FR2708202A1 - Biocompatible implant for the expression in humans or animals of certain substances. - Google Patents

Abstract

The present patent application relates to an implant characterized in that it comprises a support of biological origin allowing the biological anchoring of cells previously brought into contact with constituents capable of inducing and / or promoting their gelling, said cells being chosen for their capacity to express and secrete naturally or after recombination a determined compound, for example a compound having a therapeutic interest.

Description

A L2708202

  BIOCOMPATIBLE IMPLANT FOR EXPRESSION

  IN MAN OR IN ANIMAL OF DETERMINED SUBSTANCES

  The present application relates to a biocompatible implant for expression in humans or in

  the animal of certain substances.

  The in vivo introduction of implants capable of expressing specific substances, for example for therapeutic purposes, requires the use of effective means with regard to the therapeutic or prophylactic end sought and the capacity for the organism in which it is introduced

  the implant, to tolerate it in the more or less long term.

  The invention relates in this regard to the use of materials of biological origin, as carriers for the introduction into humans or animals, of cells, for example recombinant cells, from which it is desired to produce a molecule

  determined, in particular for therapeutic purposes.

  The preparation of implants uses such supports of biological origin, capable of being brought into contact with cells and of various factors promoting the adhesion of these cells to the support if necessary, under conditions such that the various constituents present retain their main natural functional and structural properties. These supports of biological origin can be absorbable or non-absorbable by the host in which

they are introduced.

  The subject of the invention is therefore an implant characterized in that it comprises a support of biological origin allowing the biological anchoring of cells previously brought into contact with constituents capable of inducing and / or promoting their inclusion within 'a gel-forming matrix, said cells being chosen for their capacity to express and secrete naturally or after recombination a determined compound, for example a compound having a

therapeutic interest.

  The expression "biological anchoring" means that the cells contained in the implant can be fixed to the surface of the support of biological origin or, in

  in some cases, get inside this support.

  This attachment of the cells to the support is made possible in particular by the presence of the constituents capable of inducing and / or promoting the inclusion of the cells within a matrix having the constitution

of a gel.

  This inclusion in the matrix, called gelation, allows the organization of cells into a three-dimensional structure in an amorphous environment, that is to say, not causing inflammation in vivo. The implant obtained is according to a first embodiment of the invention consisting on the one hand of a support of biological origin based on limestone, in particular based on calcium carbonate and, preferably coral and on the other hand, a gel possibly loaded with cells expressing the substance of interest, in particular recombinant cells. Preferably, the coral used is a coral with high porosity, that is to say that it has a porosity as described in French Patent No. 2,460,657; it may also be a powder of sufficient porosity to allow a solid reinforcement to be obtained with a view to the constitution of a cellular network comprising or not a biological support such as collagen. In the case where the coral is replaced - 2 - by collagen, crosslinked or not, the latter can also serve both as a support and as a matrix promoting biological gelling for the

  constitution of the cell network.

  According to an advantageous embodiment of the invention, the high porosity coral used is

spherical type.

  According to another embodiment of the invention, the implant comprises a support of biological origin forming a solid matrix, said support containing: - more or less crosslinked collagen, for example in the form of fibers or sponges as sold under the brands Hemostagen or Paroguide; - bone powder or fragment or; - polymers based on carbohydrates such as

dextran or hyaluronic acid.

  According to an advantageous embodiment of the invention, the solid frame of the original support

  biological is susceptible to resorption in vivo.

  The implant thus formed allows the formation in vivo of an individualized and stable neo-organ if necessary, in which the biological support is likely to be gradually absorbed, leaving room for a structure comprising a vascularized connective tissue of form relatively comparable to that of the implant, this neo-organ however having, where appropriate, a volume reduced relative to the volume of the original implant. An implant that is particularly preferred in the context of the invention is such that the constituents capable of inducing and / or promoting gelation of the cells are, for example, non-crosslinked collagen or

alginates.

