FR2747681A1 - Dodecahedral adeno-viral complex containing penton bases and optionally fibres - Google Patents

Abstract

Adenoviral protein complex (A) comprises: (a) 12 pentons (P), each containing at least one each of penton base (PB) and fibre (F), with P bound via PB to form a dodecahedral structure that is resistant to proteolysis and has molecular weight 4.8-6.6 MD; or (b) 12 PB forming a dodecahedron as above but with molecular weight 3.2-4 MD. (A) contain no other components of the adenoviral genome and F and PB are from the same or different adenovirus (Ad).

Description

 The present invention relates to a native or recombinant adenoviral protein complex, to a pharmaceutical composition comprising said protein complex and to their applications in the treatment (drugs) and prevention (vaccines) of human and animal diseases. Said protein complex is in particular capable of providing suitable target cells, nucleic sequences, proteins, peptides or chemical substances of interest.

 The present invention also relates to expression vectors of said protein complex.

 Adenoviruses are a family of animal DNA viruses comprising more than 90 serotypes infecting different species, including almost 50 different human serotypes. The cellular tropism of adenoviruses results in infection of the respiratory tract, gastrointestinal, urinary tract and eyes.

These viruses are responsible for 3% of all infections in humans, 10% of childhood pneumonia and 15% of intestinal infections in
The child.

 The adenovirus particle is relatively complex and includes several substructures; in particular, the external part or capsid is formed mainly of three proteins: the hexon, the base of the penton and the fiber (see FIG. 1).

 The penton, a non-covalent complex formed by a trimeric protein, the fiber and a pentameric protein, the base of the penton, forms the heights of the viral icosahedron.

 Each of the two proteins forming the penton plays a fundamental role in the infection: the fiber allows the virion to attach to a cellular receptor; the base of the penton allows the internalization of the virion, probably thanks to the interaction with cellular integrins (binding to integrin receptors, vitronectin, fibronectin, via the arg-gly-asp sequence present at the base penton) (Wickham et al., Cell, 1993, 73, 309-319). There are also observations which suggest that the base of the penton has an endosomolytic activity, allowing the escape into the cytoplasm of molecules co-internalized with the virus (Seth et al., Virus Attachment and Entry into Cells, 1986, Ed. RL

Crowell & K. Lonberg-Holm, 191-195).

 The binding of adenoviruses to cells and their internalization are therefore two separate but cooperating events.

 The ability to infect various quiescent cells makes adenovirus a vector of choice for gene therapy.

The methods commonly used are based on the repeated administration of recombinant adenoviruses, deficient for replication, carrying the target gene (PCT international applications WO 95/02697 and WO 95/14101, in the name of
Rhone-Poulenc Rorer SA).

However, the treatments recommended with such deficient adenoviruses have at least the following drawbacks:
- risk of restoration in trans of the pathogenicity of the recombinant virus in a subject treated and simultaneously infected with a wild adenovirus;
- immune and inflammatory reactions, during the repeated administration of the recombinant adenovirus, due to the massive introduction of viral particles bringing foreign proteins, which put a brake on their use as a vector in gene therapy, in man.

To avoid such drawbacks, it has been proposed to use only the penton or the penton base, independently of the rest of the adenovirus genome, to facilitate the transfer of exogenous genes into host cells (PCT International Application WO 94 / 17832, on behalf of The Scripps Research
Institute). Although such an approach has advantages over recombinant adenoviruses deficient in replication, such structures (penton or penton base) are fragile, in particular the penton base, which can be destroyed by proteolysis.

 Consequently, the Applicant has set himself the goal of providing a vector which can be used in gene therapy which better meets the needs of the practice than the vectors of the prior art, such a vector has neither the disadvantages of recombinant adenoviruses, nor the fragility of the penton or the base of the penton, as specified above.

The subject of the present invention is an adenoviral protein complex, characterized in that it is constituted either
- 12 pentons, each comprising at least one fiber and one penton base, to the exclusion of any other element constituting the genome of an adenovirus, which fiber (s) and base of the penton are derived either from the same adenovirus, or of different adenoviruses, said pentons being linked by penton bases and forming a dodecahedron structure, stable to proteolytic enzymes, which complex has a molecular weight of between 4.8.106 and 6.6.106, such complexes are called below dodecahedron-fiber complex,
- or from 12 penton bases, to the exclusion of any other constituent element of the genome of an adenovirus, which penton bases are derived either from the same adenovirus, or from different adenoviruses, and form a dodecahedron structure, stable to proteolytic enzymes and in that it has a molecular weight between 3.2.106 and 4.106; such complexes are hereinafter called dodecahedron-base complexes.

 Said complexes therefore do not comprise any element of the genome of one of said adenoviruses. Said native or recombinant dodecahedral protein complexes facilitate the transfer of foreign genes, nucleic sequences, proteins, peptides or chemical molecules into the target cells.

 According to the invention, said adenoviruses are selected from human adenoviruses and in particular adenovirus type 2 (Ad2), adenovirus type 3 (Ad3), adenovirus type 5 (Ad5), adenovirus of type 4 (Ad4), adenovirus type 7 (Ad7), adenovirus type 9 (Ad9), adenovirus type 11 (Adll), adenovirus type 15 (AdlS) or enteric adenoviruses ( Ad40 and Ad41) and avian adenoviruses.

 According to an advantageous embodiment of said recombinant dodecahedral protein complexes, at least one of its constituents (base and / or fiber) can be modified to increase the affinity with respect to a particular cell type.

