EP0787197A1 - Lignees immortalisees de cellules endotheliales cerebrales et leurs applications therapeutiques - Google Patents

Lignees immortalisees de cellules endotheliales cerebrales et leurs applications therapeutiques

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Publication number
EP0787197A1
EP0787197A1 EP95934188A EP95934188A EP0787197A1 EP 0787197 A1 EP0787197 A1 EP 0787197A1 EP 95934188 A EP95934188 A EP 95934188A EP 95934188 A EP95934188 A EP 95934188A EP 0787197 A1 EP0787197 A1 EP 0787197A1
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Prior art keywords
cells
endothelial
vector
cell lines
expression
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EP95934188A
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German (de)
English (en)
French (fr)
Inventor
Nathalie Chaverot
Pierre-Olivier Couraud
John Laterra
Jérôme QUINONERO
Françoise ROUX
Arthur Donny Strosberg
Jean-Léon TCHELINGERIAN
Lionel Vignais
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Neurotech SA
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Neurotech SA France
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Publication of EP0787197A1 publication Critical patent/EP0787197A1/fr
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to immortalized lines of mammalian cerebral endothelial cells, possibly modified, as well as to their applications as a drug for preventive or curative aim and in particular for the treatment of various troubled or primary and secondary, neurological or psychiatric diseases, including brain tumors.
  • the first trials in this area mainly concerned neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, and included intracerebral transplantation of fetal neural tissue or medullo-adrenal tissue into the brain (BJ ⁇ RKLUND A., TINS , 1991, 14, 8, 319-322).
  • primary nervous tissue of fetal origin for cell transplantation in human therapy is a source of many ethical and practical problems; an alternative to this problem is the use of primary cell lines of neural origin (neurons, glial cells, astrocytes for example) or of non-neural cell lines (fibroblasts, myoblasts, chromaffin cells of the medulo-adrenal gland, hepatocytes for example ) (GAGE FH et al., Trends Neurosci., 1991, 14, 328-333). Although adrenal, fibroblast or myoblast cell lines can effectively release active substances in vivo, they are not normally present in the nervous system, can alter the normal function of the nervous system and cause a rejection reaction.
  • the present invention relates to mammalian endothelial cell lines, characterized:
  • MHC major histocompatibility complex
  • tight junctions in that they comprise a nucleic acid fragment comprising at least one immortalizing fragment of a viral or cellular oncogene, optionally associated with at least one selection gene, and an expression vector comprising a sequence coding for a polypeptide, a protein or a viral vector, optionally associated with at least one selection gene and optionally at least one marker gene and
  • the expression vector is understood to mean any fragment of nucleic acid integrated into the genome or present at the cytoplasmic level, and capable of allowing the expression of said polypeptide, protein or viral vector.
  • the nucleic acid fragment comprising at least one immortalizing fragment of an oncogene contains the neomycin resistance gene and a fragment of the SV40 oncogene T.
  • the nucleic acid fragment comprising at least one immortalizing fragment of an oncogene contains the early E1A region of the adenovirus 2 genome and the neomycin resistance gene.
  • said expression vector is a retroviral vector, in particular an MFG vector.
  • the retroviral vector is an MFG-NB vector defective for replication.
  • Said vectors are notably described in MULLIGAN et al. (Proc. Nat. Ac. Sci. USA, 1984, 81, 6349 ⁇ 6353) and FERRY et al. (Proc Natl Acad. Sci. USA, 1990, 88, 8377-8381).
  • said endothelial cells are brain capillary cells.
  • peripheral non-immortalized peripheral vascular endothelial cells have been described, but do not constitute an appropriate vector, in that they do not constitute a pure, homogeneous and sufficient source, with a view to a reproducible application to transplants. and in that they do not exhibit the cerebral endothelial phenotype.
  • the sequence coding for a polypeptide or a protein is selected from the sequences coding for: enzymes, such as proteases, enzyme inhibitors, such as protease inhibitors, cytokines, neurotransmitters, neurotrophins, growth factors, toxins, antimetabolites, neurohormones, gangliosides, antibiotics, thrombolytic factors, coagulating factors, vasodilator or vasoconstrictor factors, hypo or hypercholesterolemic factors, factors hyper- or hypoglycemic agents or any other substance of interest.
  • enzymes such as proteases
  • enzyme inhibitors such as protease inhibitors, cytokines, neurotransmitters, neurotrophins, growth factors, toxins, antimetabolites, neurohormones, gangliosides, antibiotics, thrombolytic factors, coagulating factors, vasodilator or vasoconstrictor factors, hypo or hypercholesterolemic factors, factors hyper- or hypoglycemic agents or any other substance of interest
  • said endothelial cells advantageously comprise, as immortalizing fragment, the early E1A region of the genome of adenovirus 2 and the neomycin resistance gene and as an expression vector, a retroviral vector MFG-NB containing the nls-lacZ gene coding for ⁇ -galactosidase.
