NO330689B1 - Isolated neublastin polynucleotide, expression vector, isolated host cell, polypeptide, method for producing a polypeptide, preparation and polypeptide for use as a drug. - Google Patents

Description

Field of the invention

The present invention comprises isolated neublastin polynucleotide, expression vector, isolated host cell, polypeptide, method for producing a polypeptide, preparation and polypeptide for use as a medicine.

Background

Neurotrophic factors are naturally occurring proteins that promote survival, maintain phenotypic differentiation, prevent degeneration and increase the activity of nerve cells and tissues. Neurotrophic factors have been isolated from nervous tissue and from non-nervous tissue innervated by the nervous system, and have been classified into functionally and structurally related groups, also referred to as families, super-families or sub-families. Among the neurotrophic factor superfamilies are the fibroblast growth factor, neurotrophin, and transforming growth factor-β(TGF-β) superfamilies. Individual species of neurotrophic factors are distinguished from each other by their physical structure, their interaction with their composite receptors, and their effects on different types of nerve cells. Classified within the TGF-(3) superfamily

(Massague, et al., Trends in Cell Biology, 1994, 4, 172-178)

are glial cell line-derived neurotrophic factor ligands ("GDNF", WO 93/06116), which include GDNF, persephin ("PSP", Milbrandt et al., Neuron, 1998, 20, 245-253) and neurturin ("NTN", WO 97/08196). The ligands in the GDNF subfamily have in common their ability to induce signaling through the RET receptor tyrosine kinase. These three ligands of the GDNF subfamily differ in their relative affinities for a family of neurotrophic receptors, the GFR receptors.

Nucleotide Sequence Database EMBL, Accession Number AA844072 describes an EST nucleotide sequence with respectively 86% and 88.8% sequence identity with parts of the sequences with identification numbers 1 and 3 in the present invention.

Due to the effects of neurotrophic factors on nervous tissue, there is still a need to identify and characterize further neurotrophic factors for the diagnosis and treatment of disorders in the nervous system.

Summary of the invention

The present invention comprises isolated neublastin polynucleotide, characterized in that it is selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence SEQ ID NO: 1, b) a polynucleotide comprising an open reading frame which codes for expression of a neublastin polypeptide or a polypeptide derived therefrom, wherein the mature portion of the polypeptide has a degree of similarity to aai-aaiosi SEQ ID NO: 2 of at least 90%, c) a polynucleotide which, during hybridization under high stringency solution conditions, specifically hybridizes to

the nucleic acid having the nucleotide sequence SEQ ID NO: 1,

d) a polynucleotide comprising a nucleotide sequence that is at least 80% similar to the polynucleotide sequence indicated as SEQ ID NO: 1,

wherein the encoded polypeptide has neurotrophic activity.

Also covered by the invention is an expression vector, characterized in that it comprises the polynucleotide according to the invention.

The invention also includes an isolated host cell, characterized in that it comprises an expression vector according to the invention.

Also covered by the invention is polypeptide, characterized in that the mature part of the polypeptide has a degree of similarity with amino acids 1-105 in SEQ ID NO: 2 of at least 90%, in which the polypeptide has neurotrophic activity.

The invention also includes a method for producing the polypeptide according to the invention, characterized in that the method comprises the step of expressing the polypeptide from a neublastin neurotrophic factor polynucleotide according to the invention.

Also covered by the invention are preparations, characterized in that they comprise the polypeptide according to the invention, and a pharmaceutically acceptable carrier.

Finally, the invention comprises the polypeptide according to the invention for use as a medicine.

Additional information

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

A "neublastin polypeptide", as used herein, is a polypeptide having neurotrophic activity (e.g., as described in Examples 6, 7, 8 and 9), and includes those polypeptides having an amino acid sequence having at least 70% homology with the human "neublastin" polypeptides set forth in AA-95-AA105i SEQ ID NO: 2, AA1-AA105i SEQ ID NO: 2, AA-97-AA140i SEQ ID NO: 4, AA-41-AAno in SEQ ID NO : 4 ("pro"), AAi-AAi40i SEQ ID NO: 4, AA_8o-AAi4oi SEQ ID NO: 9 ("wild type"-pre-pro) , AA-41-AA140i SEQ ID NO: 9 (pro), AAi -AAi40i SEQ ID NO: 5 (mature 140AA) , AAi-AAn6 in SEQ ID NO: 6 (mature 116AA) , AA1-AA113i SEQ ID NO: 7 (mature 113AA) , AAi-AA^o in SEQ ID NO: 10 ( mature 14 0AA) , AAi-AAn6 in SEQ ID NO: 11 (mature 116AA) , AA1-AA113i SEQ ID NO: 12 (mature 113AA) and variants and derivatives thereof. Additionally, this invention encompasses those polypeptides having an amino acid sequence having at least 70% homology to the murine "neublastin" polypeptides set forth in AA1-AA224 in SEQ ID NO: 16.

Preferably, the C-terminal sequence in the above-identified neublastin polypeptides has an amino acid sequence as set forth in AA72-AA105i SEQ ID NO: 2 (i.e. AA107-AA140i SEQ ID NO: 9), more preferably AA41-AA105i SEQ ID NO: 2 (i.e. .AA76-AAi40i SEQ ID NO: 9), or the amino acid sequence set forth in AA191-AA224i SEQ ID NO: 16.

It is also preferred that the neublastin polypeptide retains the 7 conserved Cys residues that are characteristic of the GDNF family and the TGF-(3) superfamily.

