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WO2001000662A2 - Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques - Google Patents

Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques Download PDF

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Publication number
WO2001000662A2
WO2001000662A2 PCT/US2000/011885 US0011885W WO0100662A2 WO 2001000662 A2 WO2001000662 A2 WO 2001000662A2 US 0011885 W US0011885 W US 0011885W WO 0100662 A2 WO0100662 A2 WO 0100662A2
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Prior art keywords
hfgf
polypeptide
cells
nucleic acid
dna
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WO2001000662A3 (fr
Inventor
Jai Pal Singh
Asavari Prasad Wagle
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Eli Lilly and Co
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Eli Lilly and Co
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Priority to AU58666/00A priority Critical patent/AU5866600A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to recombinant DNA technology.
  • the invention pertains to pharmaceutical and other uses of human FGF-8 genes and their encoded proteins.
  • the compounds and compositions of the present invention can be used alone or in conjunction with other neurotrophic, neuroprotective, thrombolytic, and/or anti-thrombotic agents, in methods for treating or preventing neuronal damage caused by disease or trauma.
  • Stroke is the third leading cause of death and disability in the United States. With an excess of 500,000 new occurrences each year, strokes are responsible for about 300,000 deaths annually in the U.S. alone. Strokes are also a leading cause of hospital admissions and long-term disabilities. Accordingly, the socioeconomic impact of stroke and its attendant burden on society is immense.
  • Stroke is defined by the World Health Organization as a rapidly developing clinical sign of focal or global disturbance of cerebral function with symptoms lasting at least 24 hours. More than 85% of strokes are due to a thrombo-embolic occlusion of a cerebral artery, resulting in death of brain tissue. With complete occlusion, arrest of cerebral circulation causes cessation of neuronal electrical activity within seconds. Within a few minutes after the deterioration of the energy state and ion homeostasis, depletion of high energy phosphates, membrane ion pump failure, efflux of cellular potassium, influx of sodium chloride and water, and membrane depolarization occur. If the occlusion persists for more than five to ten minutes, irreversible damage results.
  • ischemia can be lethal, generally, ischemia is not total after a thrombotic occlusion of a cerebral vessel. Even with incomplete ischemia, however, the outcome is difficult to evaluate and depends largely on residual perfusion and the availability of oxygen.
  • ischemic event When the ischemic event is moderate it generally results in a region of collaterally perfused tissue having paralyzed neuronal function surrounding a core of severe ischemia where the infarct developed.
  • the zone of damaged brain tissue called a " penumbra” , consists of brain tissue in a state between life and death. Oftentimes, the loss of neuronal function can be reversed with restoration of adequate perfusion.
  • Cerebral infarction commonly results in deficits of motor, sensory, visual, or cognitive function. Recovery involves dendritic and axonal sprouting and new synapse formation in the damaged tissue or other hemisphere. The eventual extent of neurologic recovery depends on the patient's age and general state of health as well as on the site and size of the infarction. Impaired consciousness, mental deterioration, or aphasia, suggest a poor prognosis. About 50% of patients with moderate or severe hemiplegia, and most of those with lesser deficits, recover functionally and are ultimately able to care for their basic needs, have a clear senso ⁇ um, and can walk adequately, although use of an affected limb may be limited. Although some patients continue to improve slowly, any deficit remaining after 6 months is likely to be permanent. Furthermore, recurrence of cerebral infarction is relatively common, and each recurrence is likely to add to the neurologic disability.
  • Parkinson's disease results from the loss of dopaminergic neurons in the substantia nigra.
  • Nitric oxide (NO) and oxidative stress are thought to play important roles in the pathogenesis of many neurodegenerative disorders including neuronal dysfunction after stroke or head trauma, retinal degeneration,
  • NO Alzheimer's disease and Parkinson's disease. NO also plays a role in neuropathic pain, migraine, psychoses, angiogenesis, and vasculogenesis .
  • GDNF Glial Cell Line- Derived Neurotrophic Factor
  • Insulin and insulin- like growth factors have been shown to stimulate growth of neurites in rat pheochromocytoma PC12 cells and in cultured sympathetic and sensory neurons (Recio-Pinto et al . , J. Neurosci . , 6:1211-1219 (1986)).
  • fibroblast growth factor-2 bFGF
  • bFGF fibroblast growth factor-2 stimulates neural proliferation and growth (D. Gospodarowicz , et al . , Cell Differ., 19:1 (1986); M. A.
  • FGF-8 is composed of 215 amino acids and displays a 30-40% homology to other members of the FGF family.
  • the human FGF-8 gene has been mapped to chromosome 10q24 and is induced by androgen in breast cancer cells (Payson, et al . 1996) .
  • FGF-8 is also subject to a complex alternative splicing process that generates transcripts with coding potential for different FGF-8 isoforms (Blunt, et al . , J.
  • the mouse FGF-8 gene encodes a total of eight isoforms
  • the human gene encodes only 4 isoforms (FGF-8a, b, e, and f) due to a premature stop codon in exon IB ORF (see Figure 1; Gemel, et al . , Genomics, 35(1), 253-7, (1996)).
  • the human FGF-8a and FGF-8b are 100% identical to their mouse counterpart while the FGF-8e and FGF-8f are 98% identical to their mouse counterpart.
  • the pharmacological therapies for cerebral focal stroke and/or other neural inflictions are limited to symptomatic treatments that do not alter the development and/or fate of the damaged tissue.
  • the present invention provides compounds useful for treating, inhibiting, and/or preventing the process of neuro-degeneration caused by disease or trauma.
  • the present invention provides compounds and pharmaceutical compositions comprising human fibroblast growth factor- 8 (hFGF-8) polypeptides, or nucleic acids encoding hFGF-8 polypeptides, which possess neuronal activity.
  • the compounds and compositions can be used either alone or in conjunction with at least one other neurotrophic, neuroprotective, thrombolytic, and/or anti- thrombotic agent, such as nerve growth factor, in methods for treating or preventing neuronal damage caused by disease or trauma.
  • the compositions and methods of the present invention may be particularly useful for treating or preventing neuronal damage resulting from NO mediated toxicity.
  • compositions and methods of the present invention may be particularly useful for enhancing recovery of neuronal function.
  • the present invention provides, in one aspect, isolated nucleic acid molecules comprising polynucleotides, or complements thereof, encoding specific hFGF-8 polypeptides, as well as fragments, or variants thereof, which can be used in methods for treating or preventing neuronal damage caused by disease or trauma.
  • Nucleic acids encoding hFGF-8 full- length polypeptides, domains, fragments and/or variants thereof corresponding to at least 90-100% of the contiguous amino acids of at least one of SEQ ID NOS:4, 5, 6, and 7, are provided as non- limiting examples of the compounds and compositions of the present invention.
  • the present invention also provides methods of using said nucleic acids, vectors comprising 1-40 of said isolated hFGF-8 nucleic acid molecules, and/or host cells containing said nucleic acids and/or recombinant vectors, to produce hFGF-8 nucleic acids and/or polypeptides for use m treating or preventing neuronal damage caused by disease or trauma.
  • the present invention provides hFGF-8 polypeptides or fragments thereof which are highly potent in inducing enhanced growth of axonal and dendritic neuronal processes.
  • the present invention also provides methods of using an isolated hFGF-8 polypeptide, comprising at least one fragment, domain, or specified variant of at least 90-100% of the contiguous ammo acids of at least one of SEQ ID NOS : 4 , 5, 6, and 7 to treat or prevent neuronal damage caused by disease or trauma.
  • the present invention also provides methods of using an isolated hFGF-8 polypeptide as described herein, wherein the polypeptide further comprises at least one substitution, insertion, or deletion corresponding to portions or residues of at least one of SEQ ID NOS : 4 , 5, 6, and 7 to treat or prevent neuronal damage caused by disease or trauma.
  • the present invention also provides a method of using an isolated hFGF-8 polypeptide, as described herein, to treat or prevent neuronal damage caused by disease or trauma, and wherein said polypeptide has at least one activity, such as, but not limited to, m vi tro neurite outgrowth, in vi tro neuroprotection, in vivo neuroprotection, and in vivo recuperation.
