CZ200153A3 - Neurotrophic factors - Google Patents

Description

Neurotrophic factors

Technical field

The invention relates to polypeptide neurotrophic factors, nucleic acids encoding polypeptide neurotrophic factors

factors a antibodies, which se specific weighs on neurotrophic factors.

BACKGROUND OF THE INVENTION

Neurotrophic factors are naturally occurring factors that promote survival, maintain phenotypic differentiation, prevent degeneration, and enhance cell and tissue activity. Neurotrophic factors of tissue and from the neural tissue that is neuronal are isolated from the nerve innervated by the nervous system, and divide groups that are also functionally referred to as subfamilies. The superfamily of neurotrophic structurally related family, superfamily, or factors include fibroblast growth factor, neurotrophin, and transforming growth factor beta (TGF-β) superfamily. Individual neurotrophic factors interact by interacting with their composite receptors and effects by their physical structure, on different types of nerve cells. To the superfamily

TGF-β (Massague,

Trends in Cell Biology

1994, 4:

neurotrophic factor ligands ("GDNF, WO 93/06116), which include the obtained glial cell

GDNF, persefin line

Milbrandt et al., Neuron 1989: 20:

245-253) and neurturin ("NTN, WO

97/08196).

The ligands of the GDNF subfamily have a common ability to elicit a RET tyrosine kinase signal.

These three differ in their family of neurotrophic receptors, the GFR, by their relative affinity for the receptor ligands of the GDNF subfamily.

Due to the effects of neurotrophic factors on neuronal tissue, there remains a need to identify and characterize other neurotrophic factors for the diagnosis and treatment of nervous system disorders.

SUMMARY OF THE INVENTION

The invention relates to a novel neurotrophic factor, referred to herein as "neublastin or" NBN. Neublastin belongs to the GDNF subfamily because they have shared regions of homology with other GDNF ligands (see Tables 3 and 4 below) and because of their ability to interact with RET (see, e.g., Airaksinen et al., Mol. Cell. Neuroscience, 1999, 13, 313-). 325) neublastin is a new and unique neurotrophic factor. Unlike other GDNF ligands, neublastin exhibits high affinity for the GFRα3-RET receptor complex and unique sub-regions in its amino acid sequence.

"A neublastin polypeptide as used herein is a polypeptide that exhibits neurotrophic activity (e.g., as described in Examples 6, 7, 8, and 9) and includes polypeptides having an amino acid sequence that exhibits at least 70% homology to human" neublastin polypeptides shown in AK-gs-AKxos in SEQ ID NO: 2, AKj.AKios in SEQ ID NO: 2, AK-97-AK140 in SEQ ID NO:

4, AK-41-AK140 in SEQ ID NO: 4 ("pro"), AKi-AKi ₄₀ in SEQ ID NO: 4, ΑΚ- ₈₀ -ΑΚΐ40 ⁱⁿ SEQ ID NO: 9, ("wild type prepro") , AK-41-AK140 in SEQ ID NO: 9 (pro), AK1-AK14Q in SEQ ID NO: 5 (mature, 140AK),

AK1-AK113

AK1-AK116 in SEQ ID NO: 6 (mature, 116AK),

in sequence SEQ ID NO: 7, (maturovaný 113AK) in sequence SEQ ID NO: 10 (graduated, 140AK) in sequence SEQ ID NO: 11 (graduated, 116AK)

in sequence

SEQ ID NO:

(graduated,

113AK),

AKi-AKuo

AK! -AK ₁₁₆

AKi-AK ₁₁₃ and their variants and derivatives.

In addition, the present invention includes those polypeptides having an amino acid sequence that has this to be this. ··· · · ······· · · ·

Exhibits at least 70% homology to the mouse "neublastin polypeptides set forth in ΑΚχ-ΑΚ ₂ 24 in SEQ ID NO: 16."

Preferably, the C-terminal sequence of the above defined neublastin polypeptides has the amino acid sequence set forth in AK ₇ 2 'AKio5 in SEQ ID NO: 2 (i.e. AKi ₀₇ -AKi ₄₀ in SEQ ID NO: 9), particularly AK ₄ i-AKi ₀₅ in SEQ ID NO: 2 (i.e.

AK ₇₆ -AKi ₄ o SEQ ID NO: 9), or the amino acid sequence shown in Aki -AK2 _{91 2} 4 SEQ ID NO: 16th

It is also preferred that the neublastin polypeptide comprises 7 conserved Cys residues that are characteristic of the GDNF family and the TGF-beta superfamily.

Preferably, the neublastin polypeptide has an amino acid sequence showing homology to a foreign sequence greater than particularly greater than 95% (i.e., AK-95-AK105 in SEQ ID NO: 1).

NO:

SEQ

NO NO: 2, AK-97-AK14Q

2, AKi-AK ₁₀₅

SEQ ID NO: 4 Aki-aki q ₄ SEQ ID NO: 4, AK-4i-Aki ₄ of in SEQ ID NO: 4 AK. -80 ^- AK14Q in SEQ ID NO: 9 (λ .wild type prepro), AK- _4i -AKi _{40 of} SEQ ID NO: 9 (for), AK1-AK14Q in sequence SEQ ID NO: 5, (mature , 140ΆΚ), AK1-AK116 in sequence SEQ ID NO : 6 (matured, 116A), AKi-AK ₁₁₃ in SEQ ID NO: 7 (graduated, 113AK), ΑΚχ-AKuo in SEQ ID NO: 10 (matured, 140AK), AKi-AK _U6 in SEQ ID NO: 11 (graduated, 116AK), AKi-AK ₁₁₃ in SEQ ID NO: 12 (graduated, 113AK)) a AKI _AK-224 in SEQ ID NO : 16.

here

The term "neublastin nucleic acid," as used herein, refers to a polynucleotide that encodes a neublastin polypeptide. Thus, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons which, when exposed to suitable components required for translation, will encode a neublastin polypeptide. The neublastin nucleic acids of the invention may be RNA or DNA, for example genomic DNA or DNA that is complementary to or derived from neublastin mRNA ("cDNA").

rewrites. The neublastin nucleic acid of the invention further comprises polynucleotide molecules that specifically hybridize to a polynucleotide under stringent hybridization conditions, encoding a neublastin polypeptide. The invention also encompasses nucleic acid primers which may be used as fcfc fcfc fcfc fcfc fc tc fc tc fc ·· fcfc fcfc ·· ···· which relates to the identification, isolation and amplification of polynucleotides that encode neublastin

In certain neublastin-specific embodiments, with neublastin nucleic acids, "specific that encode the specificity of the polypeptides or fragments thereof, the invention provides certain probe primers that are used in acid hybridization, but not with other GDNF family members. The term or "specifically refers to the ability to hybridize to a neublastin nucleic acid and the inability to hybridize to a non-neublastin nucleic acid, acids, including the inability to hybridize to nucleic acids that encode only GDNF ligands (e.g., GDNF, persefin, and neurturin).

In another embodiment, the neublastin nucleic acid of the invention is identified as complementary to a polynucleotide that encodes a neublastin polypeptide, either a complementary nucleic acid sequence or that specifically hybridizes under stringent conditions by demonstrating hybridization to a polynucleotide that encodes neublastin.

Particular neublastin nucleic acids include, but are not limited to, the nucleic acid sequences described herein, designated SEQ ID NO:

SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14,

1, SEQ

SEQ ID

NO: 15, primers

SEQ ID and 32.

The neublastin nucleic acid of the invention further comprises a unique neublastin neublastin nucleic acid subregion or fragment, including, without limitation, the nucleic acid fragments depicted in Figure 8.

The neublastin nucleic acids of the invention can be used in the expression of a neublastin polypeptide, for example, by the expression of a neublastin polypeptide. The polypeptides are in vitro or by application of a neublastin nucleic acid in an animal in vivo. The neublastin acids for expression of the nucleic acid may be present in a nucleic acid vector, for example an expression vector or a cloning vector. The neublastin nucleic acid may, but is not necessarily, maintained, reproduced, transmitted or expressed as part of a nucleic acid vector.

The recombinant neublastin expression vector containing the polynucleotide sequence may be introduced into and / or maintained within the cell. The neublastin vector host cells may be prokaryotes. Alternatively, the neublastin nucleic acid may be introduced into a eukaryotic cell, for example, into a eukaryotic cell that contains suitable equipment for post-translational processing and / or appropriate organs for polypeptides to mature protein secreting polypeptides into the extracellular environment of the cell.

The invention further provides a neublastin neurotrophic factor, neublastin. The neublastin may be in the form of polypeptides or may be, for example, a multimer of two or more neublastin polypeptides, a neublastin dimer. Neublastin polypeptides also associate into multimers through intracellular structural junctions well known in the art, including, without limitation, cysteine-cysteine interactions, sulfylhydryl linkages, and non-covalent interactions. Particular neublastin polypeptides include, but are not limited to, designated SEQ ID NO:

Amino acid sequence ID

NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 a SEQ ID NO: 16.

the polypeptide of the invention is useful in the

Neublastin treatment of neuronal disorders, including but not limited to, disturbances and traumas, including but not limited to

Neublastin neurons. Peripheral nerves that have been damaged, are not limited to the nerves of the marrow or spinal cord.

the polypeptides can be used in the treatment of a neurodegenerative disease, for example, cerebral ischemic neuronal damage, neuropathy, for example peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Neublastin polypeptides may find use in treating impaired memory, for example, associated with dementia.

The Applicant has identified a nucleic acid that encodes a novel neutrophic factor referred to herein as "neublastin or" NBN. Neublastin is a member of the subclass of neurotrophic factors derived from the glial cell line (GDNF) of the transforming growth factor β (TGF-β) superfamily.

The neublastin cDNA was originally identified as follows. Using the TBLASTN 1.4.11 algorithm (Atschul et al., Nucl. Acids Res. 1997 25 3389-3402) and human persephine as a query (GenBank deposit number AF040962), two human genes were initially introduced in the high-throughput genomic sequence (HGTS) bacterial synthetic chromosomes (BAC) in GenBank AC005038 and AC005051 identified a fragment of 290 bp. AC005038 is comprised of approximately 190,000 bp of 5 contiguous sequences of contiguous sequences, and AC005051 is composed of approximately 132,000 bp of 12 contiguous sequences of contiguous sequences. The 290 bp fragment identified in the two BAC clones has been shown to have regions that are homologous but not identical to the coding region of the neurotrophic fakotre cDNA, which is human persefin.

From this 290 bp sequence, two neublastin-specific PCR primers were synthesized: "upper chain primer (SEQ ID NO: 17) and" lower chain primer (SEQ ID NO: 18)). Human fetal brain cDNA library testing was performed. Initial testing involves 96-well PCR based assays with two PCR primers (SEQ ID NOs: 17 and 18) of a Master Plate cDNA library of 500,000 cDNA clones containing approximately 5,000 clones / well. Second test based on 9 9 9 9 9 9 9 9 9 9 9 9 99 99 99 99999 on PCR was performed in the human embryonic brain cDNA library

"Sub-Plate, which contains glycerol stock solution E. coli with approximately 5 000 clones / well. Fragment the size of 102 bp (SEQ ID NO: 13) se identified in tests based on PCR on Master Plate i

Sub Plate. A positive cDNA clone (containing a 102 bp fragment) was selected, plated on two plates containing antibiotics and LB and grown overnight. A total of 96 bacterial colonies were picked from these plates and plated individually in wells on a new 96 well PCR plate containing both PCR primers (SEQ ID NOs: 17 and 18) and the required PCR amplification reagents. PCR amplification was performed and 96 individual PCR reactions were analyzed by 2% agarose gel electrophoresis. A positive colony was then identified with a clone containing a 102 bp fragment. Plasmid DNA was obtained from a positive colony containing a 102 bp fragment and sequenced. Subsequent sequence analysis revealed the presence of a full length cDNA of 861 bp (SEQ ID NO: 8). An open reading frame (ORF) of 663 bp or the coding region (CDS) identified in SEQ ID NO: 8 encodes a pre-pro-polypeptide (designated "pre-pro-neublastin") and is shown in SEQ ID NO: 9. Three variants of neublastin polypeptides were identified based on the sequence of SEQ ID NO: 9. These variants include

(and) a polypeptide comprising 140 amino acids, here marked as NBN140, which has amino acid sequence denoted by SEQ ID NO: 10, (ii) a polypeptide comprising 116 amino acids, here marked as NBN116, which has amino acid se kvenci designated SEQ ID NO: 11 and (iii) a polypeptide comprising 113 amino acids, here marked as NBN113, which has amino acid sequence

designated as SEQ ID NO: 12.

O

44 «4 4 4 · 4 · 4 4444

44444 44 444

44 4 4 4 4 444 ·

4444 4 · 4,444

9 · 44 44

The entire cDNA sequence containing

5'not translated

DNA size

782 bp, encoding 663 DNA

3'-untranslated DNA of 447 bp bp (total 1992 bp) was inserted into GenBank with accession number AF 120274.

The genomic sequence encoding neublastin was identified as follows:

In order to clone the genomic sequence encoding neublastin

another set was prepared primers. Few primers C. 1 sense primer corresponding sequence SEQ ID NO: 23 and a primer corresponding to SEQ ID NO: 24 a few primers C. 2 sense primer corresponding sequence SEQ ID NO: 25 and a primer corresponding to SEQ ID NO: 26.

contained antisense contained antisense

Using primer pair # 2, the bp DNA fragment was amplified by PCR from a human genomic DNA preparation and cloned into the pCRII vector (Invitrogen) and transformed into E. coli. The resulting plasmid was sequenced to predict the putative cDNA sequence (indicated by the size of 861 bp (coding for a protein called neublastin herein).

Similarly, using primer pair no.

has gained

PCR fragment

DNA of 870 bp size by human genomic PCR

DNA.

In this fragment, another 42 bp per region was found

3 'end of the open reading frame (ORF) compared to the size sequence

887 bp.

The genomic structure of the neoplastin gene was predicted by its comparison with the nucleic acid sequences of other neurotrophic factors by mapping the exon-intron interface.

This analysis showed that the neublastin gene has at least two exons separated by an intron of bp size.

This sequence was also used to test GenBank for EST neoplastin sequences. Three were identified with samples of AA844072, AA931637 and AA533512 in GenBak, indicating that neublastin nucleic acids are transcribed to mRNA.

Comparing the entire obtained cDNA sequence (AF 120274) and the genomic sequence present in the GenBank samples under number

AC005038 and AC005051 showed that the neublastin gene consists of at least five exons (including three coding) separated by four introns (see, for example, Figure 8). Together, the exons have the predicted full-length neublastin polypeptide amino acid sequence. It should also be noted that the 887 bp fragment was found to contain the entire coding region of pro-neoplastin. The predicted cDNA (SEQ ID NO: 3) contains an open reading frame (ORF) encoding pro-neoplastin (181 amino acid residues) that exhibits homology to the three known human proteins - persephin, neurturin, and GDNF.

Neublastin nucleic acids of the invention

The invention provides polynucleotides capable of expressing the polypeptides of the invention. Polynucleotides of the invention include DNA, cDNA, and RNA sequences as well as antisense sequences and include naturally occurring synthetic and deliberately manipulated polynucleotides. Polynucleotides of the invention also include sequences that are degenerate as a result of the genetic code, but which provide for the expression of a neublastin polypeptide.

The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases in length, preferably at least 15 bases. The term "isolated polynucleotide" refers to a polypeptide that is not immediately adjacent to it (one at the 5 'end and the other at the 3' end) of the naturally occurring genome of the organism from which it was derived. Thus, the term includes recombinant DNA that has been incorporated into an expression vector, a self-replicating plasmid or virus, or a prokaryotic or eukaryotic genomic DNA, or that exists as a separate molecule, e.g., cDNA, independent of other sequences.

Polynucleotides of the invention also include allelic variants and " mutated polynucleotides having a nucleotide sequence that differs from the nucleotide sequences present herein at one or more nucleotide positions.

In a preferred embodiment of the invention, the polynucleotide of the invention has a nucleic acid (DNA) sequence capable of hybridizing to a polynucleotide sequence shown as SEQ ID NO: 1, a polynucleotide sequence shown as SEQ ID NO: 3, a polynucleotide sequence shown as SEQ ID NO: 8 or a polynucleotide sequence as SEQ ID NO: 15, with its complementary strand or its sub-sequence under at least medium, medium / stringent, or high stringency hybridization conditions, as described in more detail below.

In another preferred embodiment of the invention, the isolated polynucleotide of the invention has a nucleic acid (DNA) sequence having at least 70 preferably at least 80

especially at least 90% and in particular o o homology with the polynucleotide sequence shown as SEQ ID NO: 1, polynucleotide sequences listed as SEQ ID NO: 3, polynucleotide sequences shown as SEQ ID NO : 8 or

a polynucleotide sequence shown as SEQ ID NO: 15.

In a most preferred embodiment, the polynucleotide has the DNA sequence shown as SEQ ID NO: 1, the DNA sequence shown as SEQ ID NO: 3, the DNA sequence shown as SEQ ID NO: 8, or the polynucleotide sequence shown as SEQ ID NO: 15.

The invention further provides novel primers and DNA sequences suitable for the identification, isolation and amplification of neublastin polynucleotides that encode the expression of neublastin polypeptides or fragments thereof. Such primers include the polynucleotides set forth in SEQ ID NOs: 17-28 and 31-32. In addition, the invention provides neublastin DNA sequences generated from these primers, including those set forth in SEQ ID NOs: 13 and 14. Further, the invention provides DNA sequences from 3 ' or 5 'untranslated regions ("UTRs") in genomic DNA flanking neublastin exons; such sequences can be used to identify, isolate and amplify neublastin polynucleotides that encode the expression of neublastin polypeptides or fragments thereof.

The 3'UTR sequences of the invention include those listed as:

nucleotides 721 to 865 SEQ ID NO: 1, nucleotides 718 to 861 SEQ ID NO: 3, nucleotides 718 to 861 SEQ ID NO: 8,

nucleotides 1647 to 2136 of SEQ ID NO: 15 and contiguous sequences comprising between 10 to 25 nucleotides, derived (i.e., falling within) them from previous sequences (which are useful, for example, as primers).

The 5'UTR sequences of the invention include those listed as:

nucleotides 1 to 10 of SEQ ID NO: 1, nucleotides 1 to 57 of SEQ ID NO: 8, nucleotides 1 to 974 of SEQ ID NO: 15, and contiguous sequences between 10 and 25 nucleotides derived (i.e., falling within) from previous sequences (which are useful, for example, as primers).

The polynucleotides of the invention may preferably be obtained by a cloning process, for example as described in Current Protocols in Molecular Biology, Jahn Wiley and Sons, Inc. . In a preferred embodiment of the invention, the polynucleotide is cloned from or derived from human genomic DNA or human brain cDNA library.

DNA sequence homology

The aforementioned sequence homology

DNA can be determined as a degree of identity between two sequences that indicates deviations of the first sequence from the second.

Homology can be conveniently determined using known computer programs such as GAP

4 45 · «··· 4 ·

4 4 · 4 · 4 · 4 · 4 · 4 · 4 444

44 · 4444 provided in the progarm package

Journal of Molecular

GCG (Needleman,

Biology 1970 48

S. B. a

Using

GAP with the following DNA sequence comparison settings:

the GAP operative operator has a value of 5.0 and the analogous GAP extension operator has a value of 0.3, the above coding region

The DNA preferably has a sequence identity of at least 70%, in particular at least 80%, preferably at least 90%, particularly at least 95%, with the (coding) portion of the CDS DNA sequence shown in SEQ ID NO: 1 or in SEQ ID NO: 3 or with the (coding) portion of the CDS DNA sequence shown in SEQ ID NO: 8, with the (coding) portion of the CDS DNA sequence shown in SEQ ID NO: 15.

The term "sequence identity" means the degree to which two polynucleotide sequences are in a particular region of comparison.

calculated by comparing two identical nucleotide-nucleotide based

The term "percent sequence identity with optimally aligned sequences in this comparison region, determining the number of positions with identical bases or I) that the nucleic paired positions appear divided by:

acids (e.g., A, in both sequences, which

T, C, the number of paired positions is given

G, U number by the total number of positions in the comparison area (i.e., window size) and the result is multiplied by 100 The term "substantial" and a percent sequence identity is obtained.

match is used to denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that exhibits at least

80% sequence identity, preferably at least

85% sequence identity often 90 to 90%

95% sequence identity, more often at least

99% sequence identity compared to a reference sequence in the comparison region.

Hybridization protocol

The polynucleotides of the invention have a nucleic acid sequence capable of hybridizing to the polynucleotide sequence shown as SEQ ID NO: 1, with the polynucleotide sequence shown as SEQ ID NO:

or polynucleotide

0 * · ·· 0 ··· ·· 000 000 «0 * 0 00000 00 0 00 0 00 ·« · 0 00 «0

00 00 0 0 «00 sequence shown as SEQ ID NO: 8 or with the polynucleotide sequence shown as SEQ ID NO: 15 or their complementary strand or their sub-sequence under at least medium, medium / stringent or high stringency hybridization conditions, as described in more detail below.

Experimental conditions suitable for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence include wetting a filter that contains DNA or RNA fragments for hybridization in 5x SSC (sodium chloride / sodium citrate, see Sambrook et al., Molecular Cloning: A Laboratory Manual) , Cold

Spring Harbor Lab., Cold Spring Harbor, NY 1989) for 10 minutes and prehybridizing the filter in 5X SSC, 5x Denhardt's solution (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab.) , NY 1989), 0.5% SDS and 100 µg / ml denatured sonified salmon sperm DNA (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY 1989), followed by hybridization in the same solution containing 10 ng / ml randomly binding [Feinberg AP and Vogelstein B, Anal. Biochem. 1983 132 6-13] ³² PdCTP-labeled probes (specific activity greater than 1x10 ⁹ cpm ^ g) for 12 hours at approximately 45 ° C. The filter is then washed twice for 30 minutes in 0.1 x SSC, 0.5% SDS buffer at a temperature of at least 60 ° C (moderately stringent conditions), preferably at least 65 ° C (moderate / very stringent conditions), in particular at least 70 ° C. ° C (stringent conditions), and in particular at least 75 ° C (stringent conditions). Molecules with which the oligonucleotide probe hybridizes under these conditions can be detected using X-ray film.

Cloned polynucleotides

In particular, the isolated polynucleotide of the invention may be a cloned polynucleotide. The term "cloned polynucleotide" means a polynucleotide or DNA sequence cloned in accordance with ·· ····

♦ standard cloning procedures currently used in gene manipulations to change the position of a DNA segment, which may in particular be a cDNA, ie enzymatically derived from RNA, from its natural position to another site where it will be reproduced.

Cloning can be carried out in any suitable manner and can include methods such as reverse transcription technology, PCR technology and the like, as well as excision and isolation of the desired DNA segment.

The cloned polynucleotide of the invention may also be referred to as a "DNA construct or" isolated DNA sequence, and may in particular be complementary DNA (cDNA).

Biological resources

The isolated polynucleotide of the invention may be obtained from any suitable source.

In a preferred embodiment of the invention, the polynucleotide of the invention is cloned from or produced from a cDNA library, e.g. , corpus callosum, hippocampus, thalamicus nucleus, subthalamicus nucleus, olfactory nucleus, putamen, substantia nigra, dorsal root ganglia, trigeminal ganglia, superior mesenteric artery or thalamus, spinal cord, heart, placenta, lung, liver, skeletal muscle , pancreas, intestines, eye, retina, dental pulp, hair follicle, prostate, pituitary or trachea.

Commercial cDNA libraries from various tissues, whether human or animal, are available, for example, from Stratagene and Clontech. The isolated polynucleotide of the invention may be obtained by standard methods, for example as described in the examples.