  - 3 - In particular, collagen, for example type I, will be used, in particular at a concentration of 1.5 mg / ml. Other constituents for gelling cells can naturally be used as long as they have the desired functional property and have the biocompatibility properties required to be introduced in vivo in humans or animals. A particular implant specific to the implementation of the invention is further characterized in that the cells are recombinant cells comprising genetic information foreign to their genome, capable of expressing this additional information in vivo and having the capacity to be tolerated immunologically by an organism to which

they would be administered.

  These cells can be modified by a recombinant vector coding for a determined polypeptide, said vector allowing the expression of foreign DNA in the cells. They can also be modified by the so-called homologous recombination methods. The vector penetrates into the cells using electroporation or the calcium phosphate precipitate or by any other method involving the entry of a nucleic acid either alone or

  via a recombinant virus for example.

  By "recombinant cells" is also meant autologous tumor cells irradiated prior to their introduction into the gel, having the capacity to maintain on their surface tumor antigens accessible to the immune system of the host in which the implant according to the invention has been introduced. By "polypeptide" is meant any amino acid sequence, whatever its size, this

  expression including proteins, and peptides.

  Likewise, the polypeptide may in reality be under

  glycosylated or non-glycosylated form.

  Advantageously, the recombinant cells contained in the implant are fibroblasts and, in

  in particular, skin fibroblasts.

  I1 can also be endothelial cells

or myoblasts.

  Preferably, these cells are autologous cells taken from a patient or a

  animal in which one wishes to introduce the implant.

  Recombinant cells can be prepared using a recombinant retroviral vector, the sequence coding for the polypeptide whose expression is sought (exogenous nucleotide sequence) being

introduced into proviral DNA.

  A provirus DNA sequence or "proviral DNA sequence" is a DNA sequence retrotranscribed from the genomic DNA of the virus and then integrated into

virus host cells.

  The DNA of the provirus is defined as thus comprising sequences coding for the gag, pol and env proteins of the retrovirus, which correspond respectively to the nucleoproteins, polymerases, proteins and

envelope glycoproteins.

  The provirus DNA also contains sequences called LTR (Long Terminal Repeat), which include regions called R, U3 and U5. These sequences of the LTR region intervene in the cycle

retrovirus replication.

  A particular implant according to the invention is characterized in that the recombinant vector is a retroviral vector comprising a provirus DNA sequence modified in such a way that: - the gag, pol and env genes of the provirus DNA have been deleted at least in part and this in order to obtain a proviral DNA incapable of replicating, this DNA not being able, moreover, to be recombined to form a wild virus, - the LTR sequence comprises a deletion in the U3 sequence, such that the transcription in the form of mRNA that it controls is reduced so

  significant, for example at least 10 times.

  This deletion makes it possible to decrease and preferably to neutralize the transcriptional effect of the LTR region, while retaining the capacity of the exogenous promoter to promote and control the expression of the exogenous nucleotide sequence contained in the

retroviral vector.

  This vector is such that the proviral DNA it

  contains advantageously from the MULV retrovirus.

  The LTR sequence of the proviral DNA has also been mutated by deletion, for example in its U3 part; this deletion affects the functions of the activator

internal of the LTR region.

  An “exogenous nucleotide sequence” according to the invention is a sequence which is not expressed naturally in a cell into which the vector, for example the retroviral of the invention, is introduced, or is insufficiently expressed therein, or which is desired get more expression than normal expression. This lack of expression or this insufficient expression results from the nature of the cell or occurs due to a pathology

  affecting this cell in a given individual.

  Likewise, the promoter used for the construction of recombinant retroviruses according to the invention is advantageously a constitutive promoter exogenous with respect to the sequence whose expression it controls in the sense that it is not naturally associated with it. The promoter of the transferred exogenous gene can also be used. The promoter used in the vector

  the invention can also be of the inducible type.

  This constitutive or inducible promoter controls the expression of the exogenous nucleotide sequence. The promoter can optionally be accompanied by a regulatory nucleic acid sequence, for example an activator.

  ("enhancer") which would regulate its activity.

  A first particular retroviral vector in the context of the invention is such that the exogenous nucleotide sequence and the exogenous constitutive promoter, as well as the proviral DNA sequence, are carried by a plasmid. For example, these sequences can be carried by the plasmid pBR322 which allows the production of DNA in a bacterium.

  view of its introduction into the packaging lines.

