Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-01-06
2001-12-04
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S091100, C435S320100, C435S455000
Reexamination Certificate
active
06326195
ABSTRACT:
The invention relates to a biocompatible implant for the expression of defined substances in man or in animals, in particular for the anchoring of the recombinant cells of the invention.
The present invention also relates to retroviral vectors for the preparation of recombinant cells capable of being implanted in vivo for a therapeutic purpose.
STATE OF THE ART
The in vivo introduction of implants capable of expressing defined substances for example for therapeutic purposes necessitates the use of efficacious agents as regards the desired therapeutic or prophylactic objective and that the organism into which the implant is introduced has the capacity to tolerate it in the relatively long-term.
The earlier international patent application published under the number 92/15676 described agents to obtain in vivo the expression of defined nucleotide sequences for the purpose of a therapeutic treatment of diseases resulting from a genetic anomaly. This international application 92/15676 proposes the use of fibroblasts genetically modified by a retroviral vector for the purpose of implanting them in the connective tissue of the skin of the subject to be treated.
The nucleotide sequence whose expression is desired in this earlier international application is placed under the control of the LTR (Long Terminal Repeat) sequence of the retroviral vector and/or under the control or inducible or constitutive exogenous promoters, the LTR sequence of the retrovirus being nonetheless conserved when the exogenous promoter is present.
SUMMARY OF THE INVENTION
The invention proposes agents making it possible to achieve in vivo the expression of a selected nucleotide sequence under conditions such that this expression is obtained over a period of several months, preferably more than 6 months, in a manner such that the product of the nucleotide sequence expressed is present in sufficient quantity and under conditions suitable for the production of a desired therapeutic effect in vivo.
The agents defined in the present application make it possible to achieve the expression and the secretion of a protein, glyco-protein or a peptide of biological interest to justify its therapeutic use in vivo.
The invention also relates to the use of biocompatible materials as supports for the introduction into man or animals of cells for example recombinant cells of the invention defined below, starting from which it is desired to produce a defined molecule in particular for therapeutic purposes.
The preparation of implants requires such supports suitable for being placed in contact with cells and various factors promoting the adhesion of these cells to the support if necessary, under conditions such that the different constituents present conserve their principal natural structural and functional properties.
These biocompatible supports whether of biological origin or not may or may not be resorbable by the host into which they are introduced.
The invention also concerns retroviral vectors having the capacity to infect cells, and in particular eukaryotic cells, as well as the recombinant cells including these vectors. The recombinant cells of the invention may be administered or implanted in a patient, thus producing in vivo a protein or any expression product of a defined nucleotide sequence inserted into the retroviral vector.
The inventors have examined which are the parameters which, at various levels, would make it possible to express in vivo a defined nucleotide sequence so as to improve the expression of the nucleotide sequence inserted in the vector in order to obtain a therapeutic effect, if necessary of long duration.
Thus, they have demonstrated that when the exogenous nucleotide sequence whose expression is desired is advantageously placed in the vector intended for the infection of the cells under the control of an inducible or constitutive exogenous promoter, the LTR sequence internal to the retroviral vector must then be partially deleted. The deletion must be sufficient to impair the transcription of the mRNA.
DETAILED DESCRIPTION OF THE INVENTION
Biocompatible Implant
The implant or neo-organ of the invention is obtained by the process which consists in the in vitro assembly of various elements in order to form an implant which is introduced preferably into the peritoneal cavity of the recipient. Other implantation sites can be used such as the perirenal space, the skin. In vivo the implant gives rise to a connective tissue formed de novo, which is vascularized and unmodified over the course of time.
In general, the implant of the invention is characterized in that it comprises:
a biocompatible support making possible the biological anchoring of cells;
cells having the capacity to express and secrete naturally or after recombination a defined substance, for example, a substance of therapeutic interest; and
a constituent capable of inducing and/or promoting the gelation of said cells.
In particular, the elements participating in the assembly of the in vitro implant are the following:
1) A rigid support. It may be prepared from different biomaterials: PTFE, coral, cross-linked collagen fibers;
2) A collagen gel. It is possible to use rat tail collagen, bovine collagen, human collagen; and
3) Genetically modified cells, preferably the recombinant cells of the present invention which are described in detail below.
Hence the object of the invention is an implant or neo-organ characterized in that it comprises a biocompatible rigid support, in particular a rigid support made of PFTE or of biological origin, making possible the biological anchoring of cells previously placed or not placed in 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 defined substance, for example a substance of therapeutic interest.
The expression “biological anchoring” means that the cells contained in the implant can bind to the surface of the biocompatible support or, in certain cases, penetrate into the interior of this support.
This binding of the cells to the support is made possible in particular by the presence of 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, permits the organization of the cells in a three-dimensional structure in an amorphous environment, not giving rise in vivo to prolonged inflammation.
According to a first embodiment of the invention, the implant obtained is constituted on the one hand of a biocompatible material such as a support consisting of a synthetic biocompatible material, in particular polytetrafluorolethylene fibers (PTFE) or a support consisting of a calcium-based material, in particular a material based on calcium carbonate, of biological origin, preferably coral and, on the other hand, a gel optionally loaded with cells expressing the substance of interest, in particular recombinant cells.
The invention also relates to a method of preparation of the implant. The method comprises the steps of:
placing 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 preceding step in order to obtain the gelation of said constituents;
culture of cells thus obtained under conditions permitting their binding to the gelled constituents; and
recovery of the implant thus obtained.
a) Implant consisting of a synthetic biocompatible material.
The object of the invention is an implant or neo-organ characterized in that it comprises cells expressing the substance of interest, in combination with polytetrafluoroethylene (PTFE) fibers and collagen. On being introduced in vivo, such an implant is capable of constituting a vascularized neo-organ, individualized within a tissue.
Advantageously, the implant such as described above comprises in addition a growth factor, for example the bFGF (basic Fibroblast Growth Factor).
Danos Olivier
Ferry Nicolas
Heard Jean-Michel
Moullier Philippe
Institut Pasteur
Nguyen Dave T.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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