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  • C12N2710/10011—Adenoviridae
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
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  • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

• the present invention relates in particular to an adenoviral fiber, mutated in the regions involved in the recognition and the binding to the natural cellular receptor of adenoviruses. It also relates to adenoviral particles carrying on their surface such a fiber, possibly combined with a ligand which gives said particles a modified, even targeted, host specificity.
• the invention is of particular interest in the context of the development of vectors usable in the context of gene therapy. Adenoviral vectors are widely used in many gene therapy applications. Highlighted in many animal species, they are not very pathogenic, non-integrative and replicate in both dividing and quiescent cells.
• 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 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 MLP (for Major Late Promoter).
• Adenoviruses have been the subject of numerous studies and numerous scientific teams have 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 proliferating. in the cells they infect.
• the defective adenoviral vectors are in particular obtained by deletion of at least the E1 region (for examples of defective adenoviral vectors, see in particular patent applications WO9428152 and WO 9412649).
• patent application WO 95/21259 describes a method for introducing a nucleic acid into a cell based on the combination of adenoviral particles and nucleic acid, more particularly naked nucleic acid. This method is mainly based on the ability of the adenoviral particle to transport molecules to the cell nucleus after endocytosis. Curiel et al. (1992 Hum. Gene Ther., 3: 147-154) and Wagner et al. (1992, Proc. Natl. Acad.
• the infectious cycle of adenoviruses is based on two essential stages.
• the early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and transcription of viral DNA.
• the replication of the genome is followed by the late phase during which the structural proteins which constitute the basis of the viral particles are synthesized. New virions are assembled in the nucleus.
• 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 infect other permissive cells.
• the fiber and the penton base of the adenoviral particle present on the surface of the capsids play a critical role in the cellular attachment of virions and their internalization (Wickham et al., 1993, Cell, 73, 309 - 319).
• the adenovirus binds to a cellular receptor (CAR) present on the surface of permissive cells via the fiber in its trimeric form (Philipson et al., 1968, J. Virol. 2, 1064 -1075; Defer et al., 1990, J. Virol. 64, 3661-3673).
• CAR cellular receptor
• the viral particle is then internalized by endocytosis thanks to the binding of the penton base to the cellular integrins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 (Mathias et al., 1994, J. Virol. 68, 6811-6814).
• 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 composed of a certain number of leaf repetitions, the number of which varies according to the serotypes considered;
• adenoviral particles for which the native fiber has been modified so as to modify their natural tropism and change the binding specificity of this fiber so that it recognizes a different cellular receptor.
• WO94 / 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 the one of the 22 repeating units of the rod. These mutants have a modified specificity for infection of adenoviral particles and 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 the cells expressing the TNF cell receptor or LDL (low density lipoprotein) receptor, respectively, present on the surface of liver cells.
• TNF tumor necrosis factor
• LDL low density lipoprotein
• WO95 / 26412 describes a fiber modified by the incorporation of a ligand at the C-terminal end.
• WO96 / 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 at the end C-terminal of an RGD peptide specific for vitronectin.
• French patent application FR2758821 (97 01 005) has demonstrated the role of the antigens of the major class I histocompatibility complex and of the fibronectin modules III as primary receptor and co-factor, respectively. adenoviruses.
• Tomko et al. (1997, Proc. Natl. Acad. Sci 94, 3352-3356) Bergelson et al.
• Xia et al. (1994, Structure 2, 1259-1270) determined the three-dimensional crystallographic structure of the adenoviral head.
• 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 R sheets are involved in the interaction with the receptor.
• the present invention provides new adenoviral fiber mutants which in particular allow viral particles to be obtained which have the following properties: (i) the adenoviral particle comprising said mutated fiber does not substantially bind to natural cellular receptors, it that is to say that the host specificity of these adenoviral particles carrying the mutated fiber is reduced, or even inhibited, compared to the host specificity of the adenoviral particles carrying the wild fiber, ie not mutated; (ii) when the adenoviral particle comprising said mutated fiber further comprises a specific ligand of 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 adenoviral particle.
• the mutated fiber does not bind substantially to the natural cellular receptors
• the fiber is modified so as to reduce or abolish its capacity of binding to the natural cellular receptor.