·· ♦ ···

Neublastin polypeptides of the invention

As mentioned above, "a neublastin polypeptide" means a polypeptide having neurotrophic activity (for example, as described in Examples 6, 7, 8, and 9) and includes those polypeptides having an amino acid sequence that exhibits at least 70% homology to the "neublastin" the polypeptide shown in AK-9 ₅ -AKi ₀₅ in SEQ ID NO: 2, AK! -if ₁₀ 5 in SEQ ID NO: 2, ₄ -AKi AK_ ₉₇ o SEQ ID NO: 4, AK-4i-AKi4o in SEQ ID NO: 4, AKi-AKi ₄₀ in SEQ ID NO: 4, AK-80-AK140 in SEQ ID NO: 9, ("wild type prepro"), AK-41-AK140 in SEQ ID NO : 9 (for), AKi-AK ₁₄₀

in sequence SEQ ID NO: 5 (graduated, 140AK), ΑΚχ-AKns in sequence SEQ ID NO: 6 (graduated, 116AK), AK1-AK113 in sequence SEQ ID NO: 7, (graduated, 113AK), AK! -AK ₁₄₀ in sequence SEQ ID NO: 10 (graduated, 140AK), ΑΚχ-ΑΚχχς in sequence SEQ ID NO: 11 (graduated, 116AK), ΑΚχ-ΑΚ ₁₁₃ in sequence SEQ ID NO: 12 (maturovaný 113AK), AK1-AK224 in sequence SEQ ID NO: 16 (mice prepro), and variants a derivatives

each of the above polypeptides.

It is preferred that the C-terminal sequence be from above

listed neublastin polypeptides had an amino acid the sequence shown in AK7 ₂ -AK ₁₀ 5 v sequence SEQ ID NO: 2 (that is AK ₁₀ 7-AK ₁₄₀ in SEQ ID NO: 9 ), in particular AK ₄ i ~ AKio5 in sequence SEQ ID NO: 2 (that is AK76 ~ AKi ₄ q in sequence SEQ ID NO: y i. Also se prefers, to make neublastin polypeptide contained 7 conservative residues Cys that are

characteristic of the GDNF family and TGF-beta superfamily.

Preferably, the Neublastin polypeptide amino acid sequence having greater than 85% homology, preferably with greater than 95% homology with the aforementioned sequences (i.e., AK-95-AK105 in SEQ ID NO: 2, ΑΚχ-ΑΚ ₁₀ 5 in SEQ ID NO 2, the AK-97-AK140 ⁱⁿ SEQ ID NO: 4, ₄₁ AK_ -AKi ₄₀ in SEQ ID

NO: 4, ΑΚχ-ΆΚχχο in SEQ ID NO: 4, ₁₄ AK_ _8O -if sequence s

• · 99 ♦ ··· 9 9 ♦ ♦ • • ♦ • 9 9 9 • · ··· • · • 9 9 • • • · «· • ♦ ♦ · • · • · • • • · 9 9 ♦ · ♦ ♦ ·· • 9 99 9

SEQ ID NO: 9 ("wild type prepro"), AK-41-AK140 in SEQ

SEQ ID NO: 9 (pro), AKi-AK ₁₄₀ in SEQ ID NO: 5 (mature 140AK), AK1-AK116 in SEQ ID NO: 6 (mature 116AK), ΑΚχ-ΑΚι ₁₃ in SEQ ID NO: 7 (mature 113AK), ΑΚ1-ΑΚ140 in SEQ ID NO: 10 (mature 140AK), Κχ-ΑΚηδ in SEQ ID NO: 11 (mature 116AK), AK1-AK113 in SEQ ID NO: 12 (mature 113AK), AK1-AK224 in SEQ ID NO: 16 (murine prepro)), and preferably any of the polypeptides with a C-terminal sequence of the above identified Neublastin polypeptides having the amino acid sequence as shown in -AKi AK _{72 0} 5 in SEQ ID NO: 2 (i.e. AK107 AK140 is-SEQ ID NO: 9), preferably even _{4 AK-10} AK 5 in SEQ ID NO: 2 (i.e., AK ₇₆ -AKi ₄₀ in SEQ ID NO: 9) or _AK-19 and AK 24 ₂ SEQ ID NO: 16.

The invention further encompasses polypeptides having an amino acid sequence having at least 70% homology to the murine "neublastin polypeptides" set forth in AK1-AK224 in SEQ ID NO: 16.

Preferred polypeptides of the invention in one embodiment represent prepro sequences (as set forth in SEQ ID NOs: 2, 4,

9, respectively. 16) for the sequence (as indicated in AK_ -AKi ₇₅ 5 ₀ SEQ ID

NO: 2 or ₄ and AK_ _aki-40 in SEQ ID NO: 4, respectively. 9) and the mature neoplastin sequence (as set forth in SEQ ID NOs: 5, 6, 7, 10, 11 or 11)

12, preferably SEQ ID NOs: 10, 11, 12).

Polypeptides of the invention include variant polypeptides. The term "variant polypeptide" means a polypeptide (or protein) having an amino acid sequence that differs from the sequence presented as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO. 7, SEQ ID NO: 9, SEQ ID NO:

10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 16 at one or more amino acid positions. Such variant polypeptides include the modified polypeptides described above, as well as conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphisms.

·· ·· * ·

As defined herein, a conservative substitution refers to the replacement of an amino acid residue with another, biologically similar residue. For example, conservative amino acid substitutions are expected to have little or no effect on biological activity, particularly when they represent less than 10% of the total number of residues in a polypeptide or protein. Conservative amino acid substitutions preferably represent changes in less than 5% of the polypeptide or protein, in particular changes less than 2% of the polypeptide or protein (for example, when using the NBN113 computation system, the most preferred conservative substitutions will represent less than 3 amino acid substitutions in the mature amino acid wild-type sequence). In a particularly preferred embodiment of the invention, there is a single amino acid substitution in the mature sequence wherein both the substituted and replacement amino acids are not cyclic.

Other examples of particularly conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another or substitution of one polar residue for another such that lysine is replaced by arginine, aspartic acid glutamic acid or asparagine glutamine and the like.

The term conservative substitution also includes the use of a substituted amino acid residue in place of the original unsubstituted amino acid residue when the antibodies raised against the substituted polypeptide also immunologically react with the unsubstituted polypeptide.

Modification of this primary amino acid sequence may result in proteins that exhibit substantially equivalent activity as compared to the unmodified polypeptide, and thus may be considered functional analogues of the parent proteins.

Such modifications may be deliberate, for example by site-directed mutagenesis, or may occur spontaneously and include splice variants, isoforms, homologues.

from other species and polymorphisms. Such functional analogs are also included in the invention.

In addition, modifications to the primary amino acid sequence may result in proteins that do not retain the biological activity of the parent protein, including dominant negative forms, etc. The dominant negative protein may interfere with the wild-type protein by binding or otherwise masking regulatory agents such as downstream or downstream components. genes that normally functionally interact with the polypeptide. Such dominant negative forms also encompass the invention.

The term "signal peptide" refers to a peptide sequence that directs a newly synthesized polypeptide to which the signal peptide is attached to an endoplasmic reticulum (ER) for further post-translational processing and distribution.

The term "heterologous signal peptide" is used in conjunction with neublastin and means a signal peptide that is not a human neublastin signal peptide, typically a signal peptide of a mammalian protein other than neublastin.

It is known in the art that human neublastin DNA sequences (either cDNA or genomic DNA) or sequences that differ from human neublastin DNA may be due to silent codon changes or codon changes that produce conservative amino acid substitutions may be used in genetic engineering. modification of cultured human cells to overexpress and secrete the enzyme.

Polypeptides of the invention also include chimeric polypeptides or cleavable fusion polypeptides in which another polypeptide is fused to the N-terminus or C-terminus of the polypeptide or fragment thereof. A chimeric polypeptide can be produced by fusing a nucleic acid sequence (or portion thereof) that encodes another polypeptide with a nucleic acid sequence (or portion thereof) of the invention.

·· · Φ · «φφφφ ··» • ♦ φ φ φ φ φφφφ

ΦΦΦΦ φ φ φ φ φ φ φ · · · · · · • • • • φ φ φ φ

Methods for producing chimeric polypeptides are standard methods. Such methods usually require linking sequences in such a way that they are both in the same reading frame, and expressing the fused polypeptide under the same promoter (s) and terminator.

The polypeptides of the invention also include truncated forms of the complete neublastin molecule. In such truncated molecules, one or more amino acids are deleted from the N-terminus or C-terminus, preferably from the N-terminus.

Amino acid sequence homology

The degree to which the test peptide shares homology with the neublastin polypeptide of the invention is determined as the degree of identity between the two amino acid sequences. A high degree of sequence identity indicates the likelihood that the first sequence was derived from the second sequence.

Homology is determined by computer analysis using, for example, a ClustalX sequence alignment computer program (Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalIX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res 1997, 25 (24): 4876-82) and standard parameters described herein. Using this program, the mature portion of the polypeptide encoded by the analogous DNA sequence of the invention exhibits a degree of identity of at least 90%, particularly at least 95%, and particularly at least 98%, to the amino acid sequence described herein in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 16.

Based on the homology assay, it is confirmed that the polypeptide of the invention belonging to the TGF-β superfamily is related to the GDNF subfamily but represents a separate member of this subfamily.

• 4,444

Bioactive polypeptides

The polypeptide of the invention may be prepared in a bioactive form, including forms of pre-pro-proteins, pro-proteins, mature proteins, glycosylated proteins, phosphorylated proteins, or any other post-translationally engineered protein.

• 444

44

444 «• · · · 4

4 44 • 444 • 44

44 • ·· • 4 44

44

444 ♦ 444

44 • 44 ·

44 • 4444

In particular, the polypeptide of the invention may be a polypeptide that is preferably glycosylated as indicated in the sequence listing.

In a preferred embodiment, the polypeptide shown as SEQ I has an asparagine residue shown as SEQ IE the amino acid sequence comprises a glycosylated asparagine residue shown as SEQ 1 the amino acid sequence comprises a glycosylated amino acid sequence comprising a glycosylated asparagine residue the amino acid sequence shown as SEQ

N-glycosylated on N-residues, according to invention D NO: 9, which in position 122, ) NO: 10, which in position 122, ) NO: 11, which position 98, or ) NO: 12, which

the asparagine residue also includes

Ig-fusion as at position 95.

The neublastin discloses contains glycosylated

This invention proteins, such as in US Patent No. 5,434,131.

In one embodiment, the invention provides an amino acid sequence depicted in SEQ or an amino acid sequence that exhibits a polypeptide, fusion, e.g.

ID NO: 2 homology of at least 85%, preferably of at least 90%, in particular of at least 98% and in particular of at least 99% with the sequence shown as SEQ ID NO: 2.

In another embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that has a homology of at least 90%, preferably at least 95%, particularly at least 98%, and particularly at least 99% with the sequence shown as SEQ ID NO: 4 .

In a third embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that has a homology of at least 90%, preferably

·· ·· ·· «· • • · • • · · « • ·· • • ··· • · • • • • • • • · • · · • · • • • • • • · • · · · • ♦ « ·· ·· ·· ·· · « ···

at least 95%, in particular at least 98%, with the sequence shown as SEQ ID NO: 5.

In a fourth embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having a homology of at least 90%, preferably at least 95%, particularly at least 98%, with the sequence shown as SEQ ID NO: 6.

In a fifth embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a homology of at least 90%, preferably at least 95%, particularly at least 98%, with the sequence shown as SEQ ID NO: 7.

The neublastin polypeptide of the invention includes allelic variants, for example, the polypeptide amino acid sequences of SEQ ID NOs: 5-7, wherein Xaa denotes Asn or Thr and Yaa denotes Ala or Pro.

In a sixth embodiment, the invention provides polypeptides having the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that has homology to the sequence of SEQ ID NO: 9 of at least 90%, preferably at least 95%, especially at least 98%.

In a seventh embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that has homology to the sequence of SEQ ID NO: 10 of at least 90%, preferably at least 95%, especially at least 98%.

In an eighth embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that has homology to the sequence of SEQ ID NO: 11 of at least 90%, preferably at least 95%, especially at least 98%.

In a ninth embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that has homology to the sequence of SEQ ID NO: 12 of at least 90%, preferably at least 95%, especially at least 98%.

• • •

In a tenth embodiment, the invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 16, which is a pre-proneublastin of murine origin, or an amino acid sequence having homology to the sequence of SEQ ID NO: 16 of at least 90%, preferably at least 95%, especially at least 98%.

In another embodiment, the polypeptide of the invention has a GDNF subfamily peptide map, i.e., amino acid residues that are underlined in Table 3.

In another embodiment, the invention provides a polypeptide encoded by a polynucleotide sequence that is capable of hybridizing under stringent conditions to the polynucleotide sequence set forth in SEQ ID NO: 1 or its complementary strand or subsequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that exhibits at least 70% homology to the polynucleotide sequence shown as SEQ ID NO: 1. In a most preferred embodiment, the polypeptide of the invention encodes a polynucleotide sequence shown as SEQ ID NO: 1.

In another embodiment, the invention provides novel polypeptides encoded by a polynucleotide sequence that is capable of hybridizing under very stringent conditions to the polynucleotide sequence shown as SEQ ID NO: 3, its complementary strand, or a subsequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that exhibits at least 70% homology to the polynucleotide sequence shown as SEQ ID NO: 3. In a most preferred embodiment, the polypeptide of the invention encodes a polynucleotide sequence shown as SEQ ID NO: 3.

In another embodiment, the invention provides polypeptides encoded by a polynucleotide sequence that is capable of hybridizing under very stringent conditions to the polynucleotide sequence shown as SEQ ID NO: 8, its complementary strand, or a subsequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence which

exhibits at least 70% homology to the polynucleotide sequence shown as SEQ ID NO: 8. In a most preferred embodiment, the polypeptide of the invention encodes the polynucleotide sequence shown as SEQ ID NO: 8.

In one embodiment, the invention provides novel polypeptides encoded by a polynucleotide sequence that is capable of hybridizing under very stringent conditions to the polynucleotide sequence shown as SEQ ID NO: 15, its complementary strand, or a subsequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence having at least 70% homology to the polynucleotide sequence shown as SEQ ID NO: 15. In a most preferred embodiment, the polypeptide of the invention encodes a polynucleotide sequence shown as SEQ ID NO: 15.

Biological origin

The polypeptide of the invention may be isolated from mammalian cells, preferably from a human or mouse cell.

In a most preferred embodiment, the polypeptide of the invention may be isolated from human heart tissue, human skeletal muscle, human pancreas, or human brain tissue, particularly nucleus caudatus or thalamus, or may be obtained from DNA of mammalian origin, as described in more detail below. .

Neurotrophic activity

The neublastin polypeptides of the invention may be used to modulate the metabolism, growth, differentiation or survival of a neural or neuronal cell. In particular, neublastin polypeptides are used to treat or ameliorate a disorder or disease in a live animal, and when the disorder or disease reacts to the activity of a neurotrophic agent. Such treatments and methods are described in more detail below.

• · • · '

Antibodies

The neublastin polypeptides or polypeptide fragments of the invention are used to produce neublastin-specific antibodies. A "neublastin-specific antibody" herein refers to an antibody, such as a polyclonal or monoclonal antibody, that is immunologically reactive with a neublastin polypeptide or polypeptide fragment, or that specifically binds to an epitope of a neublastin polypeptide.

The preparation of polyclonal and monoclonal antibodies is known in the art. In particular, polyclonal antibodies can be obtained by the method described, for example, by Green et al., Production of Polyclonal Antisera in Immunological Protocols (Manson Ed.), Humana Press, 1992, pp. 1-5, Coligan et al., "Production of Polyclonal Antisera". in Rabbits, Rats, Mice and Hamsters in Current Protocols in Immunology, 1992, Section 2.4.1, and Ed Harlow and David Lané (Eds.) in "Antibodies: A laboratory manual, Cold Spring Harbor Lab. Press 1988. Monoclonal antibodies may in particular be obtained by procedures described, for example, by Kohler and Milstein, Nature, 1975, 256: 495, Coligan et al., Current Protocols in Immunology, 1992, Sections 2.5.12.6.7 and Harlow et al. in Antibodies: A Laboratory Manual, Cold Spring Harbor, Pub., 1988, p. 726.

Briefly, monoclonal antibodies can be obtained by injection of a composition comprising an antigen, for example, into mice; whereupon antibody production is verified by collecting a serum sample, collecting the spleen to obtain B cells, fusing B cells with myeloma cells to form hybridomas, cloning hybridomas, selecting positive clones that produce antibodies against antigen, and isolating antibodies from hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established methods, including protein A Sepharose affinity chromatography, size-exclusion chromatography, and ion exchange chromatography (see, for example, Coligan et al., Current.

2.7.12 a

Protocols in Immunology,

1992,

Sections 2.9.1-2.9.3,

Barnes

Immunoglobulin G (IgG)

Methods

Press, 1992, Vol. 10, pp. 79-104.

Antibodies are on the matrix and against which they are coated.

Sections

2.7.1. Purification of Molecular Biology, Humana

The polyclonal and monoclonal may optionally be eluted from the matrix to produce antibodies.

purified, for example by binding to which the polypeptide is bound,

Antibodies that bind to a neublastin polypeptide of the invention can be prepared using intact polypeptide or fragments that contain the corresponding small peptides as immunization antigen. A polypeptide used to immunize an animal can also be obtained by recombination

DNA or by chemical synthesis and may optionally be conjugated to a carrier protein. Commonly used carrier proteins that chemically associate with the peptide include gastropod hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The fusion peptide can then be used to immunize an animal, which may be, in particular, a mouse, rat, hamster, or rabbit.

In one embodiment, the antibodies are produced using the following peptides:

Peptide 1: CRPTRYEAVSFMDVNST (amino acids 108-124 of SEQ ID NO: 9) or

Peptide 2: ALRPPPGSRPVSQPC (amino acids 93-107 of SEQ ID NO: 9).

Methods for producing antibodies using these polypeptides are described in Example 10.

We also created rabbit polyclonal antibodies against the following peptides:

peptide R27: GPGSRARAAGARGC (amino acids 30-43 SEQ ID NO: 9) peptide R2 8: LGHRSDELVRFRFC (amino acids 57-70 SEQ ID NO: 9) peptide R2 9: CRRARSPHDLSL (amino acids 74-85 SEQ ID

NO: 9)

peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO NO: 9) and peptide R31: STWRTVDRLSATAC (amino acids 123-136 SEQ ID NO: 9). IN this Western group blots recognized denatured

protein under reducing conditions only peptides R30 and R31, which are relatively near the C-terminus.

We have also identified other neublastin-derived peptides derived from the mature protein, as described below, that form predicted surface exposed loops based on the known GDNF structure (Eigenbrot and Gerber, Nat. Struct. Biol., 1997 4 435-438 ) and are thus useful in the preparation of antibodies.

Region 1: CRLRSQLVPRALGLGHRSDELVRFRFC (AK43-70 SEQ ID

NO: 9) Region 2: CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AK74-107 SEQ ID NO: 9) Region 3: CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AK108-136 SEQ ID NO:

9)

In another aspect of the invention, antibodies that specifically bind to neublastin or neublastin-derived polypeptides can be used to detect the presence of such neublastin neurotrophic, particularly in the diagnosis of conditions with neoplastin molecules according to many protocols for such FACS.

Antibodies of the invention factor in various media and / or diseases associated with the invention. Detection, including ELISA, RIA, is well known in the art and can also be used to block the effect of neurotrophic factor, and may be particularly neutralizing antibodies.

Methods of producing polypeptides of the invention

The cell containing the DNA sequence encoding the neublastin polypeptide of the invention is cultured under conditions that:

4444

4 allows production of the polypeptide, whereupon the polypeptide is recovered from the culture medium, as described in more detail below. If cells are to be genetically modified to produce a neublastin polypeptide, they can be engineered by conventional methods or by gene activation.

In accordance with conventional methods, a DNA molecule comprising a neublastin cDNA or genomic DNA sequence may be contained in an expression construct and transfected into cells by standard methods including, but not limited to, liposome, polybrene or DEAEdextran mediated transfection, electroporation, clotting calcium phosphate, introduced by microinjection or in the form of high-speed microprojectiles ("biolistics"). Alternatively, a system that introduces DNA using a viral vector can be used. Viruses known to be suitable for gene transfer include adenoviruses, adeo-associated virus, lentivirus, herpesvirus, mumps virus, poliovirus, retroviruses, Sindbis virus and vaccinia virus such as varicella virus as well as baculovirus infections in insect cells, especially SfP9 insect cells.

Alternatively, cells can be engineered using the gene activation (GA) approach as described in US Patent Nos. 5,733,761 and 5,730,376.

The term "genetically modified, as used herein with respect to cells, is intended to include cells that express a particular gene product upon introduction of a DNA molecule encoding that gene product and / or regulatory elements that direct the expression of the sequence encoding that gene product. The DNA molecule can be introduced by gene targeting, which allows the DNA molecule to be inserted into a particular site of the genome.

Recombinant expression vectors

In another embodiment, the invention provides a recombinant expression vector comprising a polynucleotide of the invention. The recombinant expression vector of the invention may be any suitable eukaryotic expression vector, recombinant

Preferred ubiquitin

Schwartz T, expression vectors are pTEJ-8, which contains a promoter (Johansen TE, Schoeller MS, Tolstoy S, Lett. 1990 267 289-294) and derivatives thereof,

A preferred commercially available eukaryotic for example a viral promoter comprising a vector

FEBS for example pUbilZ.

the expression vector is pcDNA-3 (from Invitrogen). Another preferred expression vector uses the SV40 early promoter and the major late promoter

Mol. Cell. Biol. 1985 plasmid pAD2beta, Norton and

Coffin,

281).

The invention further provides synthetic genes (syngens) with prokaryotic expression vectors and optimized codons suitable for expression in prokaryotes.

contained lower GC content (for example

Syngens were constructed such that and preferred bacterial codons were cloned into two vectors, pET19b and pMJB164, which is a derivative of pET19b. The pET19b construct is shown in Figure 14. In this construct, the sequence encoding the mature domain with the initial methionine. The construction of neublastin directly fuses with pMJB164 is shown in FIG.

15 Dec

Production cells

In another aspect, the invention provides genetically engineered cells such that the sequence of the invention comprises and / or an isolated polynucleotide of the invention. In particular, a cell according to the recombinant expression vector of the invention may be genetically engineered to provide for temporary or stable expression, overexpression, or co-expression of a polypeptide of the invention. Methods for generating temporary and stable expression are known in the art.

The polynucleotide of the invention may be incorporated into an expression vector, for example a plasmid, virus or other expression tool, and operably linked to the expression control ligation such that expression of the coding sequence is achieved under conditions that are compatible with the expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns and stop codons, all maintained in the correct reading frame of the polynucleotide of the invention to allow proper translation of mRNA. Expression control sequences may also include other components such as leader sequences and fusion partner sequences.

In particular, a constitutive or inducible promoter may be used as the promoter. When cloning into bacterial systems, inducible promoters such as pL bacteriophage λ, plac, ptrp, and bird (ptrp-lac hybrid promoter) are used. When cloned into mammalian systems, promoters derived from the mammalian cell genome, such as the ubiquitin promoter, TK promoter or metallothionein promoter, or from mammalian viruses, such as the retroviral long terminal repeat, the adenovirus late promoter, or the vaccinia virus 7.5K promoter, may be used. Promoters obtained by recombinant DNA or synthesis may also be used to effect transcription of the polynucleotide of the invention.

Suitable expression vectors typically include an origin of expression, a promoter, as well as specific genes that allow for phenotypic selection of transformed cells, and include vectors such as a T7-based expression vector for expression in bacteria (Rosenberg et al., Gene 1987, 56,125). , pTEJ-8, pUbilZ, pcDNA-3, and pMSXND, expression vectors for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 1988 263

3521), baculovirus-derived vectors for expression in insect cells and oocyte PTLN expression vector (Lorenz C, Pusch M and Jentsch TJ, Eeteromultimeric CLC chloride channels with novel properties, Proc. Natl. Acad. Sci. USA 1996, 93, 13362-) 13366).

