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Definitions

• the subject of the present invention is in particular an adenoviral fiber, mutated in a region involved in the recognition and the binding to the natural cellular receptor of adenoviruses. It also relates to viral particles, more particularly adenoviral, and pseudoparticles, carrying on their surface such a fiber, possibly combined with a ligand which confers on said particles and pseudo-particles a modified host specificity, see targeted.
• the invention is of particular interest in the context of the development of vectors or compositions which can be used in the context of the implementation of gene therapy protocols.
• Adenoviruses have long been described as a very efficient natural system for transferring biological material into target cells. This is the reason why adenoviral vectors are today widely used in many gene therapy applications.
• the adenoviral genome consists of a linear, double-stranded DNA molecule of approximately 36kb containing two inverted repeat regions (designated ITR for Inverted Terminal Repeat) surrounding the genes coding for the viral proteins.
• the early genes are distributed in 4 regions dispersed in the adenoviral genome (E1 to E4; E for early in English), comprising 6 transcriptional units provided with their own promoters.
• the late genes (L1 to L5; L for late in English) partially cover the early transcription units and are, for the most part, transcribed from the major late promoter LP (for Major Late Promoter).
• the intracellular infectious cycle of adenoviruses is well documented in the literature and is based on two essential stages:
• the late phase which follows the replication of the genome, during which the structural proteins which constitute the base of the viral adenoviral particles (or capsids) are synthesized.
• the assembly of the new viruses is then carried out in the nucleus: first, the viral proteins assemble so as to form empty capsids of icosahedral structure in which the newly formed genome is packaged.
• the released adenoviruses can then infect other permissive cells. More particularly, during infection, adenoviruses enter cells by an endocytosis process following the attachment of the adenoviral particle to a specific receptor present on the surface of permissive cells.
• the fiber and the base penton present on the surface of the adenoviral particle play a critical role in the cellular attachment of viruses and their internalization (Wickham et al., 1993, Cell, 73, 309-319).
• the adenovirus binds to a cellular receptor (in particular CAR) present on the surface of permissive cells via said fiber in its trimeric form (Philipson et al.,
• the viral particle is internalized by endocytosis thanks to the binding of the penton base to the cellular integrins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 (Mathias et al., 1994,
• adenoviruses are capable of promoting the transfer, in vitro and in vivo, of macromolecules of non-viral origin, such as for example dextrans (Otero et al., 1987, Virology, 160, 75-80), proteins (Carrasco et al., 1981, Virology, 113, 623-629; Fitzgerald et al. , 1983, Cell, 32, 607-617; Defer et al., 1990, J.
• Virol., 64, 3661-3673 a plasmid DNA associated with a ligand (Curiel et al., 1992, Hum. Gene Therapy, 3, 147-154; Cotton et al., 1992, Proc. Natl. Acad. Sci., 89, 6094-6098), possibly in the presence of an antibody neutralizing adenoviral infection (Michael et al., 1993, J Biol. Chem., 268: 6866-6869), naked nucleic acids (DNA, RNA, PNA), or optionally in the presence of cationic compounds such as cationic lipids (US Patent 5,928,944; patent application WO 95/21259).
• Adenoviruses have been the subject of numerous studies and several scientific teams have also developed adenoviral vectors defective for replication, that is to say whose genome has been manipulated so that these adenoviral vectors are incapable of dividing or proliferate in the cells they infect.
• the defective adenoviral vectors are in particular obtained by deletion of at least part of the E1 region (for examples of defective adenoviral vectors, see in particular patent applications WO 94/28152 and WO 94/12649).
• the adenoviral fiber is made up of three distinct domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200): (a) at its N-terminal end is the tail, the sequence of which is very conserved from one adenoviral serotype to another. It interacts with the penton base and ensures the anchoring of the molecule in the capsid; (b) in the center is the rod; it is a stick structure made up of a certain number of leaf repetitions, the number of which varies according to the serotypes considered;
• each monomer comprises 8 antiparallel sheets designated A to D and G to J and 6 major loops of 8 to 55 residues.
• the CD loop connects sheet C to sheet D.
• the minor sheets E and F are considered to be part of the loop DG located between sheets D and G.
• table 1 indicates the location of these structures in the amino acid sequence of the Ad ⁇ fiber as shown in sequence identifier No. 1 (SEQ ID NO: 1), the +1 representing the initiating Met residue.
• the sheets form an ordered and compact structure while the loops are more flexible.
• the four sheets A, B, C and J constitute the sheets V directed towards the viral particle.
• the other four (D, G, H and I) form the R sheets, supposed to face the cell receptor.
• the V sheets seem to play an important role in the trimerization of the structure while the sheets
• R would be involved in the interaction with the receptor.
• WO 94/10323 describes adenoviral particles of type 5 (Ad5), the fiber of which has been mutated so as to understand the sequence of an antibody fragment specific for a given antigen (of type scFv) inserted at the end of one of the 22 repeat units of the stem.
• the specificity of infection of the adenoviral particles thus mutated is modified in such a way that the adenoviruses produced are capable of binding to cells presenting the target antigen.
• US 5,543,328 describes a chimeric adenoviral fiber in which the head domain is replaced by the sequence of tumor necrosis factor (TNF), or that of the ApoE peptide, so as to redirect the binding of the modified adenoviral particles to cells expressing the TNF cell receptor or LDL (low density lipoprotein) receptor, respectively.
• TNF tumor necrosis factor
• LDL low density lipoprotein
• WO 95/26412 describes a fiber modified by the incorporation of a ligand at its C-terminal end.
• WO 96/26281 describes a chimeric fiber obtained by replacing part of the native fiber and, in particular of the head, by the equivalent part of an adenoviral fiber of another serotype and, optionally, by insertion into the C-terminus of an RGD peptide specific for vitronectin.
• French patent application FR 2758821 (97 01 005) has demonstrated the role of the antigens of the major histocompatibility class I complex and of fibronectin modules III as primary receptor and co- adenovirus factor.
• mutants of the Ad5 fiber obtained (i) by substitution of one or more amino acid (s) in these regions, said substitutions being able in particular to be selected from the following group: the glycine residue in position 443 is substituted by an aspartic acid, the serine residue in position 444 is substituted with a lysine, the leucine residue in position 445 is substituted with a phenylalanine, the alanine residue in position 446 is substituted with a threonine, the serine residue in position 449 is substituted with an aspartic acid, the glycine residue in position 450 is substituted by an asparagine or a lysine, - the threonine residue at position 451 is substituted by a lysine or a leucine, the valine residue at position 452 is substituted by an asparagine or a threonine, the alanine residue at position 455 is substituted by a phenyla
• the mutated residue is selected from threonine residues in position 404, alanine in position 406 and serine in position 408.
• the mutation carried out consists of at least one substitution of an amino acid chosen from the substitutions following: the serine residue in position 408 is substituted by a residue having at least two carboxyl groups, and in particular by a residue selected from the group consisting of aspartic acid and glutamic acid, the threonine residue in position 404 is substituted by a glycine residue, the alanine residue at position 406 is substituted by a lysine residue.
• the inventors have now identified new mutants consisting of a modification, in particular a substitution or a deletion of one or more residues from the region of the adenoviral fiber comprised between residues 491 and 505 and shown to be of interest in order to inhibit or prevent the infectivity of adenoviral particles exhibiting such a modified fiber with regard to normally permissive cells.
• the aim of the present invention is in particular to propose a new alternative making it possible to reduce the therapeutic amounts of adenoviral particles to be used and to target the infection towards the cells to be treated. This specificity is particularly advantageous when an adenoviral particle expressing a cytotoxic gene is used in order to avoid the propagation of the cytotoxic effect to healthy cells.