• Such a property can be verified by the study of the infectivity or of the cellular bond of the corresponding adenoviral particles by applying the techniques of the art, and in particular by competition experiments of infection of the virus carrying the modified fiber carried out in 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).
• a mutated fiber does not bind substantially to natural cell receptors" when the percentage of residual infection, measured by a competition experiment as disclosed in the Examples which follow, is between approximately 0 and 60%, preferably between 0 and 40% and very 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 implemented in the Examples which follow.
• the present invention has in particular the advantage of proposing new products whose properties make it possible to reduce the therapeutic amounts of adenovirus to be administered and to target the infection of the vector at the level of the cells to be treated.
• This specificity is particularly essential when using an adenoviral vector capable of expressing a cytotoxic gene in order to avoid the propagation of the cytotoxic effect to healthy and non-targeted cells.
• teachings of the present invention allow the development of other targeting systems intended for the development of methods of treatment by administration of viral or non-viral recombinant vectors.
• the present invention relates to the fiber of a modified adenovirus comprising at least one mutation at one or more residues included in the region of said fiber extending from sheet A to sheet B, and including the loop AB . More particularly, the mutations are preferably carried out at the level of one or more residues included in the AB loop.
• nucleic acid sequence is meant 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 of which said part is derived, in particular the ability to recognize and bind to 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.
• the fiber according to the present invention can derive from an adenovirus of human, canine, avian, bovine, murine, ovine, porcine or simian origin or even be hybrid and comprise fragments of various origins, including fragments of origin heterologous, ie not derived from adenoviral fiber or derived from non-adenoviral fibers.
• Concerning human adenoviruses it is preferred to use those of serotype C and, in particular, adenoviruses of type 2 or 5 (Ad2 or Ad5).
• Ad2 fiber contains 580 amino acids (aa), the sequence of which is disclosed in Hérissé et al. (1981, Nucleic Acid Res. 9, 4023-4042, incorporated by reference into the present application).
• Ad5 was determined by Chroboczek and Jacrot (1987, Virology 161, 549-554, incorporated by reference) and has 582 amino acids (see sequence identifier 1; SEQ ID NO: 1). In order to simplify the presentation of the present application, only the positions relating to Ad5 are indicated. However, it is within the capacity of those skilled in the art to identify the equivalent positions of the various sheets and loops on the basis of the sequences of adenoviral fibers of other origins. When 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.
• 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, provided that they do not affect the characteristics of the fibers proposed in the application.
• GenBank references for the adenoviral fiber sequences of the human serotype 2 (# AAA92223), 3 (# CAA26029), 5 (# M18369), 31 (# CAA54050 or 41 (# X17016).
• GenBank publications or references cited above are incorporated by reference into the present application in their entirety.
• the invention also relates to a modified fiber according to the present invention which also contains other mutations such as for example those described in patent application WO 98/44121.
• such a fiber according to the invention is characterized in that it further comprises one or more mutations in: a) CD, DG, GH, Hl and / or IJ loops and / or b) C sheets , D, G, H, I and / or J.
• mutant denotes a deletion, a substitution or even an addition of one or more residues or a combination of these possibilities.
• the adenoviral fiber according to the invention derives from a fiber of a type 5 adenovirus (Ad5) comprising all or part of the sequence as shown in the sequence identifier n ° 1 (SEQ ID NO: 1) and is characterized in that it is modified by mutation of one or more residues of the region comprised between residues 400 and 428, more particularly comprised between residues 404 and 418 and preferably comprised between residues 404 and 408 of SEQ ID NO: 1.
• Ad5 type 5 adenovirus
• the invention relates to a fiber of an adenovirus type 5 characterized in that the mutated residue is selected from threonine residues in position 404, alanine in position 406 and serine in position 408. Due to their spatial location in native fiber, these residues are capable of recognizing and / or interacting directly or indirectly with the natural cellular receptor of the adenovirus concerned.
• the mutation carried out is a substitution of at least one amino acid.
• the following examples of fiber of a type 5 adenovirus may be cited for which: 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, and / or the threonine residue in position 404 is substituted by a glycine residue and / or the alanine residue at position 406 is substituted by a lysine residue.