 Such a protein complex thus modified does not exhibit a decrease in its properties of attachment to the target cell and of internalization in said cell, on the one hand compared to the wild-type adenovirus and on the other hand compared to the complex according to the invention in which said constituents are not modified.

 For example, a sequence of attachment to other receptors, such as the CD4 receptor present on T cells, can be incorporated into the fiber by recombinant DNA techniques (ligand-fiber conjugate).

 Examples of suitable ligands that may be mentioned include the V3 loop of HIV gp120 (binding to CD4), transferrin (binding to the transferrin receptor), LDL (binding to LDL receptors), deglycosylated proteins and antibody.

A preferred dodecahedral protein complex according to the invention consists either of 12 penton bases, corresponding to those of an adenovirus selected from the group consisting of Ad3, Ad4, Ad7, Ad9, Adll and Ad15, or of 12 pentons whose sequences fibers correspond to those of any of the aforementioned human, enteric or avian adenoviruses and whose penton base sequences correspond to those of an adenovirus selected from the group consisting of
Ad3, Ad4, Ad7, Ad9, Ad11 and Ad15, preferably those of adenovirus type 3 (Ad3).

 Another preferred dodecahedral protein complex according to the invention consists of 12 pentons, whose fiber sequences correspond to those of type 2 adenovirus (Ad2) and whose penton base sequences correspond to those of adenovirus of type 3 (Ad3).

 The use of the protein complex according to the invention in place of the adenovirus virion eliminates the danger of fortuitous infection with the adenovirus and is accompanied by much weaker immune and inflammatory reactions; it is also particularly stable and the presence of the 12 bases of the penton significantly increases the lysis rate of the endosomes (faster cytoplasmic passage), compared to a structure containing only a penton or a base.

 According to another advantageous embodiment of said recombinant dodecahedral protein complex, it also comprises at least one hexon.

 The present invention also relates to a pharmaceutical composition, characterized in that it essentially comprises a dodecahedral adenoviral protein complex according to the invention and at least one other chemical substance.

 According to an advantageous embodiment of said composition, said other chemical substance is selected from the group consisting of nucleic sequences, proteins, peptides and pharmacologically active chemical substances.

 When the chemical substance is a protein, the latter preferably has immunoprotective and antigenic properties, and is particularly well suited to the preparation of a vaccine.

 When the chemical substance is a pharmacologically active chemical substance, it is in particular selected from cellular toxins, such as anticancer agents and in particular anthracyclines.

 In the latter case, the composition according to the invention has the advantage of avoiding the resistance mechanism usually encountered with anticancer agents. Indeed, the development of resistance to anti-tumor chemotherapy constitutes a major obstacle to the treatment of human cancers. This resistance is largely due to the induction of the activity of a very efficient extracellular pump which does not allow the transport of the pharmacologically active substance inside the cell.

However, surprisingly, the composition according to the invention allows the introduction of the pharmacologically active substance into the cell, bypassing this resistance mechanism.

 When the active substance is a nucleic sequence, it is selected from the genes which code for a polypeptide exhibiting therapeutic activity, the antisense sequences and the ribozymes.

 In the case of a coding sequence, it further comprises a promoter active for the expression of the polypeptide.

 According to an advantageous arrangement of this embodiment, the promoter is selected from the group consisting of constitutive promoters and inducible promoters.

Said active chemical substance can be linked to the dodecahedral protein complex according to the invention in at least two different ways:
- either via a transient bond, such as an ionic bond, which can be destroyed and thus allows the passage of the active substance in the nucleus (expression of a gene at the nucleus) (ligand selected from substances capable of producing an ionic bond with said active substance)
- Either via a stable bond, such as a covalent bond, for example, which retains the active substance in the cytoplasm, such a type of bond is particularly advantageous for the preparation of vaccine compositions (persistence of active substance in the cytoplasm) (ligand selected from the substances capable of producing a covalent bond with said active substance).

 Indeed, said chemical substance, in particular a nucleic acid sequence, a protein or a peptide, can be associated (covalently or non-covalently) with a suitable ligand.

Among the suitable ligands there may be mentioned:
(1) peptides, the N-terminal part of which comprises the N-terminal amino acid sequence of an adenovirus fiber of any serotype (zone of attachment to the protein complex according to the invention) and of which part C -terminal allows an ionic bond with the active substance to be transported in the protein complex according to the invention, for example, said C-terminal part comprises a polylysine (sequence which allows the attachment of the active substance, in particular when it is nucleic acid);
(2) peptides, the N-terminal part of which comprises the N-terminal amino acid sequence of an adenovirus fiber of any serotype (zone of attachment to the protein complex according to the invention) and of which part C -terminal allows a covalent type bond with the active substance to be transported; by way of example, said C-terminal part comprises a cysteine, which allows the attachment by covalent bond of the protein or of the nucleic acid to be transported;
(3) said ligand consists of transferrin / poly-L-lysine, which associates with said nucleic sequence or said protein, to form a nucleic acid or protein and transferrin / poly-L-Lysine conjugate, and in which the transferrin binds to transferrin receptors on the surface of target cells.

 The association of such a conjugate with the protein complex according to the invention allows better transport of the chemical, from the endosomes to the cytoplasm.

 (4) another mode of transfer of nucleic sequences or proteins is possible, using an avidin-biotin complexation reaction and an anti-fiber, anti-penton base or anti-dodecahedral protein complex antibody.