• the advantages provided by the present invention are mainly to reduce the risks of dissemination and the side effects associated with adenoviral technology. Furthermore, the teachings of the present invention allow the development of other targeting systems intended for the development of methods of treatment by administration of viral vectors, especially recombinant viral vectors, or of non-viral vectors.
• viral particles which have the following properties: (i) the viral particle comprising said mutated fiber does not bind substantially to the natural adenoviral cellular receptors, that is to say that the host specificity of these viral particles carrying the mutated fiber is reduced, or even inhibited, compared to the host specificity of the viral particles carrying the wild fiber, that is to say non-mutated;
• the viral particle comprising said mutated fiber when the viral particle comprising said mutated fiber also comprises a specific ligand for an anti-ligand, it is possible to confer on said modified particle a new tropism towards one or more specific cell types carrying on their surface said anti- ligand compared to the non-mutated viral particle.
• These new mutants of the adenoviral fiber also make it possible to obtain pseudo-particles, in particular viral or synthetic, as described below.
• the mutated fiber does not bind substantially to natural cellular receptors
• the fiber is modified so as to reduce or abolish its ability to bind to the natural cellular receptor.
• Such a property can be verified by studying the infectivity or the cellular binding of the corresponding viral particles by applying the techniques known to those skilled in the art, and in particular by competition experiments of infection of the virus carrying the fiber. modified performed in the presence of a competitor made up of all or part of the wild adenoviral fiber (for more details on this measurement technique, see the Experimental Part of this application).
• the loss of natural specificity can also be evaluated by cell attachment studies carried out in the presence of labeled viruses (for example with 3H thymidine according to the technique of Roelvink et al., 1996, J.
• a mutated fiber does not bind in a substantial way to natural cellular receptors
• the percentage of residual infection measured by a competition experiment as disclosed in the Examples which follows, is between approximately 0 and 60%, preferably between 0 and 40% and most preferably between 0 and 20%.
• the trimerization and penton-base binding properties of the mutated adenoviral fiber are not affected. These properties are easily verified according to the technique used in the Examples which follow.
• residues and “amino acids” are synonyms.
• sheets and “loops” are defined according to Xia et al. (1994, Structure 2, 1259-1270).
• virus or “adenovirus” are synonyms for "viral particles” or “adenoviral particles”, respectively.
• particle is meant a structure comprising peptides and / or lipids which is organized so as to consist of a capsid which may also contain a macromolecule (in particular viral genome, dextrans, proteins, nucleic acids (DNA, RNA, PNA, plasmid DNA, etc.).
• viral pseudo-particles or "adenoviral pseudo-particles” it is intended to denote viral or adenoviral particles which do not contain a viral or adenoviral genome, respectively (the presence of a non-viral or non-adenoviral genome not being excluded); it is also possible, in the particular case where none genome is not present, refer to so-called “empty” particles.
• non-viral pseudo-particles we mean artificial particles, produced for example by the association of amino- or carboxyterminal protein sequences, peptides or glycoproteins with lipids. Such modified lipids can then be incorporated into a liposome-like structure.
• liposomes carrying the surface glycoproteins of the influenza virus can of course further comprise a macromolecule which they transport in the target cell, and in particular a nucleic acid.
• mutation is meant a deletion, a substitution or an addition of one or more residues or a combination of these possibilities.
• such a “mutation” can also consist in a modification, in particular chemical, of at least one residue.
• modifications consist in particular of an esterification, an alkylation, PEGylation, hydroxyalkylation, ....
• nucleic acid sequence denote a fragment of DNA and / or RNA and / or PNA, double strand or single strand, linear or circular, natural isolated or synthetic, designating a precise sequence of nucleotides, modified or not, making it possible to define a fragment or a region of a nucleic acid without limitation of size. According to a preferred embodiment, it is a nucleic acid chosen from the group consisting of cDNA (complementary DNA); genomic DNA; plasmid DNA; an RNA; a viral genome.
• part of an amino acid sequence is meant an amino acid sequence comprising at least 6 consecutive amino acids, preferably 10, more preferably 15, even more preferably 20, most preferred 30, and / or having the same biological activity as the sequence from which said part is derived, in particular the ability to recognize and bind to the target cells of the virus.
• part of a nucleic sequence is meant a nucleic sequence comprising at least 18 consecutive nucleotides, preferably 30, more preferably 45, even more preferably 60, most preferably 90, and / or coding for an amino acid sequence having the same biological activity as the amino acid sequence encoded by the nucleic sequence from which said part is derived.
• elements ensuring the expression of said gene in vivo is meant the elements necessary to ensure the expression of said gene after its transfer to a target cell. These include promoter sequences and / or regulatory sequences effective in said cell, and optionally the sequences required to allow expression on the surface of target cells of said polypeptide.
• the promoter used can be a viral, ubiquitous or tissue-specific promoter or a synthetic promoter.
• promoters such as the promoters of the RSV virus (Rous Sarcoma Virus), MPSV, SV40 (Simian Virus), CMV (Cytomegalovirus) or of the vaccinia virus, the promoters of the gene coding for creatine muscle kinase, for actin, for the lung surfactant. It is also possible to choose a promoter sequence specific for a given cell type, or activatable under defined conditions (see for example US Pat. No. 5,874,534). The literature provides a great deal of information relating to such promoter sequences.
• said "nucleic acid sequence” also contains a "heterologous" gene, that is to say the origin of which is different from that of the nucleic acid sequence which contains it. Examples of such genes are given below. Otherwise, said nucleic acid sequence may comprise at least two sequences, identical or different, having transcriptional promoter activity and / or at least two genes, identical or different, located relative to each other in a contiguous, distant manner , in the same direction or in the opposite direction, provided that the function of transcriptional promoter or transcription of said genes is not affected. Similarly, in this type of nucleic acid construction, it is possible to introduce “neutral” nucleic sequences or introns which do not interfere with transcription and are spliced before the translation step.
• nucleic acid may also contain sequences required for intracellular transport, for replication and / or for integration, for transcription or translation. Such sequences are well known to those skilled in the art.
• nucleic acids which can be used according to the present invention can also be nucleic acids modified so that it is not possible for them to integrate into the genome of the target cell or nucleic acids stabilized using agents, such as for example spermine, which as such have no effect on the efficiency of the introduction of the nucleic acid into the cells.
• the fiber according to the present invention can be derived from an adenovirus of human, canine, avian, bovine, murine, ovine, porcine or simian origin, or even comprise fragments of various origins, including fragments of heterologous origin. , ie not derived from adenoviral fiber or derived from non-adenoviral fibers (we will then preferentially speak of "hybrid fiber").
• human adenoviral fiber it is preferable to refer to that derived from serotype C and, in particular, derived from adenovirus type 2 or 5 (Ad2 or Ad5).
• Ad2 fiber contains 580 amino acids (aa), the sequence of which is disclosed in Hérissé et al.
• Ad ⁇ was determined by Chroboczek and Jacrot (1987, Virology 161, 549-554) and has 582 amino acids (SEQ ID NO: 1).
• SEQ ID NO: 1 amino acids
• a person skilled in the art can identify the adenoviral fiber sequences available on databases such as GenBank and determine the equivalent positions of the different sheets, loops and residue positions as described above. For information, let us cite in particular the GenBank references for the adenoviral fiber sequences of human serotype 2 (# AAA92223), 3 (# CAA26029), 5 (#M 18369), 31 (# CAA54050 or 41 (# X17016).
• the fiber of the present invention is of animal origin, use is preferably made of bovine adenoviruses and, in particular, those of the BAV-3 strain, which have been the subject of numerous studies and the sequence of the fiber. is disclosed in application WO 95/16048.
• the fiber of the present invention may, in addition to the modifications described in the present invention, present other modifications with respect to the native sequence as far as they do not affect the characteristics of the fibers proposed in the application.
