U.S. patent application number 09/987601 was filed with the patent office on 2002-07-25 for biocompatible implant for the expression and in vivo secretion of a therapeutic substance.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Danos, Olivier, Ferry, Nicolas, Heard, Jean-Michel, Moullier, Philippe.
Application Number | 20020098223 09/987601 |
Document ID | / |
Family ID | 26230262 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098223 |
Kind Code |
A1 |
Moullier, Philippe ; et
al. |
July 25, 2002 |
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.
Inventors: |
Moullier, Philippe; (Meudon,
FR) ; Danos, Olivier; (Garches, FR) ; Heard,
Jean-Michel; (Paris, FR) ; Ferry, Nicolas;
(Paris, FR) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
INSTITUT PASTEUR
25-28, rue du Dr. -Roux
Paris
FR
F-75724
|
Family ID: |
26230262 |
Appl. No.: |
09/987601 |
Filed: |
November 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09987601 |
Nov 15, 2001 |
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09225509 |
Jan 6, 1999 |
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09225509 |
Jan 6, 1999 |
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08532814 |
Jan 19, 1996 |
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08532814 |
Jan 19, 1996 |
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PCT/FR94/00456 |
Apr 21, 1994 |
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Current U.S.
Class: |
424/426 ;
424/93.21; 424/93.7 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 14/505 20130101; C12N 2533/54 20130101; C07K 14/721 20130101;
A61P 7/00 20180101; C12N 2740/13043 20130101; C12Y 302/01076
20130101; C12N 9/2402 20130101; C12N 5/0068 20130101; A61L 27/3839
20130101; A61K 48/00 20130101; C12N 2533/18 20130101; C12Y
204/01017 20130101; A61K 38/00 20130101; A61K 9/0024 20130101; A61L
27/3804 20130101; A61L 27/3608 20130101; C12N 15/86 20130101; C12N
2740/16043 20130101; C12Y 302/01031 20130101; C12N 9/10 20130101;
A61L 27/3683 20130101; A61L 27/3604 20130101; A61L 27/3895
20130101; C12N 9/1051 20130101; A61L 27/38 20130101; A61K 35/12
20130101; C12N 2510/02 20130101; C12N 2533/30 20130101 |
Class at
Publication: |
424/426 ;
424/93.21; 424/93.7 |
International
Class: |
A61K 048/00; A61F
002/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 21, 1993 |
FR |
93/04700 |
Jul 26, 1993 |
FR |
93/09185 |
Claims
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.sup.6 to 10.sup.9, and
preferably from 5.times.10.sup.6 to 10.sup.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.
Description
[0001] 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 at the
invention.
[0002] 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
[0003] 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.
[0004] 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.
[0005] The nucleotide sequence whose expression is desired in this
earlier international application is paced under the control of the
LTR (Long Terminal Repeat) sequence of the retroviral vector and/or
under the control of inducible or constitutive exogenous promoters,
the LTR sequence of the retravirus being nonetheless conserved when
the exogenous promoter is present.
SUMMARY OF THE INVENTION
[0006] 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 aver 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] These biocompatible supports whether of biological origin or
not may or may not be resorbable by the host into which they are
introduced.
[0011] 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.
[0012] 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.
[0013] 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
[0014] The implant or neo-organ of the invention is obtained by the
process which insists 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.
[0015] In general, the implant of the invention is characterized in
that it comprises:
[0016] a biocompatible support making possible the biological
anchoring of cells,
[0017] cells having the capacity to express and secrete naturally
or after recombination a defined substance, for example, a
substance of therapeutic interest, and
[0018] a constituent capable of inducing and/or promoting the
gelation of said cells.
[0019] In particular, the elements participating in the assembly of
the in vitro implant are the following:
[0020] 1) A rigid support. It may be prepared from different
biomaterials: PTFE coral, cross-linked collagen fibers;
[0021] 2) A collagen gel. It is possible to use rat tail collagen,
bovine collagen, human collagen; and
[0022] 3) Genetically modified cell, preferably the recombinant
cell's of the present invention which are described in detail
below.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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 polytetrafluoroethylene 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.
[0028] The invention also relates to a method of preparation of the
implant. The method comprises the steps of:
[0029] placing the biocompatible support in contact with said cells
and a constituent capable of inducing and/or promoting their
gelation;
[0030] incubation Of the preparation obtained in the preceding step
in order to obtain the gelation of said constituents;
[0031] culture of cells thus obtained under conditions permitting
their binding to the gelled constituents; and
[0032] recovery of the implant thus obtained.
[0033] a) Implant Consisting of a Synthetic Biocompatible
Material.
[0034] The sect 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.
[0035] Advantageously, the implant such as described above
comprises in addition a growth factor, for example the bFGF (basic
Fibroblast Growth Factor). This growth factor promotes the
vascularization of the neo-organ when the latter is implanted in
vivo.
[0036] Preferably, the neo-organ is introduced into the peritoneal
cavity of the patent in whom it is desired to obtain the expression
of the exogenous nucleotide sequence contained in the vector of the
invention. This localization of the neo-organ is favourable for the
development of its vascularization and to its maintenance in an
individualized form in this organ. The implantation of the neo
organ comprising recombinant cells according to the invention may
be permanent or, on the contrary, temporary, it being possible in
this latter case to withdraw the neo-organ after a given time of
implantation.
[0037] Furthermore, the invention relates to a process for the
preparation at an implant defined above, comprising the steps
of
[0038] placing recombinant cells in contact with a solution of
collagen and PTFE fibers preferably previously treated with
collagen and optionally with an angiogenic growth factor,
[0039] incubation of the preparation obtained in the preceding step
in order to obtain the gelation of the collagen,
[0040] culture of the cells thus obtained for a time sufficient to
allow the cells to bind to the collagen fibers,
[0041] recovery of the implant thus obtained.
[0042] b) Implant Consisting of a Biocompatible Material of
Biological Origin.
[0043] Preferably, the material used is a high porosity coral, i.e.
it possesses a porosity such as that described in the French patent
No. 2 460 657. It may also be a powder of sufficient porosity to
make it possible to obtain a solid framework for the purpose of
constituting a cellular network comprising or not a biological
support such as collagen. In the case in which the coral is
replaced by collagen, cross-linked or not, this latter may also
serve both as support and as matrix promoting the biological
gelation for the constitution of the network of cells.
[0044] According to an advantageous embodiment of the invention the
high porosity coral used is of the spherical type.
[0045] According to another embodiment, the implant consists of a
support of biological origin forming a solid matrix, said support
containing:
[0046] collagen with variable cross-linking for example in the form
of fibers or sponges such as those sold under the trade marks
Hemostagene.RTM. or Paroguide.RTM.;
[0047] powdered or fragmented bone; or
[0048] carbohydrate-based polymers such as dextran or hyaluronic
acid.
[0049] According to an advantageous embodiment of the invention,
the solid framework of biological origin is capable of being
resorbed in vivo.
[0050] The implant thus constituted allows the in vivo formation of
an individualized and, if necessary, stable neo-organ in which the
biological support is capable of being progressively resorbed
leaving in place a structure containing a vascularized connective
tissue of a form quite comparable to that of the implant, this
neo-organ having however, optionally a reduced volume compared with
the volume of the original implant.
[0051] A particularly preferred implant in the framework of the
invention is such that the constituents capable of inducing and/or
promoting the gelation of the cells are for example uncrosslinked
collagen or alginates.
[0052] In particular, recourse will be had to type I collagen, for
example, in particular at a concentration of 1.5 mg/ml.
[0053] Other constituents for gelling the cells may naturally be
used provided that they have the desired functional property and
that they exhibit the properties of biocompatibility required in
order to be introduced in vivo in man or animals.
[0054] c) Use of the Implant of the Invention.
[0055] An implant particularly suited to the realization of the
invention is additionally characterized in that the cells are
recombinant cells carrying genetic information foreign to their
genome and capable of expressing this additional information in
vivo and having the capacity to be tolerated immunologically by an
organism to which they might be administered.
[0056] In a preferred manner, an implant according to the invention
is characterized in that the recombinant cells are modified by a
vector containing one or more exogenous nucleotide sequences,
coding for an antigen or antigenic determinant or coding for a
polypeptide or glycoprotein, soluble in serum, for sample 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 properties of an antibody or
antibody fragment.
[0057] In a preferred manner, the implant contains in addition one
or more angiogenic factors, in particular bFGF, preferably
incorporated during the placing of the biocompatible support in
contact with the cells and the constituent capable of inducing
and/or promoting their gelation.
[0058] The binding at the angiogenic factor, for example bFGF, is
promoted by the presence of the gelling agent.
[0059] This agent contributes to the stable binding of the
angiogenic factors to the support of biological origin of the
implant.
