NO321454B1 - Defective, recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell allows at least partially to inhibit cell division, uses, and pharmaceutical preparation thereof. - Google Patents

Abstract

Defective recombinant adenovirus contains a heterologous DNA sequence (I), the expression of which in a target cell contributes to the inhibition of cell division.

Description

The present invention relates to recombinant vectors of viral origin and their use. More particularly, the invention relates to recombinant adenoviruses carrying a heterologous DNA sequence whose expression in a cell, as it divides abnormally, allows at least partially inhibiting the division of the cell. The invention also relates to preparations containing these.

Cell growth is regulated in an extremely subtle way by two types of signals. Some favor a multiplication of the cell while others, on the contrary, cause them to enter a state of rest or differentiate, according to the organism's requirements.

Cancers are all characterized by a deregulation of the mechanisms that control cell division and lead to abnormal proliferation. Most often, the development of a cancer thus implies an activation of the genes that favor the multiplication of cells (genes called proto-onco genes that are activated into onco genes) and a disappearance or inactivation of the genes that inhibit cellular proliferation. The present invention offers the possibility of treating cancer by gene therapy by, to the tumoral cells, administering one or more of these genes whose expression allows at least partially inhibiting the cellular proliferation.

Gene therapy consists in correcting a defect or an abnormality (mutation, abnormal expression and so on) by introducing a genetic information into the cell or the affected organ. This genetic information can be introduced either in vitro into a cell that has been extracted from the organ and where the modified cell is then reintroduced into the organism, or directly in vivo into the intended tissue. In the second case, there are different techniques, among which should be mentioned different techniques for transfection involving complexes of DNA and DEAE-dextran (Pagano et al., "J.Virol." 1 (1967) 891), DNA and nuclear proteins (Kaneda et al., "Science" 243 (1989) 375), DNA and lipids (Felgner et al., "PNAS" 84 (1987) 7413), the use of liposomes (Fraley et al., "LBiol.Chem." 255 ( 1980) 10431), and so on. Recently, the use of mice as vectors for the transfer of genes has emerged as a promising alternative to these physical transfection techniques. In this regard, various strains have been tested for their ability to infect certain cellular populations. Particular mention should be made of the retroviruses (RSV, HMS, MMS, and so on), the HSV virus, the adeno-associated viruses and the adenoviruses.

The possibility of using gene therapy for the treatment of cancer has already been highlighted in the international application WO91/15580. This application describes the construction of retroviruses containing a gene encoding a ribozyme whose expression in cell culture can allow the destruction of an mRNA of an oncogene.

The present invention is based on the discovery that the adenoviruses constitute particularly effective vectors for the transfer and expression of therapeutic genes in tumors. In particular, the adenoviruses have the advantage of not integrating into the genome of the cells they infect, of staying there very stably, which allows to achieve a lasting therapeutic effect, and of having a very large host spectrum, which allows an application in the treatment of cancers such as affects all types of cells. Furthermore, the invention is also based on the discovery that the viruses of the adenovirus type are capable of transmitting and expressing genes capable of at least partially inhibiting cell division directly at the tumor level.

A first object of the invention thus lies in a defective, recombinant adenovirus containing a heterologous DNA sequence whose expression allows at least partially inhibiting cell division. More specifically, the present invention comprises a defective, recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell allows at least partially inhibiting cell division, characterized in that the sequence comprises:

at least one gene encoding p53 protein; and

a promoter sequence allowing expression, in the infected cell, of said gene, the promoter sequence having an origin different from that of the gene.

The present invention also includes the use of an adenovirus according to any one of claims 1 to 9 for the production of a pharmaceutical preparation intended for the therapy and/or prevention of cancer by introduction directly into the tissue to be treated.

Furthermore, the present invention comprises the use of an adenovirus according to claims 1 to 9 for the manufacture of a pharmaceutical preparation intended for the manufacture of a pharmaceutical preparation intended for the therapy or prevention of cancer by administration directly to the tumor to be treated.

Furthermore, the present invention comprises the use of an adenovirus according to claim 2 for the preparation of a pharmaceutical preparation intended for the treatment and/or prevention of cancers associated with the presence of a mutated form of p53.

The invention likewise has as its object such a defective, recombinant adenovirus for the production of a pharmaceutical preparation intended for the treatment or prevention of cancer.

Accordingly, the invention also includes a pharmaceutical preparation, characterized in that it contains one or more defective, recombinant adenoviruses according to one of claims 1 to 9.