• the contents of the publications or GenBank references cited above are incorporated by reference into the present application in their entirety.
• the invention also relates to a fiber modified as dec laughs in the present application and which also contains other mutations such as for example those described in the patent application WO 98/44121 or in the patent application benefiting from French priority N ° 99/10859 (see above in this application).
• the region of the Ad5 adenoviral fiber between the residues 491 and 505 and modified as described in the present application can of course be introduced into a heterology adenoviral fiber, that is to say ie different from the adenoviral fiber Ad5 in addition to or in substitution for the equivalent region of said heterologous fiber.
• the invention relates to a fiber of an adenovirus which is modified by mutation of one or more residues of the region between residues 491 and 505 of SEQ ID NO: 1.
• it is an adenoviral fiber comprising all or part of the fiber sequence of an adenovirus type 5 (Ad5) such as shown in sequence identifier No. 1 (SEQ ID NO: 1) and modified by mutation of one or more residues from the region between residues 491 and 505 of said sequence.
• the Ad ⁇ fiber carries at least one mutation chosen from the following mutations: the tyrosine residue at position 491 is substituted with an aspartic acid, the alanine residue at position 494 is substituted with a aspartic acid, - the valine residue at position 495 is substituted by an arginine, the glycine residue at position 496 is substituted by a serine, the phenylalanine residue at position 497 is substituted by an aspartic acid, the methionine residue at position 498 is substituted by an aspartic acid, the praline residue at position 499 is substituted by a glycine, the asparagine residue at position 500 is substituted by an aspartic acid, the alanine residue at position 503 is substituted by an aspartic acid, the tyrosine residue at position 504 is substituted with
• the invention relates to a fiber of an adenovirus type 5 characterized in that the mutated residue is selected from the residues alanine at position 494 and alanine at position 503. Due to their spatial location in the native fiber, these residues are capable of recognizing and / or interacting directly or indirectly with the natural cellular receptor of the adenovirus concerned.
• a fiber according to the invention is characterized in that it comprises, in addition to the modifications described previously, one or more mutations in: AB, CD, DG, GH, Hl and / or IJ loops and / or sheets A, B, C, D, G, H, I and / or J, and more particularly, one or more mutations as described above by the applicant in application WO 98/44121 and the patent application benefiting from French priority N ° 99/10859 (see also above).
• the amino acids forming a bend will be replaced by residues forming a similar structure such as those cited in Xia et al. 1994.
• the fiber of the present invention can also be modified by deletion.
• the deleted residues when at least one of the modifications is a deletion of at least 3 consecutive residues of a loop and / or of a sheet, the deleted residues can be replaced by residues of a loop and / or an equivalent sheet derived from a fiber of a second adenovirus capable of interacting with a cellular receptor different from that recognized by the first adenovirus.
• This makes it possible to maintain the structure of the fiber according to the invention while giving it a host specificity corresponding to that of the second adenovirus.
• the region of the fiber involved during infection and interacting with the cellular receptor is different for types 3 and 7 adenoviruses.
• an Ad ⁇ or Ad2 fiber deleted by at least 3 consecutive residues among those specified above may be substituted by residues from an equivalent region of the Ad3 fiber or Ad7 to reduce its ability to bind the Ad ⁇ receptor and thus give it a new specificity towards the cellular Ad3 or Ad7 receptor.
• the present invention also relates to a fiber of an adenovirus having a substantially reduced capacity for binding to the natural cellular receptor as presented above and nevertheless capable of trimerizing. Such a property is notably determined by the technique described in the experimental part of the application.
• the fiber according to the invention further comprises a ligand, or targeting element.
• ligand defines any entity capable of recognizing and binding or interacting, preferably with a strong affinity, with a cellular “anti-ligand” different from the natural cellular receptor of the fiber. non-mutated adenoviral (ie class I histocompatibility system antigens, fibronectin or the coxsackie virus (CAR) cell receptor.
• CAR coxsackie virus
• a ligand can be for example an antibody or an antibody fragment, a lipid, a glycolipid, a hormone, a polypeptide, a peptide of short size, a polymer (PEG, polylysine, PEI, ...), a sugar, an oligonucleotide, an antigen, a vitamin, all or part of a lectin, of the peptide JTS-1 (WO 94/40958) or else a combination of such compounds.
• antibody denotes in particular monoclonal antibodies, antibody fragments (such as for example Fab) and single chain antibodies (scFv). These names and abbreviations are conventional in the field of immunology.
• targeting elements make it possible to direct the particle or the pseudo-particle towards certain cell types or certain particular tissues (tumor cells, cells of the pulmonary epithelium, hematopoietic cells, muscle cells, nerve cells, etc.). They may also be elements facilitating the penetration into the cell or possibly lysis of the endosomes.
• they may be galactosyl residues making it possible to target the asialoglycoprotein receptor on the surface of liver cells, ligands which can interact with receptors such as growth factor receptors, cytokine receptors, lectins, adhesion proteins, it can also be an antibody fragment such as the Fab fragment, a fusogenic peptide INF-7 derived from the HA-2 subunit of the hemagglutinin of the influenza virus (Plank and al., 1994, J. Biol. Chem.
• the ligand can be an antibody fragment directed against fusine, the CD4 receptor or against a viral protein (envelope glycoprotein ) or also the part of the TAT protein of the HIV virus extending from residues 37 to 72 (Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91, 664-668).
• a tumor cell In the case of a tumor cell, the choice will be made on a ligand recognizing a specific tumor antigen (for example the protein MUC-1 in the case of breast cancer, certain epitopes of the E6 or E7 proteins of the HPV papilloma virus) or overexpressed (IL-2 receptor overexpressed in certain lymphoid tumors). If it is desired to target T cells, a T cell receptor ligand can be used. In addition, transferrin is a good candidate for liver targeting. In general, the ligands which can be used in the context of the invention are widely described in the literature and the genes coding for such ligands can be cloned by standard techniques (for example by amplification of cDNA, .. .).
• said ligand is inserted at the C-terminal end of the fiber according to the invention or as a replacement for the deleted residues when at least one of the modifications is a deletion of at least 3 residues consecutive.
• the ligand can be incorporated into the modified fiber according to the invention in different ways, and more particularly: - by cloning the nucleic sequence coding for said ligand into the nucleic sequence coding for fiber d interest changed.
• said nucleic acid sequence coding for the ligand is introduced at or near the region of the nucleic acid sequence of the fiber coding for the mutated region of said fiber; by incorporation directly on the fiber previously modified and produced, for example by chemical grafting.
• Another subject of the invention relates to a peptide fragment characterized in that it comprises the region extending from residue 491 to residue 505 of SEQ ID NO: 1 and in that said region is modified as described above.
• Such a peptide fragment has in particular the following properties: (i) when this peptide fragment is incorporated in place of a region extending from residues 491 to 505 of SEQ ID NO:
• the adenoviral particle comprising said modified fiber does not bind substantially to the natural cellular receptors of the unmodified adenoviral fiber;
• the adenoviral particle comprising said mutated fiber according to (i) further comprises a specific ligand for an anti- ligand, it is possible to confer on said modified adenoviral particle a new tropism towards one or more specific cell types carrying on their surface an anti-ligand surface in comparison with the adenoviral particle comprising said unmodified adenoviral fiber.
• the present invention also relates to a viral particle, in particular an adenoviral particle, which comprises on its surface a modified fiber or a peptide according to the invention, and optionally a ligand as defined above.
• this viral particle is devoid of functional native fiber or of any other peptide naturally involved in the binding of said viral particle to its target cell.
• a particle contains a viral genome
• a recombinant viral virus for example a recombinant adenovirus, and preferably a recombinant adenovirus defective for replication.
• the invention therefore also relates to such viruses and recombinant viruses.
