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WO1994003199A1 - Procede ameliorant la differentiation et les chances de survie des cellules precurseurs neuronales - Google Patents

Procede ameliorant la differentiation et les chances de survie des cellules precurseurs neuronales Download PDF

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Publication number
WO1994003199A1
WO1994003199A1 PCT/US1993/007167 US9307167W WO9403199A1 WO 1994003199 A1 WO1994003199 A1 WO 1994003199A1 US 9307167 W US9307167 W US 9307167W WO 9403199 A1 WO9403199 A1 WO 9403199A1
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fgf
cells
family
ngf
cntf
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PCT/US1993/007167
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English (en)
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Nancy Y. Ip
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority to AU49951/93A priority Critical patent/AU4995193A/en
Publication of WO1994003199A1 publication Critical patent/WO1994003199A1/fr

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    • 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
    • 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • A61K38/1825—Fibroblast growth factor [FGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins

Definitions

  • the present invention relates to a method of treating neuronal precursor cells to cause them to differentiate into neuronal-type cells. Such method is useful, e.g. to enhance the survival and functionality of cells used in striatal or other transplantation treatments or for the treatment of tumors.
  • the development and maintenance of the vertebrate nervous system depends on specific proteins, termed neurotrophic factors, originally defined by their ability to support the survival of neuronal populations (Snider and Johnson, 1989, Ann. Neurol. 2£:489).
  • Neurotrophic factors have also been implicated in processes involving the proliferation and differentiation of neurons (Cattaneo and McKay, 1990, Nature 347: 762-765; Lindsay and Harmar, 1989, Nature 337: 362-364), and they may play additional, thus far unexplored, roles both within as well as outside of the nervous system.
  • the first neurotrophic factor identified was nerve growth factor (NGF).
  • NGF nerve growth factor
  • NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons
  • NGF neurotrophic factor
  • NGF neurotrophic factor
  • HNGFR is phosphorylated on tyrosine in response to NGF, and apparently contains intrinsic tyrosine kinase activity
  • BDNF and NT-4 primarily use the trkB receptor (Squinto, et al. 1991 , Cell 65: 885-893); Ip, et al. 1992, Proc. Natl. Acad. Sci. U.SA. 89:3060- 30634) and NT-3 is the preferred ligand for trkC (Lamballe, et al. 1991 , Cell 66:967-979).
  • p75LNGFR has been shown to bind with low-affinity to all members of the neurotrophin family (Rodriguez-Tebar et al, 1990, Neuron 4: 487-492; Squinto, et al.
  • Ciliary neurotrophic factor was initially identified, purified, and moleculariy cloned based on its ability to support the survival of parasympathetic neurons from the chick ciliary ganglion neurons in vitro (Adler et al., 1979, Science 204:1434-1436; Lin et al., 1989, Science 246:1023-1025; Stockli et al., 1989, Nature 342:920-923).
  • CNTF shows no sequence homology, and does not appear to share any receptor components, with the NGF family of neurotrophic factors (Lin et al., 1989, Science 246_:1023-1025; Stockli et al., 1989, Nature 242:920- 923).
  • CNTF can enhance the survival of sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46), motor neurons
  • SUBSTITUTE SHEE (Blottner et al. 1989, Neurosci. Lett. 105:316-320) and hippocampal neurons (Ip et al 1991 , J. Neurosci. 11 : 3124-3134).
  • CNTF can inhibit proliferation and enhance cholinergic properties of neuronal precursors from the sympathetic ganglion (Ernsberger et al 1989,
  • Human CNTF has been moleculariy cloned (Masiakowski, et al.
  • LIF Leukemia Inhibitor Factor