  • activities can be determined by known methods (Wagner, J. A., et al . , J. of Cell Biol., 103 (4) : 1363-7 (1986); ein eta al . ,
  • Neuron 15:597-605 (1995); Neufeld, G., et al . , J. of Cell. Phys., 131 (1) .131-40 (1987); Maiese, K. , et al . , J. of Neurosci., 13 (7) : 3034-40 (1993); Cheng, B., et al . , Neuron, 7(6):1031-41 (1991); Fisher, M., et al . , J. of Cereb. Blood Flow Sc Metabol., 15(6):953-9 (1995); Jiang, N., et al . , J. Neuro.
  • the invention provides a method for inducing growth of a neuronal cell by contacting the cell with an amount of hFGF-8 polypeptide or an hFGF-8 encoding polynucleotide (in operable linkage with appropriate transcription elements) effective for inducing growth of the neuron either in vi tro or in vivo .
  • a further embodiment of the present invention provides a method for inducing migration of a neuronal cell by contacting the cell with an amount of hFGF-8 polypeptide or an hFGF-8 encoding polynucleotide effective for inducing migration of the neuron.
  • the present invention also provides a method of treating and/or preventing neuronal damage caused by a disease or trauma wherein said method comprises administering to a patient a therapeutically effective amount of a composition comprising an isolated hFGF-8 nucleic acid and/or polypeptide as described herein and a carrier or diluent.
  • the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods.
  • the invention provides a method for treating and/or preventing neuronal damage caused by disease or trauma wherein said method comprises administering a patient a therapeutically effective amount of an hFGF-8 polypeptide or an hFGF-8 encoding polynucleotide .
  • said methods are used for treating or preventing neuronal damage caused by a disease or trauma
  • said disease or trauma includes, but is not limited to, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, muscle injury, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed invertebrae disk, cervical spondylosis, plexus disorders, thoracic outlet destruction, peripheral neuropathy, such as those caused by lead, dapsone, ticks or porphyria, peripheral myelin disorders, Alzheimer's disease, Gullain-Barre syndrome, Parkinson's disease, Parkinsonian disorders, ALS, multiple sclerosis, other central myelin disorders, stroke, ischemia associated with stroke, neural paropathy, other neural degenerative diseases, motor neuron diseases, sciatic crush, neuropathy associated with diabetes, spinal cord injuries, facial nerve crush, chemotherapy- or pharma
  • the present invention provides methods for treating and/or preventing neuronal damage caused by disease or trauma comprising the administration of compositions comprising isolated, natural, recombinant, and/or synthetic nucleic acid molecules, or complements thereof, encoding at least one hFGF-8 polypeptide to a patient in need thereof.
  • An hFGF-8 polypeptide of the invention comprises at least one fragment, domain, and/or variant as a portion or fragment of an hFGF-8 protein as described herein, having neuronal activity.
  • a “polnucleotide” comprises at least 10-20 nucleotides of a nucleic acid (RNA, DNA or combination thereof) , provided by any means, such as synthetic, recombinant isolation or purification method steps.
  • complementary or “ complementarity” as used herein refer to the capacity of purine, pyrimidine, synthetic or modified nucleotides to associate by partial or complete complementarity through hydrogen or other bonding to form partial or complete double- or triple-stranded nucleic acid molecules.
  • Partial complementarity refers to association of two or more bases by one or more hydrogen bonds or attraction that is less than the complete complementarity as described above. Partial or complete complementarity can occur between any two nucleotides, including naturally occurring or modified bases, e.g., as listed in 37 CFR ⁇ 1.822. All such nucleotides are included in polynucleotides of the invention as described herein.
  • fusion protein denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.
  • polypeptide also includes such fusion proteins.
  • host cell refers to any eucaryotic, procaryotic, or fusion or other cell or pseudo cell or membrane-containing construct that is suitable for propagating and/or expressing an isolated nucleic acid that is introduced into a host cell by any suitable means known in the art (e.g., but not limited to, transformation or transfection, or the like) , or induced to express an endogenous nucleic acid encoding an hFGF-8 polypeptide according to the present invention.
  • the cell can be part of a tissue or organism, isolated in culture or in any other suitable form.
  • hybridization refers to a process in which a partially or completely single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. Hybridization can occur under conditions of low, moderate or high stringency, with high stringency preferred. The degree of hybridization depends upon, for example, the degree of homology, the stringency conditions, and the length of hybridizing strands as known in the art .
  • Reference to " isolated nucleic acid molecule (s)" intends a nucleic acid molecule, DNA, RNA, or both which has been removed from its native or naturally occurring environment.
  • recombinant nucleic acid molecules contained or generated in culture, a vector and/or a host cell are considered isolated for the purposes of the present invention.
  • isolated nucleic acid molecules include recombinant nucleic acid molecules maintained in heterologous host cells or purified (partially or substantially) nucleic acid molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro R ⁇ A transcripts of the nucleic acid molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically, purified from or provided in cells containing such nucleic acids, where the nucleic acid exists in other than a naturally occurring form, quantitatively or qualitatively.
  • isolated used in reference to at least one polypeptide of the invention describes a state of isolation such that the peptide or polypeptide is not in a naturally occurring form and/or has been purified to remove at least some portion of cellular or non-cellular molecules with which the protein is naturally associated.
  • isolated may include the addition of other functional or structural polypeptides for a specific purpose, where the other peptide may occur naturally associated with at least one polypeptide of the present invention, but for which the resulting compound or composition does not exist naturally.
  • neurite outgrowth includes dendritic and axonal outgrowth from neuronal cells.
  • neurite One of skill in the art will be familiar with the term neurite. More often, hFGF-8 activity is associated with dendritic outgrowth, and therefore the term dendritic growth or outgrowth is often used herein.
  • a " nucleic acid probe,” " oligonucleotide probe,” or “ probe” as used herein comprises at least one detectably labeled or unlabeled nucleic acid which hybridizes under specified hybridization conditions with at least one other nucleic acid. This term also refers to a single- or partially double-stranded nucleic acid, oligonucleotide or polynucleotide that will associate with a complementary or partially complementary target nucleic acid to form at least a partially double-stranded nucleic acid molecule.
  • a nucleic acid probe may be an oligonucleotide or a nucleotide polymer.
  • a probe can optionally contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe, termed a " detectable probe” or " detectable nucleic acid probe.”
  • detectable probe or detectable nucleic acid probe.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial , intrasternal , intrathecal , intrahepatic, intralesional and intracranial injection or infusion techniques .
  • a " primer” is a nucleic acid fragment or oligonucleotide which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule, e.g., using an amplification reaction, such as, but not limited to, a polymerase chain reaction (PCR) , as known in the art.
  • PCR polymerase chain reaction
  • stringency refers to hybridization conditions for nucleic acids in solution. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have much less of this effect. Stringency may be altered, for example, by changes in temperature and/or salt concentration, or other conditions, as well known in the art.
  • a non-limiting example of " high stringency” conditions includes, for example, (a) a temperature of about 42°C , a formamide concentration of about ⁇ 20%, and a low salt (SSC) concentration, or, alternatively, a temperature of about 65°C, or less, and a low salt (SSPE) concentration; (b) hybridization in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C (See, e.g., Ausubel, et al . , ed. , Current Protocols in Molecular Biology, 1987-1998, Wiley Interscience, New York, at ⁇ 2.10.3) .
  • SSC comprises a hybridization and wash solution.
  • a stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.
  • SSPE comprises a hybridization and wash solution.
  • a IX SSPE solution contains 180 mM NaCl, 9mM Na2HP04 , 0.9 mM NaH2P04 and 1 mM EDTA, pH 7.4.
  • the term " vector” as used herein refers to a nucleic acid compound used for introducing exogenous or endogenous nucleic acid into host cells.
  • a vector comprises a nucleotide sequence which may encode one or more polypeptide molecules.
  • Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non- limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated nucleic acid molecule.
