WO2005026337A2 - Adenoviral vector for infecting cells deficient in a car receptor - Google Patents

Abstract

The invention relates to an adenoviral vector provided with a tropism for Car cells and used for gene transfer. The inventive adenoviral vector comprises at least one weakly strained structural area of a capsid protein and a heterologous peptide provided with a unit (W/R)X1X2D. In a preferred embodiment, said peptide is enabled to be linked to TGF beta R and embodied in the form of the immunosuppressive peptide of a retrovirus like, for example the CKS-17 peptide. The use of the adenoviral vector for treating cancers, muscle diseases or mucoviscidosis is also disclosed.

Description

 NEW ADENOVIRAL VECTOR FOR INFECTION OF CELLS DEFICIENT OR LACKED IN CAR RECEPTORS

The subject of the invention is an adenoviral vector which has a tropism for CAR cells ^" and which is intended for gene transfer; this adenoviral vector comprises in at least one region with low structural stress of a capsid protein, a heterologous peptide comprising the motif - (W / R) XιX D -, said peptide being preferably capable of binding to TGFβR, and preferably being an immunosuppressive peptide of retrovirus such as for example the peptide CKS-17.

The invention also relates to the use of this adenoviral vector for the treatment of cancers, muscular diseases, or cystic fibrosis.

Gene therapy, which developed in the late 1980s to the early 1990s as an alternative to the treatment of serious and incurable human diseases, is now showing its usefulness in a number of clinical trials aimed at developing a treatment for inherited or acquired genetic diseases, such as certain types of cancer, coronary heart disease and cystic fibrosis.

However, during its development as a real technological field, gene therapy has revealed several limitations or defects linked to the very nature of the idea on which it is based, that is to say, effective expression of a therapeutic gene in the tissue affected by the disease in a patient.

Despite many encouraging results, it is now essential to find new technical means of effective administration of therapeutic genes on the site to be treated.

There is a need to find systems for delivery of therapeutic genes, particularly vectors, that do not elicit an immune response significant from the host. Ideally, the vector should be able to specifically deliver the therapeutic gene to the target cells in order to achieve the desired therapeutic effect.

Among the gene therapy vectors, there may be mentioned the adenoviral vectors, widely used in pre-clinical and clinical trials for the treatment of cancer. The CAR receptor (receptor common to Coxsac ievirus B3 and to adenovirus) is considered to be the primary receptor for adenovirus, and therefore adenoviral vectors. However, the efficiency of such vectors remains limited due to the low expression of the primary receptor (CAR) on the surface of many target cells.

Adenoviruses (Ad) are non-enveloped, linear double-stranded DNA viruses belonging to the family Adenoviridae. There are more than fifty different serotypes. Serotypes 2 and 5 (Ad2 and Ad5), which are responsible for often mild respiratory tract infections in humans, are the best known and constitute the basis of gene therapy vectors (Russel et al, J. Gen. Viral. 2000 , 81: 2573-2604). The adenoviral particle is composed of a core and a capsid (Figure 1).

The core consists of a 36 kb linear double-stranded DNA molecule, bordered by two origins of replication or ITR (Inverse Terminal Repeat), and four proteins: the polypeptides pV, pVII, pX and the “preTerminal Protein” (pTP) linked to the ends of the ITRs by covalent bonds.

The capsid, icosahedral in shape, is composed of 252 subunits or capsomers. The capsomers include 240 hexons (on the faces and edges) and 12 pentons (on the vertices). The hexons, trimers of the fold polypeptide, are associated with the polypeptides pVI, pVIII and pIX, elements which are essential for the cohesion of the capsid. The pentons are composed of a base (pentamer of the polypeptide pIII), and of a projection (trimer of the polypeptide pIV) called fiber, with hemagglutinating activity. , The fiber includes an anchor, a rod (varying in length depending on the serotype), and a head (C-terminal globular end).

The entry of Ad into a target cell takes place in two stages. Initially, the attachment of the virus to the surface of the target cells takes place via a strong interaction between the head of the fiber (Ad capsid protein) and the primary CAR receptor. This receptor is a transmembrane protein of 365 amino acids (46 kDa) belonging to the immunoglobulin superfamily, which plays a role in intercellular adhesion. It contains extracellular, transmembrane and cytoplasmic domains, but the extracellular domain is sufficient for attachment of the virus. After attachment of the fiber to the CAR, the base of the penton (Ad capsid protein) (by an RGD motif, Arg-Gly-Asp) interacts with secondary receptors, the integrins of the αy family (αγβ3, αγβ5 and αyβl) thus allowing the endocytosis of the viral particle in vesicles with a clathrin mantle. After endocytosis and escape of the endosome, the partially undressed viral particle migrates towards the nucleus by borrowing the network of intra-cytoplasmic microtubules (Russel et al, 2000). Recently, studies have also suggested that a glycosaminoglycan, heparan sulfate, was sufficient for the initial attachment of Ad2 and Ad5 to the target cells (Dechecchi et al, J. Virol. 2001, 75: 8772-

8780). The binding to heparan sulfates is carried out by a KKTK motif (Lys-Lys-Thr-Lys) located in the lower third of the fiber (FIG. 1).

The low level of CAR receptor expressed on the surface of target cells therefore constitutes an important limit to the use of adenovirus as a vector for gene therapy. Indeed, different cell types, and in particular muscle cells and airway epithelial cells, express the CAR receptor very weakly and are therefore only slightly transduced by Ad5. Likewise, many tumor cells (ovarian tissues, kidneys, gliomas, etc.) are refractory to Ad infection due to the absence, or low expression, of CAR (Li et al, Cancer Res. 1999, 59: 325-330). This is all the more problematic since these are the most aggressive tumors (strong tumor growth) who have this deficit. This can partly be explained by a recently described function of the CAR receptor, regulating the cell cycle (Okegawa et al, Cancer Res. 2001, 17: 6592-600).

In order to compensate for the lack of expression of the CAR receptor on the surface of certain target cell types, various strategies have been developed to provide adenoviral vectors with a CAR ^" -independent entry pathway (Krasnykh et al, Mol. Ther. 2000, 1: 391-405).

It is possible in vitro to inhibit the binding of the virus to its primary receptor by incubating the cells with soluble fiber or CAR, or even with neutralizing antibodies. Based on these observations, teams have developed tools, soluble CAR (or antibody) fused to a specific ligand, which interact with the fiber and target a membrane receptor. This approach makes it possible to target new receptors while neutralizing the natural pathway for Ad5, but its use requires the production of bi-specific molecules, and that the complex [virus-bi-functional molecule] is stable in vivo .

Other teams preferred to modify the adenoviral capsid by genetic engineering

(Krasnykh et al, 2000). This approach is possible because the three-dimensional structures of the three major capsid proteins (base and penton fiber, and hexon) have been identified, and we know how to manipulate adenoviral genomes in E. coli (homologous recombination).

The base of the penton is a pentamer, each monomer of which contains an RGD (Arg-Gly-Asp) motif which allows the attachment of the adenovirus to integrins. This RGD motif, at the apex of 2 α helices, is found within a hypervariable region which makes from 19 (Adl2) to 82 amino acids (Ad2 and 5) depending on the serotypes. This region is therefore a good candidate for the insertion of heterologous peptides in order to modify the tropism of Ad. The penton fiber has a trimeric structure. Each monomer is composed of a rod consisting of a repetition (the number of which varies according to the serotypes) of a pattern of about fifteen residues, and of a C-terminal head. This head of the fiber monomer has a globular structure, formed by 10 β sheets, separated by unstructured loops. Only one of these loops, the HI loop, does not participate in contact with the CAR receptor, and protrudes outside the virion, which makes it possible to insert peptides without altering the trirnérisation of the fiber.

Hexon, the major protein in the adenoviral capsid, is also a trimer. Each monomer contains seven hypervariable regions (HNR1 to HNR7) located within loops where the specific determinants of each serotype are found.

All these HNR regions of the hexon are located in its upper external part, which suggests that modifications can be made there without altering the three-dimensional folding of the molecule.

Different teams have genetically modified the proteins of the viral capsid so as to modify the tropism of the recombinant adenoviruses.

The document Bouri et al, 1999 (Hum Gene Ther. 1999 l; 10 (10): 1633-40) demonstrates that a genetically modified adenovirus which expresses a sequence of seven lysines at the C-terminal end of its fiber transduces the skeletal muscle cells with an efficiency far superior to that of an unmodified type 5 adenovirus (in vitro and in vivo result).

The document Su et al, 2001 (J Vase Res. 2001 Sep-Oct; 38 (5): 471-8) describes an adenoviral vector coding for a chimeric fiber protein allowing it to better transduce smooth muscle cells.

Likewise, Mizuguchi et al, 2002 (Cancer Gene Ther. 2002 Mar; 9 (3): 236-42) describes the construction of an adenovirus comprising a chimeric fiber on which an RGD motif has been inserted. This virus has a transduction capacity 25 times superior to unmodified adenovirus (in vivo experiment on a urine melanoma model).

Mizuguchi et al, 2001 (Gen Ther. 2001 8: 730-5) describes a recombinant adenovirus containing the NGR peptide inserted into the fiber, capable of infecting human glioma cells.

The international patent application published on 07/22/1999 under the number WO 99/36545 (Genzyme) describes adenoviral vectors coding for a modified capsid protein (hexon or fiber) comprising a heterologous ligand which facilitates the binding of adenovirus to the target cell.

The international patent application published on 08/27/1999 under the number WO 00/12738 (Aventis Pharma) describes adenoviral vectors coding for a modified capsid protein (hexon or fiber) comprising a heterologous ligand.

In particular, ligands are inserted into the HVR5 loop of the hexon.

Similarly, Vigne et al, 1999 (J. Virol 1999, 73: 5156-5161) describes the substitution of the HVR5 region of the Ad5 hexon with a heterologous sequence.

The international patent application published on 05/06/1997 under the number WO 97/20051 (Genvec) describes chimeric adenoviral capsid proteins (fiber and hexon) comprising a heterologous peptide sequence.

These genetic modification approaches remain limited by the very small number of peptide ligands identified, capable of modifying the tropism of the adenoviral vectors.

It would therefore be desirable to find new ligands capable of modifying the tropism of the adenoviral vectors in order to target different cell types, in particular cells lacking or deficient in CAR receptors, such as muscle cells and tumor cells. On the other hand, it would be desirable to find new ligands which are capable of modifying the tropism of the adenoviral vectors and of decreasing the specific immune response of the host simultaneously.

By aiming to control the anti-adeno virus immune responses by the insertion of an immunosuppressive peptide CKS-17 (Haraguchi et al, Cell Immunol. 1992 May; 141 (2): 388-97) of retroviral origin in proteins major capsid of adenovirus, the inventors have discovered quite unexpectedly that the viruses obtained have a new tropism: these new viruses are capable of infecting cells lacking or deficient in CAR receptors, while retaining their tropism for CAR-rich cells. In addition, these new viruses, in addition to their ability to infect these different cell types, could be less immunogenic than their predecessors, due to the immunosuppressive nature of the ligand peptide CKS-17 inserted into the capsid of the virus.

Thus, according to a first aspect, the subject of the invention is an adenoviral vector intended to ensure the expression of a DNA sequence of interest in mammalian cells, characterized in that said vector comprises in at least the one of the DNA sequences coding for regions with low structural constraint of capsid proteins, at least one DNA sequence coding for a heterologous peptide comprising the motif - (W / R) XιX D -.

In the present application, the terms "nucleic acid", "polynucleotide", "DNA sequence", "RNA sequence" are used interchangeably to designate a nucleotide sequence.

Likewise, the terms "protein", "peptide" and "polypeptide" are used interchangeably to denote an amino acid sequence.

The term “region with low structural stress” is intended to denote a capsid protein coded by an adenoviral vector a region whose acid sequence amino does not affect the three-dimensional folding of the protein

^• capsid; the heterologous DNA sequence of the invention can be inserted into this region with low structural stress without deleting the amino acid sequence from this region, or it can be inserted after this sequence from this region with low structural stress has been partly or entirely deleted. In the present application, the sequence of the motif - (W / R) XιX D - is SEQ Lu N ° 3. Preferably, the adenoviral vector according to the present invention is characterized in that the heterologous peptide is capable of binding to the receptor for the tumor growth factor β (TGFβR).

There are several types of TGFβ receptors. Type I and II receptors include a transmembrane domain, an extracellular region rich in cysteine, and an intra-cytoplasmic domain. The type III receptor has a large extracellular domain, but has only a small cytoplasmic domain, it presents TGFβ to the type II receptor, but plays no role in signaling. When the type II receptor binds TFGβ, a change in conformation takes place which facilitates the recruitment of the type I receptor to form a heterodimer. The type II receptor, via serine threonine kinase activity, phosphorylates and activates the type I receptor initiating a signal transduction cascade, notably involving the phosphorylation of SMAD 2 and 3, and leading to nuclear transcription regulation events (Hâta et al, Exp Cell Res. 2001 Mar 10; 264 (l): ll l-6). The type II receptor, the main responsible for the binding of TGFβ, has a wide distribution. In mice, this receptor is strongly expressed by skeletal muscles, lungs, heart and to a lesser extent by other organs (Lawler et al, Development 1994, 120: 165-175). In humans, this receptor is described as ubiquitous, and studies have mainly focused on the expression of these receptors in tumors. Thus, the heterologous peptide as described in the present application is preferably capable of binding to the IL-type TGFβ receptor

The TGFβR, preferably TGFβR type II, to which the heterologous peptide as described in the present application is capable of binding may be present on the surface of mammalian cells, such as murine or human cells.