  • Oncostatin M are broadly acting factors that can inhibit the proliferation and induce the differentiation of the murine myeloid leukemia cell line, M1 [Rose and Bruce, Proc. Natl. Acad. Sci. ££: 8641 -8645
  • FGF family growth factors known as fibroblast growth factors constitute a family of structurally related factors (the "FGF family") that are unrelated to the NGF family (the “neurotrophins”) or to the CNTF family of factors (Burgess and Maciag, 1989, Ann. Rev. Biochem. 58:575; Goldfarb, 1990, Cell
  • FGF-1 acidic fibroblast growth factor (aFGF); Burgess and Maciag, 1989, Ann. Rev. Biochem. 58:575
  • FGF-2 basic fibroblast growth factor (bFGF); Abraham, et al. 1986, EMBO J. 5:1523; Kurokawa, et al. 1987, FEBS Lett. 213:189]
  • FGF-3 acidic fibroblast growth factor (aFGF); Burgess and Maciag, 1989, Ann. Rev. Biochem. 58:575
  • FGF-2 basic fibroblast growth factor (bFGF); Abraham, et al. 1986, EMBO J. 5:1523; Kurokawa, et al. 1987, FEBS Lett. 213:189]
  • FGF-2 FGF-4
  • HHT FGF-4
  • FGF-7 keratinocyte growth factor (KGF); Finch, et al. 1989, Science 245:752
  • Acidic and basic fibroblast growth factor (aFGF and bFGF, respectively) in addition to their potent mitogenic properties for a variety of cells of mesodermal origin, support the survival of a number of neuronal cells (Walicke et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:3012-3016; Morrison et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7537-7541 ; Hatten et al., 1988, Dev. Biol. 125:125:280-289) including chick parasympathetic ganglion neurons.
  • the sympathoadrenal sublineage of the neural crest include the neurons of the sympathetic ganglia, SIF (small intensely florescent) cells and the chromaffin cells of the adrenal medulla (Landis and Patterson, 1981 , Trends Neurosci. 4:
  • SUBSTITUTE SHEET (Doupe, et al. 1985, J. Neurosci. 5:2143-2160).
  • a cell identified in primary cultures of embryonic rat adrenal glands appears to be the progenitor from which chromaffin, SIF and sympathetic neurons are derived.
  • the progenitor cell Under conditions, such as in the adrenal gland, where glucocorticoids are present, the progenitor cell can develop into a chromaffin cell, which is an endocrine (generally epinephrin)-secreting cell. Under conditions where glucocorticoids are absent, progenitor cells, as well as chromaffin cells, can develop into sympathetic neurons (Anderson and Axel, 1986, Cell 47:1079-1090; Anderson, 1988, NATO ASI
  • Progenitor cells of the sympathoadrenal lineage coexpress both neuronal and chromaffin cell markers.
  • Monoclonal antibodies against sympathoadrenal antigens have been used to isolate such progenitor cells from embryonic sympathetic ganglia
  • Immortalized cell lines have been established from rat sympathoadrenal progenitor cells. (Birren and Anderson, 1990; Neuron 4:189-201.) These cell lines are known as MAH (v- myc infected, adrenal-derived, HNK-1 -positive) cells (HNK -1 is a specific cell surface antibody that, within the developing adrenal gland, exclusively labels tyrosine hydroxylase positive cells of the sympathoadrenal lineage).
  • MAH v- myc infected, adrenal-derived, HNK-1 -positive cells
  • NGF in combination with a factor in heart cell- conditioned medium can induce mature chromaffin cells to differentiate into sympathetic neurons (Unsicker et al, 1978; Proc. Natl. Acad. Sci. U.S.A. 75:3498-3502; Doupe et al, 1985, J. Neurosci. 5:2119-2142). NGF also causes neuronal differentiation
  • Acidic FGF and bFGF induce similar responses to NGF in PC12 cells, including neurite outgrowth and the induction of neuron-specific mRNAs (Leonard, et al, 1987, Mol. Cell. Biol.
  • bFGF will induce cell division and neurite outgrowth from cultured neonatal rat adrenal chromaffin cells; the neurite outgrowth, but not the proliferation is inhibited by dexamethasone (Stemple, et al. 1988, Neuron 1 :517-525).
  • dexamethasone Step, et al. 1988, Neuron 1 :517-525.
  • FGF or some other similarly acting factor is the initial determinant of neuronal differentiation in the sympathoadrenal lineage, and that developing neurons acquire a dependence on NGF as they differentiate, as reflected in the induction of NGF receptor gene expression by FGF (Birren and Anderson, 1990;id).