Preferably, the neublastin polypeptide has an amino acid sequence that is more than 85% homologous, most preferably more than 95% homologous, with the above-mentioned sequences (ie AA-95-AA105i SEQ ID NO: 2, AA1-AA105i SEQ ID NO: 2, AA -97-AA140i SEQ ID NO: 4, AAi-AAi4o in SEQ ID NO: 4, AA-4i-AAi40 in SEQ ID NO: 4, AA-8o-AAi40 in SEQ ID NO: 9 ("wild type"-pre- pro) , AA-41-AA140i SEQ ID NO: 9 (pro), AA1-AA140i SEQ ID NO: 5 (mature 140AA) , AAi-AAn6i SEQ ID NO: 6 (mature 116AA) , AA1-AA113i SEQ ID NO: 7 (mature 113AA) , AA1-AA140i SEQ ID NO: 10 (mature 14 0AA) , AAi-AAu6i SEQ ID NO: 11 (mature 116AA) , AAi-AA113i SEQ ID NO: 12 (mature 113AA) ) and AA1-AA224i SEQ ID NO: 16.

A "neublastin nucleic acid" is, as used herein, a polynucleotide encoding a neublastin polypeptide. Accordingly, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons which, when exposed to the appropriate components needed for translation, will encode or code for a neublastin polypeptide. Neublastin nucleic acids according to the invention can be RNA or DNA, e.g. genomic DNA, or DNA complementary to and/or transcribed from a neublastin mRNA ("cDNA"). A neublastin nucleic acid according to the invention thus further comprises polynucleotide molecules that hybridize with specificity under strict hybridization conditions to a polynucleotide that codes for a neublastin polypeptide. This invention also relates to nucleic acid primers which can be used in the identification, isolation and amplification of polynucleotides which code for neublastin polypeptides, or fragments thereof. In certain embodiments of the invention, some of these primers are neublastin-specific probes that can be used for hybridization to a neublastin nucleic acid, but not to nucleic acids that code for the other members of the GDNF family. By "specific", "specificity" or "specifically" is meant an ability to hybridize with neublastin nucleic acid and an inability to hybridize with non-neublastin nucleic acids, including an inability to hybridize to nucleic acids uniquely encoding the GDNF ligands (eg GDNF, persephin and neurturin).

In another embodiment, a neublastin nucleic acid of the invention is one that is identified as being complementary to a polynucleotide encoding a neublastin polypeptide, either by having a complementary nucleic acid sequence or by showing that it hybridizes with specificity under stringent hybridization conditions to a polynucleotide encoding neublastin. Certain neublastin nucleic acids include, without limitation, the nucleic acid sequences shown herein designated SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 , SEQ ID NO: 29 and SEQ ID NO: 30, as well as the primers SEQ ID NOS: 17-28, 31 and 32. A neublastin nucleic acid according to the invention further comprises a unique subregion, or fragment, of a neublastin nucleic acid, including without limitation the nucleic acid fragments that are shown in Figure 8.

The neublastin nucleic acids according to the invention can be used to express a neublastin polypeptide, e.g. by expressing a neublastin polypeptide in vitro or by administering a neublastin nucleic acid to an animal for in vivo expression. Neublastin nucleic acids can be included in a nucleic acid vector, e.g. an expression vector or a cloning vector. A neublastin nucleic acid may, but need not necessarily, be maintained, reproduced, transferred or expressed as part of a nucleic acid vector. A recombinant expression vector containing a neublastin polynucleotide sequence can be introduced into and/or maintained in a cell. Cells carrying a neublastin vector may be prokaryotic. Alternatively, a neublastin nucleic acid can be introduced into a eukaryotic cell, e.g. a eukaryotic cell containing the appropriate apparatus for post-translational processing of a polypeptide into a mature protein, and/or the appropriate apparatus for secreting a polypeptide into the extracellular environment of the cell.

A neublastin neurotrophic factor, "neublastin", is further described. Neublastin may be in the form of a polypeptide, or may be a multimer of two or more neublastin polypeptides, e.g. a neublast dimer. Neublastin polypeptides are linked as multimers by means of inter-molecular structural bonds known to those skilled in the art, including without limitation cysteine-cysteine interactions, sulfhydryl bonds, and non-covalent interactions. Certain neublastin polypeptides include, without limitation, an amino acid sequence described herein designated 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 and SEQ ID NO: 16.

A neuplastin polypeptide as described herein can be used to treat a defect in a neuron, including without limitation damaged neurons and traumatized neurons. Peripheral nerves that experience trauma include, but are not limited to, nerves in the medulla or in the spinal cord. Neublastin polypeptides can be used in the treatment of neurodegenerative disease, e.g. cerebral, ischemic nerve injury; neuropathy, e.g. peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Neublastin polypeptides are further encompassed for use in the treatment of disturbed memory, e.g. memory disorders associated with dementia.