In a preferred embodiment, the cell of the invention is a eukaryotic cell, for example a mammalian cell, for example a human cell, an oocyte or a yeast cell. The cell of the invention may be a cell

without limitation, the human embryonic kidney (HEK) cell, for example

HEK 293, BHK21 cell, Chinese hamster ovary (CHO) cell, Xenopus laevis (XLO) oocyte. In another embodiment, the cell of

In another invention, a fungal cell, for example a filament cell of a preferred embodiment of the invention is an insect fungal cell.

a cell, especially a Sf9 cell. Other preferred mammalian cells of the invention are cell lines

PC12, HiB5

RN33b and human neuronal progenitor cells.

Most preferred are human cells.

Examples of primary and fibroblasts, intestinal epithelial cells epithelial cells, endothelial cells, blood elements including lymphocytes and bone secondary cells include mammary and marrow cells, glial cells, hepatocytes, keratinocytes, muscle cells, precursors of these cell types.

Examples of immortalized human neuronal cells or cell lines useful in said methods include, but are not limited to, Bowes melanoma cells (ATCC deposit number is CRL 9607), Daudi cells (ATCC deposit number is CCL 213), HeLa cells, and derivatives HeLa cells (ATCC deposit number is CCL 2, CCL2.1 and CCL2.2), HL-60 cells (ATCC deposit number is CCL 240), HT-1080 cells (ATCC deposit number is CCL 121), Jurkat cells (deposit number ATCC is TIB 152), KB carcinoma cells (ATCC deposit number is CCL 17), K-562 leukemia cells (ATCC deposit number is CCL 243), MCF-7 breast carcinoma cells (ATCC deposit number is BTH 22), MOLT- 4 (ATCC deposit number 1582), Namalwa cells (ATCC deposit number is CRL 1432), Paradise cells (ATCC deposit number is CCL 86), RPMI 8226 cells (ATCC deposit number is CRL 155), U-937 (ATCC deposit number) is CRL 1593), subline cells of 2R4 WI-38VA13 (u ATCC loading is CLL 75.1) and ovarian carcinoma cells 2780AD (Van der Blick et al., Cancer Res. 1988, 48: 5927-5932), as well as heterohybridoma cells produced by the fusion of human cells and cells of another species. Secondary strains of human fibroblasts such as WI-38 (ATCC deposit number is CCL 75) may also be used and

MRC-5 (ATCC deposit number is CCL 171).

Where the cell of the invention is a eukaryotic cell, the incorporation of the heterologous polynucleotide of the invention may be accomplished by infection (using a viral vector), transfection (using a plasmid vector), using calcium phosphate precipitation, microinjection, electroporation, lipofection or other physicochemical methods. which are known in the art.

In a preferred embodiment of the invention the isolated polynucleotide sequence of the invention and / or the recombinant expression vector of the invention is transfected into a mammalian host cell, neuronal progenitor cells, astrocyte cells, T-cells, hematopoietic stem cells, non-dividing cells or cerebral endothelial cells. a single DNA molecule capable of mediating cellular immortalization and / or transformation.

Activation of an endogenous gene in a host cell may be accomplished by introducing regulatory elements, particularly by introducing a promoter capable of causing transcription of an endogenous gene that encodes a neublastin polypeptide of the invention.

Pharmaceutical compositions

In another aspect, the invention provides novel pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide of the invention.

When used in therapy, the polypeptide of the invention may be administered in any suitable form. In a preferred embodiment, the polypeptide of the invention is incorporated into the pharmaceutical composition together with one or more adjuvants, fillers, carriers and / or diluents, and the pharmaceutical composition is prepared by one skilled in the art using conventional methods.

Such pharmaceutical compositions may comprise a polypeptide of the invention or antibodies described herein. The pharmaceutical composition may be administered alone or in combination with one or more other agents, drugs or hormones.

The pharmaceutical composition of the invention may be administered by any suitable means, including, but not limited to, oral, intravenous, intramuscular, interarterial, intramembranous, intrahecal, intraventricular, trnsdermal, subcutaneous, intraperitoneal, intranasal, anteral, superficial, sublingual or rectal, , vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intracisternal, intracapsular, intrapulmoral and transmucosal, or by inhalation.

Intrapulmonary delivery methods, devices, and drugs are described, for example, in US Patent Nos. 5,785,049, 5,780,019, and 5,775,320. Administration can be by repeated bolus injection of the formulation or by continuous intravenous or intraperitoneal administration from a reservoir that is outside (e.g. IV). bag) or inside the body (e.g., a bioerodible implant, a biogenic artificial organ, or a colony of implanted neublastin-producing cells; for example, see U.S. Patent Nos. 4,407,957, 5,798,113, and

800 828). Intrapulmonary delivery methods and devices are described, for example, in U.S. Patent Nos. 5,654,007, 5,780,014 and 5,814,607.

In particular, the administration of neublastin according to the invention can be achieved using any delivery method, including:

(a) pumps (see, for example, Annals of

Pharmacotherapy, 27: 912 (1993); Cancer, 41: 1270 (1993); Cancer Research, 44: 1698 (1984)).

(b) microcapsules (see, for example, U.S. Patent Nos. 4,352,883, 4,353,888 and 5,084,350) (c) polymer implants that provide sustained release of the active agent (see, for example, U.S. Patent No. 4,883,666) • Φ φφφφ (d) macrocapsules (see, for example, U.S. Pat.

968,733 and PCT published applications WO 92/19195,

WO 95/05452) of unprotected cell grafts to, for example, US Patent

No. 5,082,670 a

(F) subcutaneous, intravenous, intrarterial, intra-muscular, or other appropriate site injection and oral administration in the form of capsules, liquids, tablets or sustained release formulations.

In one embodiment of the invention, directly into

The CNS, preferably into the cerebral neublastin cavities, will introduce the brain parenchyma, the CNS site, the intrathecal space, particularly intrathecally.

or another suitable system assumes

In another embodiment of the invention, the introduction is by subcutaneous injection, intravenous administration or intravenous infusion.

Other useful parenteral delivery systems include ethyl vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle injection, needle-free injection, nebulizer, aerosol, electroporation, and transdermal patches.

Further details of the methods of preparation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

The active ingredient may be administered in one or more doses per day. Suitable doses range from 0.5 ng of neublastin per kilogram of body weight up to about 50 μς / kg and from about 1.0 ng / kg to up to about 100 μμ / kg daily. A neublastin-containing pharmaceutical composition should be capable of providing a local concentration of neurotrophic factor from about 5 ng / ml cerebrospinal fluid (CSF) to up to 25 ng / ml

CSF.

Of course, the dose administered must be carefully adjusted to the age, body weight and condition of the individual being treated, as well as the mode of administration, the dosage form and regimen, and the desired outcome. The precise dosage should, of course, be determined by the attending physician.

In other embodiments of the invention, the neublastin polypeptide can be administered by genetic delivery, using cell lines and vectors as described in the paragraph of the Therapeutic Method below. In order to form such therapeutic cell lines, a polynucleotide of the invention can be incorporated into an expression vector, for example a plasmid, virus or other expression tool, and operably linked to expression control sequences by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, intron splicing signals, and stop codons, all of which are maintained in the correct reading frame of the polynucleotide of the invention, thereby allowing proper translation of mRNA. Expression control sequences may also include other components such as leader sequences and fusion partner sequences.

In particular, the promoter may be constitutive or inducible.

Constitutive promoters may be synthetic, viral, or derived from the mammalian cell genome, such as the human ubiquitin promoter.

In a preferred embodiment of the invention, the therapeutic cell line will be a human immortalized neural cell line that expresses the polypeptide of the invention. In the case of implantation, between about 10 ⁵ to 10 ¹⁰ cells, in particular 10 ⁶ to about 10 ⁸ cells, are used.

• • •

Methods of treatment

The present invention, which relates to polynucleotides and proteins, polypeptides, fragments or derivatives thereof, as well as antibodies to such proteins, peptides or derivatives, can be used in the treatment or amelioration of disorders or diseases of the body of live animals and humans, diseases respond to the activity of neutrophic agents.

The polypeptides of the invention may be used directly by, for example, an injectable, implanted or ingested pharmaceutical composition suitable for the treatment of a pathological process that responds to neublastin polypeptides.

The polynucleotide of the invention, including its complementary sequences, can be used to express the neurotrophic factor of the invention. This may be accomplished by using cell lines that express such proteins, peptides or derivatives of the invention, or viral vectors that encode such proteins, peptides or derivatives thereof of the invention, or host cells that express such proteins, peptides or derivatives. These cells, vectors, and compositions can be applied in the treatment of target areas to affect the disease process that responds to neublastin polypeptides.

Suitable expression vectors can be derived from lentiviruses, retroviruses, adenoviruses, herpesviruses, and vaccinia virus and from various bacterially produced plasmids and can be used to introduce in vivo nucleotide sequences into an entire organism or target organ, tissue or cell population. Other methods include, but are not limited to, liposome transfection, electroporation, transfection with carrier peptides containing nuclear or other localization signals, and gene delivery through slow release systems. In another aspect of the invention, antisense nucleotide sequences complementary to the neublastin gene or portions thereof may be used to inhibit or enhance neublastin expression.

In another aspect, the invention relates to a method of treating or ameliorating a disorder or disease of an animal or human living body, wherein the disorder or disease is responsive to the activity of neutrophic agents.

In particular, the disorders or diseases may be nervous system damage caused by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancies or toxic agents, and genetic or idiopathic processes.

In particular, damage may occur in sensory neurons or retinal nerve cells that include neurons in the dorsal root ganglia or in any of the following tissues: geniculi ganglion, ganglia, vestibuloacoustic petrosus ganglion, and nodular cranial nerve complex, ventrolateral pole of maxillomandribular lobe and trigeminal lobe trigeminal nucleus.

In a preferred embodiment of the invention, the disease or disorder is a neurodegenerative disease that includes lesions or trauma neurons, such as traumatic lesions of peripheral nerves and / or spinal cord, cerebral ischemic neuronal injury, neuropathy, and particularly peripheral neuropathy, trauma or peripheral nerve injury, ischemic stroke, acute brain damage, acute system damage, multiple sclerosis, spinal cord, neurotoxin nerve tumors, metabolic diseases such as diabetes or reanal dysfunction and infectious agent damage, neurodegenerative damage including Alzheimer's disease,

Huntington's disease, Parkinson's disease, Parkinson's syndrome

Plus, progressive supranuclear paralysis (syndrome

SteeleRichardson-Olszewski), olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome (multi-system atrophy), guamanian parkinsonism dementia complex, amyotrophic lateral sclerosis, or • 4 44 · 4

4 4 44

4 4 4

44 other congenital or neurodegenerative diseases and memory impairment associated with dementia.

In a preferred embodiment, treatment of neurons of the sensory and / or autonomous system is contemplated. In another preferred embodiment, treatment of motor neuron disorders such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy is contemplated. In another embodiment of the invention, the use of the neublastin molecules of the invention is contemplated in promoting nerve recovery after traumatic injury. In one embodiment of the invention, it is contemplated to use a neural guide channel with a matrix containing neublastin polypeptides. Such neural guide channels are described, for example, in U.S. Patent No. 5,834,029.

In a preferred embodiment of the invention, the polypeptides and nucleic acids of the invention (and pharmaceutical compositions containing them) are used in the treatment of peripheral neuropathies. Peripheral neuropathies contemplated by treatment with molecules of the invention include trauma-induced neuropathies, for example, caused by physical damage or disease state, physical brain damage, spinal cord physical damage, stroke associated with brain damage and neurological disorders associated with neurodegeneration.

The invention also contemplates the treatment of chemotherapy-induced neuropathies (e.g., due to the introduction of chemotherapeutics such as taxol or cisplatin), toxin-induced neuropathies, drug-induced neuropathies, vitamin-deficient neuropathies, idiopathic neuropathies and diabetic neuropathies (see, e.g., U.S. Patent Nos. 5,496,804 and 5 916,555).

The invention also contemplates the treatment of non-neuropathies, monomultiplex neuropathies and polyneuropathies, including axonal and demyelinating neuropathies, using the neublastin nucleotides and polypeptides of the invention.

4444

In another preferred embodiment (and of the invention, and

Yippee

4444 • 44 • 44 44

44 · 4

4 * ·

4444 • 4

The nucleic acid polypeptides of the invention comprising) are used in the treatment of various eye disorders, which include the loss of a photoreceptor on the retina of a patient suffering from macular degeneration, retinal pigmentosis, glaucoma, and the like.

In another embodiment, the invention provides a method of preventing degenerative changes associated with the aforementioned human diseases of the vectors and disorders, by implantation into mammalian brain including or cells capable of producing a biologically active form of neublastin or a neublastin precursor, i.

molecules that can be converted into the biologically active form of neublastin by the body, or further, cells that secrete neublastin can be encapsulated, for example, into semipermeable membranes.

The cells may be cultured in vitro for transplantation or grafting into mammalian, including human brain.

according to the invention. gene encoding the embodiment

In a preferred polypeptide of the invention, it transfects into a suitable cell line, for example, an immortalized rat neural stem cell cell line, such as HiB5 and RN33b, or a human immortalized neuronal progenitor cell line, and the resulting cell line is implanted into the brain of a living body, including in the CNS secreted the therapeutic polypeptide of the invention, for example using the expression vectors described in WO 98/32869.

Methods of diagnosis and search

Neublastin nucleic acid may be used to determine whether an individual is predisposed to develop a neurological disorder resulting from a disruption of a neublastin gene, such as a defect in the neublastin allele, obtained, for example, by genetic inheritance, abnormal embryonic development, or acquired damage. DNA. The analysis may be performed, for example, by detecting deletions or point mutations in the neublastin gene or by detecting the inheritance of such predispositions of these genetic defects with specific fragment length polymorphisms (RFLPs), detecting the presence or absence of a normal neublastin gene by hybridizing a patient's nucleic acid sample with a nucleic acid probe specific neublastin gene and determining the ability of the probe to hybridize to the nucleic acid.

In particular, the neublastin nucleic acid can be used as a hybridization probe. Such hybridization assays may be used to detect, prognosis, diagnose, or monitor various conditions, disorders, or disease states associated with aberrant amount of mRNA that encodes a neublastin protein. A neublastin nucleic acid can be constructed as a "marker for neublastin neurotrophic factor-dependent physiological procedures."

but are not limited to “normal

These processes include physiological procedures (e.g. neuronal function) and pathological procedures (e.g. neurodegenerative diseases). Characterization of a sub-population of a particular patient with an abnormal amount (i.e., increased or inadequate) of neublastin protein and / or mRNA encoding neublastin can lead to the classification of a new disease. The term "abnormal amount" means an increased or decreased amount relative to the amount of a control sample or individual that does not exhibit a disorder determined in a quantitative or qualitative manner.

The neublastin nucleic acids or polypeptides of the invention may also be used in the testing and identification of neublastin analogs, including small ones, to provide a method for identifying a compound of interest that produces a neublastin-mediated biological effect. The method comprises the steps of providing a test cell which when in contact with neublastin. is induced to express a detectable product,

♦ · ·· • it ···· 99 • • * • • • • 9 9 • · and · 999 • • • 9 • • • • 9 · 9 · • • 9 9 • • • and • · • • • · • • • · ·· ·· ·· · « ···

treating the cell with a compound and detecting a detectable product. Expression of the detectable product indicates the ability of the compound to elicit a neurobiosastin-mediated biological effect.

The neublastin nucleic acids and polypeptides of the invention may further be used on DNA or protein chips or in computer programs to identify related novel gene sequences and their encoded proteins, including allelic variants and single nucleotide polymorphisms (SNPs). Such methods are described, for example, in U.S. Patent Nos. 5,795,716, 5,754,524, 5,733,729, 5,800,992, 5,445,934 and 5,525,464.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photographic representation of two northern blots tested with ³² P-labeled neublastin cDNA comparing the relative potency of neublastin gene expression in different types of adult human tissue (panel A) and different regions of the adult human brain (panel B).

Figure 2 is a photographic representation of Northern blot assayed with ³² P-labeled neublastin cDNA comparing the amount of neublastin cDNA expressed in a neublastin cDNA transfected cell line and a GDNF-cDNA transfected cell line.

· Β ·

Figure 3 is a photographic representation of western blotting that compares the potency by which neublastin protein is expressed in transfected HiB5 cells (lane 1) that are related to stably transfected HiB5 cells with neublastin cDNA (lane 2) tested either neublastin specific antibodies Ab-2 (left blot, panel A) or neublastin specific antibodies Ab-1 (right blot, panel B).

Figure 4 is a graphical representation of the effect of neublastin on the survival of cultured rat embryonic, dopaminergic, ventral mesencephalic neurons and ChaT activity in cholinergic nerve motor neurons and ChAT activity in cholinergic cranial motor neurons in serum free medium. In particular, Figure 4 is a dose response curve for recombinant GDNF for ChAT activity (dpm / hour). Figure 4B depicts ChAT activity (dpm / hour) using diluted conditioned media from either neublastin production or GDNF porducing cells. Figure 4C is a representation of the number of cells in the well of immunologically reactive cells with tyrosine hydrolase.

Figure 5 shows the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the function and survival of cultures of dopaminergic ventral mesencephalic porcine embryo neurons that are co-cultured with HiB5pUbilzNBN22 cells (neublastin) or HiB5 cells (control cells). 5A and 5B show the release of dopamine into the medium (DIV12) (dopamine concentration (pmol / ml) on day 12) and (DIV21) (dopamine concentration (pmol / ml) - day 21). Picture no

5C shows the number of immunologically reactive cells with tyrosine hydrolase in culture (TH-ir cells in culture) (DIV21).

Obrázek ž. 6 shows the effect of in vivo neublastin produced by lentivir on nigral dopamine neurons.

(

Figure 7 shows a schematic diagram of the genome structure of the zeoblastin gene that includes nucleic acid primers that can be used to identify the full length of the neublastin gene and their spatial orientation relative to the neublastin coding sequence (i.e., the gene).

Figure 8 shows neublastin-specific primers that are used to identify a cDNA clone that encodes a human neublastin plasmid that hybridizes to nucleic acids encoding neublastin polypeptides but does not hybridize to nucleic acids encoding other known GDNF family members (i.e., GDNF, persefin and neurturin).

Figure 9 shows neurotrophic activity on cultures of dissociated dorsal root nerve cells in rats at various developmental stages of polypeptides of the invention, as compared to results obtained with known neurotrophic factors (0 control experiment (no factor present), in the presence of GDNF, in the presence neurturin, in the presence of neublastin according to the invention, E12 day 12 of the embryo,

E16 embryo day 16, PO day of birth, P7 7 days after birth, P15 15 days after birth).

Figure 10 illustrates neublastin production in the CHO cell line.

Figure 11 illustrates a comparison of neublastin α binding

GDNF at GFRα-1 and GFRα-3 receptors.

Figure 12 is a photographic illustration of a western blot that illustrates R30 antipeptide antibodies and R31 antipeptide antibodies to neublastin.

Figure 13 is a gel image showing the extraction of neublastin binding to RETL3-Ig.

Figure 14 is a map of plasmid pET19b-Neublastin showing the sequence of the synthetic gene encoding neublastin.

© ·

Figure 15 is a plasmid map of pMJB164-HisNeublastin showing the sequence of the synthetic gene encoding

HisNeublastin.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Method for isolating neublastin nucleic acids

Method 1: Rapid testing for the presence of the neublastin gene in human embryonic brain cDNA.

The two genomic sequences (HGTS) identified a 290 bp fragment contained in the GenBank database (accession numbers AC005038 and AC005051) based on its homology to human persephin. Two neublastin-specific primers were synthesized from the nucleic acid sequence of the 290 bp fragment. The neublastin upper chain primer ("NBNint.sence) shows the sequence 5'-CCT GGC CAG CCT ACT GGG3 '(SEQ ID NO: 17). The neublastin lower chain primer ("NBNint.antisence") shows the sequence 5'-AAG GAG ACC GCT TCG TAG CG-3 '(SEQ ID NO: 18). These primers were used in 96-well PCR reactions.

A 96-well plate containing plasmid DNA from 500,000 cDNA clones was filled with approximately 5,000 clones per plate.

Neublastin nucleic acid was identified in three rounds of amplifications using the PCR method. Amplification increases the number of copies of the nucleic acid in the sample.

Testing the original plate

Using the 96-well PCR assay method as described above, the original human germ brain cDNA plate was tested with gene specific primers to isolate human neublastin cDNA.

30 nanograms of human embryonic brain cDNA (6 ng / μΐ, Origene e) were obtained from corresponding wells of the original plate. • · · · · · · · · ** ·

Technologies) and placed in a total volume of 25 μΐ, the solution containing the following reagents 0.2 mM of each of the two gene-specific primers previously mentioned (i.e., NBNint.sense and NBNint.antisence primers), 1x standard PCR buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTP (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories,

USA) and 0.5 units of Taq DNA polymerase (5 U / μΙ, Advanced Biotechnologies, UK).

PCR reactions were performed using the following procedure and conditions. DNA was initially denatured at 94 ° C for 3 minutes followed by 35 cycles of denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, first extension at 72 ° C after 90 seconds and final extension at 72 ° C for 5 minutes. The products of 96 individual PCR reactions were analyzed by 2% agarose gel electrophoresis using ethidium bromide. A positive 102 bp PCR product found in the wells was found to correspond to a single 96 well sub-plate.

The 102 bp nucleic acid fragment has the following sequence (SEQ ID NO: 13):

5'-CCTGGCCAGCCTACTGGGCGCCGGGCCCTGCGACCGCCCCCGGGCTCCCGGCCCGTCAG CCAGCCCTGCTGCCGACCCACGCGCTACGAAGCGGTCTCCTT-3 '

Sub-plate testing

A 96-well sub-plate containing human embryonic brain DNA was tested by PCR amplification by placing it in a well of 1 μΐ glycerol stock from the corresponding well of a 25 μ dest sub-plate containing 0.2 mM of each of the gene specific primers 1x standard PCR buffer (Buffer V, Advanced Technologies, UK), 0.2 mM dNTP (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, JSA) and 0.5 units of Taq DNA polymerase (5 U / μΙ)

Advanced Technologies, UK).

• · ··

4 _Γ · · ····· · · □

The same PCR conditions are used to test the original plate. 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide and a positive well was identified, which generated a 102 bp PCR fragment.

Column PCR

One milliliter of glycerol stock from the positive sub-plate was diluted 1: 100 in Luria (LB) soil. One milliliter and ten milliliters of said dilution was then plated on two separate agar plates containing LB soil and carbenicillin at a concentration of 100 µg / ml. LB soil plates were incubated overnight at 30 ° C. 90 colonies were picked from these plates and transferred to a new 96-well PCR plate containing 0.2 mM each of the previously mentioned gene-specific primers, 1x standard PCR buffer (Buffer V, from Advanced Biotechnologies, UK), 0 , 2 mM dNTP (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and 0.5 units of Taq DNA polymerase (at a concentration of 5 U / μΙ, from Advanced Biotechnologies, UK) with a final volume of is 25 μΐ.

The same PCR conditions are used to test the original plate. 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide. Then positive colonies containing a 102 bp fragment were identified.

Sequenced! plasmid DNA prepared from these positive colonies revealed full-length cDNA of 861 bp (SEQ ID NO: 8). The cDNA encoded pre-pro-neublastin (SEQ ID NO: 9). Automatic sequencing! DNA was performed using the BigDye® sequencing kit (PE Applied Biosystem, USA). Gel sequencing was performed on an ABI Prism 377 (PE Applied Biosystems, USA).