  • This cell line was named RBEZ by the inventors.
  • said line was deposited under the number I-1481 on October 10, 1994 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
  • said endothelial cells advantageously comprise, as immortalizing fragment, the early region E1A of the genome of adenovirus 2 and the resistance gene to neomycin and as vector, a retroviral vector pMoMuLVisisNGF coding for murine ⁇ -NGF.
  • This cell line was named RBE / NGF-4 by the inventors.
  • said line was deposited under No. 1-1482 on October 10, 1994 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
  • endothelial cells of cerebral capillaries integrate well with cerebral vascularization, are very well tolerated and release, in vivo, over a long period, the active substance which they express and find application in the preparation of a composition for the treatment of primary and secondary neurological or psychiatric disorders or diseases, including cerebral tumors or for stimulating the growth and reproduction of farm animals (poultry, sheep, cattle, pigs, equines, lagomorphs, rodents etc. ..).
  • grafted endothelial cells in accordance with the invention adopt a vascular localization.
  • vascular localization does not lead to an exacerbation of cell death and this again at 1 year after implantation.
  • the viral vector is advantageously a modified cytomegalovirus (CMV) (integrative viral vector).
  • CMV modified cytomegalovirus
  • the present invention also relates to compositions, characterized in that they comprise at least one line of cerebral endothelial cells in accordance with the invention, associated with at least one pharmaceutically acceptable vehicle.
  • compositions preferably contain between 10 4 and 10 5 endothelial cells / ⁇ l.
  • compositions can be advantageously administered intracranially, subcutaneously, intracerebroventricular route, subdural route, venous or arterial route (intracarotide, for example), intramuscular route, intrathecal route.
  • said endothelial cells can be cells of the same species as the host (allograft or homograft) or of a different species (xenograft).
  • the present invention also relates to a process for obtaining a modified cell line according to the invention, which process is characterized in that:
  • a first transfection is carried out by: culture of cerebral endothelial cells, preferably of cerebral microvessels, in a suitable culture medium, supplemented with serum and growth factors,
  • a nucleic acid fragment comprising at least one immortalizing fragment of a viral or cellular oncogene and optionally at least one selection gene, in particular a gene coding for resistance to an antibiotic , - selection of the transfected cells on a selection medium adapted to said selection gene, if necessary,
  • the transfection of step (2) is carried out with a retroviral vector in which the sequence coding for the protein to be expressed has been previously incorporated.
  • step (1) makes it possible to obtain RBE4 cells, immortalized by transfection with a plasmid containing the early region E1A of the genome of adenovirus 2 and the gene for resistance to neomycin under the control of the promoter SV40, which are deposited under the number 1-1142 with the National Collection of Cultures of Micro-organisms (CNCM) held by the Institut Pasteur.
  • CNCM National Collection of Cultures of Micro-organisms
  • the present invention also relates to a model for studying the cerebral integration of vector cells of active substances, in the brain, characterized in that it comprises a REBZ cell line, in accordance with the invention.
  • the present invention also relates to a model for studying and identifying biochemical and cellular systems of the blood-brain barrier in vitro, characterized in that it comprises at least one cell line in accordance with the invention.
  • the present invention further relates to a process for the production of a polypeptide or a protein, characterized in that it comprises the use of at least one endothelial cell line according to the invention, in a suitable bioreactor.
  • FIG. 1 illustrates the in vitro study of the expression of the NGF transgene in RBE / NGF cells, by in si tu hybridization
  • FIGS. 2 and 3 illustrate the stimulation of axonal budding of PC12 cells, obtained from the supernatant of RBE / NGF cells, in vi tro;
  • FIG. 4 illustrates the premarking of RBE4 cells before transplantation, with the nuclear dye HOECHST 33342 (bisbenzimide);
  • FIG. 5 illustrates the visualization of the cells premarked with the Hoechst dye, after transplantation into the brain of an adult rat
  • FIG. 6 illustrates the study of the morphological and functional integration of the RBEZ cells by visualizing the expression of the nlslacZ transgene and of the antigenic marker of integrity of the blood-brain barrier (BBB), the EBA (endothelial barrier). antigen);
  • FIG. 7 illustrates the ultrastructural study in electron microscopy, demonstrating the morphological and functional integration of RBEZ cells after intracerebral graft by visualization of the expression of the nls-lacZ transgene;
  • FIG. 8 illustrates the study of the morphological and functional integration of the RBEZ cells after grafting into an intracerebral tumor, by visualization of the expression of the nls-lacZ transgene;
  • FIG. 9 illustrates the identification of the nls-LacZ gene in tumors implanted with RBEZ cells, by PCR.
