Retroviral vector transducing the p 45 cytochrome gene

The present invention relates to a capsule containing cells having a diameter of 0.01 mm to 5 mm, in particular 0.1 mm to 1 mm, which is used in anti-cancer therapy using a prodrug. In addition, the invention also relates to the use of such

188 323 capsules for the preparation of medicaments, and compositions and a pharmaceutical containing the capsule.

State of the art.

Anti-cancer drugs used to treat cancer are in most cases systemically used and spread throughout the patient's body. The high systemic dose of such drugs required for treating cancer is associated with unpleasant side effects for the patient.

To overcome this problem, cancer prodrugs were used that must be metabolized or activated in the body before they become cytotoxic. Unfortunately, human tumors that contain sufficiently high levels of activating enzymes are rare. The primary site of prodrug activation is the liver and to ensure that a distant tumor receives a sufficient dose of activated drug, a very high dose of activated prodrug produced in the liver should be administered, which causes toxic side effects for the patient.

One strategy by which the problems of high systemic levels of activated drug could be bypassed would be to provide means for activating the prodrug directly or near the tumor site. Such a strategy would require cancer cells, or cells where the tumor is located, to be genetically transformed to produce large amounts of the enzymes required to metabolize this tumor prodrug. Retroviral vectors are perfectly suited to the stable delivery of genes to cells from the moment that the retrovirus will be able to integrate its own genome DNA with the host cell genome and thus daughter cells of infected cells will be carriers of retroviral vectors carrying therapeutic genes. Another benefit is that most retroviruses only infect dividing cells, which is why they are the ideal provider of genes for cancer cells.

To date, a variety of cytotoxic genes transmitted by retroviral vectors have been tested. These genes encode enzymes that cause in vivo conversions of pharmacodynamically and toxicologically inert substances, even at high doses to highly active metabolites (Connors, TA (1995), Gene Therapy 2: 702-709).

In properly planned cancer chemotherapy, prodrugs are found to be effective in the treatment of animal cancers containing high levels of activating enzymes (Connors, T. and Whisson, M. (1966), Nature 210: 866-867 and Cobb, L. et al. (1969) , Biochemical Pharmacology 18: 1519-1527). However, clinical results have been disappointing because it has been found that human cancers that contain sufficiently high levels of activating enzyme are rarely seen (Connors, T. (1986), Xenobiotica 16: 975-988). Prodrug therapy using a virus-associated enzyme (VDEPT - virally directed enzyme prodrug therapy) and the more general prodrug therapy using a gene-associated enzyme (GDEPT - gene directed enzyme prodrug therapy) show similarity as they serve to destroy cancer cells by cancer specific prodrug activation. In this case, however, the gene encoding the enzyme is either specifically targeted to malignant cells or is under the control of a specific promoter.

So far, most of the efforts directed at prodrug therapy have focused on the use of the human thymidine kinase (HSV-tk) gene Herpes Simplex as a suicide gene. Although the HSV-tk enzyme in combination with the prodrug ganciclovir (GCV) is recommended as a good system in GDEPT (Culver, K. et al. (1992), Science 256: 1550-1552, Ram, Z. et al. (1993), Cancer Research 53: 83-88 and Chen, S., Shine, H. et al. (1994), Proc. Natl. Acad. Sci. 91: 3054-3057), there are many theoretical reasons to suggest that this is undoubtedly the best combination. First, it works specifically in the S phase and does not affect the cells in the resting phase. This is because GCV monophosphate is short lived and must be present when the cells enter the S phase to be toxic. HSV-tk phosphorylates GCV to monophosphate (a reaction that cannot be carried out by mammalian enzymes), which is then phosphorylated by cellular enzymes to carbon triphosphate and is incorporated into DNA. Secondly, the active drug is triphosphate and should not be expected to diffuse freely by affecting neighboring cells. However, such effects on neighboring cells are observed both in vitro and in vivo,

188 323 although there appears to be a metabolic interaction, and in the latter case some of the effects may have an indirect nature associated with the immune component (Bi, W., Parysek, L. et al. (1993), Human Gene Therapy 4: 725- 731, Vile, R. and Hart, I. (1993) Cancer Research 53: 3860-3864 and Freeman, S., Abboud, C., et al (1993), Cancer Research 53: 5274-5283). The disadvantage is that the effect of interacting with neighboring cells is dependent on cell-cell contact. This may be due to the presence of gap joints formed by close contact between transduced and surrounding cells, which allows the transfer of phosphorylated ganciclovir.

Recently, interesting results have been reported where cells that were transfected with a gene encoding the 2B1 form of rat cytochrome P450 and then treated with cyclophosphamide (Chen, S., Shine, H., et al. (1994), Proc. Natl. Acad. Sci. 91 : 3054-3057).

Object of the invention.

The object of the invention is to provide a new tool for use in therapy.

The essence of the invention.

The subject of the invention is a capsule containing cells having a diameter of 0.01 mm to 5 mm, especially 0.1 mm to 1 mm, characterized in that it covers the cell producing cytochrome P450 and has a porous membrane permeable for prodrug particles penetrating inside the capsule and activated by cytochrome P450.

Preferably, the capsule of the invention is made of a material containing an electrolytic complex formed from such compounds as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates. Preferably, the cytochrome P450 producing cell contains a vector prepared as described in Example II, i.e. the pc3 / 2Bl vector. Equally preferably, the cytochrome P450 producing cell is a stable cell line expressing the cytochrome P450 constitutively, while the cytochrome P450 gene is under the transcriptional control of regulatory or promoter sequences or of the X-inducible target cell promoter.

The invention also relates to a pharmaceutical kit comprising a capsule and a prodrug, characterized in that said capsule covers a cell producing cytochrome P450 and has a porous membrane permeable for prodrug molecules penetrating inside the capsule and activated by cytochrome P450, wherein said capsule has a diameter from 0, 01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

Preferably, in the kit of the invention, an electrolyte complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates. Equally preferably, in the kit of the invention, the cytochrome P450 producing cell contains a vector prepared as described in Example II, i.e. the pc3 / 2Bl vector. Preferably, in the kit of the invention, the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450. Preferably, in the kit of the invention, the cytochrome P450 gene is under transcriptional control of regulatory or promoter sequences or an X-inducible target cell promoter. Preferably, in the kit of the invention, the capsules and prodrug are in the form of various formulations. Preferably, in the kit of the invention, the capsule is in a form for administration by injection and / or by implantation in target organs and / or a location near them, and the prodrug is in a form of systemic and / or local administration. Preferably, in the kit of the invention, the capsule is in a form for administration by implantation into the tissue of a breast or pancreatic tumor and / or a site nearby. Preferably, in the kit of the invention, the prodrug is cyclophosphamide and / or ifosfamide.

A further object of the invention is a pharmaceutical composition comprising an active agent and optionally a pharmaceutically acceptable carrier, characterized in that as an active agent it comprises a capsule covering a cytochrome P450 producing cell, said capsule having a porous membrane permeable for prodrug molecules penetrating inside the capsule and activated by cytochrome P450 and the capsule diameter is from 0.01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

188 323

Preferably, the pharmaceutical composition of the invention is characterized in that the capsule material comprises an electrolytic complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates.