[0060] In a particularly advantageous manner, the implant also
contains heparin or a heparin derivative such as heparan sulfate or
heparin fractions.
[0061] Heparin and its derivatives are capable of binding to the
constituents promoting gelation and thus of increasing the affinity
of the angiogenic factors for the constituents responsible for
gelation. In particular, heparin binds to collagen and thus
increases the affinity of bFGF for collagen.
[0062] The implant awarding to the invention may be employed in a
permanent or temporary fashion for use in man or animals. In fact,
its capacity to constitute in vivo an individualized neo-organ
makes it possible to withdraw it if necessary.
[0063] In a particularly valuable manner an implant according to
the invention can be used for:
[0064] the treatment of genetic diseases, in particular for the
treatment of lysosomal overload, hemophilia A or hemophilia A of
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;
[0065] 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 of a
soluble anti-viral protein;
[0066] 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 tumor
cells.
[0067] The object of the invention is also a composition
characterized in that it contains an implant according to the
invention and one or more other constituents such as for example an
antigen, an adjuvant in particular for vaccination.
[0068] The agents described in the invention thus make it possible
to envisage the treatment of genetic or acquired diseases over a
long period, more than several months, and do so without the
repeated administration of the expression product of the exogenous
nucleotide sequence whose therapeutic activity is desired
Furthermore, the agents described in the invention lead to the
production in the form of a secreted protein of a quantity of
expression product of the exogenous nucleotide sequence sufficient
to have a therapeutic effect in vivo.
[0069] Another application of the recombinant cells acceding to the
invention or compositions or implants containing them relates to
the preparation of antibodies against the expression product of the
exogenous nucleotide sequence contained in the vector introduced
into the recombinant cells. Preferably, these antibodies are
produced in vivo when the recombinant cells are implanted in an
organism.
[0070] This use makes it possible for example to produce antibodies
against an antigen neither whose amino acid sequence nor
necessarily the DNA or cDNA sequence has been identified. Depending
on the effect desired, the introduction of the recombinant vector
according to the invention will be made into a defined cell
type.
[0071] Similarly, the invention permits the in vivo production of
antibodies against a defined antigen for example for the purposes
of vaccinating a patient.
[0072] Recombinant cells such as those described below or
compositions or implants containing them may also be used for the
treatment of tumors, the exogenous nucleotide sequence contained in
the recombinant cells coding far a substance capable of promoting
or enhancing the immune response against the tumor cells.
[0073] As a particular instance, the recombinant cells utilizable
for the treatment of tumors may be cells obtained by recombination
of tumor cells taker from a patient with a retroviral vector
complying with the specifications given in the following pages.
Kit According to the Invention
[0074] The invention also includes a kit for the preparation of an
implant for achieving in vivo the expression and secretion by cells
of a substance for producing the desired therapeutic effect. The
kit of the invention usually contains:
[0075] a biocompatible support making possible the anchoring of
said cells; and
[0076] a constituent capable of inducing and/or promoting the
gelation of said cells.
[0077] In particular, the biocompatible support comprises at least
one of the ingredients chosen from among the group including PTFE
or a support of biological origin, particularly a calcium-based,
and in particular a calcium carbonate-based, support of biological
origin, and preferably coral. The constituent capable of inducing
and/or promoting the gelation of the cells is preferably collagen,
in particular type I collagen, and preferably at a concentration of
the order of 1.5 mg/ml.
[0078] The kit according to the invention may also include a DNA
comprising a sequence coding far the substance expressed and
secreted by said cells. This DNA may be used to transform the cells
taken from the patient to be treated.
[0079] More particularly, the DNA farming part of the kit is a
retroviral vector such as that described below.
[0080] The kit may also contain cells having the capacity to
express and secrete naturally or after recombination a defined
substance, far example a substance of therapeutic interest. this
deletion makes it possible to diminish and preferably to neutralize
the transcriptional effect of the LTR region by conserving the
capacity of the exogenous promoter to promote and control the
expression of the exogenous nucleotide sequence contained in the
retroviral vector.
[0081] It is also possible to envisage the deletion of all of the
sequence constituting the promoter and the enhancer. However, such
a deletion has the disadvantage of being accompanied by a
considerable diminution of the vector titers.
[0082] According to a variant of the embodiment of the invention,
the proviral sequence upstream from the exogenous promoter is the
nucleotide sequence situated between nucleotides 1 and about 1500
according to the numbing of the sequence shown in FIG. 1. This
sequence upstream from the promoter essentially lacks the entire
complement of gag, pol and env genes of the proviral sequence.
[0083] The erogenous nucleotide sequence is advantageously inserted
under the control of the exogenous promoter in the place of the
deleted gag, pol and env sequences.
[0084] When the promoter is the PGK-1 promoter described below, the
exogenous nucleotide sequence is advantageously inserted at the
BamHI site downstream from this promoter.
2) Exogenous Nucleotide Sequence
[0085] An "exogenous nucleotide sequence" according to the
invention is a sequence which is not expressed naturally in a cell
in which the retroviral vector of the invention is introduced or is
expressed there in insufficient quantity or is one which it is
desired to produce in greater quantity than is normally expressed.
This defective or insufficient expression results from the nature
of the cell or occurs because of a disease affecting this cell in a
given individual.
[0086] In a preferred manner, the "exogenous nucleotide sequence"
codes for a defined polypeptide. By "polypeptide" is meant any
amino acid sequence irrespective of its size, this expression
comprising proteins and peptides. The polypeptide may be in a
glycosylated or non-glycosylated form
Retroviral Vector and Recombinant Cells
[0087] a) Retroviral Vector
[0088] A novel recombinant retroviral vector according to the
invention is characterized in that it comprises:
[0089] a provirus. DNA sequence modified in that:
[0090] the gag, pol and env genes have been deleted at least in
part in order to obtain a proviral DNA incapable of
self-replication, this DNA in addition not being able to recombine
to form a wild-type virus;
[0091] the LTR sequence bears a deletion in the U3 sequence such
that transcription of the mRNA it controls is significantly
reduced, by at least 10 fold for example, and
[0092] this recombinant retroviral vector comprising in addition an
exogenous nucleotide sequence under the control of a promoter for
example an erogenous inducible or constitutive promoter.
[0093] The principal elements forming this vector are described
below in detail.
[0094] 1) Proviral DNA
[0095] A "proviral DNA sequence" is a sequence of DNA transcribed
from the genomic RNA of the virus when it is integrated in the host
cells of the virus The proviral DNA thus comprises sequences coding
for the gag, pol and env proteins of the retrovirus which
correspond respectively to the nucleoproteins, polymerases,
envelope proteins and glycoproteins.
[0096] The retroviral vector according to the invention is such
that the gag, pol and env genes of the proviral DNA have been
deleted at least in part in order to obtain a proviral DNA
incapable of self-replication, this DNA in addition being unable to
recombine to form a wild-type virus.
[0097] The proviral DNA also bears sequences called LTR (Long
Terminal Repeat) which bear regions called R, U3 and U5. These
sequences of the LTR region are involved in the replication cycle
of the retrovirus.
[0098] 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 internal enhancer of the LTR region.
Furthermore,
[0099] 3) Exogenous Promoter
[0100] The promoter used for the construction of recombinant
retroviruses according to the invention is advantageously exogenous
with respect to the sequence whose expression it controls in the
sense that it is not naturally associated with it. It is also
possible to use the promoter of the transferred exogenous gene
[0101] The promoter used in the vector of the invention may be of
the inducible or constitutive type This exogenous promoter controls
the expression of the exogenous nucleotide sequence The promoter
may optionally be accompanied by a regulatory nucleic acid sequence
for example an "enhancer" which would serve to regulate its
activity.
[0102] In the case of inducible promoters, promoters such as the Mx
promoter in mice, the promoters including a tetracycline operator
or even promoters regulated by hormones, in particular steroid
hormones, may also be used. As regards the so-called "constitutive"
promoters, the use of active internal promoters in resting
fibroblasts such as the PGK promoter or another promoter of the
housekeeping gene is preferred.
[0103] In a particularly advantageous manner for the construction
of the retroviral vector according to the invention, the exogenous
constitutive promoter is a promoter without a TATA box and in
particular the promoter of the phosphoglycerate kinase gene
(PGK-1). This promoter is either the mouse promoter or the human
promoter as described by Adra et al. (Gene 60, 1987, 65-74).
[0104] The insertion of the PGK-1 promoter into a retroviral vector
makes possible the transfer of the gene into skin fibroblasts for
example and the stable expression at high level and for the very
long term of this gene after the fibroblasts have been implanted in
vivo in a recipient, a mammal for example
[0105] Other constitutive promoters may be used in place of the
PGK-1 promoter. In general, recourse will be had to a promoter
active in the cells which it is desired to transform with the
retroviral vector and in particular active promoters in quiescent
fibroblasts, for example promoters preceding the genes for the
cytoskeleton. It is also possible to mention the promoter of
beta-actin. (Kort et al., 1983, Nucl. Acids Res. 11: 8287-8301) or
the promoter of the vimentin gene (Rettlez et Basenga (1987), Mol.