  • FIG. 10 illustrates the in vivo study of the expression of the NGF transgene in RBE / NGF cells, three weeks after transplantation into the nucleus basalis (basal nucleus);
  • FIG 11 illustrates the control brain structures used as internal control of hybridization in si tu of the NGF messenger, in vivo;
  • FIG. 12 illustrates the biological effect of NGF secreted by RBE / NGF cells, three weeks after grafting, at the level of the nucleus basalis;
  • FIG. 13 illustrates the biological effect of NGF secreted by RBE / NGF cells, three weeks after grafting, away from the nucleus basalis;
  • FIG. 14 illustrates the quantification of the biological effect induced by the expression of the NGF transgene at 3 and 8 weeks post-grafting and this is reflected by the surface occupied by the immunostaining p75LNGFR relative to the surface of the graft.
  • Endothelial cells of microvessels of rat brains are immortalized by transfection with the plasmid pElA-neo, containing the sequence coding for the adenovirus E1A, followed by the gene for resistance to neomycin.
  • RBE4 A clone, called RBE4, was thus obtained and its characteristics are in particular described in PCT application WO 93/06222 as well as in the articles published in J. Cell. Physiol., 1993, 155, 104-111 and J. Cell. Physiol., 1994, 159, 101-113. This clone was deposited under the number 1-1142 at the National Collection of Cultures of Micro-organisms (CNCM).
  • the calcium phosphate co-precipitation technique was used, as described in PCT application WO 93/06222 and recalled below: the transfection of said cells is carried out on the 5th passage with the abovementioned plasmid (10 ⁇ g) tale
  • the promoter SV40 in addition to the early E1A region of the adenovirus 2 genome and the neomycin resistance gene, the promoter SV40.
  • the cell line obtained has some of the characteristics of brain endothelial cells; in particular, it has an untransformed phenotype: contact inhibition, proliferation dependent on growth and adhesion factors, expression of endothelial differentiation markers (antigen related to factor VIII), agglutinin-binding site of Griffonia simplicifolia and nontumorigenicity in Nude mice.
  • these cells are stimulated by astrocytes to express the specific enzyme markers of the blood-brain barrier, namely, ⁇ -glutamine transferase and alkaline phosphatase.
  • Example 1 The RBE4 cells obtained in Example 1 are subjected to two passages per week, on an ⁇ -MEM / F10 medium (1/1; Seromed, France), supplemented with glutamine
  • a vector MFG-NB, defective for replication, and containing the lacZ gene is obtained by insertion of the sequence coding for the ⁇ -galactosidase of E. coli, fused with a sequence coding for the nuclear localization sequence (nls) of 21 amino acids, from the SV40 T antigen (Kalderon D. et al., Cell, 1984, 39, 499-509).
  • This vector MFG-NB nls-lacZ is introduced into cells producing ov-2 retroviruses (Mulligan et al., Cited above) (recombinant ov-2 retroviral infection) and makes it possible to obtain ⁇ -2-MFG- cell lines. NB.
  • retrovirus producing cells are spread in boxes at a density of 10 6 cells for boxes of 100 mm in diameter, in 7 ml of RPMI 1640 medium, supplemented with 10% fetal calf serum.
  • a volume of 6 ml of medium containing the virus is filtered and used for infection or else stored at -80 ° C. until it is used.
  • RBE4 endothelial cells are spread on a dish at a density of 10 4 cells / cm 2 and after 24 h, the virus (3 ml) is added in the presence of polybrene (10 ⁇ g / ml) for
  • the RBE4 cells are subcultured and reinfected under the same conditions.
  • RBEZ cells are sorted by FACS (fluorescent activated cell sorting; NOLAN et al., PNAS, 1988, 85, 2603-2607), using, as enzyme substrate, ⁇ -D-galactopyranoside fluorescein (FDG) .
  • FACS fluorescent activated cell sorting
  • 10 * RBEZ cells in 100 ⁇ l are incubated at 37 ° C. for 5 min, in a 5 ml polystyrene tube, before the addition of 100 ⁇ l of FDG (2 mM).
  • the cells are placed again at 37 ° C for 1 min, then on ice, and the volume is adjusted to 2 ml. 4) Detection of the expression of the transgene by visualization of the enzymatic activity of ⁇ -galartosidase, using a chromogenic substrate, X-gal.
  • the enzymatic activity is detected by incubation of the cells at 37 ° C. in a PBS buffer containing 2 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (X-gal), potassium ferricyanide. 20 mM, 20 mM potassium ferrocyanide and MgCl. 2 mM.
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside
  • ⁇ -galactosidase enzyme activity is revealed by the formation of a blue coloration. About 50-80% of RBE4 endothelial cells infected with the retrovirus stain blue during histochemistry, but the level of staining varies from cell to cell. Control (non-infected) RBE4 cells are not stained, under the same conditions.
  • the RBEZ cells obtained are cultured on a support covered with collagen in an ⁇ -MEM / F10 medium, supplemented with 10% fetal calf serum, 2mM glutamine, FGFb 1 ng / ml and G418 300 ⁇ g / ml.