Brief description of the drawings

Figure 1 is a photographic image of two northern blots probed with <32>P-labeled neublastin cDNA, comparing relative levels of expression of the neublastin gene in different human adult tissue types (panel A) and in different areas of the adult human brain (panel B). . Figure 2 is a photographic image of a northern blot probed with <32>P-labeled neublastin cDNA, where the amount of neublastin cDNA expressed in a non-transfected cell line, HiB5, is compared to the amount of neublastin cDNA expressed in a cell line transfected with neublastin cDNA, and with a cell line transfected with GDNF cDNA. Figure 3 is a photographic image of two western blots comparing the levels at which neublastin protein is expressed in untransfected HiB5 cells (lane 1) relative to a HiB5 cell line stably transfected with neublastin cDNA (lane 2), which was probed with either the neublastin-specific antibody Ab-2 (left blot, panel A) or the neublastin-specific antibody Ab-1 (right blot, panel B). Figure 4 is a graphical illustration of the effect of neublastin on the survival of cultured rat embryo dopaminergic, ventral mesencephalic neurons and ChAT activity of cholinergic cranial motor neurons in serum-free medium. In particular, Figure 4A is an illustration of the dose-response curve for recombinant GDNF on ChAT activity (dpm/hr). Figure 4B is an illustration of ChAT activity (dpm/hr) using diluted conditioned medium from either neublastin-producing or GDNF-producing cells. Figure 4C is an illustration of the number of tyrosine hydroxylase immunoreactive cells per well. Figure 5 is an illustration of the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the function and survival of slice cultures of pig embryonic dopaminergic ventral mesencephalic neurons co-cultured with either HiB5pUbilzNBN22 cells (neublastin) or HiB5 cells (control). Figure 5A and Figure 5B illustrate dopamine released into the medium at DIV12 [dopamine (pmol/ml) - day 12] and DIV21 [dopamine (pmol/ml) - day 21], respectively. Figure 5C is an illustration of the number of tyrosine hydroxylase immunoreactive cells per culture [TH-ir cells per culture] at DIV21. Figure 6 is an illustration of the effect in vivo of lentivirus-produced neublastin on nigraldopamine neurons. Figure 7 is a schematic diagram of the genomic structure of the neublast gene, including the nucleic acid primers that can be used to identify the full-length neublast gene, and their spatial orientation relative to the genomic neublast encoding sequence (ie, gene). Figure 8 is an illustration of neublastin-specific primers used to identify the cDNA clone encoding the human neublastin polypeptide that hybridizes to nucleic acids encoding neublastin polypeptides but does not hybridize to nucleic acids encoding the other known GDNF family members (ie, GDNF, persephin and neurturin). Figure 9 illustrates the neurotrophic activity in cultures of dissociated rat dorsal root ganglion cells from different developmental stages of a polypeptide described in the present invention compared to that obtained with known neurotrophic factors [0 control experiment (in the absence of factors); 1 in the presence of GDNF; 2 in the presence of neurturin; 3 in the presence of neublastin according to the invention; E12 - embryo on day 12; E16 - embryo on day 16; PO - day of birth; P7 - day 7 after birth; and P15 - day 15 after birth]. Figure 10 illustrates neublastin production from CHO cell lines. Figure 11 illustrates a comparison between neublastin and GDNF binding to GFRα-1 and GFRα-3 receptors. Figure 12 is a photographic image of a western blot illustrating R30 antipeptide antibody and R31 antipeptide antibody binding to neublastin. Figure 13 is an image of a gel showing extraction of neublastin by affinity binding to RETL3-Ig. Figure 14 is a plasmid map of pET19b-neublastin together with the sequence of the synthetic gene for neublastin. Figure 15 is a plasmid map of pMJB164-His-neublastin together with the sequence of the synthetic gene for His-neublastin.

Detailed description of the invention

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

The cDNA encoding neublastin was originally identified as follows. Using the TBLASTN 1.4.11 algorithm (Atschul et al., Nucl. Acids Res., 1997, 25, 3389-3402) and human persephin as "query" (GenBank accession number AF040962), a 290 bp fragment was initially identified in High-Throughput Genomic Sequence (HGTS) from two human bacterial artificial chromosomes (BAC) with GenBank numbers AC005038 and AC005051. AC005038 consists of approximately 190,000 bp in 5 "contigs" of disordered sequences, and AC005051 consists of approx. 132,000 bp in 12 "contigs" of disordered sequences. The 290 bp fragment identified in the two BAC clones was found to have regions that were homologous, but not identical, to a coding region in the cDNA of the neurotrophic factor, human persephin.

From this 290 bp sequence, two neublastin-specific PCR primers ("Top Stand Primer" [SEQ ID NO: 17] and "Bottom Strand Primer" [SEQ ID NO: 18]) were synthesized. Screening of human fetal brain cDNA library was performed. The initial screening comprised 96-well PCR-based screening with two PCR primers [SEQ ID NOS: 17 and 18] in a cDNA library "Master Plate" from 500,000 cDNA clones containing approx. 5,000 clones/well. A second PCR-based screening was performed on a human fetal brain cDNA library "Sub-Plate" containing E. coli glycerol standard with approx. 5,000 clones/well. A 102 bp fragment [SEQ ID NO: 13] was identified in the PCR-based screening of both "Master Plate" and "Sub Plate". A positive cDNA clone (with the 102 bp fragment) was selected, plated onto two LB/antibiotic containing plates and grown overnight. From these plates, a total of 96 bacterial colonies were selected and placed individually in the wells of a new 96-well PCR plate containing both PCR primers [SEQ ID NOS: 17 and 18] and the necessary PCR amplification reagents. PCR amplification was then performed and the 96 individual PCR reaction mixtures were analyzed by 2% agarose gel electrophoresis. The positive colony with the clone containing the 102 bp fragment was then identified. Plasmid DNA was obtained from the positive colony containing the 102 bp fragment and sequenced. Subsequent sequence analysis revealed the presence of a full-length cDNA of 861 bp [SEQ ID NO: 8]. The 663 bp open reading frame (ORF), or coding region (CDS), identified in SEQ ID NO: 8, encodes the pre-pro polypeptide (designated "pre-pro-neublastin") and is shown in SEQ ID NO : 9. Based on SEQ ID NO: 9, three variants of neublastin were identified. These variants include: (i) the 140 amino acid polypeptide designated herein as NBN14 0, which has the amino acid sequence designated as SEQ ID NO: 10; (ii) the 116 amino acid polypeptide designated herein as NBN116, having the amino acid sequence designated as SEQ ID NO: 11;

and

(iii) the 113 amino acid polypeptide designated here as NBN113, which has the amino acid sequence designated as SEQ ID NO: 12.