Such a composition uses: a dodecahedral protein complex according to the invention, an antibody which reacts either with the fiber, or with the base of the penton, or with the dodecahedral protein complex, but does not inhibit the functionality of said protein complex, a vector containing a nucleic sequence of interest in which at least one nucleotide is biotinylated, optionally linked to a marker sequence or a biotinylated protein and a chimeric protein consisting of protein A linked to streptavidin (SA-PA). Because a nucleotide or protein is biotinylated, they can bind to the chimeric protein SA-PA. In such a composition, the biotinylated DNA or the biotinylated protein binds to the chimeric protein SA-PA; the complex
DNA linked to SA-PA then binds via the PA half, to an antibody which reacts either with a fiber, or with a penton base, or with the dodecahedral protein complex. The complex product thus formed comprising PA-immobilized antibody - dodecahedral protein complex binds, via the protein complex, to the specific receptors of the latter, expressed on the surface of the target cells.

 In all cases, the exogenous nucleic acid sequence, the protein of interest or any other chemical substance, included in or associated with the composition according to the invention, enters the cell (internalization) via the protein complex. dodecahedron according to the invention.

Surprisingly, the dodecahedron-cell receptor interaction significantly increases both the internalization of the composition according to the invention and the permeability of the endosomes, which significantly increases the passage of acid. exogenous nucleic acid, protein of interest or any other chemical substance, from the endosomes to the cytoplasm, in comparison with the use of a composition containing only one penton
According to another embodiment of said pharmaceutical composition, it further comprises at least one pharmaceutically acceptable vehicle.

 Among the pharmaceutically acceptable vehicles, there may also be mentioned vehicles suitable for the desired administration route such as water, salts, dextrose, glycerol, ethanol of vegetable oils, propylene glycol, polyethylene glycol , benzyl alcohol (parenteral administration or liquid preparations), than liposomes.

 Such compositions can, in addition, contain wetting or emulsifying agents, isotonic agents, dissolving agents, stabilizers, dyes, antiseptic agents, etc.

 According to the invention, said active chemical substance is either included in said dodecahedral protein complex according to the invention, or associated with said complex; in both cases, it can be free or linked to said complex.

 The compositions according to the invention can be administered in different forms and by different routes, such as the pulmonary route (aerosol), intraperitoneal route, parenteral route or surgical implant.

The compositions according to the invention have numerous applications as medicaments, in human and veterinary medicine:
- in human and animal gene therapy, in particular in hereditary diseases involving the respiratory epithelium such as emphysema or cystic fibrosis,
- as antiviral agents (antisense sequences or ribozymes),
- as immunogenic or vaccine agents,
- as anti-bacterial, anti-cancer agents etc ...

 The present invention also relates to a composition essentially comprising an adenoviral dodecahedral protein complex, as defined above and a chemical selected from the nucleotide sequences, proteins and pharmacologically active chemicals, as a medicament.

 The present invention also relates to a composition, comprising an adenoviral dodecahedral protein complex, as defined above and a chemical substance selected from the nucleotide sequences, the immunogenic substances, and the anticancer substances, for its use in the treatment of diseases human or animal implying cells expressing receptors specific for adenovirus fibers and / or cells expressing integrin receptors, in particular integrins OCVss3 OR aVss5, such as epithelial cells, endothelial cells, blood platelets, lymphoid cells and cancer cells and releasing an effective amount of said active chemical to said cells.

 The present invention further relates to a process for the preparation of said adenoviral dodecahedral protein complex according to the invention.

Such a method preferably includes the following steps
(1) separate or simultaneous cloning of the gene (s) coding for the fiber (s) of an adenovirus, the gene coding for the penton base of an adenovirus and optionally the gene coding for l of an adenovirus, said genes being derived either from the same adenovirus or from different adenoviruses, in at least one baculovirus vector, for obtaining several recombinant plasmids containing either one gene or two genes (simultaneous expression of the two fibers in the case of an adenovirus containing two fibers per penton) or for obtaining a recombinant plasmid containing simultaneously two genes (or three genes, in the case of the use of an adenovirus containing two fibers per penton) , in the latter case, the baculovirus vector used comprises a double or triple expression cassette, whereas in the case where the protein complex according to the invention also comprises at least one hexon, said baculovirus vector can comprise a cassette multiple expression;
(2) the co-transfection of the recombinant plasmid (s) and of a linearized fragment of baculovirus DNA, in an insect cell,
(3) screening and selection of recombinant baculovirus clones expressing, separately or at the same time the fiber (s), the base of the penton and optionally the hexon;
(4) purifying the selected clones corresponding to replicable recombinant baculoviruses, containing one or more of said genes, derived from at least one adenovirus and expressing the corresponding proteins;
(5) the extraction of proteins expressed by said recombinant baculoviruses, essentially comprising the lysis of insect cells, the removal of cellular debris and nuclei by centrifugation and the recovery of the supernatant; and
(6) the purification of the dodecahedral protein complex according to the invention, by application of said supernatant (protein extract) on a sucrose gradient, the range of concentrations of which varies as a function of the molecular weight and the density of the dodecahedra obtained and recovery of the fractions, in the zone of high sucrose concentrations, for example, for complexes formed from Ad2 and / or Ad3, the sucrose gradient varies from 15 to 40%, and the fraction corresponding to a sucrose concentration of between 31 and 38%.

 According to an advantageous embodiment of said method, the insect cells of step (2) are selected from the group consisting of Spodoptera frugiperda cells and Trichoplusia ni cells.