• the modified fiber or said peptide, and optionally said ligand are identical to each other.
• (i) can be expressed by the viral genome itself, in particular when said viral particle ultimately contains such a genome.
• said genome contains the nucleic sequences necessary for the expression of said modified fiber or of a said peptide according to the invention, and optionally of a said ligand
• (ii) can be provided in trans by a complementation cell line, such as those defined below, which contains the nucleic sequences necessary for the expression of said modified fiber or of said peptide according to the invention, and optionally d 'undit ligand
• said viral particles are not adenoviral particles, and preferably are non-enveloped viral particles.
• viruses are widely described in general virology works, such as for example Fields et al. (1990, Virology, Raven Press, NY).
• the viral particle of the invention is as presented above and is characterized in that said ligand is inserted into a protein of the viral capsid other than the fiber, in particular hexon or penton in the specific case of adenoviral particles.
• said genomes and viral particles are genomes and adenoviral particles.
• the invention also relates to a viral pseudo-particle, in particular an adenoviral pseudo-particle, which comprises on its surface a mutated fiber or a peptide according to the invention, and optionally a ligand as defined above.
• said viral pseudo-particle of the invention is "empty", that is to say that it does not contain nucleic acid.
• the invention also relates to the cases in which such a viral pseudo-particle contains a macromolecule, and more particularly a nucleic acid which is not a viral or adenoviral genome.
• the use of such viral pseudo-particles is in particular illustrated in the document WO 95/21259 or US 5,928,944 cited above.
• the invention also relates to pseudo-particles which can also be called “artificial particles”.
• Such particles can in particular be produced after
• polar compounds such as lipids or glycolipids associated with amino- or carboxyterminal protein sequences, peptides or glycoproteins containing or consisting of the peptide sequences or the adenoviral fiber modified according to the invention and (ii) incorporation of said modified polar compounds into a structure of the liposome type.
• polar compounds such as lipids or glycolipids associated with amino- or carboxyterminal protein sequences, peptides or glycoproteins containing or consisting of the peptide sequences or the adenoviral fiber modified according to the invention
• incorporation of said modified polar compounds into a structure of the liposome type.
• said pseudo-particle is produced after
• lipids or cationic polymers modified so as to include in their structure such sequences or said modified adenoviral fiber.
• the literature provides a large number of examples of lipids which can be used according to the invention. Mention may also be made of the case of lipids or cationic polymers generally used as synthetic vectors for the transfer of nucleic acid into cells by transfection. By way of illustration but not limitation, they may be compounds such as those described in Feigner et al.,
• cationic compounds are capable of forming complexes (also called lipoplexes or polyplexes, or more generally posiplexes) with nucleic acids in order to allow their introduction into cells (transfection).
• complexes also called lipoplexes or polyplexes, or more generally posiplexes
• nucleic acids in order to allow their introduction into cells (transfection).
• the incorporation of such peptide or adenoviral fiber sequences modified according to the invention in said complexes allows for example to obtain posiplexes capable of targeting a cell type of interest.
• a ligand When a ligand is further included in the particle or pseudo-particle (viral or artificial), it can be chemically coupled to it.
• the variant according to which the sequences coding for the ligand are inserted within the viral and preferably adenoviral genome is preferred.
• said sequences are preferably inserted within the sequences coding for the modified fiber according to the invention, and more specifically in phase in order to preserve the reading frame.
• the insertion of the ligand coding sequence can take place anywhere in the viral genome, however the preferred insertion site is upstream of the stop codon at the C-terminus or in place of the deleted residues. It is also conceivable to introduce the ligand sequences within other viral sequences, in particular those coding for another capsid protein, such as the adenoviral hexon or penton.
• the invention relates to adenoviral particles containing a recombinant adenovirus defective for replication, ie incapable of autonomous replication in a host cell.
• the deficiency is obtained by mutation or deletion of one or more essential viral genes and, in particular, of all or part of the E1 region in the adenoviral genome. Deletions within the E3 region can be considered to increase the cloning capacities. However, it may be advantageous to keep the sequences coding for the protein gp19k (Gooding and Wood, 1990, Critical Revie s of Immunology 10, 53-71) in order to modulate the immune responses of the host.
• a recombinant virus (especially an adenovirus) of the invention comprises one or more gene (s) of interest placed under the control of the elements necessary for its (their) expression in a host cell .
• the gene in question can be of any origin, genomic, cDNA (complementary DNA) or hybrid
• polypeptide may be all or part of a polypeptide as found in nature, a chimeric polypeptide originating from the fusion of sequences of various origins, or a polypeptide mutated with respect to the sequence native with improved and / or modified biological properties.
• cytokines or lymphokines interferons and interleukins and in particular IL-2, IL-6, IL-10 or IL-12, tumor necrotizing factors (TNF), colony stimulating factors (GM-CSF, C-CSF, M-CSF ...); cellular or nuclear receptors, in particular those recognized by pathogenic organisms (viruses, bacteria, or parasites) and, preferably, by the VI H virus or their ligands; proteins involved in a genetic disease (factor VII, factor VIII, factor IX, dystrophin or minidystrophin, insulin, CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator) , growth hormones (hGH); enzymes (urease, renin, thrombin ....); enzyme inhibitors (1-antitrypsin, antithrombin III, viral protease inhibitors ...); polypeptides with anti-t
• HSV-1 HSV-1
• ricin ricin
• cholera toxin diphtheria
• immunotoxins immunotoxins
• markers ⁇ -galactosidase, luciferase .
• a recombinant adenovirus according to the invention can, in addition, comprise a selection gene making it possible to select or identify the infected cells.
• neo genes coding for neomycin phosphotransferase which confer resistance to the antibiotic G418, dhfr (Dihydrofolate Reductase), CAT (Chloramphenicol Acetyl transferase), pac (Puromycin Acetyl-Transferase) or gpt (Xanthine Guanine Phosphoribosyl Transferase) ).
• the selection genes are known to those skilled in the art.
• elements necessary for the expression of a gene of interest in a host cell is meant all of the elements allowing its transcription into RNA and the translation of an mRNA into protein.
• the promoter is of particular importance. In the context of the present invention, it can originate from any gene of eukaryotic or even viral origin and can be constitutive or regulable. Furthermore, it can be modified so as to improve the promoter activity, to suppress a region inhibiting transcription, to make a constitutive promoter regulable or vice versa, introduce a restriction site ... Alternatively, it may be the natural promoter of the gene to be expressed.
• CMV Cytomegalovirus
• RSV Raster Sarcoma Virus
• TK gene of the HSV-1 virus early SV40 virus (Simian Virus 40)
• adenoviral MLP or promoters eukaryotes of murine or human PGK (Phospho Glycerate kinase) genes
• 1-antitrypsin liver-specific
• immunoglobulins lymphocyte-specific
• a gene of interest in use in the present invention may also comprise additional elements necessary for expression (intronic sequence, signal sequence, nuclear localization sequence, transcription terminator sequence, site of initiation of the IRES or other translation ...) or its maintenance in the host cell. Such elements are known to those skilled in the art.
• the invention also relates to viral particles for which the incorporated genome is not an adenoviral genome.
• the viral genome is preferably selected from the viral genomes corresponding to non-enveloped viruses (Fields et al., 1990,
• the modified fiber or the peptide sequence of the invention can be incorporated into the particle either
• the present invention also relates to a DNA fragment coding for a fiber or a peptide fragment according to the invention, as well as to an expression vector comprising such a DNA fragment.
• any type of vector can be used for this purpose, whether it is of plasmid or viral origin, integrative or not.
• Such vectors are commercially available or described in the literature.
• a person skilled in the art is capable of adapting the regulatory elements necessary for the expression of the DNA fragment according to the invention.
