CA2158869C - Recombinant virus, process for preparing the same and use thereof in gene therapy - Google Patents

Abstract

Recombinant adenoviruses comprising a heterologous DNA sequence, preparation thereof, and use thereof for the treatment and/or prevention of cancer.

Description

RECOMBINANT VIRUSES. PREPARATION AND USE
IN GENIUS THERAPY

The present invention relates to recombinant vectors of viral origin and their use for the treatment of cancers. More particularly, she concerns recombinant adenovirus comprising a heterologous DNA sequence the expression in an abnormally dividing cell makes it possible to inhibit less partially dividing said cell. The invention also relates to preparation of these vectors and the pharmaceutical compositions containing them.

Cell growth is regulated in an extremely subtle way by two types of signals. Some promote the multiplication of cells, while that others, on the contrary, bring them into a quiescent state or make them differentiate, according to the needs of the organization. Cancers are all characterized by a disturbance of the mechanisms controlling cell division, leading to proliferation abnormal.
Most often, the development of a cancer therefore involves the activation of Genoa favoring the multiplication of cells (genes called proto-oncogenes, which are oncogenes) and the disappearance or inactivation of genes that inhibit cell proliferation. The present invention offers the possibility of treat cancers by the gene therapy, by the administration, to tumor cells, of a or many of these genes whose expression makes it possible to at least partially inhibit the proliferation cellular.

Gene therapy is cozriger deficiency or abnormality (mutation, aberrant expression) by introducing genetic information in the affected cell or organ. This genetic information can be introduced either in vitro in a cell extracted from the organ, the modified cell being then reintroduced in the body, either directly in vivo in the appropriate tissue. In this second case, different techniques exist, including various techniques of transfection involving DNA and DEAE-dextran complexes (Pagano et al., J. Virol.
(1967) 891), DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 3o 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431). More recently, the use of viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In this respect, different viruses have been tested for their ability to infect certain populations

2 cell. In particular, retroviruses (RSV, HMS, MMS, etc.), the virus HSV, the adeno-associated viruses, and adenoviruses.

The possibility of using gene therapy to treat cancers has already summer mentioned in application WO91 / 15580. This application describes the construction of retroviruses containing a gene encoding a ribozyme, the expression of which culture cell can destroy an mRNA of an oncogene.

The present invention results from the demonstration that adenoviruses are particularly efficient vectors for the transfer and the expression of therapeutic genes in tumors. In particular, adenoviruses show the advantage of not integrating into the genome of the cells they infect, stay there in a very stable way which makes it possible to obtain a lasting therapeutic effect, and to have a very wide host spectrum, which allows an application to the treatment of cancers affecting any type of cell. Moreover, the invention is also on the implementation evidence that adenovirus-like viruses are able to transfer and to express genes capable of at least partially inhibiting cell division directly to level of tumors.

A first object of the invention therefore lies in a recombinant adenovirus defective containing a heterologous DNA sequence whose expression allows inhibit at least partially cell division.
More specifically, the subject of the invention is a recombinant adenovirus defective containing a heterologous DNA sequence whose expression in a target cell makes it possible to at least partially inhibit cell division, said sequence comprising at least one gene coding for the p53 protein and a promoter sequence allowing expression in the cell infected, said gene, said promoter sequence having a different origin of that of said gene.

The invention also relates to the use of such an adenovirus Defective recombinant for the preparation of a pharmaceutical composition destiny the treatment or prevention of cancer.

2a For the purposes of the present invention, the term "defective adenovirus" refers to a adenovirus unable to replicate autonomously in the target cell.
Generally, the genorn of the defective adenoviruses used in the context of present invention is therefore devoid of at least the necessary sequences for replication of said virus in the infected cell. These regions can either be eliminated (in all or in part), either rendered non-functional or substituted by other sequences and especially by the inserted gene. Preferably, the defective virus retains However the sequences of his genome that are necessary for the encapsidation of particles viral.