 Alternatively, the co-transfection of step (2) is carried out in Spodopterafrugiperda cells and the method comprises a second step (4 ') of transfection of the purified clones of recombinant baculoviruses obtained in step (4) , in Trichoplusia ni insect cells.

 According to another advantageous embodiment of said method, the baculovirus vector comprising a double or triple expression cassette according to step (1) is a vector comprising two or three strong promoters.

 Mention may be made, for example, of the transfer vector pAcUW31 (Clontechn, which comprises (i) the polyhedrin promoter and the p10 promoter, the polyhedrin promoter being followed by a single BamHI cloning site and polyadenylation signals polyhedrin, while the p 10 promoter is followed by the unique Bgl H and EcoRI cloning sites and by a polyadenylation signal of SV40, (ii) an origin of M13 replication, (iii) an origin of pUC replication and ( iv) a reporter gene, such as luciferase, P-galactosidase or an antibiotic resistance gene (ampicillin, for example).

 According to another advantageous embodiment of said method, said linearized fragment of baculovirus DNA from step (2) is obtained from a vector containing three restriction sites Bsu36I and the lacZ gene in place of the sequence coding for polyhedrin, for example, the viral DNA vector BacPAK6 @ (Clontech), which vector is digested with the restriction enzyme Bsu36I before said co-transfection of step (2).

 According to another advantageous embodiment of said method, the Spodoptera frugiperda insect cell of step (2) is selected from the group consisting of Sf21 cells and Sf9 cells.

 According to another advantageous embodiment of said method, the purification of the clones of step (4) is carried out by the implementation of at least two successive sub-cloning.

 According to another advantageous embodiment of said method, the Trichoplusia ni insect cells of step (5) are BTI-TN-5B14 cells (High Filez from Invitrogen).

 Alternatively, the transfection of step (4 ′) comprises the co-transfection of recombinant baculoviruses which contain the two genes (or the three genes, in the case of an adenovirus containing two fibers per penton), derived from adenoviruses mentioned above, possibly associated with recombinant baculoviruses which contain only one of the two genes derived from adenoviruses.

 According to the invention, the transfection can be carried out by different methods, depending on the vector used 'calcium phosphate method, DEAE-dextran method, stable transfer method, electroporation.

 According to another advantageous embodiment of said method, step (5) of extraction of all the proteins expressed by the selected recombinant baculovirus comprises obtaining a cell extract by lysis of the Trichoplusia ni cells, by means of several freeze-thaw cycles, in a Tris 10 mM pH8 buffer, the elimination of the cellular debris and of the nuclei present in said cell extract by centrifugation for a few minutes at 7,000-10,000 g and the recovery of the supernatant.

 According to yet another advantageous embodiment of said method, step (6) of purification of the protein complex according to the invention comprises centrifugation at 121,000 g at the top of the tube and at 275,000 g at the bottom of the tube, for 5 to 7 p.m. and cold (4 "C).

 According to yet another advantageous embodiment of the method, the dodecahedral protein complex purified in step (6) is subjected to a concentration.

 The native dodecahedra are advantageously obtained by extraction of the proteins expressed by cells infected with an adenovirus (see step (5) above) and purification of the dodecahedral complex on a sucrose gradient, under the same conditions as those stated above, for the preparation of recombinant dodecahedra.

In addition to the above arrangements, the invention also comprises other arrangements which will emerge from the description which follows, which refers to examples of implementation of the process which is the subject of the present invention as well as to the accompanying drawings, in which:
- Figure 1 shows a schematic view of an adenovirus;
- Figures 2A and 2B show the results obtained from cells expressing the fiber and the base of the penton, from an Ad3 (Ad3 dodecahedron) and represent two SDS-PAGE electrophoresis gels, obtained from the fractions of the gradient sucrose: FIG. 2A: result of the protein transfer (Western blot or immuno blot); Figure 2B: result after staining with Coomassie Blue;
- Figures 3 and 4 illustrate the role of the base of the penton in the formation of the dodecahedron;
- Figures 5 and 6 show electron micrographs of dodecahedron-fiber and dodecahedron-base of Ad3 respectively, negatively stained with 1% sodium silicotungstate; these micrographs are produced from a sample of material obtained after centrifugation of the sucrose gradient varying from 31 to 38%, mixed, dialysed and concentrated;
- Figure 7 shows the intracellular accumulation (HeLa cells) of dodecahedra according to the invention;
- Figures 8 and 9 illustrate the internalization of the dodecahedral protein complex according to the invention, in HeLa cells;
FIG. 10 illustrates the internalization of a dodecahedron-base and a dodecahedron-fiber: the histograms 13 illustrate the internalization of the dodecahedron-fiber and the histograms I illustrate the internalization of a dodecahedron-base; this figure includes, on the abscissa, the quantity of viral particles / cell (= MOI or Multiplicity of Infection) and on the ordinate, the fluorescence units (arbitrary fluorescence units);
- Figures 11, 12 and 13 illustrate the transfection of a plasmid containing the gene coding for luciferase in the presence of a dodecahedron-fiber or a dodecahedron-base; they include, on the abscissa, the quantity of viral particles / cell (= MOI or Multiplicity of Infection) and on the ordinate, the quantity of relative light (= RLU or Relative Light Unit).

 It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1 Cloning, expression and purification of the adenovirus type 3 dodecahedron.

 1. PCR amplification of fiber genes and the base of the penton.

 Using PCR, it is possible to synthesize nucleotide sequences encoding useful polypeptides, which can be inserted into an appropriate vector and used to transform and express a specific cell.