• said vector will be an adenoviral vector capable of producing, under appropriate culture conditions, adenoviral particles according to the invention, namely adenoviruses or recombinant adenoviruses as described above.
• the invention also relates to a process for the preparation of adenoviral pseudo-particles according to the invention according to which: (i) the adenoviral genome coding for a modified fiber according to the invention is transfected into an appropriate cell line, for example line 293, PERC6, or a derivative or equivalent line); (ii) cultivating said transfected cell line is cultivated under conditions suitable for allowing the production of said adenovirus or of said recombinant adenovirus, and (iii) recovering the empty pseudo-particles by purifying the cell lysate on a density gradient, in particular a gradient cesium chloride for example.
• an appropriate cell line for example line 293, PERC6, or a derivative or equivalent line
• cultivating said transfected cell line is cultivated under conditions suitable for allowing the production of said adenovirus or of said recombinant adenovirus
• recovering the empty pseudo-particles by purifying the cell lysate on a density gradient, in particular
• the empty pseudo-particles sediment for example at 1.3 g / ml of cesium chloride while the recombinant adenoviruses (particles containing the adenoviral genome) sediment at 1.34 g / ml (D'Hallivin, 1995, Cur Top. Microbiol. Immunol, 199, 47-66).
• the invention also relates to a process for preparing an adenovirus or a recombinant adenovirus (adenoviral or recombinant adenoviral particles) according to the invention, according to which:
• said transfected cell line is cultured under conditions suitable for allowing the production of said adenovirus or of said recombinant adenovirus (one can also say, adenoviral particles), and
• 96022940 are particularly suitable for complementing the E1 function (Graham et al., 1977, J. Gen. Virol. 36, 59-72 or WO 97/00326, respectively).
• E1 and E2 or E4 For a double deficiency E1 and E2 or E4, one can use a line among those described in the French patent application FR 2737222 (96/04413).
• FR 2737222 96/04413
• a helper virus to complement the defective adenovirus according to the invention in any host cell or else a mixed system using complementation cell and helper virus in which the elements are dependent on each other.
• the means of propagation of a defective adenovirus are known to a person skilled in the art who can refer for example, Graham and Prevec, 1991, Methods in Molecular Biology, vol 7, p 190-128; Ed EJ Murey, The Human Press Inc.).
• the adenoviral genome is preferably reconstituted in vitro in Escherichia coli (E. coli) by ligation or even homologous recombination (see for example French application FR 2727689 (94/14470)).
• the purification methods are described in the state of the art. Mention may be made of the density gradient centrifugation technique.
• the present invention also relates to a cell line comprising either in the form integrated into the genome or in the form of an episome a DNA fragment coding for a fiber according to the invention placed under the control of the elements allowing its expression.
• Said line can be derived from a complementation cell of one or more adenoviral functions selected from those encoded by the regions E1, E2, E4 and L1-L5. It preferably derives from line 293 or from line PERC6.
• Such a line can be useful for the preparation of an adenovirus, in particular a recombinant one, the genome of which lacks all or part of the sequences coding for the fiber (so as to produce a non-functional or "redirected" fiber or not to produce fiber).
• the invention also relates to a method for producing adenoviral particles containing an adenoviral genome devoid of all or part of the sequences coding for a fiber, characterized in that:
• the said genome is transfected into a cell line presented above, (ii) the said transfected cell line is cultured under conditions suitable for allowing the production of the said adenoviral particle, and (iii) the said adenoviral particle is recovered in the culture of said transfected cell line and, optionally, (iv) purifying said adenoviral particle.
• the present invention also covers a host cell which can be infected with an adenovirus according to the invention or which can be obtained by a method according to the invention.
• a mammalian cell and, in particular, a human cell.
• It can be primary or tumor and of any origin, for example hematopoietic (totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage ...), muscular, nasal, pulmonary, tracheal, hepatic, epithelial, retinal (for example HER for Human Embryonic retinocyte) or fibroblast.
• the subject of the invention is also a composition
• a composition comprising, as therapeutic or prophylactic agent, a host cell, a viral particle, in particular adenoviral, or a pseudo-particle (viral or artificial), according to the invention or capable of being obtained by a process according to the invention, in combination with a support which is acceptable from a pharmaceutical point of view.
• composition according to the invention is, in particular, intended for the preventive or curative treatment of diseases such as genetic diseases (hemophilia, cystic fibrosis, diabetes or Duchenne, Becker's myopathy ...), cancers, such as those induced by oncogenes or viruses, viral diseases, such as hepatitis B or C and AIDS (acquired immunodeficiency syndrome resulting from HIV infection), and recurrent viral diseases, such as viral infections caused by the virus herpes.
• diseases such as genetic diseases (hemophilia, cystic fibrosis, diabetes or Duchenne, Becker's myopathy )
• cancers such as those induced by oncogenes or viruses
• viral diseases such as hepatitis B or C and AIDS (acquired immunodeficiency syndrome resulting from HIV infection)
• recurrent viral diseases such as viral infections caused by the virus herpes.
• the composition containing a viral particle, in particular adenoviral or a viral or artificial pseudo-particle of the invention further comprises a nucleic acid and a cationic substance such as for example monocationic lipids (for example DOTMA, lipofectin, DOTAP, DOPE ...), polycationic lipids (DOGS, ffransfectam, Lipofectamine, ...), cholesterol or its derivatives, phospholipids, polycarbenes ( polybrene, %), carbohydrates (DEAE-dextran, polyamino acids, ...), cationic polymers, ... It can also include neutral or uncharged lipids.
• monocationic lipids for example DOTMA, lipofectin, DOTAP, DOPE
• polycationic lipids DOGS, ffransfectam, Lipofectamine, ...)
• cholesterol or its derivatives cholesterol or its derivatives
• phospholipids polycarbenes ( polybrene, %)
• carbohydrates DEAE-dextran, poly
• said nucleic acid contains one or more gene (s) of interest placed under the control of the elements necessary for its (their) expression in a host cell and / or a selection marker as described above.
• a composition according to the invention can be produced in a conventional manner.
• the therapeutic or prophylactic agent is combined with a pharmaceutically acceptable carrier or diluent.
• a carrier or such a diluent is non-toxic to the patient. It may be a solution for injection, an isotonic solution, the pH of which is compatible with in vivo use, a solution of dextrose, glycerol, mannitol, or the like and also optionally containing dispersing agents and / or pharmacologically compatible wetting agents.
• a composition according to the invention can be administered by the local, systemic or aerosol route, in particular by the intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intrapulmonary, intranasal or intratracheal route.
• the administration can take place in single dose or repeated one or more times after a certain interval of interval.
• the appropriate route of administration and dosage vary depending on various parameters, for example, the individual or disease to be treated or the gene (s) of interest to be transferred.
• the viral particles according to the invention can be formulated in doses of between 10 4 and 1 ⁇ 1 4 pfu (units forming plaques), advantageously 10 ⁇ and 1 ⁇ 13 pfu and, preferably, 10 ⁇ and 10 ⁇ 2 pfu .
• the formulation may also include a pharmaceutically acceptable adjuvant or excipient.
• the composition according to the invention can also be formulated in the form of a solid, semi-solid preparation, in particular in the form of a gas, a tablet, a capsule, a powder, a capsule, a granule, a cream, a solution, a suppository, an aerosol, depending on the route of administration selected.
• the active ingredient can be formulated with conventional pharmaceutical carriers known to those skilled in the art.
• These supports include in particular a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, sucrose, gum arabic or the like.
• a preparation of capsules can also be obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard capsules.
• a preparation in the form of a syrup or elixir may contain the active ingredient together with a sweetener, an antiseptic, as well as a flavoring agent and an appropriate color.
• the water-dispersible powders or granules may contain the active ingredient in admixture with dispersing agents or wetting agents, or suspending agents, as well as with flavor correctors or sweeteners.