Preferably, the pharmaceutical composition of the invention is characterized in that the cytochrome P450 producing cell contains a vector prepared as described in Example II, i.e. the pc3 / 2Bl vector.

Preferably, the pharmaceutical agent of the invention is characterized in that the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450.

Preferably, the pharmaceutical agent of the invention is characterized in that the cytochrome P450 gene is under transcriptional control of regulatory or promoter sequences, or of an X-inducible target cell promoter.

Preferably, the pharmaceutical composition of the invention is characterized in that it is in a form for administration by injection and / or by implantation into target organs and / or the place near them.

Preferably, the pharmaceutical composition of the invention is characterized in that it is in a form for administration by implantation into the tissue of a breast or pancreatic tumor and / or a site nearby.

Preferably, the pharmaceutical composition of the invention is characterized in that the prodrug is cyclophosphamide and / or ifosfamide.

Another object of the invention is the use of a capsule covering a cytochrome P450 producing cell, for the manufacture of a medicament for the treatment of cancer, wherein the capsule has a porous membrane permeable to prodrug molecules penetrating into the capsule and activated by cytochrome P450, wherein the capsule has a diameter of from 0.01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

Preferably, in the use of the invention, the capsule material comprises an electrolyte complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates.

Preferably, in the use of the invention, the cytochrome P450 producing cell comprises the pc3 / 2B 1 vector.

Preferably, in the use of the invention, the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450.

Preferably, in the use of the invention, the cytochrome P450 gene is under transcriptional control of regulatory or promoter sequences, or of an X-inducible target cell promoter.

Preferably, in the use according to the invention, the capsule is in a form for administration by injection and / or by implantation into target organs and / or sites nearby.

Preferably, in use according to the invention, the capsule is in a form for administration by implantation into the tissue of a breast or pancreatic tumor and / or a site nearby.

Preferably, in the use according to the invention, the prodrug is cyclophosphamide and / or ifosfamide.

In one preferred embodiment of the use according to the invention, said medicament is a pharmaceutical kit comprising said capsule and said prodrug.

Preferably, the capsule and prodrug are in the form of various formulations in the use of the invention.

Preferably, in the use of the invention, the capsule is in a form for administration by injection and / or by implantation in target organs and / or a location nearby, and the prodrug is in a form for systemic and / or local administration.

Disclosure Description.

The disclosure contained herein includes, among others:

- a replication defective retro viral vector carrying the cytochrome P450 gene transcribed under the control of specific regulatory or yasmotoropic sequences, or, or the X-ray inducible target cell promoter;

188 323

- a replication-defective retroviral vector as above wherein the vector contains the 5'LTR region of the U3-R-U5 jteukruek; jcdna or y-clc sckwcncji selected from coding sckwcncji - nothing coding; - ecjon 3'LTR decapitating the total or partially removed ecjon U3, which replaced the replaced ecjon U3, followed by phosphooylene or other cells from carbonyl and carbon monoxide, and relieving DNA contamination. After the deugic, active lc is the phosphate and nothing is wasted by the fact that he will diffuse freeds, repaying the neighboring cells of the cell. I am saying that I am adjacent to eamóeei, looking at both in y-teo jek and in yiyo, though I am thinking that I am to share my mission, that in this last complete part of the effects may be a member of the community, a chemist, a chemist, a specialist , L. et al. (1993), Humen Gcnc Thceepy 4: 725-731, Vilc, R. and Heet, I. (1993) Cencce Rcscaech 53: 3860-3864 and Feccmen, S., Abboud, C., et al (1993), Cencce R ^ eech 53: 5274-5283). The inconvenience of this is that it separates the adjacent cells adjacent to the cell-cellular contour. This may be related to the strangeness of the staple joints formed and the close contact between the stone and the restorative chambers, which will allow the tehnero-phosphate parental patient.

Recently, data on those interested in ectultatech, where eamókach, who were ^ ζμ & ^ wenc gcncm encoding the 2B1 foemase of the rat P450 cytacheome and arterial cyclofosfemircm cell refactor (Chcn, S., Shinc, H., et al. Net. 1994) Sci. 91: 3054-3057).

Ccl invention.

Cclcm of the invention is to provide a new tool for ending up with malignancy in science.

The essence of the invention.

Pezcdmiot of the invention are key cells that are living cells that have a diameter of 0.01 mm to 5 mm, especially 0.1 mm to 1 mm, which are characterized by the fact that a yokeyye p450 producing cytochrome pellet and a full lintel eetywowenkch pez cytochrome P450.

Coexistence according to the invention is made of a metal label that contains a clolithic compound formed from these compounds: elginien and polylysine, or a chain of cclulose and polyglycol di-diyl diammonium or other polyamide products. The cell cores producing the cytochrome P450 is fully prepared according to the description in Phase II. Equally, eamóeee producing the P450 jcst cytocheome is a stable cell line conforming to the constitutive cytochrome P450, the gcn netomiest of the cytochrome P450 jcst under the reactive detection control of ecgletoric or integer epitome or target promoters. Pezcdmiatrm wynelezku jcst takżc zcstew feemeccutyczny zeyiceejący kepsułę oeez peolck, cheeekteeyzujący in that ZC ez ^ zone kepsułe pokeyye komóekę producing cytocheom P450 and pos-Ede poeowetą membrane pezcpuszczelną dle cząstcczck peolcku pezcnikejących to wnęteze kepsuły and eetkyowenych pezcz cytocheom P450 pezy the ezcczone kepsułe yosiede head from 0.01 mm to 5 mm, coresistors from 0.1 mm to 1 mm.

Coexistence, according to the invention, in the corpuscles, clcrolytic cells utilized from such compounds as elginien and polylysine or the cclulose network and polyglycol di-dimethyl diammonium chloride or other products of porous sulfonylimides and polysulfonates. Equally, coexists in the corpora according to the invention, the cell producing cytochrome P450 excipient in which it corresponds to the description of the syllabus II. Coresistors in the corpuscles according to the invention, the cell producing cytochrome P450 is a stable cell line that forms the constitutive cytochrome P450 cspecision. Coresistors in the zensts according to the invention, gcn of the cytochrome P450 jcst yod teen control of ecguletor or yeast functions, or promatoee of the target cells innueayelncego y Xmicin X. Caezystnir in place according to the invention, the miserable form I need to water incision and / or implant implantation into the target genes and / or missionaries in their vicinity, where I find that I am full of character to feed the systemist - or his missionary. Colonists put together, according to the invention, I am spoiled for immunity, for immersion in the tissue of the stomach or cancer of the thesis and / or for the mission in its vicinity. Coresistors in the corpora according to the invention, peolck jcst cyktofosfemidcm and / or ifosfamidrm.

188 323

A further object of the invention is a pharmaceutical composition comprising an active agent and optionally a pharmaceutically acceptable carrier, characterized in that as an active agent it comprises a capsule covering a cytochrome P450 producing cell, said capsule having a porous membrane permeable for prodrug molecules penetrating inside the capsule and activated by cytochrome P450 and the capsule diameter is from 0.01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

Preferably, the pharmaceutical composition of the invention is characterized in that the capsule material comprises an electrolytic complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates.

Preferably, the pharmaceutical composition of the invention is characterized in that the cytochrome P450 producing cell comprises a vector prepared as described in Example II.