  • nucleic acid molecule of the present invention encoding an hFGF-8 polypeptide can be obtained using well-known methods.
  • Nucleic acid molecules used in the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof.
  • the DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated nucleic acid molecules used in the present invention include nucleic acid molecules comprising an open reading frame (ORF) shown in at least one of SEQ ID NOS : 1 , 2, 3, and 8; nucleic acid molecules comprising the coding sequence for an hFGF-8 polypeptide; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one hFGF-8 polypeptide as described herein.
  • ORF open reading frame
  • nucleic acid molecules comprising the coding sequence for an hFGF-8 polypeptide
  • the genetic code is well known in the art.
  • the nucleic acid molecules necessary to practice an embodiment of the present invention are provided encoding the mature hFGF-8 polypeptide or the full-length hFGF-8 polypeptide lacking the N-terminal methionine.
  • the invention can also utilize an isolated nucleic acid molecule having the nucleotide sequence shown in at least one of SEQ ID NOS : 1 , 2, 3, and 8, or the nucleotide sequence of the hFGF-8 cDNA contained in at least one of the above-described deposited clones listed herein, or a nucleic acid molecule having a sequence complementary thereto.
  • Such isolated molecules are useful as probes for gene mapping by in situ hybridization with chromosomes, and for detecting transcription, translation and/or expression of the hFGF-8 gene in human tissue, for instance, by Northern blot analysis for mRNA detection.
  • nucleotide sequences identified by sequencing a nucleic acid molecule herein can be or were identified using an automated nucleic acid sequencer, and all amino acid sequences of polypeptides encoded by nucleic acid molecules identified herein can be or were identified by codon correspondence or by translation of a nucleic acid sequence identified using method steps as described herein or as known in the art. Therefore, as is well known in the art that for any nucleic acid sequence identified by this automated approach, any nucleotide sequence identified herein may contain some errors which are reproducibly correctable by resequencing based upon an available or a deposited vector or host cell containing the nucleic acid molecule using known methods.
  • Nucleotide sequences identified by automation are typically at least about 95% to at least about 99.999% identical to the actual nucleotide sequence of the sequenced nucleic acid molecule.
  • the actual sequence can be more precisely identified by other approaches including manual nucleic acid sequencing methods well known in the art.
  • a single insertion or deletion in an identified nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the identified amino acid sequence encoded by an identified nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced nucleic acid molecule, beginning at the point of such an insertion or deletion.
  • the present invention can further depend upon fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule is meant a molecule having at least 10 nucleotides of a nucleotide sequence of a deposited cDNA or a nucleotide sequence shown in at least one of SEQ ID NOS : 1 , 2, 3, and 8, and is intended to mean fragments at least about 10 nucleotides, and at least about 40 nucleotides in length, which are useful, inter alia as diagnostic probes and primers as described herein.
  • fragments such as at least about 50, 100, 120, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, and/or 4000 or more nucleotides in length, are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence (or the deposited cDNA) as shown at least one of SEQ ID NOS : 1 , 2, 3, and 8.
  • fragments which include 10 or more contiguous nucleotides from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOS : 1 , 2, 3, 8 , or consensus sequences thereof, as determined by methods known in the art (See e.g., Ausubel, supra , Chapter 7) .
  • nucleotide fragments are useful according to the present invention for screening DNA sequences that code for one or more fragments of an hFGF-8 polypeptide as described herein.
  • Such screening can include the use of so-called " DNA chips" for screening DNA sequences of the present invention of varying lengths, as described, e.g., in U.S. Patent Nos. 5,631,734, 5,624,711, 5,744,305, 5,770,456, 5,770,722, 5,675,443, 5,695,940, 5,710,000, 5,733,729, which are entirely incorporated herein by reference .
  • nucleic acid molecules useful according to the present invention may comprise a nucleic acid encoding an hFGF-8 polypeptide including, but not limited to, those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as the coding sequence of at least one signal leader or fusion peptide or of the mature polypeptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA) ; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding a polypeptide can be fused to a marker sequence, such as
  • the invention may use a polynucleotide (either DNA or RNA) that comprises at least about 20 nt, still more preferably at least about 30 nt , and even more preferably at least about 30-2000 nt of a nucleic acid molecule described herein. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly (A) of an hFGF- 8 cDNA shown in at least one of SEQ ID NOS : 1 , 2, 3, 8 , or to a complementary stretch of T (or U) resides, would not be included in a probe of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • the present invention may also utilize subsequences of full-length nucleic acids.
  • any number of subsequences can be obtained by reference to at least one of SEQ ID NOS : 1 , 2, 3, 8, or a complementary sequence, and using primers which selectively amplify, under stringent conditions to: at least two sites to the polynucleotides of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it.
  • a variety of methods for obtaining 5' and/or 3' ends is well known in the art. See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in M. A.
  • the present invention provides hFGF-8 polynucleotides having the sequence of the hFGF-8 gene, nuclear transcript, cDNA, or complementary sequences and/or subsequences thereof .
  • Primer sequences can be obtained by reference to a contiguous subsequence of a polynucleotide of the present invention.
  • Primers are chosen to selectively hybridize, under PCR amplification conditions, to a polynucleotide of the present invention in an amplification mixture comprising a genomic and/or cDNA library from the same species.
  • the primers are complementary to a subsequence of the amplified nucleic acid.
  • the primers will be constructed to anneal at their 5' terminal ends to the codon encoding the carboxy or amino terminal amino acid residue (or the complements thereof) of the polynucleotides of the present invention.
  • the primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50.
  • the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length or any range or value therein.
  • a non-annealing sequence at the 5' end of the primer (a " tail" ) can be added, for example, to introduce a cloning site at the terminal ends of the amplified DNA.
  • the amplification primers may optionally be elongated in the 3' direction with additional contiguous or complementary nucleotides from the polynucleotide sequences, such as at least one of SEQ ID NOS : 1 , 2, 3, and 8, from which they are derived.
  • the number of nucleotides by which the primers can be elongated is selected from the group of integers consisting of from at least 1 to at least 25.
  • the primers can be elongated with an additional 1, 5, 10, or 15 nucleotides or any range or value therein.
  • a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence, or to add useful sequences, such as links or restriction sites (See e.g., Ausubel, supra, Chapter 15).
  • the amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra.
  • the resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention.
  • Methods for protein synthesis from PCR derived templates are known in the art (See e.g., Ausubel, supra, Chapters 9, 10, 15; Coligan, Current Protocols in Protein Science, supra, Chapter 5) and available commercially. See, e.g., Amersham Life Sciences, Inc., Catalog '97, p. 354.
  • polynucleotide as Described Herein may also utilize isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotides disclosed herein, e.g., SEQ ID NOS : 1 , 2, 3, and 8.
  • the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides used in the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides may be genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
  • the cDNA library comprises at least 80% full- length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences .
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
  • embodiments of the present invention make use of isolated nucleic acids comprising hFGF-8 polynucleotides, wherein the polynucleotides are complementary to the polynucleotides described herein, above.
  • complementary sequences base pair throughout the entirety of their length with such polynucleotides (i.e., have 100% sequence identity over their entire length) .
  • Complementary bases associate through hydrogen bonding in double-stranded nucleic acids.
  • base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil (See, e.g., Ausubel, supra , Chapter 67; or Sambrook, supra) .
  • the isolated nucleic acids useful in the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well known in the art.
  • the nucleic acids may conveniently comprise sequences m addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidme marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the polynucleotide sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.
  • cloning and/or expression sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
  • Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (See, e.g., Ausubel, supra , Chapters 1-5; or Sambrook, supra) .
  • RNA, cDNA, genomic DNA, or a hybrid thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art (See, e.g., Ausubel, supra, Chapters 1-7; or Sambrook, supra) .
  • a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. Temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide can control the degree of stringency.
  • polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50% conveniently varies the stringency of hybridization.
  • degree of complementarity sequence identity
  • the degree of complementarity will optimally be 100%; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • RNA amplification processes include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al . ; 4,795,699 and 4,921,794 to Tabor, et al ; 5,142,033 to Innis; 5,122,464 to Wilson, et al .