Cell. Bid. 7: 1676-1685). The promoter used may lack a "TATA
box".
[0106] 4) Preferential Comstructions of the retroviral Vector
[0107] A first particular retroviral vector in the framework of the
invention is such that an exogenous nucleotide sequence and an
exogenous constitutive promoter as well as the proviral DNA
sequence are borne by a plasmid. As an example, these sequences may
be borne by the plasmid pBR322 which allows the introduction of the
DNA into the cell lines in which it is desired to produce the
vector.
[0108] According to another advantageous embodiment of the
invention a vector complying with the preceding specifications is
in addition characterized in that the proviral DNA is derived from
the Mo-MuLV retrovirus. Other retroviruses of the MuLV family may
be used and mention should be made, for example, of the HaSV or
F-MuLV retroviruses.
[0109] Preferably, the sequences of the pol and env genes of the
proviral DNA are completely deleted. In this case, the sequence of
the gag gene may also be entirely deleted or, on the other hand, be
conserved in part, provided that the proviral DNA thus constituted
is no longer capable of replication.
[0110] Advantageously, a retroviral vector such as previously
defined is characterized in that the U3 region of the LTR3'
fragment is deleted at nucleotide 2797 of FIG. 1. This deletion
corresponds to a deletion of a fragment situated 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 the LTR3' contains the
enhancer region of the LTR.
[0111] Preferably, a retroviral vector according to the invention
is a type pM48 vector derived from the Moloney virus in which the
viral "enhancer" localized in V3 has been deleted and which
contains an internal promoter active in resting fibroblasts,
preferably the PGK promoter or another promoter of the housekeeping
gene, such as the vector shown in FIG. 2, modified by the exogenous
nucleotide sequence at the BamHI site.
[0112] The retroviral vector M48 LacZ (also designated by the
expression pM48 LacZ was deposited with the CNCM (Collection
Nationale de Culture de Microorganismes, Paris-France) under the
No. I-1298 an Apr. 16, 1993.
[0113] This vector is derived from the vector pM48 and is
characterized in that it contains downstream from the exogenous
constitutive promoter a BamHI fragment of the gene for
beta-galactosidase.
[0114] This fragment of the beta-galactosidase gene may easily be
deleted and replaced by an exogenous nucleotide sequence of
interest, for example a sequence coding for a protein or a
glycoprotein capable of having therapeutic value or against the
protein product of which it would be desired for example to obtain
antibodies.
[0115] A retroviral vector of the invention may also contain a
sequence capable of enhancing the transcriptional activity of the
promoter either constitutively or inducibly. The vector may contain
an enhancer sequence upstream from the exogenous promoter.
[0116] b) Recombinant Cells
[0117] Moreover, the invention relates to recombinant cells
characterized in that they are cells having the capacity to be
tolerated immunologically by an organism in which they might be
implanted, modified by a retroviral vector complying with one of
the preceding specifications
[0118] Such cells may be cells derived from the organism into which
they are to be implanted after recombination by transduction with
the vector of the invention or cells lacking at their surface
antigens recognized by the immune system of the organism into which
they are implanted.
[0119] They may also be endothelial cells, myoblasts, muscle cells
or tumor cells irradiated or treated according to other procedures
to prevent their proliferation and taken from the patient whose
genetic make-up it is desired to modify so that they may counteract
the development of the tumor.
[0120] Preferably, these recombinant cells are recombinant
fibroblasts and in particular skin fibroblasts. Preferably, they
are autologous fibroblasts with respect to the patient in whom it
is desired to implant them after their modification by a vector
according to the invention. Fibroblasts prepared from other
organisms may be used such as for example fibroblasts isolated from
an umbilical cord. The genetically modified fibroblasts used in the
context of the present invention are not immortalized
fibroblasts.
[0121] These cells may be modified by a recombinant vector coding
for a defined polypeptide, said vector permitting the expression of
the foreign DNA in the cells. They may also be modified by the
methods of so-called homalogous recombination. The penetration of
the vector into the cells is carried out by using electroporation
or precipitation with calcium phosphate or also by any other method
implicated in the entry of a nucleic acid either alone or through
the intermediary of a recombinant virus, for example
[0122] By "recombined cells" is also meant tumor autologous cells
irradiated prior to their introduction into the gel, having the
capacity to maintain at their surface tumor antigens accessible to
the immune system of the host into which the implant according to
the invention has been introduced.
[0123] The recombinant cells of the invention are also obtained
either by infection of the cells to be modified with a retroviral
vector of the invention, or by other transduction methods involving
naked DNA DNA-proteins complex or an adenoviral vector. In the case
of an infection with a retroviral vector, the sequence coding for
the polypeptide whose expression (exogenous nucleotide sequence) is
desired is introduced into the proviral DNA.
[0124] In a preferred manner, the recombinant cells are modified by
a vector containing one or more exogenous nucleotide sequence(s)
coding for an antigen or an antigenic determinant or coding for a
polypeptide or a 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 properties of an antibody or
an antibody fragment.
[0125] As an example, this exogenous nucleotide sequence codes for
beta-glucuronidase or another lysosomal enzyme such as
alpha-iduronidase or arylsulfatase B, a coagulation factor such as
factor VIII or factor IX erythrapoietin or any active part of one
of these proteins.
[0126] Other advantages and characteristics of the invention will
become apparent in the Examples and the Figures which follow.
[0127] FIG. 1. nucleotide sequence of the vector M48;
[0128] FIG. 2 representation of the vector pM48.
EXAMPLES
Preparation of the Recombinant Vector
[0129] The transfer and expression of exogenous genetic material
makes it possible to modify experimentally the properties of the
tissue. It has been possible to suggest treatments based on this
principle for diseases due to genetic deficiencies or acquired
diseases. The example described below makes use of a retroviral
vector for introducing into cells the genetic information coding
for a secreted protein. The reimplantation in the organism of the
genetically modified cells is carried out by combining these cells
with collagen, an angiogenic factor and the framework of the coral
or cross-linked collagen type The long-term in vivo expression of
the genetic material thus transferred is obtained by using the
promoter of the murine phosphoglycerate kinase gene.
[0130] 1--Retroviral Vector
[0131] 1.1.--Properties
[0132] The retroviral vector M48 (FIG. 2) is instructed from
proviral sequences is dated from the genomic DNA of rat cells
infected with the Moloney murine leukemogenic retrovirus (Mo-MuLV).
The gag, pol and env genes which code for the proteins of the virus
were deleted and replaced by the sequences to be transmitted. The
retroviral sequences in cis necessary for the production and
maturation of the transcripts of the recombinant viral genome, for
their packaging in infectious viral particles, their reverse
transcription and their integration in the genome of the infected
cell have been conserved The sequences of the recombinant provirus
and the flanking fragments at cellular DNA are borne by the
bacterial plasmid pBR322.
[0133] The promoter of the mouse phosphoglycerate kinase (PGK-1)
gene was inserted in the place of the viral genes A unique BamHI
cloning site enables the sequences to be transmitted to be placed
in the retroviral vector under the control of this promoter.
[0134] This retroviral vector bears a deletion in the U3 sequences
of the internal LTR (Cone et al., Mol. Cell Biol. 7, 887-897, 1987)
This leads to a considerable reduction of the mRNAs being initiated
from the 5' LTR and to a predominance of mRNAs produced under the
control of the PGK-1 promoter. The use of this vector hence results
in the insertion in the genome of the target cell of an expression
cassette of the sequence of interest under the control of the PGK-1
promoter.
[0135] 1.2.--Structure of the Vector
[0136] The complete sequence of the vector M48 (FIG. 1)
comprises
[0137] Position 1 to 2019: Mo-MuLV sequences comprising the 5' long
terminal repeat (or 5' LTR or LTR 5'), the packaging sequences
.psi.+ which include a 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 SacII
linker (5' CCCGCGGG3') at position 1074 (position 626, Shinnick et
al.).
[0138] This leads to a mutation in phase with the sequence coding
for gag.
[0139] Position 2020 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).
[0140] Position 2527 to 2532: Unique BamHI site for the insertion
of sequences which will be expressed under the control of the PGK-1
promoter.
[0141] Position 2533 to 3101: Mo-MuLV sequences including 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
included between 7935 and 8113 in the numbering of Shinnick et al.
and the insertion of a linker SalI (2798 in the present
numbering).
[0142] Position 31012 to 3674: Rat genomic sequences.