  • the RBE4 cells obtained in Example 1 are subjected to two passages per week, on an ⁇ -MEM / F10 medium (1/1; Seromed, France), supplemented with 2 mM glutamine, 10% fetal calf serum and FGFb 1 ng / ml and G418
  • Example 2 The procedure is as in Example 2, using a retroviral vector pMoMuLVisisNGF, deficient for replication and in which the sequence coding for mouse ⁇ -NGF is inserted (Scott J et al., Nature, 1983, 302, 538- 540).
  • This vector introduced into ⁇ -2 producer cells, makes it possible to obtain ⁇ -2-MoMuLVisisNGF cell lines.
  • the subclones secreting ⁇ NGF are identified using a bi-site ELISA (LADENHEIM et al., J. Neurochem., 1993 , 60, 260-266).
  • a monoclonal anti- ⁇ NGF antibody called 27/21 (0.1 mg / ml in 0.05 M carbonate buffer pH 9.6), is applied to EIA / RIA Costar plates for 2 hours at 37 ° C. The plates are washed 3 times with a mixture of 50 mM Tris HCl, pH 7.4, 200 mM NaCl, CaCl. 10 mM, 0.1% Triton X-100, and 0.05% sodium azide and incubated at 4 ° C overnight in conditioned medium or ⁇ NGF standards (30-1000 pg / ml) in the same buffer supplemented with 1% bovine serum albumin.
  • ⁇ NGF is detected using the same antibody conjugated to ⁇ -D-galactosidase (400 mU / ml), after incubation for 4 hours at 37 ° C.
  • the chromogenic substrate is chlorophenol-red- ⁇ -galac-topyranoside (1 mg / ml in 100 mM Hepes medium, pH 7, 150 mM NaCl, 2 mM MgCl, 0.1% sodium azide).
  • the absorbance at 570 nm is read after 2 hours at 37 ° C.
  • Two highly positive subclones named RBE / NGF-2 and -4 are selected, as well as two less positive subclones, and this among 94 clones tested.
  • FIG. 1 illustrates the in vitro study of the expression of the NGF transgene in RBE / NGF cells, by in si tu hybridization.
  • FIG. 1A shows an intense signal in the RBE / NGF cells in culture, indicating a high level of expression of the NGF transgene.
  • FIG. 1B the absence of positivity in the non-infected RBE4 control cells is observed (x300 in 1A and 1B) (1B in phase contrast). 4) In vi tro activity of the secreted NGF:
  • the biological activity of NGF secreted in the supernatant of RBE / NGF cells is demonstrated by the property of promoting budding of neurites of rat pheochromocytoma PC12 cells.
  • the RBE / NGF cells are spread out on boxes at a density of 10 4 / cm 2 in boxes 100 mm in diameter and growth is carried out for 3 to 4 days until confluence (10 7 cells /box).
  • the medium is changed (10 ml) and the supernatants are collected after 24 hours. The results are illustrated in FIGS.
  • Figures 2 and 3 have on the abscissa the concentration of NGF (ng / ml) ( Figure 2) or the dilution rate (Figure 3; curve 1: RBE / NGF cells and curve 2: RBE4 cells) and on the ordinate, the percentage of cells carrying neurites.
  • EXAMPLE 4 Cerebral implantation of the RBE4, RBEZ and RBE-NGF cells.
  • RBE4 cells survival and integration.
  • FIG. 4 illustrates the premarking of RBE4 cells, before transplantation, with the nuclear dye HOECHST 33342 (bisbenzimide). Suspended cells are visualized under fluorine microscopy cence, under ultra-violet light. The fluorescence of the dye clearly defines the positively labeled cell nuclei (x270).
  • HOECHST 33342 bisbenzimide
  • the graft has a compact appearance with a weak and constant spreading of certain RBE4 cells, around its mass. This migration takes place essentially along the vascular network of the host, suggesting a preferential interaction between the implanted endothelial cells and the vascular elements of the host.
  • the histological colorings of the brains thus grafted show a minimum of signs of necrotic cells, and this essentially during the first week following the surgical trauma due to the transplant.
  • the cell density is homogeneous (few or no pycnotic cells).
  • the immunoreactivity GFAP glial fibrillary acidic protein
  • characteristic of astrocytes is significant, from 1 week after implantation, both around the graft and in the graft itself, indicating an infiltration of astrocytes in the latter.
  • the implanted RBE4 cells migrate and integrate into the vascular environment with sometimes a direct participation in the vascular network of the host.
  • FIGS. 5A, 5B and 5C show the cells premarked with the Hoechst dye, after transplantation into the brain of an adult rat.
  • Figure 5A shows a general view of the graft area in the brain parenchyma. Fluorescent grafted endothelial cells preferentially accumulate around vascular elements of the host brain. The asterisks mark the path of a blood vessel (x250).