More preferably, the heterologous peptide as described in the present application is capable of binding to human TGFβR type II of sequence SEQ ID No. 2 or of sequence identical to at least 60, 65, 70, 75, 80, 85, 90, 95 or 99% with the sequence SEQ ID No. 2.

Those skilled in the art are capable of identifying the heterologous peptides of the present application which are capable of binding to the receptor for TGFβ.

For example, when the TGFβ receptor is located on the surface of mammalian cells, a person skilled in the art will determine if the heterologous peptide of the present application is capable of binding to the TGFβ receptor by considering the transduction efficiency of said cells. of mammals with the adenoviral vector according to the invention. For example, when the adenoviral vector ^• of the invention is used to transduce mammalian cells deficient or lacking in CAR receptor (CAR cells ^") in vitro or in vivo, it is assumed that the adenoviral vector of the invention is capable of efficiently transduce said cells when the transduction efficiency in vitro or in vivo is at least twice as high as that obtained when CAR ^" cells are transduced with an adenoviral vector not comprising the heterologous sequence described in the present application (see Example 2, “analysis of the infectious power of viruses”, “tests of infectivity of recombinant adenoviruses” for the calculation of the transduction efficiency).

As another example of identification of the binding capacity of the heterologous peptides of the present application with the TGFβ receptor, there may be mentioned the nitrocellulose membrane analysis as described in Example 2 (“analysis of the infectious power of viruses”, “SLOT BLOT”).

The term “adenoviral vector (Ad)” is intended to denote in the present application a vector derived from an adenovirus or from a virus associated with the adenovirus, such as in particular AAV, which is intended to ensure the expression of a sequence of DNA of interest in mammalian cells.

According to a preferred embodiment, the adenoviral vector according to the invention is derived from an adeno-associated virus (AAV). The AAV genome contains open reading frames encoding the Rep (replication) and Cap (capsid) proteins. The nucleotide sequences flanking the coding regions of AAV are inverted terminal repeat (ITR) sequences, which contain palindroin sequences capable of folding to form hairpin structures functioning as primers during initiation. of DNA replication. In addition to their role in DNA replication, ITR sequences play a role in viral integration and packaging of viral nucleic acid in mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158: 97-129).

The AAV capsids have an icosahedral symmetry and are about 20 to 24 nm in diameter. They are composed of three viral proteins (VP1 (87 Kd), VP2 (73 Kd) and VP3 (61Kd), Muzyczka, 1992). The VP3 protein represents 90% of the total virion proteins; VP2 and VP1 are present at around 5% each.

The AAV virus has two modes of infection of a host cell. In the presence of "Helper" virus, the AAV enters the lyric cycle, and the viral genome is transcribed, replicated and packaged in newly formed viral particles. In the absence of the “Helper” virus function, AAV, the AAV genome integrates as a provirus in a specific region of the genome of the host cell, by recombination between the AAV ITRs and sequences of the host cell. . The specific targeting of AAV viral DNA occurs on the long arm of chromosome 19 (Rotin et al, (1990) Proc. Natl. Acad. Sci. USA 87: 2211-2215; Samulski et al., (1991) EMBO J. 10: 3941-3950). This particular feature of AAV reduces the possibility of insertional mutagenesis resulting from the random integration of viral DNA into the coding region of a host gene.

The AAV particle uses cellular receptors to bind and to infect a cell. Recently identified receptors include the heparan sulfate proteoglycan receptor as the primary receptor, and either fibroblast growth factor (FGF) or the integrin αVβ5 as secondary receptors. After binding to the cell, the viral particle follows a receptor-mediated internalization process in clathrin-coated endocytosis cell vesicles.

The AAV vector has properties that make it unique for gene therapy; for example, AAV is not associated with known diseases and is generally non-pathogenic. In addition, AAV integrates into the host chromosome in a site-specific manner (See eg, Kotin et al., (1990) Proc. Natl. Acad. Sci. 87: 2211-2215 and Samulski et al., ( 1991) EMBO J. 10: 3941-3950).

Although the AAV viral vectors are suitable for delivery of genes to target cells, they often have limited tropism for certain cell types. To date, attempts to modify the tropism have involved the introduction of a ligand peptide into the capsid. For example, Girod et al. (1999) Nature Med. 5: 1052-1056) introduced a peptide of 14 amino acids containing the RGD motif in a region of the AAV capsid of serotype 2 to modify tropism. Others have added to the N-ter terminus of VP2 single chain fragments of variable regions of a monoclonal antibody directed against CD34 (Yang et al. (1998) Hum. Gene. Ther. 9: 1929-1937 ). The major limitation of these approaches is that they require additional steps of binding to large molecules such as receptor ligands and antibodies to the virus. This increases the size of the virus as well than the cost of production. In addition, the target particles do not have a homogeneous structure, which can affect the efficiency of gene transfer. For this, there is a ^" need to generate viral vectors having a modified tropism effective for gene transfer.

According to a particularly preferred embodiment, the adenoviral vector according to the invention is derived from an adenovirus. Adenoviruses are eukaryotic DNA viruses that can be modified for the efficient delivery of nucleic acid to various types of mammalian cells. There are at least 49 serotypes among human strains. The vector Ad is preferably a vector

Ad2 or a human Ad5 vector, or an adenovirus of animal origin (international application WO 94/26914, published on 04/06/99, Rhône Poulenc Rorer). Adenoviruses of animal origin which can be used in the present invention include adenoviruses of canine origin (Manhattan or A26 / 61 strain (ATCC VR-800), for example), bovine, murine (eg Mavl, Beard et al ,

1990, Vir. 75, 81), sheep, swine and avian.

The adenoviral vector is preferably defective for replication, i.e. it is not able to replicate autonomously in the mammalian cell. For this, the adenoviral genome is modified. Modification of the viral genome may include, but is not limited to, addition of a DNA segment, rearrangement of a DNA segment, deletion of a DNA segment, substitution of 'a segment of DNA, or the introduction of damaged DNA. A segment of DNA can be as small as a nucleotide and as large as 36 Kbp (ie the approximate size of the adenoviral genome) or, alternatively, can correspond to the total amount which can be " packaged ”in an Ad virion (ie approximately 38 Kpb). Preferred modifications to the adenoviral genome are included in regions E1, E2, E3 or E4. Similarly, an adenoviral vector can be a cointegrate, i.e., the ligation of adenoviral sequences with other sequences, such as sequences from other viruses or from plasmids. Such modifications can be made using genetic manipulation techniques or by treatment with mutagens. In general, the virus which is defective for replication retains the sequences of its genome which are necessary for the packaging of the viral particles. Defective viruses, which have almost no viral genes, can also be used.

The replication-defective recombinant adenoviruses which are used in the present invention can be prepared according to any of the techniques known to those skilled in the art. They can be prepared by homologous recombination between the adenoviral genome and a plasmid which carries the DNA sequence of interest. Homologous recombination is carried out after cotransfection of the adenoviral genome and of the plasmid in Escherichia coli. The recombinant genome obtained is transfected into the transcomplementing cell line which must preferably be: (1) transformable by these elements, and (2) contain the complementation sequences of the part of the genome deleted for obtaining defective adenovirus for replication, preferably in integrated form so as to avoid the risks of recombination.

Examples of transcomplementing cell lines which can be used include, but are not limited to, cell line 293 of human embryonic kidney (Graham et al, J Gen Virol. 1977 Jul; 36 (l): 59-74 ) which contains the left part of the Ad5 genome integrated into its genome, the line PER.C6 (Fallaux et al, Hum Gène Ther. 1998 Sep 1; 9 (13): 1909-17), and cell lines which are capable of complementing the functions E1 and E4.

The general techniques for preparing adenoviral vectors and adenoviral particles for the present invention are well known to those skilled in the art, and are widely described in the literature, such as for example in Sambrook et al, "Molecular cloning: A laboratoiγ manual ”, second edition, 1989, Spring Harbor Laboratoyry Press, New York; Glover et al, "DMA cloning: a practical approach", Vol. I and II, 1985, FRL Press, Oxford; "Nucleic acid hybridization", BD Haines, SJ Higgins. (Eds), 1985, IRL Press; Haines & Higgins, "Transcription and translation: a practical approach", 1984, IRL, Oxford; "Current Protocols in Molecular Biology", F. Ausubel et al (Eds), 1993, Wiley & Sons.

The adenoviral cycle consists of an early phase and a late phase. During the early phase, the El A region is transcribed and translated, which activates the transcription of the E1B, E2, E3 and E4 regions. The transcription of the E2 region peπnet the synthesis of proteins necessary for viral replication, while the proteins of the E4 region allow the stopping of cellular protein synthesis, and favor the export of viral messengers to the cytoplasm. Finally, the proteins of the E3 region set up blocking systems against the body's immune defenses (Benihoud et al, Cur. Op. In Biotechnology 1999, 10: 255-260; Russel et al, 2000).

During the late phase, activation of a single gene (LTU) is observed under the control of the MLP (Major Late Promotor) promoter. The primary transcript obtained generates, by alternative splicing, and use of differential polyadenylation sites, 5 families of late mRNA L1 to L5, coding for the proteins of structure, assembly and maturation of the virion (FIG. 2). These neosynthesized proteins form new virions within which the adenoviral genome is packaged. Then, the virions are released by lysis of the cell membrane (Russel et al, 2000).

As an example of modification of the adenoviral genome to make the adenoviral vector deficient for replication, there may be mentioned, but not limited to, the elimination of the E1 region, coding for transcription factors essential for the initiation of the viral cycle. . This deletion therefore prevents the virus from replicating autonomously and makes it possible to insert a transgene of interest (up to 5.5 kb) without exceeding the critical size of packaging of the viral genome (105% of the genome, or 38 kb) . An additional deletion in the E3 region, which is not essential for the viral cycle, makes it possible to increase the cloning capacity to 7.5 kb. To produce these recombinant ΔE1ΔE3 vectors, it is necessary to use cell lines (lines 293 or 911) transcomplementing the El region (Benihoud et al, 1999, Russel et al, 2000).

Such ΔE1ΔE3 vectors, which can be used in the present invention, have a certain number of advantages. They infect both quiescent and dividing cells. They can be produced at high titers, of the order of 10 ¹¹ to 10 ¹² pfu. l ^"1 (plaque forming unit), enabling gene transfers in vivo. In addition, unlike the retrovirus genome, the genome of the adenoviral vectors remains in episomal form, reducing the risk of insertional mutagenesis. It should be noted, however, that this leads to a progressive loss of expression of the transgene in dividing tissues.

These ΔE1ΔE3 vectors, however, face several limitations. First, during the amplification step in the complementation line, it is possible to observe the emergence of viral particles capable of replicating (RCA, Competent Replication Adenovirus). These particles have reintegrated the E1 region into their genome, following a homologous recombination between the viral genome and the E1 region of the producer cell. They are potentially dangerous for treated patients. This problem of contamination by RCA is currently controlled by minimizing the homologies between the vectors and the propagation line (extension of the deletion

E1 of the adenoviral genome and / or new propagation line).

A second drawback stems from the fact that certain cells are capable in vivo of providing an activity called "El -like" which, by mimicking the function of the El region, induces the transcription of the E2 region and leads to the replication of the adenoviral genome. This E1 activity is responsible for the neosynthesis of early and late viral proteins potentially immunogenic in the host. These vectors are also capable of inducing a strong immune response in the host. We observe not only a cell type response (cytotoxic and helper Lp) which rapidly eliminates the cells expressing the transgene of interest, but also a humoral type response (neutralizing Ab) which renders any subsequent re-administration of the vector ineffective.

All of these problems are partly controlled by the deletion of other early genes (E2 and E4), or even of all adenoviral genes (helper-dependent vectors, Benihoud et al, 1999, Russel et al, 2000).

The adenoviral vector can for example comprise a deletion in the regions E1 and E4. Such vectors are described in international patent applications WO 95/02697 (published on 01/26/1995, Rhône Poulenc Rorer) and WO 96/22378 (published on 03/10/2000, Rhône Poulenc Rorer).

The expression DNA sequence of interest expressed in mammalian cells is intended to denote a DNA sequence which, when it is either transcribed, or transcribed and then translated, into a molecule of interest which can either be an RNA, such as in particular a ribozyme, an antisense RNA or a siRNA, or a protein, makes it possible to obtain a phenotypic change of said mammalian cells.

SiRNAs, or small interfering RNAs, are now well known to those skilled in the art and are described in the literature (see for example Shi Y., Trends Genêt. 2003 Jan; 19 (l): 9-12 ).

Preferably, the adenoviral vector according to the present invention is characterized in that the heterologous peptide is an immunosuppressant peptide of retrovirus.