Within the scope of the invention, the term "defective adenovirus" denotes an adenovirus that is not capable of replicating autonomously in the target cell. In general, therefore, the genome of the defective adenovirus used within the scope of the invention is deprived of at least the sequences that are necessary for the replication of the cell in the infected cell. These areas can either be eliminated (in whole or in part), made non-functional, or replaced with other sequences and in particular by the inserted gene. Preferably, however, the defective virus nevertheless preserves the sequences of its genome which are necessary for encapsulation of the viral particles.

There are different adenovirus serotypes whose structure and properties vary somewhat. Nevertheless, these vims are not pathogenic for humans and especially not for immunocompromised persons. Among these serotypes, it is preferred within the scope of the invention to use the adenoviruses of type 2 or 5 (Ad 2 or Ad 5). In the case of the Ad 5 adenoviruses, the sequences necessary for replication are the areas ElA and ElB.

Among tumor suppressor genes, the following genes can be mentioned in particular:

- Genp53:

The gene p53 codes for a nuclear protein of 53 kDa. The mutated form by deletion and/or mutation of this gene is implicated in the development of most human cancers (Baker et al., "Science" 244 (1989) 217). Its mutated forms are also able to cooperate with the ras oncogene to transform murine fibroblasts. The wild-type gene encoding native p53, in turn, inhibits formation of transformation sites in rodent fibroblasts transfected with different combinations of oncogenes. Later findings emphasize that the protein p53 itself can be a transcription factor and stimulate the expression of other tumor suppressor genes.

- Gene Rb:

The gene Rb determines the synthesis of a nuclear phosphoprotein of approx. 927 amino acids (Friend et coil., "Nature" 323 (1986) 643) whose function is to arrest cell division and to cause it to enter a resting phase. The inactive forms of the gene Rb have been detected in various tumors and particularly in retinoblastomas or in mesenchymal cancers such as osteosarcomas. The reintroduction of this gene into the tumoral cells where they have been inactivated results in a return to normal state and a loss of tumorigenicity (Huang et al., "Science" 242 (1988) 1563). More recently, it has been shown that the normal protein Rb, but not in its mutated forms, restrains the expression of the proto-oncogene c-fos, the gene indispensable for cell proliferation.

Genet rap IA:

The gene rap IA (also called k-revl) codes for a protein of 21 kDa, associated with the inner surface of the cytoplasmic membrane. This protein is, at higher levels, capable of reverting transformed cells under expression of the mutated ras oncogenes (Kitayama et coil., "Cell" 56 (1989) 77).

- The gene DCC:

The gene DCC codes for a protein that is homologous to the cellular adhesion proteins of the N-CAM family. This gene is frequently deleted in colon carcinomas (Fearon et al., "Science" 247 (1990)49).

The genes k-rev2 and k-rev3:

The gene k-rev2 codes for a secreted protein of 60 amino acids and the gene k-rev3 codes for a truncated version of a protein in the extracellular matrix. These two genes are able to revert NIH 3T3 cells transformed by the Ki-ras oncogene.

Other genes can also be thought of as being used for the anti-tumoral effect, in particular other tumor suppressor genes described in the literature, or any other gene whose expression product can induce cellular apoptosis.

As indicated above, the heterologous DNA sequence may carry the native tumor suppressor gene or an active derivative of this gene. Such a derivative can be obtained by mutation, deletion, substitution and/or addition of one or more base pairs in the gene sequence according to classical molecular biological techniques. The activity of the derivative thus obtained can then be confirmed in vitro on samples known to the person skilled in the art, for example as those described in the examples.

Heterologous DNA sequence may likewise comprise an anti-target gene whose expression in the target cell allows controlling the expression of the gene or transcription of cellular mRNA encoding the proteins favoring cellular proliferation. Such genes can, for example, be transcribed in the target cells into RNAs that are complementary to the cellular mRNAs and thus block their translation into protein.

Among the anti-targeting genes that can be used, more particularly any anti-targeting sequence can be mentioned which allows to reduce the production level of the oncogenes ras, much, fos, c-erb B and so on.