The entire cDNA sequence containing 782 bp 5'-untranslated DNA, 663 bp coding DNA and 447 3<1->untranslated (a total of 1992 bp) has been deposited in GenBank under accession number AF 120274.

The genomic neublast coding sequence was identified as follows: With the aim of cloning the genomic neublast coding sequence, an additional set of primers was prepared. Specifically, primer pair #1 comprised [sense = SEQ ID NO: 23, and antisense = SEQ ID NO: 24], and primer pair #2 comprised [sense = SEQ ID NO: 25, and antisense = SEQ ID NO: 26] .

Using primer pair No. 2, an 887 bp DNA fragment was amplified by PCR from a preparation of human genomic DNA and cloned into the pCRII vector (Invitrogen) and transformed into E. coli. The resulting plasmid was sequenced, and an 8 61 bp putative cDNA sequence (encoding a protein called neublastin here) was predicted (as set forth in SEQ ID NO: 3). Likewise, by using primer pair no. 1, an 870 bp DNA fragment was obtained by PCR of human genome DNA. An additional 42 bp region at the 3' end of the open reading frame (ORF) was found in this fragment, compared to the 887 bp sequence. The genome structure of the neublastin gene was predicted by comparison with the sequences in nucleic acids of other neurotrophic factors by mapping exon-intron boundaries. This analysis shows that the neublast gene has at least two exons separated by a 70 bp intron.

This sequence was also used to screen GenBank for neublastin EST sequences. Three were identified with GenBank numbers AA844072, AA931637 and AA533512, indicating that neublastin nucleic acids are transcribed into mRNA.

Comparison of the entire cDNA sequence obtained

(AF 120274) and the genome sequence present in GenBank accession numbers AC005038 and AC005051 revealed that the neublast gene consists of at least five exons (including three coding) separated by four introns (see, e.g., Figure 8). Together, the exons have a predicted amino acid sequence of a full-length neublastin polypeptide. It should also be noted that the 887 bp fragment was found to contain the entire coding region of pro-neublastin. The predicted cDNA [SEQ ID NO: 3] contains an open reading frame (ORF) encoding pro-neublastin (181 amino

acid residues), which showed homology with the three known human proteins persephin, neurturin and GDNF.

Neublastin nucleic acids

In another aspect, the invention provides polynucleotides capable of expressing the polypeptides of the invention. The polynucleotides according to the invention comprise DNA, cDNA and RNA sequences, as well as antisense sequences, and comprise naturally occurring, synthetic and deliberately manipulated polynucleotides. The polynucleotides according to the invention also comprise sequences which are degenerate as a result of the genetic code, but which code for a neublastin polypeptide after expression.

As defined herein, the term "polynucleotide" refers to a polymeric form of nucleotides that are at least 10 bases long, preferably at least 15 bases long. By "isolated polynucleotide" is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one at the 5'-end and one at the 3'-end) in the naturally occurring genome of the organism from which it is extracted. The term therefore includes recombinant DNA which is incorporated into an expression vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which is present as a separate molecule, e.g. a cDNA, independent of other sequences.

The polynucleotides according to the invention also include allelic variants and "mutated polynucleotides" which have a nucleotide sequence that differs from the nucleotide sequences shown here in one or more nucleotide positions.

In a preferred embodiment, the polynucleotide according to the invention has a nucleic acid sequence (DNA sequence) which is able to hybridize with the polynucleotide sequence shown as SEQ ID NO: 1, the polynucleotide sequence shown as SEQ ID NO: 3, the polynucleotide sequence shown as SEQ ID NO: 8 or the polynucleotide sequence shown as SEQ ID NO: 15, its complementary strand or a subsequence thereof under at least medium, medium/high or high stringency conditions, as described further below.

In another preferred embodiment, the isolated polynucleotide according to the invention has a nucleic acid sequence (DNA sequence) which is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, homologous to the polynucleotide sequence shown as SEQ ID NO: 1, the polynucleotide sequence shown as SEQ ID NO: 3, the polynucleotide sequence shown as SEQ ID NO: 8 or the polynucleotide sequence shown as SEQ ID NO: 15.

In its 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.

This invention also provides new primers and DNA sequences for the identification, isolation and amplification of neublastin polynucleotides which, after expression, code for neublastin polypeptides or fragments thereof. Such primers include the polynucleotides indicated in SEQ ID NOS: 17-28 and 31-32. Additionally, this invention provides neublastin DNA sequences generated from these primers, including those set forth in SEQ ID NOS: 13 and 14. This invention further provides DNA sequences from 3'- or 5<1->untranslated regions (" UTR") in genomic DNA flanking neublastin exons; such sequences can be used for identification, isolation and amplification of neublastin polynucleotides which, after expression, code for neublastin polypeptides or fragments thereof.

