HK1092477B - Neurotrophic growth factor - Google Patents

Description

Neurotrophic growth factor

The present application is a divisional application of an invention patent application having an application date of 14/7/1999, application number of 99810884.7, entitled "neurotrophic growth factor".

The present invention relates to neurotrophic growth factors, and in particular to the cloning and expression of a novel member of the GDNF family of neurotrophic growth factors referred to herein as "enovin" (EVN).

Introduction to the word

Neurotrophic growth factors are involved in neuronal differentiation, development and maintenance. This protein can prevent degeneration of different types of neuronal cells and promote their survival, and is therefore a potential therapeutic agent for the treatment of neurodegenerative diseases. Glial cell line-derived neurotrophic growth factor (GDNF) is the first member of an increasing subfamily of neurotrophic factors that differ structurally from neurotrophins. GDNF is a member of the transforming growth factor beta (TGF-. beta.) superfamily of growth factors characterized by a specific form of 7 highly conserved cysteine residues in the amino acid sequence (Kingsley, 1994). GDNF was originally purified using an assay based on its ability to maintain survival and function of embryonic anterolateral cerebellar dopaminergic neurons in vitro (Lin et al, 1993). Other types of neuronal cells in the Central Nervous System (CNS) or Peripheral Nervous System (PNS) can also respond to the survival of GDNF (Henderson et al, 1994, Buj-Bello et al, 1995, Mount et al, 1995, Oppenheim et al, 1995). GDNF is produced by cells as an inactive precursor that is specifically cleaved at the RXR furin recognition site to produce active (mature) GDNF (Lin et al, 1993). Exogenous administration of GDNF has potent neuroprotective effects in animal models of Parkinson's disease, a common neurodegenerative disease characterized by loss of up to 70% of dopaminergic cells in the substantia nigra of the brain (Beck et al, 1995, Tomac et al, 1995, Gash et al, 1996, Choi-Lundberg et al, 1997, Bilang-Bleuel et al, 1997).

More recently, other neurotrophic factors of the GDNF family have been discovered. Neurturin (NTN) was purified from the loop conditioned medium of Chinese Hamster Ovary (CHO) cells by a method based on its ability to promote sympathetic neuron survival in culture (Kotzbauer et al, 1996). The mature NTN protein has 57% similarity to mature GDNF. Persephin (PSP) was discovered by PCR cloning with degenerate primers using genomic DNA as a template. Mature PSP, like mature GDNF, promotes survival of the anterior cerebellar dopaminergic neurons and motor neurons in culture (Milbrandt et al, 1998). The similarity of the mature PSP protein to mature GDNF and NTN is approximately 50%. Artemin (ARTN) was discovered by DNA database search and is a survival factor for sensory and sympathetic neurons in culture (Baloh et al, 1998 b).

To accomplish downstream intracellular signaling, GDNF, NTN, PSP and ARTN require a heterodimeric receptor complex. GDNF binds to the GDNF family receptor alpha 1(GFR alpha-1; GFR alpha nomenclature Association, 1997) subunit, which is a glycosyl phosphatidyl-inositol (glycosyl PtdIns) -anchored membrane protein (sting et al, 1996, Treanor et al, 1996, Sanicola et al, 1997). The GDNF/GFR α -1 complex subsequently binds to and activates a membrane-bound tyrosine kinase, the cRET proto-oncogene (Durbec et al, 1996, Trupp et al, 1996), resulting in phosphorylation of tyrosine residues in cRET and subsequent activation of downstream signaling pathways (Worby et al, 1996). Several other members of the GFR α family of ligand-binding receptors have been identified (Baloh et al, 1997, Sanicor et al, 1997, Klein et al, 1997, Buj-Bello et al, 1997, Suvanto et al, 1997). GFR α -2 and GFR α -3(Jing et al, 1997, Masure et al, 1998, Woby et al, 1998, Naveilham et al, 1998, Baloh et al, 1998a) have been identified by several different groups. GFR α -1 and GFR α -2 are widely expressed in almost all tissues, and their expression is developmentally regulated (Sanicola et al, 1997, Widenfalk et al, 1997).

GFR α -3 is not expressed in the developing or mature central nervous system, but is highly expressed in several developing and mature sensory and sympathetic ganglia of the peripheral nervous system (Widenfalk et al, 1998, Naveilhan et al, 1998, Baloh et al, 1998 a). The 4 th family member GFR α -4 was cloned from chicken cDNA (Thompson et al, 1998). GFR α -1 is a preferred receptor for GDNF, while GFR α -2 binds to NTN preferentially (sting et al, 1996, Treanor et al, 1996, Klein et al, 1997). Chicken GFR α -4 together with cRET constitute the functional receptor complex of PSP (Enokido et al, 1998). Cells expressing both GFR α -3 and cRET have been shown to be unable to respond to GDNF, NTN or PSP (Worby et al, 1998, Baloh et al, 1998 a). Recently, ART has been shown to signal through cRET using GFR α -3 as the preferred ligand binding receptor (Baloh et al, 1998 b). Communication between neurotrophic factors and GFR α receptors in vitro is possible because GDNF binds to GFR α -2 or GFR α -3 in the presence of cRET (Sanicola et al, 1997, Trupp et al, 1998) and NTN binds to GFR α -1 with low affinity (Klein et al, 1997). In summary, GDNF, NTN, PSP and ART are part of the neurotrophic signaling system, and thus different ligand binding subunits (GFR α -1 to-4) are capable of interacting with the same tyrosine kinase subunit (cRET). These physiological relationships found in vitro have recently been shown in knockout studies (reviewed by Rosenthal, 1999) which clearly demonstrated that GDNF interacts with GFR α -1 in vivo, while NTN is a preferred ligand for GFR α -2.

The present inventors have identified, cloned, expressed, chromosomally mapped and characterized the 4 th member of the GDNF family, Enovin (EVN). The knowledge of the mature EVN protein is enhanced by the discovery of different functional and non-functional mRNA splice variants. Furthermore, we provide expression data, binding data of EVN to GFR α -3 and the in vitro effect of EVN on axonal outgrowth, as well as the protective effect on paclitaxel-induced neurotoxicity in cultures of astrocyte-differentiated SH-SY5Y human neuroblastoma cells.

Summary of The Invention

In the present application, there is provided a nucleic acid molecule encoding a novel human neurotrophic growth factor "enovin", an expression vector comprising said nucleic acid molecule, a host cell transformed with said vector, a neurotrophic growth factor encoded by said nucleic acid molecule, isolated enovin, compounds which act as agonists or antagonists of enovin, and pharmaceutical compositions comprising said nucleic acid or enovin protein or agonists or antagonists thereof.

Detailed Description

According to a first aspect of the present invention there is provided a nucleic acid molecule encoding a human neurotrophic growth factor, referred to herein as enovin, having the amino acid sequence shown in figure 21, or encoding a functional equivalent, derivative or bioprecursor of said growth factor. The nucleic acid molecule is preferably a DNA, more preferably a cDNA molecule.

The nucleic acid of the invention preferably comprises the sequence from position 81 to 419 of the sequence shown in FIG. 1, more preferably from position 81 to 422, even more preferably the complete sequence shown in FIG. 1.

Nucleic acid molecules from numbers 81 to 419 are believed to encode the sequence of the mature enovin protein that results after processing of the RXXR processing site present on the stable precursor of the enovin protein.

The invention also provides an antisense molecule capable of hybridizing to any one of the nucleic acid sequences of the invention under high stringency conditions well known to those skilled in the art.

As used herein, the stringency of hybridization refers to conditions under which nucleic acids are maintained stable. The stability of the hybrid may be reflected in the melting temperature (Tm) of the hybrid. Tm can be expressed generally by the following formula:

81.5℃-16.6(log10[Na⁺]+0.41(％G&C)-600/l

wherein 1 is the length of the hybrid in terms of number of nucleotides. For every 1% decrease in sequence homology, the Tm decreases by about 1-1.5 ℃.

Ideally, the nucleic acid molecules of the present invention can be used to express the present inventors neurotrophic growth factor in a host cell or the like using a suitable expression vector.

The expression vectors of the invention include vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of effecting expression of the DNA fragments.

Regulatory elements required for expression include a promoter sequence for binding RNA polymerase and a transcription initiation sequence for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and SD sequences for transcription initiation, as well as the initiation codon AUG. Similarly, eukaryotic expression vectors may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the initiation codon AUG, and a stop codon for ribosome cleavage. The vectors are commercially available or assembled with the disclosed sequences by methods well known in the art.

Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, upon introduction into a suitable host cell, results in expression of the DNA or RNA fragment. Suitable expression vectors are well known to those skilled in the art and include vectors capable of replication in eukaryotic and/or prokaryotic cells, as well as vectors which maintain their episomal form or which are capable of integration into the genome of a host cell.

Antisense molecules capable of hybridizing to a nucleic acid of the invention may be used as probes or as drugs or in pharmaceutical compositions.

The nucleic acid molecule of the invention may be inserted into the vector in the antisense orientation so as to be capable of producing antisense RNA. Antisense RNA or other antisense nucleic acids can be produced by synthetic methods.

Another aspect of the invention includes host cells, preferably including eukaryotic cells, more preferably mammalian cells, transformed, transfected or infected with the expression vectors of the invention.

The integration of cloned DNA into a suitable expression vector and subsequent use to transform the cells, followed by selection of transformed cells, is well known to those skilled in the art and is described in Sambrook et al (1989) molecular cloning, A laboratory Manual, Cold spring harbor laboratory Press.

Another aspect of the present invention comprises nucleic acid molecules having at least 15 nucleotides, preferably 15 to 50 nucleotides, of the nucleic acid molecules of the invention.

The sequence can be preferably used as a probe or a primer for initiating replication, or the like. The nucleic acid molecule may be produced using techniques well known in the art, such as by recombinant or synthetic methods. It can be used in a diagnostic kit or device or the like for detecting the presence of the nucleic acid of the present invention. The detection typically comprises contacting the probe with a sample under hybridization conditions and detecting the presence of any duplexes formed between the probe and any nucleic acids in the sample.

According to the invention, the probe may be anchored to a solid support. It is preferred that they are arrayed so that multiple probes can simultaneously hybridize to the same biological sample. The probes can be added dropwise to the array or synthesized in situ on the array (see Lockhart et al, Nature Biotechnology, Vol.14, 1996, 12 months, "monitoring expression by hybridization into high density oligonucleotide arrays"). A single array may contain over 100, 500, or even over 1000 different probes at different locations.

The nucleic acid molecules of the invention may also be produced by methods such as recombination or synthesis, for example, using the principle of PCR cloning, which generally involves producing a pair of primers, which may be fragments of about 10-50 nucleotides from the gene to be cloned, contacting the primers with mRNA, cDNA or genomic DNA derived from a human cell, performing a polymerase chain reaction under conditions such that the region of interest is amplified, isolating the amplified region or fragment, and recovering the amplified DNA. In general, the techniques described herein are well known in the art, as disclosed by Sambrook et al (molecular cloning: A laboratory Manual, 1989).

The nucleic acid or oligonucleotide of the invention may carry a revealing label. Suitable labels include radioisotopes such as³²P or³⁵S, enzyme labels, or other protein labels such as biotin or fluorescent labels. The label may be added to the nucleic acid or oligonucleotide of the invention and may be detected by techniques known per se.

For example, it is preferred that human allelic variants or polymorphisms of the DNA molecules of the invention can be identified by probing cDNA or genomic libraries from multiple individuals, such as different populations. In addition, the nucleic acids and probes of the invention can be used to sequence genomic DNA from a patient using techniques known in the art, such as the Sanger dideoxy chain termination method, which can preferably be used to determine any predisposition to certain diseases associated with the growth factors of the invention in a patient.

The invention also provides a transgenic cell, tissue or organism containing a transgene capable of expressing the human neurotrophic factor enovin of the invention.

The term "transgene capable of expression" as used herein refers to any suitable nucleic acid sequence that results in the expression of a neurotrophic factor having the same function and/or activity as the neurotrophic factor of the present invention. For example, the transgene may comprise genomic nucleic acid isolated from a human cell or synthetic nucleic acid, including cDNA, which may be integrated into the chromosome or maintained in an extrachromosomal state.

The transgene preferably comprises a vector of the invention comprising a nucleic acid molecule encoding the neurotrophic factor or a functional fragment of the nucleic acid molecule, a "functional fragment" of the nucleic acid being understood as a gene or cDNA fragment encoding the neurotrophic factor or a functional equivalent thereof, which fragment is capable of being expressed to produce a functional neurotrophic growth factor of the invention. Thus, for example, fragments of the neurotrophic growth factors of the invention corresponding to specific amino acid residues capable of interacting with the corresponding receptors also form part of the invention and may be used as agonists for activating the corresponding receptors of the growth factors of the invention in order to induce their growth promoting and survival sustaining effects on cells. This aspect of the invention also includes differentially spliced isoforms and transcriptional initiators of the nucleic acids of the invention.

According to the present invention, a specific nucleic acid includes not only the same nucleic acid but also any small base changes, particularly including base substitutions that result in synonymous codons (different codons encode the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid molecule" also includes complementary sequences that produce either single strand with respect to base changes.

According to another aspect of the present invention there is provided an isolated human neurotrophic growth factor encoded by a nucleic acid molecule as defined herein. The growth factor preferably comprises the amino acid sequence from positions 27-139 of the amino acid sequence of figure 1 or a functional equivalent, derivative or bioprecursor thereof.

Reference herein to "functional equivalents" is to be understood as growth factors having all the growth characteristics and functions associated with growth factor enovin. "derivatives" of enovin as defined herein are intended to include polypeptides in which certain amino acids have been altered or deleted or substituted with other amino acids and which retain the biological activity of enovin and/or which are capable of reacting with antibodies raised against the antigen produced by using the enovin of the invention.

The scope of the invention includes hybrids and modified forms of enovin, including fusion proteins and fragments thereof. For example, the hybrid and modified forms include: when certain modifications or substitutions are made to specific amino acids by methods such as point mutation, the modifications will still result in a protein that retains the biological activity of enovin of the invention. One skilled in the art can alter the particular nucleic acid sequence to produce a growth factor having the same or substantially the same characteristics as enovin.

