JP2003512841A - Human and rat FGF-20 genes and gene expression products - Google Patents

Abstract

(57) Summary The present invention relates to human fibroblast growth factor (hFGF-20), a variant thereof, and a polynucleotide encoding FGF-20. The invention also relates to diagnostic and therapeutic agents (including probes and antibodies) related to polynucleotides or proteins, methods of treating neuronal degenerative diseases such as Parkinson's disease, and cochlear disorders, including disorders that cause hearing impairment. A method of treating. The present invention also relates to rat fibroblast growth factor (rFGF-20), and to variants and polynucleotides encoding rFGF-20.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to nucleic acid sequences encoding members of the fibroblast growth factor (FGF) family and to polypeptides encoded by the nucleic acid sequences.

BACKGROUND OF THE INVENTION The substantia nigra is an area of intensive study in the brain. Interest in the substantia nigra was originally based on the finding that degeneration of dopaminergic neurons in this area results in Parkinson's disease. Furthermore, the substantia nigra was strongly involved in thought and affective disorders (1). Thus, neurotrophic factors for dopaminergic neurons in the substantia nigra are substantial clinical goals.

Glial cell line-derived neurotrophic factor (glial cell line-deri)
Ved neurotrophic factor (GDNF) is the first neurotrophic factor demonstrated to enhance survival of midbrain dopaminergic neurons (Lin, L.-F.H. et al., Science 260: 1130-11).
32 (1993)). Persepin, Artemin
emin), BDGF and NT-3 also enhance survival of midbrain dopaminergic neurons and have clinical potential in the treatment of Parkinson's disease (Milbrandt, J. et al., Neuron 20: 245-253 ( 199
8); Baloh, R .; H. Et al. , Neuron 21: 1291-1302 (
Hyman, C, et al., Nature 350: 230-232 (19).
91); Hyman, C et al. Neurosci. 14: 335-347 (1
994)). However, GDNF has been reported to be widely expressed in brain neurons (Pochon, NA et al., Eur. J. Neurosci.
9: 463-471 (1997)). Persepine was also widely expressed in several major tissues, including heart, kidney, liver and brain (Milbrand.
t, J .; Et al., Neuron 20: 245-253 (1998)). In the brain, artemin is expressed in the basal ganglia and thalamus, suggesting that artemin affects the subcortical motor system (Baloh, RH et al., Neuron.
21: 1291-1302 (1998)). BDNF and NT-3 were predominantly expressed in the hippocampus (Ernfors, P et al., Neuron 5: 511).
~ 526 (1990)). Therefore, these neurotrophic factors do not appear to be specific for dopaminergic neurons in the substantia nigra.

Prototype fibroblast growth factor (FGF), FGF-1 (aFGF) and FGF-2 (bFGF) were originally isolated from the brain and pituitary as mitogens for fibroblasts. However, FGF-1 and FGF-2 are widely expressed in developing and adult tissues and have multiple biological activities including angiogenesis, mitogenesis, cell differentiation and repair of tissue injury. (Baird, A et al., Cancer Cells 3: 239-243 (19).
91); Burgess, W .; H. Et al., Annu. Rev. Biochem. 5
8: 575-606 (1989)). According to published literature, the FGF family now consists of at least 19 members of FGF-1 to FGF-19 (Dickson, C, et al. Ann. NY Acad. Sci. 638: 18-2).
6 (1991); Yoshida, T. et al., Ann. NY Acad. Sci. 6
38: 27-37 (1991); Goldfarb, M et al., Ann. NY Ac
ad. Sci. 638: 38-52 (1991); Coulier, F et al., An.
n. NY Acad. Sci. 638: 53-61 (1991); Aarons.
on, S.S. A. Et al. , Ann. NY Acad. Sci. 638: 62-77 (
1991); Tanaka, A. et al. , Proc. Natl. Acad. Sci.
USA 89: 8928-8932 (1992); Miyamoto, M et al., M.
ol. Cell. Biol. 13: 4251-4259 (1993); Yama
saki, M. et al. J. Biol. Chem. 271: 15918-15921
(1996); Smallwood. P. M. Et al. , Proc. Natl. Ac
ad. Sci. USA 93: 9850-9857 (1996); McWhir.
ter, J. R. Et al., Development 124: 3221-3232 (
1997); Miyake, A. et al., Biochem. Biophys. Res.
Commun. 243: 148-152 (1998); Hoshikawa, M.
Et al., Biochem. Biophys. Res. Commun. 244: 187
~ 191 (1998); Ohbayashi, N. et al. J. Biol. Chem
． 273: 18161-18164 (1998); Nishimura, T et al.
Biochim. Biophys. Acta 1444: 148-151 (19
99)). FGF-3 was identified to be a common target for activation by the mouse mammary tumor virus (Dickson, C. et al., Ann. NY Aca).
d. Sci. 638: 18-26 (1991)). FGF-4 to FGF-6 are
Was identified as an oncogene (oncogene) product (Yoshida, T. et al., A.
nn. NY Acad. Sci. 638: 27-37 (1991); Goldf.
arb, M et al. , Ann. NY Acad. Sci. 638: 38-52 (19
91); Coulier, F. et al., Ann. NY Acad. Sci. 638: 5
3-61 (1991)). FGF-10 was identified from rat lung by the homology-based polymerase chain reaction (PCR) (Yamasaki, M et al., J. B.).
iol. Chem. 271: 15918-15921 (1996)). FGF-
11 to FGF-14 (FGF homologous factors (FHF) 1 to 4) are random cDNAs.
Identified from human retina by a combination of sequencing, database searching and homology-based PCR (Smallwood, PM et al., Proc. Natl. A.
cad. Sci. USA 93: 9850-985 (1996)). FGF-1
5 was identified as a downstream target of the chimeric homeodomain oncoprotein (M
cWhirter, J .; R. Et al., Development 124: 3221-
3232 (1997)). FGF-16, FGF-17, and FGF-18 were identified from rat heart and embryo, respectively, by homology-based PCR (
Miyake, A et al., Biochem. Biophys. Res. Commun
． 243: 148-152 (1998); Hoshikawa, M. et al., Bioc.
hem. Biophys. Res. Commun. 244: 187-191 (1
998); Ohbayashi, N. et al. Biol. Chem. 273: 18
161-18164 (1998)). Recently, FGF-19 was identified from human fetal brain by database search (Nishimura, T. et al., Bioch.
im. Biophys. Acta 1444: 148-151 (1999)).
They have a conserved about 120 amino acid residue core with about 30-60% amino acid identity. These FGFs also appear to play important roles in both developing and adult tissues. Therefore, there is a need in the art for additional FGF molecules that have different functions and activities than known FGFs, and for FGF molecules that are specifically expressed in regions of the brain associated with human disease.

SUMMARY OF THE INVENTION The present invention provides the following: (a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID NO: 1 or 3; (b) a variant of the polypeptide encoded by (a) A coding polynucleotide; and (c) a polynucleotide that encodes a protein expressed by the polynucleotide having the sequence of SEQ ID NO: 1 or 3, an isolated polynucleotide selected from the group consisting of: provide.

The invention further provides the use of the isolated polynucleotides or fragments thereof as diagnostic probes or primers.

The invention also includes the following: (a) a polypeptide comprising at least 6 consecutive amino acids encoded by SEQ ID NO: 1 or 3; (b) a polynucleotide encoded by a polynucleotide comprising SEQ ID NO: 1 or 3. A composition comprising: a peptide; and (c) a variant of the polypeptide of (a) or (b), a polypeptide selected from the group consisting of:

The polypeptides of the present invention are shown in SEQ ID NOs: 2 and 4. Other polypeptides include fragments of SEQ ID NOs: 2 and 4.

In certain preferred embodiments of the invention, the polynucleotide is operably linked to expression control sequences. The invention further provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with this polynucleotide sequence. The present invention also relates to full length cDNA, corresponding to SEQ ID NO: 1 or 3.
A and full length polynucleotides are provided.

The protein and polypeptide compositions of the present invention may further include a pharmaceutically acceptable carrier. Compositions comprising antibodies that specifically react with such proteins or polypeptides are also provided by the present invention.

The present invention also provides for the isolation of rare molecules expressed in progenitor cells or progeny, for use in the generation of cDNA libraries, of large or otherwise small cell populations of cells. Provide production; cells produced by the treatment can directly express growth factors or other molecules, and conditioned medium is screened in an assay for novel activity.

The present invention further comprises the isolation, self-renewal and survival of mammalian neural stem cells,
As well as the differentiation of their progeny.

The invention also provides compositions and methods that interfere with or delay the degeneration or increase the number of dopaminergic neurons, such as in the substantia nigra, in disease states including Parkinson's disease. .

The present invention further provides compositions and methods that interfere with, delay or enhance the degeneration of cells in the inner ear.

DETAILED DESCRIPTION OF THE INVENTION By virtue of its potent activity to promote growth, proliferation, survival and differentiation of a wide variety of different cell and tissue types, FGF is responsible for a number of different indications. It is being pursued as a therapeutic factor. These applications (symptoms) include musculoskeletal conditions (for example, bone fracture, ligament and tissue repair, tendonitis, bursitis); skin condition (
For example, burns, wounds, lacerations, pressure sores, slow healing ulcers); wound healing, such as tissue protection and repair during myocardial infarction and ischemia, neurological conditions (eg, neurodegenerative diseases and stroke) In treatment, in the treatment of ophthalmic diseases including macular degeneration, and the like.

The fibroblast growth factor (FGF) proteins identified to date belong to a family of signaling molecules that regulate growth and differentiation of various cell types. The importance of FGF proteins to human physiology and pathology relates, in part, to its important role in embryogenesis, vascular development and growth, and bone growth. In vitro experiments have demonstrated a role for FGF in regulating cell proliferation and division of endothelial cells, vascular smooth muscle cells, fibroblasts, and myocardial and skeletal muscle cells. Other members of the FGF family and their biological role are Crossle
y et al., Development 121: 439-451 (1995); Ohu.
chi et al., Development 124: 2235-2244 (1997).
Germel et al. , Genomics 35: 253-257 (1996);
And Ghosh et al. , Cell Growth and Different
iation 7: 1425-1434 (1996).

The FGF protein is also important for human health and disease by virtue of its role in cancer cell growth. For example, FGF-8 has been identified as an androgen-induced growth factor in breast and prostate cancer cells (Tanak).
a et al., FEBS Lett. 363: 226-230 (1995) and P.P. N
． A. S. 89: 8928-8932 (1992)).

The role of FGFs in normal development is becoming partly clear through studies of FGF reporters. Wilke, T. et al. , Dev. Dynam. 210: 41
˜52 (1997) shows that FGFR1, FGFR2, and FGFR3 transcripts are
It was found that during embryonic development in chickens, it was localized to specific areas of the head. This expression pattern was associated with regions affected by human FGFR mutants in Crouzon syndrome, a condition of abnormal intramembranous bone formation. Bellu
Ardo, N. et al. , Jour. Comp. Neur. 379: 226-246 (
1997) studied the localization of FGFR1, 2 and 3 mRNA in rat brain and found cell specificity in several brain regions. Furthermore, FG
FR1 and FGFR2 mRNAs are expressed in astroglial-reactive cells after brain lesions, supporting a role for specific FGFs in brain disease and injury. Ozawa, K. et al. , Mol. Brain Res. 41: 279-2
88 (1996) increased FGF1 and FGF-5 expression postnatally, while F
It was reported that the GF-3, FGF-6, FGF-7 and FGF-8 genes show higher expression in the late embryonic stage than in the postnatal stage.

Described herein is a new member of the FGF family, wherein the FGF protein is present in rat substantia nigra dopaminergic neurons of the rat embryo.
And expressed in cochlear tissue. The polynucleotide encoding rat FGF of the present invention has the sequence shown in SEQ ID NO: 1. The polynucleotide encoding human FGF of the present invention has the sequence shown in SEQ ID NO: 3. This rat polynucleotide contains a conserved region throughout the amino acid sequence and the known F
The region of homology shared by the polynucleotide and gene encoding the GF protein was identified as encoding a member of the FGF family.

The inventors believe that FGF-20 is a previously unidentified member of the FGF family. To date, over 19 human FGF proteins have been identified. In most cases, homologous proteins in other mammals, especially mice and rats, have also been identified. Human proteins vary to different extents with respect to amino acid sequence, receptor specificity, tissue expression pattern, and biological activity.

The FGF-20 of the present invention differs in sequence from all FGF proteins described in the publications to date. FGF-20 shares the same homology with FGF-9 and FGF-16.

As discussed herein, knowledge about the role played by various FGF proteins continues to grow, but is not yet complete.

The present invention provides that the FGF of SEQ ID NO: 1 is highly expressed in the substantia nigra dopaminergic neurons of the brain, and that human FGF-20 is involved in the development of neurological disorders (eg Parkinson's disease). And adds to this knowledge by disclosing that it may play a role in recovery from neurological disease. FGF-20 is also preferentially expressed in rat embryo (E14.5) cochlea of the inner ear.

The present invention is therefore based on the identification, isolation, sequencing and expression pattern of a new fibroblast growth factor (FGF-20).