[0143] Position 3675 to 8003: Sequences of the plasmid vector
pBR322 (position 4363 to 29) comprising the beta-lactamase gene
These sequences are modified as follows: destruction of the BamHI
site (position 7657, present numbering).
[0144] Position 8004 to 8388: Rat genomic sequences.
[0145] 1.3.--Production of the Retroviral Vector
[0146] This production is carried out by introducing the
recombinant proviral structure in a cell line in which the gag, pol
and env genes are expressed constitutively.
[0147] This line, called a transcomplementary or packaging line,
synthesizes retroviral particles lacking genomic RNA The .psi.CRIP
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 construction is introduced by transfection, the RNA
transcripts from the LTR5' are encapsidated and the cells then
produce particles capable of transmitting the recombinant genome
but which are incapable of replicating.
[0148] After transfection of the recombinant construction the cells
producing retroviral vector are selected Either a clone producing
high viral titers or a polyclonal population is used for the
preparation of the vector. The steps of isolation of a producing
clone are the following:
[0149] 1) The .psi.CRIP cells are transfected by a coprecipitate
with calcium phosphate (Graham and van der Erb, Virology, 52, 456,
1973) of the retroviral construction and the plasmid pSV2neo
(Southern and Berg, J. Mol. Appl. Gen. 1, 327-341, 1982) in a molar
ratio of 10 to 1.
[0150] 2) The cells which have integrated in a stable manner and
express the exogenous DNA are selected in the presence of 1 mg/ml
G418 (Geneticin, Gibco).
[0151] 3) The population of cells resistant to G418 in which
isolated individual clones are tested for ther capacity to produce
the retroviral vector. For that purpose, NIH/3T3 target cells are
placed in the presence of culture medium recoated from the
.psi.CRIP cells, and the transmission of the recombinant provirus
is analyzed after 48 hours. The analysis, performed on the genomic
DNA of the target cells, is made by PCR (qualitative method) or by
Southern blot (quantitative method).
[0152] 4) A library of producing cells is stored at -135.degree.
C.
[0153] 2--Preparation of the Stocks of Retroviral Vector
[0154] The culture supernatant of the packaging cells is recoated
after an incubation for 24 hours with the packaging cells producing
the retroviral vector and placed in contact with the target cells
in order to achieve the infection.
[0155] 2.1.--Bulk Preparation
[0156] A stock of a hundred ampoules containing producing cells is
stored at -135.degree. C. After being thawed, the cells are
amplified for 10 days, successively in bottles then in roller
bottles of 875 cm.sup.2, until 20 confluent rollers are obtained.
The production is then begun. It lasts 4 or 5 days. The culture
medium is DMEM containing 1 g/l of glucose, 10 mM of sodium
pyruvate and supplemented with 5% newborn calf serum (Hyclone).
Each roller bottle makes possible the conditioning of 100 ml of
medium per 24 hours. This culture supernatant is centrifuged to
remove the cell debris. The weekly production is 8 to 10 liters of
viral supernatant.
[0157] 2.2.--Concentration
[0158] The supernatant may be concentrated to increase the
infectious titer of the retroviral vector. After centrifugation,
the supernatant is dialyzed tangentially against a Sartocon
membrane of porosity 100.000 by using the Crossflow apparatus of
Sartorius. A twenty fold concentration is obtained in 3 hours. It
is accompanied by a 20 increase in the infectious titer. After
concentration, the viral preparation is used immediately or stored
at -80.degree. C. if it is not to be used for at least 2 weeks.
[0159] 3--Skin Fibroblasts
[0160] 3.1.--Isolation and Placing in Culture
[0161] The fibroblasts intended for the gene transfer are obtained
by a cutaneous biopsy performed under very careful aseptic
conditions. In the mouse, a syngeneic animal is sacrificed for this
purpose. In the large mammal, one or more fragments of skin about
10 cm.sup.2 are taken under general anesthesia. After sectioning in
strips of several mm.sup.2, the fragments are dissociated by an
enzymatic treatment. For a fragment of 10 cm.sup.2, the reaction is
carried out for 2 hours at 37.degree. C. by gentle shaking 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.sup.2 bottles at a concentration of 1
to 5.times.10.sup.6/cm.sup.2. The culture medium is RPMI 1640
supplemented with 10 to 20% fetal calf serum.
[0162] 3.2.--Infection by the Retroviral Vector
[0163] The infections with the preparation of retroviral vector are
started the day after the placing in culture although the cell
density is less than 20% of confluence They are repeated daily
following the same protocol for 4 or 5 days. After removal of the
culture medium, the cells are placed in contact with the
concentrated or unconcentrated retroviral vector preparation, bit
supplemented in all cases with 8 .mu.g/ml of polybrene The
incubation is carried out for 2 hours at 37.degree. C., then
without prior washing the viral preparation is replaced by the
culture medium.
[0164] When a vector coding for a protein detectable under the
microscope is used, it is observed that this process allows the
introduction of the foreign gene into more than 90% of the
fibroblasts. The level of secretion of the protein into the culture
supernatant may be checked during and at the end of this process
All or part of the cells may be frozen at this stage.
[0165] After infection, the cells are amplified to produce a number
sufficient far reimplantation, i.e. 2-6.times.10.sup.8 cells/kg of
body weight. The amplification is carried out on culture trays
(multitray, Nunc).
[0166] 3.3.--Harvesting of the Genetically Modified Cells
[0167] After amplification, the cells are harvested by treatment
with trypsin. After being washed and counted, they are available to
be used for the formation of a neo-organ. A sample is stored to
check the genetic transfer by Southern blot as well as the
expression and secretion of the foreign protein by a suitable
procedure
Example 1
[0168] Secretion of a lysosomal enzyme (beta-glucuronidase) in the
mouse with implants containing PTFE fibers, rat tail collagen and
skin fibroblasts modified with a retroviral vector.
[0169] 1.1.--Constituents
[0170] Three constituents are assembled in vitro. The method
described below is applicable to the construction of neo-organs of
1 ml containing 5.times.10.sup.6 to 10.sup.7 genetically modified
cells. The same proportions are used for the formation of
neo-organs of large size containing 1 to 5.times.10.sup.9
cells.
[0171] 1) Polytetrafluoroethylene fibers (Gore and associates)
sterilized by autoclaving (120.degree. C., 30 minutes) are coated
by type 1 collagen. The fibers are bathed in a 0.1% solution of rat
tail collagen (Sigma) in 0.1 N acetic acid. This treatment is
carried out under vacuum for 1 hour at room temperature in order to
expel air present between the synthetic fibers. The collagens of
mouse, bovine or human origin may be used indiscriminately. After
drying for 24 hours under a hood with laminar air flow, the fibers
thus surrounded with a fine film of collagen are incubated in the
presence of an angiogenic growth factor (basic FGF, Farmitalia,
10-20 ng/50 mg of fibers in a solution of PBS5% beta
mercaptoethanol). The fibers thus treated are washed in PBS and
stored at 4.degree. C.
[0172] 2) A solution constituted of the following ingredients is
prepared For 1 ml: 100 .mu.l 10.times.RPMI 1640 (Gibco); 12 .mu.l
7.5% bicarbonate; 8 .mu.l, 7.5 N NaOH; 2.5 1, 1M Hepes; 100 .mu.g
penicillin/glutamine (Gibco); 10 ng bFGF (Farmitalia); 1.5 mg
collagen (Bioethica); distilled water to 1 ml. This solution of
syrupy consistency is stored at 4.degree. C.
[0173] 3) After washing, the genetically modified cells are
resuspended in 100 .mu.l of RPMI 1640 with 15% fetal calf serum
[0174] 1.2.--Assembly
[0175] The in vitro assembly of the neo-organ is carried out under
sterile conditions in a hood, in the following manner:
[0176] 1) 1 mg of treated fibers are spread out in a culture dish
(Corning 24 wells plate).
[0177] 2) The genetically modified cells (100 .mu.l) are
incorporated into the collagen solution (1 ml) using a shaker,
[0178] 3) This mixture is deposited on the fibers
[0179] The entire preparation is placed in an incubator (37.degree.
C., 5% CO.sub.2) for 30 minutes in order to obtain the gelation of
the collagen. When this occurs, 1 ml of RFMI 1640 with fetal calf
serum is deposited on the gel in order to compensate the variations
in pH. Two to four hours later, the gel is delicately detached from
the rim of the culture dish with the aid of an injection needle and
transferred to a larger dish in the presence of an excess of
culture medium for a period of 12 to 24 hours. During this lapse of
time the fibroblasts included in the collagen gel bind to the
collagen fibers and this induces a retraction of the structure. It
is usual to observe a reduction of more than 50% of the initial
volume The whale is then ready to be implanted in the organism.