  • Figures 5B and 5C show a high magnification blood vessel located in the implantation area of the graft fon. In 5B, many Hoechst positive RBE4 cells integrated in the luminal (arrows) and perivascular position can be observed. In 5C, this same vessel is immunolabelled by an anti-laminin antibody (specific marker for blood vessels) (x600).
  • an anti-laminin antibody specific marker for blood vessels
  • RBEZ cells survival, integration and expression of the transgene.
  • the cells are rinsed three times in a grafting solution comprising PBS supplemented with MgCl 2 and CaCl. at 1 ⁇ g / ml and in 0.1% glucose, so as to eliminate the DMEM-SVF medium.
  • a total of 300,000 cells suspended in a grafting solution (3 .mu.l) is injected per site using a micro syringe 10 .mu.l Exmire ® having a diameter of 0.5 mm external needle.
  • the anesthetized rats are sacrificed by infusion with 150 ml of PBS and then with 300 ml of 4% PFA in a solution of PBS (0.1 M, pH 7.4) at 4 ° C.
  • the brains were cryoprotected and frozen by embedding in OCT compound ® a cut cryostat.
  • the enzymatic activity of the nls-lacZ transgene is detected by incubation at 37 ° C of the tissue in a PBS buffer containing 2 mM of 5-bromo-4-chloro-3-indolyl ⁇ -D-galactoside (X-gal , Sigma), potassium ferricyanide (20 mM), potassium ferrocyanide (20 mM) and MgCl. (2mM).
  • X-gal 5-bromo-4-chloro-3-indolyl ⁇ -D-galactoside
  • FIG. 6 illustrates the study of the morphological and functional integration of the RBEZ cells by visualization of the expression of the nls-lacZ transgene and of the antigenic marker of integrity of the blood-brain barrier (BBB), the EBA (endothelial barrier antigen ).
  • FIGS. 6A, 6B, 6C and 6D show blood vessels located at a distance from the graft area, on which RBEZ cells have migrated after transplantation.
  • nuclei of endothelial cells, expressing the nls-lacZ transgene, in luminal position (arrows) (6A, 6C) can be observed.
  • the expression of the transgene is important up to 5 weeks after implantation, but decreases afterwards.
  • the presence of implanted cells remains detectable using the aforementioned Hoechst dye.
  • the absence of specific major changes in the host's immune response to the presence of the lacZ transgene shows the good integration of these RBEZ cells.
  • RBE4 and RBEZ cells never develop tumors in vivo, because they exhibit significant stability in their phenotype.
  • Animals (n 10) were treated for an ultrastructural study of the integration of RBEZ cells in electron microscopy.
  • the animals were perfused with a PBS solution containing 2.5% PFA and 0.5% glutaraldehyde.
  • the brains are removed and left in the same fixative overnight. After rinsing, they are cut with a vibratome into sections of thickness 75 ⁇ m.
  • the X-Gal substrate was used as for optical microscopy, which, under the action of ⁇ -galactosidase, forms a dense precipitate with electrons, visible under the electron microscope.
  • a pre-identification of the graft is performed on a section before treatment with OsO 4 at 1%. These thick cups are then dehydrated and then included in the spon.
  • FIG. 7 illustrates the study of the morphological and functional integration of the RBEZ cells by visualization of the expression of the nls-lacZ transgene by electron microscopy.
  • FIG. 7A shows, in Nomarski optics, the peri-nuclear ⁇ -galactosidase labeling in the cells grafted on a semi-thin section of the brain (2 ⁇ m) (x1660). On examination by electron microscopy, the cells are observed either in the parenchyma (FIG.
  • the RBEZ cells present a normal phenotype, from the first week post-transplant (tight junction and few pinocytosis vesicles) (xl60,000 in 7B and 7C) (L: vascular light).
  • the RBEZ cells at confluence are trypsinized and resuspended in DMEM without serum, immediately before their implantation in the host animals.
  • the RBEZ cells (5,105 cells) are injected stereotaxically with a syringe (Hamilton, gauge 26, in bevel), at the level of the caudate nucleus and the putamen of Fischer 344 rats (200 to 250 g), after anesthesia .
  • the cells are injected in a volume of
  • the anesthetized Fischer rats receive 100 ⁇ l containing 10 6 RBEZ cells.
  • a test is carried out by making the same implantations (intracranial and subcutaneous) with a mixture of 9L, F98 or C6 glioma cells (10 5 cells ) and RBEZ cells, under the same conditions as above.
  • the tissues are prepared so as to make an immunohistochemical and histological study.
  • the rats are anesthetized with ether and after a thoracotomy, the right atrium is incised and a cannula is inserted into the left ventricle which is then sequentially perfused with a buffer containing 120 mM NaCl, C1 2.7 mM in a buffer phosphate pH 7.4 (1 ml / g of weight) then paraformaldehyde at 3.7% (fixer).
