Biocompatible implant for the expression and in vivo secretion of a therapeutic substance

Abstract

The invention relates to an implant obtained by assembling in vitro various elements in order to form a neo-organ which is introduced preferably in the peritoneal cavity of the recipient. The implant comprises a biocompatible support intended to the biological anchoring of cells; cells having the capacity of expressing and secreting naturally or after recombination a predetermined compound, for example a compound having a therapeutical interest; and a constituent capable of inducing and/or promoting the geling of said cells. The invention also relates to a kit for the preparation of the implant as well as to a new recombinant retroviral vector comprising a provirus DNA sequence modified in that the genes gag, pol and env have been deleted at least partially so as to obtain a proviral DNA capable of replication. The invention also relates to recombinant cells comprising the new retroviral vector.

1 . Implant characterized in that it comprises: 
 a biocompatible support permitting the biological anchoring of cells,    cells having the capacity to express and secrete naturally or after recombination a defined substance, for example a substance having therapeutic value; and    a constituent capable of inducing and/or promoting the gelation of said cells.    
     
     
         2 . Implant according to  claim 1 , characterized in that the biocompatible support includes at least me of the elements selected from the group comprising PTFE or a support of biological origin.  
     
     
         3 . Implant according to  claim 1 , characterized in that the biocompatible support is a support of biological origin of the type resorbable in vivo at least partially.  
     
     
         4 . Implant according to any one of the  claims 1  to  3 , characterized in that the biocompatible support is a calcium-based, in particular a calcium carbonate-based, support, preferably it is coral.  
     
     
         5 . Implant according to  claim 4 , characterized in that the biocompatible support is high-porosity coral.  
     
     
         6 . Implant according to  claim 3  or  claim 5 , characterized in that the high-porosity coral is a spherical coral.  
     
     
         7 . Implant according to any one of the  claims 1  to  6 , characterized in that the constituent capable of inducing and/or promoting the gelation of the cells is collagen, in particular type I collagen, preferably at a concentration of the order of 1.5 mg /ml.  
     
     
         8 . Implant according to  claim 1  or  claim 3 , characterized in that the biocompatible support is selected from: 
 cross-linked collagen, in particular in the form of fibers or sponges,  
 bone powder,  
 carbohydrate-based polymers such as dextran or hyaluronic acid.  
 
     
     
         9 . Implant according to any one of the  claims 1  to  8 , characterized in that the constituent capable of inducing and/or promoting the gelation of the cells is selected from supports based on: 
 uncross-linked collagen,  
 alginates.  
 
     
     
         10 . Implant according to any one of the  claims 1  to  9 , characterized in that the cells are recombinant cells having the capacity to be tolerated immunologically by an organism to which they are administered, modified by a nucleotide sequence coding for a defined polypeptide.  
     
     
         11 . Implant according to  claim 10 , characterized in that the recombinant cells are fibroblasts, in particular skin fibroblasts.  
     
     
         12 . Implant according to any one of the  claims 1  to  11 , characterized in that said cells are recombinant cells modified by a retroviral vector comprising a proviral DNA sequence modified in a manner such that: 
 the gag p and env genes of the proviral DNA have been deleted at least in part in order to produce a proviral DNA incapable of replicating this DNA being in addition incapable of recombining to form a wild-type virus,  
 the LTR sequence contains a deletion in the sequence U3 such that transcription of the mRNA that it controls is reduced significantly for example at least 10-fold, and the recombinant retroviral vector comprises, in addition, an exogenous nucleotide sequence under the control of a promoter for example an exogenous, constitutive or inducible promoter.  
 
     
     
         13 . Implant according to  claim 12 , characterized in that the proviral DNA of the vector is derived from the MuLV retrovirus.  
     
     
         14 . 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.  
     
     
         15 . Implant according to any one of the  claims 11  to  13 , characterized in that the U3 region of the LTR3′ fragment of the proviral DNA is deleted at the level of nucleotide 2797 of FIG. 1.  
     
     
         16 . Implant according to any one of the  claims 12  to  15 , characterized in that the exogenous nucleotide sequence is under the control of the mouse PGK-1 promoter or the human PGK-1 promoter, optionally lacking a “TATA box”.  
     
     
         17 . Implant according to any one of the  claims 12  to  16 , characterized in that the proviral sequence upstream from the exogenous promoter is the proviral nucleotide sequence situated between nucleotides 1 and about 1500 of the sequence shorn in FIG. 1.  
     
     
         18 . Implant according to any one of the  claims 12  to  17 , characterized in that the retroviral vector is the vector pM48 show in FIG. 2, modified by the insertion of the exogenous nucleotide sequence at the BamHI site.  
     
     
         19 . Implant according to any one of the  claims 1  to  10  or  12  to  18 , characterized in that the recombinant cells are tumor cells.  
     