[0180] 1.3--Implantation
[0181] The neo-organ is introduced in the peritoneal cavity in the
course of a laparotomy performed under general anesthesia. In the
mouse, the implantation is effected by inserting, without binding
the neo-organ between the midgut loops in contact with the
mesentery. In the large mammal, the neo-organ is fixed by two
points between the two layers of the omentum. Vascular connections
are established from the first days onwards. The exogenous protein
may then be released into the circulation of the recipient animal.
As from the third week following the implantation, an organ often
pediculate, encapsulated and well individualized is formed. Very
many vascular connections are visible at its surface. Inflammatory
adhesion does not exist. The examinations at 3 and 6 months show an
identical appearance. The histological analysis at this stage
reveals the presence of loose, vascularized and only slightly
inflamed connective tissue
[0182] 1.4--Treatment of Diseases of Lysosomal Overload
[0183] This process was applied to the treatment of a disease of
lysosomal overload for which a mouse model is available. It
involves a deficit of beta-glucuronidase responsible for a
mucopolysaccharidosis. The beta-glucuronidase is phosphorylated and
is thus in part transported to the lysosomes and in part secreted
in the circulation. It may be picked up by other tissues through
the intermediary of the mannose-6-phosphate receptor expressed at
the surface of many cell. This specific recovery permits a
therapeutic approach in which the continuous systemic distribution
of the missing enzyme is ensured by genetically modified cells.
[0184] Implants of 0.5 cm.sup.3 containing 20.times.10.sup.6
autologous fibroblasts are well tolerated by the mouse. The
implanted cells express the human beta-glucuronidase gene inserted
into the vector M48 under the control of the PGK promoter and
secrete the foreign protein for more than 5 months. The biological
effect is expressed by a rapid fall in the urinary elimination at
the intermediary catabolites of the degradation of the
mucopolysaccharides. The histological normalization of the tissues
of the recipient animal is spectacular, particularly in the liver
and the spleen. It is stable with time and depends on the presence
of the neo-organ whose excision induces a return to the initial
pathological state.
[0185] This process is applicable to the treatment of all of the
diseases o lysosomal overload, including Gaucher's disease for
which the supply of a soluable form of the glucocerebrosidase can
be envisaged.
Example 2
[0186] Secretary of a lysosomal enzyme (beta-glucuronidase) in the
dog with implants containing PTFE fibers, rat tail collagen and
skin fibroblasts modified with a retroviral vector.
[0187] Four dogs received from 1 to 6 neo-organs each containing
10.sup.9 autologous fibroblastsgenetically modified by means of the
vector M48-.beta.glu in order to secrete human beta-glucuronidase.
Three animals of the Labrador race weighing 21 to 29 kg and one
animal of the Beagle race weighing about 9 kg were used. The
surgical implantation performed under general anesthesia consisted
of inserting the implants between the two parietal and visceral
layers of the omentum close to the greater curvature of the
stomach. This rapid operation is accompanied by simple operative
follow-ups and makes it possible to envisage in the future an
implantation by coeliosurgery. One and a half months, four months
and six months after the implantation an exploratory laparotomy was
performed in order to make a macroscopic check-up of the implant.
The identification of the omentum was easy and the detection of the
grafted structure was made very quickly. No adhesion was recorded
and the vascularization was considerable, including large calibre
vessels in connection with the implant. The appearance of the
implant has not altered during the period co this observation. A
liver biopsy practiced at the same time made it possible to
investigate the presence of human beta-glucuronidase. The
measurement of the activity on cell extracts showed a level
equivalent to 2-3% of the endogenous canine activity which was
stable with time in the animals having received more than
2.times.10.sup.9 genetically modified autologous fibroblasts. The
revelation in situ on sections showed an activity localized in the
Kupffer cells. The results are summarized in Table I.
1TABLE I Human beta-glucuronidase absorbed by the liver of dogs in
which neo-organs were implanted Activity of human
beta-glucuronidase Number of cells % of endogenous canine activity
In situ revelation on sections Animal Weight implanted day 0 day 45
day 120 day 180 day 0 day 45 day 120 day 180 Nougatine 28 kg 0.8
.times. 10.sup.9 <0.2 3 0.8 - - +++ +/- - Eglantine 26 kg 2
.times. 10.sup.9 <0.2 2 2 1 - +++ ++ ++ Doris 21 kg 3 .times.
10.sup.9 <0.2 3 3 1 - +++ +++ ++ Cyclo 10 kg 6 .times. 10.sup.9
<0.2 nd 1 nd - nd nd nd
Example 3
[0188] Secretary of a lysosomal enzyme (beta-iduronidase) in the
mouse with implants containing PTFE fibers, rat tail collagen and
skin fibroblasts modified with a retroviral vector.
[0189] A cDNA coding for the human beta-L-iduronidase was
introduced into the vector M48 and a recombinant retrovirus was
produced in the .psi.CRIP line. Fibroblasts of nude mice were
placed in primary culture and infected with this retroviral vector.
The cells secreting the human enzyme were introduced into six
syngeneic recipients in two neo-organs each containing 10 millions
cells.
[0190] The animals were sacrificed after 35 to 77 days and the
presence of the human enzyme in their liver and spleen was verified
by means of a monoclonal antibody. An enzymatic activity equivalent
to 1-2% of the endogenous activity was demonstrated, indicating the
production of the enzyme from the cells of the neo-organ and its
detection at a distance.
Example 4
[0191] Secretary of erythropoietin (EPO) in the mouse with implants
containing PTFE fibers, rat tail collagen and skin fibroblasts
modified with a retroviral vector.
[0192] The nlslacZ gene was excised from the vector M48-nlslacZ by
BamHI digestion and replaced by a cDNA coding for mouse EPO
synthesized by PCR thus generating the vector M48EPO. A done d
.psi.CRE cells producing M48EPO retroviral particles was isolated.
A sample of fibroblasts was taken from adult mice by skin biopsy
and a primary culture was established in RPMI 1640 medium with 10%
FCS. These cells were infected by the vector M48EPO repeatedly for
the first 4 days of the culture Analysis by Southern blot showed
that the infected cells contained on average 2 copies of the M48EPO
genome per cell. Analysis by Northern blot showed the very
predominant expression of the cDNA of the EPO under the control of
the PGK-1 promoter. A biological assay of the EPO activity secreted
by these cells as well as an ELISA assay measured a secretion of 17
units of EPO per million cells per 24 hours. These cells were
amplified in culture, trypsinized, then resuspended in RPMI 1640 at
a concentration of 2.5.times.10.sup.7 to 2.times.10.sup.8 cells per
ml.
[0193] Implants were prepared 1 combining the following ingredients
in a 0.9 cm.sup.2 well: 1) PTFE filers treated previously as
described above to be coated successively with rat tail collage
(Sigma), heparin (Roche) and bFGF (Promega); 2) 1 ml of a solution
containing x mg/ml of rat tail collagen (Sigma), bFGF (10 .mu.g/ml)
in RPMI 1640 medium, 3) a volume of 10 .mu.l of the suspension of
fibroblasts genetically modified to secrete EPO. After having
obtained a homogeneous mixture, this latter is solidified by
incubation at 37.degree. C. far 30 minutes, then the gel is very
carefully separated from the walls of the culture well, transferred
to a 35 mm diameter Petri dish coated with RPMI 1640 medium
containing 10% FCS and incubated at 37.degree. C. for 3 days. A
contraction of the gel occurred during this incubation resulting in
a 50% diminution in size The implants were then inserted in the
peritoneal cavity of 14 syngeneic mice with genetically modified
fibroblasts.
[0194] The weekly measurement of the hematocrit (normal value
46+/-1.5%) showed a progressive rise towards a plateau, the level
of which varied as a function of the number of cells sting EPO in
the implants. This plateau was 80% for 2.times.10.sup.7 cells, 70%
for 10.sup.7 cells, 60% for 5.times.10.sup.6 cells, 52% for
25.times.10.sup.6 cells. It was maintained at this level during the
6 months period of observation of the animals. The concentrations
of EPO in the serum of the mice bearing implants secreting EPO
varied from 60 to 400 mU per ml (normal <20 mU/ml).
[0195] The implants constituted of PTFE, rat tail collagen and
cells genetically modified by the retroviral vector M48EPO thus
make possible a stable long-tm secretion of EPO in vivo at high
levels estimated to be between 500 and 1500 mU/kg/24 hours, i.e.
sufficient in man for the correction of the anemia due to
hemoglobinopathies such as sickle cell anemia and beta-thalassemia.
Levels 10 fold lower would be sufficient for the treatment of
anemia associated with chronic renal insufficiency.
In vivo Utilization of Implants Containing Coral as Biological
Support
[0196] 1.In the Mouse
[0197] In the C3H/He mouse compatibility experiments with
Biocoral.RTM. as implant showed that it could represent an
advantageous alternative to the synthetic supports.