  Preferably, the sequences of the pol and env genes of the proviral DNA of the retroviral vector are completely deleted. In this case, the sequence of the gag gene can also be entirely deleted or, on the contrary, be partially preserved, since the DNA of the provirus

  thus formed is no longer likely to replicate.

  Advantageously, a retroviral vector as defined above is characterized in that the U3 region of the LTR3 ′ fragment is deleted at the level of nucleotide 2797 of FIG. 1. This deletion corresponds to a deletion of a fragment located between nucleotides 7935 and 8113 according to the numbering of the sequence published by Shinnick et al (Nature 293, 543-548, 1981). This deleted fragment of the U3 sequence of

  LTR3 'contains the activating region of the LTR.

  Particularly advantageously for the production of a retroviral vector according to the invention, the exogenous constitutive promoter is a promoter without TATA box and in particular the promoter of the phosphoglycerate kinase gene (PGK-1). This promoter is either the mouse promoter as described by Adra et al (Gene 60, 1987, 65-74) or the corresponding human promoter. This promoter has the advantage of allowing a high level of expression of the exogenous nucleotide sequence. Other constitutive promoters can be used in place of the PGK-1 promoter. In general, use will be made of a promoter active in the cells which it is desired to transform with the retroviral vector and in particular promoters active in fibroblasts in the quiescent state, for example promoters preceding the genes of the cytoskeleton. We

  may also cite the promoter of the 6- gene

  actin (Kost et al 1983, Nucl. Acids. Res. 11: 8287-8301) or the promoter of the vimentin gene Rittling and Basenga (1987) Mol. Cell. Biol. 7:

1676-1685).

  The promoter used may not have a "TATA box". According to an alternative embodiment of the invention, the proviral sequence upstream of the exogenous constitutive promoter is the nucleotide sequence located between nucleotides 1 and approximately 1500 according to the numbering of the sequence represented in FIG. 1. This sequence upstream of the promoter is essentially devoid of the whole

  gag, pol and env genes of the proviral sequence.

  The exogenous nucleotide sequence is advantageously inserted under the control of the exogenous constitutive promoter, in place of the qaq, pol sequences.

and env deleted.

  The exogenous nucleotide sequence is advantageously inserted at the BamHI site downstream of the promoter. Preferably, a retroviral vector according to the invention is the vector pM48 represented in FIG. 2, modified by the nucleotide sequence exogenous at

BamHI site.

  The retroviral vector M48 LacZ, also designated by the expression pM48 LacZ, has been deposited at the CNCM (National Collection of Culture of Microorganisms,

  Paris - France) under N 1-1298, April 16, 1993.

  This vector is derived from the vector pM48 and characterized in that it contains downstream of the exogenous constitutive promoter, a BamHI fragment of the P-galactosidase gene. This fragment of the 3-galactodidase gene can easily be deleted and be replaced by an exogenous nucleotide sequence of interest, for example a sequence coding for a protein or a glycoprotein capable of being of therapeutic interest or against the protein product of which we would like to

example get antibodies.

  A retroviral vector of the invention may also contain a sequence capable of increasing the transcriptional activity of the promoter, either of

  constitutively, or inducibly.

  Preferably, an implant according to the invention is characterized in that the recombinant cells are modified by a vector containing a sequence or several sequences of exogenous nucleotides) coding for an antigen or an antigenic determinant or coding for a polypeptide or a glycoprotein, soluble in serum, for example for a polypeptide or a glycoprotein of therapeutic interest, in particular for a hormone, a protein or a structural glycoprotein or a metabolism protein or glycoprotein or a viral protein or glycoprotein or a protein with the characteristics of a

  antibody or antibody fragment.

  Preferably, the implant also contains one or more angiogenic factors, in particular bFGF. The binding of the angiogenic factor, for example bFGF, is favored by the presence of the gelling agent. This agent contributes to the stable fixation of angiogenic factors to the support of biological origin

of the implant.