In general, the heterologous DNA sequence also includes promoter sequences which allow expression of the gene or genes which are capable of partially inhibiting cell division in the target cell. These may be promoter sequences which are naturally responsible for the expression of the gene when these sequences are able to function in the infected cell. It can also be promoter sequences of a different origin (responsible for the expression of other proteins, or also synthetic ones). In particular, this may concern promoter sequences of eukaryotic or viral genes. For example, it could be promoter sequences that originate from the genome of the cell you want to infect. In the same way, it can be about promoter sequences originating from the genome of a virus, including the adenovirus used. In this connection, one can for example mention the promoters of the genes EIA, MLP, CM V, RSV, and so on. Furthermore, these promoter sequences can be modified by the addition of activation or regulatory sequences and so on. Furthermore, when the heterologous DNA sequence does not include expression sequences, these can be inserted into the defective virus genome downstream of such a sequence. It is also possible to use inducible promoters.

A heterologous DNA sequence is also conceivable in addition to the tumor suppressor gene or the anti-direction gene, a gene that codes for an antigen gene that is specific for the tumor and/or a gene that codes for a lymphokine. The combination of these genes thus allows (i) to stop cell division in a tumor and therefore to cause regression of this tumor, and (ii) to increase the immune response of the organism against this tumor.

The anti-genes specific to the tumor are anti-gene parts that appear on the surface of the tumoral cells but do not exist on the surface of non-tumoral such cells. Such anti-genes are generally used for cancer diagnosis. More recently, they have been described for the production of anti-tumour vaccines (EP 259 212). However, they have never been combined with other therapeutic genes.

Among the genes that code for the lymphokines, one can more particularly mention genes that code for the interleukins (IL-2 to IL-13), the interferons, the tumor necrosis factors, the colony-stimulating factors (G-CSF, M-CSF, GM- CSF, and so on), TGFB and so on. Furthermore, the gene that codes for lymphokine generally comprises, upstream of the coding sequence, an expression sequence and a signal sequence that directs the synthesized polypeptide into the secretory pathways of the target cell. This signal sequence can be the natural signal sequence for the lymphokine, but it can also be any other functional signal sequence, or an artificial signal sequence. Such constructions allow in particular to increase the amount of lymphokine in a very localized manner and also, in the presence of an antigen specific for the tumor, to enhance the immune response against a particular type of tumor, which gives a particularly beneficial effect. Such recombinant adenoviruses are particularly suitable for the production of anti-tumor vaccines.

The defective, recombinant adenoviruses according to the invention can be produced by any technique known to those skilled in the art (Levrero et al., "Gene" 101 (1991) 195, EP 185 573; Graham, "EMBO J." 3 (1984) 2917) . In particular, they can be produced by homologous recombination between an adenovirus and a plasmid which, among other things, carries the heterologous DNA sequence. The homologous recombination occurs after co-transfection of the adenovirus and the plasmid in a suitable cell line. The cell line used must preferably (i) be transformable with these elements, and (ii) carry sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid risks of recombination. As an example of a line can be mentioned the human embryonic kidney line 293 (Graham et al.,"J.Gen.Virol." 36 (1977) 59) which in particular, integrated into its genome, contains the left part of the genome of an Ad5 adenovirus (12 <%>)<.>

Subsequently, the adenoviruses that have multiplied are recovered and purified according to classical molecular biology techniques as shown in the examples.

The present invention likewise relates to a pharmaceutical preparation comprising one or more defective, recombinant adenoviruses as described above. Preferably, the pharmaceutical preparations according to the invention contain a pharmaceutically acceptable carrier for a formulation that is directly injectable into the tumor to be treated. This may in particular be sterile, isotonic solutions or dry preparations, particularly lyophilized, which, when added as appropriate, to sterilized water or a physiological serum, allow the constitution of injectable dissolved substances. The direct injection into a tumor to be treated is advantageous because it allows concentrating the therapeutic effect on the affected tissue.

The doses of the defective, recombinant adenoviruses used for injection can be adapted as a function of various parameters and in particular as a function of the method of administration used, the relevant pathology, the gene to be expressed or also the desired duration of the treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10<4> and 10<14> pfu/ml and preferably 1()6 to 101^ pfu/ml. The expression pfu, "plaque forming unit", corresponds to the infectivity of a virus solution and is determined by infection of a suitable cell culture and measured, generally after 48 hours, with a view to the number of colonies of infected cells. The techniques for determining the pfu value for a viral solution are well documented in the literature.

The present invention thus enables a very effective agent for the treatment or prevention of cancer. Furthermore, the treatment can be carried out on both humans and animals such as sheep and goats, cattle, domestic animals such as dogs, cats and so on, horses, fish and so on.

The invention shall be described in more detail with reference to the following illustrative examples.