  • the brains are placed in the same fixative, for 30 minutes, cryoprotected with 30% sucrose in PBS and frozen. The tissues are cut (12 ⁇ m thick) and mounted on gelatinized slides.
  • the preparations on slides are rinsed three times in PBS buffer, then incubated at 37 ° C for 1 to 2 hours in PBS containing 0.5 mg / ml of 5-bromo-4-chloro-3-indolyl- ⁇ -D -galactopyranoside (X-gal), 20 mM potassium ferric cyanide, 20 mM potassium ferrocyanide and 2 mM MgCl 2 .
  • X-gal 5-bromo-4-chloro-3-indolyl- ⁇ -D -galactopyranoside
  • 20 mM potassium ferric cyanide 20 mM potassium ferrocyanide
  • 2 mM MgCl 2 The sections incubated in the absence of X-gal substrate are used as a negative control.
  • the sections are then rinsed in PBS buffer and mounted to 90% glycerol in PBS, containing 0.02% sodium azide.
  • reaction conditions do not cause any coloration in the control animals which have not received any RBEZ cells.
  • Some sections show a positive reaction to laminin in the tumor (detection of tumor micro-vessels), to the nuclear proliferation antigen Ki67 and to the expression of markers of the blood-brain barrier, such as the endothelial glucose transporter of the type I (glut-1), after staining with X-gal.
  • the sections are digested for 15 minutes at 37 ° C. with 0.2% pepsin in 0.01 N HCl before incubation with the immunological reagents.
  • the sections are sequentially incubated with normal 1% goat serum, then either with anti-laminin rabbit antibodies, anti-glutl rabbit antibodies or anti-Ki67 rabbit antibodies, then biotinylated immunoglobulin anti-rabbit goat is added (1: 200 in PBS).
  • the sections are then incubated with an avidin-biotin-peroxidase complex (1:50 in PBS), followed by an incubation in a 50 mM Tris buffer containing 0.5 mg / ml of 3-3'-diaminobenzidine (Sigma) and 0.01% hydrogen peroxide.
  • control slides are incubated with normal rabbit serum in place of the immune serum.
  • the sections are mounted in 90% glycerol in PBS.
  • the blue product of the reaction with ⁇ -galactosidase is identified in histological sections of intracranial and subcutaneous tumors after grafting of tumor cells and RBEZ (Tables I and II). No blue reaction product is detected in tumors implanted without an RBEZ cell. Surprisingly, the grafted endothelial cells are distributed across all intracranial tumors, including marginal infiltrations, but do not appear to migrate into normal tissue.
  • FIG. 8 illustrates the integration of RBEZ cells into the tumor tissue (C6 cells) and its vascular network (arrowhead) on a section of brain tissue counter-colored with neutral red and in Nomarski optics.
  • RBEZ cells implanted in this way have the ability to integrate in an anatomically correct manner into the tumor vasculature.
  • Glut-1 type 1 glucose transporter
  • the number of RBEZ cells implanted per tumor section is quantified by computer-assisted image analysis using the imaging device (MCID) supplied by the company Imagine Research Inc. (Brock. University St Catherines, Ontario, Canada), a Hamamatsu high resolution CCD camera and a Compaq DeskPro 486/33 computer.
  • MCID imaging device
  • the total number of RBEZ cells per tumor is estimated from the number of RBEZ cells per tumor volume (adjacent section of 12 ⁇ m) and the total tumor volume is estimated from the limits of the tumor, according to two orthogonal cross-sections.
  • Tables I and II illustrate the results obtained during the implantation of the cerebral endothelial cells modified, according to the invention, in 9L gliomas.
  • Oligonucleotides complementary to DNA sequences, located on the nls-lacZ gene (5'- CGACTCCTGGAGCCCGTCAGTATC-3 ') and on the vector, downstream of the sequence 3'LTR (5'-GACCACTGATATCCTGTCTTTAAC-3'), are used as primers.
  • the PCR is carried out on genomic DNA isolated from control tumors (tumor 9L; FIG. 9, lines 2 and 3)), from experimental tumors (tumors having integrated RBEZ cells: implantation 14 days before the isolation of DNA ; Figure 9, lines 4- 6) and cell lines RBEZ ( Figure 9, line 7) and RBE4 ( Figure 9, line 8).
  • 35 amplification cycles with Taq polymerase are carried out under the following conditions: denaturation at 95 ° C, hybridization at 60 ° C and elongation at 72 ° C.
  • FIG. 9 shows the results obtained: the PCR product (400 bp) is only present in the samples containing the RBEZ cells.
  • the cells are rinsed three times in a grafting solution comprising PBS supplemented with MgCl. and CaCl 2 at 1 ⁇ g / ml and with 0.1% glucose, so as to eliminate the DMEM-SVF medium.