3'-UTR sequences according to this invention include the sequences indicated in:

nucleotides 721-865 of SEQ ID NO: 1,

nucleotides 718-861 of SEQ ID NO: 3,

the nucleotides 718-861 of SEQ ID NO: 8,

nucleotides 1647-2136 in SEQ ID NO: 15, and

contiguous sequences of between 10 and 25 nucleotides derived from (ie falling within) the above sequences (which can be used, for example, as primers).

5'-UTR sequences according to this invention include the sequences indicated in:

nucleotides 1-10 of SEQ ID NO: 1,

nucleotides 1-57 of SEQ ID NO: 8,

nucleotides 1-974 in SEQ ID NO: 15, and

contiguous sequences of between 10 and 25 nucleotides derived from (ie falling within) the above sequences (which can be used, for example, as primers).

The polynucleotides according to the invention can preferably be obtained by means of cloning methods, e.g. as described in "Current Protocols in Molecular Biology" [John Wiley & Sons, Inc.]. In a preferred embodiment, the polynucleotide is cloned from or prepared on the basis of human genome DNA or a cDNA library from human brain.

DNA sequence homology

The DNA sequence homology referred to above can be determined as the degree of identity between two sequences, indicating a deviation of the first sequence from the second. The homology can conveniently be determined using computer programs known in the art, such as GAP provided in the GCG program package [Needleman, S.B. and Wunsch, C.D., Journal of Molecular Biology, 1970, 48, 443-453]. Using GAP with the following settings for DNA sequence comparison: GAP Formation Addition of 5.0 and GAP Extender Ises Addition of 0.3, the coding region of the analogous DNA sequences referred to above exhibits a degree of identity of preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, with the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 1, or the CDS (coding) portion of the DNA the sequence shown in SEQ ID NO: 3, or the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 8, the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 15.

The term "sequence identity" refers to the degree to which two polynucleotide sequences are identical on a nucleotide-for-nucleotide basis over a particular comparison range. The term "percentage sequence identity" is calculated by comparing two optimally aligned sequences over the comparison region, determining the number of positions where the identical nucleic acid base (e.g. A, T, C, G, U or I) appears in both sequences so as to obtain the number of coincident positions, divide the number of overlapping positions by the total

number of positions in the comparison area (that is, window size) and multiply the result by 100, whereby you get

the percent sequence identity. The term "substantially identical", as used herein, denotes a characteristic size for a polynucleotide sequence where the polynucleotide comprises a sequence having at least 80% sequence identity, preferably at least 85% identity and often 90-95% sequence identity, more commonly at least 99 % sequence identity, compared to a reference sequence over a comparison range.

Hybridization procedure

The polynucleotides according to the invention are those that have a nucleic acid sequence that is capable of hybridizing with the polynucleotide sequence shown as SEQ ID NO: 1, the polynucleotide sequence shown as SEQ ID NO: 3 or the polynucleotide sequence shown as SEQ ID NO: 8, or the polynucleotide sequence shown as SEQ ID NO: 15, or their complementary strand, or a subsequence thereof under at least medium, medium/high or high stringency conditions, as described further below.

Suitable experimental conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence include presoaking the filter containing the DNA fragments or RNA to hybridize in 5 x SSC [sodium chloride/sodium citrate; cf. 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 a solution of 5 x SSC, 5 x Denhardt's solution [cf. Sambrook et al., op. eit.], 0.5% SDS and 100 ug/ml denatured, sonicated salmon sperm DNA [cf. Sambrook et al., op. eit.], followed by hybridization in the same solution containing a concentration of 10 ng/ml of an arbitrary primer [Feinberg, A.P. & Vogelstein, B., Anal. Biochem., 1983, 132, 6-13],<32>P-dCTP labeled (specific activity > 1 x 10<9>cpm/uq) probe for 12 hours at approximately 45°C. The filter is then washed twice for 30 minutes in 0.1 x SSC, 0.5% SDS at a temperature of at least approx. 60°C (medium stringency), preferably at least 65°C (medium/high stringency), more preferably at least 70°C (high stringency) and even more preferably at least 75°C (very high stringency). Molecules to which oligonucleotide probes hybridize under these conditions can be detected using an X-ray film.

Cloned polynucleotides

The isolated polynucleotide according to the invention may in particular be a cloned polynucleotide. As defined herein, the term "cloned polynucleotide" refers to a polynucleotide or a DNA sequence cloned in accordance with standard cloning procedures currently used in genetic engineering to reposition a segment of DNA which may in particular be cDNA, that is enzymatically derived from RNA, from its natural location to another location where it will be reproduced.

Cloning can be carried out using any suitable route, and can include such techniques as reverse transcriptase technology, PCR technology and the like, as well as excision and isolation of the desired DNA segment.

The cloned polynucleotide according to the invention can alternatively be called "DNA construction" or "isolated DNA sequence", and can in particular be a complementary DNA (cDNA).

Biological sources

The isolated polynucleotide according to the invention can be obtained from any suitable source.

In a preferred embodiment from which the polynucleotide according to the invention is cloned, it is produced on the basis of a cDNA library, e.g. of a cDNA library from fetal or adult brain, in particular the forebrain, hindbrain, cerebral cortex, striatum, amygdala, cerebellum, caudate nucleus, corpus callosum, hippocampus, thalamic nucleus, subthalamic nucleus, olfactory center, egg membrane, substantia nigra, spinal root ganglia, trigeminal ganglion, the large mesenteric artery or thalamus, spinal cord, heart, placenta, lungs, liver, skeletal muscles, kidneys, liver, pancreas, small intestine, eye, retina, dental nerve, hair follicle, prostate, pituitary or trachea.

