidine kinase (HSV-TK), DNA-binding domain (DBD) of poly(ADP-ribose) 30 polymerase (PARP), cytotoxic protease 2A and 3C of picorna vi- 9 ruses, preferably of enteroviruses, more preferably of group B Coxsackie viruses (CVB), in particular serotype B3. Cytosine deaminase converts 5 -fluorocytosine to 5 fluorouracil 5 which is incorporated into the DNA of replicating cells and then kills these cells. A systemic 5 -fluorocytosine treatment in connection with 'local radiotherapy leads to a specific in crease in the destruction of tumours, since cytosine.deaminase is only formed in the tumour cells so that the dreaded side ef 10 fects such as necroses/fibroses in neighbouring tissue, damage of bone marrow'and intestinal mucosa, etc. are avoided. HSV-TK acts in a similar way; this enzyme activates gancyclovir which likewise incorporates into the DNA of replicating cells 15 and destroys the DNA so that, in connection with local radio therapy, the same advantages as with CD are attained. In contrast to CD and HSV-TK, expression of DBD molecules leads to inhibition of the activity of PARP which is required for re-' 20 pairing DNA damage. In this way it is not possible to "repair" again tumour cells "predamaged" in connection with the local radiotherapy, so that they die. In contrast, the proteases 2A and 3C induce apoptosis in cells 25 and are thus cytotoxic. The inventors of the present application have now found that by the combination of firstly radiotherapy and secondly of pro teins of the above-mentioned type that are tissue-specificalily 30 expressed by the two above-mentioned promoters a selective de- 10 struction of tumours can be achieved, which is markedly more effective than the individual measures used previously. In this connection, preference is given to the therapeutic gene 5 being a fusion gene coding for a fusion protein of at least two proteins selected from the abovementioned group of proteins, the therapeutic gene, preferably between the sequence regions for the two proteins, coding for a peptide linker which pref erably comprises glycine, in particular 8-10 glycines. 10 It is advantageous here that a synergistic effect can be achieved if the therapeutic gene contains two suicide genes, with different mechanisms of action of the partners within the fusion proteins in particular producing a synergistic action. 15 The advantage of expressing fusion genes is the possibility of transferring simultaneously two different therapeutic princi ples and thus producing additive or frequently even synergistic effects in the therapy. In order for the protein domains of the fusion partners to be able to fold optimally for the applica 20 tion, it may be sensible to clone the information for a short peptide linker, preferably 8-10 glycines, between the cDNAs. Particular preference is given to the following fusion pro teins: 25 CD-linker-HSV-TK, CD-linker-DBD, CD-linker-2A, CD-linker-3C, HSV-TK-linker-DBD, HSV-TK-linker-2A, HSV-TK-linker-3C,