-3-There are different serotypes of adenovirus, including the structure and properties vary somewhat. Nevertheless, these viruses are not pathogenic for humans, and especially non-immuno-depressed subjects. Of these serotypes, use in the context of the present invention adenovirus type 2 or 5 (Ad 2 or Ad 5).
In the case of adenovirus Ad 5, the sequences necessary for replication are the regions E1A and E1B.

For the purposes of the present invention, the heterologous DNA sequence expression can at least partially inhibit cell division includes preferably at least one gene selected from tumor suppressor genes (or anti-oncogene) or any active derivative of said genes; antisense genes, which the expression in the target cell makes it possible to inhibit the expression of genes favoring division cellular;
or genes whose expression product induces apoptosis of the cell infected.

Among the tumor suppressor genes that can be used in the context of The present invention may more particularly include the following genes:

- gene p53:
The p53 gene encodes a 53 kDa nuclear protein. The mutated form by deletion and / or mutation of this gene is involved in the development of the most human cancers (Baker et al., Science 244 (1989) 217). His forms mutated are also able to cooperate with ras oncogenes for transform murine fibroblasts. The wild-type gene encoding native p53 inhibits revenge formation of foci of transformation in rodent fibroblasts transfected with various combinations of oncogenes. Recent data point out that the p53 protein could itself be a transcription factor and stimulate the expression of other tumor suppressor genes.

- Gene Rb The Rb gene determines the synthesis of a 927 nuclear phosphoprotein approximately amino acids (Friend et al., Nature 323 (1986) 643) function is to suppress cell division by bringing them into phase quiescence. of the Inactivated forms of the Rb gene have been implicated in different tumors, and especially in retinoblastomas or in mesenchymal cancers such as the osteosarcoma. The reintroduction of this gene into tumor cells where it was inactivated produces a return to normal state and a loss of tumorigenicity (Huang and coll., Science 242 (1988) 1563). Recently, it has been shown that protein Rb WO 94/24297 n PCT / FR94 / 00421

-4 -normal, but not its mutated forms, represses the expression of the proto-oncogene c-fos essential gene for cell proliferation.

- Rap gene A
The rap IA gene (also referred to as k-revl) encodes a protein of 21 kDa associated with the inner surface of the cytoplasmic membrane. This protein is capable, at high levels, of converting transformed cells expressing the Mutated ras oncogenes (Kitayama et al., Cell 56 (1989) 77).

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

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

Other genes may be used in the context of the present invention for their anti-tumor effect, and especially other tumor described in the literature, or any other gene whose expression product can induce cell apoptosis.

As indicated above, the heterologous DNA sequence may comprise the native tumor suppressor gene or an active derivative of said gene. Such a derivative may be obtained by mutation, deletion, substitution and / or addition of one or more pairs of bases in the gene sequence, according to classical biology techniques molecular. The activity of the derivative thus obtained can then be confirmed in vitro on tests known to those skilled in the art, such as those described in the examples.

Within the meaning of the invention, the heterologous DNA sequence may also be include an antisense gene whose expression in the target cell allows control gene expression or transcription of coding cellular mRNA
for some proteins promoting cell proliferation. Such genes can by example to be

-5-transcribed in the target cell into RNA complementary to cellular mRNAs and block their translation into protein.

Among the antisense genes that can be used in the context of the invention, it is possible to to quote more particularly any antisense sequence making it possible to reduce levels of production of oncogenes ras, myc, fos, c-erb B, etc.

Generally, the heterologous DNA sequence also includes promoter sequences allowing the expression of the gene (s) capable to inhibit less partially cell division in the target cell. he can this is promoter sequences that are naturally responsible for expression said gene when these sequences are likely to work in the cell infected. he can also be promoter sequences of different origin (responsible of the expression of other proteins, or even synthetic ones). In particular, he may this is promoter sequences of eukaryotic or viral genes. For example, he can this is promoter sequences from the genome of the cell that it is desired to infect.
Of same, they may be promoter sequences derived from the genome of a virus, including understood the adenovirus used. In this respect, for example, the promoters of genes E1A, MLP, CMV, RSV, etc. In addition, these promoter sequences can be modified by addition of activation sequences, regulation, etc. By elsewhere, when the heterologous DNA sequence does not contain sequences expression, it can be inserted into the genome of the defective virus downstream of such sequence. he It is also possible to use inducible promoters.