As is well known in the art, many proteins are produced in vivo, with a (pre) signal sequence at the N-terminus of the protein. Alternatively, the protein may comprise a further prosequence which represents a stable precursor of the mature protein. Typically, such pre-and pro-sequences are not required for biological activity. The enovin molecule of the invention includes not only the full length sequence shown in fig. 21, but also the sequence from position 27 to 139, which is followed by the RXXR proteolytic processing site present on this type of growth factor, and which is considered the mature sequence of enovin.

A particular protein, polypeptide or amino acid sequence of the invention includes not only the same amino acid sequence, but also isomers thereof. In addition, minor amino acid changes based on the native amino acid sequence are also included, including conservative amino acid substitutions (substitutions by amino acids related to their side chains). Also included are amino acid sequences that differ from the natural amino acids, but that produce polypeptides that are immunologically identical or similar to the polypeptides encoded by the naturally occurring sequences.

The proteins and polypeptides of the invention also include variants of the sequences, including naturally occurring allelic variants (variants that are substantially homologous to the protein or polypeptide). Substantially homologous is herein to be regarded as a sequence having at least 70%, preferably 80%, 90% or 95% amino acid homology with the protein or polypeptide encoded by the nucleic acid molecule of the invention.

Neurotrophic growth factors expressed by the host cells of the present invention are also included within the scope of the present invention.

The invention also relates to the inhibition of the neurotrophic growth factor of the invention in vivo by using antisense technology. Antisense technology can be used to control gene expression by triple helix formation or antisense DNA or RNA, both methods based on the binding of polynucleotides to DNA or RNA. For example, the part of the DNA sequence encoding the mature protein of the invention is used to design an antisense RNA oligonucleotide 10-50bp in length. A DNA oligonucleotide is designed to be partially complementary to a region of a gene involved in transcription (triple helix-see Lee et al, nucleic acids research, 6: 3073 (1979); Cooney et al, science, 241: 456 (1998); and Dervan et al, science, 251: 1360(1990)) in order to prevent transcription and production of enovin. The antisense RNA oligonucleotides hybridize in vivo to mRNA and prevent translation of mRNA molecules into enovin.

Due to the sequence similarity of the growth factors disclosed herein to previously identified growth factors of the GDNF family, enovin is also thought to promote cell survival and growth and to be useful in the treatment of diseases resulting from defects in the function or expression of the neurotrophic factors.

Thus, the nucleic acid molecule or neurotrophic factor of the present invention may preferably be used to treat or prevent a neurological disease in a patient by administering to said patient a nucleic acid molecule or growth factor of the present invention in a concentration sufficient to alleviate the symptoms of said disease. Thus, the nucleic acid molecules of the invention are useful for promoting the maintenance and survival of neuronal cells and for treating neuronal diseases or neurodegenerative conditions, including parkinson's disease, alzheimer's disease, peripheral neuropathy, amyotrophic lateral sclerosis, peripheral and central nerve trauma or injury, and neurotoxin intoxication.

Ideally, it has been observed that the neurotrophic growth factors of the present invention are capable of producing a neurotrophic or neuroprotective effect on neuronal cells or cell populations, particularly neuronal cells or cell populations that have been induced to undergo apoptosis. Thus, the nucleic acids or enovin growth factors of the invention may also be used per se for the treatment of neurodegenerative diseases such as stroke, huntington's disease, peripheral neuropathy, acute brain injury, tumors of the nervous system, multiple sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma, injury due to exposure to neurotoxins, multiple endocrine adenomatous formation, familial hirschsprung's disease, prion-related diseases, Creutzfeld-JaCob disease, by administering to a patient in need thereof an amount of said nucleic acids or enovin sufficient to reduce or prevent the symptoms of the neurological diseases described herein.

In addition, as disclosed in more detail in the examples below, enovin has been shown to accelerate the recovery of induced sensory deficits, and is a candidate for the treatment or alleviation of the painful syndrome caused by peripheral or central neurogenic components, rheumatic/inflammatory diseases and conduction disorders by administering to a patient in need thereof such a compound in a concentration sufficient to alleviate or prevent the symptoms of said disease.

Another method for treating the above neurological disorders comprises transplanting in a patient cells expressing a human neurotrophic growth factor of the present invention, such as the transgenic cells described herein.

The nucleic acid molecule of the present invention and the neurotrophic growth factor may also be added to a pharmaceutical composition at the same time as a pharmaceutically acceptable carrier, diluent or excipient thereof.

Antibodies to the neurotrophic factors of the present invention can preferably be prepared by techniques well known in the art. For example, polyclonal antibodies can be prepared by inoculating a host animal, such as a mouse, with the growth factor or epitope thereof and recovering from the immune serum. Monoclonal antibodies can be prepared according to known techniques, such as the method disclosed by Kohler R. and Milstein C. (Nature, 1975, 256, 495-497).

The antibodies of the invention may preferably be used in a method for detecting the presence of a growth factor of the invention, which method comprises reacting said antibody with a sample and identifying any protein bound to said antibody. Also provided is a kit for carrying out the method, the kit comprising an antibody of the invention, and means for reacting the antibody with the sample.

The invention also provides a kit or device for detecting the presence of a neurotrophic growth factor of the invention in a sample comprising an antibody as described above and means for reacting said antibody with said sample.

Proteins that interact with the neurotrophic growth factor of the present invention, such as their corresponding cellular receptors, can be identified by studying protein-protein interactions using a two-hybrid vector system well known to molecular biologists (Fields & Song, Nature 340: 245, 1989). This technique is based on the functional reconstitution of a transcription factor in vivo, which activates a receptor gene. More specifically, the technique involves providing a suitable host cell having a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activation domain, expressing in said host cell a first hybrid DNA sequence encoding a fragment or a first fusion of all of the nucleic acid sequence of the invention with the DNA binding domain or activation domain of said transcription factor, and expressing in said host at least a second hybrid DNA sequence, such as a library or the like, encoding a putative binding protein to be studied with the DNA binding domain or activation domain of said transcription factor, which protein is not bound to said first fusion; detecting all binding of the protein to be investigated to the protein of the invention by detecting the presence of any reporter gene product in said host cell; optionally isolating a second hybrid DNA sequence encoding said binding protein.

One example of such a technique uses GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of galactose metabolism genes and a protein binding domain. Nucleotide vectors may be constructed, one of which includes nucleotide residues encoding the GAL4 DNA binding domain. The binding domain residues may be fused to known protein coding sequences, such as the nucleic acids of the invention. Other vectors include residues encoding the binding domain of GAL4 protein. The residues are fused to residues encoding a test protein, preferably from a signaling pathway of a related vertebrate. Any interaction between the neurotrophic growth factor encoded by the nucleic acid of the present invention and the protein to be tested will result in transcriptional activation of the reporter molecule in the GAL-4 transcription deficient yeast cell into which the vector is transferred. Preferably, a reporter molecule such as β -galactosidase is activated after transcription of the galactose metabolizing gene of the yeast is restored.

The receptor for enovin identified by the present inventors is GFR α 3. Assays can therefore be performed to identify activators or antagonist compounds of enovin. The assay can also be used to bind other neurotrophic growth factors and their corresponding receptors. The identified compounds are useful for the treatment or prevention of diseases such as parkinson's disease, alzheimer's disease, neuronal diseases associated with amplified polyglutamine sequences, such as huntington's disease, peripheral neuropathy, acute brain injury, tumors of the nervous system, multiple sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma or injury caused by neurotoxins, multiple endocrine adenomatous and familial hirschsprung's disease, prion-related diseases, Creutzfeld-Jacob disease, stroke, painful syndromes caused primarily by peripheral or central neurogenic components, rheumatic/inflammatory diseases and conduction disorders, by administering to a patient said activator or antagonist in an amount sufficient to prevent or treat said neurological diseases at a concentration. The compounds may also be incorporated into pharmaceutical compositions with pharmaceutically acceptable carriers, diluents or excipients therefor.

In one embodiment, an activator or antagonist of a growth factor (e.g., enovin) can be identified by: contacting a cell tissue or organism expressing a suitable receptor and cRET with a candidate compound in the presence of said growth factor and comparing the level of activation of RET in said cell, tissue or organism with a control which has not been contacted with said candidate compound.

Another embodiment of the invention encompasses a method for identifying activators or antagonists of neurotrophic growth factor, which method comprises contacting a cell tissue or organism expressing a suitable receptor for said growth factor and cRET with a candidate compound in the presence of the growth factor, determining the level of activation of a signal kinase in a signal transduction pathway which includes said suitable receptor as a component thereof, and adding an antibody specific for said signal kinase conjugated to a reporter molecule and comparing it to a cell tissue or organism not contacted with said compound.

Another aspect of the invention includes the use of the identified compounds as antagonists of the invention in the manufacture of a medicament for the treatment of gastrointestinal disorders or conditions mediated by enhanced intestinal motility.

The compounds identified in the assays of the present invention may preferably be used to enhance gastrointestinal motility and thus may be used to treat conditions associated with impaired or impaired gastrointestinal transit.

The compounds are therefore useful in the treatment of warm-blooded animals including humans suffering from symptoms associated with impaired or impaired gastric emptying or more commonly suffering from symptoms associated with impaired or impaired gastrointestinal transit. Thus, a therapeutic method is provided for alleviating a symptom in a patient, such as gastroesophageal reflux, dyspepsia, gastroparesis, postoperative intestinal occlusion, and intestinal pseudo-obstruction.

Dyspepsia is an impairment of digestive function that can occur as a symptom of primary gastrointestinal dysfunction, particularly associated with strong muscle tone, or as a complication of other diseases such as typhlitis, galbaladder disorders, or malnutrition. Dyspepsia symptoms are such as lack of appetite, feeling of fullness, premature fullness, nausea, vomiting and abdominal distension.

Gastroparesis may be caused by an abnormality of the stomach or occur as a complication of diseases such as diabetes, progressive systemic sclerosis, anorexia, nervosa, and muscular dystrophies.

Postoperative intestinal occlusion is an obstruction or dynamic injury to the intestine caused by the tension of muscles that has been destroyed after surgery.

Intestinal pseudo-obstruction is a disease characterized by constipation, abdominal pain, and vomiting, but without the symptoms of physical injury.

Thus, the compounds of the present invention may be used to eliminate the actual causes of the disease or to alleviate the symptoms of the disease.

In addition, certain compounds that are kinetically active stimulators of the colon may be used to correct or improve intestinal transit in patients with symptoms associated with turbulent motility, such as reduced small and large bowel motility alone or in combination with delayed gastric emptying.

For the colonic kinetic use of the compounds of the present invention, a method is provided for treating a warm-blooded animal, including humans, suffering from motility disorders of the intestinal system, such as constipation, pseudo-infarction, intestinal weakness, postoperative bowel weakness, Irritable Bowel Syndrome (IBS), and drug-induced transport delays.

Antagonist compounds identified according to the test methods of the present invention are also useful for treating or preventing gastrointestinal disorders caused by enhanced intestinal motility, such as diarrhea (including secretory diarrhea, bacterially induced diarrhea, biliary diarrhea, traveler's diarrhea, and psychogenic diarrhea), Crohn's disease, spastic colon, irritable abdominal syndrome (IBS), allergic abdominal gastrointestinal allergies which manifest primarily as diarrhea.

For use of the compounds of the invention, the invention then provides a method of treating a warm-blooded animal, including humans, suffering from a gastrointestinal disorder such as Irritable Bowel Syndrome (IBS), particularly IBS diarrhoea. Thus, a method of treatment is provided for alleviating symptoms in patients suffering from conditions such as allergic abdominal syndrome (IBS), diarrhea epidemic allergic abdominal syndrome (IBS), abdominal allergies, or alleviating the pain associated with gastrointestinal allergies.

The compounds of the invention are also useful in other gastrointestinal disorders such as those associated with upper abdominal motility, and as antiemetics for the treatment of emesis, and both cytotoxic drugs and radiation-induced emesis.

For example, inflammatory abdominal diseases include ulcerative enteritis, Crohn's disease, and the like.

Yet another aspect of the invention includes a method of treating a disease mediated by expression of enovin of the invention by administering to a patient an antisense molecule or antagonist thereof of the invention in a concentration sufficient to alleviate or reduce the symptoms of the disease.

Diseases mediated by inactivation or inhibition of enovin expression may also be treated by administering to the patient a compound identified as an activator of enovin at a concentration sufficient to reduce or inhibit the symptoms of the disease.

In another aspect, the present invention provides a method of treating a disease associated with human neurotrophic growth factor enovin, said method comprising selecting a candidate compound identified as an activator or antagonist of enovin of the invention, mass producing said compound, and formulating the produced compound into a pharmaceutically acceptable carrier.

As will be more clearly understood from the examples below, enovin has successfully mitigated the paclitaxel-induced sensing defect. Therefore, enovin may play a role in the painful syndrome and conduction disorders mainly caused by peripheral and central neurogenic components, rheumatic diseases, and may play a regulatory role in the sensory process after oral, rectal and systemic administration through the epidermis, local central (e.g., epidural, intrathecal, ICV, intraplexus, intraneural). Thus, other diseases mediated by enovin can be alleviated or prevented in the same manner as described herein by administering an antisense molecule, nucleic acid, enovin protein, pharmaceutical composition, or compound identified as an activator or antagonist of the invention in an amount sufficient to alleviate or prevent the symptoms of the disease.

The therapeutic or pharmaceutical compositions of the present invention may be administered by any suitable route known in the art, including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal, or intracranial administration or applied to cells in an ex vivo treatment regimen. The administration may be rapid, such as by injection, or may require a period of time, such as by slow infusion or administration of a sustained release formulation. For treatment of tissues in the central nervous system, administration may be by injection or infusion into the cerebrospinal fluid (CSF).

Enovin may also be linked or conjugated to an agent that provides the desired pharmacological or pharmacodynamic properties. For example, it may be linked to any substance known in the art that promotes penetration or transport across the blood brain barrier, such as transferrin receptor antibodies, and administered by intravenous injection.