Isolation and Analysis of Rat cDNA Encoding FGF-20 Members of the FGF family have a conserved core of about 120 amino acid residues, which has about 30-70% amino acid identity. Have. Among the members of the FGF family, FGF-9 and FGF-16 are highly homologous (73% amino acid identity). According to the present invention, a DNA encoding a novel rat FGF has been identified. The nucleotide sequence of the entire coding region was identified by adapter ligation mediated polymerase chain reaction using rat brain cDNA as template and by cassette ligation mediated polymerase chain reaction using rat genomic DNA as template. The nucleotide sequence of the coding region allowed elucidation of the complete amino acid sequence of FGF (212 amino acids) with a conserved amino acid residue core (amino acids 62-197) (Fig. 1). Two cysteine residues that are well conserved in the FGF family are also conserved in this protein (amino acids 71 and 137) (Figure 1). This protein is tentatively named FGF-20. FGF-20 is most similar to FGF-9 and FGF-16 among the 19 members of the FGF family (respectively,
70% amino acid identity and 62% amino acid identity) (Figure 1). The unambiguous evolutionary tree of 20 members of the FGF family is shown in FIG. FGF-20 is FGF
Most closely related to -9 and FGF-16.

(Expression of FGF-20 mRNA in rat tissue) FGF-9 and FG
F-16 mRNA is preferentially expressed in rat kidney and heart, respectively (Miyamoto, M. et al., Mol. Cell. Biol. 13: 425).
1-4259 (1993); Miyake, A. et al. , Biochem. Biop
hys. Res. Commun. 243: 148-152 (1998)). FG
Expression of F-20 mRNA was analyzed by the polymerase chain reaction in brain, heart, lung,
It was tested in major tissues of adult rats including liver, kidney, and small intestine. FGF-
20 mRNA was detected in brain but was undetectable or only present at very low levels in other tissues. FGF-20 in rat brain
To confirm the expression of mRNA, rat brain poly (A) ⁺ RNA was analyzed by Northern blot analysis using a ³² P-labeled rat FGF-20 cDNA probe.
Tested. A faint but clear signal of FGF-20 mRNA was detected (Fig. 4). To confirm the integrity of poly (A) ⁺ RNA, the hybridized probe was washed from the membrane and the membrane was rehybridized with the ³² P-labeled rat β-actin cDNA probe. A strong and discrete signal of β-actin mRNA was detected, indicating that poly (A) ⁺ RNA was not degraded (FIG. 4). ^{Using 32} P-labeled FGF-20 antisense and sense cRNA probes, FGF-20 mRNA was also detected in rat embryos (E14.5), especially the cochlea of the inner ear (FIG. 10).

(Expression of FGF-20 mRNA in rat brain) FGF in rat brain
To test expression of -20 mRAN, serial coronal sections of rat brain were analyzed by in situ hybridization with ³² S-labeled antisense or sense FGF-20 cRNA probe. Specific labels separated only in the dense part of the substantia nigra were observed (Fig. 3A, C). No specific labeling was observed in other brain regions tested. Cellular localization of FGF-20 mRNA was examined microscopically at high magnification. By Nissle staining of brain sections, glial cells can be identified as small, strongly stained (dark) cells, whereas neurons are generally larger and their volume is less intense (bright) staining. Gerfen, C
． R. , Methods in Neurosciences, Academi
c Press, San Diego, CA, Vol. 1, pp. 79-97 (198).
9)). Labeled probe black grains were found in most neurons in these brain regions (FIG. 3E). In the substantia nigra dopaminergic neurons are preferentially localized to the substantia nigra compact (Fallon, JH et al., The).
Rat Mervous System, Second Edition. , Academic Pr
ess, San Diego, CA, pages 215-238 (1995)). Furthermore, neurons in the substantia nigra compact are preferentially composed of dopaminergic neurons (Fallon, JH et al., The Rat Nervous).
System, Second Edition. , Academic Press, San Diego
, CA, pp. 215-238 (1995)). FGF-20 is expected to be preferentially expressed in dopaminergic neurons in the substantia nigra pars compacta.

Preparation of Recombinant Rat FGF-20 To produce recombinant rat FGF-20, rat FGF-with 3'end extensions encoding E and His ₆ tags was generated.
Using a recombinant baculovirus containing 20 cDNA, HighFiv
e Insect cells were infected. This medium was tested by Western blot analysis with anti-E-tag antibody to detect recombinant FGF-20 in the culture medium. 26
． A major band of 5 kDa was detected in the culture medium. The observed molecular weight of the major band was consistent with the calculated molecular weight of recombinant FGF-20 (26,247).
. This result indicates that FGF-20 is secreted, but hydrophobicity index plot analysis (
Nielsen, H et al. , Protein Engineering 10: 1
-6 (1997)) showed low values in the amino terminal region of FGF-20, suggesting that FGF-20 has no signal sequence. FGF-
9 and FGF-16 do not have a typical signal sequence at the amino terminus,
FGF-9 and FGF-16 are also secreted (Miyamoto, M et al.,
Mol. Cell. Biol. 13: 4251-4259 (1993), Miy.
ake, A. et al. , Biochem. Biophys. Res. Commun. Two
43: 148-152 (1998)). Recombinant FGF-20 was purified from the culture medium by affinity chromatography using Ni-NTA agarose and analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. A 26.5 kDa protein of FGF-20 was detected.

(Neurotrophic activity of FGF-20 on rat midbrain dopaminergic neuron FGF) FGF is a local signaling molecule that acts on proximal cells (B
Urgess, W.A. H. Et al., Annu. Rev. Biochem. 58: 575
606 (1989)). Therefore, FGF-20 is expected to act on dopaminergic neurons in the substantia nigra in an autocrine and / or paracrine fashion. FGF − for cultured rat midbrain dopaminergic neurons
Twenty neurotrophic activities were tested. When dopaminergic neurons were cultured in serum-free medium for 4 days, the number of surviving dopaminergic neurons was greatly reduced. FGF-20 significantly enhanced survival of dopaminergic neurons in serum-free medium (Fig. 5A). The effect of FGF-20 on glutamate-induced neuronal death in cultured rat midbrain dopaminergic neurons was also tested. When cultured cells were exposed to 1 mM glutamate for 10 minutes, the number of surviving dopaminergic neurons was reduced. FGF-20 also significantly enhanced survival of dopaminergic neurons exposed to toxic concentrations of glutamate (
FIG. 5B).

It is expected that some FGFs are expressed in the brain and play important roles as neurotrophic factors. FGF-1 and FGF-2 are abundant in the brain (Gospodarowicz, D., Methods Enzymol. 1).
47: 106-119 (1987)) and exert a survival enhancing effect on primary cultures from various regions of the brain (Walicke, PA, J. Neuros).
ci. 8: 2618-2627 (1988)). FGF-1 is predominantly expressed on motor and sensory neurons in the midbrain and brainstem (Elde, R et al.
, Neuron 7: 349-364 (1991)). In contrast, FGF-2 is preferentially expressed in neurons in a limited area (including cingulate cortex, industium grieum, gray follicles and hippocampus), and in astrocytes in extensive areas of the brain. (Emoto, N et al., Growth Factors 2: 211-.
29 (1989); Woodward, W .; R. Et al. J. Neurosci.
12: 142-152 (1992)). FGF-5 is weakly expressed in the cerebral cortex, hippocampus and thalamus (Haub, O et al. Proc. Natl. Acad.
Sci. USA 87: 8022-8026 (1990)). FGF-9 and FGF-11 to FGF-14 are expressed in a limited area of neurons including the hippocampus, thalamus, midbrain and brainstem (Yamamoto, S. et al., Biochim. B).
iophys. Acta 1398: 38-41 (1998)). In contrast, FGF-20 of the present invention was preferentially expressed in substantia nigra dopaminergic neurons. The expression profile of FGF-20 is quite different from that of other FGFs, indicating that FGF-20 plays a unique role in the brain.

Degeneration of dopaminergic neurons in the substantia nigra causes Parkinson's disease (
Fallon, J .; H. Et al, The Rat Nervous System,
Second Edition, Academic Press, San Diego, CA, 215-
238 (1995)). Thus, neurotrophic factors for dopaminergic neurons in the substantia nigra have received considerable attention. GDNF, Persephin (Pers
ephin), Artemin, BDNF, and NT-3 are
Increases survival of midbrain dopaminergic neurons (Lin, L.-F.H. et al., Science 260: 1130-1132 (1993); Milbran
dt, J.D. Et al., Neuron 20: 245-253 (1998); Baloh.
R.K. H. Et al., Neuron 21: 1291-1302 (1998); Hym.
an, C.I. Et al., Nature 350: 230-232 (1991); Hyma.
n, C.I. Et al., J. Neurosci. 14: 335-347 (1994)). However, their expression is not limited to the substantia nigra (Pochon, NA et al., Eur.
． J. Et al., Eur. J. Neurosci. 9: 463-471 (1997);
Milbrandt, J .; Neuron 20: 245-253 (1998).
); Baloh, R .; H. Neuron 21: 1291-1302 (19).
98); Ernfors, P .; Neuron 5: 511-526 (199).
0)). In contrast, F also increases survival of dopaminergic neurons.
Expression of GF-20 is highly restricted to dopaminergic neurons in the substantia nigra compact. Therefore, FGF-20 is expected to play an important role as a neurotrophic factor for dopaminergic neurons in the substantia nigra. Therefore,
It is an important finding of the present invention that FGF-20 is the first neurotrophic factor demonstrated to be preferentially expressed in substantia nigra dopaminergic neurons.

Dopamine neurons are likely to be dysfunctional for many years before they are irreversibly damaged (Dunnett, S. B. et al., Nat).
ure 399: A32-A39 (1999)). Therefore, neurotrophic factors such as FGF-20 may be useful in preventing cell death or restoring function. FGF-20 can be administered using the gene transfer method to block degeneration. Such a method is used together with the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor). In the rat Parkinson's disease model, nanogram amounts of BDNF and GDNF were measured from transduced cells and the neuroprotective effect was on the order of 40-70% rescue of substantia nigra dopamine neurons. Therefore, transplantation with fibroblasts or fibroblast lines engineered to secrete FGF-20 of the present invention may allow secretion of this factor and rescue of nigral dopamine neurons. Alternatively, injection of the striatum or substantia nigra region with a viral vector carrying the FGF-20 gene may also have a neuroprotective effect.

In Parkinson's disease, neurodegeneration in the substantia nigra is generally slow and protracted. This suggests that early intervention, perhaps up to 4-5 years before the onset of clinical symptoms, may block or delay the degenerative process. Decreased striatal dopamine function can be detected by PET and SPECT prior to the onset of clinical symptoms, providing an opportunity for neuroprotective intervention at this early stage. In vivo imaging of dopaminergic activity in the basal ganglia with [ ¹⁸ F] fluorodopa PET can be used to monitor the progression of this disease and the impact of treatment. A progressive decrease in fluorodopa signal is observed in the brain tissue of presymptomatic and symptomatic individuals. This fluorodopa signal increases over time following treatment with substantia nigra tissue implants (D
unnett et al., supra). This technique, as well as others known in the art, can be used to measure the efficacy of the treatments described herein with FGF-20, and the clinician will determine the appropriate treatment level and regimen. I am a trader.

(FGF-20 Expression in Rat Fetal Cochlea) FGF-20 is preferentially expressed in the cochlea of the inner ear in the rat fetus (E).
14.5). This supports a role for FGF-20 in the development and maintenance of normal ear function. Other previously identified members of the FGF family contribute to normal ear growth and development. For example, sensory cells in rat cochlea transiently express FGF-1 during the time of terminal nerve stimulation transmission in the sensory epithelium (Daz).
ert et al. Cell Physiol. 177: 123-129 (1998)
)). These authors also found in vitro that spiral ganglia cultured in the presence of FGF-1 showed a dose-dependent increase in the number and length of neurites. In chicken cochlea, FGF-1 mRNA levels were increased in the cochlear sensory epithelium in response to ototoxic injury, suggesting that this FGF system may be associated with the response of the cochlear epithelium to ototoxic injury. Pickles et al., Dev. Ne
uroscience 19: 476-487 (1997). FGF-2 can help control the proliferative process during hair cell development and regeneration after trauma in rats. Zheng et al. Neuroscience 17: 216
-226 (1997). Thus, the FGF molecule plays several roles in maintaining normal cochlear development and function, and in recovering the cochlea from ototoxic damage. Absence of FGF receptor 3 contributes to inner ear defects in mice homozygous for skeletal and inner ear defects, including defects in columnar cell differentiation and Corti tunnel formation, and deep hearing loss. Colvin et al., Nat. Gen
et. 12: 390-397 (1996). It is interesting that FGF-20 of the present invention binds to FGF receptor 3c (Example 12). The fact that FGF-20 is expressed at specific stages in rat inner ear development further suggests its importance in the development of this tissue.

Therefore, FGF-20 may be suitable for treating various ear-related conditions. Currently, approximately 7.8 million Americans have mild hearing loss, 10 million have moderate hearing loss, and 2.7 million have deep or severe hearing loss.
Causes include, but are not limited to, otosclerosis; Cogan syndrome; Meniere's disease;
Pendred syndrome; diabetes-related deafness (non-insulin dependent diabetes mellitus with obesity can cause tissue changes in the cochlea, McQueen et al., J. L.
aryngol. Otol. 113: 113-118 (1999)); congenital malformations; autoimmune disease-related hearing loss; age-related hearing loss; hearing loss associated with lack of FGF receptors (Colvin et al. Nat. Genet. 12: 390-397 (
1996)): ischemia-related hearing loss; and other conditions in which cochlear structure and function play a role. Administration of FGF-20 protein or polynucleotide
By promoting survival, proliferation or differentiation of cells of the inner ear, it can be used to treat genetic, congenital and acquired disease disorders of hearing and balance.