  • FGF induces NGF dependence, this only occurs in a small portion of FGF treated MAH cells.
  • FGF promotes cell growth and survival of MAH cells in the presence of dexamethasone; removal of dexamethasone results in the death of MAH cells, although a very small percentage of the FGF treated cells can survive and differentiate in the absence of dexamethasone but in the presence of NGF.
  • SUBSTITUTE SHEET Parkinson's disease results from the degeneration of a discrete population of neurons located in the substantia nigra of the brain. Neurons in the substantia nigra produce the neurotransmitter dopamine, and form synapses with neurons in the striatum. Loss of dopamine in the striatum results in symptoms characteristic of Parkinson's, such as tremors, rigidity, difficulty in standing and slowness of movement.
  • Parkinson's has thus far been based on either treatment with the dopamine precursor L-dopa, or replacement of dopamine producing cells.
  • tissue is generally derived from fetal CNS tissue or from the adrenal gland.
  • transplantation into the substantia nigra requires the growth of axons through the brain to connect with their targets in the striatum; such growth is unlikely in the adult brain.
  • dopamine-producing cells are generally grafted closer to their targets in the striatum.
  • Chromaffin cells of the adrenal gland secrete catecholamines. Maintenance of their endocrine phenotype in the adrenal gland is under the control of the glucocorticoids. Chromaffin cells cultured in the absence of glucocorticoids are known to be able to differentiate into a neuronal-type phenotype, thus suggesting their use in transplants to innervate brain tissue (Olson, L. 1970. Histochemie 22:1-7; Olson, et al. 1980, Exp. Neurol. 70: 414-426). CNTF also promotes process outgrowth from chromaffin cells (Shults, 1992, "Parkinson's Disease"
  • the object of the present invention is to provide a method of producing sympathetic neurons from neuronal precursor cells.
  • a further object of the present invention is to provide a method of producing adrenal gland derived dopaminergic neurons.
  • Another object of the present invention is to enhance the differentiation and survival of cells used for transplantation treatment of Parkinson's disease, as well as other diseases or disorders that can be treated using transplantation therapy.
  • Another object of the invention is to provide a method of causing the differentiation of tumor cells, thereby inhibiting or retarding their growth.
  • the present invention provides a method of effecting the growth, differentiation and survival of neuronal precursor cell lines by treating such cells with a combination of a member of the FGF growth factor family in combination with one or more members
  • SUBST/TUTE SHEET of the NGF family or the CNTF family of factors are used in the context of therapies for the treatment of diseases which involve loss or a defect in neuronal tissue, such as Parkinson's disease or Alzheimers.
  • cells derived from the adrenal gland are treated with a combination of basic fibroblast growth factor and ciliary neurotrophic factor to cause such cells to differentiate and, concomitantly, become dependent on nerve growth factor.
  • Such cells are therapeutically useful for the treatment of, for example, Parkinson's disease.
  • the method of the present invention is used to treat tumor cells by promoting their differentiation and inhibiting their growth.
  • FIG. 1 Cell growth of MAH cells treated with CNTF, FGF or both factors. Open circles indicate control (untreated) cells; closed squares indicate the effect with treatment of CNTF alone; open squares indicate treatment with FGF alone; closed triangles indicate treatment with CNTF and FGF in combination.
  • MAH cells were incubated in the presence of dexamethasone (Control), plus CNTF alone, FGF alone, or a combination of both factors (CNTF+FGF) for a period of 4 days (top row). Following such a pretreatment period of 7 days, cells were washed free of the factors and dexamethasone, and NGF was added for the rest of the culture period (bottom row).
