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

• US 5,906,817 A
• Filed: 01/19/1996
• Issued: 05/25/1999
• Est. Priority Date: 04/21/1993
• Status: Expired due to Fees

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Heard, Jean-Michel, Danos, Olivier, Moullier, Philippe, Ferry, Nicolas
• Primary Examiner(s): Campell, Bruce R.
• Application Number:
  US08/532,814
• Time in Patent Office: 1,222 Days
• Field of Search: 604/891.1, 424/422, 424/423, 424/93.1, 424/93.21, 435/172.3, 435/320.1, 435/325, 435/326, 435/382, 435/395, 435/397-400, 935/62
• US Class Current: 424/93.21
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3608 Bone, e.g. demineralised bone matrix [DBM], bone powder
• CPC Class Codes: A61L 27/3683 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2533/18 Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2740/13043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/16043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0068 : General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 9/10 : Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12N 9/1051 : Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/2402 : hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 204/01017 : Glucuronosyltransferase (2.4.1.17)
• CPC Class Codes: C12Y 302/01031 : Beta-glucuronidase (3.2.1.31)
• CPC Class Codes: C12Y 302/01076 : L-Iduronidase (3.2.1.76)

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 gelling 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.

Claims

1. An implant comprising:

(a) a biocompatible support permitting the biological anchoring of cells, wherein said biocompatible support comprises high porosity coral;

(b) cells transduced by a nucleotide sequence encoding a defined polypeptide and having the capacity to express said polypeptide; and

(c) a constituent capable of inducing and/or promoting the gelation of said cells.

2. The implant according to claim 1, wherein said biocompatible support is at least partially resorbable in vivo.

3. The implant according to claim 1, wherein said high-porosity coral biocompatible support has a spherical shape.

4. The implant according to claim 1, wherein said constituent capable of inducing and/or promoting the gelation of the cells is collagen.

5. The implant according to claim 1, wherein said constituent capable of inducing and/or promoting the gelation of the cells is selected from supports based on:

uncross-linked collagen, or

alginates.

6. The implant according to claim 1, wherein said cells have the capacity to be tolerated immunologically by an organism to which they are administered, and have been transduced by a nucleotide sequence coding for a defined polypeptide.

7. The implant according to claim 6, wherein said transduced cells are fibroblasts.

8. The implant according to claim 1, wherein said transduced cells comprise a retroviral vector comprising a proviral DNA sequence in which:

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 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, and the retroviral vector comprises, in addition, an exogenous nucleotide sequence under the control of a promoter.

9. The implant according to claim 8, wherein said proviral DNA of the vector is derived from the MuLV retrovirus.

10. The implant according to claim 8, wherein said sequences of the pol and env genes of the proviral DNA are entirely deleted.

11. The implant according to claim 8, wherein the U3 region of the LTR3'"'"' fragment of the proviral DNA is deleted at nucleotide 2797 of SEQ ID NO:

1.

12. The implant according to claim 8, wherein said exogenous nucleotide sequence is under the control of the mouse PGK-1 promoter or the human PGK-1 promoter, optionally lacking a "TATA box".

13. The implant according to claim 8, wherein said proviral sequence upstream from a promoter of the exogenous nucleotide sequence is the proviral nucleotide sequence situated between nucleotides 1 and about 1500 of SEQ ID NO:

1.

14. The implant according to claim 8, wherein said retroviral vector is the vector pM48 shown in FIG. 2, comprising the exogenous nucleotide sequence at the BamHI site.

15. The implant according to claim 1, wherein said cells are tumor cells transduced with an exogenous nucleotide sequence.

16. The implant according to claim 1, wherein said cells are transduced with 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.

17. The implant according to claim 1, further comprising one or more angiogenic factor.

18. The implant according to claim 1, further comprising heparin or a heparin derivative.

19. The implant according to claim 1, comprising from 10⁶ to 10⁹ transduced cells.

20. A method of temporary or permanent delivery of a desired polypeptide to a human or animal comprising implanting into the human or animal an implant according to claim 1.

21. A method for administering cells to a human or animal comprising implanting into the human or animal an implant according to claim 1:

either for the treatment of genetic diseases, wherein an exogenous nucleotide sequence contained in the cells code for beta-glucuronidase, for the factor VIII, factor IX or erythropoietin;

or for the treatment of acquired diseases;

or for the treatment of tumors, wherein the exogenous nucleotide sequence contained in the transduced cells code for a substance capable of promoting or enhancing the immune response against the cells of the tumors.

22. A composition comprising an implant according to claim 1 with one or more substances.

23. A method of treatment of genetic diseases, acquired diseases or tumors, said method comprising the introduction into man or animals of an implant according to claim 1 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.

24. A method according to claim 23, wherein said implant is introduced in the peritoneal cavity, the peri-renal space or the skin of the patient to be treated.

25. A method of preparation of an implant according to claim 1, 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.

26. The method according to claim 25, wherein said biocompatible support is placed in contact with cells previously incorporated into a solution of collagen.

27. The method according to claim 25, wherein said biocompatible support is constituted of coral powder, previously treated with a solution of collagen or a growth factor.

31. A 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 comprising high porosity coral making possible the biological anchoring of said cells;

a constituent capable of inducing and/or promoting the gelation of said cells; and

the retroviral vector according to claim 8.

33. A 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 comprising high porosity coral making possible the biological anchoring of said cells; and

a constituent capable of inducing and/or promoting the gelation of said cells wherein said cells are transduced cells having the capacity to be tolerated immunologically by the organism to which they are administered, and wherein said cells comprise a retroviral vector according to claim 8.

28. A 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 comprising high porosity coral making possible the biological anchoring of said cells; and

a constituent capable of inducing and/or promoting the gelation of said cells.

29. The kit according to claim 28, wherein said constituent capable of inducing and/or promoting the gelation of the cells is collagen.

30. The kit according to claim 28 comprising a DNA comprising a sequence coding for the substance expressed and secreted by said cells.

32. The kit according to claim 28, comprising cells having the capacity to express and secrete naturally or after recombination a defined substance.

34. An implant comprising:

(a) a biocompatible support permitting the biological anchoring of cells, wherein said support consists of high-porosity coral,

(b) fibroblast cells transduced by a nucleotide sequence encoding a polypeptide and having the capacity to express and secrete said polypeptide; and

(c) a constituent capable of inducing and/or promoting the gelation of said cells, wherein said constituent is collagen.

35. An implant comprising:

(a) a biocompatible support permitting the biological anchoring of cells, wherein said support consists of high-porosity coral,

(b) cells transduced by a retroviral vector comprising a nucleotide sequence encoding a polypeptide and having the capacity to express and secrete said polypeptide; and

(c) a constituent capable of inducing and/or promoting the gelation of said cells, wherein said constituent is collagen.