FI118011B - A method for producing a recombinant adenovirus deficient in replication - 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

118011 1,.

A method of producing recombinant adenovirus deficient in replication

The present invention relates to recombinant DNA vectors of viral origin and their use in the treatment of cancers. More particularly, the invention relates to a method for producing a replication deficient recombinant adeno virus comprising a heterologous DNA sequence whose expression in a target cell allows at least partial inhibition of cell division, said sequence comprising: at least one gene encoding p53; and a promoter sequence that permits expression of said 15 genes in an infected cell, wherein the origin of said promoter sequence differs from the origin of said gene. The invention also relates to a process for the preparation of a pharmaceutical composition containing said virus.

Two types of signals regulate cell growth in an extremely subtle way. Some prefer cells to proliferate, while others favor them. into the nucleus or cause them to differentiate * * ·. . ·. min according to needs. Cancers are all characterized by a disruption of 1 * * cell division controlling mechanisms leading to abnormal proliferation. In most cases, the development of cancer development thus involves the activation of genes that promote cell proliferation (called genes called proto-oncogenes, which * · * * become oncogenes), and the loss or inactivation of genes that inhibit cell growth. The invention provides the possibility of treating cancers by gene therapy by introducing into the tumor cells one or more of these genes, the expression of which at least partially inhibits cell proliferation.

Gene therapy consists of repairing a defect or abnormality (mutation, abnormal expression, etc.) *: **: By introducing genetic information into a diseased cell or organ. j 2 118011 This genetic information can be introduced either into an organ extracted from an organ in vitro, after which the modified cell is reintroduced into the body, or directly into an appropriate tissue in vivo.

In this second case, there are various techniques which include different transfection techniques using DNA complexes and DEAE-dextran [Pagano et al., J, Virol. DNA and Nuclear Proteins (Kaneda i et al., Science 243: 375 (1989) 375), DNA and Lipids [Feigner et al., PNAS 84: 7413 (1987)], liposomes [Fraley et al. 10 al., J. Biol. Chem. 255: 10431 (1980)], etc. Since then, viral; The use of genes as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In this regard, the ability of various viruses to infect specific cell populations has been tested. In particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, adenolytic viruses and adenoviruses.

The possibility of using gene therapy for the treatment of cancers has already been disclosed in WO patent application 91/15580.

This patent application describes the construction of a retrovirus containing a gene encoding a ribozyme which; expression in the cell culture may make it possible to determine whether. destruction of the mRNA gene.

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. . ·. The present invention is due to the fact that adenoviruses have been shown to form highly effective vectors for the transfer and expression of therapeutic genes into tumors. Adenovirus, in particular, have the advantage that they do not integrate into the cell they infect; ; • · *. i. v * · * * genomes, they remain therein in a very stable manner, allowing a long-lasting therapeutic effect | * * • * ·· 30 and have a very broad host spectrum, which makes it * * * possible to use for all types of cell cancers:: v. in trust. In addition, the invention also relies on proving that adenovirus-type viruses are capable of transferring and expressing genes capable of inhibiting at least! * .. * · * 35 partially cell division directly at the tumor level.

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118011

A first object of the invention is a process for the preparation of a replication deficient recombinant adenovirus, characterized in what is set forth in claim 1.

The invention also relates to a method for such a defect! A list for the preparation of a pharmaceutical composition comprising a recombinant adenovirus, characterized in what is set forth in claim 7. The compositions are for the treatment or prevention of cancers.

10 In the meaning of the present invention, the expression! "defective adenovirus" means an adenovirus that is incapable of autonomous replication in a target cell. Thus, generally, within the framework of the present invention, the genome of defective adenoviruses is devoid of the sequences necessary for replication of at least the said virus in an infected cell. These regions may have been either eliminated (in whole or in part) or rendered non-functional, or substituted by other sequences, in particular by the inserted gene. Preferably, however, the defective virus retains its genome sequences necessary for the encapsulation of viral particles.

; There are different serotype adenoviruses, ·· ·. . ·. whose structure and properties vary somewhat. : • · * • · *

However, these viruses are not pathogenic to humans, * · * 25 and especially to subjects whose immune system is not impaired. Of these serotypes, adenoviruses type 2 or * * * are preferably used within the framework of the present invention.

* · '5 (Ad 2 or Ad 5). For Adenovirus Ad 5, the sequences necessary for replication are the E1A and E1B regions.