Preferably, the pharmaceutical agent of the invention is characterized in that the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450.

Preferably, the pharmaceutical composition of the invention is characterized in that the cytochrome P450 gene is under the transcriptional control of regulatory or promoter sequences or an X-inducible target cell promoter.

Preferably, the pharmaceutical composition of the invention is characterized in that it is in a form for administration by injection and / or by implantation into target organs and / or the places near them.

Preferably, the pharmaceutical composition of the invention is characterized in that it is in a form for administration by implantation into tissue of a breast cancer or pancreatic cancer and / or a site nearby.

Preferably, the pharmaceutical composition of the invention is characterized in that the prodrug is cyclophosphamide and / or ifosfamide.

Another object of the invention is the use of a capsule covering a cytochrome P450 producing cell, for the manufacture of a medicament for the treatment of cancer, wherein the capsule has a porous membrane permeable to prodrug molecules penetrating into the capsule and activated by cytochrome P450, wherein the capsule has a diameter of from 0.01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

Preferably, the use according to the invention is characterized in that the capsule material comprises an electrolytic complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates.

Preferably, the use according to the invention is characterized in that the cytochrome P450 producing cell contains a vector prepared as described in Example II.

Preferably, the use of the invention is characterized in that the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450.

Preferably, the use of the invention is characterized in that the cytochrome P450 gene is under transcriptional control of regulatory or promoter sequences, or of an X-inducible target cell promoter.

Preferably, the use according to the invention is characterized in that the capsule is in a form for administration by injection and / or by implantation in target organs and / or places near them.

Preferably, the use according to the invention is characterized in that the capsule is in a form for administration by implantation into the tissue of a breast or pancreatic tumor and / or a site nearby.

Preferably, the use according to the invention is characterized in that the prodrug is cyclophosphamide and / or ifosfamide.

Another object of the invention is the use of a cytochrome P450 producing cell capsule and a prodrug for the manufacture of a pharmaceutical kit for the treatment of cancer or other related diseases or disorders, wherein

188 323 the capsule has a porous membrane permeable to prodrug molecules penetrating inside the capsule and activated by cytochrome P450, said capsule having a diameter of from 0.01 mm to 5 mm, preferably from 0.1 mm to 1 mm.

Preferably, the use according to the invention is characterized in that the capsule material comprises an electrolytic complex formed from compounds such as alginate and polylysine or cellulose sulfate and dimethyl diallylammonium chloride, or other porous structures such as polyamides and polysulfonates.

Preferably, the use according to the invention is characterized in that the cytochrome P450 producing cell contains a vector prepared as described in Example II.

Preferably, the use of the invention is characterized in that the cytochrome P450 producing cell is a stable cell line expressing constitutive cytochrome P450.

Preferably, the use of the invention is characterized in that the cytochrome P450 gene is under transcriptional control of regulatory or promoter sequences, or of an X-inducible promoter of target cells.

Preferably, the use according to the invention is characterized in that the capsule and prodrug are in the form of various formulations.

Preferably the use according to the invention is characterized in that the capsule is in a form for administration by injection and / or by implantation in target organs and / or a place near them where they are and the prodrug is in a form of systemic and / or local administration.

Preferably, the use according to the invention is characterized in that the capsule is in a form for administration by implantation into the tissue of a breast or pancreatic tumor and / or a site nearby.

Preferably, the use according to the invention is characterized in that the prodrug is cyclophosphamide and / or ifosfamide.

Disclosure Description.

The disclosure contained herein includes, among others:

- a replication defective retroviral vector carrying the cytochrome P450 gene under transcriptional control of specific regulatory or promoter sequences, or the X-ray inducible target cell promoter;

- a replication-defective retroviral vector as above wherein the vector comprises the 5'LTR region of the U3-R-U5 structure; one or more sequences selected from coding and non-coding sequences; and a 3'LTR region containing a fully or partially deleted U3 region, wherein the deleted said U3 region is replaced by a polylinker sequence containing specific regulatory or promoter sequences, or an X-inducible target cell promoter followed by regions R and U5 characterized by having at least one coding sequence for cytochrome P450;

- a replication-defective retroviral vector as above wherein the specific regulatory or promoter sequence of the target cells is selected from one or more members of the group containing specific WAP regulatory and promoter sequences, MMTV, β-lactoglobulin and casein, pancreatic specific regulatory and promoter sequences carbon II anhydrase and β-glucokinase regulatory and promoter sequences, lymphocyte specific regulatory and promoter sequences containing specific regulatory and promoter sequences of immunoglobulins and MMTV and specific MMTV regulatory and promoter sequences giving reactivity to glucocorticoid hormones or directing expression in mammary glands;

- a replication defective retroviral vector as above, wherein said LTR regions are selected from at least one member of the group comprising LTR regions of MLV, MMTV, MSV, SIV, HIV, HTLV, FIV, FeLV, BLV and MPMV viruses;

- a replication-defective retroviral vector as above, wherein said retroviral vector is based on the BAG vector or pLXSN vector;

- a replication defective retroviral vector as above, wherein said retroviral vector is pLX2Bl, prepared as described in Example 1;

188 323

- a replication defective retroviral vector as above, wherein said retroviral vector is pc3 / 2Bl, prepared as described in Example U;

- a replication-defective retroviral vector as above wherein the retroviral sequences associated with retrovirus integration are altered or at least partially deleted;

- a replication-defective retroviral vector as above, wherein said regulatory or promoter sequences are regulated by intermediary molecules;

- the packaging cell line transfected with a replication defective retroviral vector as above, said packaging cell line comprising at least one retrovirus or recombinant retrovirus construct encoding the proteins necessary for packaging of said retrovirus vector;

- the packaging cell line as above, wherein the packaging cell line is from rodents or is of rat, canine, feline or human origin and is tissue compatible with human tissue;

- the packaging cell line as above, wherein the packaging cell line is selected from the group consisting of psi-2, psi-Crypt, psi-AM GP + E-86, PA317 and GP + envAM-12;

- a recombinant retrovirus molecule produced by culturing the appropriate packaging cell line, one of the above, under appropriate conditions, optionally followed by isolation of the produced recombinant virus particles;

- a pharmaceutical composition comprising recombinant retrovirus molecules as above, or a packaging cell line as above;

- a packaging cell line as above covered with a porous membrane capsule that is permeable to recombinant retrovirus molecules produced by said packaging cell line;

- a packaging cell line, as above, covered with capsules of cellulose sulfate and polydimethyldiallyl ammonium complex;

- a method of removing cancer cells, which comprises administering to the subject being treated a therapeutically effective amount of recombinant retrovirus particles, as above, the packaging cell line, as above, or the packaging cell line, covered with capsules, as above, and either simultaneously or at a time interval, a tumor prodrug that can be activated by cytochrome P450;

- a method as above, wherein the cancer cells are breast cancer cells or pancreatic cancer cells;

- a method as above, wherein the recombinant retrovirus molecules, the packaging cell line, or the capsule-covered packaging cell line is administered by injection or implantation into a tumor, or in a place where the tumor is located;

- the use of recombinant retrovirus particles, as above, the packaging cell line, as above, or the capsule-covered packaging cell line, as above, for the preparation of a pharmaceutical composition useful in the removal of cancer cells;

- a retroviral provirus integrated into the human genome, carrying the cytochrome P450 gene under transcriptional control of specific regulatory or promoter sequences, or the X-ray inducible target cell promoter; and

- human cells containing the cytochrome P450 gene which is under the transcriptional control of specific regulatory or promoter sequences or of the X-inducible target cell promoter.