[0198] It was in fact possible to coat Biocoral.RTM. effectively
with different ingredients of the extracellular matrix as well as
angiogenic factors. It was 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 air at room temperature for 12 to
24 hours. The presence of this element of the extra-cellular matrix
then made possible the irreversible binding of the basic fibroblast
growth factor labelled with 1251 (.sup.125I-basic Fibroblast Growth
Factor) (bFGF) to Biocoral.RTM. In this way the binding of an
average of 50 ng of bFGF to 40 mg of coral support pretreated with
murine type I collagen was achieved. Although this binding could
have also been done in the absence of pretreatment with collagen,
in this latter case it was reversible, hence of low affinity.
Collagen is thus an essential intermediary and justified for the
stable binding of angiogenic factors to Biocoral. Finally, it was
possible to increase the binding of bFGF five to ten fold when the
coating of 40 mg of Biocoral by collagen was supplemented by 200
.mu.g heparan sulfate (Sigma) (1 mg/mi solution in PBS) or 500
units of heparin (Roche) (5000 units/ml solution). 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 heparin binding domains of basic fibroblast growth
factor. Proc. Natl. Acad. Sci. USA 8 2324-2328).
[0199] Their anionic and polysulfated properties are responsible
for the strong affinity for the bFGF (Johnson, D. E., Lee, P. L.,
Lu, J., and Williams, L. T. (1990) Diverse fonrs of a receptor for
acidic and basic fibroblast growth factors Mol. Cell. Biol. 14
4728-4739). After incubation in the presence of one or other of
these components I 1 h at 4.degree. C. with shaking, the Biocoral
was dried in air at room temperature Thus heparinized, the coral
has an anti-coagulant action.
[0200] Assays of subcutaneous implantation with 40 mg of high
porosity (50%) spherical coral coated with murine type I collagen
and bFGF (=50 ng/40 mg of coral) were performed in vivo on ten
mice. The macroscopic and histological appearance of these implants
was analyzed over a period of 7 months. The results show that
Biocoral was progressively resorbed during several months and was
replaced b a structure formed of vascularized connective tissue
having the initial spherical form of the coral implant with,
however, a reduction in volume of approximately a third.
[0201] The histological analysis of this connective tissue at three
months shaved an intense vascularization and the total absence of
inflammatory cells in spite of the coral residues present. The
actual connective tissue was farmed of only slightly dense
mesenchymal cells, surrounded by newly formed collagen fibers. The
analysis at 7 months showed similar results without involution.
[0202] 2. In the Dog
[0203] In the 25 kg dog we carried cut a first implantation
experiment with 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.times.10.sup.9 genetically modified
autologous fibroblasts. The Biocoral support had been coated with
murine type I collagen and heparin (Roche). After drying, the coral
thus pretreated was incubated in the presence of an angiogenic
growth factor of the bFGF type (Farmitalia) then rinsed with PBS.
The surgical implantation performed under general anesthesia
consisted of inserting the whole implant between the two parietal
and visceral layers of the omentum close to the greater curvature
of the stomach. This rapid operation made possible simple operative
follow-ups and makes it possible to envisage in the future an
implantation by coeliosurgery. Forty five days after, an
exploratory laparotomy was performed in order to make a macroscopic
check-up of the implant. The identification of the omentum was
easy, no adhesion was noted and the detection of the coral implant,
containing the genetically modified autologous fibroblasts, was
made very quickly. It was not possible to observe any adhesion and
the vascularization was considerable, including large calibre
vessels in connection with the implant. A process of progressive
resorption of the coral was observed for at least 4 months.
[0204] 3. Other Mammals.
[0205] The creation of neo-organs by using Biocoral suited to
surgical implantation in large manuals, in particular man, with a
view to their clinical development is carried out analogously.
Biocoral can be modulated in its design, is capable of triggering a
sustained angiogenesis combined with its own resorption and,
finally, is devoid of inflammatory local and systemic 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 in the circulation
once the vascular connections are established.
Example 5
[0206] Secretary of erythropoietin (EPO) in the mouse with implants
containing fragments of coral, rat tail collagen and skin
fibroblasts modified with a retroviral vector.
[0207] In the same manner as in Example 4, mouse fibroblasts were
obtained from a skin biopsy, placed in culture and transduced with
the vector M48EPO. After amplification, the cells were trypsinized
and resuspended in RPMI 1640 medium containing 10% FCS at a
concentration of 10.sup.8 cells per ml.
[0208] Implants were prepared by combining the following
ingredients in a 0.9 any well: 1) coral powder (Inoteb) composed of
particles of porosity >45% and a granulometry between 600 and
1000 .mu.m, the pares of which had a mean diameter of 150 .mu.m,
previously treated by incubation in a solution of rat tail collagen
(1 mg/ml) for 30 minutes at room temperature, dried, then incubated
in a heparin solution (Roche) for 30 minutes at room temperature,
dried, then incubated in a 100 .mu.g/ml solution of bFGF (Promega)
for 30 minutes at room temperature, 2) 1 ml of a solution
containing 3 mg/ml of rat tail collagen (Sigma), bFGF (100
.mu.g/ml) in RPMI 1640 medium; 3) a volume at 100 .mu.l of the
suspension of fibroblasts genetically modified to secrete EPO. As
in the examples 1 to 3, a homogeneous mixture of these constituents
was solidified by incubation at 37.degree. C. for 30 minutes, then
the gel was very carefully separated from the walls of the culture
wells, transferred to a 35 mm diameter Petri dish, coated with RPMI
1640 medium containing 10% FCS and incubated at 37.degree. C. for 3
days. The implants were then inserted in the peritoneal cavity of 3
syngeneic mice with genetically modified fibroblasts.
[0209] As in Example 4, the measurement of the hematocrit of the
animal recipients showed a rapid increase reaching a plateau after
3 weeks, and which was maintained at this level for all of the
period of the observation (2 months). Implants formed of a
structured collagen gel with fragments of coral thus make possible
the maintenance In the functional state of genetically modified
fibroblasts implanted in the peritoneal cavity of recipient
animals, as well as the long-term secretion f a soluble protein in
the serum.
Example 6
[0210] Secretary of erythropoietin (EPO) in the mouse with implants
containing cross-linked collagen fibers, rat tail collagen and skin
fibroblasts modified with a retroviral vector.
[0211] A similar experiment to the Examples 4 and 5 consisted of
producing implants combining mouse fibroblasts transduced with the
M48EPO vector, rat tail collagen and a support farmed of
cross-linked collagen fibers (Imedex). The preparation was made in
the following manner: 1) the fibers dehydrated and sterilized by
radiation were first rehydrated by incubation for 12 hours at
4.degree. C. in RPMI 1640 medium. They were then incubated at roam
temperature for 30 minutes in RFMI 1640 containing 10% FCS and 100
.mu.g/ml of bFGF (Promega), then loaded into a 0.4 cm.sup.2 well to
which was added 1 ml of RPMI 1640 solution containing FCS (10%),
rat tail collagen (1 mg/ml), bFGF (100 .mu.g/ml, Promega) and
10.sup.7 fibroblasts genetically modified to secret EPO. After
solidification at 37.degree. C. far 30 minutes, the gel was then
very carefully detached from the walls of the culture well,
transferred to a 35 mm diameter Petri dish and coated with RPM 1640
medium containing 10% FCS The implants were incubated for 3 days at
37.degree. C., moderate contraction of the structure was observed,
then they were implanted in 3 syngeneic mice.
[0212] In a manner similar to that described in the Examples 4 and
5, the weekly measurement of the hematocrit showed a progressive
rise until a plateau was reached which was maintained for the
duration of the observation period (2 months). Implants formed of a
structured collagen gel with cross-linked collagen fibers thus make
possible the maintenance of the functional state of genetically
modified fibroblasts implanted in the peritoneal cavity of animal
recipients as well as the long-term secretion of a soluble protein
in the serum
Example 7
[0213] Detoxification of bilirubin by glucurono-conjugation in rats
bearing implants containing PTFE fibers, rat tail collagen and
genetically modified fibroblasts expressing the enzyme
UDP-glucuronosyl transferase. Bilirubin is a degradation product of
the haem produced continuously and in considerable quantity by the
organism.
[0214] Bilirubin is liposoluble and can penetrate passively into
cells where its accumulation is toxic The elimination of bilirubin
is achieved by glucurono-conjugation in the hepatocyes by the
enzyme bilirubin UDP-glucuronyl transferase (bil. UDPGT) which
exists in two known isoforms (1 and 2). The addition if glucuronic
add makes the molecule water soluble, non-toxic and makes possible
its excretion in the bile Complete hereditary deficit of bil. UDPGT
activity is responsible for a very serious disease (Crigler-Najjar
disease) which requires liver transplantation. There exists a model
for this disease in the rat (Gunn rat).