• the fiber of the present invention can also be modified by deletion.
• the eliminated region can concern all or part of the exposed area and, in particular of the AB loop.
• 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 infection of types 2 and 5 adenoviruses is different from that of types 3 and 7 adenoviruses.
• residues deleted from an Ad ⁇ or Ad2 fiber deleted from at least 3 consecutive residues among those specified above can be substituted by residues from an equivalent region of the Ad3 or Ad7 fiber to reduce the ability of said fiber to bind the Ad ⁇ receptor and give it a new specificity towards the receptor Ad3 or Ad7 cell.
• 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 also comprises a ligand.
• ligand defines any entity capable of recognizing and binding, preferably with a high affinity, a cellular anti-ligand different from the natural cellular receptor for adenoviral fiber, not mutated. This anti-ligand can be expressed or exposed to the surface of the cell that it is desired to target (cell surface marker, receptor, antigenic peptide presented by histocompatibility antigens, etc.), naturally or following a modification of said target intended to make it express or exhibit such an anti-ligand on its surface.
• a ligand can be an antibody or an antibody fragment, a lipid, a glycolipid, a hormone, a polypeptide, a polymer (PEG, polylysine, PEI, ...) or even a sugar.
• the term antibody notably denotes 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.
• the ligand can be an antibody fragment against fusine, the CD4 receptor or against an exposed 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.
• the ligands which can be used in the context of the invention are widely described in the literature and can be cloned by standard techniques. It is also possible to synthesize them chemically and couple them to the fiber according to the invention.
• the coupling of galactosyl residues should confer hepatic specificity due to the interaction with the asialoglycoprotein receptors.
• the preferred embodiment consists in inserting the ligand at the C-terminal end of the fiber according to the invention or in replacing the deleted residues when at least one of the modifications is a deletion of at least 3 consecutive residues.
• Another subject of the invention relates to a peptide fragment characterized in that it comprises the region extending from sheet A to sheet B and including the loop AB of a modified fiber as described above.
• a peptide fragment has in particular the following properties:
• (i) further comprises a specific ligand of 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 adenoviral particle does not including such a mutated fiber.
• the invention relates more specifically to such a peptide fragment characterized in that it is the sequence extending from residue 388 to residue 592 of a fiber of an adenovirus type 5 (Ad5) comprising all or part of the sequence as shown in sequence identifier No. 1 (SEQ ID NO: 1) and comprising at least one mutation at one or more residues in the region between residues 400 and 428.
• Ad5 adenovirus type 5
• the present invention also relates to an adenoviral particle which comprises on its surface a mutated fiber according to the invention, and optionally a ligand as defined above.
• this adenoviral particle is devoid of native functional fiber.
• the mutated fiber of the invention can be expressed by the adenoviral genome itself, in particular when said adenoviral particle contains such a genome, or brought in trans by a complementation cell line, such as those defined below.
• the adenoviral particle of the invention is as presented above and is characterized in that said ligand is inserted into a protein of the adenoviral capsid other than the fiber, in particular hexon or penton .
• said adenoviral particle of the invention is "empty", that is to say that it does not contain nucleic acid.
• the use of such viral particles is in particular illustrated in the document WO95 / 21259 cited above.
• this adenoviral particle contains an adenoviral genome, one will preferentially speak of adenoviral virus (or adenovirus) and in the particular case according to which said genome is further modified, one will more specifically speak of recombinant adenoviral virus (or recombinant adenovirus).
• adenoviral virus or adenovirus
• recombinant adenoviral virus or recombinant adenovirus
• said ligand can be chemically coupled to said adenoviral particle.
• the variant is preferred according to which the sequences coding for the ligand are inserted within the adenoviral genome, and preferably, within the sequences coding for the fiber modified according to the invention, and more specifically in phase in order to preserve the framework of reading.
• the insertion can take place anywhere.
• 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 adenoviral sequences, in particular those coding for another capsid protein, such as hexon or penton.
• the invention relates to a recombinant adenovirus defective for replication, that is to say 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 conserve the sequences coding for the protein gp19k (Gooding and Wood, 1990, Critical Reviews of Immunology 10, 53-71) in order to modulate the immune responses of the host.