Commercial cDNA libraries from many different tissues, both human and non-human, are available from e.g. Stratagene and Clontech. The isolated polynucleotide according to the invention can be obtained using standard methods, e.g. those described in the working examples.

Neublastin polypeptides

As indicated above, a "neublastin polypeptide" as used herein is a polypeptide having neurotrophic activity (eg, as described in Examples 6, 7, 8 and 9), and includes those polypeptides having an amino acid sequence having at least 70% homology with the "neublastin" polypeptides set forth in AA_95-AA105i SEQ ID NO: 2, AA1-AA105i SEQ ID NO: 2, AA-97-AA140i SEQ ID NO: 4, AA-41-AAi4oi SEQ ID NO: 4, AAi-AAi40i SEQ ID NO: 4, AA-80-AAi4oi SEQ ID NO: 9 ("wild type" prepro), AA_4i-AAi4oi SEQ ID NO: 9 (pro), AA1-AA140i SEQ ID NO: 5 ( mature 14 0AA) , AA1-AA116i SEQ ID NO: 6 (mature 116AA) , AA1-AA113i SEQ ID NO: 7 (mature 113AA) , AAi-AA140i SEQ ID NO: 10 (mature 140AA) , AAi-AA140i SEQ ID NO: 10 (mature 140AA) NO: 11 (mature 116AA), AA1-AA113i SEQ ID NO: 12 (mature 113AA), AAi-AA224i SEQ ID NO: 16 (murine pre-pro), and variants and derivatives of each of the above.

Preferably, the C-terminal sequence in the above identified neublastin polypeptides has an amino acid sequence as set forth in AA72-AA105i SEQ ID NO: 2 (i.e. AA107-AA140i SEQ ID NO: 9), more preferably AA4i-AAi05i SEQ ID NO: 2 (i.e. .AA76-AA140i

SEQ ID NO: 9).

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

Preferably, the neublastin polypeptide has an amino acid sequence that is more than 85% homologous, most preferably more than 95% homologous, with the above-mentioned sequences, that is, AA-95-AA105i SEQ ID NO: 2, AA1-AA105i SEQ ID NO: 2, AA-97-AA140i SEQ ID NO: 4, AA_4i-AAi40i SEQ ID NO: 4, AA1-AA140i SEQ ID NO: 4, AA-80-AA140i SEQ ID NO: 9 ("wild-type" prepro), AA-4i -AAi40i SEQ ID NO: 9 (pro), AAi-AA140i SEQ ID NO: 5 (mature 140AA) , AAi-AAiiei SEQ ID NO: 6 (mature 116AA) , AA1-AA113i SEQ ID NO: 7 (mature 113AA) , AA1-AA140i SEQ ID NO: 10 (mature 140AA) , AAi-AA116i SEQ ID NO: 11 (mature 116AA) , AA1-AA113i SEQ ID NO: 12 (mature 113AA) , AA1-AA224i SEQ ID NO: 16 (murine pre -pro), and preferably any of the above-mentioned polypeptides with a C-terminal sequence from the above-identified neublastin polypeptides has an amino acid sequence as set forth in AA72-AA105i SEQ ID NO: 2 (ie AA107-AA140i SEQ ID NO: 9 ), more preferably AA4i-AAi05i SEQ ID NO: 2 (ie AA76-AAi40i SEQ ID NO: 9) or AA19 1-AA224i SEQ ID NO: 16.

In addition, this invention relates to those polypeptides having an amino acid sequence having at least 70% homology to the murine "neublastin" polypeptides set forth in AAi-AA224i SEQ ID NO: 16.

Among the preferred polypeptides according to the invention are in one embodiment the pre-pro sequence (as indicated in SEQ ID NOS: 2, 4, 9 and 16 respectively), the pro sequence (as indicated in AA-75-AAi05i SEQ ID NO: 2 or AA -4i-AAi40i respectively SEQ ID NOS: 4 and 9) and the mature sequence of neublastin (as indicated in SEQ ID NO: 5, 6, 7, 10, 11 or 12, preferably SEQ ID NOS: 10, 11, 12.

The polypeptides according to the invention comprise variant polypeptides. In the context of this invention, the term "variant polypeptide" means a polypeptide (or protein) having an amino acid sequence that differs from the sequence set forth 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, in 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, the term "conservative substitutions" denoted the replacement of an amino acid residue with another, biologically similar residue. For example, one would expect that conservative amino acid substitutions have little or no effect on the biological activity, especially if they make up less than 10% of the total number of residues in the polypeptide or protein. Conservative amino acid substitutions preferably constitute changes in less than 5% of the polypeptide or protein, most preferably less than 2% of the polypeptide or protein (eg, when calculated in accordance with NBN113, most preferred conservative substitutions will constitute fewer than three amino acid substitutions in the mature wild-type amino acid sequence). In a particularly preferred embodiment, there is a single amino acid substitution in the mature sequence, where both the substituted and replacement amino acids are non-cyclic.

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

The term conservative substitution also encompasses the use of a substituted amino acid residue instead of an unsubstituted parent amino acid residue, provided that antibodies raised against the substituted polypeptide also react immunologically with the unsubstituted polypeptide.