References herein to FGF-20 refer to growth factors of any origin, which are substantially homologous and biologically equivalent to FGF-20 characterized and described herein. It should be construed as including. Such substantially homologous growth factors can be native to any tissue or species, and similarly biological activity can be characterized in any of a number of biological assay systems.

The term “bioequivalent” refers to the composition of the invention as isolated FGF-20 as described herein or recombinantly produced human FGF-20 of the invention. It is not necessary to be comparable, but it is intended to mean that some or all of the same growth characteristics may be presented in a similar fashion.

“Substantially homologous” means human FGF-20 relative to FGF-20 from any species.
It is meant that the degree of homology of is greater than between FGF-20 and any previously reported member of the FGF family.

Sequence identities or percent identities refer to two sequences in which Lasergene biocomputing software (DNASTAR, INC, Madiso) is used.
n, WI) multiple sequence alignment Clustal method (Higgin
S. et al., Cabios 8: 189-191 (1992)), with non-FGF-20 growth factors, referenced to human FGF when determining percent identity with non-human FGF-20. FG when determining percent identity
Referenced to F-20 is intended to mean the percentage of identical residues between two sequences. In this method, multiple alignments are performed in a progressive fashion, where larger and larger alignment groups are assembled with a similarity score calculated from a series of pairwise alignments. Optimal sequence alignment is the average of all scores between different residues in the alignment, determined from a residue weight table that represents the probability of a given amino acid change in two related proteins over a given evolutionary interval, Obtained by finding the maximum alignment score. Penalties for opening and extension gaps during this alignment contribute to the score. The default parameters used with this program are: Gap penalty for multiple alignments = 10; Gap length penalty for multiple alignments = 10; k-tuple value for alignment = 1; gap penalty for alignment = 3; Window value in anti-alignment = 5; diagonal saved in anti-alignment = 5. The residue weight table used in this alignment program is PAM250 (Dayhoff et al.,
Atlas of Protein Sequence and Struct
ure, Dayhoff, ed., NDRF, Washington, Vol. 5,
suppl. 3, 345, 1978).

Percentage conserved is the same as the percentage of positions where two residues represent a conservative substitution (defined in the PAM250 residue weight table as having a logarithmic difference value greater than or equal to 0.3). Calculated from the above alignment by adding the percentage of residues. Conservation refers to human FGF-20 when determining percent conservation with non-human FGF-20, and non-human FG-20
Reference is made to FGF-20 when determining percent conservation with F-20 growth factor. Conservative amino acid changes that meet this requirement are: RK; ED, YF, LM
VI and QH.

The present invention provides FGF-20 proteins or variants thereof having one or more polymers covalently attached to one or more reactive amino acid side chains. By way of example, and not limitation, such polymers are polyethylene glycol (PEG)
, Which may bind to one or more free cysteine sulfhydryl residues,
This blocks the formation of disulfide bonds and aggregates when the protein is exposed to oxidizing conditions. Furthermore, PEGylation of FGF-20 protein and / or mutein is expected to provide improved properties such as increased half-life, solubility, and protease resistance. Alternatively, the FGF-20 protein and / or mutein can be modified by covalent attachment of the polymer to free amino groups such as lysine ε or N-terminal amino groups. Suitable cysteines and lysines for covalent modification are those that are not involved in receptor or heparin binding.
In both human and rat FGF-20, the heparin binding site contains amino acids 170-186. For those skilled in the art, methods of assaying the biochemical and / or biological activity of FGF-20 will determine whether modification of a particular amino acid residue affects the activity of this protein in the desired manner. Obviously, it can be adopted.

It may be advantageous to improve the stability of FGF-20 by modifying one or more protease cleavage sites. Accordingly, the present invention provides that one or more protease cleavage sites have been modified, for example by substitution of one or more amino acids at the cleavage site to create a FGF-20 variant with improved stability.
GF-20 variants are provided. Such altered protein stability may be advantageous during protein production and / or therapeutic use.

Suitable protease cleavage sites for modification are well known to those of skill in the art and appear to vary depending on the particular application for which it is intended. For example, a typical substitution may include the substitution of lysine or arginine with other amino acids such as alanine. Loss of activity such as receptor binding or heparin binding can be tested as described herein.

FGF-20 has also been altered to fusion proteins and FGF-20 fragments and hybrids, and modified forms in which certain amino acids have been deleted or substituted, as well as one or more amino acids modified or unusual amino acids. Includes hybrids and modified forms of FGF-20, including cases and modifications such as those such as glycosylation, so long as these hybrids or modified forms retain the biological activity of FGF-20. By retaining this biological activity, it retains the same level of ability of isolated FGF-20 as described herein, or of recombinantly produced human FGF-20. It does not have to, but it does mean that neuronal survival is promoted. The fusion protein may be composed of the FGF-20 of the present invention or a fragment thereof, and the signal sequence of a heterologous protein to facilitate secretion of the protein product.

Fusion proteins comprising FGF-20 or a biologically active or antigenic fragment can be produced using methods known in the art. Such fusion proteins can be used therapeutically or can be produced to facilitate isolation and purification procedures. Histidine residues can be incorporated to allow immobilized metal affinity chromatography purification. Residue EQKLISEEDL
Contains the antigenic determinant recognized by the myc monoclonal antibody, and m
It can be incorporated to allow yc monoclonal antibody-based affinity purification. A thrombin cleavage site can be incorporated to allow cleavage of the molecule at a selected site; a preferred thrombin cleavage site consists of residues LVPRG. Purification of the molecule can be facilitated by incorporating a sequence that binds to superparamagnetic streptavidin beads, such as the residues SAWRHPQFGG. Such an embodiment is described in WO 97/25345, which is incorporated by reference.

The present invention comprises a chimeric molecule between FGF-20 and keratinocyte growth factor (KGF) (Reich-Slotky, R. et al., J. Biol. Che.
m. 270: 29813-29818 (1995)). The chimeric molecule is FGF-
20 regions and / or specific regions or fragments of KGF molecules (
For example, the FGF-20 fragment described below) may be included.

The present invention also includes fragments of FGF-20. Preferred fragments of SEQ ID NOs: 4 and 2 include: about 170 to about 186 amino acids; about 1 to about 169 amino acids; amino acids 2-21 (212 of SEQ ID NO: 2); about 1 to about 169 and about 187. To about 211 amino acids (212 of SEQ ID NO: 2), wherein amino acids about 169 and about 187 are joined by a peptide bond; and about 59 to about 193 amino acids. Such fragments can be prepared from the protein by standard biochemical methods or by expressing a polynucleotide encoding the fragment.

FGF-20 or a fragment thereof is a human serum albumin (HSA)
Alternatively, it may be produced as a fusion protein containing those portions. Such fusion constructs are suitable for enhancing expression of FGF-20 or fragments thereof in eukaryotic host cells. An exemplary HSA portion is described in US Pat.
66,883, and N-terminal polypeptides (amino acids 1-369, 1, 1) as described in publication WO 97/24445, which is incorporated herein by reference.
-419, and intermediate lengths starting with amino acid 1). Other chimeric polypeptides may include HSA proteins with FGF-20 or fragments thereof attached to each of the C- and N-termini of HSA. HS like this
The A construct is described in US Pat. No. 5,876,969, which is incorporated herein by reference.
No.

F different from native FGF-20 due to changes in the biologically active site
GF-20 molecules are also included within the scope of this invention. FGF-20 has a putative heparin binding site at amino acid residues 170-186. F that does not bind to heparin
The GF-20 molecule can be prepared by expressing DNA encoding FGF-20 with the corresponding codons deleted for amino acid residues 170-186.
Conversely, one or more additional heparin binding sites, eg, FGF-20, by expressing a DNA encoding FGF-20 in which the codon corresponding to residues 170-186 is inserted at the desired position in reading frame. Can be added to. DNA encoding FGF-20 with altered receptor binding can be similarly produced.
For example, it may be desirable to alter the receptor specificity of FGF-20 by substituting a different FGF receptor binding region for the FGF-20 receptor binding region.

FGFs herein can be isolated by cross-reactivity with antibodies to FGF-20 as described herein, or their encoding nucleotide sequences, including genomic DNA, mRNA, or cDNA. Any FGF-20 that can be isolated through hybridization of -20 or a fragment thereof with the genomic or subgenomic nucleotide sequence or the complementary sequence of the cDNA is also included within the meaning of being substantially homologous. The degenerate DNA sequence is human FGF-20.
It is also understood by one of ordinary skill in the art that, and, like the mammalian allelic variants of FGF-20, are also intended to be included within the scope of the present invention.

Growth factors are believed to act at specific receptors. According to the invention, F
The as yet unknown members of GF-20 and growth factors of this family are:
It acts through specific receptors with a well-defined distribution, as shown for other growth factor families. FGF-20 is FGF receptor 2 and F
It binds to GF receptor 3 but not to FGF receptor 1. Therefore,
Its receptor binding profile is FGF-2 and FGF-4 (these are
Binds to FGF receptor 1).

The preferred hFGF-20 of the present invention has been identified and isolated in purified form as described. HFGF-prepared by recombinant DNA technology
20 is also preferred. By "pure form" or "purified form" or "substantially purified form" is meant that the FGF-20 composition is substantially free of other proteins that are not FGF-20.

Recombinant human FGF-20 can be produced by expressing a DNA sequence encoding FGF-20 in a suitable transformed host cell. Using methods well known in the art, DNA encoding FGF-20 can be ligated into an expression vector, transformed into host cells, and conditions suitable for expression of FGF-20 by the transformed cells. Can be established.

The DNA encoding FGF-20 can be engineered to take advantage of host cell preferred codon usage. Codon usage in Pseudomonas aeruginosa is described, for example, in West et al., Nucleic Acids Res.
11: 9323-9335 (1988). Sacc
The codon usage in Haromyces cerevisiae is described, for example, in L
Lloyd et al., Nucleic Acids Res. 20: 5289-5295
(1992). Codon preference in Corynebacterium and E. coli. For comparison with E. coli priority, see Malubres et al., Gene.
134: 15-24 (1993). Drosophila
Codon usage in melanogaster is described in, for example, Akashi, G
enetics 136: 927-935 (1994). Codon usage in yeast is also shown in Figure 11, and Drosophi
Codon usage in la is shown in FIG.

Any suitable expression vector can be used to produce recombinant human FGF-20 (eg, an expression vector for use in insect cells). Baculovirus expression systems can also be used. A preferred method is expression in insect cells (eg Tr5 or Sf9 cells) using baculovirus vectors.

The present invention includes nucleic acid sequences that include sequences encoding human FGF-20. F
Sequences that are substantially the same as the nucleic acid sequence encoding GF-20 are also included within the scope of the invention. Such substantially identical sequences can be replaced, for example, with codons that are more easily expressed in a given host cell (eg, E. coli) according to well-known and standard procedures. Such modified nucleic acid sequences are included within the scope of the invention.

A particular nucleic acid sequence may be modified by one of skill in the art, and thus all nucleic acid sequences encoding the amino acid sequence of FGF-20 may be so modified as well. Accordingly, the invention also relates to a nucleic acid that hybridizes with all such nucleic acid sequences or the complement of a suitable nucleic acid sequence and that encodes a polypeptide having the neuronal cell survival promoting activity disclosed herein. Sequences are also included. The present invention also includes a nucleic acid sequence encoding a polypeptide having neuronal cell survival promoting activity, and F
Included are nucleic acid sequences that encode a polypeptide recognized by an antibody that binds GF-20. Preferred methods and epitopes for raising antibodies are described in Example 10.

The present invention also includes vectors that include expression control elements operably linked to any of the nucleic acid sequences included within the scope of the present invention. The invention also includes a host cell of any species transformed with a vector containing an expression control element operably linked to any of the nucleic acid sequences included within the scope of the invention.

Methods for producing FGF-20 are also provided herein. Preparation can be by isolation from conditioned medium from various cell types, as long as the cell type produces FGF-20. The second and preferred method is FGF-20.
By the steps of isolating or obtaining a nucleic acid sequence encoding the sequence, cloning this sequence into an appropriate vector and cell type with appropriate regulatory sequences, and expressing this sequence to produce FGF-20. Including the use of replacement methods.

Although FGF-20 has been described on the basis of its ability to enhance survival of midbrain dopaminergic neurons, the factor may act in other cell types as well. Therefore, it is considered that FGF-20 may act on other neurons.

FGF-20 is also believed to act in non-neuronal cells to promote their survival, proliferation or function. This expression is based on the activity of known growth factors. Members of the FGF family act in many cell types of different function and embryonic origin.

We herein show that FGF-20 is expressed in the brain, but not in other adult tissues, including heart, lung, liver, kidney, and small intestine. Identified. This suggests a role for FGF-20 in, for example, Parkinson's disease and other diseases of nervous tissue.

The present invention also relates to a therapeutic or pharmaceutical composition comprising FGF-20 in an effective amount for treating a patient having a neuronal disease, including Parkinson's disease, and FGF.
-20. Administering a therapeutically effective amount of -20. These compositions and methods are useful for treating numerous diseases. The compositions and methods herein may also be useful for preventing degeneration and / or promoting survival in other non-neuronal tissues as well. Those skilled in the art will appreciate that FGF-20
Various assays known in the art can readily be used to determine if is useful in promoting or functioning survival in a particular cell type (eg, neuronal cells).