• the genome of an adenovirus according to the invention can also comprise additional deletions or mutations affecting other regions, in particular the E2, E4 and / or L1-L5 regions (see for example WO94 / 28152 or WO 9412649 or Ensinger et al., 1972, J. Virol. 10, 328-339 describing the heat-sensitive mutation of the DBP gene of E2).
• a recombinant 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 (minigene lacking one or more introns). It can be obtained by conventional molecular biology techniques or by chemical synthesis. It can code for an antisense RNA, a ribozyme or an mRNA which will then be translated into the polypeptide of interest. This can be cytoplasmic, membrane or be secreted by the host cell.
• 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 native sequence exhibiting improved and / or modified biological properties.
• cytokines or lymphokines interferons ⁇ , ⁇ and ⁇ , interieukins 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
• proteins of the major histocompatibility complex of classes I or II or regulatory proteins acting on the expression of the corresponding genes proteins of the major histocompatibility complex of classes I or II or regulatory proteins acting on the expression of the corresponding genes; - polypeptides capable of inhibiting a viral, bacterial or parasitic infection or its development (antigenic polypeptides having immunogenic properties, antigenic epitopes, antibodies, transdominant variants capable of inhibiting the action of a native protein by competition ...); - toxins (thymidine kinase from herpes simplex virus 1 (TK-HSV-1), ricin, cholera toxin, diphtheria) or immunotoxins; and markers ( ⁇ -galactosidase, luciferase .).
• TK-HSV-1 herpes simplex virus 1
• ricin ricin
• cholera toxin diphtheria
• 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 be derived 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, to introduce a restriction site.
• the gene to be expressed may be the natural promoter of the gene to be expressed. Mention may be made, by way of examples, of the viral promoters CMV (Cytomegalovirus), RSV (Rous Sarcoma Virus), of the TK gene of the HSV-1 virus, early of the SV40 virus (Si ian Virus 40), adenoviral MLP or even the eukaryotic promoters of the murine or human PGK (Phospho Glycerate kinase) genes, ⁇ 1-antitrypsin (liver-specific), immunoglobulins (lymphocyte-specific).
• CMV Cytomegalovirus
• RSV Ra Sarcoma Virus
• TK TK gene of the HSV-1 virus
• adenoviral MLP early of the SV40 virus
• PGK Phospho Glycerate kinase
• 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 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 for such a fiber or such a fragment.
• Any type of vector can be used for this purpose, whether of plasmid or viral origin, integrative or not. Such vectors are commercially available or described in the literature. Likewise, those skilled in the art are able to adapt 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 particles according to the invention according to which:
• the adenoviral genome encoding a modified fiber according to the invention is transfected into an appropriate cell line, for example line 293;
• transfected cell line is cultivated under conditions suitable for allowing the production of said adenovirus or of said recombinant adenovirus, and
• the empty particles are recovered by purifying the cell lysate on a density gradient, in particular a cesium chloride gradient for example.
• the invention also relates to a method for preparing an adenovirus or a recombinant adenovirus according to the invention, according to which: - the genome of said adenovirus, recombinant or not, defective for replication or not, is transfected in a cell line cultivating said transfected cell line under suitable conditions to allow the production of said adenovirus or said recombinant adenovirus (one can also say, adenoviral particles), and said adenovirus or said recombinant adenovirus is recovered from the culture of said transfected cell line and, optionally, said adenovirus is purified.
• cell line depends, where appropriate, on the deficient functions of the adenovirus according to the invention.
• a complementation line capable of providing the defective function (s) in trans will be used.
• Lines 293 (ATCC CRL1573) or PERC6 (ECACC 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 FR2737222 (96 04413).
• auxiliary virus to complement the defective adenovirus according to the invention in any host cell or else a mixed system using complementation cell and auxiliary 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, to Graham and Prevec, 1991, Methods in Molecular Biology, vol 7, p 190-128; Ed E.J. 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 FR2727689 (94 14470)).