The term “immunosuppressive retrovirus peptide” is intended to denote a peptide coded by the genome of the retrovirus which exerts an immunosuppressive action, that is to say which decreases or suppresses the host's immune response, the host possibly being the cells that the retrovirus infects.

According to a particular embodiment, the adenoviral vector according to the invention is characterized in that the residues Xi and X ₂ are chosen, independently of one another, from the neutral residues A, V, L, I, G , S and T. Preferably, the residue Xi is G and the residue X ₂ is L.

According to yet another embodiment, the adenoviral vector according to the invention is characterized in that the heterologous peptide contains at most 24 amino acid residues. Preferably, the heterologous peptide contains between 4 and 20 amino acid residues. More preferably, the heterologous peptide contains between 7 and 18 amino acid residues. Most preferably, the heterologous peptide contains 17 amino acid residues.

Preferably, the adenoviral vector according to the present invention is characterized in that the immunosuppressive peptide of retrovirus is the peptide CKS-17 of sequence SEQ ID No 1 or any peptide of sequence identical to at least about 60% to said sequence SEQ ID No. l. Preferably, the retrovirus immunosuppressive peptide is any peptide of sequence identical to at least about 64, 70, 76, 82, 88, 94% to said sequence SEQ ID No. 1.

The peptide CKS-17 of sequence SEQ ID No. 1 is a synthetic peptide homologous to a highly conserved region of the retroviral transmembrane protein. It exerts an immunosuppressive action which results in an inhibition of the production, in vitro, in particular of interleukin 1, interleukin 2 and interferon γ by the lymphocytes and the murine monocytes (Haraguchi et al, Cell Lnmunol. 1992 May ; 141 (2): 388-97).

The DNA sequence coding for the heterologous peptide according to the invention can be inserted into at least one of the sequences of the adenoviral vector coding for an adenovirus capsid protein in regions with low stress structural, among which there may be mentioned, but limited to, the regions with low structural stress of the hexon, of the fiber or of the base of the penton, of the pVL protein pVLII or pIX.

Thus, according to one embodiment of the invention, the adenoviral vector according to the invention is characterized in that the capsid protein is chosen from hexon, the fiber or the base of penton, the protein pVI, pVIII or pLX .

The DNA sequence coding for the heterologous peptide can be included at least in one of the sequences coding for regions, with low structural stress of capsid proteins. Preferably, the DNA sequence coding for the heterologous peptide of the invention is inserted into a single region with low structural stress. It can also be inserted into at least two regions with different low structural stresses of the same capsid protein or of two or more different capsid proteins.

According to a particular embodiment, the adenoviral vector according to the invention is characterized in that the DNA sequence coding for the region with low structural stress of a capsid protein is deleted or partially deleted, and the DNA sequence encoding the heterologous peptide is inserted into the deletion site.

In general, it will be determined whether the DNA coding for the region with low structural stress should be entirely or partially deleted, in particular as a function of the heterologous peptide of the invention (size and three-dimensional structure of the heterologous peptide) and of the region with low stress. structural.

As an example of a region with low structural stress of a capsid protein encoded by the adenoviral vector, there may be mentioned, but not limited to, a hypervariable region of the penton which makes from 19 (Ad12) to 82 amino acids (Ad2 and

Ad5) according to serotypes. This region contains an RGD motif at the apex of two alpha helices. We can also cite the HI loop of the fiber, or the seven hypervariable regions (HVR1 to HVR7) of the hexon.

Preferably, the adenoviral vector according to the invention is characterized in that when the capsid protein is hexon, the region with low structural stress is the hypervariable region 5 (HVR5). Even more preferably, the DNA encoding the hypervariable region 5 (HVR5) is entirely deleted.

According to another embodiment of the invention, the adenoviral vector is characterized in that when the capsid protein is the fiber, the region with low structural stress is the HI loop. Preferably, the HI loop is deleted or partially deleted.

According to a new embodiment, the adenoviral vector according to the invention is characterized in that the heterologous peptide further comprises at least one adapter located in N-ter or in C-ter.

The term “adapter” is intended to denote, in the present application, an amino acid or an amino acid sequence located in N-ter or in C-ter of the heterologous peptide which makes it possible to obtain correct three-dimensional folding of the amino acid sequence of the protein of capsid comprising in at least one of its regions with low structural stress the heterologous peptide according to the invention.

When the adapter is an amino acid sequence, this can include 1 to 5 amino acids, preferably 3 amino acids.

Preferably, the adenoviral vector according to the invention is characterized in that the heterologous peptide comprises an adapter located in N-ter and an adapter located in C-ter. According to a particular embodiment of the invention, when the region with low structural stress into which the heterologous peptide of the invention is inserted is the HVR5 region of the hexon, the adapters are the amino acid G (Glycine) in N-ter of the heterologous peptide and the amino acids LGG (Leucine-Glycine-Glycine) in C-ter of the heterologous peptide.

According to a preferred embodiment, the adenoviral vector according to the present invention is characterized in that the DNA sequence of interest is linked in an operational manner to expression control sequences.

The expression control sequences are intended to denote the expression of the transcription and translation control sequences. Such sequences are well known and used by those skilled in the art. It is regulatory DNA sequences, such as promoters, enhancers, terminators and the like, that allow expression. of a DNA sequence of interest in a host cell. The expression of the DNA sequence of interest can be carried out under the control of any promoter / enhancer known to those skilled in the art, provided that these regulatory elements are functional in the chosen target cell for expression. Promoters that can be used to control expression of the transgene include, but are not limited to, the CMV promoter, the SV40 promoter (Benoist & Chambon, Nature. 1981 Mar 26; 290 (5804): 304-10) , the RSV promoter (Yamamoto et al, Cell. 1980 Dec; 22 (3): 787-97), the herpes thymidine kinase promoter (Wagner et al, Proc Natl Acad Sci US A. 1981 Mar; 78 (3 ): 1441-5), the metallothionein gene regulatory sequences (Brinster et al, Nature. 1982 Mar 4; 296 (5852): 39-42), and the animal transcription control regions which have specificity tissue, such as the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al, Cell. 1984 Oct; 38 (3): 639-46), the gene control region l insulin, which is active in pancreatic beta cells (Hanahan et al, Nature 1985 May 9-15; 315 (6015): 115-22), the control region of the immunoglobulin gene, which is active in cells lymphoids (Grosschedl et al, Cell. 1984 Oct; 38 (3): 647-58). The expression "operably linked to" means that the DNA sequence of interest and the expression control sequences are related to each other in a way that allows them to function as desired.

Preferably, the adenoviral vector according to the invention is characterized in that the DNA sequence of interest codes for an anti-angiogenic factor, thymidine kinase, the p53 protein, a transgene involved in a muscular disease, the gene coding for the transmembrane regulator involved in cystic fibrosis

(CFTR gene). Preferably, the anti-angiogenic factor is angiostatin or any other fragment of plasminogen, endostatin, canstatin, angiotensinogen or any other fragment derived from angiotensinogen. According to another preference, the transgene involved in a muscular disease codes for dystrophin.

When the DNA sequence of interest expresses a protein, this protein can also comprise on the N-ter side a “signal” sequence of secretion of the protein also expressed by the DNA sequence of interest.

According to a particular embodiment, the adenoviral vector according to the invention is characterized in that the DNA sequence E1 of the adenoviral vector is modified so as to allow replication of said vector only in tumor cells.

The modification can consist, for example, of a substitution or a total or partial deletion of the E1 DNA sequence. Such a modification of the E1 DNA sequence of the vector or of the adenoviral genome is well known to those skilled in the art. , and is described in particular in Barnes MN et al, Mol Cancer Ther. 2002 Apr; l (6): 435-9. Review; Lamfers ML et al, Cancer Res. 2002 Oct

15; 62 (20): 5736-42). It has been demonstrated that this sequence is modified, the adenoviral vectors obtained are capable of infecting tumor cells. According to another particular embodiment, the adenoviral vector according to the invention is characterized in that it exhibits a tropism for cells deficient or lacking in receptors common to Coxsackieviru ^' s B3 and to adenovirus (CAR receptors).

In the present application, CAR cells will be designated ^" mammalian cells lacking or deficient in CAR receptors. Conversely, cells which are not lacking or deficient in CAR receptors will be designated as CAR ⁺ cells.

Examples of mammalian cells lacking or deficient in CAR receptor (CAR cells ^") include, without limitation, tumor cells, such as glioma cells, melanoma, carcinoma, cancer of the lung, of the uterus, breast or ovary, prostate tumor, or lymphocytes, fibroblasts, lung epithelial cells, skeletal muscle, smooth muscle or myocardial cells.

As examples of mammalian cell lines lacking or deficient in CAR receptors (CAR cells ^" ), mention may be made, but not limited to, the lines CHO, L929, PC-3, LLC, B16F10, RCC law, RCC Aury, U118, RD, L6 and C2C12.

According to a particular embodiment, the adenoviral vector according to the invention is characterized in that it also comprises one or more modifications, which allows the capsid protein thus obtained to have a reduced or eliminated interaction with at least one of the primary adenovirus receptors.

In fact, the adenoviral vector of the invention, if it is capable of binding to TGFR located on the surface of mammalian cells, retains its ability to infect CAR ⁺ cells. In the present application, the receptors with which the wild-type adenovirus is capable of interacting will be designated as primary adenovirus receptors. Such receptors are generally present on the surface of mammalian cells, but they can also be present in solution in recombinant soluble form. As examples of primary adenovirus receptors, mention may be made of essential primary receptors such as the CAR receptor, heparan sulfate and the integrins!

The modification which allows the capsid protein thus obtained to have a reduced or eliminated interaction with at least one of the primary receptors can be of all types insofar as the adenoviral vector according to the invention modified in this way has a preferential tropism for CAR cells - and little or no tropism for CAR + cells.

The modification which allows the capsid protein thus obtained to have a reduced or eliminated interaction with at least one of the primary receptors of the adenovirus can consist in the ablation of the adeno viral fiber. The modification can also consist in replacing the C-teπninale part of the fiber by a targeting ligand however comprising a trimerization motif, in order to preserve the integrity of the viral capsid.

The modification can also be based on the mutagenesis of the fiber residues involved in the interactions with CAR.

Preferably, the adenoviral vector according to the invention is characterized in that the modification in at least one of the DNA sequences coding for the capsid proteins allows them to have a reduced or eliminated interaction with at least one of primary adenovirus receptors, is a deletion of the RGD motif present in the base of the penton.

According to yet another preference, the modification is inspired by the existence of different adenoviral serotypes which differ in the size of the fiber and their Ability to link or not the RAC. Thus, one can consider reducing the size of the stem of the Ad5 fiber, or replacing the fiber with that of another serotype which does not bind the CAR (under group B, Ad3) (Dechecchi et al, 2000, 268 : 382-90). For example, an Ad5 vector can be pseudotyped by an Ad3 fiber (Vigne et al.,. Gene Ther. 2003, 10: 153-62).

Thus, according to a preferred embodiment, the adenoviral vector according to the invention is characterized in that the modification consists in the substitution of the DNA sequence coding for the adenoviral fiber of a given serotype by the DNA sequence coding for an adenoviral fiber of another serotype. Preferably, the Ad5 fiber is replaced by the Ad3 fiber.

According to a particularly preferred embodiment, the adenoviral vector according to the invention is characterized in that the modification consists in the substitution of the Ad5 fiber by the Ad3 fiber and in a deletion of the RGD motif present in the base of the penton.

According to another aspect, the subject of the invention is a method for obtaining infectious adenoviral particles in production cells, said method comprising a step of transformation of said production cells by the adenoviral vector according to the present invention.

The terms “adenoviral particle”, “virion” or “adenovirus” are used interchangeably in the present application.

The term “production cell” is intended to denote in the present application any cell which makes it possible to obtain infectious adenoviral particles when it is transformed by the adenoviral vector of the invention. When the adenoviral vector is defective for replication, the cells producing infectious adenoviral particles, of the present application are the cells which provide the complementation functions (viral proteins) which are absent from the vector. adenovirus. Such cells, also called transcomplementation cells, are described above.

The subject of the invention is also the infectious adenoviral particles obtained by the method of production according to the present invention.

According to yet another aspect, the subject of the invention is a method of modifying the tropism of an adenoviral vector, comprising the insertion, in at least one of the DNA sequences coding for regions with low structural stress of proteins of capsid, of at least one DNA sequence coding for a heterologous peptide according to the invention.

Preferably, the method of modifying the tropism of an adenoviral vector according to the present invention is characterized in that the insertion step is preceded by a deletion step of all or part of the DNA sequence coding for the region with low structural stress of a capsid protein.

Preferably, the method of modifying the tropism of an adenoviral vector according to the present invention is characterized in that the capsid protein is chosen from hexon, fiber or base of penton, protein pVI, pVIII or pIX.

More preferably, the method of modifying the tropism of an adenoviral vector according to the present invention is characterized in that, when the capsid protein is hexon, the region with low structural stress is the hypervariable region 5 (HVR5) .

Most preferably, the method of modifying the tropism of an adenoviral vector according to the present invention is characterized in that the DNA sequence coding for the hypervariable region 5 (HV 5) is deleted.