Furthermore, in another embodiment of the invention, the sequence heterologous DNA comprises, in addition to the tumor suppressor gene or gene antisense, a gene encoding a tumor-specific antigen and / or a gene encoding for a lymphokine. The combination of these genes makes it possible (i) to stop cell division in a tumor and thus, to regress said tumor, and (ii), to increase the immune response of the body against said tumor.

Tumor-specific antigens are antigenic motifs that appear on the surface of tumor cells, but which do not exist at the surface of same non-tumor cells. Such antigens are generally used for the cancer diagnosis. More recently, they have been described for the realization of vaccines anti-tumor agents (EP 259,212). However, they have never been combined with other genes therapeutic as in the context of the present invention.

-6-Among the genes encoding lymphokines, mention may be made of particularly the genes coding for interleukins (IL-1 to IL-13), interferons, tumor necrosis factors, colony stimulating factors (G-CSF, M-CSF, GM-CSF), TGFB: Moreover, the gene coding for lyrnpholcin generally comprises, upstream of the coding sequence, a sequence expression and a signal sequence directing the synthesized polypeptide into the pathways of secretion of the target cell. This signal sequence can be the signal sequence natural lymphokine, but it can also be any other signal sequence functional, or an artificial signal sequence. Such constructions allow in particular to increase lymphokine levels in a very localized way, and thus, in presence of a tumor-specific antigen, to amplify the immune response against a type tumor, which gives a particularly advantageous effect. Of like recombinant adenoviruses are particularly useful for the preparation of anti-tumor vaccines.

The defective recombinant adenoviruses according to the invention can be prepared 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 to be prepared by homologous recombination between an adenovirus and a plasmid wearing among others, the heterologous DNA sequence. Homologous recombination is product after co-transfection of said adenoviruses and plasmid into a line cellular appropriate. The cell line used should preferably (i) be transformable by said elements, and (ii), comprise the sequences capable of complimenting the part of the defective adenovirus genome, preferably in an integrated form for avoid risks of recombination. As an example of a lineage, mention may be made of line of human embryonic kidney 293 (Graham et al., J. Gen. Virol 36 (1977) 59) who contains in particular, integrated in its genome, the left part of the genome of a adenovirus Ad5 (12%).
Then, the adenoviruses which have multiplied are recovered and purified according to classical techniques of molecular biology, as illustrated in the examples.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant adenoviruses as described previously. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable carrier for fortification directly injectable into the tumors to be treated. It can be in particular of WO 94/2429

7 PCT / FR94 / 00421 sterile, isotonic or dry solutions, in particular freeze-dried, which by the addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutions. Direct injection into the tumor treat is advantageous because it allows the therapeutic effect to be concentrated at the level of tissues affected.

The doses of defective recombinant adenovirus used for injection may be adapted according to different parameters, and in particular according to of the mode used, the pathology concerned, the gene to be expressed, or more the duration of the treatment sought. In general, adenoviruses recombinants according to the invention are formulated and administered in the form of doses between 104 and 1014 pfu / ml, and preferably 106 to 1010 pfu / ml. The term pfu ("plaque-forming unit") corresponds to the infectivity of a solution of virus, and is determined by infection of an appropriate cell culture, and measures, usually after 48 hours, the number of infected cell plaques. The techniques of determination of the pfu title of a viral solution are well documented in the literature.

The present invention thus provides a very effective means for the treatment or cancer prevention. In addition, this treatment may concern as well the man than any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.

The present invention will be more fully described using the examples following, which should be considered as illustrative and not limiting.