The enovin, antisense molecules, or compounds identified as activators or antagonists of enovin of the present invention may be used in the form of pharmaceutical compositions, which may be prepared by methods well known in the art. Preferred compositions include a pharmaceutically acceptable carrier or diluent or excipient, such as a physiological salt solution. Other pharmaceutically acceptable carriers including other non-toxic salts, sterile water or the like may also be used. A suitable buffer may also be present so that the composition may be lyophilized and stored in a sterile condition, or reconstituted by the addition of sterile water for subsequent use. Enovin can also be incorporated into a solid or semi-solid biologically compatible matrix that can be placed into the tissue in need of treatment.

The carrier may also contain other pharmaceutically acceptable excipients to adjust other conditions such as pH, osmotic pressure, viscosity, sterility, lipophilicity, solubility, and the like. Pharmaceutically acceptable excipients which are capable of sustained or delayed release after administration may also be included.

The enovin protein or nucleic acid molecule or compound of the invention may be administered orally. In such embodiments, it may be encapsulated and combined with a suitable carrier in a solid dosage form, as is well known to those skilled in the art.

As is well known to those skilled in the art, the particular dosage regimen may be calculated based on the patient's body surface area or volume of space occupied by the body, depending on the route of administration to be used. However, the amount of the composition actually administered is determined by a physician in view of the conditions associated with the condition to be treated, such as the severity of the symptoms, the composition to be administered, the weight and response of the patient and the chosen route of administration.

The invention will be more clearly understood from the following examples, which are purely illustrative and are described in connection with the accompanying drawings, in which:

FIG. 1: is a partial cDNA sequence of the neurotrophic factor of the present invention called enovin. The consensus sequence was obtained by PCR amplification with primers PNHsp3 and PNHap1 located on different cDNA and genomic DNA, followed by cloning and sequence analysis, and comparing the obtained sequences. The one letter code for the deduced amino acid is shown above the DNA sequence. The number of nucleotide residues is indicated to the right of the DNA sequence, and the number of amino acid residues is indicated to the right of the translated protein sequence. The putative RXR cleavage sites of the prodomain are indicated in bold and underlined. The putative origin of the mature protein is indicated by an arrow. The 7 conserved cysteine residues characteristic of all members of the TGF- β family are indicated in bold. Double-underlined below the potential N-glycosylation site,

FIG. 2: is the alignment of the putative mature protein sequences of human GDNF, NTN, PSP and EVN. The sequences were aligned using the ClustalW alignment program. Amino acids conserved in all three proteins are circled in the dark region. Residues conserved between two or three sequences are colored grey. 7 conserved cysteine residues characteristic of TGF- β family members are marked above the sequence with an asterisk. The number of amino acid residues is indicated on the right. The short lines indicate gaps introduced into the sequence in order to optimize the alignment,

FIG. 3: is a partial cDNA sequence of enovin. The consensus sequence was obtained by PCR amplification of different cDNAs (primary PCR using primers PNHsp1 and PNHap1, and nested PCR using primers PNHsp2 and PNHap2), followed by cloning and sequence analysis, and comparison of the obtained sequences. The one letter code for the translated amino acid sequence of nucleotides 30 to 284 (reading frame A) is indicated above the sequence and numbered on the right (A1 to A85). The reading frame includes a putative ATG translation initiation codon. The one letter code for the translated amino acid sequence of nucleotides 334 through 810 (reading frame B) is indicated above the sequence and numbered on the right (B1 to B159). The reading frame includes regions of homology to GDNF, NTN and PSP. The number of nucleotide residues is indicated on the right side of the DNA sequence. The putative RXR cleavage sites of the prodomain are indicated in bold and underlined. The putative origin of the mature protein is indicated by an arrow. The 7 conserved cysteine residues characteristic of all members of the TGF- β family are indicated in bold. Double-underlined are potential N-glycosylation sites.

FIG. 4 is an illustration of the chromosomal location of human enovin. (A) Schematic representation of enovin FISH mapping results. Each dot represents p31 on human chromosome 1. Dual FISH signals detected in the 3-p32 region. (B) example of enovin FISH mapping. The left photograph shows the FISH signal on chromosome 1. The right photograph shows the same mitotic image stained with 4', 6-diamidino-2-phenylindole to determine chromosome 1,

FIG. 5: indicating the expression of enovin in different human tissues. (A) The results (B) and (C) are Northern blot analysis of tissue expression of enovin. Human poly (A) -rich RNA blots were analyzed with a probe corresponding to a portion of the coding region for enovin, including the region encoding the mature enovin protein, to assess expression of enovin mRNA in different human tissues. (A) Multiple tissue northern (mtn) blots; (B) MTN blot II; (C) fetal MTN blot II. Panel (D) shows autoradiographs of human RNA main blots probed with the same enovin cDNA fragments. Panel (E) shows the position of the human tissue mRNA sample on the main blot of RNA from (D),

FIG. 6: is a representation of the overall survival of SH-SY5-Y cells after 72 hours of treatment with 10-6M paclitaxel and the effect on this survival of increasing the dose of enovin, normalized to the solvent conditions. SH-SY5-Y cells were differentiated for 5 days with 25nM staurosporine before paclitaxel addition. Data were from 6 replicates of two independent experiments. The mean and standard error are shown as well,

FIG. 7 is a graph of the effect on axotomized SH-SY5Y cells of astrocytes on axonal growth-differentiation of SH-SY5 after increasing the enovin concentration for 48 hours, normalized to the conditions of the solvent. SH-SY5-Y cells were differentiated for 5 days with 25nM staurosporine before starting the 48 hour experiment. As a positive control, the differentiation effect of 25nM staurosporine is shown. Axon lengths were calculated from at least 5000 cells, and data provided were from repeated experiments. Mean and standard error are shown.

FIGS. 8 to 18: is a schematic illustration of the effect of enovin on proliferation of various types of cells,

FIG. 19: is a schematic illustration of the effect of enovin on paclitaxel-induced sensory defects using the needle stick test. The mean scores (± 1 SEM) are given as a cumulative time of rats treated with two different doses of enovin (23 or 130 μ g/g; n ═ 10 rats/group) or vehicle/saline (n ═ 20 rats) after treatment with paclitaxel. Enovin or saline/vehicle was injected in a volume of 75 microliters under the sole of the right hindpaw.

FIG. 20: is a schematic illustration of the effect of enovin on paclitaxel-induced sensory defects using the needle stick test. The mean scores (+ -1 SEM) are given as a cumulative sum of the mean scores of rats treated with two different doses of enovin (23 or 130 microgram/g; n ═ 10 rats/group) or vehicle/saline (n ═ 20 rats) before treatment with paclitaxel over time. Enovin or saline/vehicle was injected in a volume of 75 microliters under the sole of the right hindpaw.

FIG. 21: is the DNA sequence of enovin. The consensus sequence was obtained by amplifying human prefrontal cortex cDNA and human genomic DNA with primers PNHsp5 and PNHap1, followed by cloning, sequence analysis, and comparing the resulting sequences. Only the predicted amino acid sequence of a splice variant of the enovin protein which is capable of producing a functional protein after translation is represented above the DNA sequence. The number of nucleotide residues is shown to the left of the DNA sequence, while the number of amino acid residues is shown to the right of the translated protein sequence. The 5 'and 3' splice sites detected by comparing the sequenced cDNA fragments to the genomic sequence are indicated by vertical lines that curve to the left and right, respectively, and are numbered consecutively. The putative RXR furin cleavage sites of the prodomain are indicated in bold and underlined. The putative origin of the mature protein is indicated by an arrow. The 7 conserved cysteine residues characteristic of all members of the TGF- β family are indicated in bold. Double-lined underneath the potential N-linked glycosylation sites. The 5 'and 3' splice sites are numbered and circled with a coil.

FIG. 22: indicating the expression of different enovin splice variants in human tissues. (A) Schematic representation of enovin splice variants identified by RT-PCR experiments on RNA from different human tissues with enovin-specific primers, followed by cloning and sequence analysis of the PCR products. The top line represents the scale (in bp). The second line represents the enovin genomic sequence. The translation start and stop codons, the position of the start of the mature enovin coding sequence and the positions of the 5 'and 3' splice sites are indicated (see FIG. 21). The right part of the figure shows the PCR products obtained by RT-PCR of ovarian and prefrontal cortical RNA, together with a 100bp DNA ladder (ladder). The positions of the different mRNA variants and their sizes (from start codon to stop codon) are indicated. The translated protein is shown on the left. The cDNA is indicated by a boxed portion. The dotted line indicates the spliced genomic DNA. The shaded portion indicates the mature enovin coding sequence. The dotted line indicates the start of the mature enovin coding sequence. Two transcripts capable of producing functional enovin proteins are indicated on the left with asterisks. (B) Tissue distribution of major splice variants. The photographs show the PCR fragments obtained by RT-PCR using enovin-specific primers and different human cDNAs. The four major splice variants (A-D) are indicated on the left by arrows. The size is indicated on the right, measured as a 100bp DNA ladder on a gel as a size reference.

FIG. 23: the predicted protein sequence for the long splice variant of enovin was obtained by splicing out two introns of the DNA sequence shown in figure 21. Splice sites 5 '-1 and 3' -1 are used to eliminate the first intron and splice sites 5 '-2 and 3' -3 are used to eliminate the second intron. The cDNA sequence thus obtained has an open reading frame encoding the protein shown above having 228 amino acid residues.

FIG. 24: the predicted protein sequence of another (short) splice variant of enovin was obtained by splicing out two introns from the DNA sequence shown in figure 21. Splice sites 5 '-1 and 3' -2 are used to eliminate the first intron and splice sites 5 '-2 and 3' -3 are used to eliminate the second intron. The cDNA sequence thus obtained has an open reading frame encoding the protein shown above having 220 amino acid residues. The protein sequence is 8 amino acid residues shorter than the sequence of figure 23.

FIG. 25: are graphs of results obtained from experiments designed to compare the expression levels of enovin in normal diseased tissue. The expression of enovin and GAPDH in brain tissue of multiple sclerosis and alzheimer patients is shown.

FIG. 26: is a graph of the results obtained by measuring the expression levels of enovin and GAPDH in parkinson's disease and cancer.

Storage article

Plasmid EVNmat/pRSETB comprising a DNA sequence coding for enovin has been deposited on 6/5 1999 under the provisions of the Budapest treaty on 28/4/1997 with accession number LMBP3931 at the date of the Belgian Coordinated Collections of Microorganisms (BCCM), laboratory bacteria or molecular-plasmid (LMBP) B9000, Ghent, Belgian.

Materials and methods

Material

Native Taq polymerase, ampicillin, IPTG (isopropyl. beta. -D-thiogalactopyranoside), X-GAL (5-bromo-4-chloro-3-indolyl-. beta. -D-galactopyranoside), and all restriction enzymes used were purchased from Boehringer Mannheim (Mannheim, Germany). 10mM dNTP mix was purchased from Life technologies Inc. (Gaithersburg, Md., USA). TOPO-TA kits were purchased from Invitrogen BV (Leek, Netherlands). Qiagen plasmid miniprep or median DNA purification kits, Qiaprep Spin miniprep kits, and Qiaquick gel extraction kits were purchased from Qiagen GmbH (Dusseldoff, Germany). cDNA library, Marathon^TmReady cDNA kit and human multi-tissue cDNA (MTC)^TM) Group I and group II multiple tissue Northern blots, Advantage-GC cDNA PCR kits were obtained from Clontech laboratories (Palo Alto, Calif., USA). All PCR reactions were performed in a GeneAmp PCR System 9600 cycler (Perkin Elmer, Foster, Calif., USA). LB (Luria-Bertani) medium comprises 10 g/l of tryptone, 5 g/l of yeast extract and 10 g/l of chlorideSodium. The 2 XYT/ampicillin plates comprised 16g/l tryptone, 10 g/l yeast extract, 5 g/l sodium chloride, 15 g/l agar and 100mg/l ampicillin.

Database homology search and sequence comparison.

BLAST (basic local alignment search tool; Altschul et al, 1990) searches were performed on the EMBL/GenBank human Expressed Sequence Tags (ESTs) and genomic databases updated daily using as query sequences protein sequences derived from complete human glial cell line-derived neurotrophic factor (GDNF; accession number Q99748), neurturin (NTN; accession number P39905) and persephin (PSP; accession number AF040962) cDNA.

An additional BLAST search was performed with the genomic sequence deposited under accession number AC005038 and several ESTs present in the GenBank database and showed the homology detected with this genomic sequence.

Percent identity and percent similarity between GDNF family members was calculated by pairwise comparison of sequences using the BESTFIT program (genetics computer set sequence analysis software package, version 8.0, university of Wis Conson, Madison, Wis., USA). The alignment of the DNA or protein sequences was carried out using the ClustalW alignment program (EMBL, Heidelberg, Germany).

Oligonucleotide synthesis for PCR and DNA sequencing

All oligonucleotide primers were ordered from Eurogentec (Seraing, belgium). Insert specific sequencing primers (15-and 16-mers) as well as primers used in the PCR reaction were designed manually. DNA was prepared on a Qiagen-tip-20 or-100 anion exchange column or Qiaquickspin column (Qiagen GmbH, Dusseldoff, Germany) and recovered from the column with 30. mu.l of TE-buffer (10mM Tris-HCl, 1mM EDTA (sodium salt), pH 8.0).

Both strands were sequenced using the ABI prism BigDye terminator cycle sequencing kit and applied to a biological System 377XL sequencer (Perkin Elmer, division ABI, Foster, Calif., USA)And (4) running. Will sequence^TMThe software was used for sequence assembly and manual editing (GeneCodes, AnnAlobor, MI, USA).

Cloning of novel GDNF homologs

The translated protein sequence of the DNA fragment from nucleotide 67411 to 68343 of EMBL deposit No. AC005038 was homologous to mature NTN and PSP and this fragment was used to design oligonucleotide primers for PCR amplification. The different primers used are shown in table 1.

Table 1: primers for PCR amplification of a fragment of AC 005038.