  • PCR polymerase chain reaction
  • RNA mediated amplification which uses anti- sense RNA to the target sequence as a template for double- stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek, et al , with the tradename NASBA) , the entire contents of which are herein incorporated by reference (See, e.g., Ausubel, supra, Chapter 15; or Sambrook, supra) .
  • PCR polymerase chain reaction
  • in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • examples of techniques sufficient to direct persons of skill through in vi tro amplification methods are found in Berger, Sambrook, and Ausubel (e.g., Chapter 15) supra, as well as Mullis, et al . , U.S. Patent No.
  • kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).
  • the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
  • the isolated nucleic acids required to practice the present invention can also be prepared by direct chemical synthesis by methods such as the phosphot ⁇ ester method of Narang, et al . , Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al . , Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al . , Tetra. Letts. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts.
  • the present invention can be practiced using recombinant expression cassettes comprising an hFGF-8 nucleic acid.
  • a nucleic acid sequence useful in the present invention for example a cDNA or a genomic sequence encoding a full-length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into at least one desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids comprising the present invention. These promoters can also be used, for example, m recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter hFGF-8 content and/or composition in a desired tissue.
  • isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non- heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
  • endogenous promoters can be altered m vivo or m vi tro by mutation, deletion and/or substitution.
  • a polynucleotide can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable characteristics . Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes.
  • a variety of cross- linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect and/or cleave nucleic acids. Knorre, et al . , Biochimie 67:785-789 (1985); Vlassov, et al . , Nucleic Acids Res.
  • the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of hFGF-8 polypeptides or fragments thereof by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al . , supra ; Ausubel, supra , Chapters 1-9, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host .
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs , or any other suitable promoter.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs , or any other suitable promoter.
  • the skilled artisan will know other suitable promoters.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome- binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with VAA and VAG preferred for mammalian or eukaryotic cell expression.
  • a translation initiating at the beginning e.g., UAA, UGA or UAG
  • a termination codon e.g., UAA, UGA or UAG
  • Expression vectors will preferably include at least one selectable marker.
  • markers include, e.g., ampicillin, dihydrofolate reductase, hygromycin, or neomycin (G418) resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria or prokaryotics .
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli , Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • Vectors preferred for use in bacteria include pQE70, pQE60 and pQE- 9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Preferred eucaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3 , pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan. See, e.g.,
  • Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra , Chapters 1, 9, 13, 15, 16.
  • Polypeptide (s) required to practice the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions.
  • a region of additional amino acids can be added to the N-terminus of a polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
  • peptide moieties can be added to a polypeptide to facilitate purification. Such regions can be removed prior to final preparation of a polypeptide.
  • a protein of the used in the present invention m a recombmantly engineered cell, such as bacteria, yeast, insect, or mammalian cells.
  • the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
  • the expression of isolated nucleic acids encoding a protein according to the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible) followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
  • expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
  • modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional ammo acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites, termination codons, or purification sequences .
  • hFGF-8 nucleic acids can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a polypeptide of the present invention.
  • Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
  • Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel, et al . , Nucleic Acids Res.
  • selection markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol .
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transformed with the plasmid vector DNA.
  • Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al . , Gene 22:229-235 (1983); Mosbach, et al . , Nature 302:543-545 (1983)). See, e.g., Ausubel, supra, Chapters 1-3, 16 (Sec.1); and Coligan, supra , Current Protocols in Protein Science, Units 5.1, 6.1-6.7.
  • a variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a nucleic acid useful in the present invention can be expressed in these eukaryotic systems.
  • yeast Synthesis of heterologous proteins in yeast is well known.
  • F. Sherman, et al . Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well -recognized work describing the various methods available to produce the protein in yeast.
  • Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.
  • Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known m the art and available from commercial suppliers (e.g., Invitrogen) .
  • Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a hFGF-8 protein once expressed, can be isolated from yeast by lysmg the cells and applying standard protein isolation techniques to the lysates.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques .
  • sequences encoding proteins comprising the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
  • Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter) , an enhancer (Queen, et al . , Immunol. Rev. 89:49 (1986)), and processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site) , and transcriptional terminator sequences.
  • Other animal cells useful for production of proteins of the present invention are available, for stance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7 th edition, 1992) .
  • Appropriate vectors for expressing proteins used m the present invention in insect cells are usually derived from the SF9 baculovirus.
  • suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider, J. Embryol. Exp. Morphol . 27:353-365 (1987).
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene . Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VPl intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. M. Saveria- Campo, Bovine Papilloma Virus DNA, a Eukaryotic Cloning
  • hFGF-8 polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography. Most preferably, high performance liquid chromatography ("HPLC" ) is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eucaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention can be glycosylated or can be non-glycosylated.
  • polypeptides of the invention can also include an initial modified methionine residue, in some cases as a result of host -mediated processes . Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.37- 17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • the invention may furthermore use isolated hFGF-8 polypeptides having fragments or specified variants of the amino acid sequence encoded by the deposited cDNAs , or the amino acid sequence in SEQ ID NOS : 4 , 5, 6, and 7.
  • the isolated proteins used in the present invention comprise a polypeptide encoded by any one of the polynucleotides of the present invention as discussed more fully, supra, or polypeptides which are fragments or variants thereof.
  • hFGF-8 polypeptides as described herein which possess neuroprotective, neuronal recovery, neural migration inducing, or dendritic or axonal growth promoting effects on neurons.
  • Exemplary polypeptide sequences are provided in SEQ ID NOS:4, 5, 6, and 7.
  • hFGF-8 proteins or variants thereof can comprise any number of contiguous ammo acid residues from an hFGF-8 polypeptide, wherein that number is selected from the group of integers consisting of from 90-100% of the number of contiguous residues in a full-length hFGF-8 polypeptide.
  • this subsequence of contiguous ammo acids is at least 50,
  • the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2 , 3, 4, or 5.
  • an hFGF-8 polypeptide component of the present invention can include one or more ammo acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • ammo acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of ammo acid substitutions, insertions or deletions for any given hFGF-8 polypeptide will not be more than 40, 30, 20, 10, 5, or 3, such as 1-30 or any range or value therein, as specified herein.
  • Ammo acids in an hFGF-8 polypeptide that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alan e-scannmg mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)) . The latter procedure introduces single alanine mutations at every residue in the molecule.
  • hFGF-8 polypeptides required to practice the present invention can include but are not limited to, at least one selected from SEQ ID NOS:4, 5, 6, and 7.
  • An hFGF-8 polypeptide can further comprise a polypeptide of at least one of 204, 215, 233, and 244, contiguous ammo acids of SEQ ID NOS : 4 , 5, 6, and 7, respectively.
  • An hFGF-8 polypeptide further includes an ammo acid sequence selected from one or more of SEQ ID NOS : 4 , 5, 6, and 7.
  • Non- limiting mutants that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution, insertion or deletion of SEQ ID NOS : 4 , 5, 6 , and 7.
  • the present invention also provides a composition comprising an isolated hFGF-8 nucleic acid and/or polypeptide as described herein, and a carrier or diluent.
  • the invention provides methods for stimulating nerve growth, neurite outgrowth, neuronal migration, recovery of function, and/or preventing neurodegeneration.
  • the method used to stimulate nerve growth, neurite outgrowth, neuronal migration, recovery of function, and/or preventing neurodegeneration comprises administering to a non-human animal a pharmaceutically acceptable composition comprising any of the hFGF-8 compounds described herein and a pharmaceutically acceptable carrier.
  • the amount of compound utilized in these methods is between about 0.01 and 10 mg/kg body weight/day.
  • the present invention also provides a composition
  • a composition comprising an isolated hFGF-8 nucleic acid and/or polypeptide as described herein, at least one other neurotrophic, neuroprotective, thrombolytic, and/or anti- thrombotic agent, and a carrier or diluent.