  • a grafting solution comprising PBS supplemented with MgCl. and CaCl 2 at 1 ⁇ g / ml and with 0.1% glucose, so as to eliminate the DMEM-SVF medium.
  • 300 g receive a transplant of RBE / NGF cells premarked with Hoechst dye, under deep anesthesia under stereotaxic conditions.
  • Ten animals receive stereotaxic implantation of RBE / NGF cells in the right basal nucleus.
  • a total of 300,000 cells, suspended in a graft solution (3 ⁇ l), is injected per site using a 10 ⁇ l Exmire ® micro-syringe with an external needle diameter of 0.5 mm .
  • unmodified RBE4 cells labeled with Hoechst dye are also grafted at the same time, contralateral (left side) and using the same stereotaxic levels. Coronal and horizontal sections of the transplanted brains are collected between 1 week and 12 months after transplantation. The grafts examined, visualized by the fluorescence obtained using the Hoechst dye, show a compact appearance with low cell spreading. No tumors of the RBE / NGF transplanted cells were observed.
  • the animals are anesthetized and perfused intracardiac, with a 0.1 M PBS solution, pH 7.4 at 4 ° C, followed by an infusion of paraformaldehyde at 4% in the same buffer.
  • the brains are removed and stored in the same buffer overnight at 4 ° C.
  • the brains are then stored in a PBS buffer comprising 30% sucrose for 2 days. at 4 ° C and frozen in isopentane at -60 ° C.
  • the coronal and horizontal sections (30 ⁇ m thick) are produced using a cryostat and collected in wells filled with PBS at 4 ° C. The sections are divided into different groups, to perform an immunohistochemical analysis, as well as staining with toluidine blue.
  • the sections are initially treated with PBS containing 0.4% H 2 O 2 , for 30 minutes, and rinsed in the same buffer. They are then incubated in a normal 10% serum, from the same animal as that used to produce the secondary antibodies and 0.1% Triton X-100 in PBS for 1 hour, then with one of the following primary antibodies:
  • rabbit anti-Glut-1 antibody (brain-specific glucose transporter), (1: 5000, Biogenesis); rabbit anti-GFAP antibody (1: 6000, Dako); goat anti-ChAT antibody (1: 100, Chemicon), rabbit anti-laminin antibody (1: 5000, Sigma).
  • mouse anti-p75 antibody LNGFR Low affini ty NGF receptor
  • mouse anti-CD11b antibody rat macrophages
  • mouse antibodies against rat T lymphocytes (1: 2000, clone MRC OX-52, Serotec
  • mouse anti major rat histocompatibility complex I (1: 1000, MRC clone OX-18, Serotec)
  • rat aiiti-MHC class II la) mouse antibody (1: 1000, clone MRC OX-6, Serotec
  • mouse anti-EBA antibody blood-brain barrier antigen, rat specific
  • All antibodies are diluted in PBS buffer containing 5% normal serum (donkey serum for polyclonal antibodies, and sheep serum for monoclonal antibodies) and 0.1% Triton X-100 and incubated for 36 hours with stirring at 4 ° C.
  • the cuts are rinsed and incubated with biotinylated donkey antibodies against rabbit IgG (1: 2000, Amersham), anti-goat IgG (1: 1000, Jackson Laboratories) or biotinylated sheep antibodies against mouse IgG (1: 600, Amersham) in PBS buffer containing 5% normal serum and 0.1% Triton X-100, overnight with stirring at 4 ° C.
  • NGF NGF
  • the rats are perfused with 2% paraformaldehyde in 0.1 M PBS buffer (pH 7.4, 4 ° C).
  • the collected brains are placed in this buffer for 60 minutes at 4 ° C.
  • rapid freezing of the samples is carried out by immersion in isopentane at -60 ° C.
  • the frozen brains are cut horizontally (10-14 ⁇ m), using a Microm ® cryostat, then mounted on gelatinized slides and dried at room temperature.
  • the sections are pre-hybridized for one hour at 40 ° C in a 4XSSC buffer, lX Denhardt. Hybridization is carried out in a humid chamber at 37 ° C. for 16 hours, using as a hybridization buffer a mixture of 4XSSC, 50% formamide, 10% dextran sulfate, IX Denhardt, 500 ⁇ g / ml of fragmented DNA. and denatured with salmon sperm and 100 ⁇ g / ml of yeast tRNA, containing the above-mentioned NGF probe at a final concentration of 2 ⁇ g / ml.
  • the slides are washed sequentially in 2XSSC for one hour at 20 ° C, then in IXSSC for one hour at 20 ° C, then in IXSSC for half an hour at 37 ° C and in 0.5XSSC for half -time at 20 ° C.