[0215] The rat cDNA coding for the isoform 2 (bil. UDPGT-2) and
human cDNA coding for isoform 1 (bil. UDPGT-1) were inserted into
the M48 vector after excision of the nlslacZ gene by the BamHI
enzyme, thus generating the vectors M48GT-2 and M48GT-1. In a
manner similar to that described in Examples 1, 3 and 4 samples of
Gunn rat fibroblasts were taken by skin biopsy, placed in culture
and transduced by the vector M48GT-2. The presence of, on average,
one copy of the vector genome in the transduced cells was verified
by Southern blot, and the expression under the control of the PGK
promoter was verified by Northern blot. The bil. UDPGT enzymatic
activity was detected in the microsomal extracts of the infected
fibroblasts. The cells were amplified in culture, trypsinized then
resuspended in RFMI 1640 at a concentration of 2.times.10.sup.8
cells per ml.
[0216] Implants containing 2.times.10.sup.7 genetically modified
cells were prepared in the same manner as in Examples 1, 3 and 4.
Two implants were inserted in the peritoneal cavity of each
recipient Gunn rat. As the colony of Gunn rats used is not inbred,
care was taken that each animal is reimplanted with its own cells.
The measurement of the unconjugated bilirubin concentrations in the
treated animals and in control rats of the same age showed a
significant and stable diminution (between 20 and 50%) during a
period of observation of 2 months in rats bring implants of
fibroblasts expressing the bil. UDPGT. Bile samples were collected
when the animals were sacrificed and analyzed by high pressure
liquid chromatography (HPLC). Whereas the untreated animals show a
total absence of conjugated bilirubin in the bile, the Gunn rats
bearing implants containing fibroblasts expressing the bil. UDPGT
showed peaks identified as mono and di-conjugated bilirubin. The
proportion of conjugated bilirubin attained up to 11% of the
bilirubin present in the bile.
[0217] These results indicate that rat fibroblasts transduced with
the vector M48UDPGT-1 and grafted into Gunn rats in the form of
implants combining PTFE fibers and rat tail collagen are capable of
achieving in vivo the glucurono-conjugation reaction of bilirubin,
and the conjugated bilirubin can then be eliminated in the bile by
the hepatocyes, thus making possible a partial correction of the
phenotype of the diseased animals. It is thus possible to achieve
the detoxification of biological products in vivo by means of
genetically modified fibroblasts implanted in a structured collagen
gel with PTFE fibers.
Example 8
[0218] Use of an inducible promoter (Mx) to modulate the expression
of a cDNA inserted in a retroviral vector.
[0219] In the case of implants secreting a protein continuously
into the circulation, it would be desirable to be able to exercise
control over this secretion. In order to do this a retroviral
vector derived from the vector M48 was constructed in which the
PGK-1 promoter was excised and replaced by a fragment of 250 bp
covering the region -250 to +1 of the promoter of the Mx gene of
the mouse. This promoter comprises elements of response to
stimulation by alpha and beta interferons. In the mouse the proton
Mx is expressed in response to a secretion of alpha or beta
interferon. In a manner similar to that in Examples 3, 4 and 5, the
cDNA coding for mouse EPO was inserted into this vector at the
BamHI site, thus generating the vector MxEPO. .psi.CRE cells
producing recombinant ecotropic retroviral particles containing the
genome of the vector MxEPO were isolated. Fibroblasts isolated from
a skin biopsy performed on a mouse were infected with the vector
MxEPO, and the EPO secreted into the supernatant of these cultures
was measured before and after addition of alpha interferon (1000
units per ml) in the culture medium. The basic secretion levels of
EPO were less than 0.3 units/ml/24 hours, evidence for a low
activity of the Mx promoter present in the MxEPO vector in the
absence of interferon. An increase in the secretion up to 10 fold
the basic level was measured after addition of alpha interferon.
This high secretion is maintained for at least 6 days. This
observation shows that a control may be exercised over a
transcriptional promoter inserted into a retroviral vector after
the integration of the latter in the genome of the target cell.
[0220] Implants constituted of PTFE fibers, rat tail collagen and
containing skin fibroblasts transduced with the MxEPO vector were
prepared and implanted in syngeneic mice. In the absence of other
treatment, these animal have maintained a normal hematocrit. Some
of the recipient animals were treated by the intra-peritoneal
injection of 50 mg of polyA-U (Boehringer) each 48 hours for two
weeks in order to raise the hematocrit.
Treatment of Genetic or Acquired Diseases
[0221] The process is applicable to the delivery to the serum of
any soluble protein or transmembrane proteins lacking their
membrane immobilization domain. However, it does not permit
regulation of the serum concentrations of the foreign protein. The
following applications are envisaged for genetic diseases: in
hemophilia B, the supply of factor IX); in hemophilia A, the supply
of factor VIII in the form of a protein deleted in the B region; in
beta-thalassemia, the supply of eythropoietin capable of correcting
the anemia by stimulation of fetal hemoglobin synthesis.
[0222] The supply a soluble CD4 or its derivatives coupled to
immunotoxins and immunoglobulins is conceivable in infections by
retroviruses of the HIV family. More generally, any soluble
anti-viral protein could be delivered in this manner.
Production of Antibodies
[0223] The circulation of a foreign protein in the mouse induces an
immune response marked by the appearance of specific antibodies as
we have observed for beta-glucuronidase, the soluble CD4 and
erythropoietin. The process could thus be applied to immunise
animals against the still unidentified product of a cDNA isolated
by reverse genetics or by other means. It should be possible to
obtain polyclonal or monoclonal antibodies in this manner without
necessitating the prior preparation of the protein.
[0224] The same approach can be envisaged to achieve immunizations
for a therapeutic or prophylactic purpose. It should be possible to
suggest vaccinating preparations, for example, against neutralizing
epitopes of the envelope of HIV or related viruses.
Regulation by the tet Operator/tet Repressor System
[0225] Skin fibroblasts are prepared in which two retroviral
vectors are introduced, one coding for the protein of interest
placed under the control of a promoter comprising the tet operator
elements, the other coding for the tet repressor or a related
molecule modified in order to produce an enhancer effect. Depending
on the nature of this latter element and that of the promoter
containing the operator sequences, the addition of tetracycline
will induce an enhancement of a repression of transcriptions. The
fibroblasts transduced with these two vectors are implanted in vivo
in a neo-organ and the animals are treated with tetracycline in
order to assess the induction of expression. The reporter gene is
preferably that of the mouse EPO.
Regulation by the Progresterone Receptor Modified in Order to
Induce Transcription in the Presence of RU486
[0226] When its C-terminal region is cleaved the progesterone
receptor becomes capable at stimulating transcription in the
presence of RU486. Skin fibroblasts are infected with two
retroviral vectors, one coding for the protein of interest
expressed under the control of a promoter comprising elements of
response to progesterone, the other coding for a truncated version
of the progesterone receptor. As previously, the fibroblasts
transduced by these two vectors are implanted in neo-organs in the
mouse and the animals are treated with RU486.