  • the other neurotrophic, neuroprotective, thrombolytic, and/or anti- thrombotic agents may include, but are not limited to, nerve growth factor (NGF) , insulin growth factor (IGF-1) and its active truncated derivatives such as gIGF-1, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively) , platelet-derived growth factors (PDGF) , brain-derived neurotrophic factor (BDNF) , ciliary neurotrophic factors (CNTF) , glial cell line-derived neurotrophic factor (GDNF) , neurotrophin-3 (NT-31, neurotrophin 4/S (NT-4/S) , or any of the compounds described in WO 97/36869, WO 96/41609, WO 97/16190, WO96/40633, WO 97/18828, WO 96/40140 or WO 98/13355.
  • NGF nerve growth factor
  • IGF-1 insulin growth factor
  • the most preferred neurotrophic agent in the compositions of the present invention is NGF.
  • this invention provides methods for stimulating nerve growth, neurite outgrowth, neuronal migration, neuronal recovery of function, and/or preventing neurodegeneration in a patient in need thereof.
  • said method comprises administering to the patient a pharmaceutically acceptable composition comprising any of the hFGF-8 compounds of this invention in conjunction with at least one other neurotrophic, neuroprotective, thrombolytic, and/or anti-thrombotic agent, and a pharmaceutically acceptable carrier.
  • compositions of the present invention maybe administered orally, parenterally, by inhalationspray topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • the pH of the formulation maybe adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersion wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1, 3-butanediol .
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1, 3-butanediol .
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or di-glycerides .
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long- chain alcohol diluent or dispersant, such as Ph
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers which are commonly used include lactose, corn starch, dicalcium phosphate and microcrystalline cellulose (Avicel) .
  • Lubricating agents such as magnesium stearate and talc, are also typically added.
  • useful diluents include lactose, dried corn starch and TPGS , as well as the other diluents used in tablets.
  • useful diluents include PEG400, TPGS, propylene glycol, Labrasol , Gelucire, Transcutol , PVP and potassium acetate.
  • emulsifying and suspending agents such as sodium CMC, methyl cellulose, pectin and gelatin.
  • certain sweetening and/or flavoring and/or coloring agents may be added.
  • the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration.
  • Suitable nonirritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable nonirritating excipient include cocoa butter, beeswax, gelatin, glycerin and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye or the skin. Topically-transdermal patches may also be used.
  • the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers .
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax, stearic acid, cetyl stearate, cetyl alcohol, lanolin, magnesium hydroxide, kaolin and water.
  • the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissblved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters, wax, cetyl alcohol, 2-octyldodecanol , benzyl alcohol and water.
  • the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride.
  • the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
  • compositions of this invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • the amount of either the compound or other neurotrophic, neuroprotective, thrombolytic, and/or antI-thrombotic agents (in those compositions which comprise additional neurotrophic, neuroprotective, thrombolytic, and/or anti-thrombotic agents) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • compositions of this invention should be formulated so that a dosage of between 0.01 - 10 mg/kg body weight/day of a compound of this invention can be administered. More preferably, the dosage is between 0.1 mg/kg body weight/day.
  • said agents and the FGF-8 compounds act synergistically to stimulate neurite outgrowth, nerve growth, or neuronal recovery or inhibit neurodegeneration. Therefore, the amount of any other neurotrophic, neuroprotective, thrombolytic, and/or antl -thrombotic agent in such compositions will be less than that required in a monotherapy utilizing only that factor.
  • a dosage of between 0.01 - 10 mg/kg body weight/day of each neurotrophic, neuroprotective, thrombolytic, and/or anti-thrombotic agent can be administered.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • the amount of active ingredients will also depend upon the particular compound of this invention and the neurotrophic, neuroprotective, thrombolytic, and/or antI-thrombotic agents used m the pharmaceutical composition.
  • a preferred embodiment of this aspect of the invention includes administering the FGF-8 compound and any other neurotrophic, neuroprotective, thrombolytic, and/or anti- thrombotic agent in a single dosage form when they are to be co-admmistered.
  • the pharmaceutical compositions of the present invention can be used in a method to stimulate nerve growth ex vivo.
  • the compounds or compositions described above can be applied directly to the nerve cells in culture.
  • This aspect of the invention is useful for ex vivo nerve regeneration .
  • the method of stimulating neurite outgrowth, enhancing nerve growth, or preventing neurodegeneration comprises the step of treating a patient or ex vivo nerve cells in culture with compositions of this invention as described above in the presence or absence of other neurotrophic, neuroprotective, thrombolytic, and/or anti- thrombotic agents.
  • compositions of the present invention are also useful m bioassays measuring neurite outgrowth or neuronal migration altering activities.
  • the examples discribed herein can be modified such that cell lines or cortical explants are co- cultured with cells expressing hFGF-8 polypeptides or fragments thereof.
  • a similar assays can be performed to identify small molecules (including hFGF-8 peptides or chemical molecules) that block hFGF-8' s growth promoting effects.
  • hFGF-8 may act as " kindling" in disorders such as epilepsy, by contributing to sustained and increased excitability of dend ⁇ tes through its neurite outgrowth promoting activity.
  • blocking hFGF-8's migration promoting activity might be useful for treatment of various neuronal tumors including, but not limited to, glial cell tumors.
  • the desired biological function may be obtained from polypeptides ranging in size from fragments containing a single epitope to which an antibody molecule can bind to large polypeptides which are capable of participating m the characteristic induction or programming of phenotypic changes withm a cell.
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain at least a portion of a region of a polypeptide molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a polypeptide sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked polypeptide. See, for instance, J. G. Sutcliffe, et al . , " Antibodies that react with preidentifled sites on polypeptides," Science 219:660-666 (1983) .
  • Antigenic epitope-bearmg peptides and polypeptides of the invention are useful to raise antibodies, including monoclonal antibodies, or screen antibodies, including fragments or single chain antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson, et al . , Cell 37:767-778 (1984) at 777.
  • Antigenic epitope-bearmg peptides and polypeptides of the invention preferably contain a sequence of at least five, more preferably at least nine, and most preferably between at least about 15 to about 30 am o acids contained with the ammo acid sequence of a polypeptide of the invention.
  • the epitope-bear g peptides and polypeptides of the invention can be produced by any conventional means.
  • R. A. Houghten " General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual ammo acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) .
  • This " Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten, et al . (1986).
  • hFGF-8 polypeptides of the present invention and the epitope- bearmg fragments thereof described above can be combined with parts of the constant domain of immunoglobulms (IgG) , resulting in chimeric polypeptides.
  • IgG immunoglobulms
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulms (EPA 394,827; Traunecker, et al . , Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dime ⁇ c structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric hFGF-8 polypeptide or polypeptide fragment alone (Fountoulakis, et al . , J. Biochem. 270:3958-3964 (1995)).
  • the polypeptide components or suitable fragments thereof contained in the compositions of this invention may be used in the production of antibodies.
  • the term " antibody” as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2 ' , and Fv fragments), and modified versions thereof, as well known in the art (e.g., chimeric, humanized, recombinant, veneered, resurfaced or CDR-grafted) such antibodies are capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived.
  • the instant invention also encompasses single chain polypeptide binding molecules.
  • the production of antibodies, both monoclonal and polyclonal, in animals is well known in the art.
  • Single chain antibodies and libraries thereof are yet another variety of genetically engineered antibody technology that is well known in the art. (See, e.g., R. E. Bird, et al . , Science 242:423-426 (1988); PCT Publication Nos. WO 88/01649, WO 90/14430, and WO 91/10737.
  • Single chain antibody technology involves covalently joining the binding regions of heavy and light chains to generate a single polypeptide chain. The binding specificity of the intact antibody molecule is thereby reproduced on a single polypeptide chain.
  • Antibodies included in this invention are useful in diagnostics, therapeutics or in diagnostic/therapeutic combinations.
  • polypeptides of this invention or suitable fragments thereof can be used to generate polyclonal or monoclonal antibodies, and various inter-species hybrids, or humanized antibodies, or antibody fragments, or single-chain antibodies.