• ·: * · 30 For the purposes of the present invention, a heterologous DNA sequence which, when expressed, can inhibit at least: v. partially cell division, preferably comprises at least one gene selected from tumor suppressor genes (or anti-oncogenes) or any active derivative of said genes; antisense gene * * "*: those which, upon expression in the target cell, can inhibit the expression of 4 '118011 genes favoring cell division; or genes whose expression product induces apoptosis of an infected cell.

Vegetable indispensable within the scope of the present invention; The 5 suppressor genes are:

P53 gene; l

The p53 gene encodes a 53 kDa nuclear protein. The mutagenized form of this gene by deletion and / or mutation is associated with the development of most human cancers (Baker et al., Science 244: 217 (1989)). Its mutagenized forms are also capable of cooperating with ras oncogenes to transform mouse connective tissue-forming cells. In contrast, the wild-type gene encoding native p53 inhibits the formation of trans, formation colonies in rodent connective tissue-forming cells transfected with various oncogene combinations. Recent results highlight that p53 protein itself could be a transcription factor that stimulates the expression of other tumor suppressor genes.

Other suppressive genes include: 20 Gene Rb:

The Rb gene defines approximately 927 amino acids in the nuclear phosphoprotein. synthesis (Friend et al., Nature 323: 643 (1986)), has the function of suppressing cell division, possibly; • · · Stick them to the resting phase. Inactivated form of Rb gene has been shown to be the cause of various tumors, and in particular malignant glioma of the retina or mesenchymal cancers such as bone cancers. Re-introduction of this gene • · ·! * · * * To tumor cells where it was inactive, restores normal state and produces loss of tumor formation * * ·· 30 (Huang et al., Science 242: 1563 (1988)). Recently, it has been shown that normal protein Rb suppresses, but does not; ·] ·. its mutagenized forms, the c-fos proto-oncogene, • · * ..! expression of the gene necessary for the distribution.

• · ·· ♦ »·· ·· **« i * ···· - • · i *} ·. · 4; . ·. t: i - 'i 5; '118011

Gene Rap IA:

The rap IA gene (also called k-rev1) encodes a 21 kda protein that binds to the inner surface of the cytoplasmic membrane. This protein is capable, at high levels, of reversing transformed cells expressing mutated genotoxins (Kitayama et al., Cell 56 (1989) 77). i

DCC gene:

The DCC gene encodes a protein that is homologous to the N-CAM group 10 cell attachment proteins. This gene is very frequently deleted in colorectal cancers [Fearon et al., Science 247 (1990) 49].

The genes k-rev2 and k-rev3:

The k-rev2 gene encodes a 60 amino acid differentiating pro-15 protein and the k-rev3 gene encodes a truncated form of the extracellular matrix protein. These two genes are capable of reversing NIH 3T3 cells transformed with Ki-ras oncogene. |

Other genes may be used within the scope of the present invention for their antitumor activity, and in particular other tumors described and described in the literature; suppressing genes, or any other gene that * · · i. . ·. expression may induce cell apoptosis. '' • · »

As mentioned above, heterologous DNA · · * * e. The sequence may comprise a native tumor suppressor i "*! Gene or an active derivative of said gene. Such a · · ·" * / derivative may be obtained by mutation, deletion, substitution and / or addition in the gene sequence of one or more base pairs. according to conventional techniques in molecular biology: • · * '·· 30. The activity of the derivative thus obtained can be • · ·

then confirmed in vitro by experiments known to those skilled in the art, such as those described in the Examples. I

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In the context of the invention, the heterologous DNA sequence may also comprise an antisense gene which, when expressed in a target cell, can control the expression of genes i * * "* or transcription of cellular mRNA sequences encoding 6,118,011 cell proliferation-promoting proteins. for example, such genes may be transcribed into RNA complementary to cellular mRNA sequences, thereby rendering their translation into a protein blocked. Of the antisense genes useful in the context of the invention, any antisense sequence which provides , myc,; fos, c-erb B, etc. decline in production levels

The heterologous DNA sequence also comprises promoter sequences that allow expression of one or more genes capable of at least partially inhibiting cell division in the target cell. These may include promoter sequences that are naturally responsible for the expression of said gene, insofar as | These sequences are likely to be functional contagious; cell. They may also be promoter sequences of different origins (responsible for the expression of other proteins, or even synthetic ones). In particular: they may be promoter sequences of eukaryotic or viral genes. For example, they may be promotional; sequences derived from the genome of the cell to be infected. Similarly, they can be promotional! «· ·. . ·. Rice sequences derived from the viral genome, including the adenovirus used. In this respect can be · · · *. 25 mention, for example, the promoters of genes E1A, MLP, CMV, RSV, etc. In addition, these promoter sequences may be modified by the addition of activation, regulatory, etc. sequences. In addition, when the heterologous DNA sequence does not use the expression sequences, it can be inserted by deficiency; ·· • * ·· 30 viral genome downstream of such a sequence; • · · ί> #> ί sis. It is likewise possible to use inducible promoters.