  In a particularly advantageous manner, the implant also contains heparin or a heparin derivative such as heparan sulfate or heparin fractions. Heparin and its derivatives are capable of binding to the constituents promoting gelation and thus of increasing the affinity of the angiogenic factors to the constituents allowing gelation. In particular, heparin binds to collagen and

  thus increases the affinity of bFGF for collagen.

  The implant according to the invention can be used permanently or temporarily, for use in humans or in animals. Indeed, its ability to constitute an individualized neo-organ in vivo, in which the biological support is, if necessary, at least partially absorbed, makes it possible to

  remove it by removal if necessary.

  An implant according to the invention is, in a particularly interesting way, usable for:

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  - the treatment of genetic diseases, in particular for the treatment of lysosomal storage diseases,

  hemophilia A or hemophilia B, P-

  thalassemia, the exogenous nucleotide sequence contained in the corresponding recombinant cells

  respectively to those who code for 6-

  glucuronidase for factor VIII, factor IX or erythropoietin, or for an active part of these sequences; - the treatment of acquired diseases, for example for the treatment of viral diseases, in particular for the treatment of an infection due to the HIV retrovirus for example by the expression and the secretion in the serum of soluble CD4 molecules or of an anti protein -viral soluble; - the treatment of tumors, the exogenous nucleotide sequence contained in the recombinant cells coding for a compound capable of promoting or increasing the

  immune response against tumor cells.

  The invention also relates to a composition characterized in that it comprises an implant according to the invention and one or more other constituents such as for example an antigen, an adjuvant in particular for

the vaccination.

  Other advantages and characteristics of the invention appear in the examples and the

following figures.

  - Figure 1: nucleotide sequence of the vector M48;

  - Figure 2: representation of the vector PM48.

EXAMPLES

PREPARATION OF THE RECOMBINANT VECTOR

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  The transfer and expression of exogenous genetic material makes it possible to experimentally modify the properties of a tissue. Treatments based on this principle could be offered to patients with genetic deficiencies or acquired diseases. The example described below uses a retroviral vector to introduce into cells the genetic information coding for a secreted protein. The reimplantation in the organism of genetically modified cells is carried out by associating these cells with collagen, an angiogenic factor and the reinforcement of the coral or crosslinked collagen type. The long-term in vivo expression of the genetic material thus transferred is obtained using the

  promoter of the murine phosphoglycerate kinase gene.

1 - RETROVIRAL VECTOR

  1.1 - Characteristics The retroviral vector M48 (FIG. 2) is constructed from proviral sequences isolated from the genomic DNA of rat cells infected with the murine leukemogenic Moloney retrovirus (Mo-MuLV). The gag, pol and env genes which code for the proteins of the virus have been removed and replaced by the sequences to be carried. The retroviral sequences necessary for cis were conserved for the production and maturation of transcripts of the recombinant viral genome, their packaging in infectious viral particles, their reverse transcription and their integration into the genome of the infected cell. The sequences of the recombinant provirus and the flanking fragments of cellular DNA are carried by the bacterial plasmid pBR322.

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  The mouse phosphoglycerate kinase (PGK-1) gene promoter was inserted in place of the viral genes. A unique BamHI cloning site makes it possible to place the sequences to be transported in the vector

  retroviral under the control of this promoter.

  This retroviral vector has a deletion in

  the U3 sequences of the internal LTR (Cone et al., Mol. Cel.

  Biol. 7, 887-897, 1987). This leads, in the infected cell, to a sharp decrease in mRNAs starting at the 5 'LTR, and a predominance of mRNAs produced from the PGK-1 promoter. The use of this vector therefore results in the insertion into the genome of the target cell of an expression cassette for the

  sequence of interest under the control of the PGK-1 promoter.

  1.2. - Structure of the vector The complete sequence of the vector M48 (Figure 1) includes: - Position 1 to 2019: Sequences of the Mo-MuLV including the long terminal repeat 5 '(or 5' LTR or LTR5 '), the packaging sequences a + which include part of the gag gene (position -447 to 1564 in the numbering of Shinnick et al. (Nature 293, 543-548, 1981). These sequences are modified as follows: insertion of a Sac II linker (5 'CCCGCGGG3')

  in position 1074 (position 626, Shinnick et al.).