• ♦ · · · ♦♦ · ♦♦ · ♦♦ ·

Method 2: Cloning of neublastin cDNA from human brain

Another method for amplifying a full length neublastin cDNA or cDNA fragment can be performed by RACE (rapid amplification of cDNA ends) using neublastin-specific primers (N3Nint.sence and NBNint.antisence as described above, in combination with primers specific for a vector or adapter using, for example, the Marathon cDNA Amplification Kit (Clorech Laboratories, USA, Cat # K1802-1).

Marathon-Ready human brain cDNAs (Clontech Laboratories, USA, Cat. No. 7400-1) can be used to amplify full length neublastin cDNAs. Useful full-length primers include the neublastin 5'-ATGGAACTTGGACTTGG-3 ^z (SEQ ID NO: 19) primer (NBNext.sence) and the neublastin 5'-TCCATCACCCACCGGC-3 ^z (SEQ ID NO: 20) primer (NBNext. antisence) combined with an API adapter primer included in the Marathon-Ready cDNA. An alternative 5'-CTAGGAGCCCATGCCC-3 'upper chain primer (SEQ ID NO: 28) can also be used. Another alternative lower chain primer with sequence 5 ^of -GAGCGAGCCCTCAGCC-3 ^of (SEQ ID NO: 33) can also be used. Alternative backbone primers of SEQ ID NOs: 24 and 26 may also be used.

Method 3: Cloning of neublastin cDNA from human brain

Another method of cloning neublastin cDNA is by testing human adult brain and fetal libraries with one or more neublastin probes as described above (and as shown in Figure 1). These libraries include the human brain Xgt11 (Clontech Laboratories, USA. Cat. No. HL3002b) or the human Xgt11 brain embryo (Clontech Laboratories, JSA. Cat. HL3002b).

Method 4: Rapid testing for the presence of neublastin gene in mouse embryonic cDNA 4 44 44 44 44 44 44 44

44 444 · 4444

The rapid procedure for testing for the presence of the neublastin gene was performed as follows. The original 96-well plate containing plasmid DNA from 500,000 cDNA clones was filled with approximately 5,000 clones per well. A 96-well sub-plate was filled with a E. coli glycerol stock solution containing 50 clones per well. PCR-mediated amplification was performed in three cycles to detect the presence of the neublastin gene.

Testing the original plate

To isolate mouse neublastin cDNA, the original mouse purified cDNA plate was assayed by PCR in 96 wells using gene-specific primers. The following two primers were synthesized:

1) C2 Neublastin primer (NBNint.sence): 5'GGCCACCGCTCCGACGAG-3 '(SEQ ID NO: 21); and

2) C2ub neublastin primer (NBNint.antisence): 5'GGCGGTCCACGGTTCTCCAG-3 '(SEQ ID NO: 22).

For use these two genově specific primerů se identified positive PCR product O sizes 220 bp. . Nukleová acid containing 220 bp contain e following sequences (SEQ ID NO: 13):

5'-GGCCACCGCTCCGACGAGCTGATACGTTTCCGCTTCTGCAGCGG CTCGTGCCGCCGAGCACGCTCCCAGCACGATCTCAGTCTGGCCAG CCTACTGGGCGCTGGGGCCCTACGGTCGCCTCCCGGGTCCCGGC CGATCAGCCAGCCCTGCTGCCGGCCCACTCGCTATGAGGCCGTCT CCTTCATGGACGTGAACAGCACCTGGAGAACCGTGGACCGCC-3 '

The 96-well PCR reaction was performed as follows. 30 nanograms of human embryonic brain cDNA (6 ng / μΐ, Origene Technologies) were obtained from the corresponding wells of the original plate and placed in a 25 μΐ total solution containing the following reagents 0.2 mM of each of the two previously mentioned primers specific for gene (i.e., primers C2 (NBNint.sense) and neublastin primer 1x standard PCR buffer (buffer V,

C2as (NBNint.antisence))

Advanced Biotechnologies,

Pharmacia), 0.1 M GC-Melt Taq DNA Polymerase Units · φ φ • φ q q q q q q q q q · · · ·

UK), 0.2 mM dNTP (Amersham (Clontech Laboratories, USA)) and 0.5 (5 U / μΙ, Advanced Biotechnologies,

PCR reactions were performed under conditions. DNA was initially using the following procedure and denatured at 94 ° C for 3 minutes followed by 35 cycles of denaturation at ° C for 1 minute, annealing at 55 ° C for 1 minute, first elongation at 72 ° C. seconds and final

After minutes. Products 96 by bromide electrophoresis. It was found that temperature hybridization at 55 ° C for an extension at 72 ° C of the individual PCR reactions was analyzed for 5 by agarose gel using a 220 bp ethidium positive PCR product found in the wells of the sub-plate corresponding to the single wells. .

Sub-plate testing

Human embryonic sub-plate with wells containing brain DNA was tested by PCR amplification by placing it in a well of 1 μΐ glycerol stock from the corresponding well of a 25 μ dest sub-plate containing

0.2 mM each of the gene specific primers, 1x standard PCR buffer (Buffer V, Advanced

Technologies, UK)

0.2 mM dNTP

M GC-Melt (Clontech

Laboratories,

USA) and 0.5 units of Taq DNA polymerase (5 U / μΙ)

Advanced Technologies, UK).

The same PCR conditions are used to test the original plate. 96 individual PCR reactions were analyzed for

2% agarose gel containing ethidium bromide identified a positive depression in which

A PCR fragment of 220 bp.

• fc • fcfc ·

Column PCR

One milliliter of glycerol stock from the positive sub-plate was diluted 1: 100 in Luria (LB) soil. One milliliter and ten milliliters of said dilution was then plated on two separate agar plates containing LB soil and carbenicillin at a concentration of 100 µg / ml. LB soil plates were incubated overnight at 30 ° C. 90 colonies were picked from these plates and transferred to a new 96-well PCR plate containing 0.2 mM each of the previously mentioned gene-specific primers, 1x standard PCR buffer (Buffer V, from Advanced Biotechnologies, UK), 0, 2 mM dNTP (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech

Laboratories, USA) and 0.5 Taq DNA polymerase units (at a concentration of 5 U / μΙ, from Advanced Biotechnologies, UK) with a final volume of 25 μΐ.

The same PCR conditions are used to test the original plate. 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide. Positive colonies containing a 220 bp fragment were then identified. Plasmid DNA was prepared from this positive colony. The clone was sequenced using automatic sequencing using the BigDey® sequencing kit with AmpliTaq1 DNA polymerase. Sequencing was performed on a gel using an ABI Prism 377 (PE Applied Biosystems).

The resulting sequence of this clone showed full length cDNA of 2,136 bp (SEQ ID NO: 15).

The cDNA comprises an open reading frame with the predicted amino acid sequence shown in SEQ ID NO: 16 which encodes the murine pro-neublastin polypeptide.

Example 2: Cloning of genomic neublastin

As discussed above, in two human clones

BAC identified a nucleic acid fragment of size

290 bp included in GenBank (with access number AC005038 and • 4

4 4 44 44 • ·· 4 4 4 4 4 · 4444 · 444 4 44 • 44 4 _ _Λ 44 44 44 44 444

AC005051), which contains regions of homology to persephin and flanking sequences of persephin. For the cloning of neublastin-encoding nucleic acid, additional primers described above were designed using a predicted 861 bp sequence. Two pairs of primers were used to clone a neublastin gene using PCR on genomic DNA. The two primer pairs are listed below:

Primer pair # 1

5'CCA AgC CCA CCT ggg TgC CCT CTT TCT CC3 '(sense) (SEQ ID NO: 23)

5 'CAT CAC CCA CCg gCA ggg gCC TCT Cag 3' (antisense) (SEQ ID NO: 24)

Primer pair # 2

5'gAgCCAtgCCCggCCTgATCTCAgCCCgA ggACA 3 '(sense) (SEQ ID NO: 25)

5'CCCTggCTgAggCCgCTggCTAgTgggACTCTgC3 '(antisense) (SEQ ID NO:

26).

For using a pair primers # 1 is amplified of human genomic DNA obtained from Clontech Laboratories (Cat. 6550-1) a 887 bp DNA fragment. Protocol PCR: PCR was done using the system Expand ™ High Fidelity

PCR (Boehringer Mannheim) with Buffer # 1. The PCR reaction mixture was supplemented with 5% dimethylsulfoxide (DMSO) and 17.5 pmol of each dNTP in a total volume of 50 μΐ. Temperature cycles were performed by a pre-naturing step at 94 ° C for 2 minutes. This is followed by 35 cycles of two steps at 94 ° C for 10 seconds and 68 ° C for 1 minute. Temperature cycles were determined by incubation at 68 ° C for 5 minutes. Temperature cycles were performed on a PTC-225 DNA Engine Tetrad temperature cycler (MJ Research, MA). PCR products were analyzed by gel electrophoresis on 2% agarose (FMC) and then photographed.

The 887 bp fragment was amplified from human genomic DNA with primer pair # 1 into the pCRII vector (Invitrogen) and transformed into competent E. coli XL1-Blue cells (Stratagene). The resulting plasmid, designated neublastin-2, was sequenced using thermosecvenase (Amersham Pharmacia Biotech). Sequencing products were analyzed by electrophoresis on an ALFExpress automatic sequencer (Amersham Pharmacia Biotech).

Fragments obtained by PCR amplification of human genomic DNA with a second primer pair (primer pair # 1 described above) were sequenced, with an additional 42 bp region at the 3 'end of the open reading frame. The full-length sequence was analyzed by comparing it with the nucleic acid sequences of other neurotrophic factors as well as by mapping exon-intron boundaries using gene-finding software programs that identify the likely splice site and high coding potential region using Netgene and Gene Mark software (Brunak et al. al., J. Mol. Biol. 1991 220 49-65), Borodovsky et al., Nucl. Acids Res. 1995 23 3554-62). The exon-intron boundaries were confirmed from the cDNA obtained by the rapid assay described above.

As illustrated in Figure 7, the resulting neublastin gene has two exons separated by an intron of 70 bp. The exons have the predicted amino acid sequence of full-length neublastin polypeptides. The putative cDNA (SEQ ID NO: 3) contains an open reading frame (ORF) encoding 238 amino acid residues (SEQ ID NO: 4). The neublastin-2 clone contained the entire pro-neublastin coding sequence. The amino acid sequence encoded by the gene exhibits high homology to the orane proteins, which are persefin, neurturin, and GDNF.

Example 3: Neublastin Nucleic Acid Expression Expression of Neublastin Nucleic Acids Expression of Neublastin Nucleic Acids Expression of Neublastin Nucleic Acids 9. 9

Neublastin RNA expression was detected in both neural and non-neural tissue in rodents and humans and in various developmental immature and adult stages using the methods described below.

Method for detecting RNA expression of neublastin using RT-PCR:

Based on the neublastin DNA sequence identified as SEQ ID NO: 1, the following primers were synthesized: 1) C2 5'-GGCCACCGCTCCGACGAG-3 'neublastin primer (SEQ ID NO: 21) and 2) C2as 5'-GGCGGTCCACGGTTCTCCAG-3' neublastin primer (( SEQ ID NO: 22). This primer set was used to amplify the RT-PCR fragment of DNA from whole human and human fetal mRNA. Among the DNA fragments produced by this reaction was one 220 bp fragment. The identification of this 220 bp DNA fragment confirmed that the neublastin gene was expressed in adult and fetal brain tissue. A 220 bp DNA fragment was also amplified from genomic DNA using the primers.

Method for detecting neublastin RNA expression by Northern blot hybridization

Northern blots with polyA ⁺ RNA from adult human tissue were obtained from Clontech Laboratories, USA and tested using a ³² P-labeled neublastin cDNA probe. Labeled neublastin cDNA was prepared according to the methods described in Example 1 above.

Preparation of probes: The neublastin DNA fragment (nucleotides 296-819 of SEQ ID NO: 8) was labeled with Rediprimell baptism (Amersham, Cat. No. RPN1633) suitable for use as hybridization probes under conditions recommended by the manufacturer. The DNA sample was diluted to a final concentration of 2.5 to 25 ng in 45 μΐ of 10 mM TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The DNA was then denatured by heating the sample to 95-100 ° C for 5 minutes in a water bath, rapidly cooling the sample by placing it on ice for five minutes and then briefly centrifuging to obtain the contents. on the bottom of the tube. The total amount of denatured DNA was pooled with 5 μΐ Redivue [ ³² P] dCTP (Amersham Pharmacia Biotech Ltd.) in a tube containing a buffered solution of dATP, dGTP, dTTP, Klenow exonuclease-free enzyme and a random primer in dried stabilized form. The solution was mixed by pipette 2 times and the reaction mixture was incubated at 37 ° C for 10 minutes. The labeling reaction was stopped by adding 5 μ 5 0.2 M EDTA. For use as a hybridization probe, labeled DNA was denatured into single strands by heating the DNA sample at 95-100 ° C for 5 minutes, then the DNA sample was rapidly cooled on ice for 5 minutes. The tube was centrifuged and mixed well. Finally, the single stranded DNA probe was purified using a nucleotide removal kit (Qiagen).

Hybridization procedures:

The prepared Northern blots were obtained from the aforementioned company (Multiple Tissue Northern Blots, Clontech Laboratories, USA, Cat. Nos. 7760-1 and 7769-1) and hybridized according to the manufacturer's recommendations using the ³² P-labeled neublastin probe prepared above. text. For hybridization, an ExpressHyb solution (Clontech Laboratories, USA) was used and a labeled probe at approximately 3 ng / ml was used. The ExpresHyb solution was heated to 68 ° C and stirred until all the precipitates had dissolved. Each Northern blot membrane (10x10 cm) was pre-hybridized in at least 5 ml of ExpressHyb solution at 68 ° Z for 30 minutes in a Hybaid hybridization oven according to the manufacturer's instructions. The ³² P-labeled neublastin probe was denatured at 95-100 ° C for 2 minutes and then cooled rapidly on ice. 14 microliters of labeled probe was added to 5 ml of fresh ExpresHyb solution and mixed.

The ExpresHyb solution used in pre-hybridization was replaced with 5 ml of fresh ExpresHyb solution containing the labeled probe.

DNA. The blots were incubated for 1 hour at 68 ° C in a Hybaid hybridization oven. washed several times, containing 0.05

After incubation, the blots were flushed and low stringency (2x SSC,% SDS at room temperature), then stringent 50 ° C) followed by washing with

0.1% SDS at sodium citrate, pH 7.0). The blots were exposed

Ltd) at ambient temperature (0.1x SSC containing

M NaCl / 0.3 M (20x SSC is

0.3 to (Amersham Pharmacia Biotech

Hyperfilm MP ° C using intensity plates.

The northern results are presented in FIG.

hybridization

1.

experiments are

1A (left) and 1B (right) are Northern blots of polyA ⁺ RNA that were tested with ³² P-labeled neublastin cDNA as described in Example 3. The markers represent 1.35 kb, 2.4 kb, 4.4 polynucleotides kb, 7.5 kb and 9.5 kb. The membrane shown in FIG.

IA was prepared using mRNA extracted from various adult human tissues. From the results of Northern blot hybridization analysis, it was concluded that neublastin mRNA was expressed in many adult human tissues. The strongest expression of neublastin was detected in the heart, skeletal muscles and pancreas. The membrane in Figure IB was prepared with RNA extracted from various areas of the adult's brain. In the brain of an adult, the strongest expression can be seen in the nucleus caudatus and thalamus. An approximately 5 kb mRNA transcript is the predominant form of neublastin mRNA expressed in the brain.

A method for detecting RNA expression of neublastin using in situ hybridization in tissues

The following methods can be used to measure RNA expression of neublastin in animal tissues, for example, rodent tissues with an anti-sense probe of neublastin.

• 0 ♦ · 0 «···· β0 ♦ · · · · 00 0

9,999 99,999

9 9 9 9 9 9 9 9 90

000 · 0000 000

99 99 99 00

Expression in mice

Preparation of tissue samples:

Pregnant mice (B and K Universal, Stockholm, Sweden) were sacrificed by cervical dislocation at 13.5 or 18.5 days of pregnancy. The embryos were removed by dissecting under sterile conditions and immersed immediately in a solution of 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde (PFA) for 24 to 30 hours. They were then removed from PFA and stored in PBS. Tissue was prepared by slicing immersion tissue in 30% sucrose solution and then embedded in TissueTech (O.C.T. compound, Sakura Finetek USA, Torrance, CA). Six series of coronal and sagittal sections (12 μιη) in a cryostat were prepared and thawed on a positively charged glass slide. The neonatal heads / brains (P1, P7) were fixed according to the same protocol as the embryogenic stages and further dissected brain tissue of an adult, immediately frozen on dry ice and sectioned in a cryostat without pre-fixing.

Preparation of neublastin ribosomes:

An arsenic neublastin RNA probe (neublastin ribo probe) was prepared as follows. Nucleotides 1109 to 1863 of the mouse neublastin cDNA sequence (SEQ ID NO: 15) were subionized into the BlueScript vector (Stratagene). The resulting plasmid was digested into linear DNA using EcoRI restriction endonuclease. The EcoRI DNA fragment was transcribed in vitro using RNA polymerase T3 and the RNA labeling kit with digoxigenin (DIG) according to the manufacturer's instructions (Boehringer Mannheim).

Hybridization:

The sections formed in the cryostat were fixed for 10 minutes in 4% PFA, treated for 5 minutes with proteinase K at a concentration of 10 mg / ml and gradually dehydrated in 70% and 95% ethanol for 5 and 2 minutes. They were then allowed to air dry. Hybridization Buffer (50% deionized formamide, 10% 50% dextran sulfate solution, 1% Denhardt's solution, 250 gg / ml yeast tRNA, 0.3 M NaCl, 20 mM Tris-HCl (pH8), 5 mM EDTA, 10 mM NaPO ₄ , 1% sarcosyl), which contains 1 μς / πιΐ probes labeled with DIG, was heated to 80 ° C for 2 minutes and applied to each section. The sections were then covered with parafilm and incubated at 55 ° C for 16-18 hours.

The next day the sections were washed under very stringent conditions (2x SSC containing 50% formamide) at 55 ° C for 30 minutes and then washed with RNase buffer and incubated with 20 µg / ml RNase A for 30 minutes at 37 ° C. Noc: 2 ° C. To detect the DIG-labeled probe, sections were pre-incubated with blocking solution (PBS containing 0.1% Tween-20 and 10% heat inactivated goat serum) for 1 hour and then incubated overnight at 4 ° C with alkaline phosphatase. associated with anti-DIG antibodies at a 1: 5,000 dilution (Boehringer Mannheim). The next day, each section was washed four times for 2 hours in PBS containing 0.1% Tween-20, and then washed twice for 10 minutes in NTMT buffer (100 mM NaCl, 100 mM Tris-HCl (pH 9.5), 50 ml). mM MgCl 2, 0.1% Tween-20). Sections were incubated in BM-red suspension containing 0.5 mg / ml levamisole for 48 hours. The staining reaction was stopped by washing in PBS. The sections were air dried and covered with a DPX coverslip (KEBO-lab, Sweden).

The results of the in situ hybridization reactions were presented in Table 1.

Table 1: Neublastin expression in mice

structure E13.5 E18.5 PI P7 adults front brain + + ventral midbrain - dorsal root ganglia + + spinal cord +

·· ·· ·· 4 ··· 99 9 • • • • • • * • • ··· • • • • • • · • · • · • • • · • • · • · • • • · ♦ • • · ·· 9 · 99 • 0 •

retina +++ + + + + Bulbus olfactcrius ++ + + ++ dental pulp ++ ++ + trojklanná ganglia ++ ++ ++ striatum + + ++ cortex + ++ + gyrus dentatus ++

As shown in Table 1, neublastir did not appear at 13.5 days of embryo (E13.5). expressed in the spinal cord and posterior brain and weakly in the forebrain. Neublastin expression was also detected in the developing retina and sensory ganglia (dorsal root ganglia and ganglia (V)). Outside the nervous system, a weak signal was found in the kidneys, lungs and intestine, indicating that neublastin is also expressed in the tissues.

On day 18.5 of the embryo (E18.5), neublastin was most expressed in the ganglion (V). Neublastin expression was also detected in the retina, striatum and cortex. In addition, expression was observed in the dental bed.

In Table 1, increased expression of neublastin from day E18.5 to postnatal days 1 and 7 can be seen in the cortex, striatum and trigeminal ganglion (V). Neublastin expression was strongest in the outer layers of the cortex compared to the inner layers of the cortex. On day P7, expression was found in the same structures as on day 1, but additional neublastin expression was found in the hippocampus, especially in the dentetus gyrus in the cerebel. In adult mouse brain, neublastin was strongly expressed in dentate gyrus and very little or no neublastin expression was detected in other tissues.

Expression in rats

The following experiment describes hybridization of rat tissues with a non-coding strand oligodeoxynucleotide probe labeled with alkaline phosphatase.

Preparation of tissue samples:

Rat embryos (E14) were obtained from Wistar rats (Mollegaard Breeding, Denmark) after anesthesia with pentobarbital. Postanatal rats (PO, P7 and adult) were sacrificed by decapitation. The dissected brain and whole heads were immediately immersed in chilled 0.9% NaCl, freshly frozen were cut into a 20 µm section in a cryostat (coronal and sagittal sections, 10 series).

In-network Hybridization:

Two series of sections were hybridized using an alkaline phosphatase (AP)-linked antisense oligodeoxynucleotide probe (5'-NCA GGT GGT CCG TGG GGG GCG CCA AGA CCG G-3 ') (oligo). No. 164675, DNA Technology, Denmark). This probe is complementary to bases 1140 to 1169 of the mouse neublastin cDNA (SEQ ID NO: 15).

Prior to hybridization, the sections were air dried at room temperature, heated to 55 ° C for 10 minutes and then treated with 96% ethanol at 4 ° C overnight. The sections were then air dried and then incubated in hybridization medium (5.0 pmol probe per milliliter) overnight at 39 ° C (Finsen et al., Neurosci. 1992 47 105-113, West et al. al., J. Comp. Neurol. 1996 370 11-22).

Treatment after herbidization includes four washes for 30 hours

minutes in 1 x SSC (0.15 M NaCl, temperature 55 ^; C, then follows minutes in Cris-HCl, pH 9.5 at applies AP developer. AP Developer

0.015 M sodium citrate) was washed three times at ten room temperature and then prepared immediately prior to use and containing tetrazoleum nitrate blue (NBT,

Sigma), 5-bromo, 4-chloro, 3-indole phosphate (BCIP, Sigma) and buffer containing Tris-HCl-NgCl2 pH 9.5 (described in

Finsen et al., Neurosci. 1992 47 105-113). AP development takes place in the dark at room temperature for 48 hours. The staining reaction was stopped by washing the sections with distilled water. Sections were dehydrated in acetone, softened with xylene phenol creosote (Allchem, UK), purified with xylene and overlaid using Eukitt (Bie and Berntsen, Denmark).

Control reactions include 1) treatment of RNase A sections (50 μς / γηΐ, Pharmacia, Sweden) prior to hybridization, 2) hybridization of sections with 100-fold excess of unlabeled probe, and 3) hybridization of sections with hybridization buffer alone. The results of the hybridization reactions are shown in Table 2.