Detailed description of the invention.

Cytochrome P450 forms a vast group of monooxygenases that catalyze the oxidation of a wide range of substrates. They are produced by bacteria, yeasts and higher organisms, where they play a role in the detoxification of xenobiotic substances, bioactivation reactions and in the metabolism of various endogenous compounds.

Cytochrome P450 catalyzes the hydroxylation of commonly used cyclophosphamide (CPA) and ifosfamide tumor prodrugs to their active toxic forms. Normally, the patient's endogenous cytochrome P450 gene expression is limited to the liver, so the anti-tumor effects of systemically administered CPAs depend on the progressive systemic distribution of toxic liver metabolites. This is related to the problem of toxicity as

188 323 that the activated drug affects not only the cancer but also other patient tissues such as bone marrow and kidneys.

The therapeutic approach, where the cytochrome P450 gene is selectively introduced directly into tumor cells and is subject to increased expression in these cells, allows to bypass this problem. Toxic metabolites produced by transduced tumor cells interact with surrounding, non-transduced tumor cells in a concentration-dependent manner. An additional benefit resulting from the use of the cytochrome P450 / CPA system is the lack of cytotoxic effects of dividing cells on the cells in their environment. This is because the active metabolites produced cause inter-strand cross-linking, regardless of the phase of the cell cycle. Then, during DNA synthesis, these inter-strand crosslinks contribute to cell death.

Viral vectors are the most commonly used carriers for gene transfer in current clinical practice. However, most clinical trials were ex vivo approaches where the patient's cells were isolated, modified in tissue culture and then introduced into the patient.

In the treatment of cancer, it could be performed to isolate cells from patients (cancerous or healthy cells), infect them in vitro with recombinant retrovirus molecules carrying the gene encoding the cytochrome P450, and then introduce them back to the patient near the tumor. This approach, however, is extremely labor intensive, because each patient's cell must be isolated, cultured, transduced with a gene construct, and introduced successfully, without infection due to random factors. The costs and time involved in such an approach limit its practical usefulness.

As an alternative, an approach is considered where one type of cell is infected by recombinant retrovirus molecules carrying the gene encoding cytochrome P450 and then used in the therapy of many different patients. This approach is easier to implement, assuming that you can overcome the problem of immune response. However, most cancers are not suitable for ex vivo gene therapy.

Preferably, the gene encoding cytochrome P450 is introduced in vivo into tumor cells, or into cells near the tumor.

In vivo gene delivery is associated with many new problems. First of all, you need to consider security considerations.

Selective gene expression is a major problem in in vivo gene therapy, both for safety and practical reasons. It is obvious that therapeutic genes transmitted by vectors should not be expressed in all tissues and cells without distinction, but rather only in selected target cells. This is especially important when the genes to be transferred are prodrug activating genes to destroy specific cancer cells. Destruction of other, non-target cells would of course be particularly undesirable.

Basically, the accidental integration of the proviral form of the retrovirus genome into the genome of the infected cell raises significant ethical problems, because such accidental integration can lead to the activation of proto-oncogenes and thus trigger the development of a new tumor. Most scientists would agree that the probability that a replication-defective retrovirus, such as the one currently used, will integrate into or near the cellular gene that controls cell proliferation is negligible. However, it can be presumed that the explosive expansion of a population of replication competent retroviruses resulted from one case of infection may ultimately allow multiple integrations, making such phenotypic integration realistically possible.

Retroviral vector systems are optimized to minimize replication of competent virus. Nevertheless, it is well documented that cases of recombination between components of a retroviral vector system can lead to the production of a potentially pathogenic replication competent virus and for this purpose many generations of the vector system have been constructed to minimize the risk of recombination (Salmons, B. and Gunzburg, WH (1993), Human Gene Therapy 4 (2): 129-41.

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The retroviral vector system consists of two components:

1) The retroviral vector itself, which is a modified retrovirus (plasmid vector) in which the genes encoding the viral proteins have been replaced with therapeutic genes. As the replacement of the genes encoding the viral proteins effectively damages the virus, this must be repaired by a second system component that provides the missing virus proteins to the modified retrovirus.

The second component is:

2) A cell line producing large amounts of viral proteins, although it lacks the ability to produce replication competent viruses. This cell line is referred to as the packaging cell line and contains a cell line transfected with one or more gene transfer plasmids that allow packaging of the modified retroviral vector.

To produce recombinant retrovirus particles, the retroviral vector is transfected into a packaging cell line. Under these conditions, the modified retrovirus genome containing the introduced therapeutic genes is transcribed from the retrovirus vector and packed into modified retrovirus molecules. These recombinant retrovirus particles are then used to infect cancer cells, during which the vector genome and each cytotoxic gene are integrated into the target cell DNA. A cell infected with such a recombinant virus particle cannot produce a new vector virus as there is no virus protein in these cells, while the vector DNA carrying therapeutic genes is integrated into the cell's DNA and can be expressed in the infected cell.

A number of retroviral vector systems that allow delivery of transferred cytotoxic genes have previously been described (Salmons, B. and Gunzburg, WH (1993), Human Gene Therapy 4 (2): 129-41). Most of these methods contain either limited infections to predetermined cell types, or the use of heterologous promoters to directly express linked heterologous therapeutic genes in specific tumor cells. Heterologous promoters are used that should direct the expression of the combined genes in only one type of cell in which the promoter is normally active and / or additionally controlled. Such promoters are previously introduced, in combination with therapeutic genes, into a retroviral vector in place of the gag, poi or env genes.

The retroviral LTR (Long Terminal Repeat) sequence flanking these genes is a carrier for the retrovirus promoter, which is generally non-specific as it can direct expression in a wide variety of cells (Majors, J. (1990), in "Retroviruses - Strategies of replication" (Swanstrom , R. and Vogt, R K., Eds.): Springer-Verlag, Berlin: 49-92). The literature describes the interaction between the LTR promoter and heterologous internal promoters, such as tissue specific promoters, described above. Furthermore, it is known that potent LTR-related enhancers can either independently or in combination with a retrovirus promoter affect the expression of cellular genes near the retrovirus integration site. Such a mechanism has been shown to contribute to the onset of cancer in animals (van Lohuizen, M. and Berns, A. (1990), Biochim. Biophys. Acta 1032: 213-235). These two observations encouraged the development of self-inactivating SIV (Self-Inactivating-Vectors) vectors in which retrovirus promoters are functionally inactivated in the target cell (publication WO 94/29437). Further modifications of these vectors include insertions of the promoter gene cassettes in the LTR region to create double-copy vectors (publication WO 89/11539). However, in both these vectors, the heterologous promoters introduced into either the vector or the LTR region are directly linked to the therapeutic gene.

The previously described SIN vector mentioned above, which carries the removed 3'LTR (publication WO 94/29437), additionally uses a heterologous promoter, such as in Cytomegalovirus (CMV), instead of the 5'LTR (5'LTR without U3) retroviral promoter to direct expression vector construct in the packaging cell line. The heterologous polyadenylation signal is also included in the 3'LTR (publication WO 94/29437).