 The method for producing the genes expressing the base of the penton and the fiber of the adenovirus dodecahedron according to the invention depends on the preselection of oligonucleotides as primers in a polymerase chain reaction (PCR).

The sequence of the genes for the penton base and the Ad3 fiber has been described respectively in CUZANGE et al., Gene, 1994, 146, 257-259 and
SIGNAS et al., J. Virol., 1985, 53, 672-678.

The 5 'and 3' primers used to amplify the gene coding for the penton base are 5'-GGATCCGATGAGGAGACGAGCC3 'and 5'-GGATCCYTAGAAAGTGCGGCTTG-3', respectively
The 5 'and 3' primers used to amplify the gene coding for the fiber are respectively 5 '-TTTCTTGAATTCCAGATGGC CAAGCGAGCT-3' and 5 '-AAAAGGAATTCCAATAAAAAATGTTG-3'
The cloning sites are underlined and are respectively BamH I,
BamH I, EcoR I, EcoR I. The initiation codon ATG is in bold and the stop codon TAA (or TTA in the complementary strand) is in italics.

 Fiber and base sequences of

Amplification by PCR is carried out on DNA extracts isolated from the virus, obtained by propagation in HeLa cells as described in
HORWITZ (Adenoviridae and their replication, in Virology, Fields and Knipe, eds. Raven Press, New York, 1990).

 The PCR buffer contains for example: KCI 50 mM, Tris-HCl at pH 8.3 10 mM, MgCl2 1.5 mM, gelatin at 0.001%, ATP 200 RM, dTTP 200 pLM, dCTP 200 RM, dGTP 200 ZM and 2.5 units of Thennus aquaticus DNA polymerase / 100 RI of buffer. The PCR is carried out under the following conditions: after denaturing 2 minutes at 94 "C, the PCR comprises 25 cycles of 1 min at 94" C, 1 min at 55 "C and 1 min at 72" C.

2. Cloning of PCR Products into the Intermediate Vector pCR
Script.

 The cloning vector pCR-Scripta (Stratagene, Catalog no. 211190, 1994) exhibits high DNA ligation efficiency when cloning PCR products.

 The PCR amplification products are fractionated by electrophoresis on 1% agarose gel in TAE buffer, the suitable bands are separated and the DNA is extracted according to the GENECLEAN ll procedure (niolo1, Inc.). The DNA fragments containing the genes coding for the base of the penton and the fiber are cloned, separately, in the aforementioned vector pCR-Script according to the indications of the supplier.

 The clones are then treated to obtain the DNA, according to the method described in Sambrook et al (Molecular Cloning, a Laboratorv Manquai, second edition), in the chapter concerning mini-preparations of plasmid DNA. Under these conditions, the presence of the genes coding for the fiber and the base of the adenovirus penton is confirmed, by comparing the size of the fragments obtained after digestion with BamHI and EcoRI with standard products, on agarose gel.

 The positive clones are amplified, the plasmid DNA is isolated and the genes for the fiber and the base of the penton are obtained after digestion with the same restriction enzymes (BamH I and EcoR I), in accordance with the methods of genetic engineering usually used. in molecular cloning (Sambrook et al, cited above).

 3. Cloning of the penton fiber and base genes into an expression vector.

 The double-expression vector pAcUW31 (Clontech) is used to express the dodecahedron.

 First, the penton base gene is introduced at the BamHI site of this vector, upstream of the polyhedrin promoter. The cloning is verified by hybridization with a non-radioactive probe corresponding to the base of the penton containing dUTP linked to fluorescein in accordance with the procedure of the ECLt 'kit. The orientation of the insert relative to the promoter is determined in the positive clones by a restriction analysis.

 In a second step, the gene encoding the fiber is inserted at the EcoRI site of said vector, downstream of the promoter of the p10 gene. The cloning and the orientation of the insert are checked as specified above.

 The sequence of the gene encoding the base and the gene encoding the fiber are verified in the resulting plasmid. No mutation is detected. Similarly, the vectors which carry only either the gene encoding the fiber or the gene encoding the base of the penton are prepared and analyzed as described above.

 The expression plasmids which carry either the two genes or one of the two genes of interest are transformed into a strain of E. coli TG-1, as described in HANAHAN (J. Mol. Biol., 1983, 166, 557-580) and purification of the plasmid on a large scale is carried out using well-known genetic engineering techniques (Sambrook et al, cited above).

 It is also possible, if necessary (in the case of the use of adenovirus comprising two fibers), to use a vector of triple expression, as described in AJ Belyaev and T. Roy (NAR, 1993, 21, 1219- 1223).

 4. Transfection in insect cells.

The recombinant plasmids (100 ng) are (separately) cotransfected with a linearized baculovirus (Clontech: viral DNA BacPAK6 digested by the enzyme
Bsu36I) in the presence of lipofectins (GIBCO) in Spodopterafrugidepera (Sf21) cells and Trichoplusia ni (High-Five) cells according to the technique described by KITTS et al. (NAR, 1990, 18, 19, 5667-5672). This method makes it possible to obtain a transformation efficiency greater than 80%. A few clones are chosen in each case and the expression is detected by the Western blot technique, carried out with polyclonal rabbit antibodies specific for the penton fiber and base antigens.

 5. Purification of positive clones.

The recombinant baculovirus isolates are subjected to 3 subcultures on plates, followed by a titration according to the KING and POSSEE method (The baculovirus expression system, A laboratory guide, LA KING and RD POSSEE, ed .:
CHAPMAN & HALL, 1992). The expression of the two proteins is followed by the Western blot technique as specified above.