According to another embodiment, the method of modifying the tropism of an adenoviral vector according to the present invention is characterized in that, when the capsid protein is the fiber, the region with low structural stress is the HI loop.

According to a particular embodiment, the method for modifying the tropism of an adenoviral vector according to the present invention is characterized in that the heterologous peptide according to the invention also comprises at least one adapter located in N-ter or in C- b.

According to a preferred embodiment, the method for modifying the tropism of an adenoviral vector according to the present invention is characterized in that the heterologous peptide as defined in any one of claims 1 to 6 comprises an adapter located in N- ter and an adapter located in C-ter.

According to another aspect, the subject of the invention is an in vitro method allowing preferential expression of a DNA sequence of interest in a target mammalian cell comprising bringing said cell into contact with infectious adenoviral particles according to the invention, or with the adenoviral vector according to the invention or obtained according to the method of modification of the tropism according to the invention.

Preferably, the in vitro method according to the present invention is characterized in that the mammalian target cell is deficient or lacking in CAR receptors.

Most preferably, the in vitro method according to the present invention is characterized in that the cell deficient or lacking in CAR receptors comes from a cell line chosen from the CHO, L929, PC-3, LLC, B16F10 lines, RCC law, RCC Aury, U118, RD, L6 and C2C12.

According to another aspect, the subject of the invention is a pharmaceutical composition comprising an adenoviral vector according to the present invention and an appropriate pharmaceutical excipient. The pharmaceutical composition according to the present invention can be manufactured according to various methods well known to those skilled in the art, depending on the mode of administration; this mode of administration includes administration. systemic and localized administration (such as for example aerosol administration, instillation, and oral administration). For systemic administration, injection is preferred, for example subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intrathecal, intracardiac (such as for example pericardial), intratumoral, intravaginal, intrapulmonary, intranasal, intratracheal, intravascular, intraarterial , intracoronary, or intracerebroventricular. Intramuscular or intratumoral injection is a preferred mode of administration. The administration can be carried out in a single dose or in a repeated dose one or more times after a determined time interval.

The composition according to the invention can be supplied in various forms, in particular in liquid form (for example aqueous).

The composition according to the invention also comprises an acceptable pharmaceutical excipient for the administration of the adenoviral vector according to the invention to a human or animal body. The excipient is preferably suitable for injection or is a non-toxic diluent for the human or animal organism at the dosage and the concentration used (see for example Remington's

Pharmaceutical Sciences, last Ed., Mack Publishing Co). It is preferably isotonic, hypotonic or weakly hypertonic, and has a relatively weak force, such as for example that of a sucrose solution. In addition, it may contain any suitable solvent liquids, aqueous or partially aqueous, comprising sterile, pyrogen-free water, a dispersion medium, a coating suspension, and their equivalents, or diluents (for example Tris buffer -HCl, acetate, phosphate), emulsifiers, solubilizers, emulsifiers, or adjuvants. The pH of the pharmaceutical composition is appropriately adjusted. We can cite for example, for a composition injectable, isotonic saline solutions which are preferably buffered at physiological pH (such as saline solutions with phosphate buffer, Tris buffer, mannitol, dextrose, glycerol containing or not containing proteins such as human serum albumin) . As representative examples of diluents, mention may be made of the buffer containing sucrose (for example 1M sucrose, 150 mM NaCl, ImM M9CI25 54 mg / L Tween 80, 10 mM Tris pH 8.5) and the buffer containing mannitol (for example 10 mg / ml mannitol, 1 mg / ml HSA, 20 mM Tris pH 7.2 and 150 mM NaCl).

In addition, the pharmaceutical composition according to the invention can comprise one or more stabilizing solutions such as lipids (cationic lipids, liposomes), nuclease inhibitors, hydrogels, hyaluronidase, collagenase, polymers, chelating agents. , so as to prevent degradation of the adenoviral vector in the human or animal body and / or to improve administration to the host cell. The composition may also include substances capable of facilitating the transfer of genes for specific applications, such as a polylysine and lactose gel complex to facilitate administration by the intraarterial route (Midoux et al., Nucleic Acid Res. 21 (1993), 871-878).

Preferably, the pharmaceutical composition according to the invention is intended for targeting mammalian cells deficient or lacking in CAR receptors.

According to another aspect, the subject of the invention is the use of the adenoviral vector according to the present invention for the manufacture of a medicament.

Preferably, the use according to the invention is characterized in that the medicament is intended for targeting mammalian cells deficient or lacking in CAR receptors. Preferably, the drug is intended to target CAR cells ^" which have an abnormality compared to the same healthy CAR cells ^" . The anomaly may be a different phenotype of the cell CAR ^"healthy example following the dysfunction of the expression of a gene (overexpression, underexpression, nonexpression ...).

Such an anomaly present in cells deficient or lacking in CAR receptors (CAR ^" ) can be the cause of a disease.

More preferably, the use according to the invention is characterized in that the mammalian cells deficient or lacking in CAR receptors are tumor cells, such as glioma, melanoma, carcinoma, lung cancer cells, uterus, breast or ovary, prostate tumor, bladder tumor, or lymphocytes, fibroblasts, lung epithelial cells, skeletal muscle cells, smooth muscle or myocardium.

The adenoviral vector according to the invention, and the medicament which is derived therefrom, is intended to prevent or to treat any disease present following an abnormality of the CAR cells ^" , such as for example, but not limited to, a cancer, lung disease, a disease related to a dysfunction of the immune system or muscle disease.

Most preferably, the use according to 1 invention is characterized in that the medicament is intended for preventing or treating a subject suffering from breast, uterus, prostate, lung, skin, ovary, colorectal cancer, leukemia, cystic fibrosis or myopathy.

According to another aspect, the subject of the invention is a recombinant adenoviral capsid protein comprising the insertion into at least one region with low structural stress of at least one heterologous peptide as defined in the present application.

According to yet another aspect, the invention relates to a mammalian cell transformed with an adenoviral vector according to the present invention. According to a last aspect, the subject of the invention is a method of treatment of a subject suffering from a disease present following an abnormality of CAR cells ^" , said method, comprising the administration to said subject of the adenoviral vector according to the invention, infectious adenoviral particles according to the invention or the pharmaceutical composition according to the invention. Preferably, said disease is cancer of the breast, uterus, prostate, lung, skin, ovary, colorectal cancer, leukemia, cystic fibrosis or myopathy.

The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope.

LEGENDS OF FIGURES

Figure 1: Structure of the adenoviral capsid.

The right part, corresponding to the enlargement of the penton, indicates the areas of interaction with cellular receptors.

Figure 2: Organization of the adenoviral genome.

The two black boxes at the ends represent the LTRs. The early genes are designated by the letter E for Early, and the late gene by the letter L for Late.

Figure 3: Modification of the hexon of a recombinant adenovirus by double homologous recombination in E. colL

Figure 4: Analysis of the infectious power of recombinant adenoviruses for β-galactosidase, with wild capsid (AE18) or modified in hexon (SE2) by insertion of the peptide CKS-17.

CAR ⁺ (Hela) or CAR ^" (L929) cells are infected at different MOIs. The expression of β-galactosidase in transduced cells is evaluated by colorimetry, after fixation of the cells, or quantified on cell lysates by luminometry (activity β-Gal in RLU.μg ^"1 protein).

Figure 5: Specific binding of the SE2 virus on the TGFβRII.

Different concentrations of recombinant TGFβRII (1 μg, 0.5 μg and 0.25 μg), and of soluble CAR (0.5 μg), are deposited on the membrane. The specific binding of viruses, SE2 or AE18, is revealed by immunodetection using anti-Ad Ab.

Figure 6: Biochemical characterization of soluble receptors.

Western blots of Hela supernatants not infected (Ni.) Or infected with recombinant Ad / TGFβRII-Fc (A) or Ad / CAR ^" Fc (B) Ad, followed by immunodetection with anti-receptor Ab (TGFβRII or CAR) or anti-IgGl.

Figure 7: Purified TGFβRII-Fc protein.

SDS-PAGE gel electrophoresis, followed by Coomassie blue staining of the purified TGFβRII-Fc protein by affinity chromatography on a protein A column. Figure 8: Capacity of the SE2 virus [Hexon / CKS-17] at infect kidney tumor cells. The primary culture Aury (RCC) is infected with different MEs. The expression of β-galactosidase is evaluated, after fixation of the cells, by colorimetry, or quantified on cell lysates by luminometry (RLU.μg ^"1 of proteins). Figure 9: Expression of primary Ad5 receptors and TGFβRII on the surface of different lines.

The level of receptor expression (integrins αV, αγβ ₃ , α, γβ ₅ , TGFβRII and CAR) is analyzed, by flow cytometry, after labeling the cells with anti-receptor Ab (CAR and Integrins) coupled to FITC, or biotinylated TGFβ (TGFβRII). Figure 10: Ability of recombinant capsid modified (SE2) or wild type (AE18) Ad to infect tumors in vivo.

Pre-established tumors (LLC cells) are injected with 2.10 ⁹ pV of SE2 virus [Hexon / CKS-17] or of AE18 control virus (wild capsid). (A) The expression of β-galactosidase in the transduced cells is observed on D + 3 pi., Under a microscope (X50) on sections after immunodetection, or (B) quantified (RLU.μg ^{" l} ) on D + 2 pL, in tumor lysates. The points correspond to the individual tumor results and the bars to the means. (A) and (B) come from independent experiments. Figure 11: Capacity of the SE2 virus [Hexon / CKS-17] at infect muscle lines.

L6 and C2C12 cells are infected at different MOIs. The expression of β-galactosidase in the transduced cells is evaluated, after fixation, by colorimetry or quantified on cell lysates by luminometry (RLU.μg ^"1 ). Figure 12: Infectious power of different recombinant Ad mutants for the LacZ gene. Hela and L929 cells are infected at different MOIs by the viruses [Hexon / CKS-17] AE95SE2 and SE2, and the control viruses AE95 and AE18. The expression of β-galactosidase in transduced cells is evaluated on whole cells by X-gal staining Figure 13: Modification of the capsid proteins allowing the natural interactions between Ad5 and its receptors to be broken.

The SE2 virus [Hexon / CKS-17] has been modified in order to delete the RGD motif from the base of the penton and / or pseudotyped by the Ad3 fiber. EXAMPLES

EXAMPLE 1: MATERIALS

CDNA:

The human TGF-β type II receptor and that of the CAR receptor cDNAs were provided respectively by Dr X.-F. Wong (Duke University, USA) and Dr. E. Vigne (Aventis, Vitry).

Vectors:

- Expression plasmid:

SE17: this plasmid, derived from pCEP4 (Invitrogen), has the cDNA sequence coding for the constant part (Fc) of murine immunoglobulins of the IgG1 type, cloned under the control of the immediate early promoter of the cytomegalovirus (CMV) and of the late polyadenylation site of the virus SV40. This vector is used to construct soluble receptors.

Shuttle vector

pCA350: this vector, developed in the laboratory, carries the kanamycin resistance gene (Km ^R ), a multiple cloning site as well as the left part (5 ') of the genome of the human adenovirus type 5 (Ad 5 ), from 1TTR 5 'to the pIX gene.

In this sequence, the E1 region (position 382 to 3513 of Ad5) is eliminated to allow the insertion of an expression cassette. The origin of R6Kγ conditional replication restricts replication of this plasmid to E. coli bacteria expressing the pir (pir ⁺ ) gene.

pSE2: this plasmid contains the sequences 19244 to 19643 and 19686 to 20084 of PAd5 which border the hypervariable region No. 5 (HVR5) of the hexon. Between these sequences is inserted the sequence of the retroviral peptide CKS-17. This plasmid shuttle also includes the origin of replication colEl, the kanamycin resistance gene (Km ^R ) and the sacB gene, the product of which metabolizes sucrose into a compound toxic to E. coli (Vigne & al., 1999).

- Recombination vector:

pOSE1700: this vector contains the tetracycline resistance gene (Tet ^R ), the origin of replication RK2 and the right part (3 ') of the genome of Ad 5, of the sequence from pLX to 1TTR 3' ( Dr. J.-J. Robert, Aventis). It also has a 2kb deletion in the E3 region. The pLX sequence allows homologous recombination between the shuttle vector pCA350 and the vector ρOSE1700. Thus, the genome of Ad5, deleted from the El and E3 regions, with the transgene of interest inserted in the El region is reconstructed. This new plasmid is called pAd.

- Adenoviral episomes:

AE18c: episome containing the genome of Ad 5 ΔE1ΔE3, in which the lacZ gene is cloned in place of the El region under the control of the “immediate early” promoter of the cytomegalovirus (CMV) and of the late polyadenylation signal of the SV40 virus. It contains a tetracycline resistance marker (Tet ^R ) (Vigne et al,

1999).

SE2c: episome identical to AE18, modified by homologous recombination to insert the retroviral peptide CKS-17 into the HVR5 region of the hexon.