Figure explanation:

Figure 1: Illustration of the vector mp53wtI-.CMV

Figure 2: Illustration of the vector mp53pIX.CMV.

General molecular biology techniques.

The classical methods used in molecular biology, for example preparative extraction of plasmid DNA, centrifugation of plasmid DNA in a cesium chloride gradient, electrophoresis on agarose or acrylamide gel, purification of fragments of DNA by electroelution, extraction of proteins with phenol or phenol chloroform, precipitation of DNA in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and so on, are well known to those skilled in the art and abundantly described in the literature [Maniatisk T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor , N.Y., 1982; Ausubel F.M. et al., (eds.), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987]. The plasmids of the type pBR322, pUC and the phages from series M13 are of commercial origin (Bethesda Research Laboratories).

For the ligations, the DNA fragments can be separated according to size by electrophoresis on agarose or acrylamide gel, extraction with phenochloroform, precipitation in ethanol and incubation in the presence of DNA ligase by phage T4 (Biolabs) according to the supplier's recommendations.

The filling of the overhanging 5' ends can be carried out with the Klenow fragment of DNA polymerase I of E.coli (Biolabs) according to the supplier's specifications.

The destruction of the protruding 3' ends is carried out in the presence of DNA polymerase of phage T4 (Biolabs) which is used according to the manufacturer's recommendations. The destruction of the protruding 5' ends is carried out by treatment with the nuclease Sl.

The in vitro directed mutagenesis with synthetic oligo-deoxynucleotides can be carried out according to the method developed by Taylor et al., ["Nucleic Acids Res." 13. (1985) 8749-8764] using the kit obtainable from Amersham.

The enzymatic amplification of the DNA fragments by the so-called PCR technique ["Polymérase-catalyzed Chain Reaction", Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. et Faloona F.A., "Meth. Enzyme." 155 (1987) 335-350] can be carried out by using a "DNA thermal cycler" (Perkin Eimer Cetus) according to the manufacturer's specifications.

The verification of the nucleotide sequences can be carried out by the method developed by Sanger et al., ["ProcNatl. Acad.Sci." USA, 74 (1977) 5463-5467] using the kit available from Amersham.

Examples

El. Construction of the mp53wtI-CMV vector carrying the p53 gene under the control of the cytomegalovirus promoter (Figure 1).

The eukaryotic expression vector mp53wtI-CMV is constructed from plasmid pUC19 by inserting: of a promoter region of viral origin corresponding to the early cytomegalovirus (CMV) promoter. This region is surrounded in the vector by unique restriction sites EcoRI-SphI in the connection CMV/pUC and BamHI in the connection CMV/p53. The presence of unique sites flanking the promoter region allows the replacement of the CMV region with any other promoter. A second series of vectors is then obtained in which the gene p53 is placed under the control of an inducible promoter:

the promoter of metallothionein, inducible by heavy metals (cadmium and zinc);

of a sequence of 1173 bp corresponding to the cDNA encoding the protein p53 from mouse in its wild form (Zakut-Houri et al., "Nature" 36 (1983) 594). In this construction, the suppressor gene is in the form of cDNA, that is, deprived of introns. This corresponds in particular to reducing the size of the vector. Furthermore, it has been verified that they achieved

expression levels are comparable in the presence or absence of introns;

of a polyadenylation signal for the late virus SV-40 genes corresponding to a highly efficient polyadenylation signal. Two unique restriction sites SalI and HindIII are located downstream of the polyadenylation signal. These sites have allowed insertion of plX regions by adenoviruses (cf. E3).

E2. Activity in vitro of the vector mp53wtI-CMV.

The functionality of the vector mp53wtI-CMV has been confirmed in vitro by transient expression in HeLa cells. For this purpose, the vector is introduced into the cells by transfection and, 40 hours later, protein p53 is dosed by immunofluorescence and immunoprecipitation. The results obtained show that more than 50% of the transfected cells induce substantial amounts of protein p53.

E3. Construction of vector mp53pIX.CMV.

The plasmids used to form the recombinant adenoviruses that express gene p53 by homologous recombination are constructed as follows: The eukaryotic expression vector mp53pIX.CMV is constructed by inserting the sequence pDC from the genome of the adenoviruses between the Sali and EcoRI sites of mpl3wtl-.CMV. The sequence pIX is isolated from the recombinant plasmid pLTR-figal pIX (Stratford-Perricaudet et al., "J.Clin.Invest." 90 (1992) 626) by fermentation with the enzymes EcoRV and HindIII.