  • FIG. 3 CNTF, FGF and NGF collaborate to make postmitotic neurons that are neurofilament-immunopositive.
  • A. MAH cells were pretreated with CNTF and FGF for 1 week, and were then washed free of the factors and dexamethasone. The cells were then cultured in the presence of
  • NGF for 1 week to 5 weeks. The pictures were taken from the same field at the various times indicated to demonstrate these cells are postmitotic.
  • SUBSTITUTE SHEET B Neurofilament staining was performed on MAH cells at the end of a 7 day pretreatment with CNTF, FGF or both factors, or followed the addition of NGF.
  • MAH cells were treated with CNTF, FGF or both factors for 7 days, and gene expression for LNGFR, trkA and c-myc were examined.
  • the present invention is based, in part, on the discovery by applicants that the treatment of neuronal precursor cells with a combination of members from the structurally and functionally unrelated neurotrophic factor and growth factor families results in the differentiation of the cells to a neuronal, NGF-dependent phenotype.
  • neuronal precursor cells are treated with a combination of one or members of the FGF family selected from the group consisting of FGF-1 , FGF-2,
  • FGF-3, FGF-4, FGF-5, FGF-6 and FGF-7 in conjunction with one or more factor from the CTNF family, which includes CNTF, Oncostatin M and LIF, or from the NGF-family, which includes NGF, BDNF, NT-3, and NT-4 .
  • CTNF family which includes CNTF, Oncostatin M and LIF
  • NGF-family which includes NGF, BDNF, NT-3, and NT-4 .
  • the member of the FGF, CNTF or NGF family used may consist of the mature protein, or an analog or biologically active derivative thereof.
  • Members of a family are those factors that share either a common receptor component, such as CNTF and LIF, or members that share a common receptor.
  • Neuronal precursors include any cell capable of differentiating into a neuronal type cell.
  • neuronal precursors include both progenitor cells, as well as cells such as, for example, chromaffin cells and SIF cells, which, although not considered progenitor cells for sympathetic neurons, can be converted into neuronal type cells given the appropriate signals.
  • Neuronal precursors include immortalized human or animal cell lines or genetically engineered cells capable of differentiating and expressing a neuronal phenotype in response to a neurotrophic or growth factor.
  • Neuronal precursors may be isolated, for example, from embryonic ganglia using specific markers such as tyrosine hydroxylase or by isolating cells that display both neuron- and chromaffin-cell specific markers (Carnahan and Patterson, 1991 ,
  • ciliary neurotrophic factor is used, in combination with FGF, to promote the differentiation of adrenal gland derived precursor cells, such as MAH cells.
  • Co- treatment with these factors results in conversion of a large number of cells to a neuronal phenotype, as measured by expression of neurofilament.
  • cotreatment of the cells results in the marked induction of trkA and p75LNGFR and concomitant reduction in c-myc expression, indicating enhanced receptivity of the cells to ⁇ GF.
  • Cells treated according to the invention may be used, for example, in place of fetal cells or adrenal chromaffin cells, in transplantation therapy for Parkinson's disease.
  • Transplanted chromaffin cells are known to be able to differentiate into a neuronal-type phenotype and to innervate brain tissue (Olson, L. 1970. Histochemie 22:1-7; Olson, et al. 1980, Exp. ⁇ eurol. 70: 414-426).
  • administration of ⁇ GF, in conjunction with adrenal medullary implant is known to lead to better cell survival and more functional effect (Strombert, et al. 1985. Exp. Brain Res. 60:335-349; Strombert, et al. 1985, Cell Tissue Res.
  • Cells pretreated according to the present invention would express ⁇ GF binding receptors upon transplantation, and, therefore would be expected to have a better chance of surviving within the striatal environment. Accordingly, the present invention contemplates pretreatment of C ⁇ TF/bFGF treated cells with ⁇ GF prior to transplant, or co-injection of ⁇ GF