Legend of figties Figure 1: Representation of the vector mp53wtI-.CMV
Figure 2 Representation of the vector mp53pIX.CMV
General techniques of molecular biology The methods conventionally used in molecular biology such as preparative extractions of plasmid DNA, DNA centrifugation plasmid in cesium chloride gradient, electrophoresis on agarose gels or acrylamide, the purification of DNA fragments by electroelution, extraction of proteins phenol or phenol-chloroform, the precipitation of DNA in a salt medium by WO 94/24297 ~ 1 QQQL 'Q PCT / FR94 / 00421

-8-ethanol or isopropanol, transformation into Escherichia coli, etc.
... are good known to those skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory Cold Spring Harbor, NY, 1982; Ausubel FM et al. (eds), "Current Protocols in Molecular Biology, "John Wiley & Sons, New York, 1987].
Plasmids of the pBR322, pUC and M13 series are of commercial origin (Bethesda Research Laboratories).
For ligations, the DNA fragments can be separated according to their cut by electrophoresis in agarose or acrylamide gels, phenol extracts or by a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA
T4 phage ligase (Biolabs) according to the supplier's recommendations.
The filling of the prominent 5 'ends can be performed by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to specifications from the supplier. The destruction of the prominent 3 'ends is carried out in presence T4 phage DNA polymerase (Biolabs) used according to the recommendations of maker. The engagement of the prominent 5 'ends is effected by a treatment provided by the nuclease SI.
In vitro directed mutagenesis by synthetic oligodeoxynucleotides can be be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.
Enzymatic amplification of DNA fragments by the so-called PCR technique Lolymerase-catalyzed Reaction Chain, Saiki RK et al., Science 230 (1985) 1354; Mullis KB and Faloona FA, Meth. Enzym. ] (1987) 335-3501 can be performed using a "DNA thermal cycler" (Perkin Elmer 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. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467]
in using the kit distributed by Amersham.

Exempl es El. Construction of the vector mp53wtl-CMV carrying the p53 gene under the control of the cytomegalovirus promoter (Figure 1).

The eukaryotic expression vector mp53% vtI-CMV was constructed from the plasmid pUC19, by insertion:

WO 94/24297 'm. 21 CQQ ~ (~ PCT / FR94 / 00421

-9 a promoter region of viral origin which corresponds to the promoter early cytomegalovirus (CMV). This region is surrounded in the vector of websites of unique EcoRI-SphI restrictions at the CMV / pUC and BamHI junction at the junction CMV / p53. The presence of unique sites flanking the promoter region allows of replace the CMV region with any other promoter. A second series of vectors is thus obtained in which the p53 gene is placed under the control of a sponsor inducible: the metallothionine promoter, inducible by metals heavy (cadmium and zinc).
a sequence of 1173 bp corresponding to the cDNA coding for the protein p53 of mice in its wild form (Zakut-Houri et al., Nature 36 (1983) 594). In this construct, the suppressor gene is in the form of cDNA, that is to say free introns. This allows in particular to reduce the size of the vector. By elsewhere it has been verified that the expression levels obtained are comparable in the presence or in the absence of introns.
the polyadenylation signal of the late genes of the SV40 virus, which corresponds to a very effective polyadenylation signal. Two sites of restriction unique SalI and HindIII are located downstream of the polyadenylation signal. These sites have allowed the insertion of the pIX regions of the adenovirus (Cf E3).

E2. In vitro activity of vectetir nip53% i, tI-CMV

The functionality of the mp53wt1-CMV vector has been confirmed in vitro by transient expression in HeLa cells. For this, the vector has been introduced in cells by transfection and, 40 hours later, the p53 protein was assayed by immunofluorescence and immunoprecipitation. The results obtained show that more 50% of transfected cells induce significant levels of protein p53.

E3. Vector construction mp53pIX.CMV

Plasmids used to generate, by homologous recombination, the Recombinant adenoviruses expressing the p53 gene were constructed as follows:
The eukaryotic expression vector mp53pIX.CMV was constructed by inseRion of the pIX sequence derived from the genome of the adenovirus between the sites Sall and EcoRl of mpl3wtI-.CMV. The pIX sequence was isolated from the plasmid Recombinant pLTR- (3gal pIX (Stratford-Perricaudet et al., J. Clin Invest.
(1992) 626) by digestion with EcoRV and HindIII enzymes.