AE74c, DB6c and AE95c are adenoviral episomes derived from AE18c. They respectively contain a deletion of the RGD motif from the base of the penton, the replacement of the Ad5 fiber by the Ad3 fiber, or these two modifications combined (collaboration with Dr E. Vigne, Aventis, Vitry). Recombinant adenoviruses:

AE18 and SE2 are ΔE1ΔE3 viruses obtained from the AE18c and SE2c episomes. SE2 contains the retroviral peptide CKS-17 inserted into the hexon. The experiments presented below were carried out using different stocks of AElδ (2 stocks) and SE2 (4 stocks).

Oligonucleotides:

Amplification of the extracellular domain of receptors

The oligonucleotides framing the cDNAs of the TGFβRII and CAR receptors were synthesized and then purified by HPLC (ESGS, Evry). Their use in PCR reactions makes it possible to obtain sequences coding for the extracellular domain of receptors.

Specific oligonucleotides Target cDNA cDNA produced

SE50A: SEQ IDN ° 4 TGFβRπ TGFβRH Human SE50B: SEQ IDN ° 5 Extra-cellular domain

SE51A: SEQ IDN ^O 6 CAR CAR Human SE51B: SEQ ΓDN ° 7 Extra-cellular domain

These oligonucleotides present, at the start of the sequence, a unique enzyme restriction site (underlined) which facilitates the cloning of the amplified fragment in the expression vector SE17.

Sequencing or PCR analysis of expression vectors:

Oligonucleotide Nucleotide sequence Targeted sequence CMVGi SEQ ID N ° 8 Promoter CMV SE17A SEQ IDN ° 9 Start sequence Fc IgG IgGASl SEQ ID N ° 10 Fc IgG The oligonucleotides SE50A, SE50B, SE51A and SE51B described above, were also used for sequencing.

Screening of recombinant adenoviral genomes:

Oligonucleotide Nucleotide sequence Targeted sequence Hex SEQ ID No. Π ^' hexon SE2B SEQ ΓDN ° 12 CKS-17

Bacterial strains:

XLl blue: strain of E. coli not expressing the DNA repair enzyme RecA (RecA ^" ), used for the cloning of expression vectors, and the amplification of shuttle plasmids which have the colΕl origin.

Texl: E. coli recA ^" , expressing the pir protein (Pir ⁺ ), which allows the replication of shuttle plasmids which have the R6Kγ origin.

G4977: E.coli recA, not expressing polA (Pol T), which does not allow replication of plasmids having the colΕl origin. It is used for homologous recombination steps during the construction of adenoviral episomes (pAd).

JM83: E.coli recA, Pir ^" , which does not allow replication of the plasmids having the R6Kγ origin. It is used for the homologous recombination step during the construction of the pAds.

Top 10: E.coli recA ^" , used to amplify adenoviral episomes (pAd).

Cell lines:

Cell types of various origins have been used. The cells were cultured in RPMI 1640 medium, Minimum Eagle Medium (MEM), Dubelcco's MEM (DMEM) or F12 (HAM) supplemented with 10% fetal calf serum (LifeTechnologies ™), or as recommended by ATCC or ECACC.

Cells 293 (ATCC CRL-1573): line derived from human embryonic kidney cells modified by the insertion of DNA fragments (position 1 to 4344) from Ad

.5. It is thus capable by transcomplementation of providing the E1 activity necessary for the amplification of the adenoviruses ΔE1ΔE3.

911 cells: line derived from human embryonic retinoblasts modified by transfection of a DNA fragment containing regions 79 to 5789 of the Ad 5 genome. This line is therefore also capable of producing the proteins of the El region (Benihoud and al, 1996).

HeLa cells (ATCC CCL-2): line from a human tumor of the cervix used for the production of recombinant proteins.

The other cell lines and primary cultures used in the experiments are described in Table I.

ATCC or ECACC cell lines Histological type Species

L929 CCL-1 Fibroblast Mouse

CHO CCL-61 Hamster Ovary

PC-3 CRL-1435 Male Prostate

LLC CRL-1642 Lung Mouse

B16-F10 CRL-6475 Melanoma Mouse

RCC Law E. Angevin, IGR, Villejuif Kidney tumors Male Aury

U118 HTB-15 Glioma Man

RD CCL-136 Male Rhabdomyosarcoma

L6 CRL-1458 Myoblast - Skeletal muscle Rat

C2C12 CRL-1772 Myoblast - Skeletal Muscle Mouse Table I: Primary cultures and cell lines

Antibodies and Serums

Primary antibodies:

Antibody Specificity Origin Isotype Coupling Reference L5 viral particle Rabbit polyclonal None Vine et al 1999 1070-05 IgGl mouse Goat Polyclonal HRP SouthernBiotech AF-241-NA TGFβRII human Goat IgG None R&D Systems sc-10313 CAR human Goat IgG None Santa Cruz Upstate RmcB CAR human IgGl mice None biotechnology AMF7 Human α _v integrins IgGl FITC mouse Beckman-Coulter MAB 1976F Human α _v β ₃ integrins Human IgGl FITC Chemicon MAB1961F Human ocy integrins IgGl FITC Chemicon 555748 control IgGl FITC mouse Beckton-Dickinson Secondary Antibodies

Antibody Specificity Origin Isotype Coupling Reference M35201 IgG + M mouse Goat F (ab ') ₂ FITC CalTag 605-703-002 IgG goat Donkey IgG HRP Rockland

Recombinant proteins:

Recombinant proteins Origin Characteristics Reference

CAR extra cellular domain Recombinant Human Dr Curiel, Alabama

TGFβRII (241-R2) Recombinant Human R&D Systems

TGFβ-biotin Human Recombinant R&D Systems

Human TGF / 3RII: recombinant protein formed from the 159 amino acids of the extracellular domain of the type II receptor to TGF- / 31 (Lin & al., 1992). The protein contains 136 amino acids, after cleavage of 23 a. at. constituting the signal peptide (R&D Systems).

EXAMPLE 2: METHODS Construction of shuttle plasmids:

- Construction of soluble receptors: TGF ^ RII-Fc and CAR ^' Fc:

Construction of cDNAs coding for receptor antagonists:

To obtain the cDNA of the antagonists, the sequences corresponding to the extra-cellular domains of the TGFβRII, or of the CAR receptor, were amplified using different oligonucleotides (0.5 μM) (cf. table example 1, "oligonucleotides", "Amplification of the receptor extracellular domain") by 30 PCR cycles (denaturation 94 ° C, pairing 60 ° C, elongation 72 ° C) with 1U of DNA polymerase (DyNAzyme EXT, Finnzymes) in the presence of 0.2 mM dNTP, MgCl ₂ l, 5mM. The ^1st PCR cycle is paired at 50 ° C. These PCRs were produced from plasmids containing the complete TGFβRII sequence or that of the extracellular domain of CAR.

An aliquot of the amplification is analyzed on. agarose gel, the rest of the preparation is freed from dNTP (QlAquick PCR purification, Qiagen) and digested with suitable restriction enzymes to release ends compatible with the expression vector. These digested amplification products are deposited on agarose gel and then eluted (QlAquick Gel Extraction, Qiagen) and quantified by electrophoresis on agarose gel.

Linearization of the plasmid:

The vector containing the Fc sequence of the murine IgG1s in the CMV-polyA expression cassette is linearized by the restriction enzymes Bam HI and Xho I (1U / μg of DNA). The vector is then dephosphorylated by calf intestinal phosphatase (CLP lOU / reaction) in order to prevent its recircularization and to promote ligation with the digested PCR fragment. After electrophoresis on agarose gel, the strip corresponding to the linearized vector is cut to avoid contamination by the circular vector. The plasmid DNA is purified (QlAquick Gel Extraction, Qiagen) then analyzed and quantified after electrophoresis on agarose gel.

Ligation and transformation:

For the construction of soluble receptors, the corresponding PCR fragment is ligated with the linearized plasmid SE17 (Amp ^R ), overnight at 16 ° C with a unit of phage T4 DNA ligase (Biolabs) in the presence of ATP 1 WMO. An aliquot of the ligations is introduced into XL1 -Blue bacteria by electroporation (25μF, 200Ω, 12.5 kv / cm ² ). After a one hour incubation at 37 ° C with shaking, permitting the expression of the ampicillin resistance marker, the bacteria are spread on Luria Bernati medium (LB) supplemented with ampicillin (LB-Ampi 50 μg / ml) and incubated overnight at 37 ° C. Screening of transformants:

The bacterial colonies obtained are amplified overnight at 37 ° C. with stirring in LB-Ampi medium (50 μg / ml). The plasmid DNA is prepared from the bacterial cultures by alkaline lysis followed by purification on an ion-exchange column fixing the DNA (Kit Wizard Plus SV minipreps, Promega). The plasmids obtained are analyzed by enzymatic digestion (framing and orientation) to verify the insertion of the fragment.

Production of expression plasmids:

For each cloning, two recombinant plasmids are amplified in LB-Ampi medium overnight at 37 ° C. with stirring. The DNA is extracted by alkaline lysis followed by purification on an ion exchange column (Qiafilter Plasmid midi kit,

Qiagen). The concentration and the purity of the preparation are determined by measuring the absorbance at 260nm and 280nm. The recombinant plasmids are checked by enzymatic digestion then by sequencing using oligonucleotides (ESGS, Evry).

Obtaining the recombinant shuttle plasmid

After enzymatic digestion with the Notl and Sali enzymes, the expression cassette [CMV-antagonist-polyA] is isolated from the expression plasmid (SE17 containing CAR ^' Fc or TGFβRII-Fc) on agarose gel, then eluted following protocol

QlAquick Gel Extraction (Qiagen). In parallel, the shuttle vector pCA350 (Km ^R ) is linearized between ITR-ψ and the pIX sequence by the same enzymes, then dephosphorylated. An aliquot of the DNA corresponding to the purified expression cassette [CMV-PCR product-polyA] is ligated with the shuttle vector. The ligation is introduced by electroporation into Texl bacteria. After an incubation of hi at 37 ° C allowing expression of resistance to kanamycin, the bacteria are spread on an LB-Kanamycin dish (LB-K 50 μg / ml) and incubated at 37 ° C overnight.

The screening of the transformants and the quantitative preparation of the DNA of the shuttle plasmid are carried out in an identical manner to the expression vectors, only the selection medium used varies (LB-Km 50 μg / ml). When the enzymatic digestions do not make it possible to discriminate the recombinant plasmids, amplification by PCR (94 ° C, 50 ° C, 72 ° C) with 1.25U of Taq DNA polymerase (Promega) in the presence of 0.2 mM dNTP, MgCl ₂ l, 5 mM and 0.5 μM of oligonucleotides (cf. table example 1, “oligonucleotides”, “sequencing or PCR analysis of expression vectors”) is carried out directly on an isolated colony or on plasmid DNA. The oligonucleotides used surround the cloning site and thus make it possible to identify the clones having inserted the expression cassette in the orientation corresponding to its transcription from 1TTR 5 ′ to pLX.

Construction of recombinant adenoviral genomes (backbones):

Two methods based on homologous recombination make it possible to construct, by modifying them, adenoviral genomes. A first rapid method uses simple homologous recombination but only allows modification of the 5 ′ part of the Ad genome, which contains the transgene (JJ. Robert, Aventis). The second, longer method makes it possible to modify by homologous double recombination any sequence of the Ad genome (J. Crouzet, Aventis).

- Preparation of competent bacteria containing an adenoviral episome:

For simple homologous recombination, the plasmid pOSE1700, a resistant tetracycline, coding for the right part (pIX to 1TTR 3 ′) of the Ad 5 genome is introduced by thermal shock into JM83 bacteria. After an incubation of hi at 37 ° C allowing the expression of resistance to tetracycline, the bacteria are spread on LB-Tet dishes (12 μg / ml) and incubated overnight at 37 ° C. An isolated colony is amplified to prepare competent bacteria [JM83 + pOSE1700]. For the homologous double recombination, the Tet ^R adenoviral episomes (AE74, DB6 or AE95) are introduced by thermal shock into competent bacteria G4977. After one hour of incubation in Luria Bernati medium (LB) with shaking at 37 ° C allowing expression of resistance to tetracycline, the bacteria are spread on LB-tetracycline (12 μg / ml) and incubated overnight at 37 ° vs. An isolated colony (containing the adenoviral episome) is amplified to prepare competent bacteria.

- Homologous recombination:

In the simple homologous recombination method, the shuttle plasmid derived from pCA350 (Km) is introduced, by thermal shock, into the competent bacteria [JM83 + pOSE1700]. After 1 hour of incubation at 37 ° C. allowing the expression of the markers of resistance to kanamycin and to tetracycline, the bacteria are amplified in LB-Tet-Km medium (respectively 12 μg / ml and 50 μg / ml) overnight at 37 ° C with stirring. The bacteria Tet ^R Km ^R having thus achieved homologous recombination (at the level of the sequence coding for pLX) between the genome of the shuttle plasmid and the recombination vector pOSE1700 are thus selected. This event allows the emergence of a ^3rd plasmid pAd, encoding an adenovirus genome containing the expression cassette for the transgene of interest. The non-incorporated shuttle vectors are incapable of replicating in the chosen bacterial strain while kanamycin makes it possible to eliminate the unprocessed bacteria [JM83 + pOSE1700]. The DNA of the pAds is prepared from bacterial cultures by the alkaline lysis technique followed by phenol-chloroform extraction, the size of the plasmids making purification on the ion exchange column ineffective.