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In addition, in another embodiment of the invention, the heterologous DNA sequence comprises, in addition to a tumor suppressor gene, a gene encoding a tumor specific antigen and / or a gene encoding a lymphokine. Yh-; By sequencing these genes, it is in fact possible to (i) stop cell division in the tumor and thereby obtain regression of said tumor, and (ii) increase the body's immune response against said tumor. : 4 5 Tumor specific antigens are antigenic motifs present on the surface of tumor cells but not on the same non-tumor cells. Such antigens are generally used in cancer diagnostics. They have now been described for the preparation of antitumor vaccines-10 (EP 259,212). However, they have never been combined with other therapeutic genes, as in the context of the present invention.

More specifically, among the genes encoding lymphokines, genes encoding interleukins 15 (IL-1 to IL-13), interferons, tumor necrosis factors, pe-, 1-bag stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), * TGF5, etc. In addition, the gene encoding the lymphokine generally comprises an expression sequence upstream of the coding sequence and a signal sequence that directs the synthesized polypeptide to the target cell secretory pathways. This signal sequence may be the natural signal sequence of lymphokine, but it may also be any other function; ♦ · · .. *. thionic signal sequence or artificial signal mix ··· quency. In particular, such constructs allow for a very local increase in lymphokine levels * '** and thus, in the presence of tumor-specific antigen, ♦ · ♦ * ·· / enhances the immune response against the particular type of tumor. which has a particularly beneficial effect. Such recombinant adenoviruses are particularly useful ··! • * ·· 30 for the manufacture of anti-tumor vaccines.

• · Defective recombinant adeno-adeno- described here: *] ·. viruses may be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) * 195) EP 185 573; Graham, EMBO J. 3: 2917 (1984)). In particular, they can be prepared by homologous DNA fusion between * ί * 'ί adenovirus and a plasmid comprising, inter alia, a heterologous DNA sequence-8 118011. Co-transfection of said adenovirus and a plasmid into a suitable cell line. The cell line used should preferably (i) be transformable with said elements, and (ii) comprise sequences capable of complementing part of the defective adenovirus genome, preferably in an integrated form, to avoid the risks of DNA fusion. Graham et al., J. Gen. Vi-10 rol. 36 (59) (1977)], containing in particular the left side of the adenovirus Ad5 genome integrated into its genome part i s (12%).

The amplified adenoviruses are then harvested and purified according to conventional molecular biology techniques i: 15, which are illustrated in the Examples. :; ;

The pharmaceutical compositions of the present invention comprise one or more defective recombinant adenoviruses as described above. Preferably said pharmaceutical compositions; The compositions comprise a liquid carrier which is pharmaceutically acceptable in a composition that can be directly injected into a tumor to be treated. Different types of • · * • specifically to become sterile, isotonic solutions, or dry, especially freeze-dried compositions which when added; • * * *>; 25 cases of sterilized water or physiological serum, respectively, allow the preparation of solutions for injection; ♦ · · · * · * tuss. Direct injection into the tumor to be treated is advantageous because it enables the therapeutic effect to be concentrated at the level of the affected tissues. 1 ·· • * ·· 30 Dosage of recombinant adenovirus deficient virus used for injection may be adjusted differently; ; · [·, As a function of such parameters, and in particular the output • · · 'used? van, the pathology in question, the gene to be expressed, or; • · T as a function of the desired duration of treatment. Overall ....

The 35 recombinant adenoviruses described herein are assembled and! administered in doses of 104 to 1014 cfu / ml, and preferably 9 to 10 10 cfu / ml. The expression "cfu" ("Plaque Forming Unit") corresponds to the infectivity of the viral solution and is determined by infecting a suitable cell culture and measuring plaque counts of infected cells, usually after 48 hours.Technologies for determining the cytokine titer of virus solution have been extensively documented in the literature;

The deficient adenovirus described above provides a very effective means of treating or preventing cancers.

In addition, this treatment can apply to humans as well as 10 animals, such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.

The present invention will be more fully illustrated by the following examples, which are to be considered as illustrative rather than limiting.

15 Explanation of figures

Figure 1: Representation of vector mp53wtI-.CMV.

Figure 2: Representation of vector mp53pIX.CMV.