 6. Purification of the protein.

The kinetics of the expression of dodecahedron is followed in the two types of cells for 5 days, on the adherent cells, cultured at 27 "C on medium.
TC 100 GIBCO containing 5% fetal calf serum (Sf21 or Sf9 cells) or on TC 100 GIBCO medium containing 10% fetal calf serum (High Fivs49 cells).

Since the protein expression yield is significantly higher in HighFive cells, large-scale protein expression is achieved only in these cells.

 Attempts to increase the level of expression by co-transfection in insect cells with a baculovirus carrying the two genes and different quantities of baculovirus carrying only one gene were not significant.

 3 days after infection with the recombinant baculovirus (multiplicity of infection 5), Higil-Five cells are harvested in a 10 mM Tris buffer pH 8, containing protease inhibitors.

 Cell lysis is carried out by implementing 3 freeze-thaw cycles in the same Tris buffer. The lysis extract is freed of solid elements (cell debris and nuclei) by centrifugation for 5 min at 7,000-10,000 g, then is subjected to a sucrose gradient varying from 15 to 40% (11 ml) in a gradient buffer comprising 10% glycerol, 150 mM NaCI, 10 mM Tris-HCl pH 7.4 and 2 mM EDTA.

 After 18 hours of centrifugation at 4 "C in a Beckman SW 41 rotor, at 121,000 g at the top of the tube and at 275,000 g at the bottom of the tube, the sample thus treated is recovered from above, in fractions of 800 μl. .

 Under these conditions, the dodecahedron is recovered in the fractions containing 31-38% of sucrose. These fractions are mixed and dialyzed against a gradient buffer without sucrose and glycerol.

 The dodecahedron is concentrated by centrifugation on Centritrep (Amicon).

 Figures 2A and 2B illustrate the results obtained after purification of the dodecahedron: they are obtained from aliquots of each fraction, obtained from the sucrose gradient as described above, denatured with heat in the presence of SDS at 1% and electrophoresis in two 10% SDS-polyacrylamide gels; the proteins are analyzed on one of the gels by protein transfer, followed by a reaction with specific anti-fiber and anti-penton base antibodies (Western blot) (FIG. 2A) and on the second gel, by staining with Coomassie Brilliant Blue. The fractions located at the top of the tube contain the bases of the pentons and free fibers and the fractions recovered in the zone 31-38% of sucrose, contain the dodecahedral protein complex according to the invention.

 FIG. 5 represents an electron micrograph of negatively colored Ad3 dodecahedron fiber; the upper figure illustrates a field containing a certain number of dodecahedra in different orientations, while the figures numbered 2, 3 and 5 illustrate dodecahedra on a support film according to their different axes of symmetry (of order 2, of order 3 and 5, respectively); in the figure illustrating the axis of symmetry of order 5, 10 fibers are visible, while the 11th and 12th fibers are probably in the vertical plane relative to the plane of the paper.

 FIG. 6 represents an electron micrograph of negatively colored Ad3 dodecahedronase; the upper figure illustrates a field containing a certain number of dodecahedra in different orientations, while the figures numbered 2, 3 and 5 illustrate dodecahedra on a support film according to their different axes of symmetry (of order 2, of order 3 and order 5, respectively).

 These fractions contain spherical particles consisting of pentons (Figure 5), associated in dodecahedron, via their bases.

 In Figures 5 and 6, the different axes of symmetry are easily identifiable.

 The diameter of the dodecahedron with the fibers (between the opposite heads of 2 fibers) is 49 nm, while the diameter of the base part (from the upper part of a base to the upper part of the opposite base) is 27 , 8 nm (diameter almost identical to the diameter of the fiber-free dodecahedron (27.5 nm)). The pentons of the dissociated dodecahedra that can be found have a size of 21.4 + 1 nm (n = 24), measured from the lower part of the base to the end of the fiber head.

 The interior of the dodecahedron consists of a cavity having a diameter of about 6-7 nm.

7. Role of the base of the penton in the formation of the dodecahedron
Dodecahedra formed from penton bases and fibers can be formed in insect cells, not only through the co-expression process from a baculovirus carrying two genes, but also from a co -infection with two baculoviruses, each of which carries a gene (FIGS. 3A, B).

 It is this approach that was chosen to study the role of the penton base in the formation of the dodecahedron.

When an Ad2 base is used with an Ad2 fiber or a fiber
Ad3, the proteins obtained in high density sucrose fractions, contain unstructured aggregates of pentons (Figure 4D) or bases (Figure 4E).

 Chimeric adenovirus pentons can be formed in vitro, by incubation of purified fibers and penton bases derived from different serotypes.

 From such a method, dodecahedra consisting only of bases were obtained. After incubation of a dodecahedron made only of Ad3 bases with native Ad2 fibers, isolated from cells infected with Ad2, dodecahedra are obtained carrying the heterologous fibers inserted (FIG. 4).

 These experiments show that as regards the study of the adenoviruses Ad2 and Ad3, the formation of a dodecahedron depends on the presence of the base Ad3 and not of the base Ad2.

 When the base of the Ad3 penton is expressed alone in the baculovirus system, it is found almost exclusively in the dodecahedral form (Figure 3C).

 The formation of chimeric dodecahedron, when Ad2 fibers are inserted into a preformed dodecahedron-base, confirms the previous results concerning the formation of chimeric pentons.