The purpose of the present invention is to construct a safe retroviral vector that contains the cytochrome P450 gene as a therapeutic agent. This new vector

188 323 carries heterologous, constitutive, inducible or tissue specific regulatory and / or promoter sequences in the 3'LTR region, which, after infection, are duplicated and translocated to the 5'LTR fragment in the target cell. In this way, the introduced promoter in the infected cell controls the expression of the cytochrome P450 gene, which is introduced into the vector. Such a vector does not self-inactivate, but instead promoter exchange occurs, thanks to which it can be called the ProCon vector, i.e. the Promoter Conversion vector. The principles and benefits of the ProCon system are described in detail in WO 96/07748.

As promoter conversion (Promoter Conversion) does not cause self-inactivation, the retroviral vector will be transcriptional active in the target cell. In addition, both LTR regions will contain a large number of heterologous promoter / enhancer sequences in the target cell. This will reduce the likelihood that the vector integrated in the target cell will be inactivated for longer periods as described for conventional vectors (Xu, L., Yee, JK et al. (1989), Virology 171: 331-341) and will also reduce the possibility of recombination with endogenous retroviral sequences to produce a potentially pathogenic replication competent virus, which will increase system security.

According to the invention, the 5'LTR region of the retrovirus vector construct is unmodified and expression of the viral vector in the packaging cell line is driven by the normal U3 promoter. Normal retrovirus polyadenylation is allowed and no heterologous polyadenylation signals are included in the 3'LTR sequence. This is important for the development of in vivo therapy strategies as normal physiological regulation of the virus, through a normal viral promoter, and possibly also including normal polyadenylation control, will prevail for a longer period in vivo, while the packaging cells will produce recombinant viruses.

In order to achieve the above and other purposes, the invention provides a retroviral vector undergoing conversion of a promoter containing the 5'LTR region of the U3-R-U5 structure; one or more coding sequences selected from the group of genes known as cytochrome P450 genes; and the 3'LTR region comprising the fully or partially deleted U3 region, wherein said removed U3 region is replaced by a heterologous promoter followed by the R and U5 region.

Said promoter can be either constitutive, such as the immediate early promoter / enhancer of the Cytomegalovirus (CMV) virus, inducible by glucocorticoid hormones (e.g. the MMTV promoter) or by specific target cells.

Specific regulatory and promoter sequences of target cells are selected from one or more elements of each gene, but in this embodiment, they may be derived from promoters of regulatory sequences and promoter of carbon anhydrase II and β-glucokinase, specific regulatory sequences and promoter lymphocytes containing regulatory sequences and promoter anhydrases carbon II and β-glucokinase, specific lymphocytic regulatory and promoter sequences of Whey Acidic Protein, Whey Acid Protein (WAP), Mouse Mammary Tumor Virus (MMTV), Mouse Nipple Cancer Virus, specific regulatory and promoter sequences of β-lactoglobulin and casein, specific pancreatic regulatory and promoter sequences containing immunoglobulin and MMTV regulatory and promoter sequences, and specific MMTV regulatory and promoter sequences, giving reactivity to glucocorticoid hormones or directing expression in mammary glands. Other promoters include, for example, CD4, CD34 and IL2 promoters. Said regulatory and promoter sequences preferably regulate expression of said retroviral vector.

It turns out that the WAP promoter region, necessary for the specificity of expression in the mammary glands, is a restriction fragment of 320 base pairs XhoI / XbaI (-413 to -93) (Kolb, AF, Gunzburg, WH, Albang, R., Brem, G. Erfle, V., and Salmons, B. (1995), Biochem. Biophys. Res. Commun. 217, 1 ^ 045-1052). In addition, individual experiments indicate that the 0.6 Kb Pstl mMtV promoter fragment (Salmons, B., Groner, B., Calberg Baca, CM, and Ponta, H. (1985), Virology 144: 101-114) may play a role in regulating expression specificity

188 323 in mammary glands, exhibited by MMTV (Kolb, AF, Gunzburg, WH, Albang, R., Brem, G, Erfle, V., and Salmons, B. (1995) Biochem. Bioptys. Res. Commun. 217,1045 -1052).

The LTR regions are selected from at least one member of the group containing the LTR Murinr Leukaemia Virus (MLV) (Mouse Leukemia Virus), Mouse Mammary Tumour Virus (MMTV) (Mouse Cancer Nipple Virus), Murine Sarcoma Virus (MSV) (Mouse Sarcoma Virus) , Simian Immonsdoficiency Virus (SlV) (Human Immunodeficiency Virus), Human Immo] nsreiicieccc Virus (HIV) (Human Immunodeficiency Virus), Human T-cell Lookaemie Virus (HTLV) (T Cell Leukemia Virus), Folino Immonodąficiency Viaop (FIV) (Reduced Cat Immunity Virus), Felinr Leukaemia Virus (FELV) (Cat Leukemia Virus), Bovino Leokaemia Virus (BLV) (Bovine Leukemia Virus) and Massn-PfizorMonkec Virus (MPMV).

The aetaoviaus vector is preferably based on the LXSN vector (Miller, AD and Rosman, GJ (1989), Biotechniqoes 7: 980-990), pBAG (Price, J., Turner, D. et al. (1987), Proc. Natl. Acad. Sci. USA 84: 156-160) or a hybrid of both.

The sequence encoding the therapeutic gene can be any C4-chromium P450 gene, but the most preferred is the rat cctschaomo P450 form 2B1 gene defined by FujiKuricama, Y., Mizukami, Y., et al. (1982) Proc. Natl. Acad. Sci. USA 79: 2793-2797.

In another embodiment of the invention, a retroviral vector system comprising a retroviral vector, as described above, is provided as the first component and packaging cell line comprising at least one retrovirus or recombinant retrovirus construct encoding the proteins necessary to coat said viral vector.

The packaging cell line is preferably selected from members of the group comprising ψ-2, y-Crypt, ψ / AM, Gp + E-86, PA 317 and GP + envAM-12, or each of the lines transfected with the recombinant construct, which will allow expression surface proteins of other enveloped viruses.

The invention also contains mRNAs derived from the rotrowirupowogs vector of the invention.

In the packaging cell line, expression of the aeta-viral vector is regulated by normal non-constructively retasvirus promoters contained in the U3 region. In contrast, as soon as the vector enters the target cell, promoter conversion occurs, and the P450 gene is expressed by tissue-specific or induced selected promoters introduced into the ProCon vector. Not only every tissue-specific promoter can be included in a system, which is selectively delivered to a wide range of different types of cells, but in addition, after conversion, the structure and properties of the retrsvirus vector no longer resemble viral. This has extremely important security implications.

This vector system will be used to generate zroksmbinswαnrgs virus that can be used to infect cancer or normal cells, both in vitro and in vivo.

Recombinant rotaoviruses that have been purified or concentrated can be protected by adding the appropriate amount of regulatory buffer to the environment containing the recombinant retrovirus to obtain an aqueous suspension. The prescribed buffer is an aqueous solution containing a saccharide as a high molecular weight add-y ™ ^ compound and a buffering component in water. The aqueous solution may also contain one or more amino acids.