In certain circumstances it may be desirable to adjust or reduce the amount of FGF-20 expressed. Therefore, in another aspect of the invention, FGF-20
Antisense oligonucleotides can be made and one or more FGF-20
Methods involving administering antisense oligonucleotides are used to reduce the expression level of FGF-20 by cells. The FGF-20 antisense oligonucleotide has a nucleotide sequence that interacts with a specific complementary nucleic acid sequence involved in the expression of FGF-20 through base pairing to reduce the expression of FGF-20. Refers to an oligonucleotide. Preferably, FGF-20
The specific nucleic acid sequence involved in the expression of is a genomic DNA molecule or mRNA molecule encoding FGF-20. The genomic DNA molecule may include the regulatory region of the FGF-20 gene, or the coding sequence for the mature FGF-20 protein. In the context of FGF-20 antisense oligonucleotides and methods therefor, the term "complementary to a nucleotide sequence" is such that it allows hybridization to that sequence in cells, ie under physiological conditions. It is meant to be sufficiently complementary to the sequence. The FGF-20 antisense oligonucleotide preferably comprises a sequence comprising about 8 to about 100 nucleotides, and more preferably the FGF-20 antisense oligonucleotide comprises about 15 to about 30 nucleotides. The FGF-20 antisense oligonucleotide also has a nucleotide degrading property.
various modifications that confer resistance to radiation (eg modified internucleoside linkages (Uhlmann and Peyman, Chemical Rev.
articles 90: 543-548 1990; Schneider and B.
anner, Tetrahedron Lett. 31: 335 (1
990), which are incorporated by reference), modified nucleobases and / or sugars, etc.).

Therapeutic or pharmaceutical compositions of the invention are for example intravenous, subcutaneous, intramuscular, transdermal,
It may be administered by any suitable route known in the art, including subarachnoidally or in the brain. Administration can be either rapid, such as by injection, or over a period of time, such as by slow infusion or administration of a slow-release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into cerebrospinal fluid (CSF). When FGF-20 is intended to be administered to cells in the central nervous system, the administration will cross the blood brain barrier.
It may employ one or more agents that can facilitate the penetration of

FGF-20 can be linked or conjugated with an agent that provides the desired pharmacological or pharmacodynamic properties. For example, FGF-20 can be coupled to any substance known in the art to promote penetration or transport across the blood brain barrier, such as an antibody to the transferrin receptor, and administered by intravenous injection. Obtain (eg Friden et al., Science 259:
373-377 (1993), which is incorporated by reference).
In addition, FGF-20 can be stably linked to a polymer (eg, polyethylene glycol) to obtain the desired properties of solubility, stability, half-life and other pharmaceutically advantageous properties. (For example, Davis et al., Enzyme Eng. 4: 1.
69-73 (1978); Burnham, Am. J Hosp. Pharm.
51: 210-218 (1994), which are incorporated by reference).

The compositions are usually used in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation uses a physiological saline solution vehicle, but other pharmaceutically acceptable carriers (eg,
Other non-toxic salts in physiological concentrations, 5% glucose in water, sterile water, etc.)
It is contemplated that can also be used. It may be desirable for a suitable buffer to be present in the composition. Such solutions can, if desired, be stored in lyophilized and sterile ampoules and are ready for reconstitution by the addition of sterile water for the prepared injection. The initial solvent may be aqueous or non-aqueous. FGF-20 can also be incorporated into a solid or semi-solid, biocompatible matrix that can be implanted in tissues in need of treatment.

The carrier may also be other pharmaceutically acceptable for modifying or maintaining pH, osmolality, clay, clarity, color, sterility, stability, degradation rate, or formulation odor. Excipients can be included. Similarly, the carrier may still include other pharmaceutically acceptable excipients to modify or maintain release or absorption or penetration through the blood brain barrier. Such excipients may be for parenteral administration, either in unit dosage form or in multiple dosage form, or for direct injection into the cerebrospinal fluid by continuous or periodic infusion. It is the substance normally or customarily used to prescribe amounts.

Dose administration can be repeated depending on the pharmacokinetic parameters of the dosage form used and the route of administration.

It is also intended that certain formulations containing FGF-20 be administered orally.
Such formulations are preferably encapsulated and formulated in a solid dosage form with a suitable carrier. Some examples of suitable carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, Gelatin, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil and the like. The formulations may additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The composition can be formulated to provide a rapid, sustained or delayed release of the active ingredient after administration to a patient by using procedures well known in the art. The formulation may also include substances that reduce proteolytic degradation and enhance absorption, such as detergents.

Depending on the intended treatment regimen, it may be desirable to control the release rate of the FGF-20 protein or variants thereof, providing long-term treatment while minimizing dosing frequency. Such a treatment regimen is, for example, where the FGF-20 protein is found to be relatively unstable, so that effective levels of localized concentration of active protein are found for a period of time that is insufficient. , May be desirable. Thus, for example, performing repeated frequent injections for a particular disease is neither desirable nor practical. The major advantages of such sustained release systems are the targeted local delivery of the drug at a constant rate, the relatively few drugs required to treat the disease state, the minimal possible side effects. And enhanced treatment effect. Also, these forms of delivery systems are unstable in vivo and may protect drugs that normally require frequent dosing intervals. Under such circumstances, sustained release is such as encapsulation of FGF-20 conjugated heparin-sepharose beads to form a preparation of heparin-alginate microspheres or FGF-20 PLG microspheres. , Can be achieved by one of the methods readily available in the art.

Heparin-alginate microspheres have been successfully used for delivery of basic fibroblast growth factor to tissues (Lopez et al., Journal of Ph).
armalogy and Experimental Therapeu
tics 282 (l): 385-390 (1997)). Similarly, alginate / heparin sepharose microspheres and films are used as drug carriers to control the release of basic FGF-saponin conjugates to control the release in small doses. Addition of heparin to a solution of bFGF avoids loss of activity with changing pH or increasing temperature. For example, Gospodarowicz et al., J.
． Cell. Physiol. 128: 475-484 (1986).

As disclosed herein, FGF-20 has a heparin binding domain at residues 170-186. Thus, binding of FGF-20 to heparin can be used to enhance its stability, either during in vivo expression or administration or during various stages of protein purification in vitro. Thus, according to the present invention, heparin can be added to FGF-20 solution and its activity assayed by the methods disclosed herein.

FGF-20-conjugated heparin-sepharose beads may be encapsulated in calcium alginate microspheres and allow for controlled release of heparin-stabilized FGF-20 protein. For example, microspheres may contain sodium alginate and FGF-
It can be constructed by dropping a mixed solution with 20 linked heparin-Sepharose beads into a hardening solution of calcium chloride. Spheres are formed instantaneously when this mixture enters the curing solution. Microsphere size can be adjusted by passing FGF-20 conjugated heparin-Sepharose beads through a reduced cross-section cylinder (eg, via a hypodermic needle).

The encapsulation efficiency can be determined by comparing the amount of growth factor encapsulated with the amount originally present in the solution. For example, FGF-20 is 3M Na
Cl solutions can be used to strip the heparin-Sepharose beads, and functional activity assays can be performed.

A particular dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space occupied. This dose will also be calculated depending on the particular route of administration chosen. Further refinement of the calculations required to determine the appropriate dosage for treatment can be tolerated by those of skill in the art. Such a calculation
In view of the activity disclosed herein in the target cell assay preparations, one of skill in the art can do without undue experimentation. The exact dosage will be determined in combination with standard dose response studies. The amount of composition actually administered will depend on the context (conditions to be treated, the choice of composition to be administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the choice of the situation). It depends on the practitioner's administration (including the route of administration).

In one embodiment of the invention, FGF-20 is FGF-20 or FG.
Transplanting a vector or cell capable of producing a biologically active form of the F-20 precursor (ie, a molecule that can be readily converted by the body into the biologically active form of FGF-20) into a patient. Can be administered therapeutically. In one approach, FGF-20 secreting cells can be encapsulated in a semipermeable membrane for implantation in a patient. The cell can be a cell that normally expresses FGF-20 or its precursor, or a cell that can be transformed to express FGF-20 or its precursor. If the patient is human, cells of human origin and FG
It is preferred that F-20 is human FGF-20. However, the formulations and methods herein can be used for veterinary as well as human applications, and the term "
"Patient" as used herein is intended to include human and veterinary patients.

The cells can be expanded ex vivo for use in transplantation or implantation in a patient (Muench et al., Leuk. & Lymph. 16: 1-11 (199).
4) (this is incorporated by reference)). In another embodiment of the present invention, F
GF-20 is used to promote ex vivo expansion of cells for transplantation or implantation. The current method uses a bioreactor culture system containing factors such as erythropoietin, colony stimulating factors, stem cell factors, and interleukins to generate hematopoietic precursors for erythrocytes, monocytes, neutrophils, and lymphocytes. Expand cells (Verfaillie, Stem Cells 12: 466-476 (
1994) (which is incorporated by reference)). These stem cells can be isolated from human donor bone marrow, human peripheral blood, or cord blood cells. Expanded blood cells are used to treat patients lacking these cells either as a result of certain disease states or as a result of high-dose chemotherapy for the treatment of malignancies (
George, Stem Cells 12 (Appendix 1): 249-255 (19).
94) (which is incorporated by reference)). In the case of post-chemotherapy cell transplantation, autologous tissue transplantation expands cells ex vivo using a method that also works to remove bone marrow cells prior to chemotherapy and to purge malignant details, and this expansion. Cells can be carried out by transplanting them back into this patient after chemotherapy (for review, see Rummel and Van Zant, J. Hemato).
therapy 3: 213-218 (1994), which is incorporated by reference). Since FGF-20 is expressed in neurons, F
It is believed that GF-20 may function to avoid or slow the degradation of dopaminergic neurons (eg, substantia nigra).

In many settings, it is desirable to determine the level of FGF-20 in a patient. The identification of FGF-20 in addition to the data herein showing FGF-20 expression provides the basis for the conclusion that the presence of FGF-20 serves normal physiological functions associated with cell proliferation and cell survival. To do. Indeed, other neurotrophic factors are known to play a role in neural and non-neural tissue function. (Sc
ully and Otten, Cell Bol. Int. 19: 459-469
(1995); Otten and Gadient, Int. J. Devl. Ne
urosciences 13: 147-151 (1995), which are incorporated by reference. Endogenously produced FGF-20 may also play a role in certain disease states, especially in the presence of cellular degeneration such as neurodegenerative conditions or neurodegenerative diseases. Other neurotrophic factors are known to change during disease states. For example, in multiple sclerosis, the level of NGF protein in cerebrospinal fluid is increased during the acute phase of the disease (Bracci-Laudiero et al., Neuroscience Lett. 147: 9-12 (1992) (this is a reference). , And in systemic lupus erythematosus, there is a correlation between inflammatory expression and serum NGF levels (Bracci-Lau).
diero et al., NeuroReport 4: 563-565 (1993), which is incorporated by reference).

Given that FGF-20 is expressed in adult neurons and cochlear cells during embryonic development, perhaps the levels of FGF-20 could be altered under various conditions, and quantification of FGF-20 levels was Provide clinically useful information. Further, in the treatment of neurodegenerative conditions, a composition comprising FGF-20 can be administered and achieve a specific target level of FGF-20 in serum, cerebrospinal fluid, or any desired tissue component. Is probably desirable. Therefore, FGF-20 in patients
It is advantageous to be able to monitor the level of Thus, the patients of the invention also provide a method for detecting the presence of FGF-20 in a sample derived from a patient.

The term “detection”, as used herein in the context of detecting the presence of FGF-20 in a patient, results in an amount of FGF-20 or an amount of FGF-20 in the patient.
-20-determination of the ability to express, differentiation of FGF-20 from other growth factors, judgment of prognosis and prognosis regarding possible outcome of degenerative disease, FGF over time as a measure of status status- It is intended to include 20 levels of monitoring as well as monitoring of FGF20 levels to determine a preferred treatment regimen for the patient.

To determine the presence of FGF-20 in a patient, a sample is obtained from the patient. This sample can be used for tissue, biopsy, blood, plasma, serum, CSF
It may be a sample such as. FGF-20 is expressed in neural tissue, as discussed in Example 8. A sample for detecting FGF-20 can be taken from this tissue. When assessing peripheral levels of FGF-20, it is preferred that the sample is a blood, plasma or serum sample. FG in the central nervous system
When assessing F-20 levels, preferred samples are those obtained from cerebrospinal fluid or neural tissue.

In some instances, it may be desirable to determine whether the FGF-20 gene is intact in the patient or tissue or cells within the patient. An intact FGF-20 gene is meant to have no alterations in the gene such as point mutations, deletions, insertions, chromosome disruptions, chromosome rearrangements, etc., where such alterations are FGFs. The production of -20 can be altered or its biological activity, stability, etc. can be altered leading to susceptibility to disease processes or cytopathic conditions. Thus, in one embodiment of the invention, a method for detecting and characterizing any alteration in the FGF-20 gene is provided. This method uses FGF-20 cD
Providing an oligonucleotide comprising NA, FGF-20 genomic DNA, or a fragment thereof or a derivative thereof. A derivative of an oligonucleotide is a derivative of oligonucleotide in that the derivatized sequence hybridizes to the FGF-20 gene with sufficient sequence homology to the sequence from which it was derived. Is substantially the same as the sequence from which it is derived. The derived nucleotide sequence need not be physically derived from the nucleotide sequence, but can be made in any manner including, for example, chemical synthesis or DNA replication or reverse transcription or transcription.