  • MAH cells were maintained in culture as previously described (Birren et. al., 1990, Neuron 4: 189-201 ). Briefly, cells were plated onto dishes precoated with poly-D-lysine (100 microgram/ml) and laminin (10 microgram/ml), at a low density of 5,000/35mm well. The medium used was modified L15-C02 medium supplemented with 10% fetal bovine serum (FBS) and dexamethasone ( ⁇ micromolar). Cells were treated with FGF (10ng/ml) alone, CNTF (10ng/ml) alone, or a combination of the two factors (10ng/ml) for 7 days in the presence of dexamethasone. the cells were then washed free of factors and dexamethasone, and maintained in the presence of NGF for 1-5
  • 3H-thvmidine incorporation assay and cell counts For 3H-thymidine incorporation assay, MAH cells were treated with CNTF, FGF, or both factors for 1-4 days, and 3H- thymidine (NEN-NET-027E purchased from New England Nuclear, MA) was added at a final concentration of 1 microcurie/ml and incubated for 4 hours at 37°C. Cells were then washed three times with phosphate-buffered saline (PBS), lysed with NaOH
  • neurofilament staining cells were rinsed, and blocked with normal serum prior to incubation with primary antibody specific for neurofilament (RT97, 1 :1000 dilution) at 4°C overnight. Cells were then incubated with the biotinylated secondary antibody, followed by avidin:Biotin:Peroxidase complex, and nickel-sulfate intensification of DAB reaction.
  • RNA samples were plated at a density of 100,000- 300,000 on 100mm dishes, and treated with CNTF, FGF, or both factors for 7 days.
  • Total RNA was prepared by the guanidinium thiocyanate method (Chomczynski, 1987, Anal. Biochem. 162: 156-159).
  • Ten micrograms of RNA from each sample were electrophoresed on a formaldehyde agarose gel, transferred to a nylon membrane (MSI), and hybridized to 32P-probes labelled by random oligo-priming (Stratagene).
  • the probes used included p75LNGFR, trkA and c-myc.
  • SUBSTITUTE SHEET an antibody specific for neurofilament revealed that untreated or CNTF treated MAH cells did not express neurofilament. In contrast, a very small percentage of FGF treated MAH cells expressed neurofilament protein, whereas a substantial portion of MAH cells treated with both FGF and CNTF displayed neurofilament expression. Similarly, examination of a number of relevant gene markers revealed that co-treatment with both CNTF and FGF resulted in marked induction of trkA and P75LNGFR mRNA, and a concomitant reduction in c-myc expression (Figure 4). Analysis of these protein and RNA markers defines changes in gene expression which distinguish FGF/CNTF co-treatment from treatments involving either factor alone. Thus combined treatment with FGF/CNTF resulted in inhibition of proliferation as well as enhanced neuronal phenotypic expression in an adrenal gland derived precursor cell.

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Abstract

Un procédé permet d'améliorer la différentiation des cellules précurseurs neuronales en cellules neuronales dépendantes du NGF (facteur de croissance des nerfs). Ce procédé consiste à traiter lesdites cellules précurseurs avec une combinaison d'un membre de la famille FGF (facteur de croissance des fibroblastes) et d'un membre de la famille CNTF (facteur neurotrophique ciliaire) pour renforcer leur efficacité et leur chances de survie dans une thérapie de transplantation destinée au traitement de maladies telles que la maladie de Parkinson.
PCT/US1993/007167 1992-08-04 1993-07-30 Procede ameliorant la differentiation et les chances de survie des cellules precurseurs neuronales WO1994003199A1 (fr)

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AU49951/93A AU4995193A (en) 1992-08-04 1993-07-30 Method of enhancing differentiation and survival of neuronal precursor cells

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US07/925,556 1992-08-04

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