• the purification methods are described in the state of the art. Mention may be made of the density gradient centrifugation technique. According to an alternative process, it is also possible to construct "empty" adenoviral particles artificially by the association of the carboxy or amino-terminal ends of proteins, peptides or glycoproteins of the adenoviral capsid with lipids. Such modified lipids, incorporating in particular the peptide fragments of the invention, can then be incorporated into a liposome. Such a technique has been described by Tikchonenko et al., 1988, Gene, 63, 321-330 in the case of liposomes carrying on their surface glycoproteins of the influenza virus.
• 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 fiber or not to produce fiber).
• the invention further 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: said genome is transfected into a cell line presented below. above, said transfected cell line is cultured under conditions suitable for allowing the production of said adenoviral particle, and said adenoviral particle is recovered in the culture of said transfected cell line and, optionally, said adenoviral particle is purified.
• 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. It is advantageously 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 ...), muscle, nasal, pulmonary, tracheal, hepatic, epithelial or fibroblast.
• hematopoietic totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage
• muscle nasal, pulmonary, tracheal, hepatic, epithelial or fibroblast.
• the subject of the invention is also a composition
• a composition comprising, as therapeutic or prophylactic agent, a host cell, an adenoviral particle or an adenovirus, in particular a recombinant one, according to the invention or capable of being obtained by a method according to the invention. , in combination with a pharmaceutically acceptable carrier.
• 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 diseases recurrent viral infections, such as viral infections caused by the herpes virus.
• 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)
• diseases recurrent viral infections such as viral infections caused by the herpes virus.
• a composition according to the invention can be produced in a conventional manner.
• a therapeutically effective amount of the therapeutic or prophylactic agent is combined with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier is non-toxic to the patient. It may be a solution for injection, an isotonic solution, the pH of which is compatible with use in vivo, a solution of dextrose, glycerol, mannitol, etc.
• a composition according to the invention can be administered by the local, systemic or aerosol, in particular intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intrapulmonary, intranasal or intratracheal. 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 10 14 pfu (units forming plaques), advantageously 10 5 and 10 13 pfu and, preferably, 10 6 and 10 12 pfu .
• the formulation may also include a pharmaceutically acceptable adjuvant or excipient.
• 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 composition 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 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 composition with a diluent and by pouring the mixture obtained into soft or hard capsules.
• a preparation in the form of a syrup or elixir may contain the composition 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 composition 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.
• composition can also be formulated in the form of microcapsules, optionally with one or more additive supports.
• present invention also relates to a composition characterized in that it further comprises at least one compound selected from a naked nucleic acid or a nucleic acid combined with at least one cationic compound.
• the adenoviral particles according to the invention can also be complexed with synthetic or natural compounds.
• Such adenoviral particles and their use are for example described in O'Riordan et al., 1999, Human Gene Therapy, 10, 1349-1358 or in patent application WO98 / 44143. The content of these documents is incorporated by reference into the present request.
• the present invention relates to the use of a peptide fragment, of an adenoviral particle, of an adenovirus or of a host cell according to the invention or of an adenovirus capable of being obtained by a process. according to the invention, for the preparation of a medicament intended for the treatment of the human or animal body.
• the medicament 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 in which a therapeutically effective amount of an adenovirus or a host cell according to the invention is administered to a patient in need of such treatment.
• the NM522 strain (Stratagene) is suitable for the propagation of phage vectors M 13.
• the amplification techniques by PCR are known to the skilled person (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 Genebank 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 with a host tropism towards cells expressing the GRP receptor (for gastrin releasing peptide in English).
• the plasmid pTG6593 is derived from p poly II (Lathe et al., 1987, Gene 57, 193-
• 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 et al., 1995, Gene Ther. 2, 660-668).
• the oligonucleotide oTG7001 (SEQ ID NO: 3) is used for this purpose.
• the Hin ⁇ ⁇ - Sma ⁇ 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 genome fragment adenoviral 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 of positions 21562 to 35935.
• pTG4600 from which the BstEW fragment is isolated (nt 24843 to 35233).
• 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.
• 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 presence of the GRP peptide at the adenoviral fiber makes it possible to target the cells expressing on their surface the GRP receptor.
• 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 Northem-blot .