 Figures 3 and 4 illustrate the role of the base of the penton in the formation of the dodecahedron. Western blot analysis of the different fractions of the sucrose density gradient is carried out as illustrated in Example 1: FIG. 3A: coexpression of the base and the Ad3 fiber, from a baculovirus; FIG. 3B: coexpression of the base and of the Ad3 fiber, from a coinfection with two baculoviruses, each carrying one of the genes; FIG. 3C: expression of the base Ad3; FIG. 4D: expression of the Ad2 base and of the Ad3 fiber, from a coinfection with two baculoviruses each carrying a gene; FIG. 4E: expression of the base Ad2; FIG. 4F: in vitro formation of a dodecahedron by incubation of a dodecahedron-base (Ad3), preformed in vivo, with native Ad2 fibers.

EXAMPLE 2 Intracellular accumulation of dodecahedra.

 Figure 7 illustrates the intracellular accumulation of dodecahedron. HeLa cells are grown in plates containing 96 wells (Falcon) at 5,104 cells / well.

Increasing amounts (30 to 500 ng) of dodecahedron are added to each well and incubated with cells for 3 hours at 4 "C or for 2 hours at 37" C in 3% PBS-BSA buffer. The cells are washed twice with
PBS, then fixed and permeabilized with cold methanol. These cells are then incubated with antibodies directed either against the fiber (1: 500) or against the base of the penton (1: 500) and then incubated with an anti-rabbit antibody conjugated to fluorescein (1: 100; Cappel) . The resulting fluorescence is measured with a fluorescence measurement system (CytofluorTM 2350 Millipore).

 Figures 7, 8 and 9 illustrate the internalization of the dodecahedron in HeLa cells. FIG. 7 which includes on the abscissa the amount of dodecahedron (in ng) and on the ordinate the intensity of fluorescence, illustrates these results (curve -A-: incubation at 37 "C in the presence of anti-fiber antibodies; curve -A -: incubation at 37 "C, in the presence of anti-base antibodies; curve -O-: incubation at 4" C, in the presence of antibase antibodies).

 To make the photos illustrated in Figures 8 and 9, portions of 5,104,104 HeLa cells are cultured in DMEM medium containing 10% fetal calf serum, on strips (diameter: 1.2 cm). Each slide is incubated for 2 hours at 4 "C with 1 μg of dodecahedron in 50 RI of PBS-BSA at 3%. The cells are washed twice with cold PBS and incubated at 37" C in PBS-BSA at 3% for 0 to 45 min.

The cells are fixed and permeabilized by methanol treatment for 5 min at -20 C. The coverslips are then incubated for 1 hour at 37 "C with an antibody against the fiber (1: 200, 50 μl in PBS-BSA at 3% / strip), washed twice with
PBS, then incubated for 30 min with an antibody conjugated to fluorescein (1: 250, 50 µl in 3% PBS-BSA / coverslip). After a final washing in PBS, the slides are mounted on a microscope plate with a drop of 1,4-diazabicyclo [2.2.2.] Octane (Sigma) at 50 mg / ml in 50% glycerol -PBS at 50%. The observations are made on a confocal microscope MRC600 (Bio-Rad).

 Both Figure 7 and Figures 8 and 9 show that after incubation at 4 C (Figure 7, curve -O-), there is only attachment of the dodecahedral protein complex to said cells, while at 37 C (Figure 7 , curves -A- and -A-), there is internalization of said complex.

 Figures 8 and 9 illustrate more particularly the fate of the dodecahedral protein complex (or dodecahedron) in HeLa cells: at 40C, the dodecahedron is on the surface of the cells (visible after staining with an anti-fiber antibody, see Figure 7); when the HeLa cells are transferred to 37 "C, Figures 8 and 9 illustrate the evolution of the internalization of the dodecahedron, as a function of time: up to 5 minutes, the image is very similar (no internalization) , after 10-15 minutes of incubation (figure 8), a massive transfer of the dodecahedron is observed in the vicinity of the nuclear membrane. A longer incubation (20-45 minutes, figure 9) shows a more diffuse signal, which illustrates cytoplasmic evacuation of the dodecahedron Similar results are obtained when the fate of the dodecahedron is followed with an anti-base serum.

 A similar experiment is carried out with a dodecahedron made up solely of Ad3 bases. No attachment or internalization is observed by confocal electron microscopy when using a molar ratio comparable to that used in the previous experiment. However, when larger amounts of dodecahedron-base are used, internalization is observed. Quantities of dodecahedron-base 10 to 20 times greater are necessary to obtain a degree of internalization equivalent to that obtained with the dodecahedron-fiber.

Figure 10 illustrates the results obtained after one hour of internalization. Similar results are obtained after 30 minutes of internalization.

 These experiments show that the absence of fiber can be compensated by larger amounts of dodecahedron-base, however, the presence of fiber gives the dodecahedron greater efficiency, at the time of cell entry.

 These results are notably linked to the fact that the binding affinity of the fiber is 30 times greater than that of the base of the penton Ad2 (Kds of 1.7 and 55 nM, respectively).

At the temperature at which attachment and internalization of the virus is carried out, it has been shown that the dodecahedron Ad3 attaches to cells.
HeLa and that 10 to 20 minutes, at the temperature suitable for the entry of the virus, the dodecahedron is found in the cytoplasm, targeted on the cytoplasmic surface of the nuclear membrane. The overall kinetics seem to be similar to that of the first stages of a viral infection, when 20 minutes after attachment, approximately 50% of the incoming viral inocium (Ad5) is at the periphery of the nucleus.