• Suppositories are used for rectal administration which are prepared with binders that melt at rectal temperature, for example cocoa butter or polyethylene glycols.
• the active principle can also be formulated in the form of microcapsules, optionally with one or more additive supports.
• the viral particles, in particular adenoviral, or the pseudo-particles according to the invention can also be complexed or associated with synthetic compounds or natural as described in the introductory part of this application, in O'Riordan et al., (1999, Human Gene Therapy, 10, 1349-1358) or in patent application WO 98/44143 or US patent 5,928,944 .
• the content of these documents is incorporated into the present request by reference.
• the present invention relates to the use of a peptide fragment, of a modified adenoviral fiber, of a viral particle, in particular adenoviral, of a pseudo-particle or of a host cell according to the invention or of an adenovirus capable of being obtained by a method according to the invention, for the preparation of a medicament intended for the treatment of the human or animal body.
• the drug can be administered directly in vivo (for example by intravenous injection, in an accessible tumor, in the lungs by aerosol ).
• the invention also extends to a treatment method according to which a therapeutically effective amount of an adenovirus or of a host cell or of a pharmaceutical composition according to the invention is administered to a patient in need of such treatment .
• a therapeutically effective amount is such that it makes it possible to observe a desired effect in the organism treated which can be followed by various techniques well known to those skilled in the art.
• a desired effect may consist of the transfer of a nucleic acid into the target cells of the host.
• Such a transfer can be evaluated by any method demonstrating either said transfer or the expression of a gene encoded by said nucleic acid (by polymerase chain reaction, PCR, by hybridization analyzes by Northern or by Southern, by immunodetection of the encoded peptide .).
• PCR polymerase chain reaction
• hybridization analyzes by Northern or by Southern, by immunodetection of the encoded peptide .
• the invention relates to the use of a peptide fragment, of a modified adenoviral fiber, of a viral particle, in particular adenoviral, of a pseudo-particle or of a host cell according to the invention.
• invention or an adenovirus capable of being obtained by a method according to the invention to allow the transfer of a nucleic acid (plasmid, DNA, RNA, PNA, viral genome, etc.) of interest into target cells , in vivo or in vitro.
• the invention relates to antibodies specifically directed against a modified adenoviral fiber according to the invention.
• Such antibodies, and especially monoclonal antibodies, are easily obtained by immunization of animals immunocompetent with the peptide sequences or the fibers modified according to the invention in accordance with the usual practices in the field of the production of antibodies from a peptide or an identified polypeptide.
• Such antibodies, and in particular specific antibodies can be useful for the implementation, for example, of monitoring the treatment of patients to which the particles or pseudo-particles of the invention are administered. They can also be used for the preparation of a composition intended for administration in vivo in the patient treated with the particles or pseudo-particles of the invention in order to allow the interruption of said treatment.
• An antibody can also be modified to allow its detection, for example by incorporating a marker or an enzyme capable of reacting with a substrate, in particular a chromogenic substrate. Such applications are well known in the field of diagnostics.
• the NM522 (Stratagene) strain is suitable for the propagation of phage vectors M13.
• Amplification techniques by PCR are known to those skilled in the art (see for example PCR Protocols-A guide to methods and applications, 1990, published by Innis, Gelfand, Sninsky and White, Académie Press Inc).
• the technique used consists of filling the protruding 5 'ends with the large fragment of DNA polymerase I from E. coli (Klenow).
• the Ad5 nucleotide sequences are those used in the Genbank database under the reference M73260.
• the cells are transfected according to standard techniques well known to those skilled in the art. Mention may be made of the calcium phosphate technique (Maniatis et al., Supra), but any other protocol can also be used, such as the DEAE dextran technique, electroporation, methods based on osmotic shock, microinjection or methods based on the use of cationic lipids. As for the growing conditions, they are classic.
• EXAMPLE 1 Construction of an adenovirus having a host tropism towards cells expressing the GRP receptor (for qastrin releasing peptide in English).
• the plasmid pTG6593 is derived from p poly II (Lathe et al., 1987, Gene 57, 193-201) by introduction of the complete gene coding for the Ad ⁇ fiber in the form of an EcoRI-Smal fragment (nucleotides (nt) 30049 to 33093).
• the Hind ⁇ - Sma ⁇ fragment (nt 31994-33093) is isolated and cloned into M13TG130 (Kieny et al., 1983, Gene 26, 91-99) digested with these same enzymes, to give M13TG6526.
• the latter is subjected to a directed mutagenesis using the oligonucleotide OTG7000 (SEQ ID NO: 2) (Sculptor kit, in vitro mutagenesis, Amersham) in order to introduce an adapter coding for a spacer arm of 12 amino acids of PSASASASAPGS sequence.
• the mutated vector thus obtained, M13TG6527 is subjected to a second mutagenesis making it possible to introduce the sequence coding for the 10 residues of the GRP peptide (GNHWAVGHLM; Michael and al., 1995, Gene Ther. 2, 660-668).
• the oligonucleotide OTG7001 (SEQ ID NO: 3) is used for this purpose.
• the H / ⁇ dlll-S al fragment is isolated from the mutated phage M13TG6528 and introduced by the homologous recombination technique (Chartier et al., 1996, J. Virol. 70, 4805-4810) into the plasmid pTG6590 carrying the adenoviral genome fragment Ad5 extending from nt 27081 to 35935 and linearized by Mun ⁇ (nt 32825).
• the Spel-Scal fragment (carrying nt 27082 to 35935 of the Ad5 genome modified by introduction of the spacer arm and of the GRP peptide) is isolated from the preceding vector designated pTG8599 then is exchanged for the equivalent fragment of pTG6591 previously digested with these same enzymes.
• pTG6591 comprises the wild adenoviral sequences from positions 21562 to 35935.
• pTG4600 from which the BsfEII fragment (nt 24843 to 35233) is isolated.
• the vector pTG4601 is generated.
• a cassette allowing the expression of the LacZ gene is introduced in place of the adenoviral region E1 by homologous recombination between the plasmid pTG4601 linearized by C / al and a BsrG ⁇ -Pst ⁇ fragment comprising the LacZ gene coding for ⁇ -galactosidase under control of the MLP promoter of Ad2 and the polyadenylation signal of the SV40 virus.
• This fragment is isolated from the vector pTG8526 containing the 5 ′ end of the viral genomic DNA (nt 1 to 6241) in which the E1 region (nt 459 to 3328) is replaced by the LacZ expression cassette. Its construction is within the reach of the skilled person.
• the final vector is designated pTG4628.
• the corresponding viruses AdTG4601 and AdTG4628 are obtained by transfection of the adenoviral fragments released from the plasmid sequences by Pacl digestion in line 293.
• AdTG4601 carries the complete Ad5 genome in which the fiber gene comprises at its 3 ′ end an arm spacer followed by the GRP peptide.
• the recombinant virus AdTG4628 further carries the expression cassette for the LacZ reporter gene under the control of the adenoviral promoter MLP.
• the expression of the messengers coding for the latter is studied in 293 cells and in Swiss-3T3 murine cells (Zachary et al., 1985, Proc. Natl. Acad. Sci. USA 82, 7616-7620) by Northern-blot .
• a mixture of 2 DNA fragments complementary to the sequence coding for the GRP receptor, labeled by conventional techniques with the 32p isotope is used.
• the fragments are produced by reverse PCR from cellular RNA. totals using the oligonucleotides OTG10776 (SEQ ID NO: 4) and oTG10781 (SEQ ID NO: 5) (Battey et al., 1991, Proc. Natl. Acad. Sci.
• the competitor consists of the head of the Ad ⁇ fiber produced in E. coli, the properties of which have been shown to bind to the adenoviral cellular receptor (Henry et al., 1994, J. Virol 68, 5239-5246).