Amplification of cDNAs from different human tissues (fetal brain, whole fetus, prostate or lung Marathon-Ready) with primers PNHsp3 and PNHap1^TMcDNA (Clontech laboratories), prefrontal cortex, hippocampus and cerebellum cDNA) and a 502bp fragment on human genomic DNA. Based on the genomic sequence from the EMBL/GenBank database (accession number AC005038), the G + C content of the fragment to be amplified is expected to be 76%. Thus, amplification was optimized for amplification of GC-rich DNA sequences using the Advantage-GC cDNACR kit (Clontech laboratories, Palo Alto, Calif., USA). The PCR reaction was carried out in a total volume of 50. mu.l containing 1 XGC cDNA PCR reaction buffer, 0.2mM dNTP, 1MGC-MELT^TM200nM primers PNHsp3 and PNHap1, 1 microliter Advantage KlenaTaq polymerase mix, and 1-5 microliter cDNA or 0.5 microgram genomic DNA. The sample was heated to 95 ℃ for 5 minutes and cycled for 35 cycles with parameters of 45 seconds at 95 ℃, 1 minute at 58 ℃, 40 seconds at 72 ℃, and 7 minutes at 72 ℃ as the last step. Finally, 2.5U of native Taq DNA polymerase was used to add the A-overhang. The PCR products were analyzed on a 1% (w/v) agarose gel in 1 XTAE buffer (40mM Tris-acetate, 1mM EDTA (sodium salt), pH 8.3). Cutting off the gelPCR fragments of size (495bp) were purified using the Qiaquick gel extraction kit. The PCR fragment was sequenced to confirm its identity and cloned into the plasmid vector pCR2.1-TOPO using the TOPO TA kit according to the manufacturer's protocol. Approximately 20ng of the purified fragment was mixed with 1. mu.l of pCR2.1-TOPO vector in a total volume of 5. mu.l. The reaction mixture was incubated at room temperature (20 ℃) for 5 minutes. 2 microliter of the reaction mixture was transferred into TOP 10F' competent cells (Invitrogen BV) by heat shock transformation and plated on 2 XYT/ampicillin plates supplemented with 10mM IPTG and 2% (w/v) X-gal for blue-white selection. After overnight growth white colonies were picked from the plates, grown in 5 ml LB medium supplemented with 100mg/l ampicillin, and plasmid DNA was prepared using the Qiaprep Spin miniprep kit. The presence of an insert of the expected size was confirmed by restriction digestion with EcoRI. Plasmid inserts from several positive clones were sequenced and the sequences obtained were compared using the ClustalW alignment program.

To obtain additional coding sequences for the novel GDNF homologues, a fragment with the predicted size of 931bp based on the EMBL/GenBank sequence (accession number AC005038) was amplified by PCR using the primers PNHsp1 and PNHap 1. The PCR reaction was carried out in a total volume of 50. mu.l containing 1 XGC cDNA PCR reaction buffer, 0.2mM dNTP, 1M GC-MELT^TM200nM of primers PNHsp3 and PNHap1, 1 microliter of Advantage KlenaQ polymerase mix, and 1-5 microliter of either cDNA from cerebellum, prefrontal cortex or hippocampus or 0.5 microgram of genomic DNA. The sample was heated to 95 ℃ for 5 minutes and cycled for 35 cycles with parameters of 45 seconds at 95 ℃, 1 minute at 58 ℃, and 1 minute 30 seconds at 72 ℃, with the last step being 7 minutes at 72 ℃. The PCR products were analyzed on a 1% (w/v) agarose gel in 1 XTAE buffer (40mM Tris-acetate, 1mM EDTA (sodium salt), pH 8.3). A second round of amplification was performed with nested primers (PNHsp2 and PNHap 2). 1 microliter of the reaction product of the first round of amplification is used in a total volume of 50 microliter containing 1 XGC cDNA PCR reaction buffer, 0.2mM dNTP, 1M GC-MELT^TMPrimers PNHsp2 and PNHap2 at 200nM, and 1 μmLiter Advantage Klenaq polymerase mix. The sample was heated to 95 ℃ for 5 minutes and cycled for 35 cycles with parameters of 45 seconds at 95 ℃, 1 minute at 58 ℃, 1 minute at 72 ℃ for 30 seconds, and 7 minutes at 72 ℃ for the last step. Samples were analyzed on a 1% (w/v) agarose gel in 1 XTAE buffer. The PCR fragment of the expected size (870bp) was excised from the gel and purified using the Qiaquick gel extraction kit. The PCR fragment was sequenced to confirm its identity, treated with 2.5U Taq polymerase and cloned into the plasmid vector pCR2.1-TOPO using the TOPO TA cloning kit according to the manufacturer's instructions. Approximately 20ng of the purified fragment was mixed with 1. mu.l of pCR2.1-TOPO vector in a total volume of 5. mu.l. The reaction mixture was incubated at room temperature (20 ℃) for 5 minutes. 2 microliter of the reaction mixture was transferred into TOP 10F' competent cells by heat shock transformation and plated on 2 XYT/ampicillin plates supplemented with 10mM IPTG and 2% (w/v) X-gal for blue-white selection. Plasmid DNA grown in 5 ml LB medium containing 100mg/l ampicillin and prepared using the Qiaprep Spin miniprep kit. The presence of an insert of the expected size was confirmed by restriction digestion with EcoRI. The sequences obtained were compared using the ClustalW alignment program.

Analysis of enovin Gene expression by RT-PCR analysis

Oligonucleotide primers PNHsp3 and PNHap1 (see Table 1) were used for specific PCR amplification of a 502bp fragment of enovin. PCR amplification was performed with human multi-tissue cDNA (MTC) normalized to the mRNA expression levels of six different housekeeping genes^TM) The groups were performed. PCR amplification with enovin-specific primers was performed in a total volume of 50. mu.l containing 5. mu.l of cDNA, 1 XGC cDNA PCR reaction buffer, 0.2Mm dNTP, 1M GC-MELT^TM400nM of primers PNHsp3 and PNHap1, and 1 microliter Advantage Klenaq polymerase mix. The sample was heated to 95 ℃ for 30 seconds and cycled for 35 cycles with parameters of 30 seconds at 95 ℃ and 30 seconds at 68 ℃. The samples were analyzed on a 1.2% (w/v) agarose gel in 1 XTAE buffer (40mM Tris-acetate, 1mM EDTA (sodium salt), pH8.3), andimages of the ethidium bromide-stained gel were obtained using the Eagle Eye II camera system (Stratagene, La Jolla, Calif., USA).

Similarity searches of the daily updates of the EMBL/GenBank database with human neurturin and persephin protein sequences resulted in a genomic DNA sequence encoding a putative novel protein similar to the neurotrophic factors GDNF, NTN and PSP, which was named Enovin (EVN). Other database homology searches with genomic DNA sequences flanking the region encoding enovin resulted in several Expressed Sequence Tag (EST) clones from different human tissues (prostate epithelium [ accession No. AA533512(ID1322952) ], lung carcinoma [ accession No. AA931637] and parathyroid tumor [ accession No. AA844072 ]). These clones contained DNA sequences outside the region of homology to GDNF, PSP or NTN, but demonstrated that enovin mRNA was expressed in normal and tumor tissues.

Initial PCR amplification with genomic sequence based primers (PNHsp3 and PNHap1) yielded approximately 500bp fragments from fetal, fetal brain, prostate, prefrontal cortex, hippocampus, cerebellum cDNA and from genomic DNA, but not lung cDNA. Cloning and sequence analysis of the fragment resulted in a 474bp DNA sequence which was translated into a predicted protein sequence of 139 amino acid residues, including 7 conserved cysteines (Kingsley, 1994) characteristic of all members of the transforming growth factor beta (TGF- β) protein family (FIG. 1). The sequence also contains a RXR motif (Barr, 1991) for cleavage of the prodomain (RAAR, amino acid positions 23-26), with a similar cleavage site present at the equivalent position of the prodomain sequence on GDNF, NTN and PSP protein sequences. Cleavage of the enovin prodomain is presumed to occur at this site in vivo. The mature EVN protein sequence contains 113 amino acid residues (residues 27-139 in fig. 1) and has a calculated molecular weight of 11965Da and an isoelectric point of 11.8. A potential N-glycosylation site is present in the mature sequence (NST at amino acid position 121-. Furthermore, several regions conserved between the known mature forms of the neurotrophic factors GDNF, NTN and PSP are also present in enovin (fig. 2). Table 2 summarizes the results of comparing the mature protein sequences of GDNF family members by the BESTFIT program. Percent identity and percent similarity are shown. The GDNF, NTN, PSP, and EVN maturation sequences used for this comparison begin with the first amino acid after the RXXR cleavage site.

Table 2: pairwise comparisons of mature human GDNF family members were performed using the BESTFIT program.

As can be seen from the above comparison, the mature enovin protein is associated to a higher degree with persephin and neurturin than with GDNF.

Amplification, cloning and sequence analysis of a larger fragment of the enovin DNA sequence derived from prefrontal cortex cDNA using primers based on the genomic EMBL/GenBank sequence (accession number AC005038) gave a 819bp sequence (FIG. 3). The sequence includes a putative ATG start codon at nucleotide positions 30-32 and includes an open reading frame (reading frame A in FIG. 3) that extends to a stop codon at nucleotide position 285-287. The translated protein sequence of this region does not show similarity to any known proteins in the database. Translation of the cDNA sequence in the second reading frame (reading frame B in FIG. 3) resulted in a predicted protein sequence of 159 amino acid residues. The sequence includes an RXR cleavage site (positions B43-B46; nucleotide positions 460-471) and corresponds to the mature enovin sequence (positions B47-B159; nucleotide positions 472-810). The open reading frame includes an RXR cleavage site, and the mature enovin coding sequence extends from nucleotide position 334 (which is preceded by an in-frame stop codon) to a stop codon at position 811-813, but does not include the ATG codon encoding the initiating methionine residue. Similar to persephin (Milbrandt et al, 1998), we hypothesize that there is an unspliced intron in the major portion of the mRNA transcript derived from the EVN gene. GDNF and TNT also have an intron in their corresponding prodomain coding regions (Matsushita et al, 1997, Heuckeroth et al, 1997).

To assess the presence of different mRNA transcripts of enovin, RT-PCR experiments were performed with the 5 ' end primer of the enovin coding sequence located 5 ' to the possible upstream ATG start codon (primer PNHsp5[ 5'-GCA AGC TGC CTC AAC AGG AGG G-3' ] and nested primer PNHsp6[ 5'-GGT GGG GGAACA GCT CAA CAA TGG-3' ] and the primer located 3 ' end (primer PNHap1 and nested primer PNHap2[ see Table 1 ]) experiments were performed with various tissue cDNA sets (Clontech MTC sets I and II), fetal heart cDNA library (Clontech) and cDNAs from human cerebellum, hippocampus or prefrontal cortex (Masure et al, 1998). microliter primary PCR reactions were performed with the primers PNHsp5 and PNHap1 under GC-rich conditions (avataggeGC-PCR kit, Clontech) for 30 rounds (95-30 seconds, 60-30 seconds, 72-1 minutes) using the primers PNHsp6 and PNHap2 under the same conditions 30 nested PCR reactions were performed. The resulting PCR products were analyzed on a 1.5% agarose gel and the size distribution ranged from. + -. 350bp to. + -. 800 bp. Several bands on the gel were purified and the PCR fragment was directly sequenced. Some of the purified PCR products were also cloned into the vector pCR2.1-TOPO (TOPO-TA cloning kit, Invitrogen) and then re-sequenced. The sequence molecules confirmed the presence of different mRNA molecules containing the enovin sequence. The obtained fragment sequences were compared to the genomic enovin sequence, which enabled us to determine several possible 5 'and 3' splice sites present on the genomic sequence (fig. 21). All these splice sites correspond to the consensus sequence of the donor and acceptor splice sites (Senapathy, P.Shapiro, M.B. & Harris, N.L. (1990) splice junctions, branch point sites, and exons: sequence statistics, characterization, and use in genome engineering. methods of enzymology 183, 252-. The different enovin splice variants identified and their presence in different human tissues are summarized in FIG. 22. Only two of the five sequenced transcripts were able to produce functional enovin proteins after translation from the ATG start codon. These two transcripts encode proteins with 228 or 220 amino acids, predicted signal peptides with 47 and 39 amino acid residues, respectively, and the predicted protein sequences for these two variants are shown in fig. 23 (long variant) and fig. 24 (short variant). The long variant can be deduced from the DNA sequence of FIG. 21 by excision of the first intron at positions 5 '-1 and 3' -1 and the second intron at positions 5 '-2 and 3' -3. Upon open reading frame translation, the expected protein sequence of FIG. 23 is available. The shorter variant can be deduced from the DNA sequence of FIG. 21 by excision of the first intron at positions 5 '-1 and 3' -2 and the second intron at positions 5 '-2 and 3' -3. After translation of the open reading frame, the predicted protein sequence of fig. 24 was obtained.

The longest transcript appeared to be most abundant in most tissues, judged by the intensity of the bands in fig. 22B. Shorter transcripts produce frameshifts, resulting in a translated protein lacking the mature enovin amino acid sequence homologous to GDNF, NTN and PSP. The two smallest transcripts even lack the mature coding sequence portion, including 2 of the 7 highly conserved cysteine residues. FIG. 22B shows the distribution of the major splice variants in different human tissues. Functional enovin mRNA is expressed in almost all tissues tested, including brain, heart, kidney, liver, lung, pancreas, skeletal muscle, colon, small intestine, peripheral blood leukocytes, spleen, thymus, prostate, testis, ovary, placenta, and fetal heart. In certain human tissues (e.g., cerebellum, hippocampus), only non-functional transcripts can be amplified by PCR. To the best of our knowledge, the occurrence of non-functional mRNA transcripts to such an extent has not been previously disclosed. The biological significance of this finding has yet to be investigated. Although the expression of NTN and PSP in different tissues is not well understood, its expression levels appear to be lower, and its expression is also more limited to certain tissues (Kotzbauer et al, 1996, Milbrandt et al, 1998).