 In the case of cellular entry of the Ad3 dodecahedron, the integrity of the dodecahedron and its components is not compromised either during the first 20 minutes or later (Figures 8 and 9).

EXAMPLE 3 Transfection of human cells with luciferase engineering using the dodecahedron Ad3 with or without fiber and a bifunctional peptide.

- Preparation of the peptide
The bifunctional peptide synthesized has 20 amino acids corresponding to the N-terminal part of the Ad3 fiber and 20 lysines on the C-terminal side, which will give a polycation capable of fixing polyanions like DNA.

polypeptide sequence: AKRARLSTSFNPVYPYEDES (K) 20.

- Preparation of the plasmid containing the DNA to be transported (active substance)
The plasmid encoding the luciferase reporter gene (pGL3control vector) (Promega) is produced in E. coli JM109 and purified by Qiagen.

- Transfection
The transfection of the cells is carried out as follows, for three types of cells (HeLa (FIG. 11), Hep2 (FIG. 12) and A549 (FIG. 13)):
a 24-well dish (Falcon) is seeded with 5,104 cells / well. The following day, the culture medium is removed and the cells are incubated with 200 μl of DMEM medium according to the following conditions:
a preincubation for fifteen minutes of the peptide with the plasmid pGL3, then addition of the dodecahedron for another fifteen minutes before putting everything on the cells,
b preincubation for fifteen minutes of the peptide with the dodecahedron, then addition of the plasmid pGL3 for another fifteen minutes, before putting everything on the cells,
c no preincubation, addition of the peptide, the plasmid pGL3 and the dodecahedron, directly on the cells.

These manipulations are made with a constant amount of peptide (1 Fg / well) and of plasmid (0.9 Rg / well). The fiber-containing dodecahedron is used at a ratio of 106 or 5,106 molecules per cell and that containing only the base at 5,106 molecules per cell. After 1 hour the inocula are removed and the wells are rinsed with DMEM-10% FCS and incubated for 48 hours in this medium. The lysis of the cells is carried out with 70 μl of RLB buffer according to the protocol described by the manufacturer (Promega). 20 μl fractions are counted on the luminometer according to the kit
Promega (Manual and Technical Bulletin provided by Promega).

 The results obtained are illustrated in Figures 11, 12 and 13.

 In these figures, the histogram sets A correspond to the use of a fiber dodecahedron at a concentration of 106 particles / cell, the histogram sets B correspond to the use of a fiber dodecahedron at a concentration of 5.106 particles / cell and the sets of C histograms correspond to the use of a dodecahedron-base at a concentration of 2.5.106 particles / cell.

 In each of these sets, the histogram l corresponds to the transfection of cells in the presence of a dodecahedron containing the plasmid pGL3, but in the absence of the bifunctional peptide, the histograms a, b and c correspond to the conditions set out above .

 As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described in a more explicit manner, on the contrary it embraces all of them. variants which may come to mind of the technician in the field, without departing from the scope, or the scope, of the present invention.

Claims (8)

CLAIMS - either of 12 penton bases, to the exclusion of any other constituent element of the genome of an adenovirus, which penton bases are derived either from the same adenovirus, or from different adenoviruses, and form a stable, dodecahedron structure to proteolytic enzymes and in that it has a molecular weight between 3.2.106 and 4.106. - either of 12 pentons, each comprising at least one fiber and one penton base, to the exclusion of any other constituent element of the genome of an adenovirus, which fiber (s) and base of the penton are derived either from the same adenovirus, either of different adenoviruses, said pentons being linked by the penton bases and forming a dodecahedron structure, stable to proteolytic enzymes, which complex has a molecular weight of between 4.8.106 and 6.6.106,  1 ") Adenoviral protein complex, characterized in that it consists of: Adenovirus type 9 (Ad9), adenovirus type 11 (Ad11), adenovirus type 15 (Ad15) or enteric adenoviruses (Ad40 and Ad41) and avian adenoviruses.  2 ") Dodecahedral protein complex according to claim 1, characterized in that said adenoviruses are selected from human adenoviruses and in particular adenovirus type 2 (Ad2), adenovirus type 3 (Ad3), adenovirus type 5 (Ad5), adenovirus type 4 (Ad4), adenovirus type 7 (Ad7),  3) Dodecahedral protein complex according to claim 1 or claim 2, characterized in that at least one of its constituents (base and / or fiber) can be modified to increase the affinity for a type particular cell.  4) Dodecahedral protein complex according to any one of claims 1 to 3, characterized in that it further comprises at least one hexon.  5) Pharmaceutical composition, characterized in that it essentially comprises an adenoviral dodecahedral protein complex according to any one of claims 1 to 4 and at least one other chemical substance.  6) Composition according to claim 5, characterized in that said chemical substance is associated with a suitable ligand selected from substances capable of producing an ionic bond with said chemical substance and substances capable of producing a covalent bond with said chemical substance.  7) Composition according to claim 6, characterized in that said ligand is selected from peptides whose N-terminal part comprises the N-terminal amino acid sequence of an adenovirus fiber of any serotype and whose part C-terminal allows an ionic bond with the chemical and in particular comprises a polylysine.  8) Composition according to claim 6, characterized in that said ligand is selected from peptides whose N-terminal part comprises the N-terminal amino acid sequence of an adenovirus fiber of any serotype and whose part C-terminal allows a covalent type bond with the chemical and in particular comprises a cysteine.