In the homologous double recombination method, the shuttle plasmid pSE2, containing the peptide CKS-17 in hexon, is introduced by thermal shock into competent bacteria [G4977 + adenoviral episome Tet ^R ]. After an incubation of one hour allowing the expression of genes for resistance to antibiotics, the bacteria are spread on LB-Tet-Km medium in order to select the Tet ^R Km ^R bacteria containing the co-integrate (adenoviral episome in which the shuttle plasmid is inserted by homologous recombination) (cf. FIG. 3). The

- non-incorporated plasmids are eliminated because they do not replicate in the chosen bacterial strain and the bacteria [G4977 + Tet ^R episome] are killed by kanamycin. After amplification of the bacterial colonies, the episomal DNA is prepared by alkaline lysis, followed by phenol-chloroform extraction (the size of the episomes makes the use of ion exchange columns ineffective). The integration of the plasmid into the episome (co-integrate step) is verified by enzymatic digestion.

The colonies containing the co-integrate are cultured in LB-Tet medium. To promote the second homologous recombination event, the bacteria are spread on LB-Tet medium containing 5% sucrose which makes it possible to select the bacteria having eliminated the sacB gene after a new homologous recombination in the co-integrate at the level of the modified region of the genome. The cointegrate can be resolved in two ways to give: - either the initial adenoviral episome, - or the adenoviral episome containing the new nucleotide sequence inserted into the adenoviral genome (see Figure 4).

The identification of the colonies having the recombinant episome is made by PCR amplification (94 ° C, 50 ° C, 72 ° C) with 1.25 units of Extrapol II (Eurobio) in the presence of 0.2 mM dNTP, MgCl ₂ l, 5 mM and 0.5 μM of oligonucleotides, directly from an isolated colony.

The recombinant episomes are extracted by alkaline lysis from cultures of different clones and then analyzed by enzymatic digestion or PCR. - Amplification of the recombinant adenoviral episome:

The recombinant adenoviral episome (pAd) is introduced into Top10 competent bacteria by electroporation (25μF, 200Ω, 12.5kv / cm ² ). These bacteria, being deficient in repair by homologous recombination, allow stable propagation of the recombinant adenoviral episome. After a one hour incubation at 37 ° C with shaking, during which the antibiotic resistance markers are expressed, the bacteria are spread on LB + antibiotic medium and incubated overnight at 37 ° C. After amplification of 2 bacterial colonies, the recombinant episomes (pAd) are analyzed by enzymatic digestion or by PCR in order to confirm their structure.

A colony containing the recombinant adenoviral genome is then amplified in 500 ml of ad hoc medium overnight at 37 ° C. with stirring. DNA is extracted in three stages. It is released from the bacteria by alkaline lysis, rid of the bacterial genome by treatment with exonuclease III and finally purified on an ion exchange column (Large-Construct Kit, Qiagen). Its concentration and purity are determined by measuring the absorbance at 260nm and 280nm. The structure of pAd is verified by enzymatic digestion.

Obtaining recombinant adenoviruses:

Transfection:

The recombinant adenoviral genome is released from the episome (pAd) by enzymatic digestion (Pac I) and purified by phenol-chloroform extraction. This DNA (5 to 10 μg) is introduced into 293 cells by lipofection (Effectene, Qiagen) then, after 24 hours, the medium is changed. When the cells round off and take off (cytopathic effect due to the virus about 8 days after transfection), they are harvested and then lysed by freeze / thaw cycles thus allowing the release of the virus. After centrifugation to remove the cellular debris, the supernatant containing the virus is recovered.

- Amplification and purification of the virus:

The viral titer (expressed in viral particles per mL, pV.ml ^"1 ) of the transfection supernatant is measured by HPLC (Blanche et al, 2000). This titer makes it possible to define the optimal volume necessary for the various amplifications (Multiplicity Of Infection = ME, 100 pV. cell ^"1 ). The viruses are amplified on 293 cells until a satisfactory viral titer is obtained (10 ¹⁰ pV.ml ^{" 1} ). Then, 293 cells (approximately 5.10 ⁸ to 70% confluence) are infected to obtain a stock of virus. The cells are harvested 40 to 72 hours after infection, centrifuged and then lysed by freezing and thawing cycles. A new centrifugation (4000 rpm) removes cell debris, then the supernatant containing the virus is deposited on a gradient of cesium chloride (CsCl) buffered at pH 7.4 (density 1.25 and 1.4), then ultracentrifuged at 18 ° C, 2 h at 35,000 rpm. The virus band at the interface of the 2 densities is removed and then ultracentrifuged at 18 ° C, in CsCl (density 1.34), 24 h at 35000 rpm. The purpose of these ultracentrifugation steps is to concentrate the virus and to separate it from the immature proteins and empty capsids present in the supernatant. The virus is desalted on a Sephadex G-25M column (Pharmacia Biotech) by elution with PBS. Glycerol (7% final) is added to the suspension of purified viruses allowing it to be stored at -80 ° C.

- Titration:

The quantification of the number of viral particles is obtained by HPLC (in pV.ml ^{" l} ) by selective retention on a Q sepharose XL anion exchange column.

(Amersham Pharmacia), elution around 20 mM NaCl and comparison with a standard viral suspension (Blanche & al., 2000). At the same time, the quantification of the number of infectious viral particles is obtained by measuring the pfu (unit forming the range). 911 cells are infected with different dilutions of virus and incubated for 1 h at 37 ° C. After removing the suspension, add a layer of 1% agarose mixture (FMC, Sea Plate), MEM IX nutrient medium (Eurobio), 1% glutamine and 2% SVF. The cells are fed the same way every 3-4 days. After 14 days, the lysis ranges are counted for each dilution. The viral titer in pfu.ml ^"1 is obtained by relating this number of ranges to the volume of infection and to the dilution factor.

Biochemical characterization of soluble receptors: - Detection of soluble receptors by western blot:

Soluble antagonists are produced after infection of 10 ⁷ HeLa cells with the corresponding recombinant adenovirus, at an MOI of 1000 pV, in DMEM 2% FCS medium. After 24 hours of infection, the cells are washed with PBS and returned to culture in a medium devoid of serum for 4 days. After centrifugation, the supernatant is heated 30 min at 56 ° C to inactivate the remaining viruses.

The supernatants (soluble antagonists) are diluted in Laemmli 1 ×, DTT 0, 1 mM to be heated to 95 ° C. After an electrophoresis (115mA, 110V) carried out on 10% NuPage Bis-Tris gel (Novex) in MOPS buffer (3- (N-morpholino) propane sulfonic acid), the proteins (18μl of supernatant) are transferred (150mA, 18V or 3mA cm ² ) on a nitrocellulose membrane

(porosity 0.45 μM, Schleicher & Schuell) during 1 h 30 min. The membrane is then saturated with PBS 0.1% tween (PBST) -5% milk. After several washes in PBST, it is incubated for 1 hour in the presence of the first antibody diluted in PBST-2% milk. Following further washes, a secondary antibody, coupled to peroxidase, is deposited in PBST-2% milk on the membrane for 1 hour. After washing, the fixed antibodies are revealed by an X-ray exposure which highlights the emission of photons released by the transformation of a substrate, luminol, by peroxidase (ECL ™, Amersham Pharmacia Biotech).

- Purification of soluble receptors:

The purification of the soluble antagonists is carried out using culture supernatants of HeLa cells (approximately 800 ml of culture starting from 8.10 ⁸ cells) infected with the appropriate recombinant adenoviruses. The proteins are then precipitated with ammonium sulphate (50%) overnight at 4 ° C. After a 20 min centrifugation at 10,000 rpm, the precipitate is taken up in PBS to be dialyzed against PBS, at 4 ° C. The dialyzed solution is then adjusted with NaCl

3M final, 10mM boric acid (pH 8.9). The supernatants are deposited (flow rate 1 ml / min) on a column of Protein A (Hi Trap rProtein A, Parmacia Biotech) which retains the soluble antagonists by interaction between protein A and their part Fc. After washing (25ml) in 3M NaCI buffer, 10mM sodium borate (pH 8.9), the recombinant proteins are recovered, by fraction of 500μl, thanks to an acid elution (lOOmM glycine, pH 5) which will then be neutralized by 50μl of Tris 1M, pH 8. The purified proteins are dialyzed (Slide-A-Lyzer Dialysis Products, Pierce) against PBS and the amount of protein (μg.mi ^"1 ) present in the dialysate is measured by a Bradford test. The purity of the preparation is analyzed on 10% Nu-Page Bis-tris gel colored with Coomasie Blue.

Analysis of the infectious power of viruses:

- SLOT BLOT:

Different amounts of protein are deposited, in 200 μl of PBS, on a nitrocellulose membrane (Hybond ™ ECL ™, Amersham Pharmacia Biotech). The membrane is then saturated with PBS-milk 5%, at 4 ° C overnight. After two washes of the membrane with 0.05% PBS-NP40, the membrane is incubated for 4 hours at room temperature with 20 ml of ¹⁰ pV-mi ^" 10 viral solution in PBS, 0.05% NP40, milk 0 0.5% After washing, the membrane is incubated for 2 hours at room temperature with an anti-Ad Ab (serum L5, example 1, “antibodies and sera”, “primary antibodies”) at 1/2000 ^e in PBS. -NP40 0.05% -milk 0.5%.

After washing, the fixed Ab are revealed (cf. example 1, “antibodies and sera”, “secondary antibodies”).

- Infectivity tests of recombinant adenoviruses:

All tests are performed in duplicate, using two different viral stocks for each type of virus each time.

12-well culture plates are seeded with 3 to 10 × 10 ⁴ cells per well in 2 ml of suitable medium. When the cells reach 90% confluence (10 ⁵ to 10 ⁶ cells per well) they are infected at different MOIs (10 ² , 10 ³ , 10 ⁴ pV cell ^"1 ) with the different recombinant adenoviruses (carrying the lacZ transgene) , in a volume of 400 μl of medium without serum. After 1 h at 37 ° C. 2 ml of ad hoc medium supplemented with 10% of S VF are added for each well. The entry of the virus is evaluated after 24 hours of infection.

- Colorimetric detection: The infection medium is removed and the cells are fixed (formaldehyde 1%, glutaradéhyde 0.2%), then rinsed with PBS.

| 3-galactosidase activity is revealed by adding the X-gal substrate (5-bromo-4-chloro-3-indolyl- / 3-D-galactoside 0.4 mg / ml, potassium ferrocyanide 4mM, potassium ferricyanide 4mM , MgCl ₂ 2 mM) overnight at 37 ° C. The next day, after washing with PBS, the number of transduced cells is evaluated by macroscopic observation of the blue coloration.

- Luminometric detection: The infection medium is removed and the cells are washed with PBS and then dissolved by lOOμl of lysis buffer (100 M potassium phosphate pH 7.8, 0.1% newt, 0.2 mM DTT, 1 cocktail anti-protease (Roche) for 10 ml), 30 min at 4 ° C. The cell lysate is centrifuged, 5 min at 10,000 rpm, in order to remove the cellular debris. The β-gal activity is measured for 5 seconds (in RLU = Relative Light Unit) on an aliquot of the cell lysate (Luminescent beta-galactosidase detection Kit II, Clontech) (Lumat meter Lumat LB 9501, Berthold). The amount of total protein present in the cell lysate is measured (in μg) by a Bradford test (Protein Assay, Bio-rad).

The β-galactosidase activity is related to the quantity of total proteins present in the cell lysate (RLU.μg ^"1 ). The measurements of the RLU and the quantity of proteins are carried out in duplicate.

- FACS:

The cells are detached using PBS-EDTA. The levels of membrane receptors (CAR, Integrins, Integrins αyβ ₃ , Integrins αyβ ₅ and TGFβRII) are determined on the cells (500,000) after incubation with an Ac directly coupled to FITC, or with an uncoupled Ac, for 45 min to 4 ° C, then with the II ^T antibody coupled to FITC (30 min, at 4 ° C). The negative control is carried out by labeling the cells not treated with a non-specific antibody, or, in the case indirect labeling, omitting the first Ab (example 1, "antibodies and sera", "primary antibodies" and "secondary antibodies").

The measurement of the membrane TGFβRII level is carried out using the fluorokine kit _: (R&D), the cells are briefly (100,000) are incubated with human TGFβ-1 coupled to biotin (60 min at 4 ° C), followed incubation with avidin coupled to FITC (30 min, at 4 ° C). The negative control is carried out by incubating the cells with an irrelevant protein also coupled to biotin.

The washes are made in PBS or in the ad hoc buffer. The fluorescence intensity is measured on fixed cells (PBS-Formaldehyde 4%) or not, using a cytofluorometer (FacsCalibur ^® ).