  • the techniques for producing antibodies are well known to skilled artisans. (See, e.g., Colligan supra; Monoclonal Antibodies: Principles & Applications, Ed. J. R. Birch & E. S. Lennox, Wiley-Liss (1995) .
  • a polypeptide used as an immunogen may be modified or administered in an adjuvant, by subcutaneous or intraperitoneal injection into, for example, a mouse or a rabbit.
  • spleen cells from immunized animals are removed, fused with myeloma or other suitable known cells, and allowed to become monoclonal antibody producing hybridoma cells in the manner known to the skilled artisan.
  • Hybridomas that secrete a desired antibody molecule can be screened by a variety of well known methods, for example ELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al . Exp. Cell Res. 175:109-124 (1988); Monoclonal Antibodies: Principles & Applications, Ed. J. R. Birch & E . S . Lennox, Wiley-Liss (1995) ; Colligan, supra) .
  • labeled antibodies are desirable.
  • Procedures for labeling antibody molecules are widely known, including for example, the use of radioisotopes , affinity labels, such as biotin or avidin, enzymatic labels, for example horseradish peroxidase, and fluorescent labels, such as FITC or rhodamine (See, e.g., Colligan, supra) .
  • Labeled antibodies are useful for a variety of diagnostic applications.
  • the present invention relates to the use of labeled antibodies to detect the presence of an hFGF-8 polypeptide.
  • the antibodies could be used in a screen to identify potential modulators of an hFGF-8 polypeptide.
  • the antibody or compound to be tested is labeled by any suitable method.
  • Competitive displacement of an antibody from an antibody-antigen complex by a test compound such that a test compound-antigen complex IS formed provides a method for identifying compounds that bind HPLFP.
  • the present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as a mouse) the germ cells and somatic cells of which contain nucleic acid genomic DNA according to the present invention which codes for at least one hFGF-8 polypeptide.
  • a transgenic non-human eukaryotic animal preferably a rodent, such as a mouse
  • At least one hFGF-8 nucleic acid can be introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably the 1-1000 cell or oocyte, stage, and preferably not later than about the 64 -cell stage.
  • transgene as used herein, means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of at least one hFGF-8 polypeptide in the transgenic animal
  • hFGF-8 nucleic acid can be introduced into a cell or genome of the animal embryo so as to be chromosomally incorporated and expressed according to known methods .
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the an hFGF-8 polypeptide nucleic acid of the present invention such as animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic animal, are also intended to be withm the scope of the present invention.
  • Chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention, which may be used for testing expression of at least one hFGF-8 polypeptide m human tissue and/or for testing the effectiveness of therapeutic and/or diagnostic agents associated with delivery vectors which preferentially bind to an hFGF-8 polypeptide of the present invention.
  • Methods for providing chimeric non-human mammals are provided, e.g., in U.S. Serial Nos.
  • 4,736,866 (hereby entirely incorporated by reference) for producing transgenic non-human mammals may be used for the production of a transgenic non-human mammal of the present invention.
  • the various techniques described in U.S. patent Nos. 5,454,807, 5,073,490, 5,347,075 and 4 , 736 , 866 may also be used.
  • Animals carrying at least one hFGF-8 polypeptide and/or nucleic acid can be used to test compounds or other treatment modalities which may prevent, suppress or cure a pathology relating to at least one hFGF-8 polypeptide or hFGF-8 nucleic acid. Such transgenic animals will also serve as a model for testing of diagnostic methods for the same diseases. Transgenic animals according to the present invention can also be used as a source of cells for cell culture.
  • Example 1 Expression and Purification of an hFGF-8 Polypeptide in E. coli
  • the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., Chatsworth, CA) .
  • pQE60 encodes ampicillin antibiotic resistance (" Ampr” ) and contains a bacterial origin of replication (" ori” ), an IPTG inducible promoter, a ribosome binding site (“ RBS” ), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid (“ Ni-NTA” ) affinity resin sold by QIAGEN, Inc., and suitable single restriction enzyme cleavage sites.
  • a DNA fragment encoding a polypeptide can be inserted in such a way as to produce that polypeptide with the six His residues (i.e., a " 6 X His tag” ) covalently linked to the carboxyl terminus of that polypeptide.
  • a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.
  • the nucleic acid sequence encoding the desired portion of an hFGF-8 polypeptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers (based on the sequences presented, e.g., as presented in at least one of SEQ ID NOS:l, 2, 3, and 8), which anneal to the amino terminal encoding DNA sequences of the desired portion of an hFGF-8 polypeptide and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
  • the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of an hFGF-8, e.g., as presented in at least one of SEQ ID NOS : 1 , 2, 3, and 8, according to known method steps.
  • SEQ ID NOS : 1 , 2, 3, and 8 amino acids sequences
  • the point in a polypeptide coding sequence where the 5' primer begins can be varied to amplify a desired portion of the complete polypeptide shorter or longer than the mature form.
  • the amplified hFGF-8 nucleic acid fragments and the vector pQE60 are digested with appropriate restriction enzymes and the digested DNAs are then ligated together.
  • Insertion of the hFGF-8 DNA into the restricted pQE60 vector places an hFGF-8 polypeptide coding region including its associated stop codon downstream from the IPTG- inducible promoter and in- frame with an initiating AUG codon.
  • the associated stop codon prevents translation of the six histidine codons downstream of the insertion point.
  • E. coli strain Ml5/rep4 containing multiple copies of the plasmid pREP4 , which expresses the lac repressor and confers kanamycin resistance (" Kanr" ), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing hFGF-8 polypeptide, is available commercially from QIAGEN, Inc. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin.
  • Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constructs are grown overnight (" O/N” ) in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) .
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250.
  • the cells are grown to an optical density at 600 nm (“ OD600" ) of between 0.4 and 0.6.
  • IPTG Isopropyl -b-D-thiogalactopyranoside
  • the cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8.
  • the cell debris is removed by centrifugation, and the supernatant containing the hFGF-8 is dialyzed against 50 mM Na-acetate buffer pH6 , supplemented with 200 mM NaCl.
  • a polypeptide can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH7.4 , containing protease inhibitors.
  • the protein is made soluble according to known method steps. After renaturation the polypeptide is purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column is used to obtain pure hFGF-8 polypeptide. The purified polypeptide is stored at 4°C or frozen at -40°C to -120°C.
  • Example 2 Cloning and Expression of an hFGF-8 Polypeptide in a Baculovirus Expression System
  • the plasmid shuttle vector pA2 GP is used to insert the cloned DNA encoding the mature polypeptide into a baculovirus to express an hFGF-8 polypeptide, using a baculovirus leader and standard methods as described in Summers, et al . , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 polypeptide and convenient restriction sites such as BamHI , Xba I and Asp7l8.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell- mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide .
  • baculovirus vectors are used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in- frame AUG as required.
  • Such vectors are described, for instance, in Luckow, et al . , Virology 170:31-39.
  • the cDNA sequence encoding the mature hFGF-8 polypeptide in the deposited or other clone, lacking the AUG initiation codon and the naturally associated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non- limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of an hFGF-8 polypeptide, e.g., as presented in at least one of SEQ ID NOS : 1 , 2, 3, and 8, according to known method steps.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit (e.g., " Geneclean, " BIO 101 Inc., La Jolla, CA) .
  • the fragment then is then digested with the appropriate restriction enzyme and again is purified on a 1% agarose gel. This fragment is designated herein " FI” .
  • the plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit (" Geneclean” BIO 101 Inc., La Jolla, CA) . This vector DNA is designated herein " VI” .
  • E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human hFGF-8 gene using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing the hFGF-8 gene fragment will show amplification of the DNA. The sequence of the cloned fragment is confirmed by DNA sequencing.
  • This plasmid is designated herein pBac hFGF-8.
  • Five ⁇ g of the plasmid pBachFGF-8 is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ( " BaculoGoldTM baculovirus DNA” , Pharmingen, San Diego, CA) , using the lipofection method described by Feigner, et al . , Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
  • 1 ⁇ g of BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBac hFGF-8 are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies, Inc., Rockville, MD) . Afterwards, 10 ⁇ l Lipofectin plus 90 ⁇ l Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop- wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution.