Recombinant rotovirus can also be protected in purified form. More specifically, before adding the prescribed buffer, the crude reconstituted retroviros described above can be purified by passing through filters, then concentrated by, for example, a cross flow thickener system (Filtron Technolsgc Corp., Noatbsaoogh, MA). In one embodiment, DNAza is added to the concentrate to digest exogenous DNA. After digestion, the solution is then diafiltered to remove excess environmental components and to obtain a mixed-up rrtaovirus in the desired buffered solution. The diafiltrate is then passed through a Sephadex S-500 gel column, after which the purified recombinant virus is eloated. An appropriate amount of prescribed buffer is added to the solution to achieve the desired final concentration of ingredients as well as to minimally dilute the recombinant retrovirus, and then the aqueous solution is stored at -70 ° C or dried immediately. As stated above, the prescription buffer is an aqueous solution containing a saccharide, a high molecular weight structural additive compound, and a water buffering component. The aqueous solution may also contain one or more amino acids.

Crude recombinant retrovirus can also be purified by ion exchange chromatography. Generally, the crude recombinant retrovirus is purified by passing through a filter, and the filtrate is introduced into a column containing a highly sulfonated cellulosic substrate. The recombinant virus is eluted from the column in a purified form due to the use of a buffer with a high salt concentration. The high salt buffer is then exchanged for a more favorable buffer by passing the eluate through an inverted molecular sieve column. Then the appropriate amount of the prescribed buffer, discussed above, is added to the purified recombinant virus and the aqueous suspension is either immediately dried or stored, preferably at -70 ° C.

The aqueous suspension in raw or purified form can be dried by freeze drying or evaporation at room temperature. More specifically, freeze-drying consists of the steps of cooling an aqueous suspension below the glass transition temperature or below the eutectic point of the aqueous suspension and removing water from the cooled suspension by sublimation to form a freeze-dried retrovirus. In lyophilized form, the recombinant retrovirus is stable and can be stored at -20 ° C to -25 ° C, as discussed in more detail below.

In the evaporation method, water is removed from the aqueous suspension at room temperature by evaporation. Water can also be removed by spray drying.

The aqueous solution used in the recipe, as described earlier, consists of saccharide, high molecular weight additive compound, buffering component and water. The solution may also contain one or more amino acids. The combination of these components serves to maintain the activity of the recombinant retrovirus during freezing and lyophilization, or drying by evaporation.

The high molecular weight addition compound serves to prevent virus aggregation during freezing and provides its structural substrate in a freeze-dried or dried state. In the context of the present invention, structural addition compounds are believed to have "high molecular weight" if it exceeds 5000 m in. The preferred high molecular weight addition compound is human serum albumin. Amino acids, if present, are used to further protect virus infectivity under cooling and thawing of aqueous suspensions. In addition, amino acids are used to further protect virus infectivity during sublimation of the cooled aqueous suspension and when it is in a lyophilized state.

The buffering component is used to buffer the solution by maintaining a relatively constant pH. Many different buffers can be used, depending on the desired pH range, preferably between pH 7.0 and 7.8.

Aqueous solutions for the preparation of recombinant retroviruses are described in detail in WO-A2-96121014.

In addition, it is preferred that the aqueous solution contains a neutral salt to determine the appropriate iso-osmotic salt concentration in the final, recombinant retrovirus solution.

Lyophilized or dehydrated retroviruses may be reconstituted using a variety of substances, but preferably they are reconstituted using water. In special cases, dilute salt solutions can be used to ensure the isotonicity of the final solution. In addition, it may be beneficial to use aqueous solutions containing ingredients known to increase the activity of the reconstituted retrovirus. Such components contain cytokines such as IL-2, polycations, such as protamine sulfate, or other components that increase the transduction efficiency of the reconstituted virus. The lyophilized or dehydrated recombinant retrovirus can be reconstituted in any convenient volume

188 323 water or reconstituting agents that allow a significant, and preferably complete, dissolution of the freeze-dried or dehydrated sample.

Recombinant retrovirus molecules can be administered to a wide range of sites such as, for example, human organs or cancer sites. In other embodiments, the recombinant retrovirus may be administered orally, intravenously, orally / sublingually, intraperitoneally or subcutaneously. The daily dose depends on the method of administration, the form in which it is administered, the indications for administration, the body and body weight of the subject being treated, and then the preferences and experience of the attending physician.

The administration methods described herein are carried out simply by using a needle, catheter or similar tools directly. Particularly, in some embodiments of the invention, one or more doses may be administered directly.

In one embodiment of the invention, the packaging cells will be placed in capsules. For effective treatment, the virus producing cells must survive a long period of time in the target organ after implantation, and the virus must be produced and released from the packaging cells during this period. In this way, the virus producing packaging cells could in effect form a small virus producing point located at the site of administration. This would allow efficient delivery of the recombinant virus in vivo. Alternatively, infected cells either normal or of human origin, or from other species, will be covered in capsules and implanted, representing a small prodrug conversion point located close to or in the tumor mass.

The long-term effectiveness of this approach depends on (1) protecting cells against the host's immune system, which normally eliminates infected or virus-producing cells, and especially if such cells come from other species, as is usually the case with cells producing retroviral vectors and (2) survival cells at the application site for a longer period of time, which may require vascularization.

It has been found that continuous production of the retroviral vector by implanted packaging cells can occur due to adequate coverage, prior to implantation, of packaging cells producing virus with semi-permeable membrane capsules. In addition, it was found that such capsules implant well into the host's body, are vascularized, and do not elicit an immune response or inflammation of the host. Such conditions, together with the semi-permeability of the capsule membranes, will allow long-term delivery of the retrovirus vector in vivo.

Capsule coating technology has been developed, used to cover capsules for packaging cells that produce the virus and cells infected with the virus or normal, cellulose-based material. Using this technique, up to 1O¹⁰, and preferably 10⁵-10⁷cells are covered with capsules from an electrolyte complex (e.g., from alginic fibers and polylysine, or more preferably from cellulose sulfate and dimethyl diallylammonium chloride) or other porous materials (such as polyamides, polysulfonates). The capsules obtained have different diameters between 0.01 and 5 mm, but preferably 0.1 and 1 mm. As a result, capsules can contain a different number of cells. The capsule is semi-permeable due to pores that are large enough to allow viruses or prodrug molecules to pass, but small enough to prevent access of immune system cells, thereby reducing the immune response directed against these cells. Capsules and cells covered with capsules are stored in a normal cell culture environment (the type of which depends on the lines covered with capsules), under standard conditions of humidity, temperature and CO2 concentration.

After a suitable residence time in culture (normally not less than 1 hour and not more than 30 days), the capsules containing the cells can be surgically implanted into different areas of the body either directly or with a syringe.

Cyclophosphamide or ifosfamide may be administered to the host at different times after implantation of the capsule-covered cells, both locally and systemically. Cells infected with the virus that directs the expression of the cytochrome P450 gene will convert these pro-drugs into active metabolites; which will cause alkylation and crosslinking of DNA. Also, cells that carry and direct the expression of the cytochrome P450 gene (such as capsule-covered infected cells, or capsule-covered packaging cells) will

188 323 also catalyze this conversion. In one embodiment of this invention, these capsule-coated infected or packaging cells will be either slowly dividing cells or cells treated with mitomycin C, low doses of radiation, or other agents to prevent cell division, to prevent these cells from being cytotoxic prodrug action.