DBD

linker-2A and DBD-linker-3C. The order of the fusion partners within a fusion protein may also be reversed. 30 11 Overall, preference is given to the vector being based on a vi rus vector, in particular on an adenovirus vector or an adeno associated virus vector (AAV). 5 The invention further relates to a retrovirus vector coding for the novel vector. Thus, the novel gene therapy system may be introduced both via conventional viral or non-viral vectors and also via a retrovi 10 rus vector into the organism in which it has a substantially more selective effect on the tumour than has previously been possible. Furthermore, preference is given to providing, between the pro 15 moter and the therapeutic gene, a positive-feedback system which is driven by the promoter and controls. by itself the ex pression of the therapeutic gene, the positive-feedback system preferably comprising the T7 promoter and the gene for T7 RNA polymerase, and preference is furthermore given to the promoter 20 controlling the gene for T7 RNA polymerase and the T7 promoter controlling the therapeutic gene, with furthermore a second ex pression unit being provided, which contains the T7 RNA poly merase under the control of the T7 promoter. 25 This system of positive feedback causes increased expression of the therapeutic gene in the target cells, with the promoter, i.e. the telomerase or cyclin-A promoter making sure that the positive-feedback system is "triggered" only in the target cells. This positive feedback is based on the finding that the 30 T7 promoter is silent in eukaryotic cells without said T7 RNA 12 polymerase, thus providing a very safe system which multiplies the therapeutic effect without displaying side effects. In a first expression unit the telomerase promoter, for exam 5 ple, controls T7 RNA-polymerase expression. The second expres sion unit then contains the T7 promoter which again controls T7 RNA-polymerase expression. In this way, production of T7 RNA polymerase is increased via positive feedback. Since in the third expression unit the therapeutic gene is under the control 10 of the T7 promoter, T7 RNA-polymerase expression by positive feedback also increases expression of the therapeutic gene. It is understood that the features mentioned above and those still to be illustrated below can be used not only in the com 15 binations indicated in each case but also in other'combinations or on their own, without leaving the context of the present in vention. Further features and advantages of the invention arise from the 20 following description of preferred embodiments. The examples below are illustrated on the basis of the attached drawing in which: 25 Fig. 1 shows viral vectors for expressing therapeutic genes in tumour cells; and Fig. 2 shows viral vectors- with positive-feedback expres sion of therapeutic genes in tumour cells. 30 13 Example 1: Generation of the building blocks for the viral vectors 5 1.1 Telomerase promoter: The telomerase ,promoter sequence is known; Horikawa et al., loc. cit. and Takakura et al., loc. cit. The promoter may be amplified from cells (e.g. HeLa tumour cells) via PCR using, for example, the fol lowing primers: 10 Forward primer: ATC AGC TTT TCA AAG ACA CAC Reverse primer: CGC GGG GGT GGC CGG GGC CAG 1.2 Cyclin-A promoter: The cyclin-A promoter sequence is likewise known; Henglein et al., loc. cit. The promoter may be amplified from cells, for example HeLa tumour cells, via PCR using the following primers: Forward primer: CGT GTT AAA TAA TTT ATG CAC 20 Reverse primer: CAC TGC TCC CGG GAG TGG ACG 1.3 T7 promoter: The T7 RNA-polymerase promoter (approx. 30 bp) may be obtained from plasmid pCR-Script (Strata 25 gene) by BssHI/KpnI digest. The T7-promoter and T7 RNA . polymerase sequences are described, for example, in Dunn and Studier, J. Mol. Biol. (1983), Volume 166, 477-535; GenBank Accession No. V01146, JO2518, X00411. 30 1.4 Cytosine deaminase (CD): The CD gene (1.3 kb) may be obtained from plasmid pcDNA3-CD of the applicant by 14 BamHI/NotI digest. The CD sequence has been described, for example, by Austin and Huber, Mol. Pharmacol. (1993), Volume 43, 380-387; GenBank Accession No. S56903. 5 1.5 Herpes simplex virus thymidine kinase (HSV-TK): The HSV TK gene (1.1 kb) can be obtained from plasmid pCDTK of the applicant by BamHI/BglI digest. The HSV-TK sequence is described by Suzutani et al. in Microbiol. Immunol. (1995), Volume 39, 787-794; GenBank Accession No. 10 AB009255. 1.6 DNA-binding domain (DPD) of poly(ADP-ribose) polymerase (PARP): The DBD (1.1 kb) may be obtained from plasnid pPARP6 by XbaI/SalI digest; KUpper et al., J. Biol. Chem. 15 (1990), Volume 265, 18721-18724. 1.7 CVB3 protease 2A: The sequence coding for the cytotoxic protease 2A is obtained from plasmid pIND-2A of the ap plicant by Pme digest. 20 1.8 CVB3 protease 3C: The sequence coding for' the cytotoxic protease 3C is obtained from plasmid pIND-3C of the ap plicant by Pme digest. 25 Klump et al. describe the complete CVB3-cDNA sequence in J. Vi rol. (1990), Volume 64, 1573-1583; GenBank Accession No. M33854; protein 2A: Nucleotide 3304-3744; protein 3C: Nucleo tide 5362-5910. 30 1.9 Fusion genes: A fusion gene means the continuous sequence of a therapeutic gene composed of several, preferably 15 two, cDNAs which are expressed via a single promoter to give a continuous fusion protein. In this way it is pos sible to transfer simultaneously two therapeutic princi ples, thus resulting in additive or synergistic effects in the therapy. In order for the protein domains of the fusion partners to be able to fold optimally for application, the infor mation for a short peptide linker, preferably for gly o cine, in particular 8-10 glycines, is cloned between the cDNAs. In this way, the following fusion genes are generated: CD-linker-HSV-TK, CD-linker-DBD, CD-linker-2A, CD-linker 3C, HSV-TK-linker-DBD, HSV-TK-linker-2A, HSV-TK-linker 3C, DBD-linker-2A and DBD-linker-3C. The order of. the fu sion partners within a fusion gene may also be reversed. 1.10 T7 RNA polymerase (T7 Pol): The T7 Pol cDNA (2.6 kb) originates from plasmid pAR3132 of the applicant and con tains codons 11-883 of the T7 RNA-polymerase gene. The sequences of the building blocks have been deposited with the PubMed gene bank of the National Library of Medicine .5 (http://ww4.ncbi.nlm.nih.gov/Pub-Med/).