Typically, patient genomic DNA is isolated from a patient-derived cell sample and digested with one or more restriction endonucleases such as, for example, TaqI and AluI. Using the Southern blot protocol, which is well known in the art, this assay determines whether a patient or a particular tissue in a patient has an intact FGF-20 gene or an FGF-20 gene abnormality. It

Hybridization to the FGF-20 gene denatures chromosomal DNA to obtain single stranded DNA; contacting this single stranded DNA with a gene probe associated with the FGF-20 gene sequence; and DNA hybridized to detect chromosomal DNA containing at least part of human FGF-20 gene
-Including the step of identifying the probe.

The term “probe,” as used herein, refers to a structure that comprises a polynucleotide that forms a hybrid structure with a target sequence due to the complementarity of the probe sequence with the sequence in the target region. Say. Oligomers suitable for use as probes may contain a minimum of about 8-12 contiguous nucleotides, preferably a minimum of about 20 contiguous nucleotides, which are complementary to the target sequence.

The FGF-20 gene probe of the present invention comprises a DNA oligonucleotide or R
It can be a NA oligonucleotide and can be made by any method known in the art such as, for example, excision, transcription or chemical synthesis. The probe can be labeled with any detectable label known in the art, such as a radioactive or fluorescent label or an enzymatic marker. Labeling of the probe can be accomplished by any method known in the art such as PCR, random priming, end labeling, nick translation and the like. One of skill in the art will also recognize that other methods that do not use labeled probes can be used to determine hybridization. Examples of methods that can be used to detect hybridization include Southern blotting, fluorescence in situ hybridization, and single-stranded conformational polymorphisms using PCR amplification.

Hybridization is typically at 25 ° -45 ° C, more preferably 3 ° C.
It is carried out at 2 ° to 40 ° C, more preferably 37 ° to 38 ° C. The time required for hybridization is about 0.25 to about 96 hours, more preferably about 1
To about 72 hours, and most preferably about 4 to about 24 hours.

FGF-20 gene abnormalities can also be detected by using PCR methods and primers flanking or within the FGF-20 gene. PCR methods are well known in the art. Simply put, this method
It is carried out by amplifying the target sequence with two oligonucleotide primers capable of hybridizing to a nucleic acid sequence flanking the target sequence within the FGF-20 gene. The term "oligonucleotide primer" as used herein refers to a short strand of DNA or RNA ranging in length from about 8 bases to about 30 bases. The upstream and downstream of the primer are typically about 20 to about 30 base pairs in length and hybridize to flanking regions for replication of the nucleotide sequence. Polymerization is catalyzed by DNA polymerase in the presence of deoxynucleotide triphosphates or nucleotide analogs to produce double-stranded DNA molecules. The duplexes are then separated by any denaturing method including physical, chemical, or enzymatic methods. Usually, the nucleic acid is heated (typically about 80 ° C to 105 ° C).
At a temperature range of about 1 to about 10 minutes).
used. This process is repeated for the desired number of cycles.

A primer is selected that is substantially complementary to the strand of DNA to be amplified. Therefore,
The primer need not reflect the exact sequence of the template. However, it must be sufficiently complementary to selectively hybridize to the strand to be amplified.

After PCR amplification, the DNA sequence containing FGF-20 or prepro-FGF-20 or fragments thereof is then directly sequenced and analyzed by sequence comparison using the sequences disclosed herein. Identify alterations that can alter activity levels, expression levels, etc.

In another embodiment, a method for detecting FGF-20 is provided based on the analysis of tissues expressing the FGF-gene, as described in the examples. The method involves hybridizing the polynucleotide to mRNA from a tissue sample that normally expresses the FGF-20 gene. This sample is
Obtained from a patient suspected of having an abnormality in the FGF-20 gene or the FGF-20 gene of a specific cell.

A sample is obtained from the patient to detect the presence of mRNA encoding the FGF-20 protein. The sample may be from blood or a tissue biopsy sample. The sample can be treated to extract the nucleic acid contained therein. Nucleic acids obtained from this sample are subjected to gel electrophoresis or other size separation techniques.

The sample mRNA is contacted with a DNA sequence that serves as a probe to form a hybrid duplex. The use of labeled probes as described above allows detection of this resulting duplex.

When a cDNA encoding the FGF-20 protein or a derivative of this cDNA is used as a probe, false positives (which are actually intact and functional F
High stringency conditions can be used to avoid (hybridization and unambiguous detection of FGF-20 nucleotide sequences in the absence of the GF-20 gene). When using sequences derived from the FGF-20 cDNA, relatively low stringency conditions can be used, but are a less preferred approach due to the potential for false positives. Hybridization stringency is determined by a number of factors during the hybridization and during the wash procedure, including temperature, ionic strength, length of time and concentration of formamide.

In order to increase the sensitivity of detection of mRNA encoding FGF-20 protein in a sample, the technique of reverse transcription / polymerization chain reaction (RT / PCR) has been applied to FGF-2.
It can be used to amplify cDNA transcribed from mRNA encoding 0 protein. RT / PCR methods are well known in the art and can be performed as follows. Total cellular mRNA is isolated, eg, by standard guanidine isothiocyanate methods, and total RNA is reverse transcribed. This reverse transcription method involves the synthesis of DNA based on an RNA template using a reverse transcriptase and a 3'primer. Typically, this primer contains an oligo (dT) sequence. The cDNA thus produced is then amplified using the PCR method and FGF-20 specific primers. (Bellyavsky et al., Nucl. Acid Re.
s. 17: 2919- 2932 (1989); Krug and Berger, M.
methods in Enzymology 152: 316-325, Aca
demic Press, NY, 1987 (these are incorporated by reference).
).

The polymerase chain reaction method is carried out as described above with two oligonucleotide primers that are substantially complementary to the two flanking sequences of the DNA segment to be amplified.

After amplification, the PCR products are then electrophoresed and detected by ethidium bromide staining or phosphoimaging.

The present invention further provides methods for detecting the presence of FGF-20 protein in a sample obtained from a patient. Any method in the art for detecting proteins can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, immunochemical methods, binding agent-ligand assays, immunohistochemistry techniques, agglutination assays and complement assays. (For example, Ba
sic and Clinical Immunology, 217-262, Si
tes and Terr, ed., Appleton & Lange, Norway
k, CT, 1991, which is incorporated by reference). Antibody F
FGF-20 labeled with and reacted with an epitope of GF-20 protein
A binding agent comprising the step of competitively displacing a protein or a derivative thereof-
Ligand immunoassay methods are preferred. Preferred antibodies are prepared according to Example 11.

As used herein, a derivative of the FGF-20 protein is intended to include a polypeptide in which certain amino acids have been deleted, substituted, or changed with modified or unusual amino acids, Here, the FGF-20 derivative is FG-20.
Bioequivalent to F-20, this polypeptide derivative cross-reacts with antibodies raised against the FGF-20 protein. Cross-reactivity means that the antibody reacts with an antigen other than those that induce its formation.

Many competitive and non-competitive protein binding immunoassays are well known in the art. Antibodies used in such assays can be unlabeled, eg, as used in agglutination tests, or labeled for use in a wide variety of assay methods. Labels that can be used include radioimmunoassay (RIA)
, Enzyme immunoassays (eg enzyme-linked immunosorbent assay (ELIS
A)), radionuclides, enzymes, fluorescent agents, chemiluminescent agents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes etc. for use in fluorescent immunoassays and the like.

Polyclonal or monoclonal antibodies directed against the FGF-20 protein or an epitope thereof can be made for use in immunoassays by any of a number of methods known in the art. An epitope provides a reference to an antigenic determinant of a polypeptide. An epitope can include 3 amino acids in a spatial conformation that is unique to the epitope. Generally, an epitope consists of at least 5 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. One approach for preparing antibodies to a protein is the selection and preparation of the amino acid sequence of all or part of the protein, chemical synthesis of that sequence and injection into a suitable animal (usually rabbit or mouse). See Example 11).

Oligopeptides can be selected as candidates for the preparation of antibodies to the FGF-20 protein based on the oligopeptides in the hydrophilic region (and thus probably exposed to the mature protein). The oligopeptide for raising an antibody comprises consecutive amino acids at positions 176-189 or 56-70 of SEQ ID NO: 4. These oligopeptides are RDGARSKRHQKFTH (SEQ ID NO: 5) and QLAHLHGILRRRQLY (SEQ ID NO: 6). Further oligopeptides are, for example, Antigenicity Index of We
lling (GW et al., FEBS Lett. 188: 215-218 (19).
85) (incorporated herein by reference)).

Antibodies to FGF-20 can also be raised against oligopeptides that include one or more of the conserved regions as identified herein, so that the antibodies are in another family. Can cross-react with members of. Such antibodies can be used to identify and isolate members of other families.

Methods for the preparation of FGF-20 protein or its epitope include:
Examples include, but are not limited to, chemical synthesis, recombinant DNA technology or isolation from biological samples. Chemical synthesis of peptides is, for example, the classical Maryfield method of solid phase peptide synthesis (Merrifeld, J., Am. Chem.
Soc. 85: 2149 (1963), which is incorporated by reference) or an FMOC strategy (EI du P) on a rapid automated multiple peptide synthesis system.
ont de Nemours Company, Wilmington, De
Laware) (Caprino and Han, J Org. Chem. 37:
3404 (1972), which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting the antigen followed by subsequent boosts at appropriate intervals. The animals were bled and the serum was usually FGF-20 purified by ELISA.
It is assayed for proteins or by bioassays based on their ability to block the action of FGF-20 on neurons or other cells. When using an avian species (eg, chicken, turkey, etc.), the antibody can be isolated from the yolk of the egg. Monoclonal antibodies can be prepared following the method of Milstein and Kohler by fusing splenocytes from mice immunized with serially replicating tumor cells such as myeloma cells or lymphoma cells. (M
ilstein and Kohler, Nature 256: 495-497,
1975; Gulfre and Milstein, Methods in En.
Zymology: Immunochemical Techniques 7
3: 1-46, edited by Langone and Banatis, Academic P.
ress, 1981, which are incorporated by reference). The hybridoma cells so formed are then cloned by limiting dilution and the supernatant assayed for antibody production by ELISA, RIA, or bioassay.

The unique ability of antibodies to recognize and specifically bind target proteins provides an approach for treating protein overexpression. Accordingly, another aspect of the invention provides a method of preventing or treating a disease involving overexpression of the FGF-20 protein by treating the patient with an antibody specific for the FGF-20 protein.

Antibodies specific for the FGF-20 protein (either polyclonal or monoclonal) may be produced by any suitable method known in the art, as discussed above. For example, a mouse or human monoclonal antibody can be produced by hybridoma technology, or the FGF-20 protein, or an immunologically active fragment thereof, or an anti-idiotype antibody, or fragment thereof, can be administered to an animal, It can induce the production of antibodies capable of recognizing and binding the FGF-20 protein. Such antibodies may be derived from, but are not limited to, any antibody class and any antibody subclass including: IgG, IgA, IgM, IgD, and IgE, or IgY in the case of avian species.

The polynucleotides of the invention and the polypeptides encoded by the corresponding full-length genes can be used to screen peptide, protein, small molecule, and phage display libraries, and Peptides and other known methods can be used to identify analogs or antagonists.

Native FGF polypeptides may play a role in cancer. For example,
FGF family members can induce significant morphological transformation of NIH3T3 cells and show strong tumorigenicity in nude mice. Angiogenic activity was demonstrated by FGF family members. Therefore, inhibitors of FGF may be used to treat cancer.

A library of peptides can be synthesized according to the methods disclosed in US Pat. No. 5,010,175 and PCT number WO 91/17823. Mixtures of peptides are prepared, which are then screened to identify peptides that exhibit the desired signaling and receptor binding activity, as briefly described below. In accordance with the method of the '175 patent, a suitable peptide synthesis support (eg, resin) is coupled to a mixture of appropriately protected and activated amino acids. The concentration of each amino acid in the reaction mixture is equilibrated or adjusted inversely with the coupling reaction rate so that the product is an equimolar mixture of amino acids coupled to the starting resin. The bound amino acids are then deprotected and reacted with another equilibrated mixture of amino acids to form an equimolar mixture of all possible dipeptides. This process is repeated until a peptide mixture of the desired length (eg hexamer) is formed. Note that it is not necessary to include all amino acids in each step: some steps may include only 1 or 2 amino acids (eg, if a particular amino acid is known to be essential at a given position). Therefore, the complexity of the mixture can be reduced. After the peptide library synthesis is complete, the mixture of peptides is screened for binding to the selected polypeptide. The peptide is then tested for its ability to inhibit or enhance activity. Peptides exhibiting the desired activity are then isolated and sequenced.

The method described in PCT number WO 91/17823 is similar. However, instead of reacting the synthetic resin with a mixture of activated amino acids, the resin
Divide into 0 equal parts (or into many parts corresponding to the number of different amino acids added in the process), and each amino acid is individually coupled to that part of the resin. The resin portions are then combined, mixed, and again divided into many aliquots for reaction with the second amino acid. In this manner, each reaction can be easily completed. In addition, separate "subpools" may be maintained by processing the portions in parallel, rather than combining all the resins at each step. This simplifies the process of determining which peptide is responsible for any observed receptor binding or signaling activity.