Recombinant expression of Enovin in escherichia coli structure of Enovin expression plasmid

Using primer PNHsp4 and PNHap2 (Table 1) A414 bp PCR fragment from human genomic DNA was amplified and cloned into the vector pCR2.1-TOPO using TA-cloning (Invitrogen). The sequence of the insert was confirmed by sequence analysis. The clone containing the insert with the consensus sequence of enovin (clone 36) was used for the subsequent construction of an expression plasmid. Two primers were designed containing appropriate restriction sites at their 5' ends. Forward primer PNHexp-sp1 (5' -GC)G GAT CCGGCT GGG GGC CCG GGC A-3 ') contains a BamHI site (underlined) and the reverse primer PNHexp-ap1 (5' -GC)C TCG AGTCAG CCC AGG CAG CCG CAG G-3') contains an XhoI restriction site (also underlined). The primers were used to amplify a 343bp fragment encoding mature enovin from clone 36 (positions 81-422 in FIG. 1). The PCR reaction was carried out in a total volume of 50. mu.l containing 1 XGC cDNA PCR reaction buffer, 0.2mM dNTP, 1MGC-MELT^TM200nM primers PNHexp-sp1 and PNHexp-ap1, 1 microliter AdvantageKlenaTaq polymerase mix, and 10ng plasmid DNA from clone 36. The sample was heated to 94 ℃ for 5 minutes and cycled for 25 cycles with parameters of 45 seconds at 94 ℃, 1 minute at 58 ℃, 30 seconds at 72 ℃, and 7 minutes at 72 ℃ as the last step. The resulting 50 microliters of product was purified using the Qiaquick PCR purification kit (Qiagen) and the DNA was eluted in a volume of 30 microliters. 25 microliters of this purified product was digested with 10U BamHI and 10U XhoI in 1 Xbuffer B (Boehringer Mannheim) at 37 ℃ for 1 hour in a reaction volume of 30 microliters. The electrophoresis was performed on a 1% (w/v) agarose gel in 1 XTAE buffer 40mM Tris-acetate, 1mM EDTA (sodium salt), pH8.3), and then the predicted fragment of 353bp was excised from the gel and purified using the Qiaquick gel extraction kit, and the resulting fragment was ligated to the vector pRSET B (Invitrogen) linearized with BamHI and XhoI. The insertion of the resulting plasmid construct (Hevnmat/pRSETB) was confirmed by full sequence analysis. The resulting structure encodes a protein of 146 amino acids with a predicted molecular weight of 15704Da, including an NH 2-terminal 6 xhis-tag fused into the frame of the mature enovin coding sequence. The NH 2-terminal amino acid sequence of the obtained protein is

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPAGGPGS (mature enovin sequence in bold, underlined with 6 His tags).

Expression of enovin in BL21(DE3) E.coli cells

Recombinant production of enovin protein was essentially performed according to Neurturin production as disclosed by Creedon et al (1997) with modifications. To produce recombinant enovin protein, plasmid hEVNMat/pRSETB was transferred into E.coli strain BL21(DE3) (Novagen) and grown to an OD600 of about 0.5 in 2 XYT/ampicillin-medium (16g/l tryptone, 10 g/l yeast extract, 5 g/l sodium chloride, and 100mg/l ampicillin) at 30 ℃ (225rpm) or 37 ℃ (300rpm), followed by addition of IPTG to a final concentration of 0.2mM to induce expression. After 3 hours of induction, the cell pellet was harvested by centrifugation, washed with phosphate buffered saline, centrifuged and stored frozen. For purification and refolding, the cell pellet was resuspended in sonication buffer (20mM Tris-HCl, pH8.0, 300mM sodium chloride, 1mM 2-mercaptoethanol, protease inhibitor (Complete)^TMProtease inhibitor cocktail tablets (boehringer mannheim, 1 tablet per 50 ml buffer) and 1mg lysozyme per 500 mg cell pellet). Cells were disrupted by sonication and inclusion bodies were harvested by centrifugation. The inclusion bodies were dissolved in buffer A (8M urea, 20mM Tris-HCl, pH7.6, 200mM sodium chloride, 1mM 2-mercaptoethanol) and incubated at 37 ℃ for 30 minutes. Then add Ni-NTA resin (nickel nitrilotriacetic acid, Qiagen). After shaking for 40 min at 37 ℃ the sample was washed 1 time with buffer A and loaded onto a5 ml NI-NTA column. The column was washed successively with 10 x column volume of buffer a, 10 x column volume of buffer a at pH 7.2 and 10 x column volume of buffer a +10mM imidazole at pH 7.2. Enovin was washed from the column with 4 column volumes of buffer A +200mM imidazole pH 7.2.

Enovin refolding was performed by stepwise overnight dialysis at 4 ℃ in renaturation buffer (0.1M sodium phosphate, 0.15M sodium chloride, 3. mu.M cysteine, 0.02% Tween-20, 10% glycerol, 0.01M Tris-HCl, pH8.3) containing reduced amounts of urea (6M to 4M to 3M to 2M to 1M to 0.5M to 0M urea) at each step. The purified protein was aliquoted, stored at-20 ℃ and further used for functional experiments.

Chromosomal mapping of the Enovin Gene

A3.3 kb fragment of the Enovin gene from cerebellar cDNA was amplified using primers EVN (7) -sp1 (5'-TTC GCG TGT CTA CAA ACT CAA CTC CC-3') and PNHap1 (5'-GCA GGAAGA GCC ACC GGT AAG G-3') designed according to the sequence of EMBL accession number AC 005038. The PCR reaction was carried out in a total volume of 50. mu.l containing 1 Xexpand Long Template PCR reaction buffer (Boehringer Mannheim), 0.5mM dNTP, 1M GC-MELT^TM(Clontech), primers EVN (7-sp1) and PNHap1 at 400nM, and cerebellar cDNA at 1 μ l. First, after 2 minutes of reaction at 94 ℃, 0.75. mu.l of Expand Long Template polymerase (Boehringer Mannheim) was added and 10 cycles were performed, at 94 ℃ for 10 seconds, at 58 ℃ for 30 seconds, and at 68 ℃ for 3 minutes. Then, 20 additional cycles of reaction were carried out, and the time at 68 ℃ was extended by 20 seconds for each 1 cycle. Also included was a final 7 minutes at 68 ℃. The resulting 3.3kb fragment was purified after electrophoresis on a 0.8% agarose/TAE gel and cloned into the vector pCR2.1-TOPO using TA-cloning (Invitrogen). Full sequence analysis of one cloned 3.3kb insert confirmed the obtainment of a cDNA sequence corresponding to the genomic sequence in the EMBL database (accession number AC 005038). No intron was spliced out of the cDNA obtained from the cerebellar cDNA.

Chromosome mapping studies are essentially performed according to the published Fluorescein In Situ Hybridization (FISH) assay (Heng et al, 1992, Heng & Tsui, 1993). Human lymphocytes were cultured at 37 ℃ for 68-72 hours prior to treatment with 0.18 mg/ml 5-bromo-2' deoxyuridine (BrdU) to synchronize the cell cycle in this cell population. The synchronized cells were washed and incubated at 37 ℃ for an additional 6 hours. Cells were harvested and slides prepared using standard methods including hypotonic treatment, fixation and air drying. The 3.3kb probe for Enovin was biotinylated and detected using FISH. Slides were baked at 55 ℃ for 1 hour, treated with RNase, and denatured in 70% formamide prepared in 2 NaCl/Cit (20 NaCl/Cit is 3M NaCl, 0.3M disodium citrate, pH7.0) at 70 ℃ for 2 minutes, then dehydrated with ethanol. The probes were denatured prior to loading onto the denatured chromosome slides. Slides were washed after overnight hybridization and FISH signals and 4 ', 6-diamidino-2-phenylindole bands were recorded on photographic films, respectively, and alignment of FISH mapping data to chromosomal bands was achieved by overlapping FISH signals and 4', 6-diamidino-2-phenylindole band chromosomes (Heng & Tsui, 1993). Under the conditions used, the hybridization efficiency of this probe was approximately 72% (of the 100 examined mitotic pictures, 72 appeared on a pair of said chromosomes). Since the 4', 6-diamidino-2-phenylindole band was used to identify the particular chromosome, the signal from the probe was aligned with the short arm of chromosome 1. The detailed position of the 10 photographs was further determined based on the overall results (fig. 4A). Under the conditions used, no other sites were found by FISH detection, and therefore, it was considered that Enovin was located at p31.3-p32.3 of human chromosome 1. An example of the plotting result is shown in fig. 4B.

From the genetic map data of the national center for Biotechnology information (NCBI, http:: www.ncbi.nlm.nih.gov/genemap), it can be concluded that the genomic clone containing the Enovin sequence (EMBL accession number AC005038) is located between the markers D1S2843 and D1S417 of chromosome 1. It corresponds to the region p31.1-p32.3 of chromosome 1, confirming the data obtained by FISH analysis.

Tissue distribution of Enovin determined by Northern blot and dot blot analysis

Poly (A) -rich RNA containing 2. mu.g from different human tissues (Clontech laboratories, Palo Alto, Calif., USA; MTN) was used according to the manufacturer's instructions^TMBlot I, MTN^TMBlot II and fetal MTN^TMNorthern blotting of blot II)And alpha³²A897 bp Enovin fragment labeled with a random primer for P-dCTP (High Prime kit, Boehringer Mannheim) was hybridized. This fragment was obtained by PCR amplification of prefrontal cortex cDNA using the primers PNHsp1 and PNHap1 and was then cloned into the vector pCR2.1-TOPO, which contains 897bp of the Enovin sequence up to the stop codon and contains the entire coding sequence of the mature Enovin protein.

Enovin mRNA was detected as a major transcript of approximately 4.5kb (FIGS. 5A-C). Enovin mRNA can be expressed in a variety of tissues, most notably in heart, skeletal muscle, pancreas, and prostate. Some smaller transcripts are present in tissues such as placenta (4kb, 2.4kb and 1.6kb) and prostate (4kb and 1.6 kb). In fetal tissues, a major 2.4kb transcript is present in the liver and in lesser amounts in the lungs. Other transcripts are also present in fetal kidney, liver, lung and brain.

In addition, RNA master blots (Clontech laboratories) containing poly (A) -rich RNA from different human tissues and developmental stages were hybridized with 897bp Enovin probe. The poly (A) -rich RNA samples used to prepare the blots were normalized by the manufacturer with respect to the mRNA expression levels of 8 different housekeeping genes. Enovin mRNA was ubiquitously expressed, but the highest mRNA content appeared in the prostate, pituitary, trachea, placenta, fetal lung, pancreas and kidney (fig. 5D + E).

Construction of GFR alpha-IgG-Fc fusion vector

cDNA regions of GFR α -1, GFR α -2 and GFR α -3 (encoding amino acids 27-427, 20-431 and 28-371, respectively) which do not include sequences encoding a Signal peptide and a COOH-terminal hydrophobic region involved in GPI-anchoring were cloned in frame onto an expression vector Signal pIg plus (R & D systems Europe, Inc.). The protein produced by expression of these constructs included a 17 amino acid NH 2-terminal CD33 signal peptide, a GFR α protein region and a 243 amino acid COOH-terminal human IgG1-Fc fusion domain. CHO cells were transfected with GFR α fusion constructs and stably transfected cells were selected with 500 μ G418. Permanent clones were screened with anti-Fc antibodies. To purify GFR α fusion protein, cells were grown in serum-free medium and the medium was collected after every 3 days. The medium was centrifuged and applied to a protein a column (protein a Sepharose, Pharmacia biotechnologies). Bound proteins were eluted with 0.1M sodium citrate pH3.0 and collected into 1M Tris buffer, pH 8.4. The protein concentration was estimated by absorbance at 280nm, using an extinction coefficient of 1.5. This purified soluble GFR α -1 to-3 Fc fusion protein was used for subsequent binding studies.

Surface plasmon resonance analysis

Surface Plasmon Resonance (SPR) experiments were performed using a BIAcore3000 instrument at 25 ℃. Analysis was performed using enovin and NGF as immobilized ligands. The carboxylated matrix of the F1 sensor chip was first activated for 10 minutes with a 1: 1 mixture of 400mM N-ethyl-N- (dimethylaminopropyl) -carbodiimide and 100mM N-hydroxy-succinimide. Recombinant enovin and NGF dissolved in 10mM acetate buffer, ph4.5, were then loaded onto the activated surface at a flow rate of 5 μ l/min. The unoccupied reactive groups were deactivated with 1M ethanolamine hydrochloride. For binding experiments, soluble GFR α 1-3-Fc was superfused at a concentration of 10-100nM in 10-100nM HEPES buffered saline solution (150mM sodium chloride, 3.5mM EDTA, 0.05% P-20, 10mM HEPES, pH7.4) at a flow rate of 10. mu.l/min. Binding was detected for 3 minutes and dissociation was detected for 1 minute, followed by regeneration with 5mM sodium hydroxide. Dissociation was initiated by superfusion with HEPES buffered saline. Binding rates (k) were calculated using BIAcore evaluation software 3.0_a) Dissociation rate (k)_d) And equilibrium dissociation constant (K)_DWith K_d/K_aCalculation).

Results

SPR was used to determine the binding of soluble GFR α 1-3 to immobilized enovin. Specific binding to enovin can only be detected with soluble GFR α 3. GFR α 1 and GFR α 2 cannot bind to immobilized enovin. The binding of GFR α 3 observed is specific because it is not able to bind NGF. Specific binding of TrkA-Fc (NGF receptor) to immobilized NGF, but not to immobilized enovin, was detected in a separate control experiment.

From the binding curves obtained with three different concentrations of GFR α, the following constants in table 3 were derived. The above results demonstrate that GFR α 3 specifically binds to enovin.

TABLE 3

Since GDNF, NTN, PSP all promote the maintenance and survival of different types of nerve cells, enovin is predicted to have the same biological effect on nerve cells and may have similar effects on other types of cells. Thus, the enovin protein is expected to be useful in the treatment of neurological diseases in general, including parkinson's disease, alzheimer's disease, peripheral neuropathy, Amyotrophic Lateral Sclerosis (ALS), huntington's disease, peripheral neuropathy, acute brain injury, nervous system tumors, multiple sclerosis, peripheral nerve trauma or injury, and injury from exposure to neurotoxins.

Enovin can also be used in several aspects of neuroprotection. Given its effect on the survival of different neural cell populations and on the elongation of axons observed on our SH-SY5Y cell model, we believe that this compound may have neuroprotective and neuroregenerative uses.