The expression vector mp53pIX.CMV obtained in this way (Figure 2) has a unique HindIII site downstream of the insert pIX that allows a linearization of the construct

Ofr. E4).

E4. Construction of a defective, recombinant adenovirus carrying the p53 gene under the control of the CMV promoter.

The mp53pIX.CMV vector is linearized and co-transfected with a defective adenoviral vector into helper cells (line 293) that carries in trans the functions encoded by the E1 (E1 A and E1 IB) regions of the adenovirus.

Adenovirus Ad.p53 is obtained by homologous recombination in vivo between the mutant adenovirus Ad-dl324 (Thimmappaya et al., "Cell" 31 (1982) 543) and the vector mp53pIX.CMV, according to the following protocol: after linearization with the enzyme HindIII plasmid mp53pDC.CMV and adenovirus dl 324 are co-transfected into line 293 in the presence of calcium phosphate thereby allowing homologous recombination. The recombinant adenoviruses thus formed are selected by purification on plates. After isolation, the recombinant adenovirus DNA is amplified in the 293 cell line, leading to a culture supernatant containing unpurified, recombinant, defective adenovirus with a titer of approx. 1010 pfu/ml.

The viral particles are then purified by centrifugation over a cesium chloride gradient according to known techniques (see especially Graham et al., "Virology" 52 (1973) 456). Adenovirus Adp53 can be preserved at -80°C in 20% glycerol.

The ability of adenovirus Adp53 to infect cells in culture and to express a biologically active form of wild-type p53 in the culture medium has been demonstrated by infection of human lmjen 293 cells. The presence of p53 in the culture supernatant is then detected using a monoclonal antibody which is specific for p53.

These studies allow us to show that the adenovirus well expresses a biologically active form of GAD67 in cell culture.

Claims (16)

1. Defective, recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell allows at least partially inhibiting cell division, characterized in that the sequence comprises: at least one gene encoding p53 protein; and a promoter sequence allowing expression, in the infected cell, of said gene, the promoter sequence having an origin different from that of the gene. 2. Defective, recombinant adenovirus according to claim 1, characterized in that it contains a sequence that codes for the wild-type protein p53 under the control of a heterologous promoter. 3. Defective, recombinant adenovirus according to claim 1 or 2, characterized in that the promoter sequence is a promoter sequence originating from the genome of the infected cell. 4. Defective, recombinant adenovirus according to claim 1 or 2, characterized in that the promoter sequence is a synthetic sequence. 5. Defective, recombinant adenovirus according to claim 1 or 2, characterized in that the promoter sequence is a sequence of viral origin, selected from the promoter sequence of the genes CMV or RSV. 6. Defective, recombinant adenovirus according to claim 1 or 2, characterized in that it contains a sequence that codes for the wild-type protein p53 under the control of an early promoter of cytomegalovirus. 7. Adenovirus according to any one of claims 1 to 6, characterized in that it is deprived of the regions of its genome necessary for replication in the target cell. 8. Adenovirus according to claim 7, characterized in that it is an Ad5 type adenovirus. 9. Adenovirus according to any one of claims 1 to 8, characterized in that the heterologous DNA sequence further comprises a gene that codes for an antigen that is specific for the tumor and/or a gene that codes for a lymphokine. 10. Use of an adenovirus according to any one of claims 1 to 9 for the production of a pharmaceutical preparation intended for the therapy and/or prevention of cancer by introduction directly into the tissue to be treated. 11. Use of an adenovirus according to claims 1 to 9 for the manufacture of a pharmaceutical preparation intended for the manufacture of a pharmaceutical preparation intended for the therapy or prevention of cancer by administration directly to the tumor to be treated. 12. Use of an adenovirus according to claim 2 for the preparation of a pharmaceutical preparation intended for the treatment and/or prevention of cancers associated with the presence of a mutated form of p53. 13. Use according to claims 10 or 11 for the production of an anti-tumoral vaccine. 14. Pharmaceutical preparation, characterized in that it contains one or more defective, recombinant adenoviruses according to one of claims 1 to 9. 15. Pharmaceutical preparation according to claim 14, characterized in that it is available in injectable form. 16. Pharmaceutical preparation according to claim 14, characterized in that it comprises between 10<4> and 10<*4> pfu/ml and preferably between 10^ to 10<*0> pfu/ml of defective, recombinant adenoviruses.