• a mixture of 2 DNA fragments complementary to the sequence coding for the GRP receptor, labeled by conventional techniques with the 32 P isotope is used.
• the fragments are produced by reverse PCR from Total cellular RNA using the oligonucleotides oTG10776 (SEQ ID NO: 4) and oTG10781 (SEQ ID NO: 5) (Battey et al., 1991, Proc. Natl. Acad. Sci. USA 88, 395-399; Corjay et al., 1991, J. Biol. Chem. 266, 18771-18779).
• the intensity of the mRNA detected is much greater in the case of Swiss-3T3 cells than in 293 cells, indicating the overexpression of the GRP receptor by the murine line.
• 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 integrins ⁇ j ⁇ ⁇
• OTG 11991 SEQ ID N ° 13 Mutagenesis with m3TG6527 to give M13TG13265.
• 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 are used.
• 0TGHO68 allow the generation of an Xho ⁇ -Sma ⁇ fragment (ranging 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 oTG 11070 (SEQ ID NO: 11).
• the 3 fragments digested with the appropriate enzymes are then religated to give an H / nc / III-SmaI 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 BstB ⁇ site in the region targeted by conventional mutagenesis techniques.
• the homologous recombination between pTG4609 linearized with BstB ⁇ and the preceding H / ncflll-SmaI fragment generates the plasmid pTG4225 carrying the wild-type E1 region.
• Its equivalent carrying the LacZ expression cassette pTG4226 is obtained by homologous recombination with pTG4213 digested with ⁇ s.BI.
• the AdTG4225 and AdTG4226 viruses can be produced conventionally by transfection of an appropriate cell line, for example overexpressing the EGF receptor.
• 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 AB region (amino acid 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 modify the tropism of the corresponding adenoviruses. . - Replacement in the AB loop of the serine in position 408 by the glutamic acid residue of serotype 3 using the oligo oTG12499 (SEQ ID NO 14);
• Mutagenesis can be carried out on the vector M13TG6526 or M13TG6528.
• the first carries the wild H / ⁇ c / 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 pTG4225 (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 (PAGE-SDS).
• the different proteins are detected by staining with silver nitrate.
• the fiber is specifically revealed by performing a western 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 trimeric fiber is capable of binding the base penton (Novelli and Boulanger, 1991, supra) and to be incorporated into the particle, detection of the protein in the above experiment indicates that the modified fiber is still capable of forming trimers.
• 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.
• An effective infection in the presence of saturated concentrations of the wild peptide indicates an infection independent of the binding to natural primary receptors. This suggests a greatly reduced affinity of the modified fiber for its receptors.
• EXAMPLE 6 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 ⁇ H / ndlll-X ⁇ ol 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) -X / .al-EGF-BstGI-hexon (nt19699-20312).
• the fragment nt19043 to 19647 is obtained by PCR amplification from the plasmid pTG3602 with the oligonucleotides oTG11102 (SEQ ID NO: 17) and oTG11103 (SEQ ID NO: 18).
• the nt19699 to 20312 fragment is amplified from the same DNA with the oligonucleotides oTG11104 (SEQ ID NO: 19) and oTG11105 (SEQ ID NO: 20).
• EGF is cloned from the cDNA using the oligonucleotides oTG11106 (SEQ ID NO: 21) and OTG11107 (SEQ ID NO: 22) 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 Hinu ⁇ - Xho ⁇ digestion and replaced in their genomic context by homologous recombination.
• We can use the vector pTG3602, pTG4607, pTG4629 linearized by Sgfl or a vector carrying the adenoviral genome deleted from the fiber sequences (like 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 initiating codon but not extending to the other adenoviral ORFs.
• the procedure is as follows: the 5 'adenoviral fragment of the deletion (nt 30564 to 31041) is amplified by PCR using the primers OTG7171 and oTG7275 (SEQ ID NO: 23 and 24). Amplification of the 3 ′ fragment (nt 31129 to 33099) uses the primers oTG7276 and OTG7049 (SEQ ID NO: 25 and 26).
• the PCR fragments are digested with Xho ⁇ and ligated before being introduced by homologous recombination into the vector pTG6591 linearized by ⁇ / c / el, 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.

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