The following examples will illustrate the invention in further detail. These examples do not limit the scope of the present invention in any way, since of course modifications and substitutions obvious to those skilled in the art are possible.

Example I.

This example describes the construction of a retroviral expression vector for intra-tumor infection that contains the rat cytochrome P450 2B1 gene. .

The expression vector pLX2B1, shown in Figure 1, was constructed by ligation of fragments obtained from the plasmid pLX125 and pSW1 (Kedzie, KM, Escobar, GY, Grimm, SW, He, YA, Pepperl, DJ, Regan, JW, Stevens, JC, and Halpert, JR (1991), J. Biol. Chem. 266 (33): 2215-2). Plasmid pLX125 was prepared as described in publication PCT / EP96 / 04447).

Plasmid pLX125 was linearized with Hpal and the resulting blunt ends were dephosphorylated with calf intestinal phosphatase. DNA was purified by separation on a 1% agarose gel, cut and prepared according to the Qiaquick protocol (Qiagen). After precipitation in ethanol, the DNA was resuspended in water.

The cloning vector pSW1 was digested with Smal and HincII to obtain two blunt-ended fragments. The pickling mixture was separated on a 1% agarose gel. The shortest fragment (1.5 kb) containing rat cytochrome P450 2B1 cDNA (Fuji-Kuriyama, Y., Mizukami, Y. et al. (1982) Proc. Natl. Acad. Sci. USA 79: 2793-2797), was excised and eluted according to Qiaquick DNA extraction protocol, precipitated in ethanol and resuspended in water.

7.6 fMoles of pLX125 and 24 fMole of the Smal / Hindll fragment of pSW1 were mixed together and ligated for 3 days at 12 ° C, using T4 ligase (Boehringer). The ligase was inactivated at 65 ° C for 10 min. and DNA was precipitated in butanol in a 10x volume of butanol. The precipitated DNA was resuspended in water and was electroporated into DH10B (Gibco) bacteria. Ampicillin resistant colonies were selected, DNA prepared and assayed for digestion with SspBIZSalI, BamHl / SspBI, PvuI and BamHl. The final valid plasmid was designated pLX2B1 (see Fig. 1).

Lipofection.

One day before lipofection 3x10⁶ retrovirus packaging PA317 cells (Miller, AD and Buttimore, C. (1986), Mol. Cell. Biol. 6: 2895-2902) were placed in 10 cm Petri dishes or culture dishes. On the day of infection, 4 pg pLX2Bl were mixed with 300 μΐ serum free medium. At the same time, 45 pl of Lipofectamine (Gibco BRL) was mixed with 300 pl of serum-free medium. The plasmid containing solution was added to the Lipofectamine mixture and was incubated for 45 min. After 35 min cells were washed once using 6 ml serum-free medium. 2.4 ml of serum-free medium was added to the lipofection mixture, and the resulting 1 ml was placed on prepared cells. After 5 hours 3.5 ml of Dulbecco modified Eagles medium containing 20% ​​FCS was added. The next day, the cells were digested, diluted 1:20 and mounted in 100 mm dishes. After 24 hours the environment was replaced by another environment containing the neomycin G418 analogue. The cell population was isolated and expression of cytochrome P450 was analyzed.

The supernatant from this cell population was used to infect CK target cells. 1 ml of virus-containing supernatant from 5x106 cells was filtered through a 0.45 pm filter followed by addition of 1x106 CK target cells in the presence of 8 pg / ml polybrene. After 4 hours 1ml of Dulbecco modified Eagles with 10% fetal calf serum was added.

The next day, the cells were trypsinized, diluted and 24 hours. later placed in a selection medium containing an additional 400 pg / ml G418. After 2 weeks, G418 resistant colonies were isolated and tested for cytochrome P450 activity

188 323

2B1. 2x10⁴ cells were placed on a 3 cm plate and exposed to ifosfamide at a concentration between 0 and 5 mM. Higher sensitivity of cytochrome P450 2B1 was observed in cells infected with retrovirus compared to control, uninfected cells.

Capsule coating.

The resulting packaging cells that produce the retroviral vector are covered with capsules, as described in Example 2 in WO 97/01357.

Implantation.

The resulting capsules are surgically introduced through a key hole, close to either the transplanted or spontaneous BALB / c or GR mouse tumor. About 6 capsules with a diameter of 1 mm are introduced into each surgical site. The treatment site is closed with 1 suture. Mice were then given cyclophosphamide or ifosfamide locally by intra-tumor injection of 100 μΐ from a 20 mg / ml solution or intraperitoneally using systemic concentrations of 130 mg CPA / kg body weight and 40-60 mg IFO / kg body weight for up to 10 weeks. During this period, tumor size and macroscopic appearance were monitored daily. Mice were then sacrificed, tissue containing inserted capsules and tumor removed, and histological sections for light and electron microscopy prepared. These sections clearly showed good implantation of the capsules, vascularization and lack of evidence of the presence of lymphocytes as an indicator of cellular immune response. These sections also showed no sign of cell death or necrosis inside the capsules. In contrast, the tumor showed necrosis and the reduction in tumor size was clearly visible macroscopically during the test period.

Example II

This example describes the construction of stable cell lines that constitutively direct the expression of rat cytochrome P450 2B1.

The expression vector pc3 / 2B1 was constructed by ligation of fragments obtained from the plasmid pcDNA3 (Invitrogen) and pSW1 (Kedzie, KM, Escobar, GY, Grimm, SW, He, YA, Pepperl, DJ, Regan, JW, Stevens, JC, and Halpert, JR (1991), J. Biol. Chem. 266 (33): 2215-21).

The plasmid pcDNA3 was digested with XhoI / XbaI and the fragments thus obtained with sticky ends were dephosphorylated using calf intestine phosphatase. The vector backbone DNA was purified by separation on a 1% agarose gel, excised and prepared using the Qiaquick protocol (Qiagen). After precipitation in ethanol, the DNA was resuspended in water.

The pSW1 cloning vector was digested using XhoI and XbaI to obtain two fragments. The pickling mixture was separated on a 1% agarose gel. The shortest fragment (1.5 kb) containing rat cytochrome P450 2B1 cDNA (Fuji-Kuriyama, Y., Mizukami, Y. et al. (1982), Proc. Natl. Acad. Sci. USA 79: 2793-2797) was excised and was eluted according to Qiaquick DNA extraction protocol, then precipitated in ethanol and resuspended in water.

8.3 fMoles of the skeletal pcDNA3 and 24.8 fMoles of the XhoI / XbaI fragment of the pSW1 vector were mixed together and were ligated for 1 day at 12 ° C using T4 ligase (Boehringer). The ligase was then inactivated at 65 ° C for 10 min and the DNA was precipitated in butanol in 10 times the volume of butanol. The precipitated DNA was resuspended in water and electroporated into DH10B (Gibco) bacteria. Ampicillin resistant colonies were selected, DNA prepared and digested with EcoRI, BamHI, EcoRV and XhoI. The final valid plasmid was designated as pc3 / 2B1.