  • the hybridized probe labeled with digoxigenin, is detected using an immunoenzymatic detection kit (Boehringer-Mannheim), according to the manufacturer's instructions. Control procedures are carried out in parallel, either by digestion of the mRNAs with RNase A (20 ⁇ g / ml for 30 minutes at 37 ° C.), or by competition with an excess of unlabeled probe (excess of the order of 40) in the hybridization mixture. Diluting the probe to 0.5 ⁇ g / ml gives a weak but specific signal. The absence of a signal is observed when the NGF probe is not put on during hybridization.
  • FIG. 10 illustrates the in vivo study of the expression of the NGF transgene in RBE / NGF cells, three weeks after transplantation into the nucleus basalis (basal nucleus) and
  • FIG. 11 illustrates the control brain structures used as internal control of the in situ hybridization of the NGF messenger, in vivo.
  • FIGS. 10A and 10B show a graft (G) of RBE / NGF cells strongly expressing the NGF transgene detected by in situ hybridization. This expression of the transgene remains as strong 3 weeks after the intra-cerebral transplant.
  • FIG. 10C visualizes a control graft (G) of uninfected RBE4 cells, grafted in the contralateral hemisphere, which shows no positive NGF signal (xl30 in 10A); (x270 in 10B and 10C, with transmitted interference contrast).
  • FIG. 11A illustrates the neuronal detection of NGF, in the fronto-parietal cortex and FIG. 11B illustrates the detection of NGF in the hippocampus (x260 in 11A); (x65 in 11B).
  • the modified cell line is grafted as specified above in the basal nucleus, in which the cholinergic neurons exhibit a very sensitive response to NGF .
  • These cholinergic neurons in addition to their expression of the enzyme ChAT, can also be characterized by significant immunoreactivity to the p75LNGFR receptor. The latter makes it possible to visualize the cholinergic fibers as well as their cellular somas, especially during studies of axonal regeneration.
  • the reaction budding detected by immunoreactivity p75LNGFR is observed at least up to three weeks, at the level of the RBE / NGF grafts.
  • the biological effect of NGF produced by grafts on cholinergic neurons of the basal nucleus is localized in this region and does not exceed the limits of the latter.
  • FIG. 12 illustrates the biological effect of the NGF secreted by the RBE / NGF cells, three weeks after the graft, at the level of the nucleus basalis (NB) (action on the promotion and maintenance of the reactive axonal regrowth of the cholinergic neurons damaged after transplantation).
  • NB nucleus basalis
  • FIG. 12A a general view of the form of an RBE / NGF graft, placed in the NB, is displayed, using the Hoechst premarking.
  • 12B, 12D and 12F the effect of NGF produced by endothelial cells on axonal regrowth is visualized by the strong immunoreactivity of these axonal processes for the p75 receptor for NGF. This axonal regrowth takes place all along the graft (G) and has a strong reactivity around certain blood vessels (arrows).
  • non-infected control RBE4 grafts not infected with the NGF retroviral construct, placed in the NB of the contralateral hemisphere, are unable to promote and maintain reactive axonal regrowth of NB cholinergic neurons (x65 in A, B, C, D, E and F, horizontal plane).
  • FIG. 13 illustrates the biological effect of the NGF secreted by the RBE / NGF cells, three weeks after the graft, at a distance from the nucleus basalis and illustrates the directional growth of the growing extensions of the NB cholinergic neurons, along the graft up to at the level of the dorsal striatum.
  • FIGS. 13 illustrates the biological effect of the NGF secreted by the RBE / NGF cells, three weeks after the graft, at a distance from the nucleus basalis and illustrates the directional growth of the growing extensions of the NB cholinergic neurons, along the graft up to at the level of the dorsal striatum.
  • 13A and 13B illustrate a horizontal section passing through the dorsal part of the graft in the striatum.
  • the RBE / NGF cells are visualized by the Hoechst nuclear dye.
  • 13B the same section was made in transmitted light, showing a reactive axonal regrowth visualized by the anti-p75 NGF receptor antibody (x100 in 13A and 13B).
  • a quantification of the biological effect induced by the expression of the NGF transgene was undertaken according to the method described by Gundersen HJG et al. (APMIS, 1988, 96, 379-394), by calculating the area occupied by the p75LNGFR immunostaining at the implantation sites of the RBE / NGF and RBE4 cells. The ratio of this area to that of the graft was calculated at 3 and 8 weeks post-implantation and presented in Figure 14 where the area occupied by positive p75LNGFR structures (expressed as a percentage relative to that of the graft) is plotted on the ordinate.
  • immunohistochemical labeling was carried out, using macrophage markers, the major histocompatibility complex and lymphocytes.
  • the above data show that both RBE4 cells alone, RBEZ cells and RBE / NGF cells survive and integrate after transplants.
  • the RBEZ and RBE / NGF cells are capable of expressing and / or secreting the product of said transgene which, in the case of NGF, has the capacity to induce a biological effect in the brain.

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