In such cases, for example, subpools each containing 1 to 2,000 candidates are exposed to one or more polypeptides of the invention. Each subpool that produces a positive result is then resynthesized and reassayed as a group of smaller subpools (sub-subpools) containing, for example, 20-100 candidates. Positive sub-
The subpools can be resynthesized as individual compounds and finally assayed to determine peptides that exhibit high binding constants. These peptides can be tested for their ability to inhibit or enhance native activity. PCT number WO91
/ 7823 and US Pat. No. 5,194,392 (incorporated herein by reference) are directed to parallel automation such that all syntheses and resynthesis can be done in days. It allows the preparation of such pools and subpools by the techniques described.

Peptide agonists or antagonists are screened using any available method (eg signal transduction, antibody binding, receptor binding, mitogen assay). Assay conditions are ideally those in which native activity is demonstrated in vivo (ie, under physiological pH, temperature, and ionic strength).
Should be similar to. Suitable agonists or antagonists exhibit potent inhibition or enhancement of native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native polypeptide may require concentrations above the native concentration, while inhibitors that may irreversibly bind to the polypeptide are at concentrations in the order of the native concentration. Can be added.

The availability of hFGF-20 and rFGF-20 indicates that conventional application of high throughput screening methods (HTS) allows the identification of small and low molecular weight compounds that inhibit the binding of FGF-20 to its receptor. To The HTS method is
In general, it relates to techniques that allow rapid assaying of lead compounds for therapeutic potential. HTS technology involves robotic manipulation of test materials, detection of positive signals and interpretation of data. Lead compounds can be identified by the incorporation of radioactivity assays or by optics that rely on absorbance, fluorescence or luminescence as a readout. Gonzalez, J .; E. Curr. Opin. Biot
hch. 9: 624-631 (1998). Assays for detecting the interaction between FGF molecules and FGF receptors are described, for example, in Blunt, A .; G. ,
J. Biol. Chem. 272: 3733-3738 (1997), and such assays can be adapted to determine whether a candidate molecule can inhibit the interaction between FGF-20 and its receptor.

For example, by competing with FGF-20 for receptor binding, FGF-2
Model systems are available that can be adapted for use in high throughput screens for compounds that inhibit the interaction of FGF-20 with the receptor to which 0 binds. Sarubbi et al., Anal. Biochem. 237: 70-75 (199
6) describes a cell-free, non-isotopic assay to discover molecules that compete with natural ligands for binding to the active site of the IL-1 receptor. Martens
, C. Et al., Anal. Biochem. 273: 20-31 (1999) describes a general particle-based non-radioactive method, in which a labeled ligand binds to its receptor immobilized on the particle; Reduce the presence of molecules that compete with the labeled ligand for receptor binding.

Therapeutic FGF-20 polynucleotides and polypeptides of the present invention can be utilized in gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (generally Jolly, Cancer Gen.
e Therapy 1: 51-64 (1994); Kimura, Human.
Gene Therapy 5: 845-582 (1994); Connel
ly, Human Gene Therapy 1: 185-193 (1995)
); And Kaplitt, Nature Genetics 6: 148-1.
53 (1994)). Gene therapy vehicles for delivery of constructs containing the coding sequences of therapeutic agents of the invention can be administered either locally or systemically. These constructs may utilize viral or non-viral vector approaches. Expression of such coding sequences may be induced by the use of endogenous mammalian or heterologous promoters. Expression of the coding sequence can be constitutive or regulated.

The present invention may utilize recombinant retroviruses constructed to carry or express selected nucleic acid molecules of interest. Retroviral vectors that can be utilized include those described below: EP 0 415 731; WO 90 /
07936; WO 94/03622; WO 93/25698; WO 93 /
25234; US Pat. No. 5,219,740; WO 93/11230; WO
93/10218; Vile and Hart, Cancer Res. 53:
3860-3864 (1993); Vile and Hart, Cancer R
es. 53: 962-967 (1993); Ram et al., Cancer Res.
53: 83-88 (1993); Takamiya et al. Neurosci.
Res. 33: 493-503 (1992); Baba et al., J. Am. Neurosu
rg. 79: 729-735 (1993); U.S. Pat. No. 4,777,127;
GB Patent No. 2,200,651; and EP 0 345 242. Preferred recombinant retroviruses include those described in WO 91/02805.

Packaging cell lines suitable for use with the retroviral vector constructs described above can be readily prepared (eg, PCT Publications WO 95/30763 and WO
92/05266), and for the production of producer cell lines (also called vector cell lines) for producing recombinant vector particles. In a particularly preferred embodiment of the invention, the packaging cell line is of a recombinant retrovirus that is made from a human (eg, HT1080 cell) or mink parental cell line, thereby surviving inactivation in human serum. Enable production.

The present invention also uses alphavirus-based vectors that can function as gene delivery vehicles. Such vectors are, for example, Sindbis virus vector, Semliki Forest virus (ATCC VR-67; ATCC VR-
1247), Ross River virus (ATCC VR-373; ATCC VR-12).
46) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC
VR-1250; ATCC VR-1249; ATCC VR-532) can be constructed from a wide variety of alphaviruses. Representative examples of such vector systems include US Pat. Nos. 5,091,309 and 5,217,879.
And PCT Publication No. WO 92/10.
578; WO 94/21792; WO 95/27069; WO 95/27.
044; and those described in WO 95/07994.

The gene delivery vehicles of the present invention may also utilize parvoviruses such as adeno-associated virus (AAV) vectors. A representative example is WO 93/09239.
Srivastava, Samulski et al. Vir. 63: 3822-
3828 (1989); Mendelson et al., Virol. 166: 154-
165 (1988); and Flotte et al. N. A. S90: 10613
-10617 (1993).

AAV vectors may be suitable for administering FGF-20 to treat deafness. For example, Lalwani et al., Gene Ther. 3: 588 ~
592 (1996) used AAV to obtain in vivo expression of a heterologous gene in the cochlea of guinea pigs.

Representative examples of adenovirus vectors include those described below: Berkner, Biotechniques 6: 616-627 (
Biotechniques); Rosenfeld et al., Science 25.
2: 431-434 (1991); WO 93/19191; Kolls et al., P.
． N. A. S. : 215-219 (1994); Kass-Eisler et al., P.
． N. A. S. 90: 11498-11502 (1993); Guzman et al.,
Circulation 88: 2838-2848 (1993); Guzma
n et al., Cir. Res. 73: 1202-1207 (1993); Zabner.
Et al., Cell 75: 207-216 (1993); Li et al., Hum. Gene
Ther. 4: 403-409 (1993); Cailaud et al., Eur. J
． Neurosci. 5: 1287-1291 (1993); Vincent et al., Nat. Genet. 5: 130-134 (1993); Jaffe et al., Na.
t. Genet. 1: 372-378 (1992); and Levrero et al.,
Gene 101: 195-202 (1992). Exemplary adenovirus gene therapy vectors that can be used in the present invention also include those described below: WO94 / 12649, WO93 / 03769; WO93 / 19191.
WO94 / 28938; WO95 / 11984 and WO95 / 00655.
Curiel, Hum. Gene Ther. 3: 147-154 (1992)
Administration of killed adenovirus-ligated DNA as described in Section 1.

Other gene delivery vehicles and methods can be utilized. This includes: Polycationic enriched DNA linked or unlinked to killed adenovirus alone (eg Curiel, Hum. Gene Ther. 3: 1).
47-154 (1992)); ligand-ligated DNA (eg, Wu, J. Bio).
l. Chem. 264: 16985-16987 (1989));
Eukaryotic cell delivery vehicle cells (eg, US patent application Ser. No. 08 / 240,030 filed May 9, 1994 and US patent application Ser. No. 08 / 404,796).
No.); Deposition of photopolymerized hydrogel material; US Pat. No. 5,149,6.
Portable gene transfer particle gun described in US Pat. No. 5,206.
, 152 and WO 92/11033; nuclear charge neutralization or fusion with cell membranes. A further approach is described by Philip, Mol. Ce
ll Biol. 14: 2411-2418 (1994), and Woffen.
din, Proc. Natl. Acad. Sci. 91: 1581-1585 (
1994).

Naked DNA may also be utilized. An exemplary method for introducing naked DNA is WO90
/ 11092 and US Pat. No. 5,580,859. Uptake efficiency can be improved by the use of biodegradable latex beads. The latex beads coated with DNA are efficiently transported into cells after the initiation of endocytosis by the beads. The method can be further improved by treatment of the beads to increase hydrophobicity, and thereby facilitate disruption of endosomes and facilitate release of DNA into the cytoplasm. Liposomes that can serve as gene delivery vehicles are described in US Pat. No. 5,422,120; PCT Patent Publication W
O95 / 13796; WO94 / 23697; WO91 / 14445; and E
P0 524 968.

Additional non-viral delivery suitable for use is described by Woffendin et al., Proc.
Natl. Acad. Sci. USA 91 (24): 11581-11585.
Including a mechanical delivery system such as the approach described in (1994). In addition, the coding sequences and expression products of such can be delivered through a precipitate of photopolymerized hydrogel material. Other conventional methods of gene delivery that can be used to deliver coding sequences include: Use of a portable gene transfer particle gun as described, for example, in US Pat. No. 5,149,655; US Pat. Use of ionizing radiation for activation of transferred genes as described in 206,152 and PCT patent publication WO 92/11033.

FGF can be reversed or prevented by loss of function, inappropriate function / number, cells or tissues with abnormal function or death whose function or survival can be prolonged / rescue, and treatment with FGF. It is associated with diseases characterized by abnormalities.

Loss of lung or bronchial or alveolar cell or function, healing of lung or bronchial injury, lung failure, emphysema / chronic obstructive pulmonary disease, asthma, sequelae of infectious or autoimmune diseases, pulmonary arterial or venous hypertension Sequelae, pulmonary fibrosis, premature infants (immaturi
Lung disease of ty), and cystic fibrosis are conditions that are amenable to treatment with FGF.

Ischemic vascular disease may be easy to treat with FGF-20, where the disease is characterized by inappropriate blood flow to organs. Treatment can induce therapeutic angiogenesis or preserve cell function / survival (myocardial ischemia / myocardial infarction, peripheral vascular disease, renal artery disease, stroke). Cardiomyopathy characterized by loss of function or death of cardiomyocytes or supporting cells of the heart (congestive heart failure, myocarditis)
Can also be treated with FGF-20, as well as musculoskeletal disorders characterized by loss of function of skeletal muscle cells, bone cells or supporting cells, its inappropriate function or its death. Examples include skeletal myopathy, bone disease and arthritis.

FGF-20 polynucleotides and polypeptides may aid in the correction of birth defects resulting from loss of the FGF-20 molecule or its function (heart, lungs, brain, limbs, kidneys, etc.). FGF-20 polynucleotides and polypeptides may also help correct such defects, where the defects cause hearing loss due to cochlear defects.

Treatment of wound healing (caused by either trauma, disease, medical or surgical treatment, including regeneration of cell populations and tissues depleted by these treatments) is FG
Yet another use of F-20 polypeptides and polynucleotides. Examples include liver regeneration, surgical wound healing, re-endovascularization of damaged blood vessels.
healing of trauma wounds, healing of ulcers caused by blood vessels,
Examples include bone loss such as metabolic diseases, loss of cells due to inflammatory diseases, and the like.

FGF-20 can also be used in a screen to identify drugs for the treatment of cancers that contain overactivity of this molecule, or to identify new targets that are useful in identifying new drugs.

For all of the above-described embodiments, the clinician will allow the FGF-
20 polypeptides or polynucleotides, antibodies to FGF-20, or small molecules such as peptide analogs or antagonists are determined to be the most appropriate form of treatment. All of these forms are within the scope of the invention.

Preferred embodiments of the present invention are described in the examples below. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the invention, as disclosed herein. It is intended that the specification, together with the examples, be considered as exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

EXAMPLES Example 1 RNA Preparation—RNA Extraction Kit (Pharmacia Biotech, U.)
RNA was prepared from adult rat brain using ppsala, Sweden). Poly (A) + RNA was added to oligo (dT) -cellulose (type 2, Collabo)
rational Biomedical Products, Bedford, M
It was prepared using H.A.

Example 2 Isolation and Analysis of Rat FGF-20 cDNA-Rat FGF-9 (17)
And FGF-16 (21) consensus amino acid sequence, FEEN, respectively
Presents all possible codons corresponding to WY and THFLPR, 5 each
DNA was amplified from rat genomic DNA by 30 cycles of the polymerase chain reaction (PCR) in 25 μl of a reaction mixture containing pmol / μl sense and antisense degenerate primers. This amplified product was labeled with sense degenerate primers and antisense to present different consensus amino acid sequences for rat FGF-9 (17) and FGF-16 (21), all possible codons corresponding to ENWYNT and HQKFTH, respectively. Further amplification by PCR was performed with each of the degenerate primers. Expected size (about 1
Amplified DNA of 50 base pairs was added to pGEM-T DNA vector (Prom
Ega, Madison, Wisconsin). The nucleotide sequence of this cloned DNA was used as a DNA sequencer (Applied Bi
ossystems, Foster, California).
To determine the coding region of the novel FGF cDNA, Marathon c
DNA amplification kit (Clontech, Palo Alto, California)
The coding region was amplified from cDNA synthesized from rat brain poly (A) ⁺ RNA by adapter ligation mediated polymerase chain reaction using ia). To determine the amino-terminal region, the DNA encoding this region was cloned into L
A PCR in vitro cloning kit (TaKaRa, Kyoto, Ja
pan) and cassette ligation mediated polymerase chain reaction (I
Segawa, Y. Et al., Mol. Cell. Probes 6: 467-475
(1992)), and amplified from rat genomic DNA. Polymerase chain reaction in the presence of 5% dimethyl sulfoxide (Villarreal, XC, et al., A,), using FGF specific primers containing 5'and 3'noncoding sequences.
nal. Biochem. 197: 362-367 (1991)), F
A cDNA encoding the entire coding region of GF was amplified from rat brain cDNA,
It was then cloned into the pGEM-T DNA vector. The apparent evolutionary relevance of members of the FGF family was analyzed using sequence analysis software, Genetyx (
Software Development Co. , Tokyo, Japan
) Using the unweighted pair-group method (unweig) with the arithmetic mean method.
Hed pair-group method).