Table 2: Neublastin expression in rats

structure E14 P0 / P1 P7 adults front brain + + ventral midbrain - dorsal root ganglia ++ spinal cord + retina + bulbus olfactorius (+) ++ + + small cerebellum + ++ + trojklanná gar.glia ++ + + striatum + + (+) cortex (+) ++ ++ + hyppocampus (+) + + ++

On embryo day (E14), neublastin was poorly expressed in rat embryos in the forebrain, hindbrain and spinal cord. Neublastin mRNA was also detected in the eyes (retina), in the dorsal root ganglia, in the truncated gnaglia (V) and in the kidneys, lungs, hearts, liver and intestines. Neublastin expression in the cortex and striatum was found in neonatal rats (PO). Neublastin expression was also detected in the bulb ofaktorius and in the hippocampus. In Ί days old rats (P7), this neublastin was expressed in cerebral cortex, stratum, olfactorius, and cerebellum. The signal was also detected in the hippocampus. Very low or no neublastin expression was detected in most areas of the brain in adult rats. Weak signals were detected in the thalamic nucleus and appreciable expression of neublastin was detected in the hippocampus.

Example 4: Neublastin polypeptides

The open reading frame or coding region (CDS) identified in SEQ ID NO: 8 encodes a pre-pro-polypeptide (designated pre-pro-neublastin). The amino acid sequence predicted based on the open reading frame is shown

in SEQ ID NO: 9. Based on SEQ ID NO NO: 9 se identified three variants of neublastin polypeptides. These variants include: and) a polypeptide designated as NBN140, who has the amino acid sequence designated as SEQ ID NO: 10, (ii) a polypeptide designated as NBN116, who has the amino acid sequence designated as SEQ ID NO: 11 a (iii) a polypeptide designated as NBN113, who has the amino acid sequence designated as SEQ ID NO: 12.

Similarly, based on the coding region (CDS) as set forth in SEQ ID NO: 3, which encodes a pre-pro-polypeptide having an amino acid sequence (designated as SEQ ID NO: 4),

identified three variants neublastin. These variants include: and) a polypeptide that has amino acid sequence designated SEQ ID NO: 5, (ii) a polypeptide that has amino acid sequence designated SEQ ID NO: 6 a

* * 1 φ φ · φ φ φ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (iii) a polypeptide having the amino acid sequence designated SEQ ID NO: 7.

Based on the ClustalW (1.75) based multi-sequence alignment, SEQ ID NO: 9 aligned with amino acid sequences

GDNF, persephine and neurtutin. This arrangement is shown in Table 3.

Table 3: Amino acid sequence comparison of neublastin, neurturin and GDNF neurturin - whole neublastin persefin - whole GDNF _ human - whole neurturin - whole neublastin persefin - whole GDNF _ human - whole neurturin - whole neublastin persefin - whole

GDNF _ human - all neurturin - all neublastin persefin - whole

GDNF _ human - whole ____________________MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPA

MELGLGGLSTliSHCPWPRRQPALWPTLAALALLSSVAEASLGSAPRSPAPREGPPP

----- MKL · WDWAVCLVLL · HTASAFPLPAGKRPPEAPAEDRΞL · GRRRAPFΆLSSDS

LVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWAGRPPGPRRRAGPRRR VLASPAGHLPGGRTARWCSGRARRPPPQPSRPAPPPPAPPSALPRGGRAARAGGPG -MAVGKFL·LGSLL·LLΞLQL GQGWGPDARGVPVADGEFSSΞQVΆKAGGTWL · · GTHRPL NMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKG

RARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEA-AARVYDLGLRR

SRARAAGARGCRLRSOLVPVRALGLGHRSDELVRFRFCSGSCRR-ARSPHDLSLAS

ARLRRALSGPCQLWSLTLSVAELGLGYASEEKVIlTOCAGSCPRGARTQHGIALAIi

RRGQRGKNRGCVLTAIHLNVTDLCTjGYETKEELiraYCSGSCDA-AETTYDKILKN * · * ♦ · * '* · ** · *' ^: '' **** JJ. · · * *

LRQRRRLRRE --- RVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSAREGACVllgagalrpppgsrpvsqpccrptrye-avsfmdvwstwrtvdrlsatacgclg

LQGQGRAHGG -------- PCCRPTRYT-DVAFLDDRHRWQRLPOLSAAACGCGG

LSRNRRLVSD ---- KVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI * * * * *. where * indicates a position that shows a single completely conserved residue.

the symbol: indicates that one of the following "strong groups" is fully conservative: -STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

symbol. indicates that one of the following "weak groups" is completely conservative: -CSA, ATV, SAG, STNK, STPA, SGND,

SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

Based on the alignment of the amino acid sequences shown in Table 3, it can be seen that the conserved cysteines have neublastin having seven residues in positions that are conserved in the superfamily

TGF-β.

in one preferred embodiment of the invention the preferred neublastin polypeptide comprises (seven) cysteine residues conserved as in SEQ ID NO: 2 at position 8, 35, 39, 72, 73,

101 and 103 or as SEQ ID NO: 4 and at positions 43, 70, 74,

107, 108

136 and 138. These seven conserved residues appear in the TGF-β super-family, forming three intramonomeric disulfide bonds (considered, for example, in SEQ ID NO: 2 between cysteine residues 8 to 73, 35 to 101 and 39 to 103, and for example in SEQ ID NOs: 4 and 9 between cysteine residues 43 to 108, 70 to 136 and 74 to 138) and a single monomeromeric disulfide bond (referred to, for example, in SEQ ID NO: 2 between cysteine residues 72 to 72 and, for example, SEQ ID NO. 4 and 9 between cysteine residues 107-107), which together with the large beta chain region form a conserved structural motif for the TGF- [beta] superfamily (Daopin et 192).

Due to the arrangement of this neublastin, it is a member of the subfamily (LGLG-FR (Y / F) of CSGSC-QxCCRP-SAxxCGC, GDNF, underlined in Table 3).

The homology of neubl GDNF was calculated and the result is shown in Table al., Proteins 1993 17, 176 The sequence showed that

GDNF neutrophic factors peptide map of the istin subfamily with other family members # 4 below.

Table 4: Homology of Neublastin Polypeptides with Other GDNF Family Members

mature NBN140 protein mature NBN113 protein homology full-length peptide homology homology full-length peptide homology neurotrophic conformity overlap high homology conformity conformity overlap high homology conformity

factor (if) (if) GDNF 34% 137 48% 31.9% 36% 111 52% 29, 5% (47/137) (67/137) (41/111) (59/111) NTN 48% 127 56% 36.9% 49% 114 57% 44.7% (61/127) (72/127) (56/114) (66/114) PSP 44% 125 56% 36, 9 45% 111 57% 44,3% (55/125) (71/125) (51/111) (65/111) IHA 31% 81 - 25.2% 31% 81 - 22.5% (25/81) (25/81) TGF-β2 23% 73 - 18.5 23% 73 - 20.2% (17/73) (17/73)

GDFN = neurotrophic factor derived from the glial cell line

NTN = neurturin

PSP = persefin

IHA = Inhibin

TF ~ P2 = transforming growth factor beta 2 high homology indicates that one of the following "strong groups" is conservative: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Example 5: Neublastin production

Neublastin was produced in eukaryotic and prokaryotic cells as described below.

Full-length cDNA expression vectors encoding neublastin were incorporated into the eukaryotic expression vector pUbilZ. This vector was created by cloning the human UbC promoter into a modified version of pcDNA3 / Zeo. Untreated pcDNA3.1 / Zeo is available from Invitrogen. The modified form of pcDNA3.1 / Zeo is smaller than the parent vector because the gene encoding ampicillin (from position 3 933 to 5 015) and the sequence from position 2 838 to 3 134. have been removed. In this modified version of pcDNA3.1 / Zeo, the CMV promoter Replaced by the UbC promoter of pTEJ-8 (see

Johansen et al., FEBS Lett. 1990, 267, 289-294), the resulting plasmid being designated pUbilZ.

Expression in mammalian cells

The pUbilZ vector containing the neublastin coding sequence was then transfected into a mammalian HiB5 cell line consisting of immortilized rat nerve cells (Renfranz et al., Cell, 1991, 66, 713-729). Several HiB5 cell lines stably expressing neublastin (as determined by RT-PCR) were generated. In one of these stable lines, expression of HiB5pUbilzNBN22 was confirmed by hybridizing total RNA on a Northern blot with a ³² P-labeled neublastin probe. The results of these studies are shown in Figure 2. HiB5pUbilzNBN22 was then used as neublastin source in neublastin neutrophic activity.

Figure 2 shows the expression of neublastin cDNA in the HiB5pUbilzNBN22 clone (i.e. Northern blot was assayed using a ³² P-labeled neublastin cDNA probe of the invention as described above). The blot was prepared by total RNA extraction from untransfected HiB5 cells, HiB5pUbilzNBN22 cells, and HiB5pUbilzGDNF14 cells. The positions of the 28S and 18S rRNA bands corresponding to the size of 4.1 kb and 1.9 kb are indicated on the blot.

As shown in Figure 3, antibodies raised against neublastin-derived polypeptides also recognize a protein of approximately 13 kD in conditioned media that originates from the clone HiB5pUbi1zNBN22, but not from untransfected HiB5 cells (Example 6).

The predicted molecular weights of the unmodified (e.g., lack of post-translational modifications) neublastin polypeptides NBN140 (SEQ ID NO: 10), NBN116 (SEQ ID NO: 11) and NBN113 (SEQ ID NO: 12) were determined to be 14.7 kD, 12 , 4 kD and 12.1 kD.

• • •

Methods

Northern blots containing total RNA (10 μα) from untransfected HiB5 cells and clone HiB5pUbilzNBN22 were prepared by 0.8 agarose gel electrophoresis in formaldehyde and blotted onto a nylon membrane (Duralone, Stratagene). The blot was hybridized and washed as described in Example 3 with a 1.3 kb ³² P-labeled probe prepared by random labeling and covering SEQ ID NO: 8 and other nucleotides from 5'UTR and 3'UTR neublastin cDNAs. The blot was exposed to Hyperfilm MP (Amersham) at -80 ° C using signal strength plates.

Conditioned medium from HiB5pUbilzNBN22 or untransfected Hib5 cells was incubated overnight in serum-free medium supplemented with N2 supplement (Life Technologies Cat. No. 17502048), concentrated and separated on 15% polyacrylic gels (Amersham Pharmacia Biotech, Cat. 80-1262-01). Proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech, Cat. No. RPN-303F) and non-specific protein binding sites were blocked with 5% fat-free milk powder in PBS with 0.1% Tween-20. Membranes were incubated overnight with polyclonal neublastin antibodies (1: 1000), followed by incubation with secondary anti-rabbit IgG antibodies (Amersham Pharmacia Biotech, Cat. No. NA 934). Immunological staining was visualized using enhanced chemoluminescence (ECL) (Amersham Pharmacia Biotech, Cat. No. RPN2109) or ECL + (Amersham Pharmacia Biotech, Cat. No. RPN2132) according to the manufacturer's instructions (Amersham).

The results of these experiments are shown in Figure 3. Figures 3A and 3B are representations of neublastin protein expression in transfected HiB5 cells. Culture medium derived from non-transfected HiB5 cells cultured overnight (lane 1) or from a HiB5 clone stably transfected with neublastin cDNA (lane 2) was concentrated, *

as described above. Culture medium • 4 • 4 • 4 • 4 • 4 • 4

4 · • 4 • 4

4 ·

Then, the various Ab-1 and Ab-2 described in Example 10 were analyzed by Western polyclonal antibodies that are specific for neublastin. Proteins of molecular weight were detected in the transfected cells obtained

This protein appeared in the case of untransfected cells by transfer using two culture antibodies, approximately 15 which medium

HiB5.

The cloned cDNA encoding the neublastin of another eukaryotic expression eukaryotic expression vector in Johansen et al., FEBS Lett.

has also been incorporated into a vector, for example

TEJ-8 (described

1990 267 289-294) or pcDNA-3 (Invitrogen) and the result is transfected into an alternative cell, for example, Chinese hamster ovary cells with an expression plasmid line such as (CHO), HEK293, COS,

PC12 or RN33b cells or human neural stem cells. Stable neublastin-expressing cell lines are used, for example, in the production of a neublastin protein.

Expression in CHO cells

Construction of plasmid pJC070.14

In order to express neublastin cDNA in Chinese hamster ovary cells, a cDNA fragment encoding the prepreg form of human neublastin was inserted into the mammalian expression vector pEAG347 to generate plasmid pJC070.14. The vector pEAG347 contains the tandem SV40 early promoter and the adenoviral major late promoter (obtained from plasmid pAD2beta, described in Norton and Coffin, Mo Cell Biol. 1985, 5, 281) and a single NotI cloning site followed by SV40 late termination transcription signals and poles A signal (obtained from plasmid pCMVbeta, see MacGregor and Caskey, Nucl. Acids. Res. 1989, 17, 2365). In addition, the vector pEAG347 contains a plasmid backbone derived from the vector pUC19 and a dhfr obtained from pSV2dhfr for MTX selection and amplification in transfected CHO cells.

Plasmid pJCO70.14 was generated in two steps. First, a fragment encoding the human neublastin preproform was isolated from plasmids pUbilZ-NBN using pCR with oligonucleotides KD2-824 5 'AAGGAAAAAAGCGGCCGCCA TGGAACTTGG ACTTGGAGG3' (SEQ ID NO: 31), KD2-825 5'TTGCGCC 32) and PFU polymerase. In the second step, partial Not-1 digestion of plasmids pJC069 was performed to produce a 685 bp fragment (containing the neublastin gene), which was cloned into the Not-1 restriction site of plasmids pEAG347 to give plasmids pJC070.14. The transcription of the neublastin gene in plasmids pJC070.14 was driven by the adenoviral major late promoter.

Establishment of neublastin expressing CHO cell lines

200 µg of vector pJC070.14 was linearized by restriction endonuclease digestion with Mlu-1. The DNA was then extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1) and precipitated with ethanol. Linearized DNA was resuspended in 20 mM Hepes pH7.05, 137 mM NaCl, 5 mM Kel, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose (HEBS) and introduced into ~ 4E7 CHO dukx B1 (dhfr-) cells ( p23) by electroporation (280V and 960 μΓ). After electroporation, the cells were further cultured in C + + conditioned Eagle's medium (MEM) supplemented with 10% embryonic bovine serum (FBS) for 2 days. The cells were then trypsinized and not plated on a 100 mm plate (100,000 cells / plate) in aMEM culture medium (which does not contain ribo- and deoxyribonucleotides) supplemented with 10% dialyzed FBS for 5 days. The cells were then plated in 100 mm diameter plates at a density of 100,000 cells per plate and harvested in 200 nM metatroxate. Resistant colonies were isolated and plated on 6-well plates. The conditioned culture media from each clone was assayed using a specific assay suitable for the determination of neublastin as described below. 12 clones expressing the highest amount of neublastin were introduced into bottles

T162 and subsequently tested. Figure 10 shows that CHO cell lines produced neublastin in the range of 25 to 50 ng / ml.

Ternary complex test suitable for neublastin

The presence of neublastin in the culture medium of CHO cell line supernatants was tested using the modified form of the ternary complex assay described by Sanicola et al. Why. Nati. Acad, Sci USA 1997, 94, 6238).

In this assay, GDNF-like molecules are evaluated for their ability to mediate binding between the extracellular region of RET and various GFRα1, GFRα2, and GFRα3 receptors. Soluble forms of RET and co-receptors have been formed as fusion proteins. Fusion protein between rat RET extra-cellular region and placenta! alkaline phosphatase (RET-AP) and a fusion protein between the extracellular region of rat GFRα (described in WO 9744356, Nov. 27, 1997) and the human IgG1 Fc region (rGFRα1-Ig), as described in Sanicola et al., Why. Nati. Acad. Sci. USA 1997 94 6238.

To generate a fusion protein between the mouse GFRα3 extra-cellular region and the human IgG1 Fc region (mGFRα3-Ig), the DNA fragment encoding amino acids 1 to 359 of mouse RETL3 was ligated with a fragment containing the human IgG1 Fc region and cloned into pEAG347 expression vector to create a plasmid pGJ144. Plasmid pGJ144 was transfected into Chinese Hamster Ovary (CHO) cells to produce a stable fusion protein producing cell line, which was purified on an immunoaffinity column on protein A Sepharose using standard methods. If a GDNF-like molecule can mediate co-receptor binding to RET in this assay, then the RET-AP fusion protein is stored on the plate and the amount that is stored is measured using chemoluminescent alkaline phosphatase.

substrate suitable for

Microlite-1 ELISA plates (Dynex Technologies) were coated with 1 μρ / πιΐ goat antibodies specific for human Fc in 50 mM bicarbonate / carbonate at pH 9.6 for 16 hours. Plates were emptied and filled with 300 μΐ of a 1% I-block solution (Tropix) in TBS / 0.5% Tween-20 (TBST) for 1 hour. After washing three times with TBST, the wells were filled with 100 µg rGFRalIg or mGFRα3-Ig at a concentration of 1 µg / ml diluted in conditioned culture medium, derived from culturing 293 EBNA cells expressing the RET-APOD fusion gene. 100 μΐ of conditioned media originating from neublastin CHO clones was then added to the upper well of the well column and two-fold serial dilutions were made in each well well and incubated for 1.5 hours at room temperature. The plates were then washed three times with TBST and two times with 200 mM Tris pH9.8, 10 mM MgCl 2 (buffer).

CSPD). The wash solution was then replaced with 425 μΜ CSPD (Tropix) in CSPD buffer containing 1 mg / ml Saphire chemiluminescence enhancer and incubated for 30 minutes at room temperature. Chemoluminescence was measured using a Dynatech luminometer.

In initial experiments, it was investigated whether neublastin produced by CHO cell lines mediated the binding of GFRα1 or GFF.alpha. To the extracellular region of RET. As shown in Figure 11, conditioned CHO cell culture medium clone # 53 showed a strong signal in the ternary complex assay when the rGFRα1-Ig fusion protein was included indicating that neublastin binds to GFRα3 but not to GFRα1. This evolution is different from neublastin derived from GDNF as shown in Figure 11, with GDNF binding to

But not on GFRα3. No signal was observed with the fusion protein co-receptor when the conditioned medium from the parent CHO cell line or culture medium was tested.

To quantify the potency of neublastin expression in CHO cell lines, a standard curve starting at 1 ng / ml was prepared using rGFRal-Ig and GDNF. Neublastin concentrations for the different cell lines were then calculated using a standard curve. The amounts of neublastin produced by the five CHO cell lines are shown in Figure 10. Because this estimation depends on the tested assumption that the binding affinity between GDNF and GFRα is similar to the binding affinity between neublastin and GFRα3, the amount being approximate.

Neublastin analysis from CHO cell line supernatants

To further analyze the neublastin produced by CHO cell lines, the protein was extracted from the culture medium using the GFRcc3-Ig fusion protein and analyzed by Western blotting using polyclonal antibodies raised against neublastin peptides.

In the first experiment, neublastin was extracted using mGFRα, 3-Ig, which is attached to the beads of sepharose. mGFRα3-Ig was attached to Sepharose beads using conditions recommended by the manufacturer (Pharmacia Inc). 100 μΐ mGFRα3-Igsepharose was added to the conditioned culture medium samples from the CHO cell line serving as a negative control or from neublastin produced by cell line # 16 with a volume of 1 ml. The suspensions were incubated for two hours with stirring. Each suspension was centrifuged and the supernatant was removed, followed by three washes with 1 ml of 10 mM HEPES, 100 mM NaCl, pH7.5. Each resin was resuspended in 100 μΐ 2x concentrated reducing buffer and heated to 100 ° C for 5 minutes. 20 μΐ of sample supernatant buffer and 10 μΐ of molecular weight standard (FMC) were applied to each well of 10 to 20% SDS-PAGE gel (Owl Scientific). The gel was electrophoresed at a constant current of 40 mA for 72 minutes.

For Western blot analysis, the protein was electroblotted onto a nitrocellulose membrane (Schleicher and Schuell) on a Hofer Scientific in 10 mM CAPS, 10% methanol, 0.05% SDS, pH11, 2 (for 45 minutes at a constant current of 400 mA). After transfer, the nitrocellulose filter was removed from the cassette and the molecular weight markers were visualized by staining with a solution of 0.1% Ponceau S in 1% acetic acid for 60 seconds. The membrane was cut into two sections and excess dye was removed by gentle stirring in distilled water. The membranes were blocked in 2% non-fat dry milk in TBS overnight at 4 ° C. The membranes were incubated individually with two affinity purified anti-neublastin peptide antibodies at a concentration of 1.0 μρ / ml in 2% non-fat dry milk in TBS.

The membranes were washed three times for 10 minutes at room temperature

TBS-Tween and incubated with anti-rabbit

IgG-HRP conjugate (Biorad) at a 1: 5,000 dilution for 30 minutes.

The membranes were washed three times for 10 minutes in TBS-Tween and developed with ECL substrate (Amersham).

As shown in the figure

12. Both types of antibodies were detected in proteins extracted from neublastin produced by the cell line

CHO, specific lanes (lanes 2 and 4).

These bands were compared to those seen in proteins extracted from the cell line that served as a negative control (lane 1).

The molecular weight of the lower species is approximately 13 kD and probably represents the mature region of neublastin that results from pro-domain withdrawal. This cleavage can occur after any of the three residues of the Arg -__ (for example, -RXXR> 1 · -) preproneublastin protein to produce either 140 ak or 113 ak as shown in SEQ ID NO: 10, 11 or 12. that the predicted molecular weights of the unmodified (that is, the lack of post-translational modifications) of the neublastin polypeptides

NBN140 (SEQ ID NO: 10), NBN116 (SEQ ID NO: 11), and NBN113 (SEQ.

ID NO: 12) are 14.7 kD, 12.4 kD and 12.1 kD. Further analysis is required to confirm the structure of these species as well as other neublastin-specific bands.

In a second experiment, neublastin was extracted with hGFRα3-Ig on ELISA plates. To generate a fusion protein between the extracellular domain of the human hGFRoc3 DNA fragment (described in WO97 / 44356, November 27, 1997) and the human IgG1 Fc domain (hGFRα3-Ig), the DNA fragment encoding amino acids 1 to 364 of human GFRα.3 was ligated to a fragment that contains the human IgG1 Fc region and cloned into the CH269 expression vector described by Sanicola et al., Proc. Nati. Acad. Sci USA 1997, 94, 6238). The fusion protein encoded by this plasmid was transiently expressed in 293 cells with Epstein-Barr virus encoded nuclear antigen (EBNA) and purified on an immunoaffinity column on protein A sepharose using standard methods.

Six wells of a 96 well plate were coated overnight at 4 ° C with goat anti-human IgG (Fcγ-specific fragment, Jackson Immunologics) at 25 mg / ml in PBS (300 ml / well) · Wells were blocked for 1 hour at room temperature 400 ml of 1% BSA in PBS. After 3 hours of washing with PBST (PBS + 0.05% Tween 20), 300 ml hGFRα3-Ig (10 mg / ml in PBS containing 0.1 0 BSA) was added to each well. The plate was incubated for 1 hour at room temperature and the plate was shaken vigorously (200 rpm) to maximize binding. The wells were then emptied and washed three more times with PBST. To each of the three wells was added 250 mL of conditioned medium from the CHO cell line serving as a negative control or neublastin producing CHO # 25 cell line. The plate was incubated for 3 hours at room temperature and mixed gently (300 rpm).

The wells were then washed twice with PBST.

added 25 ml of non-reducing buffer. Laemli was stirred for 5 minutes to wash out

300 rpm). Content transferred

Into the first well and the plate were rapidly bound proteins (1 in the minor well and the protein bound procedure was repeated to wash out in the second or third well. After addition (final concentration is analyzed for 5% polyacrylamide minutes and gel).

In the case of western blot analysis for nitrocellulose.

proteins were transferred

The membrane was blocked and probed with 5% non-fat dry milk, PBST, and washed

PBST.