     
         20 . Implant according to any one of the  claims 1  to  19 , characterized in that the recombinant cells are modified by a vector containing one or more exogenous nucleotide sequences coding for an antigen or an antigenic determinant or coding for a polypeptide or glycoprotein soluble in the serum, for example a polypeptide or a glycoprotein of therapeutic interest, in particular a hormone, a structural protein or glycoprotein or a metabolic protein or glycoprotein or a viral protein or glycoprotein or a protein having the characteristics of an antibody or an antibody fragment.  
     
     
         21 . Implant according to any one of the  claims 1  to  20 , characterized in that it contains in addition one or more angiogenic factors, in particular bFGF.  
     
     
         22 . Implant according to any one of the  claims 1  to  21 , characterized in that it contains heparin or a heparin derivative.  
     
     
         23 . Implant according to any one of the  claims 1  to  22 , characterized in that it contains from 10 6  to 10 9 , and preferably from 5×10 6  to 10 7  recombinant cells.  
     
     
         24 . Use of an implant according to any one of the  claims 1  to  23 , in a permanent or temporary fashion, for the implantation in man or animals.  
     
     
         25 . Use of an implant according to any one of the  claims 1  to  23 : 
 either for the treatment of genetic diseases, in particular for the treatment of diseases of lysosomal overload, hemophilia A or hemophilia B, beta-thalassemia, the erogenous nucleotide sequence contained in the recombinant cells corresponding respectively to those which code for beta-glucuronidase, for the factor VIII factor IX or erythropoietin, or for an active part of these sequences;  
 or for 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 secretion into the serum of soluble CD4 molecules or a soluble anti-viral protein;  
 or for the treatment of tumors, the exogenous nucleotide sequence contained in the recombinant cells coding for a substance capable of promoting or enhancing the immune response against the cells of the tumors.  
 
     
     
         26 . Composition characterized in that it contains an implant according to any one of the  claims 1  to  23  with one or more substances, in particular an antigen or an adjuvant.  
     
     
         27 . Method of treatment of genetic diseases, acquired diseases or tumors, said method comprising the introduction into man or animals of an implant according to any one of the  claims 1  to  23  for a period of time sufficiently long to allow the cells included in said implant to produce in vivo a peptide, a protein or a glycoprotein having a therapeutic effect on the disease to be treated.  
     
     
         28 . Method according to  claim 7 , characterized in that said implant is introduced in the peritoneal cavity, the peri-renal space or the skin of the patient to be treated.  
     
     
         29 . Method of preparation of an implant according to any one of the  claims 1  to  23 , said method comprising the steps of: 
 placing of the biocompatible support in contact with said cells and a constituent capable of inducing and/or promoting their gelation;  
 incubation of the preparation obtained in the previous step in order to obtain the gelation of said constituents;  
 culture of the cells thus obtained under conditions allowing them to bind to the gelled constituents, and  
 recovery of the implant thus obtained.  
 
     
     
         30 . Method according to  claim 29 , characterized in that the biocompatible support is placed in contact with cells previously incorporated into a solution of collagen.  
     
     
         31 . Method according to  claim 29  or  30 , characterized in that the biocompatible support is constituted of PTFE fibers or coral powder, previously treated with a solution of collagen or a growth factor.  
     
     
         32 . Recombinant retroviral vector characterized in that it comprises: 
 a proviral DNA sequence modified in a manner such that: 
 the gag, pol and env genes of the proviral DNA have been deleted at least in part in order to produce a proviral DNA incapable of replication, this DNA being in addition unable to recombine for form a wild-type virus,  
 the LTR sequence bears a deletion in the U3 sequence such that transcription of mRNA that it controls is reduced significantly, for sample by at least 10 fold, and  
   the recombinant retroviral vector comprising in addition an exogenous nucleotide sequence under the control of a promoter for example an exogenous, inducible or constitutive promoter.    
     
     
         33 . Retroviral vector according to  claim 32 , characterized in that the modified proviral DNA sequence, the exogenous nucleotide sequence and the exogenous promoter are borne by a plasmid.  
     
     
         34 . Retroviral vector according to  claim 1  or  claim 33 , characterized in that the proviral DNA is derived from the MuLV retrovirus.  
     
     
         35 . Retroviral vector according to any one at the  claims 32  to  34 , characterized in that the sequences for the pol and env genes of the proviral DNA are entirely deleted.  
     
     
         36 . Retroviral vector according to any one of the  claims 32  to  35 , characterized in that the U3 region of the LTR3′ fragment is deleted at the level of nucleotide 2797 of FIG. 1.  
     
     
         37 . Retroviral vector according to any one of the  claims 32  to  36 , characterized in that the exogenous nucleotide sequence is under the control of the mouse PGK-1 promoter or the human PGK-1 promoter, optionally lacking a “TATA box”.  
     