The above conclusions are based on the following observations. Paclitaxel was able to induce neuronal apoptosis in NGF-differentiated PC12 rat pheochromocytoma cells (proposed by Nuydens et al), and thus paclitaxel-induced cytotoxicity was characterized by neuronal apoptosis detected by DNA fragmentation, Annexin V labeling and bc1-2 protection. Therefore, by extension, paclitaxel is presumed to induce apoptosis in differentiated SH-SY5Y cells. It has been demonstrated that Enovin can attenuate this cell death and thus can reverse apoptosis of nerve cells in general.

Thus, the compounds are useful in the treatment of the following neurodegenerative diseases in which apoptosis has been observed: stroke (Hakim1998), parkinson's disease (Marsden et al, 1998), alzheimer's disease (NAGY et al, 1998), huntington's disease (Wellington et al, 1997), neurotrauma (Smirnova et al, 1998), peripheral neuropathy (Srinivisan et al, 1998).

As an example of the last clinical evidence, we have demonstrated that this neurotrophic factor indeed protects differentiated SH-SY5Y human neuroblastoma cells from paclitaxel-induced cytotoxicity.

Method for measuring viability

Cell viability was determined by adding 100 μ l of 2, 3-bis [ 2-methoxy-4-nitro-5-thiophenyl ] -2H-tetrazole-5-carboxanilide (XTT, Sigma) in DMEM (37 ℃) supplemented with 0.02mM phenazine methosulfate (PMS, Sigma) to each well. The plates were then incubated at 37 ℃ for 2.5 hours. The optical density was read at 450nm (molecular device) using 650nm as a control. The XTT experiments are based on the conversion of the tetrazolium salt XTT to the red formazan product. The reaction is carried out with a mitochondrial enzyme.

Method of neural differentiation

1. Differentiation in human neurofibroma SH-SY5Y cells

SH-SY5Y cells were differentiated for 5 days with 25nM staurosporine. The effect of Enovin was determined 72 hours after the start of the experiment (see Jalava et al, "protein kinase inhibitor Staurosporine induces mature neural phenotype in SH-SY5Y human neuroblastoma cells via the independent a, b, z PKC pathways", J. cell physiology, 155, 301-312 (1993)).

2. Determination of axonal extension

The morphological changes of neurons were automatically quantified by the following method. Briefly, glutaraldehyde was added directly to the medium at an appropriate time and left at room temperature for 30 minutes. This ensures that the morphology of the cells at that point in time reflects the actual situation. The cells were observed in transmitted light mode on an Axiovert microscope (Zeiss Oberkochen, Germany) equipped with a Marzhauser scanning stage driven by an Indy workstation (Silicon Graphics, Mountain View, USA). Images were acquired with a MX5 video camera (HCS). Approximately 3000 cells were evaluated in 64 parallel images to form an 8 x 8 image square. The actual alignment ratio of the images ensures that axons can pass from one image field of view to another. Axonal extension labeled with polyclonal tau antibody was automatically detected with a systematic error free detector with a curvilinear structure (Steger 1998). The analysis software was able to automatically calculate the total cell body area, the number of cell bodies and the total axon length.

To investigate the effect of Enovin on different cell types, 2 experiments were performed. One is a DNA synthesis experiment and the other is a chemotaxis experiment.

DNA Synthesis experiments

Cells including human epidermal fibroblasts (39SK), Human Umbilical Vein Endothelial Cells (HUVEC), Human Smooth Muscle Cells (HSMC), human chondrocytes, and rat osteoblasts were maintained in DMEM containing 10% FBS (39-SK, HSMC, rat osteoblasts) or in defined media (chondrocytes and HUVECs) at 37 ℃ in 5% carbon dioxide and 95% air. For DNA synthesis experiments, cells were seeded at 5000 cells/well in DMEM containing 10% FBS on 96-well tissue culture plates and cultured for 24 hours. The medium was then replaced with DMEM containing various concentrations of Enovin and 0.1% BSA (for 39-SK, osteoblasts, HSMC, chondrocytes) or DMEM containing various concentrations of Enovin and 0.5% FBS (for HUVEC), and the cells were cultured for 24 hours. Then, with 100. mu.l, 5% FBS and 0.1. mu.i³H]100 μ l of DMEM with thymidine instead of the medium. After 2 hours of pulse labeling, cells were fixed with methanol/acetic acid (3: 1, vol/vol) at room temperature. Washing with 80% methanolFixed cells were 2 times. The cells were lysed in 0.05% trypsin (100. mu.l/well) for 30 minutes and then in 0.5% SDS (100. mu.l/well) for another 30 minutes. An aliquot of the cell lysate (180 μ l) was combined with 2 ml of scintillation cocktail and the radioactivity of the cell lysate was measured with a liquid scintillation counter (Wallac 1409).

Chemotaxis experiments

Cells were maintained as described in the "DNA synthesis experiments". Enovin chemotactic activity was analyzed in a 12-well modified Boyden chamber (McQuillan, D.J., Handley, C.J., Campbell, M.A., Bolis, S., Milway, V.E., Herington, A.C., (1986), "promotion of proteoglycan synthesis in cultured bovine articular cartilage by serum and insulin-like growth factor-I," J.Biochem.240: 423-430). Cells were trypsinized with 0.05% trypsin and 0.5mM EDTA and resuspended in DMEM. To the bottom well of the Boyden chamber, 150 microliter aliquots of medium containing various concentrations of Enovin were added. A polycarbonate membrane (8 microns) coated with 0.1 mg/ml type I collagen was placed over the bottom well and the top well was then assembled. A 100 μ l aliquot of cells (70,000 cells/ml) was added to the top well. After 6 hours of incubation, the device was detached. Cells remaining on top of the membrane were removed. The membranes were fixed with 10% formaldehyde for 15 minutes, then stained with Gill's strong hematoxylin, cells were counted under a microscope (250-fold magnification), and the average number of cells in 5 regions per well was used. Each experiment was repeated at least 4 times. Results are expressed as a fold of control (DMEM with 0.1% BSA).

As shown by the results in FIGS. 8-18, Enovin had no effect on the proliferation of each of the cells used, or on the migration of HUVEC cells (FIG. 14), as described above. Enovin has an effect on SH-SY5Y neuroblastoma cells. This demonstrates the selective effect of Enovin on neural cells.

GDNF and NTN have been shown to signal through a signaling complex consisting of the ligand binding subunit GFR α -1 or GFR α -2 and the signal subunit cRET protein tyrosine kinase. Enovin is expected to exert its biological effect through a similar signaling complex consisting of a GFR α binding partner (GFR α -1, GFR α -2, the newly identified preschool receptor GFR α -3, or other yet unidentified GFR α family member) and cRET or other signaling partners. In fact, our binding data demonstrate that Enovin binds specifically to GFR α -3.

In humans, germline mutations on GDNF or cRET can lead to several disease phenotypes including multiple endocrine adenomatous and familial Hirschsprung's (SHCR) (Romeo et al, 1994, Edery et al, 1994, Angrist et al, 1996). Both of these diseases are associated with intestinal weakness, with the congenital megacolon being the most common cause of congenital ileus in infants. Interestingly, GDNF or cRET knockout mice exhibit pathological symptoms very similar to renal hypoplasia and enteric ganglion cell deficiency (Sanchez et al, 1996; Moore et al, 1996; Pichel et al, 1996). Enovin may be associated with similar diseases of the gut or kidney, or, because it is ubiquitously expressed, plays an important role in the development of other peripheral organs of the body.

The interaction of a ligand and its receptor is usually achieved by interactions resulting from specific bonds between specific residues on the two proteins. Fragments of a protein can be used as activators to activate its receptors, resulting in growth promotion and survival maintenance of cells. Thus, portions of Enovin or peptides synthesized from the Enovin protein sequence can be used as activators or antagonists to modulate its receptor GFR α -3. Hybrid growth factors consisting of GDNF, NTN or PSP or any other neurotrophic or growth factor with Enovin moieties may be produced using peptide synthesis or recombinant techniques to obtain novel synthetic growth factors with novel characteristics.

To examine whether Enovin can alter the paclitaxel-induced sensory deficit in rats after plantar injection into rats, two preliminary experiments were performed. In the first experiment, it was examined whether a single treatment with Enovin could reverse the paclitaxel-induced sensory defect, while in the second experiment, Enovin prevented the development of the paclitaxel-induced defect.

Recovery of paclitaxel-induced sensory abnormalities over time

Method of producing a composite material

Male Sprague-Dawley rats weighing 300-340 g were used. The animals were housed individually and allowed free access to food and water. Before starting the experiment, the animals were placed in standard observation cages and after an adaptation period of 15 minutes, the needle reflex was assessed. To achieve this, the plantar surface of the right paw of the animal was stimulated with a needle and the response to the needle was counted as either with (score 1) or without (score 0). Repeating the above process 3 times in each period, wherein the time interval between two consecutive acupuncture reactions is 1 min; thus, the needling experiment included 3 measurements of response to needling. Only rats with a normal response in all 3 acupuncture were used for this experiment.

The animals were injected daily in the sole of the right hind paw with 50 microliters of paclitaxel (3 mg/ml paclitaxel, dissolved in cremophor and dehydrated alcohol plus water) in the morning for 3 consecutive days. In the morning of the following day, the needle stick response was again evaluated and animals were screened for no response to 3 needle sticks. The animals were randomly divided into small groups (n-10/group) and injected with 75 μ l of vehicle, saline or 23 or 130 μ g/ml Enovin in the sole of the right hind foot. Since no difference was found between the animals treated with vehicle and saline, the two groups were combined (control group) and the needling experiments were performed on days 1, 4, 5 and 7 after the last treatment, in the morning (8-9 am) and in the afternoon (3: 30-4:30 pm). The last needle puncture experiment was performed in the morning on day 8. For each animal, the cumulative score for the response to the needle stick was measured over time. Since a total of 9 needle-punching experiments (each comprising 3 needle-punching) were performed after the last drug treatment, the maximum score obtained after the total time of the experiment was 27.

Results

Repeated injections of paclitaxel on the sole of the foot for 3 consecutive days resulted in an acute inflammatory response in most animals, making them deficient in response to the needle stick stimulus. Plantar injection of saline or vehicle did not affect paclitaxel-induced defects. Only 4 out of 20 controls showed at least 1 response to 3 punctures at the time of the first assay, and the mean (± SEM) puncture score of the first assay control was 0.25(± 0.12); this is different from the case at the beginning of the experiment when the average score was 3.0 (+ -0.0) because all animals responded to the needle stick. Control reactivity was compromised even 8 days after assay, with 11 out of 20 rats having at least 1 response with an average puncture score of 0.75(± 0.18). In this control group, none of the rats showed a normal response to 3 needle punctures. The cumulative score of the control pricks over time is given in fig. 19. Since the animals were subjected to 9 experiments over a period of 8 days, the highest score achieved by 3 needle sticks in each experiment was 27. As shown in the graph, the injection of saline or vehicle intraplantar failed to reverse the paclitaxel-induced deficit over the time of the experiment. The mean total cumulative score for the controls at the end of the experiment was 5.10 (+ -0.87); it is 18.9% of the highest score to be achieved.

After the first measurement, a single plantar injection of 75 μ l of 23 μ g/ml Enovin resulted in at least one response in 4 out of 10 rats with an average needle puncture score of 0.70 (+ -0.33). On day 8, all 10 animals responded to the needle at least once, and a normal response occurred in 5 out of 10 rats. The mean puncture score for this group was 2.20(± 0.29) on day 8. The mean cumulative score at the end of the 8 day assay was significantly higher compared to the control (Mann-Whitney U-experiment, two-tailed, p < 0.01), reaching a mean needle score of 14.50 (+ -1.96) (FIG. 19). This result was 53.7% of the highest score.

In addition, plantar injection of 130 μ g/ml Enovin had improved efficacy over the control. At the first assay after 130. mu.g/ml Enovin injection, 6 out of 10 rats responded at least once with a mean needle puncture score of 1.10 (+ -0.35). On day 8, all 10 animals responded to the needle stick at least once with an average needle stick score of 2.60 (+ -0.22). Of 10 rats, 8 responded normally to 3 needle punctures. At the end of the experiment, the average cumulative total needle puncture score for this group was 17.20 (+ -1.94). This result was 63.7% of the total score possible and was a significant improvement (p < 0.01) over the control.

Prevention of paclitaxel-induced sensory defects over time

Method of producing a composite material

Male Sprague-Dawley rats weighing 300-340 g were used. The animals were housed individually and allowed free access to food and water. Before starting the experiment, the animals were placed in a standard observation cage and after an acclimation period of 15 minutes, the needling reflex was evaluated. To achieve this, the plantar surface of the right paw of the animal was stimulated with a needle and the response to the needle was counted as either with (score 1) or without (score 0). Repeating the above process 3 times in each period, wherein the time interval between two consecutive acupuncture reactions is 1 min; thus, the needling experiment included 3 measurements of response to needling. Only rats having a normal response in all 3 punctures were used in the experiment (puncture score of 3). After the control assay was performed, the animals were randomly divided into small groups (n-10/group) and injected with 75 μ l of vehicle, saline or 23 or 130 μ g/ml Enovin in the sole of the right hind foot. Since no difference was found between the animals treated with vehicle and saline, the two groups were combined (control group) and the animals were injected daily for 3 consecutive days with 50 μ l paclitaxel (3 mg/ml paclitaxel, dissolved in cremophor and dehydrated alcohol plus water) in the plantar region of the right hind leg. Acupuncture experiments were performed on days 1, 4, 5 and 7 after paclitaxel treatment, in the morning (8-9 am) and in the afternoon (3: 30-4:30 pm). The last needle puncture experiment was performed in the morning on day 8. For each animal, the cumulative score for the response to the needle stick was measured over time. Since a total of 9 needle prick experiments (each comprising 3 needle pricks) were performed after paclitaxel treatment, the maximum score obtained after the total time of the experiment was 27.

Results

Plantar injection of saline or vehicle prior to paclitaxel treatment did not inhibit paclitaxel-induced defects in the needle stick experiments. At the first test after paclitaxel, 8 out of 20 rats had at least one response to needle sticks with an average needle stick score of 0.60 (+ -0.18). At day 8, there was still a defect induced by paclitaxel, with only 8 of 20 animals responding, with an average score of 0.8(± 0.25). There were standardized needle prick reflexes on 2 animals. The cumulative acupuncture score also decreased over time, with an average of 6.55(± 1.08) which was 24.3% of the highest score (fig. 20).