  This leads to a mutation in phase with the

coding sequence for gag.

  - Position 2020 to 2526: Promoter of the mouse gene coding for phosphoglycerate kinase (position -524 to -20, before the translation start codon,

  according to Adra et al. Gene 60, 65-74, 1987).

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  - Position 2527 to 2532: Single BamHI site for the insertion of sequences which will be expressed under the

PGK-1 promoter control.

  - Position 2533 to 3101: Sequences of Mo-MuLV comprising the 3 'end of the env gene (32 codons) and the LTR3'. These sequences are modified as follows: deletion of the U3 sequences of the LTR between 7935 and 8113 in the numbering of Shinnick et al. and insertion of a Sal I linker (2798 in the

numbering present).

  - Position 3102 to 3674: Genomic sequences of rats. - Position 3675 to 8003: Sequences of the vector plasmid pBR322 (position 4363 to 29) comprising the 3-lactamase gene. These sequences are modified as follows: destruction of the BamH I site (position

7657, numbering present).

  - Position 8004 to 8388: Genomic sequences of rats. 1.3. - Production of retroviral vector This production is carried out by introducing the recombinant proviral structure into a cell line where the gag, pol and env genes are expressed constitutively. This so-called transcomplementing or packaging line synthesizes retroviral particles lacking genomic RNA. The TCRIP line, derived from NIH / 3T3 mouse fibroblasts is used here (Danos and Mulligan Proc. Natl. Acad. Sci (USA) 85, 6460-6465, 1988). When the recombinant construct is introduced by transfection, the RNAs transcribed from LTR5 ′ are packaged and the cells

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  then produce particles capable of transmitting

  the recombinant genome but unable to replicate.

  After transfection of the recombinant construct, the cells producing the retroviral vector are selected. For the vector preparation, a clone producing high viral titers is used.

  high viral titers, a polyclonal population.

  The stages of the isolation of a producer clone are as follows: 1) The WCRIP cells are transfected with a calcium phosphate coprecipitate (Graham and Van Der Erb, Virology, 52, 456, 1973) of the retroviral construction and of the plasmid pSV2neo (Southern and Berg, J. Mol. Appl. Gen. 1, 327-341, 1982), in a report

10 to 1 molar.

  2) The cells which have stably integrated and which express the exogenous DNA are selected in the presence

  G418 at 1 mg / ml (Geneticin, Gibco).

  3) The population of cells resistant to G418 o of the isolated individual clones are tested for their capacity to produce the retroviral vector. For this, NIH / 3T3 target cells are placed in the presence of the culture medium collected from 4CRIP cells, and the transmission of the recombinant provirus is analyzed after 48 hours. The analysis, carried out from the genomic DNA of the target cells, is done by PCR (qualitative method) or by Southern blot (quantitative method). 4) A bank of producer cells is

kept at - 135 C.

  2 - PREPARATION OF RETROVIRAL VECTOR STOCKS

  The culture supernatant of the packaging cells is collected after a 24 hour incubation on the packaging cells producing the

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  retroviral vector, and contacted with cells

targets for infection.

  2.1. - Mass preparation A stock of a hundred ampoules containing producer cells is kept at -135 C. After thawing, the cells are amplified for 10 days, successively in a bottle then in rollers of 875 cm2, until obtaining 20 confluent skates. The

  production is then started. It lasts 4 or 5 days.

  The culture medium is DMEM containing 1 g / l of glucose, 10 mM of sodium pyruvate, and added with 5% of newborn calf serum (Hyclone). Each roller allows the packaging of 100 ml of medium per 24 hours. This culture supernatant is centrifuged to remove cell debris. The production

  weekly is 8-10 liters of viral supernatant.