Neublastin is reacted with

R31) generated against two polyclonal antibodies by neublastin peptides (v (R30 and 1 gg / ml)) tested by electrochemoluminescence followed by reaction with goat anti-rabbit antibodies conjugated to HRP

13, five neublastin-specific bands (BioRad) were detected in the extracted neublastin.

proteins

As shown in FIG.

from a CHO producing cell line (lane 2). The two lower bands are very similar to those seen in Figure 12. The lower band is likely to represent the mature region of neublastin formed after pro-domain cleavage.

Subsequent analysis (data not shown) of the bars in Figure no.

shows that deglycosylation with the PGNase F band, which corresponds to a molecular weight of 18 kD, reduces the molecular weight of the band to the size corresponding to the lowest band on the gel in Figure 13. This indicates that neublastin can be produced as a glycosylated protein in mammalian cells.

Neublastin expression in E. coli.

In order to express the neublastin gene in E. coli, lower GC syngens and preferred E. coli codons were constructed. Syngen was cloned into two vectors pET19b and pMJB164, which are derivatives of pET19b. The pET19b construct is shown in Figure 14. In this construct, the sequence encoding the mature neublastin region (NBN113) is directly fused to the initiating methionine. The construct with pMJB164 is shown in Figure 15. In this construct, the mature region of neublastin is fused to a histidine tag (i.e., 10 histidines) separated by an entarokinase cleavage site. The initiating methionine lies before the histidine tag.

Nucleotide sequence encoding neublastin in Figure 14

ATGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTGTCG TCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACCGTTCCGA CGAACTAGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCACGTTCTCCG CATGATCTATCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGAČCGCCGCCG GGATCTAGACCTGTATCTCAACCTTGTTGTAGACCTACTAGATACGAAGCAGTAT CTTTCATGGACGTAAACTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGC ATGTGGCTGTCTAGGATGATAATAG [SEQ ID NO: 29]

Nucleotide sequence encoding neublastin with his-tag in Figure 15

ATGGGCCATCATCATCATCATCATCATCATCATCACTCGAGCGGCCATATCGACG ACGACGACAAGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGGACGT GGCTGTCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACAC CGTTCCGACGAACTAGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCAC GTTCTCCGCATGATCTATCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGACC GCCGCCGGGATCTAGACCTGTATCTCAACCTTGTTGTAGACCTACTAGATACGAA GCAGTATCTTTCATGGACGTAAACTCTACATGGAGAACCGTAGATAGACTATCTG CAACCGCATGTGGCTGTCTAGGATGATAATAG [SEQ ID NO: 30]

Example 6: Effect of Neublastin on Rat Germ Dopaminergic Neurons Survival and ChAT Activity

In this series of experiments, the effect of conditioned media from Neublastin-producing HiB5pUbilzNBN22 cells was determined as described above.

Preparing cultures * ·· »

Ventral mesencephalon or spinal cord was obtained from E14 rat embryos in chilled Hanks buffered salt solution (HBSS). Tissue pieces were incubated in sterile filtered 0.1% trypsin (Worthington) and 0.05% DNase (Sigma) in HBSS at 37 ° C for 20 minutes. Tissue pieces were then washed four times in HBSS + 0.05% DNase and ground using a 1 ml automatic pipette. The suspension was then centrifuged at 600 rpm. for 5 minutes and the pellet was resuspended in serum-treated culture medium (SCM, DMEM with 10% fetal calf serum). Total cell counts were determined with tryptophan blue staining and plated at 100,000 cells / cm ² in a eight-well (Nunc) chamber coated with poly-L-lysine suitable for dopaminergic neuron estimation or at 200,000 cells / cm ² on plates with 48 wells (Nunc) for ChAT activity measurement. Cells were incubated in SCM in an atmosphere of 5% CO ₂ /95% O ₂ and 95% humidity at 37 ° C for 24-48 hours and then the medium was changed to serum free medium (SEM) and neurotrophic factors were added.

Cells suitable for determining the survival of dopaminergic neurons were left for 5 days in SFM with trophic factor and then fixed for 5 minutes in 4% PFA and stained by immunohistochemistry to determine the presence of tyrosine hydroxylase.

Cells suitable for determining ChAT activity were left with SFM for three days and then lysed in HBSS and 0.1% Triton X-100 and immediately frozen on dry ice until measurement of Chat activity.

To add a trophic factor:

The conditioned medium was obtained from non-transfected HiB5 control cells or neublastin-producing HiB5 cells (HIB5βUb from NBN22) or GDNF (HIB5βUb from GDNF-L17). HiB5pUbilzNBN22 produces approximately 20 ng GDNF per 24 hours out of 10 ⁵ cells as determined by GDFN-ELISA for 99

9 9

9 999

9 4> 9

9 9 9

99 • 9 ·

99

99 • 9 99

99

9 · r

999

99 • 99 ·

99

99999 conditioned medium that was collected from cell culture. Cell lines were cultured overnight with DMEM and 1% FCS and the supernatant was collected and stored at -20 ° C until use. Supernatants were diluted 1:50 in SFM and then added to the cells. Separate wells were then treated with supernatant derived from control HiB5 cells (1:50) and purified recombinant rat GDNF (from 0.03 to 10 ng / ml).

The results of these experiments are shown in Figure 4. Figure 4A to 4C are illustrations of the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the survival of cultured germinal dopaminergic ventral mesencephalic neurons and ChAT activity in cholinergic cranial nerve motor neurons in serum-free medium as described in Example 5.1 above.

Figure 4A shows a dose response curve for recombinant GDNF to ChAT activity (dpm / hour) measured in DIV5 in serum free cultures that were generated from E14 ventral midbrain (i.e., HiB5, GDNF 0.03 ng / ml, GDNF 0.1 ng / ml, GDNF 0.3 ng / ml, GDNF 1 ng / ml, GDNF 10 ng / ml, GDNF 100 ng / ml).

Figure 4B shows ChAT activity (dpm / hour) measured at DIV5 in serum free cultures that initially originated from E14 ventral midbrain. Diluted conditioned culture medium derived from the cultivation of Neublastin (neublastin) or HiB5GDNFL-17 producing GDNF (GDNFL-17) cells as shown in the figure (i.e. neublastin 1:10, neublastin 1) was added: 50, GDNF 1-17 1: 5C).

Figure 4C shows the number of immunoreactive cells with tyrosine hydrolase per well (TH + cell count / well) in DIV7 in serum-free cultures that originated from E14 rat ventral midbrain. Diluted conditioned culture medium from either untransfected HiB5 cells (HiB5) or HiB5pUbilzNBN22 cells producing neublastin (neublastin) or recombinant GDNF at various concentrations was added as shown (i.e., HiB5 1:10, HiB5 1): 40, GDNF 0.1 ng / ml, GDNF 10 ng / ml, GDNF 100 ng / ml and neublastin 1:40).

The conditioned culture medium obtained from 1:40 diluted neublastin-transfected HiB5 cells significantly increases the number of TH immunoreactive cells per well compared to control (untransfected) cells.

HiB5 at equivalent and lower dilutions (1:10 and 1:40) (shown, for example, in Figure

4B). The increase in TH immunoreactive cells is comparable to the increase observed at the maximum concentration of GDNF (10 ng / ml). This indicates that neublastin secreted into the culture medium has an effect on the survival of the dopaminergic neuron population from rat germinal ventral midbrain.

Unlike

GDNF secreted from transfected cells

Hib5 conditioned medium from HiB5 cells transfected with neublastin showed no effect on another population of nerve cells in the same culture of cholinergic neurons (shown, for example, in Figure 4A).

Example 7: Effect of Neublastin on the Survival of Cultures in a Thin Layer of Porcine Gland Dopaminergic Neurons.

In this experiment, the effect of co-culture of neublastin-producing HiB5pUbilzNBN22 cells with a culture of medium brain neurons in a thin layer of porcine embryos was examined.

Preparation of cultures:

Ventral midbrain (VM) was isolated from porcine embryos (E28, n = 12) under sterile conditions. It was cut into 400 µm slices and placed in chilled Gey's equilibrium salt solution (GiBco) with glucose (6.5 mg / ml). Tissue slices were cultured by the phase interface method of L. Stoppini, P.A.

Buchs, D. Muller, and J. Neurosci. Methods 1991 37 173-182).

The strips were placed on a semi-porous membrane (Millipore, 0.3 µπι, 8 slices per membrane corresponding to one VM) placed as inserts on 6-well plates (Costar) with serum-containing culture medium (Gibco BRL). Each well contained 1 ml of culture medium (50% Optimem, 25% horse serum, 25% Hank's equilibrium salt solution (all from Gibco) supplemented with D-glucose to a final concentration of 25 mM. On day 0, 7,000 tissue slices were applied. transfected HiB5pUbilzNBN22 cells (neublastin) or 7000 untransfected HiB5 cells (control) Co-cultures were first cultured in an incubator at 33 ° C for 48 hours, allowing Hib5 cells immortalized with a temperature-sensitive oncogene to multiply and then placed in the incubator. at 37 ° C, where cell differentiation occurred The culture medium was changed twice a week, neither antimitotic nor antibiotic was used at any stage.

Determination of dopamine by HPLC

Culture media was collected on days 12 and 21 and the presence of dopamine was analyzed by HPLC with electrochemical detection (W.N. Slooth, J.B.P. Gramsbergen, J. Neurosci Meth. 1995 60 141-49).

Tissue processing and immunohistochemistry

On day 21, cultures were fixed in 4% paraformaldehyde in phosphate buffer for 60 minutes, dehydrated in 20% sucrose solution for 24 hours, frozen, cut into 20 μπι (4 series) sections in a cryostat, and coated. on gelatin-coated microscope slides. One series of sections was immunologically stained to detect the presence of tyrosine hydrolase (TH). Sections were washed in 0.05 M Tris-buffered saline (TBS, pH7.4) containing 1%

Triton X-100 for 3x15 minutes and incubated with 10% bovine serum (FBS, Life Technologies) in TBS for 30 minutes. The tissue was then incubated for 24 hours at 4 ° C with monoclonal mouse anti-TH antibodies (Boehringer Manheim) diluted 1: 600 in TBS with 10% FBS. After washing in TBS with 1% Triton X-100 for 3x15 minutes, sections were incubated for 60 minutes with biotinylated anti-mouse IgG (Amersham) diluted 1: 200 in TBS with 10% FBS. Sections were then washed in TBS with 1% Triton X-100 (3x15 minutes) and incubated for 60 minutes with streptavidin peroxidase (Dako) diluted 1: 200 in TBS with 10% FBS. After washing in TBS (3x15 minutes), bound antibodies were visualized by treatment with 0.05% 3,3diaminobenzidine (Sigma) in TBS containing 0.01% H 2 O 2.

Finally, the sections were dehydrated in alcohol, cleaned with xylene and covered with a slide from the Eukitt set.

Cell count and morphometric analysis

Determination of the number of TH immunologically responsive neurons was performed using light field microslopy (Olympus). Only cells showing intense staining with a well-preserved cell structure and a separate nucleus were counted. The estimate was based on the number of cells in every fourth section using an x20 lens. Cell counts were adjusted by Abercrombie counting twice (M. Abercrombie, Anat. Rec. 1946, 94, 239-47), using the average core diameter in Th-ir neurons (6.6 ± 0.2 μιη) , N = 30). The nucleus size was estimated using a neuronal search system (Neurolucida, MicroBrightField, Inc).

The results of these experiments are shown in Figure 5. Figures 5A to 5C are illustrations of the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the function and survival of dopaminergic neuronal cultures of ventral porcine germs co-cultured with the middle brain either with cells

HiB5pUbilzNBN22 (neublastin) or with HiB5 cells (control cells) as described above. 5A and 5B illustrate dopamine released into the culture medium in DIV12 (dopamine / pmol / ml) - day 12) and DIV21 (dopamine (pmol / ml) - day 21). Figure 5C illustrates the number of cells in culture that immunologically react with tyrosine hydroxylase (THir cell in culture) in DIV21.

On day 12, HPLC analysis showed that the culture medium of HiB5-neublastin cultures contained 84% more dopamine compared to the HIB5-C cultures (Figure 5A). At day 21, this difference was 78% (Figure 5B) and the cell count shows that HiB5-neublastin cultures contained 66% more neurons immunologically reactive with tyrosine hydroxylase than the HIB5-C cultures (P <0.05) (Figure # 5). 5C). This indicates that neublastin secreted from the HiB5pUbilzNBN22 clone has the potential to affect the survival of porcine embryonic dopaminergic neurons.

Example 8: Survival of dorsal root ganglion cells in serum free culture medium

This example shows the neutrophic activity of neublastin polypeptides as compared to known neurotrophic factors.

Female mice waiting for pups were sacrificed by cervical dislocation. The embryos were conditioned for the culture as follows.

Electrolytically sharpened tungsten needles were used to cut the dorsal root ganglia from the indicated stage of C57% B16 mice (Mollegaard Breeding, Denmark). The ganglia of the germs were incubated for 5 minutes at 37 ° C with 0.05% trypsin (Gibco / BRL) Hanks' equilibrium salt solution without calcium and magnesium. Postnatal ganglia were treated with collagenase / dispase at a concentration of 1 mg / ml for 30 to 45 minutes and then with trypsin / DNase at a concentration of 0.25% for 15 minutes. After removal of the trypsin solution, the ganglia were washed once with 10 ml of DMEM containing 10% heat-inactivated horse serum and gently triturated over fire-sterilized

With a pasteur pipette, a single cell suspension is formed.

These cells were plated on 24-well plates (Nunc), which had been pre-coated with polyornitine (0.5 mG / ml, overnight) and laminin (20 mg / ml for 4 hours, Gibco / BRL). Neurons were incubated at 37 ° C in a humidified incubator at 5% CO ₂ in a defined culture medium containing Hams F14 supplemented with 2 mM glutamine, 0.35% bovine serum albumin, 60 nG / ml progesterone, 16 mg / ml putrescine 400 ng / ml L-thyroxine, 38 ng / ml sodium selenite, 340 ng / ml triiodothyronine, 60 mg / ml penicillin and 100 mg / ml streptomycin.

After 48 hours of incubation, the nerons were clearly distinguished according to their bipolar morphology by phase contrast optics. The percent survival of neurons in the absence or presence of trophic factors (added to the culture medium before the neurons are plated at 10 ng / ml) or conditioned medium from Neublastin-producing HiB5pUbilzNBN22 cells was estimated by determining the number of neurons in wells after 48 hours. kutlivace.

The results of these experiments are shown in Figure 9, where:

represents a control experiment (in the absence of factors) represents experiments in the presence of GDNF, represents experiments in the presence of neurturin, represents experiments in the presence of neublastin according to the invention,

E12 represents data from experiments performed using DRG cells isolated from embryos on day 12,

E16 represents data from experiments performed using DRG cells isolated from embryos on day 16,

PO represents data from experiments performed using DRG cells isolated on the day of birth, ♦ • 999 ··· 9 9 9 · 9 «

99999 «9 9 99 * · · 999 9 9« 9 9

* .. ** .. ·.

P7 represents the data of experiments performed using cells isolated 7 days after birth and

P15 represents data from experiments performed using DRG cells isolated 15 days after birth.

These results clearly show that the neurotrophic factor of the invention shows activities comparable to or even higher than those of well established neurotrophic factors.

Example 9: Effects of Neublastin on Nigral Dopamine Neurons in Vivo

To test the ability of neublastin (neublastin) to protect adult adult nigral dopamine neurons (DA) against 6-hydroxydopamine-induced degeneration, a rat model of Parkinson's disease (Sauer and Oertel, Neuroscience, 1994, 59, 401-415) and transfer was used. lentiviral neublastin gene.

Lentivir production

To produce a lentiviral transfer vector encoding neublastin, pHR'-neublastin, a 1331 bp BamHI fragment from the neublastin cDNA was subcloned into the BamHI / BglII restriction site of vector pSL30 (Invitrogen). A 1519 bp BamHl / XhoI fragment was isolated from this construct and ligated to a BamHl / XhoI pHR 'restriction site that carries a posttranslational fragment of the steppe moth hepatitis virus (Zufferey R, Donello JE, Trono D, Hope TJ). : Woodchuck hepatitis virus posttranscriptional regulators element enhancements expression of transgenes delivered by retroviral vectors, "J. Virol. 1999 73 (4) 2886-2892). To generate pHR-GDNF, a 701 bp BamHI / XhoI fragment from pUbilz-GDNF was ligated to a BamHI / XhoI restriction site pHR '.

Lentiviral vector production is described in the publication

Zufferey R.,

Nagy D.,

Mandel Rj, Naldini L.,

Trono D .:

"Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo, Nat. Biotechnol. 1997, 15 (9) 871-875. Transfer constructs and helper plasmids pR8.91 and pMDG were cotransfected into 293T cells. Virions released into the culture medium were collected 48 and 72 hours after transfection. To increase virus concentration, the culture medium was centrifuged for 1.5 hours at 141,000 g and the pellets were dissolved in DNEM. The control titer in 293T cells carrying the green fluorescent protein (GFP) gene is 10 ⁸ transform units (TU) per milliliter, as determined by GFP fluorescence. The RNA slot transfer method (von Schwedler U, Song J, Aiken C, Trono D) was used to determine the viral particle titer: "Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells, J. Virol. 1993, 67, (8) 4945-4955). There are 10 times fewer particles in the GDNF and neublastin supernatant compared to the GFP supernatant.

Surgical procedures

All animal procedures were performed in accordance with the Ethics Committee for the use of laboratory animals at the University of Lund.

A total of 21 young adult Sprague-Dawley rats (B and K Universal, Stockholm, Sweden) were used. Animals were maintained in a 12 hour light and dark cycle with free access to food and water. Retrograde labeling and 6-OHDA lesions were performed 3 weeks prior to lesion formation (Sauer and Oertel, Neuroscience 1994, 59, 401-415). Under equithesin anesthesia (0.3 ml in 100g), rats were bilaterally injected with a 0.2 μΐ 2% solution (dissolved in 0.9% NaCl) of a retrograde fluorine-gold detection additive (FG, Fluorochrome, Inc. Englewood, CO). ). Injections were made using a 2 μΐ Hamilron syringe at the coordinates: AP = +0.5 mm, HL = ± 3.4 mm relative to bregma, DV = -5.0 relative to bre ··· · • · · · 84 ····· 84 ·········· · · · · dů k k dů dů dů dů dů dů dů dů dů dů dů dů dů dů a dů dů a a dů dů dů dů a a a a a a a a a a Furthermore, after five minutes of injection, 0.05 μΙ / min was introduced before the needle was withdrawn.

days for FG injection, animals received a total of 5 doses (1 microliter per dose) of the lentiviral vector carrying the green fluorescent protein (GFP), neublastin, or GDFN gene. Four doses were introduced into the striatum using two needles according to the following coordinates AP = +1.0 mm, ML = -2.6 mm, DVi = 5.0, DV2 = -4.5 mm and AP = 0.0 mm, ML = -3.7 mm, DVi = -5.0 mm, DV ₂ = -4.5 mm. Supranigral insertion was performed at AP = -5.2 mm, ML = -2.0 mm, DVi = -6.3 mm. Teeth spacing set to -

2.3 mm.

days after retrograde labeling and 7 days after lentiviral injections, the animals were re-anesthetized and given a 10 μ Hamilton Hamilton syringe with a single dose of 20 μg 6-OHDA (Sigma, calculated as the free base and dissolved in 3 μΐ ice-cold saline supplemented with 0 (02% ascorbic acid) introduced into the right striatum at the same position as the FG dose. The injection rate was 1 μΙ / min, the needle was withdrawn after another three minutes.

Tissue Processing:

days after 6-OHDA administration, the animals were deeply anesthetized with chloral hydrate and transcardially perfused with saline (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were sectioned and fixed for 3-4 hours and transferred to 25% sucrose / 0.1 M phosphate buffer for 48 hours. Five series of 40 μιη stratium and nigra (SN) substrates on a freezing microtome were cut.

Quantitative estimation of dopaminergic neurons in SN

The number of FG-labeled neurons in SN was estimated by a blinded observation device as described by Sauer and

Oertel, Neurcscience 1994, 59, 401-415). Three were used. 4 < * &quot; &quot; &quot; &quot; &quot; B consecutive sections arranged around the center of the media terminal nuclei of the optical tract (MTN, -5.3 in the atlas according to Paxinos and Watson (1997) ) and all labeled / stained neurons placed laterally to the MTN at 40X magnification (n = 6-7 per group) were counted. FG-labeled neurons were included if bright fluorescent at epiluminescence at 330 nm, exhibited a neuronal profile and prolonged at least one neuritic process.

The lesions were injected with lentivir carrying GFP on the lesion side. The number of FG-positive nigral neurons decreased to 18% of that obtained in the intact section. In contrast, animals injected with lenti-neublastin show almost complete protection for FG-positive nigral neurons (89%). This protection is as effective as in LentiGDNF-treated animals, where 87% of retrograded neurons were retained on the lesion side. This suggests that neublastin is a potent survival factor for adult nigral dopamine neurons from lesions and that its activity is the same as GDFN.

Figure 6 is an illustration of the effect of in vivo neublastin produced by lentivir on nigral dopamine neurons. SN in female Sprague Dawley rats was labeled retrogradely with fluorolate (FG) 3 weeks prior to a single injection of 6-hydroxydopamine (β-OHDA) into the right striatum. One week prior to the introduction of β-OHDA, animals were injected with lentoviral vectors that express neublastin (neublastin), GDNF (GDNF), or green fluorescent protein (GFP) as shown in the figure. 21 days after 6-OHDA injections, the number of FG-labeled neurons on both sides of the striatum was determined. The figure shows the percentage (% FG lesions / intact) of FG-labeled neurons at the lesion site (right side) versus intact (left) side of the striatum in three groups of animals.

Example 10: Production of antibodies

4 · ♦ ······ · 4 4 · · 4 • 444 4 4 4 4 4

86 4 4 4 4 • 44 · 44 To prepare antibodies against neublastin, two rabbits were immunized with either peptide 1: CRPTRYEAVSFMDVNST (amino acids 108 to 124 of SEQ ID NO: 9) or peptide 2: ALRPPPGSRPVSQPC (amino acids 93 to 107 of SEQ ID NO: 9) conjugated to a carrier protein at three intervals weeks. At weeks 0, 3, 6 and 10, they were immunized for each peptide

two rabbits a week and

The rabbits were bled. Blood from the second donation was purified on a peptide affinity column.

The antibodies were named Ab-1 and Ab-2 in accordance with the peptide.

Western blot:

2x10 ⁶ HiB5 cells were stably transfected with neublastin cDNA (Hib5pUbilzNBN22) or untransfected cells

HiB5 were incubated overnight in nitrogen-free serum culture medium (GIBCO).

The concentration of the cutter medium was increased in a small concentrator using a membrane separating molecules of molecular weight kDa (Millipore, Bedford, MA).

Concentrated samples were added to 5x Laemmli concentrated sample buffer and heated to 95 ° C for 5 minutes. Samples were separated by SDS 15% polyacrylic gel electrophoresis and transferred to PVDF membranes. The remaining protein binding sites were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20. Membranes were incubated overnight with neublastin antibodies by incubation with horseradish peroxidase-conjugated secondary anti-rabbit or mouse IgG antibodies (1: 2000).

Immunological staining of chemolumimiscence Plus was visualized using enhanced (ECL +) according to the manufacturer's instructions (Amersham). The results of these experiments are shown in Figure 3 and described in Example 5.

Rabbit polyclonal antibodies against the following peptides were also generated using standard methods:

peptide R27: GP3SRARAAGARGC (amino acids 30 to 43

SEQ ID NO: 9)

peptide R28: LGHRSDELVRFRFC (amino acids 57 to 70 SEQ ID NO: 9), peptide R29: CRRARSPHDLSL (amino acids 7 4 to 85 SEQ ID NO: 9), peptide R30: LRPPPGSRPVSQPC (amino acids 94 to 107 SEC ) ID NO: 9) a peptide R31: STWRTVDRLSATAC (amino acids 123- 136 SEQ ID NO: 9) · Only peptides R30 and R31, which are relatively near C-

ends, recognize the denatured protein under western blotting conditions.