Modifications of this primary amino acid sequence can result in proteins that have essentially equivalent activity compared to the unmodified counterpart polypeptide, and thus can be considered functional analogues of the parent proteins. Such modifications may be intentional, e.g. as in site-directed mutagenesis, or they can occur spontaneously, and include splice variants, isoforms, homologues from other species and polymorphisms. Such functional analogues are also covered according to the invention.

Modifications of the primary amino acid sequence can also result in proteins that do not retain the biological activity of the parent protein, including dominant negative forms etc. A dominant negative protein can act on the wild-type protein by binding to or otherwise sequestering regulatory agents, such as upstream or downstream components, which normally functionally react with the polypeptide. Such dominant negative forms are also covered according to the invention.

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

A "heterologous signal peptide", as used herein in the context of neublastin, means a signal peptide other than the human neublastin signal peptide, usually the signal peptide of a mammalian protein other than neublastin.

Those skilled in the art will recognize that the human neublastin DNA sequence (either cDNA or genomic DNA) or sequences that differ from human neublastin DNA due to either silent codon changes or codon changes resulting in conservative amino acid substitutions can be used to genetically modify cultured human cells so that they will overexpress and secrete the enzyme.

Polypeptides according to the present invention also include chimeric polypeptides or cleavable fusion polypeptides where another polypeptide is fused at the N-end or C-end of the polypeptide or fragment thereof. A chimeric polypeptide can be produced by fusing a nucleic acid sequence (or part thereof) which codes for another polypeptide to a nucleic acid sequence (or part thereof) according to the present invention.

Techniques for making chimeric polypeptides are standard techniques. Such techniques usually require joining the sequences in such a way that they are both in the same reading frame, and expressing the fused polypeptide under the control of the same promoter(s) and the same terminator.

Polypeptides according to the present invention also comprise truncated forms of the full-length neublastin molecule. In such truncated molecules, one or more amino acids have been split from the N-terminus or the C-terminus, preferably the N-terminus.

Amino acid sequence homology

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

Homology is determined using a computer analysis, such as, without limitation, the ClustalX computer assembly program [Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G.: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools; Nucleic Acids Res., 1997, 25 (24):4876-82], and the default parameters proposed herein. Using this program, the mature portion of a polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity of at least 90%, more preferably at least 95%, most preferably at least 98%, with the amino acid sequence shown herein 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.

Based on the homology determination, it is confirmed that the polypeptide according to the invention, which belongs to the TGF-(3 superfamily), is related to the GDNF subfamily, but constitutes a separate member of this subfamily.

Bioactive polypeptides

The polypeptide of the invention can be provided in any bioactive form, including the form of pre-pro-proteins, pro-proteins, mature proteins, glycosylated proteins, phosphorylated proteins or any other post-translationally modified protein.

The polypeptide according to the invention may in particular be an N-glycosylated polypeptide which is preferably glycosylated in the N-residues indicated in the sequence list.

In a preferred embodiment, the polypeptide according to the invention has the amino acid sequence indicated as SEQ ID

NO: 9, with a glycosylated asparagine residue in position 122; the amino acid sequence shown as SEQ ID NO: 10, with a glycosylated asparagine residue at position 122; the amino acid sequence shown as

SEQ ID NO: 11, with a glycosylated asparagine residue at position 98; or the amino acid sequence shown as SEQ ID NO: 12, with a glycosylated asparagine residue at position 95.

This invention also encompasses neublast fusion proteins, such as Ig fusions, as described e.g. in US Patent No. 5,434,131.

In one embodiment, the invention provides a polypeptide with the amino acid sequence shown as SEQ ID NO: 2, or an amino acid sequence that is at least approx. 85%, preferably at least approx. 90%, more preferably at least approx. 98% and most preferably at least approx. 99%, homologous to the sequence shown as SEQ ID NO: 2.

In another embodiment, the invention provides a polypeptide with the amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence that is at least 90%, more preferably at least 95%, even more preferably at least 98%, most preferably at least 99%, homologous to the sequence shown as SEQ ID NO: 4.

In a third embodiment, the invention provides a polypeptide with the amino acid sequence according to SEQ ID

NO: 5, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 5.

In a fourth embodiment, the invention provides a polypeptide with the amino acid sequence according to SEQ ID

NO: 6, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 6.

In a fifth embodiment, the invention provides a polypeptide with the amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 7.

The neublastin polypeptide comprises allelic variants, e.g. the polypeptide amino acid sequences according to SEQ ID NOS: 5-7, where Xaa denotes Asn or Thr, and Yaa denotes Ala or Pro.

In a sixth embodiment, a polypeptide is provided with the amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 9.

In a seventh embodiment, a polypeptide is provided with the amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 10.

In an eighth embodiment, a polypeptide is provided with the amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence which is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 11.

In a ninth embodiment, a polypeptide is provided with the amino acid sequence according to SEQ ID NO: 12, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 12.

In a tenth embodiment, there is provided a polypeptide having the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98%, homologous to the sequence shown as SEQ ID NO: 16, which is a pre-pro-neublastin of murine origin.

In another embodiment, the polypeptide has the GDNF subfamily fingerprint, that is, the amino acid residues underlined in Table 3.

In a further embodiment, a polypeptide encoded by a polynucleotide sequence is provided which is capable of hybridizing under conditions of high stringency with the polynucleotide sequence shown as SEQ ID NO: 1, its complementary strand or a subsequence thereof. In a preferred embodiment, the polypeptide according to the invention is encoded by a polynucleotide sequence that is at least 70% homologous with the polynucleotide sequence shown as SEQ ID NO: 1. In its most preferred embodiment, the polypeptide according to the invention is encoded by the polynucleotide sequence shown as SEQ ID NO: 1.