• the monolayer cells are previously incubated for 30 min in the presence of PBS or of increasing concentrations of recombinant Ad5 head (0.1 to 100 ⁇ g / ml) in DMEM medium (Gibco BRL) supplemented with 2% fetal calf serum ( FCS).
• the AdTG4628 virus the fiber of which contains the GRP peptide
• the recombinant AdLacZ virus (Stratford-Perricaudet et al., 1992, J. Clin. Invest. 90, 626-630) which carries a native fiber gene is used as a control and under the same experimental conditions.
• the cells are then fixed and the expression of the LacZ gene evaluated (Sanes et al., 1986, EMBO J. 5, 3133-3142).
• the number of blue cells is representative of the effectiveness of the viral infection.
• Competitive inhibition results in a reduction in the number of stained cells compared to an uninfected control (PBS).
• EXAMPLE 2 Construction of an adenovirus exhibiting a tropism towards tumor cells expressing mucins
• EXAMPLE 3 Construction of an adenovirus exhibiting a tropism towards tumor cells expressing ⁇ 4 ⁇ 1 integrins
• EXAMPLE 4 Construction of an adenovirus having a host tropism towards cells expressing the EGF receptor (Epidermal Growth Factor in English).
• This example describes a fiber carrying the EGF sequences at its C-terminal end.
• the oligonucleotides OTG11065 (SEQ ID NO: 6) and oTG11066 (SEQ ID NO: 7) are used to amplify a Hind ⁇ - Xba ⁇ fragment from the plasmid M13TG6527.
• the oligonucleotides OTG11067 (SEQ ID NO: 8) and 0TGHO68 (SEQ ID NO: 9) make it possible to generate an Xho ⁇ -Sma ⁇ fragment (ranging from the stop codon to nt 33093) from M13TG6527.
• the complementary EGF DNA obtained from ATCC (# 59957), is amplified in the form of an Xho ⁇ -Xba ⁇ fragment using the oligonucleotides oTG 11069 (SEQ ID NO: 10) and OTG11070 ( SEQ ID NO: 11).
• the 3 fragments digested with the appropriate enzymes are then religated to give a Hind ⁇ - Sma ⁇ fragment containing the EGF fused at the C-terminal end of the fiber.
• the same homologous recombination procedure as that described in Example 1 is applied to place this fragment in its genomic context.
• the cloning steps can be simplified by introducing a single Ss BI site into the region targeted by conventional mutagenesis techniques on the M13TG6526 matrix using the oligo oTG7213 (SEQ ID NO: 57).
• the plasmid carrying the total genome of the modified Ad ⁇ pTG4609 is obtained. Its equivalent carrying the LacZ expression cassette in place of the E1 region is obtained as previously described in Example 1 and is named pTG4213.
• the homologous recombination between pTG4609 or pTG4213 linearized by ⁇ sfBI and the preceding Hind ⁇ - Sma ⁇ fragment generates the plasmid pTG4225 and pTG4226 carrying the E1 region respectively. wild or the LacZ expression cassette.
• the AdTG4225 and AdTG4226 viruses can be produced conventionally by transfection of an appropriate cell line, for example overexpressing the EGF receptor. To test the specificity of infection of these viruses, murine fibroblast cells NR6 and NR6-hEGFR cells expressing the human EGF receptor can be used. Competitions with the recombinant Ad ⁇ head or with EGF make it possible to evaluate the intervention of natural cellular receptors and EGF to mediate virus infection.
• the mutation of the adenoviral fiber region involved in the interaction with the natural cellular receptor has been undertaken in order to eliminate the ability of the fiber to bind its natural receptor and the addition of a ligand will modify the tropism of corresponding adenoviruses.
• the Ad5 fiber sequences encoding the region extending from residues 443 to 462 have been subjected to various mutations.
• the deletion of sheet D implements the mutagenesis oligonucleotide oTG7414 (SEQ ID NO: 12) and the deletion of the CD loop the oligonucleotide oTGA (SEQ ID NO: 13).
• the oligonucleotide oTGB (SEQ ID NO: 14) allows the deletion of the CD loop and of sheet D.
• oligonucleotides contain a ⁇ amHI site making it possible to easily detect the mutants and, also, to insert the sequences coding for a ligand, for example the EGF peptide.
• Another series of modifications consists in replacing these deleted regions by the equivalent sequences originating from the Ad3 fiber. Indeed, numerous data show that Ad ⁇ and Ad3 do not bind to the same receptor, so that such a substitution should abolish infection mediated by the Ad5 receptor and target the cells carrying the Ad3 receptor.
• This target region of the adenoviral head was also modified by a series of point mutations: - replacement of the elbow aa GSLA in elbow aa DKLT: oTGC
• G443 in D oTGE (SEQ ID NO: 20), - L44 ⁇ in F: oTGF (SEQ ID NO: 21),
• OTGE to I introduce mutations in the CD loop of the adenoviral fiber on amino acids which are non-conservative between Ad ⁇ and Ad3 whereas oTGJ to K relate to amino acids of the D sheet not committed in a hydrogen bond stabilizing the structure.
• Mutagenesis can be carried out on the vector M13TG6 ⁇ 26 or M13TG6528.
• the first carries the wild Hind ⁇ - Sma ⁇ fragment and the second this same fragment modified by the insertion of GRP sequences.
• the plasmids carrying the adenoviral genome can be reconstituted as described above for the plasmids pTG422 ⁇ (wild E1) and pTG4226 (LacZ in place of the E1 region).
• Viruses are generated by transfection of 293 cells or of cells overexpressing the ligand-binding receptor concerned. Such cells can be generated by transfection of the corresponding complementary DNA.
• Cells which do not naturally express the natural cellular receptor for adenoviruses are preferably used, for example the Daudi line (ATCC CCL213).
• EXAMPLE 6 Modifications of the fiber head to eliminate the binding to the natural cellular receptor.
• the mutation of the AB region (residues 404-418) of the adenoviral fiber was undertaken in order to eliminate the ability of the fiber to bind its natural receptor and the addition of a ligand will make it possible to modify the tropism of the corresponding adenoviruses.
• Mutagenesis can be carried out on the vector M13TG6526 or M13TG6528.
• the first carries the wild H / n III-SmaI fragment and the second this same fragment modified by the insertion of GRP sequences.
• the plasmids carrying the adenoviral genome can be reconstituted as described previously for the plasmids pTG422 ⁇ (wild E1) and pTG4226 (LacZ in place of the E1 region) (by homologous recombination with the plasmid pTG4609 or else pTG4213).
• Viruses are generated by transfection of cells 293, 293 expressing wild fiber (Legrand et al., 1999; J.
• Virol., 73, 907-919 or else cells overexpressing the ligand-binding receptor concerned.
• Such cells can be generated by transfection of the corresponding complementary DNA.
• Cells which do not naturally express the natural cellular receptor for adenoviruses are preferably used, for example the Daudi line (ATCC CCL213).
• the viruses purified after amplification in 293 cells are deposited on 10% acrylamide gel under denaturing conditions (SDS-PAGE).
• the different proteins are detected by staining with silver nitrate.
• the fiber is revealed specifically by performing a westem-blot using a serum directed against the head of the Ad ⁇ fiber (Henry et al., 1994, supra).
• An intense signal at the expected size indicates that the viruses incorporate stoichiometric amounts of the protein of interest. Since only the trimeric fiber is capable of binding the penton base (Novelli and Boulanger, 1991, supra) and of being incorporated into the particle, detection of the protein in the above experiment indicates that the modified fiber is still capable of forming trimer.