  • Sf9 insect cells ATCC CRL 1711
  • the plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27°C for four days.
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, according to known methods.
  • An agarose gel with " Blue Gal” (Life Technologies, Inc., Rockville, MD) is used to allow easy identification and isolation of gal -expressing clones, which produce blue- stained plaques.
  • a detailed description of a " plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies, Inc., Rockville, MD, page 9-10). After appropriate incubation, blue stained plaques are picked with a micropipettor tip (e.g., Eppendorf) .
  • a micropipettor tip e.g., Eppendorf
  • the agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ⁇ l of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C.
  • the recombinant virus is called V-hFGF-8.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS .
  • the cells are infected with the recombinant baculovirus V-hFGF-8 at a multiplicity of infection (" MOI" ) of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available, e.g., from Life Technologies, Inc., Rockville, MD) . If radiolabeled polypeptides are desired, 42 hours later, 5 mCi of 35S- methionine and 5 mCi 35S-cysteine (available from Amersham) are added.
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation.
  • the polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled) . Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide.
  • Example 3 Cloning and Expression of hFGF-8 in Mammalian Cells
  • a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI , HIVI and the early promoter of the cytomegalovirus (CMV) . However, cellular elements can also be used (e.g., the human actin promoter) .
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3. l/Hygro (+/-) (Invitrogen) , PSVL and PMSG (Pharmacia, Uppsala, Sweden) , pRSVcat (ATCC 37152) , pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) .
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Ze
  • Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt , neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded polypeptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al . , Biochem. J. 227:277-279 (1991); Bebbington, et al . ,
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al . , Cell 41:521-530 (1985)).
  • LTR strong promoter
  • CMV-enhancer Boshart, et al . , Cell 41:521-530 (1985)
  • Multiple cloning sites e.g., with the restriction enzyme cleavage sites BamHI , Xbal and Asp7l8, facilitate the cloning of the gene of interest.
  • the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, phFGF-8 HA is made by cloning a cDNA encoding hFGF-8 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.) .
  • the expression vector pcDNAI/amp contains: (1) an E . coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eucaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an " HA" tag to facilitate purification) or HIS tag (see, e.g, Ausubel, supra) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • HA hemagglutinin fragment
  • HIS tag see, e.g, Ausubel, supra
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin polypeptide described by Wilson, et al . , Cell 37:767-778 (1984) .
  • the fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizes the HA epitope.
  • pcDNAIII contains, in addition, the selectable neomycin marker.
  • a DNA fragment encoding the hFGF-8 is cloned into the polylinker region of the vector so that recombinant polypeptide expression is directed by the CMV promoter.
  • the plasmid construction strategy is as follows.
  • hFGF-8 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of hFGF-8 in E. coli.
  • suitable primers include those based on the coding sequences presented in at least one of SEQ ID NOS : 1 , 2, 3, and 8, as they encode hFGF- 8 polypeptides as described herein.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme (s) and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) , and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the hFGF- 8 -encoding fragment .
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook, et al . , Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expression of hFGF- 8 by the vector.
  • hFGF- 8 -HA fusion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow, et al . , Antibodies: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) .
  • the cells are labeled by incubation in media containing 35S-cysteine for 8 hours.
  • the cells and the media are collected, and the cells are washed and lysed with detergent -containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson, et al .
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody.
  • the precipitated polypeptides then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls .
  • Plasmid pC4 is used for the expression of hFGF-8 polypeptide.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146) .
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • MTX methotrexate
  • DHFR gene If a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s) . Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome (s) of the host cell.
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec.
  • LTR long terminal repeat
  • telomeres a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al . , Cell 41:521-530 (1985)). Downstream of the promoter are BamHI , Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • CMV cytomegalovirus
  • high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI .
  • Clontech' s Tet-Off and Tet-On gene expression systems and similar systems can be used to express the hFGF- 8 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt , G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate .
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete hFGF-8 polypeptide is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non- limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of an hFGF-8, e.g., as presented in at least one of SEQ ID NOS : 1 , 2, 3, and 8, according to known method steps.
  • the amplified fragment is digested with suitable endonucleases and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • 5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 400 ⁇ g/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 400 ⁇ g/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) .
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M) . The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 ⁇ M. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
  • Northern blot analysis is carried out to examine hFGF-8 gene expression in human tissues, using methods described by, among others, Sambrook, et al . , cited above.
  • a cDNA probe containing the entire nucleotide sequence of an hFGF-8 polypeptide (SEQ ID NO:l) is labeled with 32 P using the RediprimeTM DNA labeling system (Amersham Life Science) , according to the manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN-100TM column (Clontech Laboratories, Inc.), according to the manufacturer's protocol number PT1200-1. The purified and labeled probe is used to examine various human tissues for hFGF-8 mRNA.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • Example 5 Directed Mutagenesis of hFGF-8 Polypeptides to Provide DNA Encoding Specified Substitutions, Insertions or Deletions of SEQ ID NO: 2 Using the Polymerase Chain Reaction
  • PCR polymerase chain reaction
  • SEQ ID NO: 2 e.g., SEQ ID NO:l, or any sequence described herein
  • This technology can be used as a quick and efficient method for introducing any desired sequence change into the DNA of interest .
  • Unit 8.5 of Ausubel, supra contains two basic protocols for introducing base changes into specific DNA sequences.
  • Basic Protocol 1 as presented in the first section 8.5 of Ausubel, supra (entirely incorporated herein by reference) , describes the incorporation of a restriction site and Basic Protocol 2, as presented below and in the second section of Unit 8.5 of Ausubel, supra, details the generation of specific point mutations (all of the following references in this example are to sections of Ausubel, et al . , eds., Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-1999)).
  • An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
  • the PCR procedure described here can rapidly, efficiently, and/or reproducibly introduce any desired change into a DNA fragment. It is similar to the oligonucleotide-directed mutagenesis method described in UNIT 8.1, but does not require the preparation of a uracil - substituted DNA template.
  • the mam disadvantage of PCR-generated mutagenesis is related to the fidelity of the Taq DNA polymerase.
  • the mutation frequency for Taq DNA polymerase was initially estimated to be as high as 1/5000 per cycle (Saiki et al . , 1988) . This means that the entire amplified fragment must be sequenced to be sure that there are no Taq-derived mutations.
  • the entire amplified fragment can usually be sequenced from a single primer. If the fragment is somewhat longer, it is best to subclone the fragment into an M13 -derived vector, so that both forward and reverse primers can be used to sequence the amplified fragment.
  • oligonucleotides UNITS 2.11 & 2.12
  • phosphorylation of oligonucleotides UNIT 3.10
  • electrophoresis of DNA on nondenaturing agarose and low gelling/melting agarose gels UNITS 2.5A & 2.6
  • restriction endonuclease digestion UNIT 3.1
  • ligation of DNA fragments UNIT 3.16
  • transformation of E. coli UNIT 1.8
  • plasmid DNA miniprep UNIT 1.6
  • DNA sequence analysis UNIT 7.4
  • oligonucleotide primers Prepare template DNA (see Basic Protocol 1, steps 1 and 2). Synthesize (UNIT 2.11) and purify (UNIT 2.12) the oligonucleotide primers (primers 3 and 4 in Fig. 8.5.2B).
  • the oligonucleotide primers must be homologous to the template DNA for more than 15 bases. No four-base " clamp” sequence is added to these primers.
  • the primer sequences are based on a DNA encoding the hFGF-8 polypeptide sequence of interest including at least one substitution, insertion or deletion of SEQ ID NOS : 4 , 5, 6, and 7. Phosphorylate the 5' end of the oligonucleotides (UNIT 3.10) . This step is necessary because the 5' end of the oligonucleotide will be used directly in cloning. Amplify DNA and prepare blunt-end fragments
  • ALTERNATE PROTOCOL INTRODUCTION OF A POINT MUTATION BY SEQUENTIAL PCR STEPS
  • the two fragments encompassing the mutation are annealed with each other and extended by mutually primed synthesis; this fragment is then amplified by a second PCR step, thereby avoiding the blunt -end ligation required in Basic Protocol 2.