  2.2. - Concentration The supernatant can be concentrated to increase the infectious titer of the retroviral vector. After centrifugation, the supernatant is dialyzed tangentially against a Sartocon membrane with a porosity of 100,000, using the Crossflow device from Sartorius. A concentration of 20 times is obtained in 3 hours. It is accompanied by a 20-fold increase in infectious titers. After concentration, the viral preparation is used immediately, or stored at -80 C if the period of use is

more than 2 weeks.

3. SKIN FIBROBLASTS

  3.1. - Isolation and cultivation The fibroblasts intended for gene transfer are obtained by a skin biopsy carried out under very careful aseptic conditions. In mice, a

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  syngeneic animal is sacrificed for this use. In large mammals, one or more skin fragments of approximately 10 cm2 are removed during general anesthesia. After cutting into shreds of a few mm2, the fragments are dissociated by an enzymatic treatment. For a 10 cm 2 fragment, the reaction is carried out for 2 hours at 37 ° C. with gentle agitation in 50 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, 100 mg of collagenase (Worthington), 500 U of dispase (Collaborative Research Inc.). After centrifugation and washing, the cells are counted, then seeded in 150 cm 2 flasks at a concentration of 1 to 10 × 10 6 / cm 2. The culture medium is RPMI 1640

  supplemented with 10 to 20% fetal calf serum.

  3.2. - Infection with the retroviral vector Infections with the retroviral vector preparation are started the day after culture, when the cell density is less than 20% confluence. They are repeated daily, following the same protocol for 4 or days. After removal from the culture medium, the cells are brought into contact with the non-concentrated or concentrated retroviral vector preparation, in

  all cases, added with 8gg / ml polybrene.

  The incubation is carried out for 2 hours at 37 ° C., then, without prior washing, the viral preparation is

  replaced by culture medium.

  When a vector coding for a protein detectable under microscopic examination is used, it is found that this method allows the introduction of the foreign gene into more than 90% of fibroblasts. The level of protein secretion in the culture supernatant can be monitored during and after this

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  process. Cells can be frozen in

part or all at this stage.

  After infection, the cells are amplified until a sufficient number for reimplantation is obtained, i.e. 2-6x108 cells / kg of body weight. Amplification is carried out in

  culture trays (multitray, Nunc).

  3.3. - Collection of genetically modified cells. After amplification, the cells are collected by treatment with trypsin. After washing and counting, they are available to be used for the formation of a neo-organ. A sample is kept to control genetic transfer by Southern blot, as well as the expression and secretion of the foreign protein

by an appropriate technique.

  IN VIVO USE OF IMPLANTS INCLUDING CORAL

AS BIOLOGICAL SUPPORT

  1. In mice In C3H / He mice, compatibility experiments with Biocoral as an implant have shown that it could represent an alternative

  advantageous compared to synthetic supports.

  The Biocoral could indeed be effectively covered with different elements of the extra cellular matrix as well as angiogenic factors. It has been shown that murine type I collagen at a concentration of 0.5 mg / ml in 0.001% acetic acid adheres and covers the coral support after drying in ambient air for 12 to 24 hrs. The presence of this element of the extracellular matrix then allowed the irreversible binding of basic factor of

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  growth of fibroblasts labeled with 125I 125I-basic Fibroblast Growth Factor (bFGF) in Biocoral. Fixing was thus carried out on average of 50 ng of bFGF on 40 mg of coral support pretreated with murine type I collagen. Although this connection could also have been carried out in the absence of collagen pretreatment, it was in this reversible case therefore of low affinity. Collagen is therefore an essential and justified intermediary for the stable fixation of angiogenic factors in Biocorail. Finally, the binding of bFGF could be increased from five to ten times when the covering of 40 mg of Biocoral with collagen was supplemented by heparan sulfate (Signa) 200 g (1 mg / ml solution in PBS) or heparin (Roche) 500 units (solution at 5000 units / ml). These two components of the extracellular matrix have a known affinity for collagen (Baird, A., Schubert, D., Ling, N., and Guillemin, R. (1988) Receptor and heparine binding domains of basic fibroblast growth

  factor. Proc. Natl. Acad. Sci. USA 85, 2324-2328).