WO 00/01815

-Λ

• · · · · · · fc ^'of PCT / bK ^ / * 00384 ·

SEQUENCE LISTING <160> 33 <170> PatentIn Ver. 2.0 <210> 1 <211> 865 <212> DNA <213> Homo sapiens

<221> CDS <222> (120). . (719) <220}

<221> 5 'UTR <222> (1). . (119) <220}

<221> 3'UTR <222> (721) .. (865) <220>

<221> sig_peptide <222> (120) .. (179) <220>

<221> mat_peptide <222> (405) .. (719) <220>

<221> misc_struccure <222> (661). . (663) <223> CARBOHYD: Glycosylated Asparagine at Asn87

<221> mission — struc-ure <222> (426). . (623) <223> DISULFID - Cys8-Cys73 disulfide bridge <220>

<221> misc_struccure <222> (507). . (707) <223> DISULFID: Cys35-Cys01 disulfide bridge <220>

<221> misc_structure <222> (519) .. (713) <223> DISULFID: Cys39-Cysl03 Disulfide Bridge

<221> misc_structure <222> (616) .. (619) <223> DISULFID: Cys72-Cys72

SUBSTITUTE SHEET (rule 26)

to <400> 1 ctaggagccc tgatctcagc ccgaggacag cccctccttg aggtccttcc 60

WO 00/01815 to I to

PCT7DK99 / 00383 * *

119 tccccaagcc cacctgggtg ccctctttct ccctgaggct ccacttggtc tctccgcgc

atg Met -95 cct For gcc Ala ctg Leu rgg ccc acc Thr ctg gcc Leu Ala get Ala ctg get Leu Ala -85 ctg Leu ctg agc agc Ser -80 167 Trp Pro -90 Leu Ser gtc gca gag gcc ccc ctg ggc tcc gcg ccc cgc agc cct gcc ccc cgc 215 Wall Ala Glu Ala Ser Leu Gly Ser Ala For Arg Ser For Ala For Arg -75 -70 -65 gaa ggc ccc ccg cct gtc ctg gcg tcc ccc gcc ggc cac ctg ccg ggg 263 Glu Gly For For For Wall Leu Ala Ser For Ala Gly His Leu For Gly -60 -55 -50 gga cgc acg gcc cgc tgg tgc agt gga aga gcc cgg cgg ccg cgc cgc 311 Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg For Arg Arg -45 -40 -35 aga cac ttc tcg gcc cgc gcc ccc gcc gcc tgc acc ccc atc tgc tet 359 Arg His Phe Ser Ala Arg Ala For Ala Ala cys Thr For Ile Cys Ser -30 -25 -20 tcc ccg cgg gtc cgc gcg gcg cgg ctg ggg ggc cgg gca gcg cgc tcg 407 Ser For Arg Wall Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser -15 -10 -5 -1 1 ggc agc ggg ggc gcg ggg tgc cgc ctg cgc tcg cag ctg gtg ccg gtg 455 Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gin Leu Wall For Wall 5 10 15 Dec cgc gcg ctc ggc ctg ggc cac cgc tcc gac gag ctg gtg cgt ttc cgc 503 Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Wall Arg Phe Arg 20 May 25 30 ttc tgc acc ggc ccc tgc ccg cgc gcg cgc tet approx cac gac ctc agc 551 Phe Cys Thr Gly Ser Cys For Arg Ala Arg Ser For His Asp Leu Ser 35 40 45 ctg gcc agc cta ctg ggc gcc ggg gcc ctg ega ccg ccc ccg ggc tcc 599 Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For For For Gly Ser 50 55 60 65 cgg ccc gtc agc oag ccc tgc tgc ega ccc acg cgc tray gaa gcg gtc 647 Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg Tyr Glu Ala Wall 70 75 80 tcc ttc atg gac ctc aac agc acc tgg aga acc gtg gac cgc ctc tcc 695 Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall Asp Arg Leu Ser 85 90 95 gcc acc gcc tgc cgc tgc ctg ggc tgagggctcg ctccagggct ttgcagactg 749 Ala Thr Ala Cys Gly Cys Leu Gly

100 105

SUBSTITUTE SHEET (rule 25)

WO 00/01815 - 10 - 3 - ·· · · ♦ · · ♦ · · · · • · · · • · · · • · · · agagtcccac • · ···· · · • · · · 9 * · · In · · · · · PCY7DK55 / 00383 · * · gacccttacc ggtggctctt cctgcctggg accctcccgc tagccagcgg 809 cctcagccag ggacgaaggc ctcaaagctg agaggcccct gccggtgggt gatgga 865

<210> 2 <211> 200 <212> PRT <213> Homo sapiens <400> 2

Met -95 For Ala Leu Trp Pro -90 Thr Leu Ala Ala Leu Ala -85 Leu Leu Ser Ser -80 Wall Ala Glu Ala Ser Leu Gly Ser Ala For Arg Ser For Ala For Arg -75 -70 -65 Glu Gly For For For Wall Leu Ala Ser For Ala Gly His Leu For Gly -60 -55 -50 Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg For Arg Arg -45 -40 -35 Arg His Phe Ser Ala Arg Ala For Ala Ala Cys Thr For Ile Cys Ser -30 -25 -20 Ser For Arg Wall Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser -15 -10 -5 -1 1 Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gin Leu Wall For Wall 5 10 15 Dec Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Wall Arg Phe Arg 20 May 25 30 Phe Cys Thr Gly Ser cys For Arg Ala Arg Ser For His Asp Leu Ser 35 40 45 Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For For For Gly Ser 50 55 60 65 Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg Tyr Glu Ala Wall 70 75 80 Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall Asp Arg Leu Ser 85 90 95 Ala Thr Ala Cys Gly Cys Leu Gly

100 ALIGN! <210> 3 <211> 861 <212> DNA <213> Homo sapiens <220>

SUBSTITUTE SHEET (rule 26) wo 00/01815: i::

9 4 *

Ύ <221> CDS <222> (7) .. (717) <220>

<221> 5'UTR <222> (1) .. (6) <220>

<221> 3’UTR <222> (718). . (861) <220>

<221> sig_peptide <222> (7) .. (174) <220>

<221> mat_peptide <222> (298) ... (717) <220>

<221> mat_peptide <222> (370) .. (717) <220>

<221> mat_peptide <222> (379) .. (717) <220>

<221> misc_structure <222> (661). (663) <223> CARBOHYD: glycosylated Asparagine as Asn.122 <220>

<221> misc_structure <222> (424) .. (622) <223> DISULFID: Cys43-Cysl08 Disulfide Bridge <220>

<221> misc_structure <222> (504). . (705) <223> DISULFID: Cys70-Cys36 disulfide bridge <220>

<221> misc_structure <222> (517) .. (711) <223> DISULFID: Cys74-Cysl38 Disulfide Bridge <220>

<221> misc_struccure <222> (616) .. (618) <223> DISULFID: Cys07-Cys07 interchain disulfide bridge <400> 3 gagccc atg ccc ggc ctg Ser Ala Arg Gly Gin Pro Leu -95 -90 ♦ 4 * ♦ 9 4 4 • f * 4 4 4 Í J f l ^ 9 9, W) 384. 'Ctt gag 48

Leu Glu

SUBSTITUTE SHEET (ru 26) »· • 4 · ·

WOOO / D1815 • W · W

PG? T> K <W? 00384 * ‘

gtc Val ctt Leu cct For ccc For -80 caa Gin gcc Ala cac His ctg Leu ggt Gly -75 gcc Ala ctc Leu ttt Phe ctc Leu cct For -70 gag Glu gct Ala 96 approx ctt ggt ctc tcc gcg cag cct gcc ctg tgg ccc acc ctg gcc gct 144 For Leu Gly Leu Ser Ala Gin For Ala Leu Trp For Thr Leu Ala Ala -65 -60 -55 ctg gct ctg ctg agc agc gtc gca gag gcc tcc ctg ggc tcc gcg ccc 192 Leu Ala Leu Leu Ser Ser Wall Ala Glu Ala Ser Leu Gly Ser Ala For -50 -45 -40 cgc agc cct gcc ccc cgc gaa ggc ccc ccg cct gtc ctg gcg tcc ccc 240 Arg Ser For Ala For Arg Glu Gly For For For Wall Leu Ala Ser For -35 -30 -25 -20 gcc ggc cac ctg ccg ggg gga cgc acg gcc cgc tgg tgc agt gga aga 288 Ala Gly His Leu For Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg -15 -10 -5 gcc cgg cgg ccg ccg ccg cag cct tet cgg ccc gcg ccc ccg ccg cct 336 Ala Arg Arg For For For Gin For Ser Arg For Ala For For For For -1 1 5 10 gca ccc approx tet gct ctt ccc cgc ggg ggc cgc gcg gcg cgg gct ggg 384 Ala For For Ser Ala Leu For Arg Gly Gly Arg Ala Ala Arg Ala Gly 15 Dec 20 May 25 ggc ccg ggc aac cgc gct cgg gca gcg ggg gcg cgg ggc tgc cgc ctg 432 Gly For Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu 30 35 40 45 cgc tcg cag ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc cac cgc tcc 480 Arg Ser Gin Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser 50 55 60 gac gag ctg gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgc gcg 528 Asp Glu Leu Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala 65 70 75 cgc tet approx cac gac ctc agc ctg gcc agc cta ctg ggc gcc ggg gcc 576 Arg Ser For His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala 80 85 90 ctg ega ccg ccc ccg ggc tcc cgg ccc gtc agc cag ccc tgc tgc ega 624 Leu Arg For For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg 95 100 ALIGN! 105 ccc acg cgc tray caa gcg gtc tcc ttc atg gac gtc aac agc acc tgg 672 For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr Trp 110 115 120 125 aga acc gtg gac cgc ctc tcc gcc aac ccc tgc ggc tgc ctg ggc 717 Arg Thr Wall Asp Arg Leu Ser Ala Asn For Cys Gly Cys Leu Gly 130 135 140

SUBSTITUTE SHEET (Rule 26) • · ·· · * «* ·« · * ♦> · · · * ^{«·: ϊ ..: ..:} 'p ^ Woo * ₃₈ i ^..:.:.

WO 00/01815

tgagggctcg ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctggg 777 accctcccgc agagtoccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctg 837 agaggcčcct gccggtgggt gatg 861

<210> 4.

<211> 237 <212> PRT <213> Homo sapiens

<400> 4 Ser Ala Arg Gly Gin Pro Leu Leu -85 Glu Wall Leu Met For Gly Leu -95 ' le For For Gin Ala His Leu Gly Ala Leu Phe Leu For Glu Ala For Leu -80 -75 -70 Gly Leu Ser Ala Gin For Ala Leu Trp For Thr Leu Ala Ala Leu Ala -65 -60 -55 -50 Leu Leu Ser Ser Wall Ala Glu Ala Ser Leu Gly Ser Ala For Arg Ser -45 -40 -35 For Ala For Arg Glu Gly For For For Wall Leu Ala Ser For Ala Gly -30 -25 -20 His Leu For Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg -15 -10 -5 Arg For For For Gin For Ser Arg For Ala For For For For Ala For -1 1 5 10 15 Dec For Ser Ala Leu For Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly For 20 May 25 30 Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser 35 40 45 Gin Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu 50 55 60 Leu Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser 65 70 75 For His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg 80 85 90 95 For For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg For Thr 100 ALIGN! 105 110 Arg Tyr Glu Ala 3.1 Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr 115 120 125 Wall Asp Arg Leu Ser Ala Asn For Cys Gly Cys Leu Gly 130 135 140

SUBSTITUTE SHEET (rule 26) ·

9 9 9 9 9 9 9 9 9

9 99 9 9 9 9

PCT / DK99 / 00384

WO 00/01815

<210> 5 <211> 140 <212> PRT <212> Homo sapiens <220>

<223> Wherein Xaa at position 134 designs Asn or Thr, and Yaa at position 135 designs Ala or Pro

<400> 5 Gin , 1 Pro Ser Arg 5 Pro Ala For 10 For For Pro Ala For 15 Dec For Ser Ala Leu For Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly For Gly 20 May 25 30 Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gin 35 40 45 Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu 50 55 60 Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser For 65 70 75 80 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For 85 90 95 For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg 100 ALIGN! 105 110 Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall 115 12 0 125 Asp Arg Leu Ser Ala Xaa Yaa Cys Gly Cys Leu Gly 130 135 140

<210> 6 <211> 117 <212> PRT <213> Homo sapiens <220>

<223> Wherein Xaa at position 110 designs Asn or Thr and Yaa at position 111 designs Ala or Pro <400> 6

Ala Ala Arg Ala Gly Gly For Gly Asn Arg Ala Arg Ala Ala Gly Ala 1 5 10 15 Dec Arg Gly Cys Arg Leu Arg Ser Gin Leu Wall For Wall Arg Ala Leu Gly 20 May 25 30 Leu Gly His Arg Ser Asp Glu Leu Wall Arg Phe Arg Phe Cys Ser Gly 3 5 40 45

SUBSTITUTE SHEET (rule 26) 9

WO 00/01815

• 9 9 9 9 9 9 9

PCWK5W00384 · * ⁴

Ser Cys Arg Arg Ala Arg Ser 55 For His Asp Leu Ser 60 Leu Ala Ser Leu 50 Leu Gly Ala Gly Ala Leu Arg For For For Gly Ser Arg For Wall Ser 65 70 75 80 Gin For Cys Cys Arg For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp 85 90 95 Wall Asn Ser Thr Trp Arg Thr Wall Asp Arg Leu Ser Ala Xaa Yaa Cys 100 ALIGN! 105 110 Gly Cys Leu Gly

115 <210> 7 <211> 113 <212> PRT <213> Homo sapiens <220>

<223> Wherein Xaa at position 107 by Asn or Thr, and Yaa at position 108 by Ala or Pro

<400> 7 Ala Gly 1 Pro Gly 5 Asn Arg Ala Arg Ala Gly Ala Ala Gly Cys 10 15 Dec Arg Leu Arg Ser Gin Leu Wall For Wall Arg Ala Leu Gly Leu Gly His 20 May 25 30 Arg Ser Asp Glu Leu Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg 35 40 45 Arg Ala Arg Ser For His Asp Leu Ser Leu Ala Ser Leu Leu cly Ala 50 55 60 Gly Ala Leu Arg For For For Gly Ser Arg For Wall Ser Gin For Cys 65 70 75 80 Cys Arg For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser 85 90 95 Thr Trp Arg Thr Wall Asp Arg Leu Ser Ala Xaa Yaa Cys Gly Cys Leu 100 ALIGN! 105 110

Gly

<210> 8 <211> 861 <212> DNA <213> Homo sapien <220> <221> CDS <222> (58). . . (717)

SUBSTITUTE SHEET (rule 26)

WO 00/01815

<220>

<221> 5'UTR <222> (1) .. (57) <220>

<221> 3'UTR <222> (718) .. (861) <220>

<221> sig_peptide <222> (58). (174) <220>

<221> mat_peptide <222> (298) .. (717) <220>

<221> mat_peptide <222> (370). . (717) <220>

<221> mat_peptide <222> (379) .. (717) <220>

<221> misc_structure <222> (661). (663) <223> CARBOHYD: glycosylated asparagine at Asn.122

<221> misc_structure <222> (424) .. (622) <223> DISULFID: C-ly43-Glyl08 disulfide bridge

<221> misc_structure <222> (504). . (705) <223> DISULFID: Gly70-Glyl36 Disulfide Bridge <220>

<221> misc_structure <222> (517) .. (711) <223> DISULFID: Gly74-Glyl38 disulfide bridge

<221> misc_structure <222> (616). . (618) <223> DISYFID: Glyl07-Glyl07 interchain disulfide bridge <400> 8

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

-80 -75 -70 -6 * 5

SUBSTITUTE SHEET (rule 25)

WO 00/01815 · 00 «

0 9 00 '0 · 0 9 0 * • * w · · 0

PCT / DKW00385 · *

cct For agg Arg cgg Arg cag Gin cct For -60 gcc Ala ctg Leu tgg Trp ccc For acc Thr -55 ctg Leu gcc Ala get Ala ctg Leu get Ala -50 ctg Leu 153 ctg age age gtc gca gag gcc tec ctg ggc tec gcg ccc ege age cct 201 Leu Ser Ser Wall Ala Glu Ala Ser Leu Gly Ser Ala For Arg Ser For -45 -40 -35 gcc ccc ege gaa ggc ccc ccg cct gtc ctg gcg tec ccc gcc ggc cac 249 Ala For Arg Glu Gly For For For Wall Leu Ala Ser For Ala Gly His -30 -25 -2 0 ctg ccg ggg gga ege acg gcc ege tgg tgc agt gga aga gcc cgg cgg 297 Leu For Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg -15 -10 -5 -1 ccg ccg ccg cag cct tet cgg ccc gcg ccc ccg ccg cct gca ccc approx 345 For For For Gin For Ser Arg For Ala For For For For Ala For For 1 5 10 15 Dec tet get ctt ccc ege ggg ggc ege gcg gcg cgg get ggg ggc ccg ggc 393 Ser Ala Leu For Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly For Gly 20 May 25 30 age ege get cgg gca gcg ggg gcg cgg ggc tgc ege ctg ege tcg cag 441 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gin 35 40 45 ctg gtg ccg gtg ege gcg ctc ggc ctg ggc cac ege tec gac gag ctg 489 Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu 50 55 60 gtg cgt ttc ege ttc tgc age ggc tec tgc ege ege gcg ege tet approx 537 Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser For 65 70 75 80 cac gac ctc age ctg gcc age cta ctg ggc gcc ggg gcc ctg ega ccg 585 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For 85 90 95 ccc ccg ggc tec cgg ccc gtc age cag ccc tgc tgc ega ccc acg ege 633 For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg 100 ALIGN! 105 110 tray gaa gcg gtc tec ttc atg gac gtc aac age acc tgg aga acc gtg 681 Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall 115 120 125 gac ege ctc tec gcc acc gcc tgc ggc tgc ctg ggc tgagggctcg 727 Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 130 135 140

ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctggg accctcccgc 787 agagtcccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctg agaggcccct 847 accggtgggt gatg 861

SUBSTITUTE SHEET (rule 25)

99 · ♦ ·· ···· · 9 9 9 9 9 ··· 9 9 · · 9 * · 9 * 9 9 999 9 · * φ φ WO 00/01815 PC * f / 1) 9/00384 ¹

-X <210> 9 <211> 220 <212> PRT <213> Homo Sapper.s <400> 9

Met Glu Leu Gly Gly Leu Ser Thr Leu -70 Ser His Cys For Trp -65 -80 -75 For Arg Arg Gin For Ala Leu Trp For Thr Leu Ala Ala Leu Ala Leu -60 -55 -50 Leu Ser Ser Wall Ala Glu Ala Ser Leu Gly Ser Ala For Arg Ser For -45 -40 -35 Ala For Arg Glu Gly For For For Wall Leu Ala Ser For Ala Gly His -30 -25 -20 Leu For Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg -15 -10 -5 -1 For For For Gin For Ser Arg For Ala For For For For Ala For For 1 5 10 15 Dec Ser Ala Leu For -Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly For Gly 20 May 25 30 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gin 35 40 45 Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu 50 55 60 Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser For 65 70 75 80 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For 85 90 95 For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg 100 ALIGN! 105 110 Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall 115 120 125 Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 130 135 140

<210> 10 <211> 140 <212> PRT <213> Homo sapier.s <220> <221> CARBOHYD <222> (122)

SUBSTITUTE SHEET (rule 26) • 99 * 9 9 »4 9

9 9 9

WO 00/01815 <223> glycosylated asparagine

9 9 99 • · 99 ♦ 9 99 • 9 9 9 94

PŤŤ / DKÍ9 / 00384 * *

<400> 10

For 1 For For Gin For 5 Ser Arg Pro Ala For 10 For For For Ala Pro 15 Dec For Ser Ala Leu For Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly For Gly 20 May 25 30 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gin 35 40 45 Leu Wall For Wall Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu 50 55 60 Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser For 65 70 75 80 His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg For 85 90 95 For For Gly Ser Arg For Wall Ser Gin For Cys Cys Arg For Thr Arg 100 ALIGN! 105 110 Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr Trp Arg Thr Wall 115 120 125 Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

130 135 140 <210> 11 <211> 116 <212> PRT <213> Homo sapiens <220>

<221> CARBOHYD <222> (98) <223> Glycosylated asparagine <400> 11

Ala Ala Arg Gly Gly Gly Ser Arg Ala Arg Ala Ala Gly Ala 15 Dec 1 5 10 Arg Gly Cys Arg Leu Arg Ser Gin Leu Wall For Wall Arg Ala Leu Gly 20 May 25 30 Leu Gly His Arg Asp Glu Leu Wall Arg Phe Arg Phe Cys Ser Gly 35 40 45 Ser Cys Arg Arg Ala Arg Ser For His Asp Leu Ser Leu Ala Ser Leu 50 55 60 Leu Gly Ala Gly Ala Leu Arg For For For Gly Ser Arg For Wall Ser 65 70 75 80

SUBSTITUTE SHEET (rule 25) ♦ «0 ·· 0 · ·> 0 * 0 000 0 000»

WO 00/01815

I ire 000 0 00 . 0 «PCWDK 9/00384» * <3 -

Gin For Cys Cys Arg 85 For Thr Arg Tyr Glu 90 Ala Wall Ser Phe Met 95 Asp Wall Asn Ser Thr 100 ALIGN! Trp Arg Thr Wall Asp 105 Arg Leu Ser Ala Thr 110 Ala Cys Gly Cys Leu Gly

115 <210> 12 <211> 113 <212> PRT <213> Homo sapiens <220>

<221> CARBOHYD <222> (95) <223> Glycosylated asparagine <400> 12

Ala Gly Gly Pro Arg Gly Ser Ala Ala Ala Ala Gly Ala Ala Gly Cys 1 5 10 15 Dec Arg Leu Arg Ser Gin Leu Wall For Wall Arg Ala Leu Gly Leu Gly His 20 May 25 30 Arg Ser Asp Glu Leu Wall Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg 35 40 45 Arg Ala Arg Ser For His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60 Gly Ala Leu Arg For For For Gly Ser Arg For Wall Ser Gin For Cys 65 70 75 80 Cys Arg For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser 85 90 95 Thr Trp Arg Thr Wall Asp Arg Leu Ser Al a Thr Ala Cys Gly cys Leu

100 105 110

Gly <210> 13 <211> 102 <212> DNA <213> Homo sapiens <400> 13 gene. properties Sp.

102

SUBSTITUTE SHEET (rule 25)

WO 00/01815

· ♦ »

PCŤZbK ^ / 00384 * · <400> 14

ggccaccgct ccgacgagct gatacgtttc cgcttctgca gcggctcgtg ccgccgagca 60 cgctcccagc acgatctcag tctggccagc ctactgggcg ctggggccct acggtcgcct 120 cccgggtccc ggccgatcag ccagccctgc tgccggccca ctcgctatga ggccgtctcc 180 ttcatggacg tgaacagcac ctggagaacc gtggaccgcc 220

<210> 15 <211> 2136 <212> DNA <213> Murinae gen. properties Sp.