General Techniques in Molecular Biology Methods commonly used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA using cesium chloride gradient, agarose or acrylamide gel electrophoresis, DNA-J! purification of fragments by electroelution, extraction of proteins with phenol solution or phenol-chloroform, precipitation of DNA in a saline environment with ethanol or isopropanol, transformation into Escherichia coli, etc., are well known to the person skilled in the art and they are extensively described in the literature (Maniatis, T., et al., "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, Cold, * 30 Spring Harbor, NY, 1982; Ausubel, F.M., et al. (ed.), * · * ··· 'Current Protocols in Molevular Biology', John Wiley & * * *: Sons, New York, 1987].

• ·; ***. Plasmids of pBR322, pUC and M13 phages of the series ··· *. are of commercial origin (Bethesda Research Laborato- • · * ···· 35 Ries).

* • · · · · • * 118011: ίο

For ligation, DNA fragments may be electrophoretically separated on size on agarose or acrylamide gels, extracted with phenol solution or mixture phenol-chloroform, ethanol precipitated and then incubated in the presence of T4-5 phage DNA ligase (Biolabs) | following the recommendations.

Filling the protruding 5 'ends can be accomplished with the Klenow fragment of E. Coli DNA polymerase I according to the supplier's specifications. Destruction of the protruding 3'-10 ends is performed in the presence of T4 phage DNA polymerase (Biolabs), which is used according to the manufacturer's recommendations. Destruction of the protruding 5 'ends is accomplished by controlled treatment with S1 nuclease.

Targeted mutagenesis with in vitro synthetic oligodeoxynucleotides can be performed according to the method developed by Taylor et al. rNucl. Acids Res. 13 '8749-8764 (1985)] using a packaging marketed by Amersham. ! Enzymatic amplification of DNA fragments for PCR [Polymerase-catalyzed Chain Reaction, Saiki, R.K.,; .

et al. , Science 230: 1350-1354 (1985); Mullis, K.B., and. Faloona, F.A., Meth. Enzym, 155: 335-350 (1987)]. The technology can be carried out using a "DNA- • · · # * j'f heat recycler" (Perkin Elmer Cetus, DNA Thermal Cycler) according to the manufacturer's specifications. 7 • · · ***; Nucleotide sequence verification can be performed according to the method developed by Sanger et al. [Proc.

V * Natl. Acad. Sci. USA 74: 5463-5477 (1977)]

Packaging marketed by Amersham.

• · • * ·· 30 Examples: El. Construction of the mp53wtI-CMV vector comprising the p53 gene in the control of the cytomegalovirus promoter (Fig. 1) • · *; ** Eukaryotic expression vector mp53wtI-CMV was constructed from pUC19 by inserting:; '* * · 11 118011: j

A promoter region of viral origin corresponding to the early cytomegalovirus (CMV) promoter. This area surrounded by single restriction sites at EcoRI-Sph1 at CMV / pUC and BamHI at 5 at CMV / p53. The presence of single sites that touch the promoter region allows the CMV region to be replaced by any other promoter. This results in another set of vectors in which the p53 gene is placed under the control of an inducible promoter: a metallothionine promoter induced by heavy metals (cadmium and zinc). 1117 bp sequence corresponding to cDNA encoding murine p53 protein in its wild form (Zakut-Houri et al., Nature 36: 594 (1983)). In this construct, the suppressing gene is in the form of cDNA, i.e. introne-15 and. In particular, this makes it possible to reduce the size of the vector. In addition, it has been verified that the expression levels obtained are comparable in the presence or absence of introns.

Polyadenylation signal of late genes of virus SV40, which corresponds to a highly efficient polyadenylation signal. Two single restriction sites Sali and

HindiII are located downstream of the polyadenylation signal. ta. These sites enabled the adenovirus plX regions! 1 • · ·. ·. insertion (cf. E3). ; • · * • · · m E2. In vitro activity of mp53wtI-CMV vector · · · 25 The functionality of the vector mp53wtI-CMV was confirmed by I vt • · ·; · *; by in vitro transient expression in HeLa cells. For this * * *: · ** · vector was introduced into cells by transfection, and 40 hours later, p53 protein was determined by immunofluorescence and immunoprecipitation. The results obtained show that more than 50% of the transfected cells induce significant levels of p53 protein.

; · * ·, E3. Construction of mp53pIX.CMV • ·

The plasmids used to generate the recombinant *: * 35 DNA adenoviruses expressing the p53 gene by homologous DNA combination were constructed as follows:! * MI r ***** ":: ^, j; ''! '' 118011 i2: ..

The eukaryotic expression vector mp53pIX.CMV was constructed by inserting the adenovirus genome-derived sequence pIX between SalI and EcoRI mpl3wtl-. <; .CMV together. The sequence pIX was isolated from recombinant DNA plasmid pLTR-Bgal pIX [Stratford-Perricaudet et al., J.