Intra-tumor gene transfer

C57B16 mice (females, 8 to 10 weeks) are first injected subcutaneously with 2.10 ⁶ LLC cells (in 100 μl of PBS). When the tumors reach a size of approximately 5 mm in diameter (approximately 15 days), an intratumoral injection of virus is carried out (4.10 ¹⁰ pV in 50 μl of PBS). In a first experiment, the mice are sacrificed 3 days after the viral administration. The tumors are removed and fixed in an aqueous solution of GlyoFixx (5%) - EtOH (10%). After inclusion in paraffin, the tumors are cut (5 μm) and the β-galactosidase is revealed using an anti-β-galactosidase rabbit Ab (Cappel), followed by an incubation with a second HRP Ab (Power Vision , Microm). The peroxidase activity is then revealed by DAB, then the slides are stained with Mayer's hematoxilin. In a second experiment, the mice are sacrificed 2 days after viral administration, then the tumors are removed and frozen at -80 ° C. They are then homogenized with a ml of lysis buffer (cf. example 2, "analysis of the infectious power of viruses", "infectivity tests of recombinant adenoviruses") using the FastPrep ™ System (Bio 101). After centrifugation at 10,000 rpm, 15 min at 4 ° C, the β-Gal activity is measured by luminometry in the supernatants. EXAMPLE 3: ANALYSIS OF THE INPUT MECHANISMS OF THE MODIFIED CAPSIDE VIRUS. SE21HEXON / CKS-171

A new independent CAR ^' entry route:

In order to check that the insertion of the CKS-17 peptide into hexon does not alter the infectious power of the SE2 virus, we carried out infectivity tests in vitro. The entry of adenoviruses into a cell being mainly conditioned by the interaction of the fiber with the cellular receptor CAR, we used cells expressing this receptor (HeLa). The infections were carried out at different multiplicities of infection (MOI of 10 ² , 10 ³ and 10 ⁴ pV. Cell ^"1 ). The quantity of transduced cells is evaluated, at 24 h pi, by measuring the expression of the lacZ reporter gene carried by the adenoviral genome (X-gal staining and / or β-gal activity expressed in RLU.μg ^"1 of proteins).

FIG. 4 shows that the quantity of CAR ⁺ cells transduced by the SE2 virus [hexon CKS-17] is comparable to that observed for the control virus AE18 (wild hexon). The quantitative analysis of 5-gal activity, obtained on lysates of infected cells, confirms the results of the colorimetric tests (Figure 4). For example, at the ME of 10 ³ pV.cell ^"1 , we did not observe any difference

significant difference between the activity of β-galactosidase in cells infected with the SE2 virus (6.5.10 ⁵ RLU.μg ^"1 ) compared to that of cells infected with 1ΑE18 (7.4.10 ⁵ RLU.μg ^{" 1} ) .

On the contrary, the infectivity tests carried out on negative CAR cells (L929, Vigne et al, 2003) show that the SE2 virus returns effectively to the MOI of 10 ⁴ pV.cells ^"1 unlike the control virus AE18 (X-gal staining Likewise, the measurement of the β-gal activity indicates that the SE2 virus enters from the MOI of 10 ³ pV cells ^"1 , with an input 20 times more effective than that of the control virus AE18, at 10 ⁴ pV . cells ^"1 (4.1.10 ⁵ RLU.μg ^{" 1} against 2.0.10 ⁴ RLU.μg ^"1 ). These results indicate that the SE2 virus retains the capacity to infect CAR ⁺ cells and that the insertion of the CKS-17 peptide into hexon does not modify the interactions of the fiber with the CAR receptor. In addition, they demonstrate that the insertion of the peptide CKS-17 into the hexon, endows the virus with a new independent CAR ^′ entry pathway.

Characterization of the new entry route:

- Role of the type II receptor to TGFβ:

First, we analyzed whether the SE2 virus was able to bind to the recombinant TGFβRII. Different concentrations of CAR and recombinant TGFβRII were immobilized on nitrocellulose membranes before incubation with the viruses AE18 and SE2. The attached viruses were revealed by specific anti-Ad Abs.

The results indicate that the AE18 and SE2 viruses interact well with the CAR receptor (Figure 5). In addition, the SE2 virus, but not the AE18 control, is capable of interacting with the TGFβRII immobilized on the membrane (FIG. 5). The labeling is specific since its intensity is proportional to the amount of TGFβRII deposited.

- Production of soluble receptors:

Construction of recombinant adenoviruses:

To demonstrate the role of the TGF / 3 type II receptor in the demonstrated entry pathway, it is necessary to show that competition with TGFj8RII effectively inhibits infection of CAR cells ^" (eg L929). This implies having sufficient soluble recombinant proteins available. We have therefore chosen to construct a recombinant adenovirus encoding a soluble TGFβRII. At the same time, we have constructed a recombinant adenovirus encoding a

Soluble CAR to neutralize the Ad5 fibers of the SE2 virus, and test its capacity to enter CAR ^" -independently in different CAR ⁺ cell types. The sequences encoding the extracellular part of human TGFβRII and CAR were amplified by PCR and cloned in phase with the constant part (Fc) of murine IgG1. Chimeric cDNAs, under the control of the early promoter. immediate CMV and the late polyadenylation site of the SV40 virus, encode proteins which can be dimerized by the formation of disulfide bridges between two fusion proteins at the level of the Fc parts. The presence of the Fc part gives the extra-cellular domain greater stability (increased half-life).

Once confirmed by sequencing, the plasmid expression cassettes

(encoding TGFβRII-Fc and CAR ^' Fc) were isolated and introduced into a shuttle vector which, after homologous recombination in E. coli with the plasmid carrying the 3' part of the adenoviral genome, makes it possible to obtain a recombinant adenoviral genome (ΔΕ1ΔΕ3) (cf. example 2, “construction of the recombinant adenoviral genomes (backbones)”). The adenoviral genomes were transfected into cells complementing the E1 region to produce the corresponding viruses. Thus, we isolated the recombinant adenoviruses Ad / TGFβRÏÏ-Fc and Ad / CAR ^" Fc. These two viruses were amplified to obtain viral stocks. These stocks were purified on a cesium chloride gradient, and titrated by both measurement of total viral particles (pV.ml ^"1 determined in HPLC), and by measurement of infectious particles (prù-ml ^{" 1} ).

Biochemical characterization of soluble receptors:

In order to check that the two recombinant Ad constructs encode fusion receptors, we analyzed the expression of these receptors in supernatants of HeLa cells infected with these Ad, by electrophoresis followed by a western blot revealed by an Ac directed against the Fc part of the soluble receptors. No protein is detected in the supernatants of uninfected HeLa cells. In contrast, culture supernatants from cells infected with recombinant Ad,

Ad / TGFβRII-Fc and Ad / CAR ^" Fc, have a majority molecular weight (PM) band of 41.6 and 52 kDa respectively (FIG. 6). membranes and revelation using Ac directed specifically against the extracellular domain of each of the receptors (TGFβRII or CAR), we observed the presence of a single band, of expected size, for each type of supernatant (figure 6). The detection of soluble receptors in culture supernatants, both by their specific part and their common part Fc, indicates that the proteins expressed by the Ad7TGFβRII-Fc and Ad / CAR ^" Fc are secreted fusion proteins.

Purification of TGFβRII-Fc and CAR Fc:

The HeLa cells were infected with Ad / TGFβRII-Fc or Ad / CAR ^" Fc. The harvested supernatants, containing the TGFβRII-Fc protein in large quantities, were precipitated with ammonium sulphate, dialyzed and purified by affinity chromatography on a protein A column. A first experiment allowed us to obtain 1 mg of purified recombinant protein from a liter of supernatant

(figure 7). The degree of purity of the preparation of TGFβRÏÏ-Fc is visualized on SDS-PAGE gel after staining with Coomassie blue. An additional band around 90 kDa is observed on the gel, it could be aggregates. The production of conditioned supernatants containing CAR ^' Fc is currently underway. These proteins, purified in quantity, will allow us to inhibit, by competition, the cellular receptors TGFβRII and / or CAR in the infectivity tests.

EXAMPLE 4 CAPACITY OF THE SE2 VIRUS TO INFECT CELLS

TUMORALS:

Transduction of tumor cells in vitro:

Different tumor lines were infected with the SE2 virus or the AE18 control virus, and the gene transfer was evaluated by X-gal staining. After infection with the SE2 virus, the melanoma (B16F10, mouse), glioma (Ul 18, male), lung cancer (LLC, mouse), prostate tumor lines (PC3, male), and ovarian cancer (CHO, hamster) show a greater blue coloration than that observed after infection with the AE18 control virus (data not shown). This is also the case for a primary culture

•.; (Aury) and a line (Law) from kidney tumors of patients (supplied by Dr E. Angevin, IGR). When the β-gal activity is measured for this primary culture (Aury), it is observed that at the ME of 10 ³ pV.cells ^"1 , the β-gal activity after infection with SE2 is 2.5.10 RLU.μg ^" , whereas for cells infected with AE18 it is only 3.6.10 ⁴ RLU.μg ^{" 1.} The value of the β-gal activity is underestimated at the MOI of 10 ⁴ pV cells. ^"1 for the SE2 virus, due to the saturation of the luminometer (*, figure 8).

All the results obtained by the infectivity tests are summarized in Table II where is represented, for different MOI, the transduction index. This is defined at a given MOI, as the ratio of the β-gal activity (RLU.μg ^"1 ) observed for SE2 over that generated by the AE18 virus. For all tumor cells, a transduction index is observed greater than 4 (and up to 71.2 for CHO), with the exception of the LLC tumor, the index of which increases only at the ME of 10 ⁴ pV.cells ^"1 . Thus, the primary kidney cultures (Aury) are particularly transduced by the SE2 virus (transduction index of 41.7 and 59.9 respectively at 10 ² and 10 ³ pV cells ^"1 ).

In parallel with these transduction tests, we analyzed, by flow cytometry, the expression of the Ad5 receptors (CAR and integrins) on the one hand, and that of TGFβRII on the other hand. In the case of integrins, the markings were carried out with Ab directed against the integrins of the family αy as a whole, or against the heterodimers αγβ ₃ or αγβ ₅ . These heterodimers, but also otyβi, are directly involved in the entry by endocytosis of the Ad5 particle (Russell, 2000, Benihoud et al, 1999). In HeLa cells which are efficiently transduced by the wild-capsid AE18 control virus (FIG. 4), there is a high expression of CAR and of the integrins, in particular αyβ ₅ , but also of TGFβRII (FIG. 9). In the PC3 line, CAR is absent and the expression of integrins is very low. In the Law line, there is a medium and relatively weak expression of the CAR of the integrins αyβ ₃ and αγβ ₅ . The two lines on the other hand, like HeLa strongly express TGFβRII which could explain the strong transduction index observed in these cells (Table II). A systematic study of the expression of the different receptors, by flow cytometry, on a wider panel of cells, including cells not expressing TGFβRII, is necessary to demonstrate that the efficiency of cell transduction is correlated with the expression of TGFβRII.

Cell lines Primary culture ME PC3 CHO LLC Law Aury (pV cell- ¹ ) (prostate) (ovary) (lung) (kidney) (kidney) 10 ² 7.1 4.1 2.1 8.5 41.7 10 ³ 5.8 71.2 2.7 12.2 59.9 10 ⁴ 4.6 22.3 33.4 5.9 saturation

Table II: Index of transduction of different tumor cells

Transduction of tumor cells in vivo:

In order to analyze the capacity of the SE2 virus to transfer genes in vivo into tumors, we pre-established tumors by subcutaneous injection of LLC cells in mice. The SE2 virus, or the AE18 control virus, was injected into the tumors when they had a size of approximately 5 mm in diameter (2.10 ⁹ pV. tumor ^"1 ). In one experiment, the animals were sacrificed for 3 days after infection of the tumors, these were fixed, and the β-gal was detected by immunohistology. A greater number of cells expressing the β-gal is observed in the sections carried out on the tumors of animals injected with the SE2 virus than in that of the animals injected by the AE18 control (FIG. 10A). In another experiment, the animals were sacrificed at 2 days pi and the activity of β-gal was measured in the lysate of these tumors. β-gal expression is higher in the group infected with the SE2 virus than in the group infected with the AE18 control. Indeed, the average of the SE2 group (3.5 ± 3.10 ⁴ RLU.μg ^"1 ) is approximately three times higher than that of the AE18 group (1.3 ± 0.7.10 ⁴ RLU.μg ^{" 1} ) (Figure 10B) .

These results show that the insertion of CKS-17 into the hexon makes it possible to ensure better gene transfer in tumors in vivo. It will be particularly interesting to test gene transfer in a tumor type with a higher transduction index (for example Aury cells, index - 59.9 to 10 ³ pV cells ^"1 ).

EXAMPLE 5 CAPACITY OF THE SE2 VIRUS TO INFECT MUSCLE CELL LINES:

Muscle cells are particularly difficult to infect with Ad, because of their low expression of CAR, so we analyzed whether the SE2 virus returned more efficiently to these cells than the AE18 control virus. In the L6 (rat) or C2C12 (mouse) myoblasts, the expression of β-gal after infection with the SE2 virus is greater, at all MOIs, than that observed for the control virus AE18 (FIG. 11). At the ME of 10 ⁴ pV.cells ^"1 , the SE2 virus generates a β-gal activity, respectively 52 and 81 times, greater than that of the control virus AE18 (FIG. 11). The expression of the β-gal also has was measured in rhabdomyosarcoma RD (man) .It is on average 4 times greater than that induced by the control virus whatever the MOI (data not shown). These results highlight the interest of studying, in a mouse model, virus capacity

SE2 to transfer genes into muscles.