Lipofection. _from

Before the day of transfection 3x10^at cat kidney cells were placed on 100 mm plates. On the transfection day, 4 μg pc3 / 2B1 was mixed with 100 μΐ serum free medium. At the same time, 15 μΐ Lipofectamine was mixed with 100 μl serum-free medium. The plasmid containing solution was added to the Lipofectamine mixture and was incubated for 45 min. After 35 min cells were washed once in 2 ml serum-free medium. 800 μΐ of serum-free medium was added to the lipofection mixture and the resulting 1 ml was placed on prepared cells. After 6 hours 1 ml DMEM (Glutamax) with 10% FCS was added. The next day, the cells were digested with trypsin, diluted 10 × 100 and plated on 100 mm plates. After 24 hours the environment has been replaced with neomycin. After 14 days, neomycin resistant colonies were isolated and tested for the presence and activity of the vector.

Feline kidney cells expressing cytochrome P450 were 10-fold more sensitive to ifosfamide or cyclophosphamide compared to wild-type cells.

An additional effect can be demonstrated on wild-type cat kidney cells as well as on Rin5 cells derived from pancreatic cancer. This was shown by co-cultivating cat kidney cell clones containing the cytochrome P450 2B1 gene, either with wild type non-cytochrome-producing cat kidney cells or with Rin5 cells derived from rat pancreatic cancer when ifosfamide was added. Non-cytochrome CK and Rin cells were killed by the toxic metabolites of ifosfamide, which were produced and released by cat kidney cells containing cytochrome P450 2B1. Titration carried out on cat kidney cells containing one clone of cells that possess the cytochrome P450 2B1 genes showed that even a small amount of ifosfamide, such as 0.25 mM, causes specific toxic effects on these cells, and 1-2 mM causes the death of all cells . Polymerase Chain Reaction (PCR) analysis showed that cat kidney cell clones containing the cytochrome P450 2B1 gene acquired DNA from the P450 2B1 gene construct. Subsequent biochemical analysis of the clones using enzymatic dealkylation of 7-pentoxyresorufm by cytochrome P450 2B1 revealed that genetically modified cells produce cytochrome P450 2B1.

Capsules containing these cells were produced as described in Example 1 and implanted into mice close to the tumor. After administration of cyclophosphamide or ifosfamide, administration efficiency was assessed as above.

A retroviral expression vector containing the gene encoding rat cytochrome P450 2B1 can also be prepared as described in Example III below:

Example III.

Plasmid pLX125 was first partially digested with the XhoI restriction enzyme to obtain a vector that was linearized at position 3547. Such a linear plasmid was further digested with the SspBI restriction enzyme to remove the short fragment within the pLX125 polylinker. A correctly cut vector fragment appeared in the preparation gel as the largest band. Using the Quiaex (Qiagen) protocol, the DNA from this band was eluted and gel purified.

In order to obtain the rat cytochrome P450 2B1 gene, the HTC line liver cancer cells were lysed in a solution D (4 M guanidine thiocyanate, 25 mM sodium citrate pH 7, 0.5% sodium N-lauNlostucosinate, 0.1M 2-mercaptoethanol) and whole RNA was extracted by adding 1/15 volume of a 3 M sodium acetate solution, saturated phenol and 1/5 volume of chloroform / acetyl alcohol (49: 1) were added in the same volume of water, and the whole mixture was vigorously mixed. After 15 min on ice, the extract was centrifuged 20 min. At 4 ° C and 10,000 g. RNA in the aqueous phase was precipitated in one volume of isopropanol for 30 min. at -20 ° C and centrifuged at 10,000 g at 4 ° C. The pellet was rinsed in 70% ethanol and left at room temperature for 15 min. After 5 min centrifugation at 4 ° C and 10,000 g, the pellet was dried in a vacuum dryer and redissolved in 0.5% SDS solution.

Extracted RNA was reverse transcribed using a cDNA synthesis protocol (Pharmacia).

The resulting cDNA was used as a template for PCR. Primers were designed to contain the SspBI restriction site (underlined) in the left-hand primer (5'-AAGCCTGTACACTGGAGAGCATGCAC-3 ') and the XhoI site (underlined) in the right-hand primer (5'-CGATTACTCGAGaCCTGGCTGCG1CA-3'). Both primers had additional bases at the 5 'end to increase cleavage efficiency by appropriate restriction enzymes. The product with 1562 base pairs was cut with XhoI and SspBI to obtain three fragments.

The longest fragment (1545 base pairs) containing the C4-chromium P450 gene was ligated with the XhoI / SspBI digested pLX125 plasmid.

A stable cell line expressing constitutive expression of the 2B1 form of rat cytochrome P450 can also be prepared as described in Example V below:

188 323

Example IV

To obtain mRNA from form 2B1 of the rat cytochrome P450 gene, 4-week-old female rats were sacrificed, the liver was removed, and immediately frozen in liquid nitrogen. The frozen liver was placed in sterile, filtered GTC buffer (6 M guanidine isothiocyanate, 5 mM sodium citrate, 0.1 M 2-mercaptoethanol, 0.5% sodium N-lauryl sarcosinate) and homogenized at room temperature. To separate RNA, the liver extract was placed on a cesium chloride medium (5.7 M cesium chloride, 0.1 M EDTA) and centrifuged in a rocker rotor at 20 ° C and 32,000 rpm. through the night. After complete removal of the supernatant, the deposited RNA was re-dissolved in 10 mM Tris pH 7.5, chilled on ice, and precipitated overnight at -20 ° C in 1/15 volume of 3 M sodium acetate and 2.5 volumes of ethanol. The RNA was then centrifuged for 40 min. at 8000 rpm and 4 ° C, and the dried pellet was resuspended in sterile water.

The extracted RNA was reverse transcribed using a cDNA synthesis protocol (Pharmacia). The resulting cDNA was used as a template for the following PCR. The primers were designed so that they had an EcoRI restriction site in the left hand primer (5'-CGTGCGGAATTCGGCGGATTCAGCAT-3 ') and an EcoRVI site in the right hand primer (5'-ATAACGGATATCACCTGGCTGCCTCA-3'). Both primers had additional bases at the 5 'ends for greater efficiency of cutting enzymes. Amplified DNA containing 1588 base pairs was digested with EcoRI and EcoRV to obtain three fragments.

The longest fragment (1572 base pairs) containing the rat cytochrome P450 2B1 gene was ligated with the EcoRI / EcoRV digested pcDNA3 plasmid (Invitrogen).

Example V

Construction of the ProCon vector, containing the rat cytochrome P450 gene, which is under the transcriptional control of the WAP promoter.

The extracted RNA, prepared in Example IV, was reverse transcribed using a cDNA synthesis protocol (Pharmacia). The resulting cDNA was used as a template in the PCR reaction. The primers were designed to contain a BamHI restriction site (underlined) in the left-hand primer for example

5'-AAGCCGGATCCCTGGAGAGCATGCAC-3 ') and a BamHI site (underlined) in the right-hand primer (for example, 5'-CGATTAGGATCCCTGCCTCA-3'). Both primers had additional bases at the 5 'ends for greater cleavage efficiency by the respective enzymes. The product containing 1562 base pairs was digested with BamHI and the resulting fragment containing the cytochrome P450 gene was ligated with the BamHI digested pWAP.6 plasmid (see PCT Application No. PCT / EP96 / 03922).

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