Pretreatment with Enovin 23 μ g/ml attenuated paclitaxel-induced needle defects. On day 1, 8 of 10 animals had at least one reaction and the mean needle punch score was 1.70 (+ -0.40). On day 8, all animals had a response with an average score of 2.50 (+ -0.27). Here, 7 animals showed normal responses to all needling treatments. For the response accumulated over time (fig. 20), the mean total score improved significantly (p < 0.01) from the control level to 18.40(± 1.73), which is 68.1% of the maximum.

Comparable results were obtained after pretreatment with 130 μ g/ml Enovin. In this case, 6 out of 10 animals had a response in the first experiment, and the average puncture score was 1.70 (+ -0.31). On day 8, all animals responded to the needle stick at least once, with an average score of 2.40(± 0.22), and half of the animals responded normally 3 times. In terms of cumulative score, the average score obtained on day 8 was 17.70(± 1.92), accounting for 65.5% of the total score.

The experimental series show that single plantar injection of Enovin can weaken paclitaxel-induced sensory defects, which is determined by a needling experiment. When the drug was used before and after paclitaxel, activity was observed.

Enovin is a likely candidate for painful syndromes primarily caused by peripheral and central nervous components, rheumatic/inflammatory diseases, and conduction disorders, and can play a regulatory role in the sensing process after transdermal, external, topical, central (e.g., epidural, intrathecal, etc.) and systemic use.

In addition, Enovin can be used as a diagnostic tool to screen for the pathophysiological changes that occur at the sites.

The expression of Enovin mRNA in normal and diseased tissues was compared.

The expression of Enovin mRNA was quantitatively analyzed using the ABI Prism7700 sequence detection system (TaqMan; Perkin Elmer) using a proprietary technique developed and implemented by Pharmagene laboratories, Inc. (Royston) of UK. The system uses a fluorescent probe to generate a sequence-specific fluorescent signal during PCR. The probe is an oligonucleotide to which a fluorescent label and a quencher dye are attached, and is located between the forward and reverse PCR primers. In the intact case, the intensity of the fluorescent label is suppressed by the quencher dye. If the probe forms part of a replication complex, the fluorescent label will be cleaved from the quencher by the 5 'to 3' exonuclease activity inherent in Taq polymerase. In the reaction, the enhancement of the fluorescence labeling signal is direct evidence of the accumulation of PCR products. The initial copy number of the mRNA target sequence (Cn) -the point at which the fluorescence signal exceeds the background range is determined by determining the number of separate PCR rounds (Ct) at which the PCR product was initially detected. The amount of target mRNA in each sample was determined by comparing the experimental Ct values to a standard curve.

RNA preparation and quality control

Total RNA was isolated from intact and sub-dissected tissues using Tri-Zol reagent (Life technologies, Gaithersburg, Md., USA) according to the manufacturer's protocol. Quality control methods for all RNA samples included assessment of integrity (intact 18S and 28S ribosomal RNA) and determination of the presence of high (actin) and low (transferrin receptor) transcripts.

Primer/probe design

Design of a pair of primers and TaqMan probes for amplification of specific sequences from Enovin

Primer 1: 5 'ACGGTTCTCCAGGTGCTGT 3'

Primer 3: 5 'TGCTGCCGACCCACG 3'

And 5, probe: 5 'CTACGAAGCGGTCTCCTTCATGGACG 3'

In addition, a pair of primers and TaqMan probe were designed, which span one intron, and were used to amplify a portion of the human GAPDH gene

Primer 2: 5 'CAGAGTTAAAAGCAGCCCTGGT 3'

Primer 4: 5 'GAAGGTGAAGGTCGGAGTCAAC 3'

And 6, probe 6: 5 'TTTGGTCCGTATTGGGCGCCT 3'

Probe 5 is labeled with the fluorescent agent FAM and probe 6 is labeled with the fluorescent agent VIC.

DNase treatment of Total RNA

For each tissue of the experiment, 2.2ug of total RNA was digested in a 20 microliter volume of 1 XDase buffer (Gibco BRL) with 2 units of RNase-free DNase (Gibco BRL) at room temperature for 15 minutes. The reaction was stopped by adding 2. mu.l of 25mM EDTA solution. The sample was then incubated at 65 ℃ for 10 minutes to inactivate the enzyme.

Synthesis of first cDNA Strand

For each experimental tissue, 100ng of total RNA was used as template for the first cDNA strand synthesis. The RNA was heated to 72 ℃ for 5 minutes in a volume of 4 ml in the presence of 50nM primer 1 and 2, 1 XPCR buffer II (Perkin Elmer) and 5mM magnesium chloride, and slowly frozen to 55 ℃. After all other reagents were added, 6 ml of the reaction was incubated at 48 ℃ for 30 minutes, followed by an enzyme inactivation step at 90 ℃ for 5 minutes. The final reaction conditions were as follows: 1 XPCR buffer II, 5mM magnesium chloride, 1mM dATP, dTTP, dGTP, dCTP, 12.5 units of MuLV reverse transcriptase (Gibco BRL).

PCR amplification of the first cDNA Strand

The cDNA derived from 100ng of total RNA from each sample was PCR amplified in one reaction to identify the target transcript and the GAPDH transcript. The final primer/probe concentrations for the target were 300nM primer 1, 300nM primer 3 and 200nM probe 5, and GAPDH 20nM primer 2, 20nM primer 4 and 100nM probe 6. The final concentration of the other reagents in the reaction was 4.5% glycerol, 1 XTAQMan buffer A (Perkin Elmer), 6.25mM magnesium chloride, 430M dATP, dUTP, dGTP, dCTP, 2.5 units AmpliTaq Gold. The PCR amplification was performed in the ABI7700 sequence detection system, with the initial enzyme activation step being performed at 94 ℃ for 12 minutes, followed by 45 cycles of 94 ℃ for 15 seconds and 60 ℃ for 1 minute (minimum cycle time).

Detected diseases and tissues

The expression of Enovin mRNA in tissues of disease patients and normal control individuals was compared (fig. 25 and 26). The diseases and related tissues that have been studied are given in the table below. For each disease, 3 lesion samples and 3 control samples were analyzed.

Disease and disorder	Tissue 1	Tissue 2	Tissue 3
				Alzheimer's disease	Temporal cortex	Sea horse body	Posterior scalp cortex
Multiple sclerosis	Cerebrospinal fluid	Periventricular white matter	Cerebellum
				Parkinson's disease	Black texture	Hard core	Cerebellum
Cancer treatment	Adenocarcinoma of colon	Adenocarcinoma of mammary duct	Squamous cell carcinoma of lung

Statistical analysis

For each set of 3 tissues, the mean and standard error of the Ct values (which are normally distributed) were calculated and then 10 according to the formula Cn^{((Ct-40.007)/-3.623)}Converted to Cn values. Error analysis (ANOVA) was performed on the Ct values and the mean Enovin mRNA expression levels were compared between normal and diseased tissues.

FIGS. 25 and 26 show the mean Enovin mRNA copy number (. + -. SD; n.3) in lesion and control tissues. Statistical analysis showed that the expression level of Enovin in periventricular white substance was significantly increased in multiple sclerosis patients (p ═ 0.013). The internal GAPDH control showed no significant difference (p ═ 0.79). Although the expression level of Enovin in periventricular white mass in normal tissues was low (on average 270 copies per 100ng total RNA and 200000 copies of GAPDH), the expression level was 3-fold higher in patients with multiple sclerosis (825).

Only one other diseased tissue showed a significant difference from the normal control: in ductal adenocarcinoma of the breast, the expression level of Enovin mRNA was 6-fold higher (6000 versus 1000; p 0.007), although the value of GADPH control was also significantly increased (165000 versus 44000; p 0.03), possibly representing a general increase in mRNA content.

In summary, we found that the amount of Enovin mRNA was upregulated in periventricular white mass in multiple sclerosis patients.

Enovin mimetics on the GFR α 3/cRET receptor complex were screened using a phospho-specific antibody cell-based ELISA.

The methods are also useful for identifying activators or antagonists of other neurotrophic factor receptors, such as GFR α 1, GFR α 2, GFR α 4, TrkA, TrkB, and TrkC.

Experiment of

Using this assay, we can identify neurotrophic growth factor activating or antagonistic compounds by measuring activation of key signaling kinases that are activated in the neurotrophic pathway or by measuring activation of cRET receptor kinases. The activation is determined by detecting the amount of phosphorylated kinase or receptor kinase with a phosphorus-specific antibody. We used NIH3T3 cells that transiently or permanently expressed TrkA, TrkB, TrkC, GFR α 1/cRET, GFR α 2/cRET, GFR α 3/cRET, or GFR α 4/cRET.

Activation of p42/p44 MAP kinase, PKB kinase, c-jun, CREB, JAN/SAPK kinase and other kinases was detected using commercially available phospho-specific antibodies. In addition, activation of cRET can be abolished with a phosphorus-specific cRET antibody.

The method used was as follows:

NIH3T3 cells were plated in 96 wells in 10% bovine serum, and cells had to reach 80% confluency before stimulation.

The next day, medium was replaced with serum-free medium and cells were starved for 18-24 hours.

After starvation, cells were stimulated with compounds and neurotrophic factors as positive controls (concentration of neurotrophic factor 10 ng/ml).

Cells were fixed with 4% formaldehyde prepared from PBS for 20 min at 4 ℃.

Wash cells with 200 μ l PBS/0.1% Triton for 5 min, 3 times.

0.6% H in 100. mu.l in PBS₂O₂Cells were quenched for 20 min with 0.1% Triton.

Wash cells with 200 μ l PBS/0.1% Triton for 5 min, 3 times.

Cells were blocked with 100 μ l 10% fetal bovine serum/0.1% Triton prepared in PBS for 60 min.

-incubating the cells with a phosphorus-specific antibody overnight in 50 μ l of 5% BSA/PBS/0.1% Triton at 4 ℃. The antibody dilution is determined experimentally, and a range of 1: 100 to 1: 250 is suggested.

Wash cells with 200 μ l PBS/0.1% Triton for 5 min, 3 times.

Incubation with a 1: 100 dilution of a second HRP-crosslinked antibody in 50. mu.l of 5% BSA/PBS/0.1% Triton for 1 hour at room temperature.

Wash cells with 200 μ l PBS/0.1% Triton for 5 min, 3 times.

Dissolve 1 piece of OPD (Sigma) in 25 ml buffer (3.65 g citric acid-H)₂O and 5.9 g disodium hydrogen phosphate-2H₂O, dissolved in 0.5 l of water, pH5.6) and 12.5. mu.l of H is added₂O₂. To each well 50. mu.l was added and incubated on a shaker for 15 minutes (200rpm) covered with aluminum foil.

The reaction was stopped with 25. mu.l of sulfuric acid.

Determination of 0D on an ELISA reader_490-650。

Cerebellar dopaminergic neural cultures

Neural cultures

Neural cultures were prepared from the anterior cerebellum of the rat fetus by enzymatic and mechanical dispersion. Tissues were collected and washed in an iced phosphate buffered saline solution free of calcium and magnesium ions containing 0.6% glucose (PBSG) and incubated with PBSG containing 0.1% trypsin for 30 minutes at 37 ℃. At 2.510⁵Cell/cm density the cell suspension was plated onto 96-well NUNC tissue culture plates. Previously, culture plates were coated with poly-L-ornithine and CDM containing 10% fetal calf serum. The cultures were maintained in Chemically Defined Medium (CDM) comprising a 1: 1 mixture of Dulbecco's modified Eagles medium and F12 nutrient solution supplemented with glucose (0.6%), glutamine (2mM), sodium bicarbonate (3mM), HEPES (5mM), insulin (25. mu.g/ml), human transferrin (100. mu.g/ml), putrescine (60. mu.g/ml), sodium selenate (30. mu.g), streptomycin (100. mu.g/ml), and penicillin (100 IU/ml).

Treatment with neurotrophic factors

Neurotrophic factors were dissolved in 0.5% bovine serum albumin as a mother liquor. Neurotrophic factors were added 3 hours after the start of plating and 5 days after culture. The same amount of 0.5% bovine serum albumin was added to the control wells.

High affinity dopamine uptake

Dopamine uptake was determined after 10 days. For uptake, cells were washed 2 times with pre-warmed PBS supplemented with glucose (5mM), ascorbic acid (100mM), and pargyline (100mM) and pre-incubated with the same solution for 10 min. Using a composition containing 50nM³H]Phase of DAThe same solution was changed to the preculture solution and the culture was continued at 37 ℃ for 15 weeks. Uptake was terminated by 3 quick washes with iced PBS. The accumulated [ 2] is released by incubation with acidified ethanol at room temperature for 30 minutes³H](ii) dopamine. After addition of 4 ml of scintillation fluid (Packard ultima gold MV), the radioactivity was determined with a Packard scintillation counter. Nonspecific uptake was determined by addition of 20 μ M cocaine.

Cells were grown for 10 days with or without Enovin. The untreated control was set to 100%. Results were obtained in 1-5 independent experiments.