16 Example 2: Preparation of recombinant adenoviruses Recombinant adenoviruses are prepared by using, for example!, the El/E3-deleted adenovirus-5 system of Vogelstein; He et al., PNAS (1998), Volume 95, 2509-2514. The cDNA to be expressed, i.e. the therapeutic gene, is cloned into vector pShuttle (6.7 kb). The promoter (telomerase or cy clin-A) intended for application is cloned in front of the cDNA and a polyadenylation signal is cloned behind the cDNA. The newly generated plasmid is transformed together with the pAdEasyl helper plasmid into the recombination-competent bacte rial strain BJ5183. Homologous recombination of the overlapping- shuttle- and 5 helper-vector sequences results in a recombinant adenoviral genome which is isolated from the bacteria and transformed into the recA~ strain HB101 for preparative processing. Plasmid ma terial is then isolated from the said bacteria on the prepara tive scale and purified via caesium chloride centrifugation. !0 The plasmid material obtained is transfected into the El expressing helper cell line 911; Fallaux et al., Hum. Gene Ther. (1996), Volume 7, 215-222. After transfection with the recombinant adenoviral genome, the cells are overlaid with soft agar. The virus then propagates in transfected cells and a 25 plaque is formed from which the recombinant viruses can be iso lated by freeze-thaw lysis. Expression can be detected after two days but no cytopathic-effect (CPE) is apparent yet. Virus stocks are obtained by infecting new 911 cells with the appro priate recombinant adenoviruses. After approx. four days, the CPE has fully formed. The cells are disrupted in a Dounce ho 30 mogenizer, cell debris is pelleted by short centrifugation and the viruses present in the supernatant are purified via caesium 17 chloride density-gradient centrifugation. The adenovirus vector obtained in this way is depicted in Fig. 1, top, and the abbreviations used are explained in the legend 5 to the figures. Example 3: Preparation of recombinant adeno-associated vi ruses (AAV) An example of the system used here is the system of Samuiski, comprising two plasmids plus adenovirus; Snyder et all. "Production of recombinant adeno-associated viral vectors'. Current Protocols in Human Genetics. New York: John Wiley and Sons (1996), 12.1.1-12.1.24. 5 The cDNA to be expressed, i.e. the therapeutic gene, is cloned together with regulatory sequences (promoter and poly-A signal) into a vector containing only AAV-2 terminal repeats. These re peats are the minimum Cis-regulatory sequences required for replication and packaging; Xiao et al., J. Virol. (1997), Voi ume 71, 941-948. The vector is generated by excising. the rep/cap sequence via XbaI digest from plasmid pSub201 (Human Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA). The termi nal repeats of in each case 0.18 kb remain in the vector. The building blocks intended for the particular gene Ctransfer system, such as promoter, cDNA of the therapeutic gene and polyadenylation signal, are then cloned into said vector. The vector plasmids thus generated are transfected into 2 9 3-cells, 0 in each case together with the pAAV/Ad (Human Gene Therapy Cen ter) helper plasmid which provides the AAV structural and non- 18 structural proteins (cap and rep) in trans. On the next day, wild-type adenovirus is added as a helper for AAV replication to the cells at an MOI of 3. Two to three days after transiec tion/infection, the cytopathic effect (CPE) is well visible and 5 recombinant AAV can be obtained from the cells. The caesium chloride purification method is carried out as de scribed by Snyder et al., lc. cit. Essential elements of this AAV purification are three times freeze-thaw lysis of the in 10 fected cells plus ultrasound treatment to release the viruses, ammonium sulphate precipitation to remove cellular proteins, purification of the AAV particles on a CsCl gradient by ultra centrifugation, dialysis of the purified AAV fractions by PBS and heat-inactivation of contaminating adenoviruses by incuba tion at 56 0 C for 15 minutes (AAV is not inactivated by this 15 treatment). The AAV vector obtained in this way is depicted in Fig. 1, cen tre. 20 Example 4: Preparation of recombinant retroviruses An example of a system which may be used here is the system from Clonetech (Heidelberg). This system comprises shuttle vec tors, for example pLNCX (6.2 kb), which can be propagated via transformation into bacteria and also a helper cell line, the 25 RetroPack PT67 line, which enables transcomplementation of the vectors to virions. Prior to using the pLNCX shuttle vector, the CMV promoter is removed from this vector in order to be replaced thereafter by 30 the promoter of choice, i.e. the telomerase promoter or the cy clin-A promoter. The abovementioned therapeutic genes or fusion 19 genes are then cloned into the multiple cloning sequence with polyadenylation signals. The recombinant vector is transfected into the packaging cell line PT67. Transfected cells can be selected for by using the antibiotic G418. As a result, recombinant retroviruses are pro duced, which are obtained from cells and cell culture super natant via methods analogous to those described in Examples 2 and 3 and which can be purified by caesium chloride density gradient centrifugation. The retrovirus vector obtained in this way is depicted in prin ciple in Fig. 1, bottom. Examole 5: Adenovirus vector with positive-feedback system Fig. 2 depicts an adenovirus vector which was prepared as de scribed in Example 2. However, instead of a single promoter and a single therapeutic gene, this vector contains three expres sion units, the first of which contains T7 RNA polymerase under D the control of the telomerase promoter. The second expression unit likewise contains T7 RNA polymerase but under the control of its own promoter, the T7 promoter. Finally, the third ex pression unit contains the therapeutic gene under the control of the T7 promoter. 25 When target cells are infected, first the telomerase promoter causes expression of T7 RNA polymerase (first expression unit). The T7 RNA polymerase generated "switches on" the T7 promoter in the second expression unit, so that this T7 promoter, too, causes T7 RNA-polymerase production. This results in a posi tive-feedback system, and the more T7 RNA polymerase is gener 30 ated, the more T7 promoter is switched on.