Sequence CWU 1
1
1 1 8388 DNA mus musculus, Mo-MuLV, and other 1 tgaaagaccc
cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60
ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
120 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
tcagggccaa 180 gaacagatgg aacagctgaa tatgggccaa acaggatatc
tgtggtaagc agttcctgcc 240 ccggctcagg gccaagaaca gatggtcccc
agatgcggtc cagccctcag cagtttctag 300 agaaccatca gatgtttcca
gggtgcccca aggacctgaa atgaccctgt gccttatttg 360 aactaaccaa
tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 420
aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
480 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct
cgctgttcct 540 tgggagggtc tcctctgagt gattgactac ccgtcagcgg
gggtctttca tttgggggct 600 cgtccgggat cgggagaccc ctgcccaggg
accaccgacc caccaccggg aggtaagctg 660 gccagcaact tatctgtgtc
tgtccgattg tctagtgtct atgactgatt ttatgcgcct 720 gcgtcggtac
tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga 780
gttcggaaca cccggccgca accctgggag acgtcccagg gacttcgggg gccgtttttg
840 tggcccgacc tgagtccaaa aatcccgatc gttttggact ctttggtgca
ccccccttag 900 aggagggata tgtggttctg gtaggagacg agaacctaaa
acagttcccg cctccgtctg 960 aatttttgct ttcggtttgg gaccgaagcc
gcgccgcgcg tcttgtctgc tgcagcatcg 1020 ttctgtgttg tctctgtctg
actgtgtttc tgtatttgtc tgagaatatg ggcccgcggg 1080 ccagactgtt
accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140
cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga
1200 atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
acctcatcac 1260 ccaggttaag atcaaggtct tttcacctgg cccgcatgga
cacccagacc aggtccccta 1320 catcgtgacc tgggaagcct tggcttttga
cccccctccc tgggtcaagc cctttgtaca 1380 ccctaagcct ccgcctcctc
ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440 ttcgaccccg
cctcgatcct ccctttatcc agccctcact ccttctctag gcgccaaacc 1500
taaacctcaa gttctttctg acagtggggg gccgctcatc gacctactta cagaagaccc
1560 cccgccttat agggacccaa gaccaccccc ttccgacagg gacggaaatg
gtggagaagc 1620 gacccctgcg ggagaggcac cggacccctc cccaatggca
tctcgcctac gtgggagacg 1680 ggagccccct gtggccgact ccactacctc
gcaggcattc cccctccgcg caggaggaaa 1740 cggacagctt caatactggc
cgttctcctc ttctgacctt tacaactgga aaaataataa 1800 cccttctttt
tctgaagatc caggtaaact gacagctctg atcgagtctg ttctcatcac 1860
ccatcagccc acctgggacg actgtcagca gctgttgggg actctgctga ccggagaaga
1920 aaaacaacgg gtgctcttag aggctagaaa ggcggtgcgg ggcgatgatg
ggcgccccac 1980 tcaactgccc aatgaagtcg atgccgcttt tcccctcgag
aattctaccg ggtaggggag 2040 gcgcttttcc caaggcagtc tggagcatgc
gctttagcag ccccgctggc acttggcgct 2100 acacaagtgg cctctggcct
cgcacacatt ccacatccac cggtagcgcc aaccggctcc 2160 gttctttggt
ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc 2220
gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc
2280 gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
cagcggccaa 2340 tagcagcttt gctccttcgc tttctgggct cagaggctgg
gaaggggtgg gtccgggggc 2400 gggctcaggg gcgggctcag gggcggggcg
ggcgcgaagg tcctccggag cccggcattc 2460 tgcacgcttc aaaagcgcac
gtctgccgcg ctgttctcct cttcttcatc tccgggcctt 2520 tcgaccggat
ccggcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc 2580
tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat
2640 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
tgtaggtttg 2700 gcaagctagc ttaagtaacg ccattttgca aggcatggaa
aaatacataa ctgagaatag 2760 agaagttcag atcaaggtca ggaacagatg
gaacagggtc gaccctagag aaccatcaga 2820 tgtttccagg gtgccccaag
gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc 2880 agttcgcttc
tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac 2940
aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
3000 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
ggagggtctc 3060 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt
tatgtgtcat aaatatttct 3120 aattttaaga tagtatctcc attggctttc
tactttttct ttttattttt ttttgtcctc 3180 tgtctccatg tgttgttgtt
gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 3240 tttttaaaga
tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt 3300
gaccttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat
3360 gagctatcat tttggtatat tgattgattg attgattgat gtgtgtgtgt
gtgattgtgt 3420 ttgtgtgtgt gattgtgtat atgtgtgtat ggttgtgtgt
gattgtgtgt atgtatgttt 3480 gtgtgtgatt gtgtgtgtgt gattgtgcat
gtgtgtgtgt gatgtgttag tgtatgattg 3540 tgtgtgtgtg tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg tgtgtgttgt gtatatatat 3600 ttatggtagt
gagaggcaac gctccggccc aggcgtcagg ttggtttttg agacagagtc 3660
tttcacttag cttgaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
3720 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
ggaaatgtgc 3780 gcggaacccc tatttgttta tttttctaaa tacattcaaa
tatgtatccg ctcatgagac 3840 aataaccctg ataaatgctt caataatatt
gaaaaaggaa gagtatgagt attcaacatt 3900 tccgtgtcgc ccttattccc
ttttttcggg cattttgcct tcctgttttt gctcacccag 3960 aaacgctggt
gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 4020
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
4080 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
gacgccgggc 4140 aagagcaact cggtcgccgc atacactatt ctcagaatga
cttggttgag tactcaccag 4200 tcacagaaaa gcatcttacg gatggcatga
cagtaagaga attatgcagt gctgccataa 4260 ccatgagtga taacactgcg
gccaacttac ttctgacaac gatcggagga ccgaaggagc 4320 taaccgcttt
tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 4380
agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
4440 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa 4500 tagactggat ggaggcggat aaagttgcag gaccacttct
gcgctcggcc cttccggctg 4560 gctggtttat tgctgataaa tctggagccg
gtgagcgtgg gtctcgcggt atcattgcag 4620 cactggggcc agatggtaag
ccctcccgta tcgtagttat ctacacgacg gggagtcagg 4680 caactatgga
tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4740
ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
4800 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
atcccttaac 4860 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
gatcaaagga tcttcttgag 4920 atcctttttt tctgcgcgta atctgctgct
tgcaaacaaa aaaaccaccg ctaccagcgg 4980 tggtttgttt gccggatcaa
gagctaccaa ctctttttcc gaaggtaact ggcttcagca 5040 gagcgcagat
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 5100
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
5160 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc 5220 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca 5280 ccgaactgag atacctacag cgtgagctat
gagaaagcgc cacgcttccc gaagggagaa 5340 aggcggacag gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc 5400 cagggggaaa
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 5460
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
5520 cctttttacg gttcctggcc ttttcgtggc cttttgctca catgttcttt
cctgcgttat 5580 cccctgattc tgtggataac cgtattaccg cctttgagtg
agctgatacc gctcgccgca 5640 gccgaacgac cgagcgcagc gagtcagtga
gcgaggaagc ggaagagcgc ctgatgcggt 5700 attttctcct tacgcatctg
tgcggtattt cacaccgcat atggtgcact ctcagtacaa 5760 tctgctctga
tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt 5820
catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
5880 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
gtcagaggtt 5940 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
agctcatcag cgtggtcgtg 6000 aagcgattca cagatgtctg cctgttcatc
cgcgtccagc tcgttgagtt tctccagaag 6060 cgttaatgtc tggcttctga
taaagcgggc catgttaagg gcggtttttt cctgtttggt 6120 cactgatgcc
tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg 6180
agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
6240 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
ctcagggtca 6300 atgccagcgc ttcgttaata cagatgtagg tgttccacag
ggtagccagc agcatcctgc 6360 gatgcagatc cggaacataa tggtgcaggg
cgctgacttc cgcgtttcca gactttacga 6420 aacacggaaa ccgaagacca
ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca 6480 gtcgcttcac
gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg 6540
ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
6600 aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
tggatatgtt 6660 ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
aattgattgg ctccaattct 6720 tggagtggtg aatccgttag cgaggtgccg
ccggcttcca ttcaggtcga ggtggcccgg 6780 ctccatgcac cgcgacgcaa
cgcggggagg cagacaaggt atagggcggc gcctacaatc 6840 catgccaacc
cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt 6900
ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg
6960 tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
gccgccggaa 7020 gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
tcgcgaacgc cagcaagacg 7080 tagcccagcg cgtcggccgc catgccggcg
ataatggcct gcttctcgcc gaaacgtttg 7140 gtggcgggac cagtgacgaa
ggcttgagcg agggcgtgca agattccgaa taccgcaagc 7200 gacaggccga
tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc 7260
gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
7320 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
caagggcatc 7380 ggtcgacgct ctcccttatg cgactcctgc attaggaagc
agcccagtag taggttgagg 7440 ccgttgagca ccgccgccgc aaggaatggt
gcatgcaagg agatggcgcc caacagtccc 7500 ccggccacgg ggcctgccac
catacccacg ccgaaacaag cgctcatgag cccgaagtgg 7560 cgagcccgat
cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 7620
gcgccggtga tgccggccac gatgcgtccg gcgtagagcg ccacaggacg ggtgtggtcg
7680 ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact
gggcggcggc 7740 caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag
aaattgcatc aacgcatata 7800 gcgctagcag cacgccatag tgactggcga
tgctgtcgga atggacgata tcccgcaaga 7860 ggcccggcag taccggcata
accaagccta tgcctacagc atccagggtg acggtgccga 7920 ggatgacgat
gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta 7980
actgtgataa actaccgcat taaagctttg cttaggagtt tcctaataca tcccaaactc
8040 aaatatataa gcatttgact tgttctatgc cctaggggga ggggggaagc
taagccagct 8100 ttttttaaca tttaaaatgt taattccatt ttaaatgcac
agatgttttt atttcataag 8160 ggtttcaatg tgcatgaatg tcgcaatatc
ctgttaccaa agctagtata aataaaaata 8220 gataaacgtg gaaattactt
agagtttctg tcattaacgt ttccttcctc agttgacaac 8280 ataaatgcgc
tgctgagaag ccagtttgca tctgtcagga tcaatttcca ttatgccagt 8340
catattaatt actagtcaat tagttgattt ttgacatata catgtgaa 8388
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