  Their anionic and poly-sulfated characteristics are responsible for the strong affinity for bFGF

  (Johnson, D.E., Lee, P.L., Lu, J., and Williams, L.T.

  (1990) Various forms of a receptor for acidic and basic fibroblast growth factors. Mol. Cell. Biol. 10, 4728-4739). After incubation in the presence of one or other of these components, 1 hr at 4 ° C. with shaking, the Biocorail was dried in ambient air. So

  heparinized, coral has an anticoagulant action.

  Subcutaneous implantation tests of spherical coral of 40 mg, with high porosity (50%), covered with murine type I collagen and bFGF (= 50 ng / 40 mg of coral) were carried out in vivo on a ten mice. The macroscopic and histological aspect of these implants was analyzed over a period of 7 months. The

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  results show that the Biocoral was gradually absorbed in a few months and gave way to a structure formed of vascularized connective tissue having the initial spherical shape of the coral implant with, however, a reduction in volume of approximately one third. Histological analysis of this connective tissue at three months showed intense vascularization and the complete absence of inflammatory cells compared to the coral residues. The connective tissue itself was made up of sparse mesenchymal cells, surrounded by neoformed collagen fibers. Analysis at 7 months showed similar results without involution. 2. In dogs: In a 25 kg dog, we carried out a first experiment of implantation of a neo-organ, the external part of which made of porous coral (50%) made it possible to retain a retracted collagen gel containing 1 to 2 x 109 genetically modified autologous fibroblasts. The Biocoral support had been covered with

  murine type I collagen and heparin (Roche).

  After drying, the coral thus pretreated was incubated in the presence of angiogenic growth factor of

  bFGF type (Farmitalia) then rinsed in PBS.

  The surgical implantation performed under general anesthesia, consisted of inserting the assembly between the two parietal and visceral sheets of the omentum apron near the large curvature of the stomach. This rapid gesture allowed simple operative follow-ups, and suggests the possibility of implantation by laparoscopic surgery in the future. Forty five days later, an exploratory laparotomy was

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  performed in order to practice a macroscopic assessment of the implant. Updating the omentum apron was easy, without adhesion and locating the coral implant, containing the autologous fibroblasts

  genetically modified, happened very quickly.

  No adhesion could be noted, and the vascularization was important, including

  large caliber vessels in connection with the implant.

  A process of gradual resorption of the coral has been

observed for at least 4 months.

  3. Other mammals: The creation of new organs, using Biocoral, suitable for surgical implantation on large mammals, especially in humans, for a

  clinical development is performed analogously.

  The Biocoral is modular in its conception, capable of triggering sustained angiogenesis associated with its own absorption and finally devoid of systemic and local inflammatory effects. Such a biomaterial advantageously serves as receptacle and skeleton for a type I collagen gel containing genetically modified autologous cells capable of secreting a therapeutic factor into the circulation.

  once the vascular connections are established.

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Claims (23)