Example 3 Expression of FGF-20 mRNA in rat embryo inner ear-FG in rat embryo
The expression of F-20 was tested. Serial cross sections (E14.5) of rat embryos were examined by in situ hybridization with ³⁵ S-labeled FGF-20 antisense and sense cRNA probes. This fragment was counterstained with hematoxylin and eosin. Bright-field and dark-field photographs of this fragment show FGF-2.
0 is predominantly expressed in the cochlea of the inner ear.

Example 4 Northern Blot Analysis-Poly (A) ⁺ RNA from adult rat brain (10 μg)
Were dissolved in denaturing agarose gel containing formaldehyde (1%) and transferred overnight to a nitrocellulose membrane in 20 × SSC (1 × SSC: 0.15M NaCl / 0.015M sodium citrate). ³² P-labeled FGF-20 c
Random primer labeling kit (Pha
rmcia Biotech, Uppsala, Sweden) and deoxycytidine 5 ′-[α- ³² P-] triphosphate (about 110 TBq / mmol) (I
CN Biomedicals Inc. , Costa Mesa, Calif
ornia). The membrane was incubated in a hybridization solution containing the labeled probe (22) described above and an X-ray imaging analyzer (BAS 2000, Fuji Photo Film Co., Tokyo).
o, Japan). In order to confirm the integrity of poly (A) ⁺ RNA, the probe hybridized on the membrane was treated with 0.5 × SSC at 100 ° C. for 5 minutes.
Wash with 0.01M EDTA (pH 8.0) and 1 at 60 ° C.
0.05 × SSC (0.01 M EDTA (pH 8.0) and 0.
1% SDS was included). The ³² P-labeled rat β-actin cDNA probe (about 410 base pairs) (Nudel, U. et al., Nuclei) was washed with the washed membrane.
c Acids Res. 11: 1759-1771 (1983)).

Example 5 In Situ Hybridization-Adult Wistar rat brain was frozen in powdered dry ice and sagittal sections were cut in a 16 μm cold bath and thawed onto poly-L-lysine coated slides. Attached and stored at -85 ° C until hybridized. ³⁵ S-labeled rat FGF-20 antisense or sense cRNA probe was used to bind uridine 5′-α using SP6 RNA polymerase or T7 RNA polymerase (TaKaRa, Kyoto, Japan), respectively.
[ ³⁵ S] thiotriphosphate (about 30 TBq / mmol) (Amersha
, Buckinghamshire, England). Fragments,
Tested by in situ hybridization with a labeled probe as described above (Yamasaki, M. et al., J. Biol. Chem. 271:
15918-15921 (1996)).

Example 6 Preparation of Recombinant Rat FGF-20--E-tag (G
(APVPYPDPREPR) and His ₆ tag (HHHHHH) -encoding rat FGF20 cDNA with a DNA fragment (75BP) was transferred to the transfer vector DNA pBacPAK9 (Clontech, Palo Alto,
(California). FGF-20 cDNA having a tag sequence
Recombinant baculovirus containing recombinant pBacPAK9 and Bsu36
I-digested expression vector, BacPAK6 (Clontech, Palo Alt
o, California) were obtained by co-transfection of Sf9 cells. High Five insect cells were infected with the resulting recombinant baculovirus and serum-free medium EX-CELL 400 (JRH Bioscience).
ces, Lenexa, Kankas) at 27 ° C. for 65 hours. The culture medium is dialyzed against phosphate buffered saline (PBS) and 20 mM
Applied to a column of Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany) in PBS containing imidazole and 0.5 M NaCl. The column was loaded with P containing 20 mM imidazole and 0.5 M NaCl.
After washing with BS, recombinant FGF-20 was treated with 20 mM imidazole and 0.5
Elute from the column with PBS containing M NaCl, and 100 μg / ml BSA
Bio-Gel P-6DG (Bio-Rad Lab., H in PBS containing
ercules, California).

Example 7 Detection of Recombinant FGF-20 by Western Blotting Analysis Culture medium or rat recombinant FGF-20 was treated with sodium dodecyl sulfate (
SDS) -polyacrylamide gel (12.5%) separation by electrophoresis and nitrocellulose membrane (Hybond-ECL, Amersham, Buck).
inghamshire, England). This membrane was treated with anti-E tag antibody (1: 500) (Pharmacia Biotech, Uppsala, Sw).
eden). Proteins with E-tags were visualized as described (Hoshikawa, M. et al., Biochem. Bio.
phys. Res. Commun. 244: 187-191 (1998)).

(Example 8) Rat midbrain cultured cells-The lower midbrain was excised from a rat embryo (E16.5).
Midbrain blocks were washed 10 times with Hanks solution and mechanically dissociated without enzymatic treatment. Cultured midbrain cells were prepared essentially as described (Sa
wada, H .; Et al., J. Neurosci. Res. 43: 503-510 (1
996)). The culture medium was 0.2% sodium carbonate, 0.1% glucose, 0.
Eagl supplemented with 029% L-glutamine and 0.238% HEPES
e consisted of minimal essential medium (EMEM). Cultured cells were incubated at 37 ° C in culture medium containing 10% fetal calf serum. From day 5 of culture, cells were incubated in culture medium containing 10% horse serum.

Example 9 Testing Neurotrophic Activity of FGF-20 on Midbrain Dopaminergic Neurons--Day 8 cells in culture were treated with 0.2% sodium carbonate, 0.1% glucose,
Eagle's Minimal Essential Medium (EM) supplemented with 0.029% L-glutamine, 0.238% HEPES and 10% horse serum or 0.1% bovine serum albumin.
(EM) in the presence or absence of FGF-20 for 4 days, then fixed on day 12 with fresh 4% paraformaldehyde for 30 minutes. Day 8 cells in culture were incubated for 24 hours in the presence or absence of recombinant rat FGF-20 and then treated with 1 mM glutamate for 10 minutes. Cultured cells were further incubated in medium without FGF-20 and 1 mM glutamate, then on day 12 3% with 4% paraformaldehyde.
Fixed for 0 minutes. Fixed cells are washed with PBS for 15 minutes, then 0.2
% Triton X-10 for 30 minutes. Cells were treated with anti-tyrosine hydroxylase (TH) antibody (Eugene Tech, Ridgefield Par).
k, New Jersey), essentially as described (Sawada, H.
． Et al., J. Neurosci. Res. 43: 503-510 (1996)),
Immunostained. The number of cultured dopaminergic neurons was assessed by counting the cells stained with anti-TH antibody.

Example 10 Isolation and Analysis of Human FGF-20--The coding region of human FGF-20 DNA was labeled with P using primers specific for FGF-20 and λgt10 DNA.
Amplified from a human brain cDNA library (λgt10) by CR. The nucleotide sequence of the cDNA encoding the carboxy-terminal 112 amino acids of human FGF-20 was determined and is shown in FIG. Rat and human FGF-20
The amino acid sequence of is shown in FIG.

Example 11 Preparation of Antisera to FGF-20 by Immunization with Rabbit FGF-20 Peptide--Synthesis of Peptide Sequences Corresponding to Selected Contiguous Amino Acids of Human FGF-20 Protein, and Keyhole limpet as described (l
impet) bound to chemocyanin (KLH) (Harlow and Lan)
d, Antibiodies: A Laboratory Manual, 1998.
． Cold Spring Harbor Laboratory, New Y
ork, NY). The KLH binding peptide is used to immunize rabbits. Antisera are tested for specificity for FGF-20 and cross-reactivity with other FGF proteins.

[0146]   An exemplary peptide sequence is:

[0147]

[Equation 1] .

Example 12 Binding of FGF-20 to Recombinant Extracellular Domains of FGFR-1c, FGFR-2c and FGFR-3c-Recombinant FGF-20 was used as a sensor chip CM5 (A.
It was fixed to the Mersham Pharmacia Biotech). FGFR
Binding of the recombinant extracellular domains of -1c, FGFR-2c and FGFR-3c to FGF-20 on the chip was performed using BIACORE 2000 System (Am
ersham Pharmacia Biotech) was used for analysis. Equilibrium dissociation constants were calculated using BIA evaluation software (Amersham Pharmaci).
a Biotech).

[0149]

[Table 1] As shown in Table 1, FGF-20 binds to FGF receptors 2 and 3, but not FGF receptor 1. Therefore, FGF-20 is
Members of the FGF family that bind to F receptor 1 (FGF-2 and FG
It may exhibit biological effects not found in F-4).

All patents, published patent applications and publications cited herein are incorporated by reference as if fully set forth herein.

While certain preferred embodiments have been described herein, such embodiments are to be construed as limiting the scope of the invention except as set forth in the claims below. Is not intended.

[Brief description of drawings]

FIG. 1 is an amino acid sequence comparison of rat FGF-20, rat FGF-9 and FGF-16. Numbers indicate amino acid positions of FGF-9, FGF-16 and FGF-20. Asterisks indicate identical amino acid residues in this sequence.

FIG. 2: Apparent evolutionary tree of 20 members of the FGF family. The length of each horizontal line is proportional to the degree of amino acid sequence difference. mFGF, rFGF and hFGF are
Mouse FGF, rat FGF, and human FGF are shown, respectively.

[Figure 3]   Localization of FGF-20 mRNA in rat brain. (A, B) Coronal part, ³⁵ S-labeled FGF-20 antisense (A) and sense (B) probes
And was exposed to X-ray film for 10 days. section
A is adjacent to section B. Scale bar = 0.5 cm (C, D). section
Dip A and B in liquid emulsion and after 3 weeks crystal violet
Counterstained. Darkfield photographs of SNCs in Sections A and B
Shown in C and D respectively. The white particles in the dark field photograph are FG.
The localization of F-20 mRNA is shown. Scale bar = 50 μm. SNC: Black matter dense part

FIG. 4: Expression of FGF-20 mRNA in rat brain. Rat brain poly (A) + RN
A (10 μg) was electrophoresed on a denaturing agarose gel containing formaldehyde (1%) and transferred to a nitrocellulose filter. ³² P-labeled rat FGF-2
Hybridization was performed with 0 or β-actin cDNA probes. 28S and 18S indicate the positions of 28 and 18S rRNA, respectively.

FIG. 5. FGF-20 enhances survival of midbrain dopaminergic neurons. (A)
FGF-2 on survival of midbrain dopaminergic neurons in serum-free medium
0 effect. Cultured midbrain cells were supplemented with medium supplemented with 10% horse serum (control),
Alternatively, the cells were incubated for 4 days in serum-free medium supplemented with FGF-20, and then the number of surviving dopaminergic neurons was determined. (B) 24 hours F
GF-20, then no addition (control) or 10 with 1 mM glutamic acid
After processing for minutes, the effect. Cultured cells were further incubated in medium in the absence of glutamate and FGF-20 for 3 days, then the number of surviving dopaminergic neurons was determined.

[Figure 6]   FIG. 6 shows the DNA sequence of rat FGF-20 (SEQ ID NO: 1).

[Figure 7]   FIG. 7 shows the amino acid sequence of rat FGF-20 (SEQ ID NO: 2).

FIG. 8 shows the DNA sequence (SEQ ID NO: 3) and amino acid sequence of human FGF-20 (SEQ ID NO: 3).
SEQ ID NO: 4) is shown.

FIG. 9: Amino acid sequence of human FGF-20 (SEQ ID NO: 4) and rat FGF
1 shows an alignment of the -20 amino acid sequence (SEQ ID NO: 2).

[Figure 10]   FIG. 10 shows that E. The codon usage of E. coli is shown.

FIG. 11 shows yeast codon usage. The first column of information on each line of this table shows the three letter code for the amino acid. The second column shows the ambiguous codon for that amino acid. Column 3 lists the number of occurrences of that codon in this gene (from which this table will be edited). The fourth column lists the expected number of occurrences of that codon per 1000 codons in the gene, whose codon usage is the same as that edited in the codon frequency table. The last column shows the fraction of occurrences of this codon in the synonymous codon family.