Clin. Invest. 90: 626] by digestion with EcoRI and HindIII.

The expression vector thus obtained, mp53pIX.CMV (Figure 2), has a single HindIII site downstream of the insert 10 pIX, allowing linearization of the structure (cf. E4.).

E4. Construction of a deficient recombinant adenovirus comprising the p53 gene under the control of the CMV promoter: The vector mp53pIX.CMV is linearized and cotransfected with the defective adenoviral vector into adrenal cells (cell line 293), which introduce the trans adenovirus regions E1 (E1A and E11B).

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Adenovirus Ad.p53 is obtained by homologous DNA combination in vivo between adenovirus mutant Ad-dl324 (Thim-20 mappaya et al., Cell 31, 543 (1982)) and the mp53pIX.CMV vector according to the following protocol: enzyme. Following Hindlll linearization, the plasmid mp53pIX.CMV and • * * '. ·; (?. Adenovirus dl324 are co-transfected into cell line 293 in the presence of calcium * Φ 4 phosphate to allow homologous DNA integration * * · 25. - ** '* viruses are selected by plaque purification.

DNA of the recombinant recombinant adenovirus is amplified in ♦ · · * · * * cell line 293, resulting in a culture supernatant containing unpurified deficient recombinant adenovirus with a titer is about 1010 cfu / ml.

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Viral particles are then purified by centrifugation with a 1 * * club using a cesium chloride gradient according to known techniques (see, in particular, Graham et al., Virology 52, 456 (1973)). Adenovirus Adp53 can be stored at -80 ° C. glycerol.

ί 13 118011

The ability of Adenovirus Ad-p53 to infect cells in culture and to express the biologically active form of wild-p53 in culture medium was demonstrated by infecting cells of human cell line 293. The presence of p53 in culture supernatant-5 was then detected by a monoclonal antibody specific for p53.

These studies make it possible to show that adenovirus expresses well the biologically active form of GAD67 in cell culture.

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Claims (9)

118011 1 14 A method for producing a replication deficient recombinant adenovirus comprising; A heterologous DNA sequence, the expression of which in the target cell permits at least partial inhibition of cell division, said sequence comprising: at least one gene encoding p53; j and 10 a promoter sequence allowing expression of said gene in an infected cell, wherein said gene. the origin of the promoter sequence differs from the origin of said gene, characterized by co-transfecting a suitable cell line with replication deficient adenovirus and a plasmid containing said heterologous DNA sequence to allow homologous DNA fusion and screening, isolating, amplifying, and recovering adenoviruses. A method according to claim 1, characterized in that a defective recombinant adenovirus is produced which contains a sequence encoding wild-type protein p53 in the control of a heterologous promoter. Φ · · * · * 25 3. A method according to claim 2, characterized in that a defective recombinant adenovirus containing a sequence encoding a wild-type protein is produced. p53 under the control of the cytomegalovirus early promoter. j · * · .. 30 A method according to any one of claims 1 to 3, characterized in that an adenovirus, ··· mSm, free of the regions of its genome necessary for its replication in the target cell is produced. The method of claim 4, wherein the adenovirus is an adenovirus of type · 5 · 5. i * ·:., / g 118011 A method according to any one of claims 1 to 5, characterized in that an adenovirus having a heterologous DNA sequence further comprising a gene encoding a tumor specific antigen and / or a gene encoding lymphokine is produced. 7. A method of preparing a pharmaceutical composition comprising: (A) a replication deficient recombinant adenovirus comprising a heterologous DNA sequence, the expression of which in a target cell permits at least partial inhibition of cell division, said sequence comprising: (i) at least one gene that encodes a protein! ph 3; and 15 (ii) a promoter sequence which permits expression of said gene in an infected cell, wherein: the origin of said promoter sequence is different from the origin of said gene, and j (B) a pharmaceutically acceptable carrier, characterized by co-transfecting an appropriate cell line -:. and the said heterologous DNA sequence contains * **! to achieve homologous DNA fusion with the plasmid, and to screen, isolate, amplify, and harvest replication-deficient recombinant adenoviruses, and • * *: with a pharmaceutically acceptable carrier. '* · * V A method according to claim 7, characterized in that a pharmaceutical composition is prepared in a form for injection. . ***. A process according to claim 8, characterized in that a pharmaceutical composition comprising 104 to 1014, preferably 106 to 1010, is prepared. recombinant · ··· 35 DNA adenoviruses deficient in cfu / ml. • · · · ······ · · 1! 118011;