  1. Implant characterized in that it comprises a support of biological origin allowing the biological anchoring of cells previously brought into contact with constituents capable of inducing and / or promoting their gelation, said cells being chosen for their ability to express and secrete naturally or after recombination a determined compound, for example a compound having a therapeutic interest. 2. Implant according to claim 1, characterized in that the support of biological origin is of the type   absorbable in vivo, at least partially.   3. Implant according to claim 1 or claim 2, characterized in that the support of biological origin is a support based on limestone, in particular calcium carbonate, preferably in that it is coral.   4. Implant according to claim 3, characterized in that the support of biological origin is coral high porosity.   5. Implant according to claim 3 or claim 4, characterized in that the coral to   high porosity is spherical coral.   6. Implant according to any one of   claims 1 to 5, characterized in that the   constituents capable of inducing and / or promoting gelation of the cells are collagen, in particular type I collagen, preferably to a   concentration of the order of 1.5 mg / ml.   7. Implant according to claim 1 or claim 2, characterized in that the support of biological origin is chosen from: - 22 -   - crosslinked collagen, in particular in the form of fibers or sponges, - bone powder, - polymers based on carbohydrates such as dextran or hyaluronic acid.   8. Implant according to any one of the claims   1 to 7, characterized in that the constituents capable of inducing and / or promoting gelling of the cells are chosen from supports based on: - non-crosslinked collagen, - alginates.   9. Implant according to any one of claims   1 to 8, characterized in that the cells are recombinant cells having the capacity to be tolerated immunologically by an organism to which they would be administered, modified by a nucleotide sequence   encoding a specific polypeptide.   10. Implant according to claim 9, characterized in that the recombinant cells are fibroblasts,   especially skin fibroblasts.   11. Implant according to any one of   claims 1 to 10, characterized in that the vector   recombinant is a retroviral vector comprising a provirus DNA sequence modified in such a way that: - the gag, pol and env genes of the provirus DNA have been deleted at least in part in order to obtain an incapable proviral DNA to replicate, this DNA not being able, moreover, to be recombined to form a wild virus, - the LTR sequence comprises a deletion in the U3 sequence, such that the transcription in the form of mRNA which it controls is significantly reduced , for example at least 10 times, and the recombinant retroviral vector further comprises an exogenous nucleotide sequence under the control of a - 23 -   promoter, for example a constitutive promoter or inducible, exogenous.   12. Implant according to claim 11, characterized in that the proviral DNA of the vector comes from Mo-MuLV retrovirus.   13. Implant according to claim 11 or claim 12, characterized in that the sequences of the pol and env genes of the proviral DNA are entirely deleted. 14. Implant according to any one of   claims 11 to 13, characterized in that the region   U3 of the LTR3 'fragment of proviral DNA is deleted at   level of nucleotide 2797 of FIG. 1.   15. Implant according to any one of   claims 11 to 14, characterized in that the sequence   of exogenous nucleotides is under the control of the mouse PGK-1 promoter or the human PGK-1 promoter, if applicable if necessary without "TATA box".   16. Implant according to any one of   claims 11 to 15, characterized in that the sequence   proviral upstream of the exogenous constitutive promoter is the nucleotide sequence of the provirus, located between nucleotides 1 and approximately 1500 of the sequence shown in Figure 1.   17. Implant according to any one of   claims 11 to 16, characterized in that the vector   retroviral is the vector pM48 represented in FIG. 2, modified by the insertion of the nucleotide sequence exogenous to the BamHI site.   18. Implant according to any one of   claims 1 to 9 or 11 to 17, characterized in that   the recombinant cells are tumor cells.   19. Implant according to any one of   claims 1 to 18, characterized in that the cells   are modified by a vector containing a - 24 -   sequence or several sequences of exogenous nucleotides, coding for an antigen or an antigenic determinant or coding for a polypeptide or a glycoprotein, soluble in serum, for example for a polypeptide or a glycoprotein of therapeutic interest, in particular for a hormone , a structural protein or glycoprotein or a metabolism protein or glycoprotein or a viral protein or glycoprotein or protein having the characteristics of an antibody or antibody fragment. 20. Implant according to any one of   Claims 1 to 19, characterized in that it contains   additionally one or more angiogenic factors, in particular of bFGF.   21. Implant according to any one of   Claims 1 to 20, characterized in that it contains   heparin or a heparin derivative.   22. Therapeutic medium comprising, as active principle, an implant according to any one of   claims 1 to 22 for processing   - or genetic diseases, in particular for the treatment of lysosomal overload diseases, hemophilia A or hemophilia B, P-thalassemia, the exogenous nucleotide sequence contained in the recombinant cells corresponding respectively to those which encode 3-glucuronidase for factor VIII, factor IX or erythropoietin, or for an active part of these sequences; - or acquired diseases, for example for the treatment of viral diseases, in particular for the treatment of an infection due to the HIV retrovirus for example by the expression and the secretion in the serum of soluble CD4 molecules or of an anti-protein soluble viral; - 25 -   - or tumors, the exogenous nucleotide sequence contained in the recombinant cells encoding a compound capable of promoting or increasing the   immune response against tumor cells.   23. Composition characterized in that it comprises   an implant according to any one of claims 1 to   21, with one or more substances, in particular a antigen or adjuvant. - 26 -