     
         38 . Retroviral vector according to any one of the  claims 32  to  36 , characterized in that the proviral sequence upstream from the exogenous promoter is the proviral nucleotide sequence situated between the nucleotides 1 and about 1500 of the sequence shown in FIG. 1.  
     
     
         39 . Retroviral vector according to  claim 37 , characterized in that the exogenous nucleotide sequence is inserted at the BamHI site downstream from the exogenous constitutive PGK-1 promoter.  
     
     
         40 . Retroviral vector according to any one of the claims  32  and  39 , characterized in that it is the pM48 vector shown in FIG. 2, modified by the insertion of the exogenous nucleotide sequence at the BamHI site.  
     
     
         41 . Retroviral vector according to any one of the  claims 32  to  40 , characterized in that it contains at the BamHI site downstream from the exogenous constitutive promoter a BamHI fragment of the gene of beta-galactosidase.  
     
     
         42 . Retroviral vector acceding to any one of the  claim 32  to  41 , characterized in that it contains in addition upstream from the exogenous constitutive promoter an enhancer sequence.  
     
     
         43 . Recombinant cells characterized in that they are cells having the capacity to be tolerated immunologically by the organism to which they are administered, modified by a retroviral vector according to any one of the  claims 32  to  42 .  
     
     
         44 . Recombinant cells according to  claim 43 , characterized in that they are recombinant fibroblasts, in particular skin fibroblasts.  
     
     
         45 . Recombinant cells according to  claim 43 , characterized in that they are tumor cells modified by a retroviral vector according to any one of the  claims 32  to  42 .  
     
     
         46 . Recombinant cells according to any one of the  claims 43  to  45 , characterized in that the exogenous nucleotide sequence which they contain codes for a protein whose expression is desired, in particular a protein soluble in the serum.  
     
     
         47 . Recombinant cells according to any one of the  claims 43  to  46 , characterized in that the exogenous nucleotide sequence which they contain codes for beta-glucuronidase.  
     
     
         48 . Use of the recombinant cells according to any one of the  claims 43  to  47  for the treatment of a disease capable of being corrected by the expression and secretion into the serum of a patient of the exogenous nucleotide sequence contained in these cells.  
     
     
         49 . Use of the recombinant cells according to any one of the  claims 43  to  47  for the treatment of genetic diseases, in particular for the treatment of diseases of lysosomal overload, hemophilia A or hemophilia B, beta-thalassemia, the exogenous nucleotide sequence contained in the recombinant cells corresponding respectively to those which code for beta-glucuronidase for the factor VIII, factor IX or erythropoietin or for an active part of these sequences.  
     
     
         50 . Use of the recombinant cells according to any one of the  claims 43  to  47  for the treatment of acquired diseases, for Ocala 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 secretion into the serum of soluble CD4 molecules or of a soluble anti-viral protein.  
     
     
         51 . Use of the recombinant cells according to any one of the  claims 43  to  47  for the preparation of antibodies against the expression product of the exogenous nucleotide sequence contained in the recombinant cells.  
     
     
         52 . Use of the recombinant cells according to any one o the  claims 43  to  47  for the treatment of tumors, the exogenous nucleotide sequence contained in the recombinant cells coding for a substance capable of promoting or enhancing the immune response against the cells of the tumor.  
     
     
         53 . Recombinant cells according to  claim 52  such as obtained by recombination of tumor cells with a retroviral vector according to any one of the  claims 32  to  42 .  
     
     
         54 . Kit for the preparation of an implant to achieve the in vivo expression and secretion by cells of a substance to produce a desired therapeutic effect, said kit containing: 
 a biocompatible support making possible the biological anchoring of said cells; and    a constituent capable of inducing and/or promoting the gelation of said cells.    
     
     
         55 . Kit according to  claim 54 , characterized in that the biocompatible support comprises at least one of the elements selected from the group including PTFE or a support of biological origin, in particular a calcium-based, in particular a calcium carbonate-based, support of biological origin, preferably coral.  
     
     
         56 . Kit according to  claim 54  or  55 , characterized in that the constituent capable of inducing and/or promoting the gelation of the cells is collagen, in particular type I collagen, preferably at a concentration of the order of 1.5 mg/ml.  
     
     
         57 . Kit according to  claim 54 , characterized in that it contains a DNA comprising a sequence coding for the substance expressed and secreted by said cells.  
     
     
         58 . Kit according to  claim 57 , characterized in that it contains a retroviral vector according to any one of the  claims 32  to  42 .  
     
     
         59 . Kit according to any one of the  claims 54  to  58 , characterized in that it contains cells having the capacity to express and secrete naturally CT after recombination a defined substance for example a substance having a therapeutic value.  
     
     
         60 . Kit according to  claim 59 , characterized in that the cells are recombinant cells a according to anyone of the  claims 43  to  47 .