• the use of the modified fiber to allow the entry of the corresponding mutated virus can be studied by carrying out the competition experiments using a recombinant head as described above in Example 1B. Effective infection in the presence of saturated concentrations of wild peptide indicates infection independent of receptor binding natural primaries. This suggests a greatly reduced affinity of the modified fiber for its receptors.
• EXAMPLE 7 Insertion of the ligand into a capsid protein other than the fiber in combination with one of the modifications of the aforementioned fiber.
• This example describes the insertion of the EGF ligand into the hexon capsid protein.
• the corresponding adenovirus has lost its capacity for attachment to the natural cellular receptor.
• Its genome may for example include a modified fiber gene or be devoid of at least part of the fiber sequences.
• a transfer plasmid is constructed for homologous recombination covering the region of the Ad ⁇ genome coding for hexon (nt 18842-21700).
• the Ad ⁇ Hin ⁇ ⁇ - Xho ⁇ fragment (nt 18836-24816) is cloned into pBSK + (Stratagene) digested with these same enzymes to give the plasmid pTG4224.
• the sequences encoding the EGF peptide are introduced into the hypervariable loop L1 of the hexon by creation of chimeric fragments by PCR: hexon (nt19043-19647) -X6al-EGF- ⁇ stGI- hexon (nt19699-20312).
• the fragment nt19043 to 19647 is obtained by PCR amplification from the plasmid pTG3602 with the oligonucleotides 0 OTG11102 (SEQ ID NO: 28) and OTG11103 (SEQ ID NO: 29).
• the nt19699 to 20312 fragment is amplified from the same DNA with the oligonucleotides OTG11104 (SEQ ID NO: 30) and oTG11105 (SEQ ID NO: 31).
• EGF is cloned from the cDNA using the oligonucleotides OTG11106 (SEQ ID NO: 32) and oTG11107 (SEQ ID NO: 33) making it possible to ⁇ put the coding sequence of EGF in phase with hexon.
• the PCR products are digested with the appropriate enzymes and then religated.
• the chimeric fragment can then be inserted by homologous recombination into the plasmid pTG4224 linearized with Nde ⁇ (nt 19549), to give pTG4229.
• the sequences coding for the modified hexon can be obtained by 0 Hin ⁇ ⁇ - Xho ⁇ digestion and replaced in their genomic context by homologous recombination.
• One can use the vector pTG3602, pTG4607, pTG4629 linearized by Sgf ⁇ or a vector carrying the adenoviral genome deleted sequences from the fiber (as pTG4607 described below) or expressing a modified fiber.
• the adenoviral genome incapable of producing a functional native fiber is obtained by a deletion touching the initiator codon but not extending to the other adenoviral ORFs.
• the procedure is as follows: the adenoviral fragment at ⁇ 'of the deletion (nt 30664 to 31041) is amplified by PCR using the primers oTG7171 and oTG727 ⁇ (SEQ ID NO: 34 and 3 ⁇ ). Amplification of the 3 ′ fragment (nt 31129 to 33099) uses the primers oTG7276 and OTG7049 (SEQ ID NO: 36 and 37).
• the PCR fragments are digested with Xho ⁇ and ligated before being introduced by homologous recombination into the vector pTG6591 linearized by Nde ⁇ , to give pTG4602. Then the BstEW fragment isolated from the latter is subjected to homologous recombination with the vector pTG3602 digested with Spel. We obtain pTG4607.
• the vector pTG4629 is equivalent to pTG4607, but also carries the LacZ expression cassette in place of E1.
• the corresponding viruses can be obtained after transfection of cells 293, 293 expressing wild fiber (Legrand et al., 1999, supra) or cells overexpressing the EGF receptor.
• the study of the specificity of infection can be carried out as described previously using the EGF as a competitor (see Example 1).
• EXAMPLE 8 Construction of an adenovirus exhibiting a tropism with regard to cells expressing particular glycoproteins, namely heparan sulfate qlycoproteins
• This example describes a fiber carrying seven lysine residues located following the linker polypeptide (PSASASASAPGS) coded by SEQ ID NO: 2, at the C-terminal end and which confer the property of binding to glycoproteins called "heparan sulfate glycoproteins" .
• the oligonucleotide oTG12125 (SEQ ID NO: 43) is hybridized to the construct M13TG6527 in order to generate by mutagenesis the construct M13TG6670.
• the homologous recombination step between the linearized plasmid pTG4213 by cleavage with SsfBI and the H / ⁇ III-SmaI fragment of M13TG6670 makes it possible to obtain the plasmid pTG4274.
• the corresponding adenoviral virus AdTG4274 is obtained after transfection of said plasmid in the complementation line 293 and culture under the usual culture conditions, ⁇
• competition experiments with a recombinant adenovirus can be performed (Example 1).
• EXAMPLE 9 Construction of adenoviruses having a host specificity 0 directed to tumor cells expressing mucins.
• This example describes the production of a fiber carrying at its C terminal end the peptide EPPT (described in US Pat. No. 6,691,693) which confers specificity with regard to the mucins overexpressed on the surface of certain 6 tumor cells.
• Construction M13TG6627 is subjected to a mutagenesis step directed using the oligonucleotide (SEQ ID NO: 44) in order to generate the construction M13TG6672.
• the plasmid pTG4278 is obtained according to the procedure described above. The introduction of the mutation into the viral genome is carried out as described in Example 8.
• Virus 0 AdTG4278 is produced after transformation and culture in line 293.
• Competitive experiments of viral infection using the adenoviral head and with the soluble peptide EPPT shows that it is possible to assess the implication natural cellular receptors and mucins in the infectious process of adenovirus. ⁇
• Example 10 Construction of an adenovirus exhibiting specificity with respect to tumor cells expressing the ⁇ 4 ⁇ 1 integrins.
• This example describes an adenoviral fiber carrying the peptide sequence LDV 0 (see US 6,628,979) at its C-terminal end in order to confer on the protein the ability to bind to expressing the ⁇ 4 ⁇ 1 integrins which are overexpressed on the surface of certain tumor cells.
• the vector M13TG6627 is subjected to a mutagenesis step directed using OTG11991 (SEQ ID NO: 4 ⁇ ) in order to generate the modified vector M13TG 13265.
• the incorporation of this mutation into the viral genome is carried out according to the protocol described in Example 6.
• the virus is then produced using the complementation line 293. Competition experiments are carried out according to the protocol described in Example 1 using the fiber head of 'Ad ⁇ produced in E.
• Example 11 Modification of the fiber head so as to eliminate the binding to its natural cellular receptor.
• the mutagenesis is carried out using the vectors M13TG6626, ⁇ M13TG6628, M13TG6670, M13TG6572 or M13TG13265 described in the previous examples.
• the plasmids carrying the adenoviral genome are produced as indicated above and the viral particles are obtained by transfection and culture in: the cell line 293, 0 - or the cell line 293 expressing the wild adenoviral fiber (Legrand et al., 1999, J. Virol, 73, 907-919), or cells overexpressing the receptor corresponding to the ligand used. Such cells can be generated by transfection with the corresponding cDNA.
• ⁇ cells which do not express the natural adenoviral receptor are preferably used, such as for example the Daudi (ATCC CCL213) or CHO (ATCC Ccl61) line.
• the modified viral particles are deposited on a 10% SDS polyacrylamide gel.
• the different viral proteins are revealed by silver staining.
• the fiber protein is then specifically detected by "western blot" using antibodies directed against the adenoviral head (Henry et al., 6 1994, supra). In the present case, a strong signal is observed for a band whose migration corresponds to the expected size. This clearly shows that the viruses incorporated during their production a stoichiometric amount of mutated fiber. Since only the fiber in its trimeric form can bind the penton base and then be packaged (Novelli 0 and Boulanger, 1991, supra), the presence of this signal at the expected position indicates that the fiber modified according to the invention is always capable of trimerizing.

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