20 In this way, expression of the therapeutic gene which here is under the control of a T7 promoter is increased. In this system, the telomerase promoter thus controls expression of the therapeutic gene not directly, as in Examples 2-4, but indirectly via the 5 intermediate step of the positive-feedback system of T7 promoter and T7 RNA polymerase. Since the T7 promoter is silent in eukaryotic cells without the T7 RNA polymerase which usually is not found there, the therapeutic gene consequently can be expressed only in those cells in which the telomerase promoter is active, 10 i.e. especially in tumor cells. Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components 15 or groups thereof.

Claims (20)

1. A vector for treating tumors by gene therapy, the vector comprising a DNA sequence comprising a) a first sequence encoding T7 RNA polymerase under control of a 5 tissue-specific promoter that is a telomerase catalytic subunit promoter or a cyclin-A promoter; b) at least one therapeutic gene under control of a first T7 promoter, wherein the therapeutic gene encodes a protein selected from the group consisting of: cytosine deaminase (CD), herpes simplex-virus thymidine kinase 10 (HSV-TK), DNA-binding domain (DBD) of poly(ADP-ribose) polymerase (PARP), cytotoxic protease 2A and 3C; or a fusion protein comprising at least two proteins selected from the group of proteins; and c) a second sequence encoding T7 RNA polymerase under control of second T7 promoter. 15

2. The vector of Claim 1, wherein the therapeutic gene codes for a protein selected from the group of proteins consisting of: cytosine deaminase (CD), herpes simplex-virus thymidine kinase (HSV-TK), DNA-binding domain (DBD) of poly(ADP-ribose) polymerase (PARP), cytotoxic protease 2A and 3C.

3. The vector of Claim 2, wherein said cytotoxic protease 2A and 3C is from 20 picornaviruses.

4. The vector of Claim 2, wherein said cytotoxic protease 2A and 3C is from group B Coxsackie viruses.

5. The vector of Claim 2, wherein said cytotoxic protease 2A and 3C is from group B Coxsackie viruses, serotype B3. 25

6. The vector of Claim 1, wherein the therapeutic gene is a fusion gene coding for a fusion protein of at least two proteins selected from the group of proteins. 22

7. The vector of Claim 6, wherein the therapeutic gene between the sequence regions for the two proteins codes for a peptide linker.

8. The vector of Claim 7, wherein the linker comprises glycine.

9. The vector of Claim 8, wherein the linker comprises between 8-10 glycines. 5

10. The vector of Claim 7, wherein the fusion gene is selected from the group consisting of: CD-linker-HSV-TK, CD-linker-DBD, CD-linker-2A, CD-linker-3C, HSV-TK-linker-DBD, HSV-TK-linker-2A, HSV-TK-linker-3C, DBD-linker-2A and DBID-linker-3C.

11. The vector of Claim 1, comprising a vector selected from a virus vector, an 10 adenovirus vector and an adeno-associated virus (AAV) vector.

12. A retrovirus, coding for a vector of Claim 1.

13. A method for treating tumors by gene therapy, the method comprising the step of administering to an individual in need of such therapy a vector of Claim 1.

14. The method of Claim 13, for treating neoplastic disorders.

15 15. The method of Claim 13, further comprising the step of treating the individual with radio-therapy.

16. The method of Claim 13, further comprising the step of treating the individual with a therapy with cytostatics.

17. A method for treating benign tumors, the method comprising the step of 20 administering to an individual in need of such treatment the vector of Claim 1, wherein the tissue-specific promoter is the cyclin-A promoter.

18. The method of Claim 17 for treating recurring stenoses. 23

19. Use of a vector of any one of Claims 1 to 11 in the manufacture of a medicament for treating tumors by gene therapy.

20. Use according to Claim 19, for treating neoplastic disorders. HEARTBIOSYSTEMS GMBH WATERMARK PATENT & TRADE MARK ATTORNEYS P21157AU01