[Fig. 12]   FIG. 12 shows the Drosophila codon usage.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/10 A61P 27/16 4H045 25/16 37/00 27/16 C07K 14/50 37/00 16 / 22 C07K 14/50 C12N 1/15 16/22 1/19 C12N 1/15 1/21 1/19 C12P 21/02 H 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12P 21/02 5/00 A C12Q 1/68 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS) , MW, MZ, SD, SL, S Z, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW F terms (reference ) 4B024 AA01 AA11 BA01 BA43 CA01 GA11 HA12 4B063 QA01 QA18 QQ42 QR32 QR55 QS34 QX01 4B064 AG13 CA19 CC24 DA0 1 DA13 4B065 AA91Y AA93Y AB01 AC14 BA02 CA24 CA25 CA44 CA46 4C084 AA01 AA07 AA13 CA18 CA23 CA70 NA14 ZA02 ZA14 ZA34 ZA36 ZB07 ZC35 ZC52 ZC54 4H045 AA10 AA11 BA10 CA40 DA01 EA20 EA50 FA74

Claims (52)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) a polynucleotide encoding the amino acids of about 1 to about 211 of SEQ ID NO: 4; (b) of about 2 to about 211 of SEQ ID NO: 4. (C) a polynucleotide encoding the amino acids of about 1 to about 169 of SEQ ID NO: 4; (d) a polynucleotide encoding the amino acids of about 187 to about 211 of SEQ ID NO: 4; (e) A polynucleotide encoding about 170 to about 186 amino acids of SEQ ID NO: 4; (f) a polynucleotide encoding about 59 to about 193 amino acids of SEQ ID NO: 4; (g) at least 30 contiguous sequences of SEQ ID NO: 4. A polynucleotide encoding an amino acid; (h) the amino acid sequence of a fragment of the polypeptide shown as amino acids 1-211 of SEQ ID NO: 4 A polynucleotide encoding the polynucleotide, wherein the fragment has fibroblast growth factor activity; (i) (a), (b), (c), (d), (e), ( f), (g) or (h
And (j) (a), (b), (c), (d), (e), (f), (g) or (h).
An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: a polynucleotide that is at least 90% identical to the polynucleotide of. 2. An isolated nucleic acid molecule comprising 20 to 600 contiguous nucleotides from the coding region of SEQ ID NO: 3. 3. The isolated nucleic acid molecule of claim 2, which is SEQ ID NO: 3.
An isolated nucleic acid molecule comprising 60 to 400 contiguous nucleotides from the coding region of. 4. The isolated nucleic acid molecule of claim 3, which is SEQ ID NO: 3.
An isolated nucleic acid molecule comprising 200 to 300 contiguous nucleotides from the coding region of. 5. An isolated nucleic acid molecule, said nucleic acid molecule comprising a polynucleotide encoding the polypeptide, wherein, except for at least one conservative amino acid substitution, said polypeptide is: (A) about 1 to about 211 amino acids of SEQ ID NO: 4; (b) about 2 to about 211 amino acids of SEQ ID NO: 4; (c) about 170 to about 186 amino acids of SEQ ID NO: 4; (d) sequence Amino acids from about 1 to about 169 and from about 187 to about 211 of number 4, wherein the amino acids at positions about 169 and about 187 are linked by peptide bonds; and (e) of SEQ ID NO: 4. An isolated nucleic acid molecule having an amino acid sequence selected from the group consisting of about 59 to about 193 amino acids. 6. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is DNA. 7. A method of making a recombinant vector, the method comprising inserting the nucleic acid molecule of claim 1 into a vector operably linked to a promoter. 8. A recombinant vector produced by the method according to claim 7. 9. A method of producing a recombinant host cell, the method comprising the step of introducing the recombinant vector of claim 8 into a host cell. 10. A recombinant host cell produced by the method of claim 9. 11. A recombinant method of making a polypeptide, the method comprising:
A method comprising culturing the recombinant cell according to claim 10 under conditions such that the polypeptide is expressed and recovering the polypeptide. 12. An isolated polypeptide, which polypeptide comprises: (a) about 1 to about 211 amino acids of SEQ ID NO: 4; (b) about 2 to about 211 amino acids of SEQ ID NO: 4. (C) about 170 to about 186 amino acids of SEQ ID NO: 4; (d) about 1 to about 169 and about 187 to about 211 amino acids of SEQ ID NO: 4 at positions about 169 and about 187. An amino acid that is at least 95% identical to an amino acid selected from the group consisting of: (a) an amino acid wherein said amino acid is linked by a peptide bond; and (e) about 59 to about 193 amino acids of SEQ ID NO: 4. Polypeptide. 13. An isolated polypeptide, wherein at least 1
Except for one conservative amino acid substitution, the polypeptide has: (a) about 1 to about 211 amino acids of SEQ ID NO: 4; (b) about 2 to about 211 amino acids of SEQ ID NO: 4; (c) a sequence. (D) about 1 to about 169 and about 187 to about 211 amino acids of SEQ ID NO: 4, wherein the amino acids at positions about 169 and about 187 are peptide bonds. An isolated polypeptide having an amino acid sequence selected from the group consisting of: (a) an amino acid linked by; and (e) about 59 to about 193 amino acids of SEQ ID NO: 4. 14. An isolated polypeptide, which polypeptide comprises: (a) about 1 to about 211 amino acids of SEQ ID NO: 4; (b) about 2 to about 211 amino acids of SEQ ID NO: 4. (C) about 170 to about 186 amino acids of SEQ ID NO: 4; (d) about 1 to about 169 and about 187 to about 211 amino acids of SEQ ID NO: 4 at positions about 169 and about 187. An amino acid selected from the group consisting of: (a) an amino acid wherein said amino acids are linked by peptide bonds; and (e) about 59 to about 193 amino acids of SEQ ID NO: 4. 15. An epitope-bearing portion of the polypeptide of SEQ ID NO: 4. 16. The epitope-carrying portion according to claim 15, comprising 10 to 50 consecutive amino acids of SEQ ID NO: 4. 17. The epitope-carrying portion according to claim 15, which comprises amino acids RDGARSKRHQKFTH (SEQ ID NO: 5) and QLAHLHGILRR.
An epitope-bearing portion comprising a polypeptide selected from the group consisting of RQLY (SEQ ID NO: 6). 18. An isolated antibody that specifically binds to the polypeptide of claim 12. 19. An isolated antibody that specifically binds to the polypeptide of claim 13. 20. An isolated antibody that specifically binds to the polypeptide of claim 14. 21. A pharmaceutical composition comprising the polypeptide of claim 12 in combination with a pharmaceutically acceptable carrier. 22. A method of providing a nutritional supplement to cells of a patient in need thereof, the method comprising the step of providing the patient with a polynucleotide encoding the polypeptide of SEQ ID NO: 4. A method comprising the step of administering 23. The method of claim 22, wherein the patient suffers from Parkinson's disease. 24. A method of providing a nutritional supplement to cells of a patient in need thereof, said method comprising the step of administering to said patient a composition comprising the polypeptide of SEQ ID NO: 4. A method of including. 25. The method of claim 24, wherein the patient suffers from Parkinson's disease. 26. The method of claim 25, wherein the patient suffers from a substantia nigra. 27. A method of reducing a disease state in the brain of a human patient, the method comprising:
Wherein the disease state consists of slowing the degeneration of dopaminergic neurons in the human patient, restoring the function of the dopaminergic neurons and increasing the number of the dopaminergic neurons. Ameliorated by at least one method selected from the group, said method comprising the step of administering to said patient a pharmaceutically effective composition comprising a polypeptide having the amino acid sequence of SEQ ID NO: 4. 28. A method of alleviating a disease state in the brain of a human patient, the method comprising:
Wherein the disease state consists of slowing the degeneration of dopaminergic neurons in the human patient, restoring the function of the dopaminergic neurons and increasing the number of the dopaminergic neurons. Ameliorated by at least one method selected from the group, said method comprising administering to said patient a pharmaceutically effective composition comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4. . 29. The method of claim 28, wherein the polynucleotide has the sequence of SEQ ID NO: 3. 30. An mRN encoding FGF-20 in a patient-derived sample.
A kit for detecting the presence of A, the kit comprising a polynucleotide having at least 20 contiguous nucleotides of the nucleic acid of claim 1, packaged in a container. 31. A kit for detecting the presence of FGF-20 polypeptide in a sample from a patient, the kit packaged in a container.
A kit comprising the antibody according to 1. 32. A method of alleviating a cochlear-related disease state in a human patient, wherein the disease state slows cochlear degeneration in the human patient and restores the normal structure of the cochlea. Maintaining the normal structure of the cochlea,
Is mitigated by at least one method selected from the group consisting of:
A method comprising administering to said patient a pharmaceutically effective composition comprising a polypeptide having the amino acid sequence of SEQ ID NO: 4 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4. 33. The method of claim 32, wherein the disease state is otosclerosis; Kogan's syndrome; Meniere's disease; Pendred's syndrome; diabetes-related hearing impairment; congenital malformation; autoimmune disease. Related hearing impairment; Age related hearing impairment; FG
A method selected from the group consisting of hearing loss associated with F receptor deficiency; and ischemia-related hearing impairment. 34. An isolated nucleic acid molecule, the nucleic acid molecule comprising: (a) a polynucleotide encoding amino acids from about 1 to about 212 of SEQ ID NO: 2; (b) about SEQ ID NO: 2. (C) a polynucleotide encoding the amino acids of about 1 to about 169 of SEQ ID NO: 2; (d) a polynucleotide encoding the amino acids of about 187 to about 212 of SEQ ID NO: 2; (E) a polynucleotide encoding the amino acids of about 170 to about 186 of SEQ ID NO: 2; (f) a polynucleotide encoding the amino acids of about 59 to about 193 of SEQ ID NO: 2; A polynucleotide encoding 30 consecutive amino acids; (h) a fragment of the polypeptide set forth as amino acids 1-212 of SEQ ID NO: 2 A polynucleotide encoding the amino acid sequence of claim 1, wherein the fragment has fibroblast growth factor activity; (i) the amino acids of the fragment of the polypeptide shown as amino acids 1-212 of SEQ ID NO: 2. A polynucleotide encoding a sequence, wherein the fragment has fibroblast growth factor activity; (j) (a), (b), (c), (d), (e) , (F), (g), (h) or (i) the polynucleotide complement; and (k) (a), (b), (c), (d), (e), (f), An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: a polynucleotide that is at least 90% identical to the polynucleotide of (g), (h) or (i). 35. An isolated nucleic acid molecule comprising 20-600 contiguous nucleotides from the coding region of SEQ ID NO: 1. 36. The isolated nucleic acid molecule of claim 35, comprising 60-400 contiguous nucleotides from the coding region of SEQ ID NO: 1. 37. The isolated nucleic acid molecule of claim 36, comprising 200 to 300 contiguous nucleotides from the coding region of SEQ ID NO: 1. 38. An isolated nucleic acid molecule, wherein the nucleic acid molecule comprises a polynucleotide encoding the polypeptide, wherein, except for at least one conservative amino acid substitution, the polypeptide is: (A) about 1 to about 212 amino acids of SEQ ID NO: 2; (b) about 2 to about 212 amino acids of SEQ ID NO: 2; (c) about 170 to about 186 amino acids of SEQ ID NO: 2; (d) sequence Amino acid from about 1 to about 169 and about 187 to about 212 of number 2, wherein the amino acids at positions about 169 and about 187 are linked by a peptide bond; and (e) of SEQ ID NO: 4. An isolated nucleic acid molecule having an amino acid sequence selected from the group consisting of about 59 to about 193 amino acids. 39. The isolated nucleic acid molecule of claim 34, wherein the nucleic acid molecule is DNA. 40. A method of making a recombinant vector, the method comprising inserting the nucleic acid molecule of claim 34 into a vector operably linked to a promoter. 41. A recombinant vector produced by the method of claim 40. 42. A method of producing a recombinant host cell, the method comprising the step of introducing the recombinant vector of claim 41 into a host cell. 43. A recombinant host cell produced by the method of claim 42. 44. A recombinant method of making a polypeptide, the method comprising:
44. A method comprising the steps of culturing the recombinant cell of claim 43 under conditions such that the polypeptide is expressed and recovering the polypeptide. 45. An isolated polypeptide, the polypeptide comprising: (a) about 1 to about 212 amino acids of SEQ ID NO: 2; (b) about 2 to about 212 of SEQ ID NO: 2. (C) about 170 to about 186 amino acids of SEQ ID NO: 2; (d) about 1 to about 169 and about 187 to about 212 amino acids of SEQ ID NO: 2 at positions about 169 and about 187. An amino acid that is at least 95% identical to an amino acid selected from the group consisting of: (a) an amino acid wherein said amino acid is linked by a peptide bond; and (e) about 59 to about 193 amino acids of SEQ ID NO: 2. Polypeptide. 46. An isolated polypeptide, wherein at least 1
Except for one conservative amino acid substitution, the polypeptide has: (a) about 1 to about 212 amino acids of SEQ ID NO: 2; (b) about 2 to about 212 amino acids of SEQ ID NO: 2; (c) a sequence. (D) about 1 to about 169 and about 187 to about 212 amino acids of SEQ ID NO: 2, wherein said amino acids at positions about 169 and about 187 are peptide bonds. An isolated polypeptide having an amino acid sequence selected from the group consisting of: (a) an amino acid linked by; and (e) about 59 to about 193 amino acids of SEQ ID NO: 2. 47. An isolated polypeptide, which polypeptide comprises: (a) about 1 to about 212 amino acids of SEQ ID NO: 2; (b) about 2 to about 212 of SEQ ID NO: 2. (C) about 170 to about 186 amino acids of SEQ ID NO: 2; (d) about 1 to about 169 and about 187 to about 212 amino acids of SEQ ID NO: 2 at positions about 169 and about 187. An amino acid selected from the group consisting of: (a) the amino acids of SEQ ID NO: 2, wherein the amino acids are linked by peptide bonds; and (e) the amino acids of about 59 to about 193 of SEQ ID NO: 2. 48. An epitope-bearing portion of the polypeptide of SEQ ID NO: 2. 49. The epitope-bearing portion of claim 48, which comprises 10-50 consecutive amino acids of SEQ ID NO: 2. 50. An isolated antibody that specifically binds to the polypeptide of claim 45. 51. An isolated antibody that specifically binds to the polypeptide of claim 46. 52. An isolated antibody that specifically binds to the polypeptide of claim 47.