Reference to the literature

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Abbreviation list

BLAST basic local alignment search tool

bp base pair

cDNA complementary DNA

CNS central nervous system

EST expression sequence markers

EVN enovin

GDNF glial cell line-derived neurotrophic factor

GFR alpha GDNF family receptor alpha

GPI sugar phosphatidylinositol

MTC multiple tissue cDNA

NTN neurturin

PCR polymerase chain reaction

PNS peripheral nervous system

PSP persephin

RT-PCR reverse transcription PCR

TGF-beta transforming growth factor beta

FISH fluorescence in situ hybridization

MTN multiple tissue northern

NGF nerve growth factor

SPR surface plasmon resonance

Sequence listing

<110> Jensen pharmaceutical Co., Ltd

<120> neurotrophic growth factor

<130>50936/002

<140>PCT/EP99/05031

<141>19990714

<150>9815283.8

<151>19980714

<150>09/248,772

<151>19990212

<150>09/327,668

<151>19990608

<160>15

<170>PatentIn Ver. 2.0

<210>1

<211>339

<212>DNA

<213> human

<400>1

gctgggggcc cgggcagccg cgctcgggca gcgggggcgc ggggctgccg cctgcgctcg 60

cagctggtgc cggtgcgcgc gctcggcctg ggccaccgct ccgacgagct ggtgcgtttc 120

cgcttctgca gcggctcctg ccgccgcgcg cgctctccac acgacctcag cctggccagc 180

ctactgggcg ccggggccct gcgaccgccc ccgggctccc ggcccgtcag ccagccctgc 240

tgccgaccca cgcgctacga agcggtctcc ttcatggacg tcaacagcac ctggagaacc 300

gtggaccgcc tctccgccac cgcctgcggc tgcctgggc 339

<210>2

<211>474

<212>DNA

<213> human

<400>2

cgccgccgca gccttctcgg cccgcgcccc cgccgcctgc acccccatct gctcttcccc 60

gcgggggccg cgcggcgcgg gctgggggcc cgggcagccg cgctcgggca gcgggggcgc 120

ggggctgccg cctgcgctcg cagctggtgc cggtgcgcgc gctcggcctg ggccaccgct 180

ccgacgagct ggtgcgtttc cgcttctgca gcggctcctg ccgccgcgcg cgctctccac 240

acgacctcag cctggccagc ctactgggcg ccggggccct gcgaccgccc ccgggctccc 300

ggcccgtcag ccagccctgc tgccgaccca cgcgctacga agcggtctcc ttcatggacg 360

tcaacagcac ctggagaacc gtggaccgcc tctccgccac cgcctgcggc tgcctgggct 420

gagggctcgc tccagggctt tgcagactgg acccttaccg gtggctcttc ctgc 474

<210>3

<211>113

<212>PRT

<213> human

<400>3

Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys

1 5 10 15

Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His

20 25 30

Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg

35 40 45

Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala

50 55 60

Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys

65 70 75 80

Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser

85 90 95

Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu

100 105 110

Gly

<210>4

<211>139

<212>PRT

<213> human

<400>4

Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro Ser

1 5 10 15

Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly Ser

20 25 30

Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln Leu

35 40 45

Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Val

50 55 60

Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro His

65 70 75 80

Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro

85 90 95

Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr

100 105 110

Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp

115 120 125

Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

130 135

<210>5

<211>819

<212>DNA

<213> human

<400>5

gagtttcccc tccacacagc taggagccca tgcccggcct gatctcagcc cgaggacagc 60

ccctccttga ggtccttcct ccccaagccc acctgggtgc cctctttctc cctgaggctc 120

cacttggtct ctccgcgcag cctgccctgt ggcccaccct ggccgctctg gctctgctga 180

gcagcgtcgc agaggcctcc ctgggctccg cgccccgcag ccctgccccc cgcgaaggcc 240

ccccgcctgt cctggcgtcc cccgccggcc acctgccggg taggtgagag ggcgaggggg 300

cggggcgggg ctggcccggg acaccgcgcg tgactgggtc tcattccagg gggacgcacg 360

gcccgctggt gcagtggaag agcccggcgg ccgccgccgc agccttctcg gcccgcgccc 420

ccgccgcctg cacccccatc tgctcttccc cgcgggggcc gcgcggcgcg ggctgggggc 480

ccgggcagcc gcgctcgggc agcgggggcg cggggctgcc gcctgcgctc gcagctggtg 540

ccggtgcgcg cgctcggcct gggccaccgc tccgacgagc tggtgcgttt ccgcttctgc 600

agcggctcct gccgccgcgc gcgctctcca cacgacctca gcctggccag cctactgggc 660

gccggggccc tgcgaccgcc cccgggctcc cggcccgtca gccagccctg ctgccgaccc 720

acgcgctacg aagcggtctc cttcatggac gtcaacagca cctggagaac cgtggaccgc 780

ctctccgcca ccgcctgcgg ctgcctgggc tgagggctc 819

<210>6

<211>85

<212>PRT

<213> human

<400>6

Met Pro Gly Leu Ile Ser Ala Arg Gly Gln Pro Leu Leu Glu Val Leu

1 5 10 15

Pro Pro Gln Ala His Leu Gly Ala Leu Phe Leu Pro Glu Ala Pro Leu

20 25 30

Gly Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala

35 40 45

Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser

50 55 60

Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly

65 70 75 80

His Leu Pro Gly Arg

85

<210>7

<211>159

<212>PRT

<213> human

<400>7

Leu Gly Leu Ile Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg

1 5 10 15

Ala Arg Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro

20 25 30

Ala Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly

35 40 45

Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu

50 55 60

Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser

65 70 75 80

Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala

85 90 95

Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala

100 105 110

Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg

115 120 125

Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp

130 135 140

Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

145 150 155

<210>8

<211>1188

<212>DNA

<213> human

<400>8

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcaggt gagtggttct cccagtgact cctacctggt 120

actgaggaaa ggcggcttga ctggtgaggg agagcagggc ttggcttggg cagcggttag 180

gtgtgggagg gaaaatggtc agggagggac caggtgaatg ggaggaggag cgggacttct 240

ctgaatggtc ggtgcactca ggtgattcct cccctgggct cccagaggca gcaaacccat 300

tatactggaa cctaggccct tcctgagttt cccctccaca cagctaggag cccatgcccg 360

gcctgatctc agcccgagga cagcccctcc ttgaggtcct tcctccccaa gcccacctgg 420

gtgccctctt tctccctgag gctccacttg gtctctccgc gcagcctgcc ctgtggccca 480

ccctggccgc tctggctctg ctgagcagcg tcgcagaggc ctccctgggc tccgcgcccc 540

gcagccctgc cccccgcgaa ggccccccgc ctgtcctggc gtcccccgcc ggccacctgc 600

cgggtaggtg agagggcgag ggggcggggc ggggctggcc cgggacaccg cgcgtgactg 660

ggtctcattc cagggggacg cacggcccgc tggtgcagtg gaagagcccg gcggccgccg 720

ccgcagcctt ctcggcccgc gcccccgccg cctgcacccc catctgctct tccccgcggg 780

ggccgcgcgg cgcgggctgg gggcccgggc agccgcgctc gggcagcggg ggcgcggggc 840

tgccgcctgc gctcgcagct ggtgccggtg cgcgcgctcg gcctgggcca ccgctccgac 900

gagctggtgc gtttccgctt ctgcagcggc tcctgccgcc gcgcgcgctc tccacacgac 960

ctcagcctgg ccagcctact gggcgccggg gccctgcgac cgcccccggg ctcccggccc 1020

gtcagccagc cctgctgccg acccacgcgc tacgaagcgg tctccttcat ggacgtcaac 1080

agcacctgga gaaccgtgga ccgcctctcc gccaccgcct gcggctgcct gggctgaggg 1140

ctcgctccag ggctttgcag actggaccct taccggtggc tcttcctg 1188

<210>9

<211>228

<212>PRT

<213> human

<400>9

Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp

1 5 10 15

Pro Arg Arg Gln Ala Pro Leu Gly Leu Ser Ala Gln Pro Ala Leu Trp

20 25 30

Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser Val Ala Glu Ala Ser

35 40 45

Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg Glu Gly Pro Pro Pro

50 55 60

Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly Gly Arg Thr Ala Arg

65 70 75 80

Trp Cys Ser Gly Arg Ala Arg Arg Pro Pro Pro Gln Pro Ser Arg Pro

85 90 95

Ala Pro Pro Pro Pro Ala Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg

100 105 110

Ala Ala Arg Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala

115 120 125

Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly

130 135 140

Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly

145 150 155 160

Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu

165 170 175

Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser

180 185 190

Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp

195 200 205

Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys

210 215 220

Gly Cys Leu Gly

225

<210>10

<211>220

<212>PRT

<213> human

<400>10

Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp

1 5 10 15

Pro Arg Arg Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu

20 25 30

Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro

35 40 45

Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His

50 55 60

Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg

65 70 75 80

Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro

85 90 95

Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly

100 105 110

Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln

115 120 125

Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu

130 135 140

Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro

145 150 155 160

His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro

165 170 175

Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg

180 185 190

Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val

195 200 205

Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

210 215 220

<210>11

<211>766

<212>DNA

<213> human

<400>11

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcaggc tccacttggt ctctccgcgc agcctgccct 120

gtggcccacc ctggccgctc tggctctgct gagcagcgtc gcagaggcct ccctgggctc 180

cgcgccccgc agccctgccc cccgcgaagg ccccccgcct gtcctggcgt cccccgccgg 240

ccacctgccg gggggacgca cggcccgctg gtgcagtgga agagcccggc ggccgccgcc 300

gcagccttct cggcccgcgc ccccgccgcc tgcaccccca tctgctcttc cccgcggggg 360

ccgcgcggcg cgggctgggg gcccgggcag ccgcgctcgg gcagcggggg cgcggggctg 420

ccgcctgcgc tcgcagctgg tgccggtgcg cgcgctcggc ctgggcGacc gctccgacga 480

gctggtgcgt ttccgcttct gcagcggctc ctgccgccgc gcgcgctctc cacacgacct 540

cagcctggcc agcctactgg gcgccggggc cctgcgaccg cccccgggct cccggcccgt 600

cagccagccc tgctgccgac ccacgcgcta cgaagcggtc tccttcatgg acgtcaacag 660

cacctggaga accgtggacc gcctctccgc caccgcctgc ggctgcctgg gctgagggct 720

cgctccaggg ctttgcagac tggaccctta ccggtggctc ttcctg 766

<210>12

<211>742

<212>DNA

<213> human

<400>12

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcagcc tgccctgtgg cccaccctgg ccgctctggc 120

tctgctgagc agcgtcgcag aggcctccct gggctccgcg ccccgcagcc ctgccccccg 180

cgaaggcccc ccgcctgtcc tggcgtcccc cgccggccac ctgccggggg gacgcacggc 240

ccgctggtgc agtggaagag cccggcggcc gccgccgcag ccttctcggc ccgcgccccc 300

gccgcctgca cccccatctg ctcttccccg cgggggccgc gcggcgcggg ctgggggccc 360

gggcagccgc gctcgggcag cgggggcgcg gggctgccgc ctgcgctcgc agctggtgcc 420

ggtgcgcgcg ctcggcctgg gccaccgctc cgacgagctg gtgcgtttcc gcttctgcag 480

cggctcctgc cgccgcgcgc gctctccaca cgacctcagc ctggccagcc tactgggcgc 540

cggggccctg cgaccgcccc cgggctcccg gcccgtcagc cagccctgct gccgacccac 600

gcgctacgaa gcggtctcct tcatggacgt caacagcacc tggagaaccg tggaccgcct 660

ctccgccacc gcctgcggct gcctgggctg agggctcgct ccagggcttt gcagactgga 720

cccttaccgg tggctcttcc tg 742

<210>13

<211>603

<212>DNA

<213> human

<400>13

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcaggg ggacgcacgg cccgctggtg cagtggaaga 120

gcccggcggc cgccgccgca gccttctcgg cccgcgcccc cgccgcctgc acccccatct 180

gctcttcccc gcgggggccg cgcggcgcgg gctgggggcc cgggcagccg cgctcgggca 240

gcgggggcgc ggggctgccg cctgcgctcg cagctggtgc cggtgcgcgc gctcggcctg 300

ggccaccgct ccgacgagct ggtgcgtttc cgcttctgca gcggctcctg ccgccgcgcg 360

cgctctccac acgacctcag cctggccagc ctactgggcg ccggggccct gcgaccgccc 420

ccgggctccc ggcccgtcag ccagccctgc tgccgaccca cgcgctacga agcggtctcc 480

ttcatggacg tcaacagcac ctggagaacc gtggaccgcc tctccgccac cgcctgcggc 540

tgcctgggct gagggctcgc tccagggctt tgcagactgg acccttaccg gtggctcttc 600

ctg 603

<210>14

<211>489

<212>DNA

<213> human

<400>14

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcagcc tgccctgtgg cccaccctgg ccgctctggc 120

tctgctgagc agcgtcgcag aggcctccct gggctccgcg ccccgcagcc ctgccccccg 180

cgaaggcccc ccgcctgtcc tggcgtcccc cgccggccac ctgccggcgg ctcctgccgc 240

cgcgcgcgct ctccacacga cctcagcctg gccagcctac tgggcgccgg ggccctgcga 300

ccgcccccgg gctcccggcc cgtcagccag ccctgctgcc gacccacgcg ctacgaagcg 360

gtctccttca tggacgtcaa cagcacctgg agaaccgtgg accgcctctc cgccaccgcc 420

tgcggctgcc tgggctgagg gctcgctcca gggctttgca gactggaccc ttaccggtgg 480

ctcttcctg 489

<210>15

<211>350

<212>DNA

<213> human

<400>15

ctgatgggcg ctcctggtgt tgatagagat ggaacttgga cttggaggcc tctccacgct 60

gtcccactgc ccctggccta ggcggcagcg gctcctgccg ccgcgcgcgc tctccacacg 120

acctcagcct ggccagccta ctgggcgccg gggccctgcg accgcccccg ggctcccggc 180

ccgtcagcca gccctgctgc cgacccacgc gctacgaagc ggtctccttc atggacgtca 240

acagcacctg gagaaccgtg gaccgcctct ccgccaccgc ctgcggctgc ctgggctgag 300

ggctcgctcc agggctttgc agactggacc cttaccggtg gctcttcctg 350

Claims (5)

1. Use of a cell expressing a human neurotrophic factor, referred to as enovin, and having an amino acid sequence as set forth in SEQ ID NO: 4, respectively.

2. Use according to claim 1, said cell being a transgenic cell comprising a transgene capable of expressing a human neurotrophic factor as defined in claim 1.

3. The use of claim 1, wherein the amino acid sequence of the human neurotrophic factor is as set forth in SEQ id no: 3, respectively.

4. The use according to any one of claims 1 to 3, wherein the neurological disease is selected from any one of the group consisting of: parkinson's disease, alzheimer's disease, neurological diseases associated with amplified polyglutamine sequences, peripheral neuropathies, acute brain injury, nervous system tumors, multiple sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma, injury caused by neurotoxins, multiple endocrine adenomatous formation, familial hirschsprung, prion-related diseases, Creutzfeld-Jacob disease, stroke, painful syndromes of predominantly peripheral or central neurogenic components, rheumatic inflammatory diseases and conduction disorders.

5. The use according to claim 4, wherein the neurological disorder is Huntington's disease.