<220>

<221> CDS <222> (975) .. (1646) <400> 15

gcggccgcga attcggcacg agggcgtctc gctgcagccc gcgatctcta ctctgcctcc 60 tggggtcttc tccaaatgtc tagcccccac ctagagggac ctagcctagc cagcggggac 120 cggatccgga gggtggaccg gccaggtgag ccctgaaagg tggggcgggg cgggggcgct 180 ctgggcccca ccccgggatc tggtgacgcc ggggctggaa tttgacaccg gacggcggcg 240 ggcaggaggc tgctgaggga tggagttggg ctcggccccc agatgcggcc cgcgggctct 300 gccagcaaca agtccctcgg gccccagccc tcgctgcgac tggggcttgg agccctgcac 360 ccaagggcac agaccggctg ccaaggcccc acttttaact aaaagaggcg ctgccaggtg 420 cacaactctg ggcatgaccc acttgagctt cgggggaaag cccagcactg gtcccaggag 480 aggcgcctag aaggacacgg accaggaccc ctttggtatg gagtgaacgc tgagcatgga 540 gtggaaggaa ctcaagtoac tactttctcc aaccaccctg gtaccttcag ccctgaagta 600 cagagcagaa gggtcttaga agacaggacc acagctgtgt gagtctcccc cctgaggcct 660 tagacgatct ctgagctcag ctgagctttg tttgcccatc tggagaagtg agccattgat 720 tgaccttgtg gcatcgccaa ggaacaggtc ctgccaagca cctaacacag agagcaaggt 780 tctccatcgc agctaccgct gctgagttga ctctagctac tccaacctcc tgggtcgctt 840 cgagagactg gagtggaagg aggaataccc caaaggataa ctaactcatc tttcagtttg 900 caagctgccg caggaagagg gtggggaaac gggtccacga aggcttctga tgggagcttc 960 tggagccgaa agg atg gaa gg ctt gca gag Met Glu Leu Gly Leu Ala Glu 5 cct act gca For Thr Ala 10 . ttg tcc. Leu Ser 1010 cac tgc ctc His Cys Leu 15 Dec : cgg cct agg tgg cag tca gcc tgg. Arg Prc Arg Trp tgg approx acc Trp Pro Thr 25 cta gct Leu Ala 1058

SUBSTITUTE SHEET (rule 26) · · ·

WO 00/01815

gtt Val cta Leu 30 gcc Ala ctg Leu ctg Leu agc Ser tgc Cys gtc Wall aca Thr gaa Glu gct Ala tcc Ser 40 ctg Leu gac Asp approx For atg Met 1106 tec cgc agc CCC gcc gct cgc gac ggt ccc tea ccg gtc ttg gcg ccc 1154 Ser Arg Ser For Ala Ala Arg Asp Gly For Ser For Wall Leu Ala Pro. 45 50 55 60 CCC acg gac cac ctg cct ggg gga cac act gcg cat ttg tgc agc gaa 1202 For Thr Asp His Leu For Gly Gly His Thr Ala His Leu Cys Ser Glu 65 70 75 aga acc ctg ega ccc ccg cct cag tet cct cag ccc gca ccc ccg ccg 1250 Arg Thr Leu Arg Prc For For Gin Ser For Gin For Ala For For For 80 85 90 cct ggt CCC gcg ctc cag tet cct ccc gct gcg ctc cgc ggg gca cgc 1298 For Gly For Ala Leu Gin Ser For For Ala Ala Leu Arg Gly Ala Arg 95 100 ALIGN! 105 gcg gcg cgt gca gga acc cgg agc agc cgc gca cgg acc aca gat gcg 1346 Ala Ala Arg Ala Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala 110 115 120 cgc ggc tgc cgc ctg cgc tcg cag ctg gtg ccg gtg agc gcg ctc ggc 1394 Arg Gly Cys Arg Leu Arg Ser Gin Leu Wall For Wall Ser Ala Leu Gly 125 130 135 140 cta ggc cac agc tcc gac gag ctg ata cgt ttc cgc ttc tgc agc ggc 1442 Leu Gly His Ser Ser Asp Glu Leu Ile Arg Phe Arg Phe Cys Ser Gly 145 150 155 tcg tgc cgc ega gca cgc tcc cag cac gat ctc agt ctg gcc agc cta 1490 Ser Cys Arg Arg Ala Arg Ser Gin His Asp Leu Ser Leu Ala Ser Leu 160 165 170 ctg ggc gct ggg gcc cta cgg tcg cct ccc ggg tcc cgg ccg atc agc 1538 Leu Gly Ala Gly Ala Leu Arg Ser For For Gly Ser Arg For Tle Ser 175 180 185 cag CCC tgc tgc cgg ccc act cgc melt gag gcc gtc tcc ttc atg gac 1586 Gin For Cys Cys Arg For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp 190 195 200 gtg aac agc acc tgg agg acc gtg gac cac ctc tcc gcc act gcc tgc 1634 Wall Asn Ser Thr Trp Arg Thr Wall Asp His Leu Ser Ala Thr Ala Cys 205 210 215 220

1686 ggc tgt ctg ggc tgaggatgat ctatctccaa gcctttgcac actagaccca

tgtgttgccc tacctggaac agctccaccg ggcctcacta accaggagcc tcaactcagc 1746 aggatatgga ggctgcagag ctcaggcccc aggccggtga gtgacagacg tcgtcggcat 1806 gacagacaga gtgaaagaeg tcggaaccac tgaccaacag tcccaagttg ttcatggatc 1866 ccagctctac agacaggaga aacctcagct aaagagaact cctctgggag aatccagaaa 1926 tggccctctg tcctggccaa tgaattttga agagatatat atacatatat acattgtagt 1986 cgcgttgctg gaccagcccg tgctgaaacc agtcccgtgt tcacttgtgg aagccgaagc 2046 cetatttatt atttctaaat tatttattta ctttgaaaaa aaacggccaa gtcggcctcc 2106

SUBSTITUTE SHEET (rule 25)

WO 00/01815 ··

ctttagtgag ggttaat- “g tgatcccggg

2136 <210> 16 <211> 224 <212> PRT <213> Murinae gen. properties Sp.

<400> 16

Glu Leu Gly Leu Ala Glu For Thr Ala Leu Ser His Cys Leu Arg 1 5 10 15 Dec For Arg Trp Gin Ser Ala Trp Trp For Thr Leu Ala Wall Leu Ala Leu 20 May 25 30 Leu Ser Cys Wall Thr Glu Ala Ser Leu Asp For Met Ser Arg Ser For 35 40 45 Ala Ala Arg Asp Gly For Ser For Wall Leu Ala For For Thr Asp His 50 55 60 Leu For Gly Gly His Thr Ala His Leu Cys Ser Glu Arg Thr Leu Arg 65 70 75 80 For For For Gin Ser For Gin For Ala For For For For Gly For Ala 85 90 95 Leu Gin Ser For For Ala Ala Leu Arg Gly Ala Arg Ala Ala Arg Ala 100 ALIGN! 105 110 Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala Arg Gly Cys Arg 115 120 125 Leu Arg Ser Gin Leu Wall For Wall Ser Ala Leu Gly Leu Gly His Ser 130 135 140 Ser Asp Glu Leu Badly Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg 145 150 155 160 Ala Arg Ser Gin His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly 165 170 175 Ala Leu Arg Ser For For Gly Ser Arg For Ile Ser Gin For Cys Cys 180 185 190 Arg For Thr Arg Tyr Glu Ala Wall Ser Phe Met Asp Wall Asn Ser Thr 195 200 205 Trp Arg Thr Wall Asp His Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 210 215 220

<210> 17 <211> 18 <212> DNA <213> Artificial Sequence

SUBSTITUTE SHEET (rule 26) · · rule rule rule rule rule rule rule rule rule rule rule rule rule rule rule rule rule

... · PGT / dK99 / 00384 · · ♦

WO 00/01815

<220>

<223> Description of Artificial Sequence: PCR primer <400> 17 cctggccagc ctactggg <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 18 aaggagaccg cttcgtagcg <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 19 atggaacttg gacttgg <210> 20 <211> 16 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR Primer <400> 20 tccatcaccc accggc <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 21 ggccaccgct ccgacgag 18 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence

SUBSTITUTE SHEET (rule 26) PCTflDK $ 9 / b038 * 4 .. '.

WO 00/01815

<220>

<223> Description of Artificial Sequence: PCR primer <400> 22 ggcggtccac ggttctccag <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR Primer <400> 23 ca.agcccac ctgggtgccc tctttctcc <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 24 catcacccac cggcaggggc ctctcag <210> 25 <211> 35 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 25 gagcccatgc ccggcctgat ctcagcccga ggaca <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 26 ccctggctga ggccgctggc tagtgggact ctgc <210> 27 <211> 31 <212> DNA <213> Artificial Sequence

SUBSTITUTE SHEET (rule 25)

WO 00/01815

Μ -X <220>

<223> Descriptior. of Artificial Sequence: Hybridization

Probe <400> 27 ncaggtggtc cgtggggggc gccaagaccg g <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220>

<223> Descriptior. of Artificial Sequence: PCR primer <400> 28 ctaggagccc atgccc

ΚβΤΛ) Κ99 / 003 * 8 <1 · * <210> 29 <211> 351 <212> DNA <213> Homo sapiens <400> 29

atggctggag gaccgggatc tcgtgctcgt gcagcaggag cacgtggctg tcgtctgcgt 60 tctcaactag tgccggtgcg tgcactcgga ctgggacacc gttccgacga actagtacgt 120 tttcgttttt gttcaggatc ttgtcgtcgt gcacgttctc cgcatgatct atctctagca 180 tctctactag gagccggagc actaagaccg ccgccgggat ctagacctgt atctcaacct 240 tgttgtagac ctactagata cgaagcagta tctttcatgg acgtaaactc tacatggaga 300 accgtagata gactarctgc aaccgcatgt ggctgtctag gatgataata G 351 <210> 30 <211> 414 <212> DNA <213> Homo sapiens <400> 30 atgggccatc atcatcatca tcatcatcat catcactcga gcggccatat cgacgacgac 60 gacaaggctg gaggaccggg atctcgtgct cgtgcagcag gagcacgtgg ctgtcgtctg 120 cgttctcaac tagtgccggt gcgtgcactc ggactgggac accgttccga cgaactagta 180 cgttttcgtt tttgttcagg atcttgtcgt cgtgcacgtt ctccgcatga tctatctcta 240 gcatctctac taggacccgg agcactaaga ccgccgccgg gatctagacc tgtatctcaa 300 ccttgttgta gacctactag atacgaagca gtatctttca tggacgtaaa ctctacatgg 360 agaaccgtag atagaccatc tgcaaccgca tgtggctgtc taggatgata atag 414

SUBSTITUTE SHEET (ruie 25) · «····

WO 00/01815

<210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 31 aaggaaaaaa gcggccgcca tggaacttgg acttggagg <210> 32 <211> 39 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: PCR primer <400> 32 ttttttcctt ggcggccgct cagcccaggc agccgcagg 39 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Primer <400> 33 gagcgagccc tcagcc 16

SUBSTITUTE SHEET (RULE 26)

Claims (9)

PATENT CLAIMS An isolated neublastin nucleic acid comprising any sequence of SEQ ID NO: 1, 3, 8 or 13 and which encodes the expression of a neublastin polypeptide having at least 70% homology to SEQ ID NO: 2, 4, 5, 6, 7, 9, 10, 11, or 12. 2. A nucleic acid sequence comprising an open reading frame which encodes the expression of a neublastin polypeptide or a derived bioactive polypeptide that has at least 70% homology to SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11, or 12 . The nucleic acid sequence of claim 2, wherein the bioactive polypeptide is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 10, 11 or 12. A nucleic acid that specifically hybridizes under very stringent solution hybridization conditions to the nucleic acid of claims 1 to 3. A nucleic acid comprising a nucleic acid sequence that is complementary to the nucleic acid of claim 4. A method of using a nucleic acid according to any one of claims 1 to 5, characterized in that it comprises the step of inducing expression in a cell of a polypeptide encoded by said nucleic acid. 7. The method of claim 6, further comprising the step of administering to said animal nucleic acid and causing said polypeptide to be expressed in said animal. A vector comprising the nucleic acid of any one of claims 1 to 5. The vector of claim 8, wherein the vector is an expression vector. A method of using a vector characterized by t ·· ·· • · · • · · · · • · · · ·· · ♦ • ♦ ··· · • · · • ♦ · • ♦ · φ ♦ · · • · · · • · · • · · ·· · ♦♦ according to claim 9, i m that include e step causing expression of said polypeptide encoded by said nucleic acid from said nucleic acid. A cell transformed with a nucleic acid according to any one of claims 1 to 5. The cell of claim 11, wherein the cell is selected from the group consisting of mammalian cells, fungal cells, yeast cells, insect cells, and bacterial cells. The cell of claim 12, wherein the cell is a Chinese hamster ovary cell. The cell of claim 12, wherein the cell is a cell derived from a mammalian central nervous system. A neurotrophic factor neublastin polypeptide comprising any of the amino acid sequences of SEQ ID NO: 2, 4, 5, 6, 7, 9, 10, 11 or 12. The polypeptide of claim 15, wherein the polypeptide is glycosylated. The polypeptide of claim 15, wherein the polypeptide is encoded by the nucleic acid of any one of claims 1 to 5. A method for preparing polypeptides according to any one of claims 15 to 16, comprising the step of expressing said polypeptides from neublastin neurotrophic factor nucleic acid. The method of claim 18, comprising culturing a cell comprising said neublastin neurotrophic factor nucleic acid in a culture medium that allows the production of said polypeptides. The method of claim 19, further comprising the step of recovering said polypeptides from said culture medium. The purified polypeptide obtained by the method of claim 20. ·· ♦ ··· 22nd A composition comprising the polypeptide of any one of claims 15 to 15 17 or 21 and a pharmaceutically acceptable carrier. 23. A polypeptide with an amino acid sequence, at least 90% homology to any sequence that it exhibits from the group 24. comprising SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11 A method of administering a polypeptide according to any one 25. 26. 27 Mar: or 12. of claims 15 to 17 and 21, characterized in that the introduction comprises the step of said polypeptide in a cell culture In vitro method or in claim The method by mammalian groups in vivo. 24, said application comprising, according to claim 24, severe injury, disease, systemic application. said mammal having a condition comprising cerebral trauma of the brain, peripheral Huntington amyotrophic lateral sclerosis a The method of claim 24, in the disease, ischemic neuropathy selected from neuronal Alzheimer Parkinson's memory impairment. a disease characterized in that said mammal is afflicted with a neuronal disorder in the peripheral nervous system, medulla or spinal cord. 28. A method of treating a neurodegenerative disease or disorder in an animal, comprising administering to said animal one or more neublastin nucleic acids of the group comprising SEQ ID NOs: 1, 3, 8, or 13. 29. A method of treating a neurodegenerative disease or disorder in an animal comprising administering one or more of the neublastin polypeptides listed in the group comprising SEQ ID NOs: 2, 4, 5, 6, 7, 7, 9, 9, 9 9, 10, 11 or 12 of said animal. An antibody that binds to any polypeptide identified in the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11, or 12. ·· ···· Ml Ό ' The antibody of claim 30, wherein the antibody is a monoclonal antibody. 32. A method of determining whether a neurodegenerative disease or disorder in an animal is associated with altered activity of neublastin neurotrophic factor polypeptides, comprising: a) contacting a biological sample originating from said animal with an antibody according to claims 30 or 31, (b) determining whether an immune complex is formed between said antibody and said protein; c) comparing the amount of said immune complex formed in said sample with the amount of said immune complex formed in a corresponding biological sample from a patient not suffering from said neural condition; and d) determining based on said comparison whether said disease or disorder is associated with an altered level of activity of said neublastin neurotrophic factor polypeptide. A nucleic acid comprising any of the sequences set forth in the group comprising SEQ ID NOs: 17, 18, 19, 20, 23, 24, 25, 26 or 28. A method of producing any of the polypeptides set forth in SEQ ID NO: 2, 4, 5, 6, 7, 9, 10, 11 or 12, characterized in that it comprises culturing a cell that contains any of the nucleic acid sequences listed in the group comprising SEQ ID NOs: 1, 3, 8 or 13 under conditions allowing production of the polypeptides and recovering the polypeptides from the culture medium. 35. A method of producing a neublastin polypeptide according to any one of claims 15, 23, or 26, comprising: (a) introducing into the cell a polynucleotide that encodes the expression of neublastin polypeptides or introducing a &quot; Z &quot; from the regulatory sequence by homologous recombination into the cell such that the regulatory sequence regulates expression of the endogenous neublastin gene to form a neublastin producing cell; b) culturing the neublastin-producing cell under culture conditions that result in the expression of the neublastin polypeptide. 36. A neublastin polypeptide comprising (a) the Cys framework residues characteristic of the GDNF family and the TGF-β superfamily; and b) at least 70% homology to any sequence listed in the group comprising SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11 or 12, wherein said neublastin polypeptide exhibits neurotrophic activity. 37. The neublastin polypeptide of claim 36, having the C terminal amino acid sequence set forth in AK72-AK105 of SEQ ID NO: 2. 38. The neublastin polypeptide of claim 36, having the C terminalminal amino acid sequence set forth in AK41-AK105 of SEQ ID NO: 2. The neublastin polypeptide of any one of claims 36, 37 or 38, wherein said homology to any of the sequences listed in the group comprising SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11, or 12 is greater than 85 %. The neublastin polypeptide of any one of claims 36, 37 or 38, wherein said homology to any of the sequences listed in the group comprising SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11, or 12 is greater than 95 %. 41. A neublastin polypeptide having any sequence set forth in the group comprising SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11 or 12, and variants thereof with conservative amino acid substitutions. 42. The neublastin polypeptide of claim 41, wherein the conservative amino acid substitution represents less than 10% of the total number of residues in the polypeptide. The neublastin polypeptide of claim 41, wherein the conservative amino acid substitution represents less than 2% of the polypeptide. The neublastin polypeptide of claim 41, wherein the conservative amino acid substitutions represent a single amino acid substitution in the mature sequence, wherein both the replaced and replacement amino acids are not cyclic. 45. An isolated nucleic acid sequence as set forth in any one of SEQ ID NOs: 17, 18, 19, 20, 23, 24, 25, 26, or 28. An isolated nucleic acid sequence that comprises nucleotides 721 to 865 of SEQ ID NO: 1, nucleotides 718 to 861 of SEQ ID NO: 3, or nucleotides 718 to 861 of SEQ ID NO: 8. An isolated nucleic acid sequence consisting of 10 to 25 contiguous nucleotides falling within or derived from any of the sequences of claim 46. An isolated nucleic acid sequence comprising nucleotides 1 to 10 of SEQ ID NO: 1 or nucleotides 1 to 57 of SEQ ID NO: 8. An isolated nucleic acid sequence consisting of 10 to 25 contiguous nucleotides falling within or derived from any of the sequences of claim 48. A method for identifying, isolating or amplifying a neublastin nucleic acid sequence comprising the use of the nucleic acids of any one of claims 45 to 49 as primers or probes. 51. A neublastin nucleic acid comprising any of SEQ ID NOs: 1, 3, 8, or 13 and encoding the expression of a neublastin polypeptide having at least 70% homology to SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10 , 11 • 44 * • 4 · 4 · 4 · 4 · 4 4 44 4 4 4 4 44 ♦ · 4 «4 4 · 4 4 4 4 4 44 • 44 4 4 44 4, 444 or 12, wherein the nucleic acid is isolated by the method of claim 50. An isolated nucleic acid sequence comprising the sequence shown in SEQ ID NO: 13. 53. A synthetic gene encoding a neublastin polypeptide, wherein the gene has the sequence shown in SEQ ID NO: 29 or 30. 54. A neublastin peptide having any of the following sequences: GPGSRARAAGARGC (AK30-43 of SEQ ID NO: 9), LGHRSDELVRFRFC (AK57-70 of SEQ ID NO: 9), CRRARSPHDLSL (AK74-85 of SEQ ID NO: 9), LRPPPGSRPVSQPC (AK94-107 of SEQ ID NO: 9), STWRTVDRLSATAC (AK123-136 of SEQ ID NO: 9), CRLRSQLVPVRALGLGHRSDELVRFRFC (AK43-70 of SEQ ID NO: 9), CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AK74-107 of SEQ ID NO: 9), CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AK108-136 of SEQ ID NO: 9), CRPTRYEAVSFMDVNST (AK108-124 of SEQ ID NO: 9) or ALRPPPGSRPVSQPC (AK93-107 of SEQ ID NO: 9). 55. An antibody raised against any of the peptides of claim 54. 56. The neublastin polypeptide of claim 36, wherein said peptide comprises seven conserved cysteine residues, such as in SEQ ID NO: 2 in positions 8, 35, 39, 72, 73, 101 and 103 or SEQ ID NO: 4 and 9 in positions 43, 70, 74 107 108, 136 and 138. 57. Set, n z and no to you m that include e in one or more containers a substance selected from the group of comprising a neublastin polypeptide according to any one of claims 15, 23 or 36, an anti-neublastin polypeptide antibody, one or more nucleic acid probes capable of hybridizing to neublastin RNA, or at least one pair of nucleic acid primers capable of initiating amplification of at least a portion of a neublastin gene. • 9 · ♦ ♦ · ···· 99 • IN • * · ♦ · • E • 9 • • 9 9 • a • • • · • « • • • • 9 9 9 9 9 • • • ·· 9 99 99 ·· 999 58. A method for diagnosing or screening for the presence of a disease or disorder characterized by an amount of a neublastin polypeptide according to claims 15, 23 or 36 or a predisposition for a subject, comprising measuring an amount of said aberrating any development thereof by the neublastin polypeptide, RNA encoding a neublastin neublastin polypeptide in from a subject, wherein increasing or decreasing the amount of neublastin polypeptide, neublastin RNA, or functional functional activity of the polypeptide, or 59. the activity of a neublastin polypeptide in a sample relative to the amount of neublastin polypeptide, neublastin RNA, or functional activity of neublastin found in an analogous sample of a subject not suffering from a disease or disorder or showing no predisposition to developing a disease or disorder indicates the existence of the disease or disorder or predisposing to disease. A method for testing a purified neublastin polypeptide according to any one of claims 15, 23 or 36, or a derivative or fragment thereof, or a modulator of their activity, for activity in treating or preventing disease, comprising: a) measuring changes in phenotype, genotype, behavior, survival or the cell's or cell's differentiation of cells or test animals, which cells or animals are derived from or exhibit characteristics associated with the disease, these cells or animals having been in contact with or administered a neublastin polypeptide, derivative, fragment or modulator, b ) comparing said changes with phenotype, genotype, behavior, survival or proliferation in cells or animals not in contact with, or not being administered to, a neublastin polypeptide, derivative, fragment or modulator. 60. A method for treating peripheral neuropathies in a mammal comprising administering a therapeutically effective amount of a neublastin polypeptide according to any one of claims 15, 23 or 36, wherein said peripheral neuropathy is selected from the group consisting of trauma-induced, chemotherapy, toxin, vitamin deficiency, idiopathic neuropathy and diabetic neuropathy. 61. The method of claim 24, wherein the neublastin is delivered directly to the central nervous system. 62. The method of claim 24, wherein the neublastin is administered systemically by subcutaneous injection, intravenous administration or intravenous infusion. A method of using the one or more nucleic acid sequence of any one of claims 1 to 5 or 45 to 49 in a computer program for identifying, isolating, or detecting new nucleic acid sequences. A fixed substrate or DNA chip comprising one or more of the nucleic acids of any one of claims 1 to 5, 33, or 45 to 49. Use of a fixed substrate or DNA chip according to claim 64 in the identification, isolation or detection of new nucleic acid sequences. Use of a sequence of one or more polypeptides according to any one of claims 15 to 17, 21, 23 or 36 to 44 in a computer program for identifying, isolating or detecting new nucleic acid sequences. 67. A method for identifying a promising compound that induces a neuroblastin-mediated biological effect, comprising: a) preparing a test cell, wherein in said cell when in contact with the neublastin polypeptide of any one of them Α1Ψ / 7 of claims 15, 23 or 36, inducing the expression of a detectable product, b) exposing the cell to a selected compound; and c) detecting a detectable product, wherein said expression of the detectable product indicates the ability of the selected compound to induce said neuroblastin-mediated biological effect.