All of the tests that we have carried out show that the SE2 virus very effectively infects muscle cells on the one hand, and on the other hand, many tumor lines and primary cultures. EXAMPLE 6: ABLATION OF NATURAL TROPISM OF AD5:

We have shown on CAR cells ^" that the SE2 viras has a new entry pathway, however this virus remains capable of interacting with its natural receptors (CAR, heparan sulfates and integrins). In order to be able to study in vivo In the interest of the new entry route, it is necessary to prevent the sequestration of the virus in organs rich in CAR (for example the liver), by abolishing the native interactions of Ad5 with its natural receptors.

Construction of adenoviral genomes:

To break the interactions of the Ad5 capsid with its receptors, CAR but also the heparan sulfates, the Ad5 fiber can be replaced by that of Ad3 which recognizes an unidentified receptor, and does not interact with the heparan sulfates (Figure 1, Dechecchi et al, 2000). For this, we modified, by homologous recombination in E. coli, the recombinant Ad genomes for LacZ, deleted in the base of the penton, of the RGD integrin binding motif (AE74), pseudotyped by the fiber Ad3 (DB6) or combining these two mutations (AE95) (Vigne & al., 2003 10: 153-62), by inserting into the hexon the sequence of the peptide CKS-17 (FIG. 13).

After construction in E. coli, the modified adenoviral genomes (AE74SE2, DB6SE2 and AE95SE2), and their respective unmodified controls (AE74, DB6 and AE95), were transfected into cells complementing the E1 region to produce the corresponding viruses. The viruses are all detected by HPLC in the transfection or amplification supernatants, which shows that all the constructs are infectious. The AE95 [Fiber Ad3-ΔRGD] and AE95SE2 [Fiber Ad3-ΔRGD-Hexon / CKS-17] viruses were amplified to obtain purified viral stocks. AE95 and AE95SE2 are produced in titles comparable to the control viruses AE18 and SE2. The stocks of the other viruses are being produced. Characterization of the infectious power of Ad mutants:

Infection tests were carried out with the capsid viruses modified by insertion into the hexon of the motif CKS-17 (AE95SE2 and SE2), and their control (AE95 and AE18), and revealed by X-gal staining. In HeLa cells (CAR ⁺ , Integrins ^"1" , cf. FIG. 10), the same transfer efficiency of the LacZ gene (blue coloration) is observed for the AE18 and SE2 viruses, whatever the MOI (FIG. 12). On the other hand, the entry of 1ΑE95 (Ad3 fiber) is much reduced compared to AE18 (wild Ad5 fiber). Despite this, like the AE18 and SE2 viruses, the AE95SE2 virus is able to enter these cells efficiently (Figure 12).

L929 cells (CAR ^" , rntégrines +), are little infected by the AE18 and AE95 viruses, but the presence of the CKS-17 motif in the capsid of the AE95SE2 virus gives it an important infectious power, similar to that of the SE2 virus (FIG. 12 These results indicate that the CKS-17 motif in hexon is capable of providing a virus deleted for its interactions with primary TAd5 receptors (CAR, heparan sulfates and integrins) with a new pathway. AE95SE2 virus no longer has the integrin binding motif, these results demonstrate that the new entry pathway through TGFβRII is independent of integrins.

EXAMPLE 7: CONCLUSIONS & OUTLOOK:

The work presented focused on the study of a recombinant adenovirus (SE2), with capsid modified by insertion into the hexon of the peptide motif CKS-17, of retroviral origin. Our objective was to identify the potential target cells of this retargeted virus and to suppress the interactions of this virus with the natural Ad5 receptors.

First, we observed that the SE2 virus carrying the mutation [Hexon / CKS-17] remains capable of infecting CAR ⁺ cells (HeLa) but transduces more efficiently than the control virus (AE18 with wild capsid) cells not expressing this receptor. This demonstrates the existence of a new route of entry. This path seems to involve TGFβRII since this vector binds to recombinant TGFβRII. To prove the role of TGFβRII in the new entry pathway, we constructed a recombinant Ad encoding a soluble TGFβRII (TGFβRII-Fc). This vector allowed the purification of a fusion protein in large quantities (from supernatants of infected cells) essential for carrying out competition tests to inhibit the new entry pathway. The role of TGFβRII could also be demonstrated by evaluating the susceptibility of cells, overexpressing (transfectants) or under-expressing (interfering RNA) TGFβRII, to be transduced by the SE2 virus [Hexon / CKS-17].

At the same time, we studied the ability of the virus to transduce cell types of histological origins and different species in vitro. These tests show that the SE2 virus, compared to the wild capsid virus AE18, is particularly effective in infecting muscle cells on the one hand (up to

81 times more), and tumor cells on the other hand (up to 70 times more). By flow cytometry, we have shown that two different tumors, much more strongly transduced by the SE2 virus than by the AE18 control virus, do not express the CAR receptor, and weakly the integrins, but express TGFβRII. This study must be extended in order to correlate the gain in infectivity of the SE2 virus with the level of membrane TGFβRII in cells, by including in particular cells not expressing TGFβRII.

We have shown that the insertion of CKS 17 into hexon makes the virus capable of transducing better than the virus controlling pre-established tumor cells in mice. It should be noted that these results were obtained with the LLC tumor, which exhibits a high transduction index only at the MOI of 10 ⁴ pV. Cell ^"1. However, we have shown that certain types of tumors (Aury for example) may have a higher transduction index at lower MOIs. These tumors will be good candidates for in vivo experiments. The SE2 virus [Hexon / CKS-17] has a new entry pathway, but remains capable of infecting cells through the natural CAR pathway. In order to control the tropism of this virus, we have broken down the interactions of this virus with the natural Ad5 receptors (CAR and heparan sulfate _. And / or integrins) by pseudotyping this virus with an Ad3 fiber incapable of interacting with the receiver

CAR and heparan sulfate and / or by deleting the RGD motif, from the base of the penton, responsible for binding to integrins. Viruses carrying both these mutations and the CKS-17 motif in hexon have. been produced. The infection tests carried out show that the AE95SE2 virus [Fiber Ad3-ΔRGD-Hexon / CKS-17] retains the new entry path, which demonstrates that the endocytosis of this virus is _. independent of integrins.

Claims

claims

1. Adenoviral vector intended to ensure the expression of a DNA sequence of interest in ^" mammalian ^" cells, characterized in that said vector comprises in at least one of the DNA sequences coding for the regions to weak structural constraint of capsid proteins, at least one DNA sequence coding for a heterologous peptide comprising the motif- (W / R) X _t XD -.

2. Adenoviral vector according to claim 1, characterized in that the heterologous peptide is capable of binding to the receptor for the tumor growth factor β (TGFβR).

3. Adenoviral vector according to claim 1 or 2, characterized in that the heterologous peptide is an immunosuppressive peptide of retrovirus.

4. Adenoviral vector according to any one of claims 1 to 3, characterized in that the residues Xi and X ₂ are chosen, independently of one another, from the neutral residues A, V, L, I, G , S and T.

5. Adenoviral vector according to any one of claims 1 to 4, characterized in that the residue Xi is G and the residue X ₂ is L.

6. Adenoviral vector according to any one of claims 1 to 5, characterized in that the heterologous peptide contains at most 24 amino acid residues.

7. Adenoviral vector according to any one of claims 1 to 6, characterized in that the immunosuppressive peptide of retrovirus is the peptide CKS-17 of sequence SEQ ID No. 1 or any peptide of sequence identical to at least 60% to said sequence SEQ LD N ° 1.

8. Adenoviral vector according to any one of claims 1 to 7, characterized in that the capsid protein is chosen from hexon, fiber or penton base, protein pVI, pVIII or pIX.

9. Adenoviral vector according to any one of claims 1 to 8, characterized in that the DNA sequence coding for the region with low structural stress of a capsid protein is deleted or partially deleted, and the DNA sequence encoding the heterologous peptide is inserted into the deletion site.

10. Adenoviral vector according to any one of claims 1 to 9, characterized in that when the capsid protein is hexon, the region with low structural stress is the hypervariable region (HVR5).

11. Adenoviral vector according to claim 10, characterized in that the DNA sequence coding for the hypervariable region 5 (HVR5) is deleted.

12. Adenoviral vector according to any one of claims 1 to 11, characterized in that when the capsid protein is the fiber, the region with low structural stress is the HI loop.

13. Adenoviral vector according to any one of claims 1 to 12, characterized in that the heterologous peptide further comprises at least one adapter located in N-ter or in C-ter.

14. Adenoviral vector according to any one of claims 1 to 13, characterized in that the heterologous peptide comprises an adapter located in N-ter and an adapter located in C-ter.

15. Adenoviral vector according to any one of claims 1 to 14, characterized in that the DNA sequence of interest is operably linked to expression control sequences.

16. Adenoviral vector according to any one of claims 1 to 15, characterized in that the DNA sequence of interest codes for an anti-angiogenic factor, thymidine kinase, the p53 protein, a transgene involved in a muscular disease , the gene encoding the transmembrane regulator involved in cystic fibrosis (CFTR gene).

17. Adenoviral vector according to claim 16, characterized in that the anti-angiogenic factor is angiostatin or any other fragment of plasminogen, endostatin, canstatin, angiotensinogen or any other fragment derived from angiotensinogen.

18. Adenoviral vector according to claim 15, characterized in that the transgene involved in a muscular disease codes for dystrophin.

19. Adenoviral vector according to any one of claims 1 to 18, characterized in that the DNA sequence E1 of the adenoviral vector is modified so as to allow replication of said vector only in tumor cells.

20. Adenoviral vector according to any one of claims 1 to 19, characterized in that it presents a tropism for mammalian cells deficient or lacking in receptors common to Coxsackievirus B3 and to adenovirus (CAR receptors).

21. A method for obtaining in vitro infectious adenoviral particles in production cells, said method comprising a step of transformation of said production cells with the adenoviral vector according to any one of claims 1 to 20.

22. Infectious adenoviral particles obtained by the method according to claim 21.

23. Method for modifying the tropism of an adenoviral vector, comprising the insertion, in at least one of the DNA sequences coding for regions with low structural stress of capsid proteins, of at least one sequence of DNA coding for a heterologous peptide as defined in any one of claims 1 to 7.

24. Method according to claim 23, characterized in that the insertion step is preceded by a deletion step of all or part of the DNA sequence coding for the region with low structural stress of a capsid protein .

25. Method according to claim 23 or 24, characterized in that the capsid protein is chosen from hexon, fiber or penton base, protein p VI, pVIII or pIX.

26. Method according to any one of claims 23 to 25, characterized in that when the capsid protein is hexon, the region with low structural stress is the hypervariable region 5 (HVR5).

27. Method according to any one of claims 23 to 26, characterized in that the DNA sequence coding for the hypervariable region 5 (HVR5) is deleted.

28. Method according to any one of claims 23 to 27, characterized in that when the capsid protein is the fiber, the region with low structural stress is the HI loop.

29. Method according to any one of claims 23 to 28, characterized in that the heterologous peptide as defined in any one of claims 1 to 7 further comprises at least one adapter located in N-ter or in C- b.

30. Method according to any one of claims 23 to 29, characterized in that the heterologous peptide as defined in any one of claims 1 to 7 comprises an adapter located in N-ter and an adapter located in C-ter .

31. An in vitro method allowing preferential expression of a DNA sequence of interest in a mammalian target cell comprising bringing said cell into contact with infectious adenoviral particles according to claim 22, or with the adenoviral vector according to any of claims 1 to 20 or obtained according to any of claims 23 to 30.

32. Method according to claim 31, characterized in that the mammalian target cell is deficient or lacking in CAR receptors.

33. Method according to claim 32, characterized in that the cell deficient or lacking in CAR receptors comes from a cell line chosen from the lines CHO, L929, PC-3, LLC, B16F10, RCC law, RCC Aury, U118 , RD, L6 and C2C12. .

34. A pharmaceutical composition comprising an adenoviral vector according to any one of claims 1 to 20 and an appropriate pharmaceutical excipient.

35. Pharmaceutical composition according to claim 34 intended for targeting mammalian cells deficient or lacking in CAR receptors.

36. Use of the adenoviral vector according to any one of claims 1 to 20 for the manufacture of a medicament.

37. Use according to claim 36, characterized in that the medicament is intended for targeting mammalian cells deficient or lacking in CAR receptors.

38. Use according to claim 37, characterized in that the mammalian cells deficient or lacking in CAR receptors are tumor cells, such as glioma, melanoma, carcinoma, lung cancer, uterus cells, breast or ovary, prostate tumor, bladder tumor, or lymphocytes, fibroblasts, lung epithelial cells, skeletal muscle cells, smooth muscle or myocardium.

39. Use according to any one of claims 36 to 38, characterized in that the medicament is intended for preventing or treating a subject suffering from breast cancer, uterus, prostate, lung, skin, ovary, colorectal cancer, leukemia, cystic fibrosis or myopathy.

40. A mammalian cell transformed with an adenoviral vector according to any one of claims 1 to 20.