The expression vector mp53pIX.CMV thus obtained (FIG.
single site HindIII downstream of the pIX insert which allows linearization of the construction (cf E4.).

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

The vector mp53pIX.CMV is linearized and cotransfected with a vector adenoviral deficiency, in helper cells (line 293) providing trarr functions encoded by the adenovirus regions E 1 (E 1 A and E 11 B).
The Ad.p53 adenovirus is obtained by homologous recombination in vivo between Ad-d1324 mutant adenovirus (Thimmappaya et al., Cell 31 (1982) 543) and the vector mp53pIX.CMV, according to the following protocol: after linearization by the enzyme HindIII
plasmid mp53pIX.CMV and adenovirus d1324 are co-transfected in the line 293 in the presence of calcium phosphate, to allow recombination counterpart. The recombinant adenoviruses thus generated are selected by plate purification. After isolation, the DNA of the recombinant adenovirus is amplified in cell line 293, which leads to a supernatant of culture containing the unpurified recombinant defective adenovirus about 1010 pfu / ml.

The virus particles are then purified by gradient centrifugation of cesium chloride according to known techniques (see in particular Graham et al., Virology 52 (1973) 456). Adenovirus Adp53 can be stored at -80 C in 20 %
glycerol.

The ability of Ad-p53 adenovirus to infect cells in culture and express in the culture medium a biologically active form of p53 wild has been demonstrated by infection of cells of the human 293 line. The presence of p53 in the culture supernatant was then evidenced by means of a monoclonal antibody specific for p53.

These studies show that the adenovirus expresses biologically active p53.

Claims (15)

1. Defective recombinant adenovirus containing a sequence of heterologous DNA whose expression in a target cell makes it possible to inhibit at at least partially cell division, said sequence comprising - at least one gene encoding the p53 protein and, - a promoter sequence allowing the expression, in the infected cell, of said gene, said promoter sequence having an origin different from that of said gene. 2. Defective recombinant adenovirus according to claim 1 containing a sequence coding for the wild-type p53 protein under control from a heterologous promoter. 3. Defective recombinant adenovirus according to claim 2 containing a sequence coding for the wild-type p53 protein under the control of cytomegalovirus early promoter. 4. Adenovirus according to one of claims 1 to 3 characterized in that it lacks the regions of its genome which are necessary for its replication in the target cell. 5. Adenovirus according to claim 4, characterized in that it It is an Ad 5 type adenovirus. 6. Adenovirus according to one of claims 1 to 5 characterized in that the heterologous DNA sequence further comprises a gene encoding a tumor-specific antigen or a gene encoding a lymphokine. 7. Use of an adenovirus according to one of claims 1 to 6 for the preparation of a pharmaceutical composition intended for the treatment or prevention of cancers by direct administration in the tumor to be treated. 8. Use of an adenovirus according to claim 2 for the preparation of a pharmaceutical composition intended for the treatment or prevention of cancers associated with the presence of a mutated form of p53. 9. Use according to claim 7 for the preparation of a anti-tumor vaccine. 10. Pharmaceutical composition comprising one or more defective recombinant adenoviruses according to one of Claims 1 to 6. 11. Pharmaceutical composition according to claim 10 characterized in that it is in injectable form. 12. Pharmaceutical composition according to claim 11 characterized in that it comprises between 10 4 and 10 14 pfu/ml adenovirus defective recombinants. 13. Pharmaceutical composition according to claim 12, characterized in that it comprises between 10 6 and 10 10 pfu/ml adenovirus recombinants. 14. Use of a defective recombinant adenovirus containing a heterologous DNA sequence comprising a gene encoding the protein p53, for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of cancer. 15. Use of a defective recombinant adenovirus containing a heterologous DNA sequence coding for the wild-type p53 protein for the preparation of a pharmaceutical composition intended to induce apoptosis cellular.