  • This strategy is outlined in Figure 8.5.3.
  • Basic Protocols 1 and 2 of Ausubel, supra. Prepare template DNA (see Basic Protocol 1, steps 1 and 2). Synthesize (UNIT 2.11) and purify (UNIT 2.12) the oligonucleotide primers (primers 5 and 6 in Fig.
  • hFGF-8 polypeptide sequence of interest including at least one substitution, insertion or deletion of SEQ ID NOS:4, 5, 6, and 7.
  • the oligonucleotides must be homologous to the template for 15 to 20 bases and must overlap with one another by at least 10 bases. The 5' end does not have a " clamp" sequence.
  • Amplify the template DNA and generate blunt -end fragments see Basic Protocol 2, steps 4 and 5) . Purify the fragments by nondenaturing agarose gel electrophoresis (UNIT 2.5A) . Resuspend in TE buffer at 1 ng/ ⁇ l .
  • each flanking sequence primer (each 1 ⁇ M final) 10 ⁇ l lOx amplification buffer 10 ⁇ l 2 mM 4dNTP mix H 2 0 to 99.5 ⁇ l
  • Digest the DNA fragment with the appropriate restriction endonuclease for the flanking sites (UNIT 3.1) . Purify the digested fragment on a low gelling/melting agarose gel (UNIT 2.6). Subclone into an appropriately digested vector. Transform recombinant plasmid into E. coli (UNIT 1.8) . Prepare DNA by plasmid miniprep (UNIT 1.6) . Analyze the amplified fragment portion of the plasmid DNA by DNA sequencing (UNIT 7.4) to confirm the point mutation. This is critical because the Taq DNA polymerase can introduce additional mutations into the fragment (see Critical Parameters) .
  • Example 6 FGF-8 Induced Neurite Outgrowth in PC12 Cells
  • a neurite outgrowth assay can be performed with cells or cell lines derived from neuronal tissue essentially as described by Lyons et al . , (1994) . Briefly, PC12 cells are maintained at 37'C and 5% CO z in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat- mactivated horse serum, 5% heat inactivated fetal bovine serum (FBS) , and 1% glutamate .
  • DMEM Dulbecco's modified Eagle's medium
  • the cells are then plated at 105 per well in 96 well plates coated with 5 m/cm2 rat tail collagen and allowed to attach overnight.
  • the medium is then replaced with DMEM, 2% heat -inactivated horse serum, 1% glutamate, 1-5 ng/ml of NGF (Sigma) and varying concentrations of the FGF-8 compound (0.001 nM - 100 ⁇ M) .
  • the background control culture is administered with 105 ng/ml of NGF alone without compound. Positive control culture's are administered with high concentration of NGF (50 ng/ml) .
  • Example 7 FGF-8 Induced Neurite Outgrowth in Human Neuronal SHSY-5Y Cells
  • Stimulation of neurite outgrowth by FGF-8 can also be demonstrated using the human neuronal SHSY-5Y cell line (ATCC) .
  • SHSY-5 Y cells are cultured and maintained in a 1:1 mixture of Eagle's Minimum Essential Medium and Ham's F-12 medium containing 10% feral bovine serum.
  • cell are plated in chamber coverslips coated with Metrigel (Collaborative Research Inc.) at a density of 20,000 cells per chamber.
  • FGF-8b R&D Systems, Inc. was added at various concentrations (0.001 nM - 100 ⁇ M) . After 48 to 72 hours, cells were fixed with 5% Zn-formaline and stained with H&E stain.
  • Neurite outgrowth was evaluated by phase contrast microscopy and can be scored as in Example 6.
  • Neurite outgrowth can also be determined by measuring the expression of any known molecular marker of new axonal growth, including but not limited to the neuronal growth- associated protein 43 (GAP43) by any means know in the art.
  • GAP-43 is a phosphoprotein component of the neuronal membrane and growth cone that is selectively upregulated during new axonal growth in both the peripheral and central nervous systems.
  • GAP-43 has previously been used as a reliable marker of new axonal growth during brain development, and following brain injury or ischemia (Kawamata et al . , (1997(a)).
  • Such axonal sprouting is likely to be accompanied by new dendritic sprouting and synapse formation in the intact uninjured brain. This in turn leads to enhanced recovery from neuronal damage as a result of disease of physical trauma.
  • Immunostaining with axonal specific protein GAP43 followed by fluorescence microscopy according to known procedures shows that FGF-8 compounds resulted in a significant increase in neurite outgrowth over background control cultures (see Figure 2) .

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Abstract

L'invention concerne des compositions pharmaceutiques et des procédés relatifs au traitement des troubles neurologiques. Les compositions considérées renferment au moins un polypeptide hFGF-8, ou au moins un acide nucléique isolé, ou son complément, qui code au moins un polypeptide hFGF-8. L'invention concerne également des anticorps et des chiméres hFGF-8, des vecteurs d'expression hFGF-8, des cellules hôtes et des animaux transgéniques, ainsi que des procédés relatifs à leur élaboration et à leur utilisation.
PCT/US2000/011885 1999-06-29 2000-06-21 Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques Ceased WO2001000662A2 (fr)

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AU58666/00A AU5866600A (en) 1999-06-29 2000-06-21 The use of human fgh-8 polypeptides as neurotrophic agents

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7414019B2 (en) * 2002-10-04 2008-08-19 Auckland Uniservices Limited FGF-8 methods of use
US7935342B2 (en) 2006-02-02 2011-05-03 Rinat Neuroscience Corp. Methods for treating obesity by administering a trkB antagonist
WO2015121457A1 (fr) 2014-02-13 2015-08-20 Westphal Sören Fgf-8 destiné à être utilisé dans le traitement de maladies ou de troubles de l'homéostasie énergétique
US20190240179A1 (en) * 2015-10-31 2019-08-08 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of rxr agonists and thyroid hormones

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003199A1 (fr) * 1992-08-04 1994-02-17 Regeneron Pharmaceuticals, Inc. Procede ameliorant la differentiation et les chances de survie des cellules precurseurs neuronales
WO1994020125A1 (fr) * 1993-03-12 1994-09-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Traitement des affections des neurones moteurs a l'aide du facteur de croissance des fibroblastes 5 (fgf-5)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7414019B2 (en) * 2002-10-04 2008-08-19 Auckland Uniservices Limited FGF-8 methods of use
US7935342B2 (en) 2006-02-02 2011-05-03 Rinat Neuroscience Corp. Methods for treating obesity by administering a trkB antagonist
US10532083B2 (en) 2014-02-13 2020-01-14 Technische Universität München Methods for inducing differentiation or conversion of white adipocytes and/or preadipocytes to brown adipocites using FGF8
WO2015121457A1 (fr) 2014-02-13 2015-08-20 Westphal Sören Fgf-8 destiné à être utilisé dans le traitement de maladies ou de troubles de l'homéostasie énergétique
US10806713B2 (en) 2015-10-31 2020-10-20 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10588881B2 (en) 2015-10-31 2020-03-17 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10695312B2 (en) 2015-10-31 2020-06-30 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10702489B2 (en) 2015-10-31 2020-07-07 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US20190240179A1 (en) * 2015-10-31 2019-08-08 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of rxr agonists and thyroid hormones
US10842764B2 (en) 2015-10-31 2020-11-24 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10857117B2 (en) 2015-10-31 2020-12-08 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10973791B2 (en) 2015-10-31 2021-04-13 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10980760B2 (en) 2015-10-31 2021-04-20 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10980759B2 (en) 2015-10-31 2021-04-20 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US10980761B2 (en) 2015-10-31 2021-04-20 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones
US11065219B2 (en) 2015-10-31 2021-07-20 Io Therapeutics, Inc. Treatment of nervous system disorders using combinations of RXR agonists and thyroid hormones

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