In yet another embodiment, novel polypeptides encoded by a polynucleotide sequence are provided which are capable of hybridizing under conditions of high stringency with the polynucleotide sequence shown as SEQ ID NO: 3, its complementary strand or a subsequence thereof. In a preferred embodiment, the polypeptide according to the invention is encoded by a polynucleotide sequence which is at least 70% homologous to the polynucleotide sequence shown as SEQ ID NO: 3. In its most preferred embodiment, the polypeptide according to the invention is encoded by the polynucleotide sequence shown as SEQ ID NO: 3.

In yet another embodiment, novel polypeptides encoded by a polynucleotide sequence are provided which are capable of hybridizing under high stringency conditions with the polynucleotide sequence shown as SEQ ID NO: 8, its complementary strand or a subsequence thereof. In a preferred embodiment, the polypeptide according to the invention is encoded by a polynucleotide sequence that is at least 70% homologous with the polynucleotide sequence shown as SEQ ID NO: 8. In its most preferred embodiment, the polypeptide according to the invention is encoded by the polynucleotide sequence shown as SEQ ID NO: 8.

In yet another embodiment, novel polypeptides encoded by a polynucleotide sequence are provided which are capable of hybridizing under conditions of high stringency with the polynucleotide sequence shown as SEQ ID NO: 15, its complementary strand or a subsequence thereof. In a preferred embodiment, the polypeptide according to the invention is encoded by a polynucleotide sequence that is at least 70% homologous to the polynucleotide sequence shown as SEQ ID NO: 15. In its most preferred embodiment, the polypeptide according to the invention is encoded by the polynucleotide sequence shown as SEQ ID NO: 15.

Biological origin

The polypeptide according to the invention can be isolated from mammalian cells, preferably from a human cell or from a cell of murine origin.

In a highly preferred embodiment, the polypeptide according to the invention can be isolated from human heart tissue, from human skeletal muscle, from human pancreas or from human brain tissue, in particular from the caudal nucleus or from the thalamus, or it can be obtained from DNA of mammalian origin, as discussed further below.

Neurotrophic activity

Neublastin polypeptides according to the invention can be used to moderate the metabolism, growth, differentiation or survival of nerve or neuron cells. In particular, neublastin polypeptides are used to treat or alleviate a disorder or disease in a living animal, e.g. a human, where the disorder or disease responds to the activity of a neurotrophic agent. Such treatments and procedures are described in more detail below.

Antibodies

Neublastin polypeptides or polypeptide fragments according to the invention are used to produce neublastin-specific antibodies. As used herein, a "neublastin-specific" antibody is an antibody, e.g. a polyclonal antibody or a monoclonal antibody, which is immunoreactive against a neublastin polypeptide or polypeptide fragment, or which binds with specificity to an epitope on a neublastin polypeptide.

The production of polyclonal and monoclonal antibodies is well known in the art. Polyclonal antibodies can in particular be obtained as described e.g. by Green et al.,

"Production of Polyclonal Antisera" in Immunochemical Protocols (Manson, ed.); Humana Press, 1992, pp. 1-5; by Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters" in Current Protocols in Immunology, 1992, section 2.4.1, and by Ed Harlow and David Lane (eds.) in "Antibodies: A Laboratory Manual", Cold Spring Harbor Lab. Press, 1988. Monoclonal antibodies can particularly be obtained as described e.g. by Kohler & Milstein, Nature, 1975, 256:495; Colligan et al. in Current Protocols in Immunology, 1992, Sections 2.5.1-2.6.7, and Harlow et al. in Antibodies: A Laboratory Manual, Cold Spring Harbor Pub., 1988, p. 726.

Briefly, monoclonal antibodies can be obtained by injecting e.g. mice with a preparation comprising an antigen, verify the presence of antibody production by removing a serum sample, remove the spleen to obtain B lymphocytes, fuse the B lymphocytes with myeloma cells to produce hybridomas, clone the hybridomas, select positive clones that produce the antibodies against the antigen, and isolate the antibodies from the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridoma cultures using many different well-established techniques, including protein A-Sepharose affinity chromatography, size exclusion chromatography and ion exchange chromatography, see e.g. Colligan et al. in Current Protocols in Immunology, 1992, Sections 2.7.1-2.7.12, and Sections 2.9.1-2.9.3; and Barnes et al.: "Purification of Immunoglobulin G (IgG)" in Methods in Molecular Biology; Humana Press, 1992, vol. 10, pp. 79-104. Polyclonal or monoclonal antibodies can optionally be purified further, e.g. by binding to and eluting from a matrix to which the polypeptide against which the antibodies were raised is bound.

Antibodies that bind to the neublastin polypeptide according to the invention can be prepared by using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide used to immunize an animal can be obtained by means of recombinant DNA techniques with or by means of chemical synthesis, and can optionally be conjugated to a carrier protein. Commonly used carrier proteins that are chemically linked to the peptide include albumen shell hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA) and tetanus toxoid. The linked peptide can then be used to immunize the animal, which can in particular be a mouse, a rat, a hamster or a rabbit.

In one embodiment, antibodies are prepared 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 raised rabbit polyclonal antibodies against the following peptides:

peptide R27: GPGSRARAAGARGC (amino acids 30-43 i

SEQ ID NO: 9);

peptide R28: LGHRSDELVRFRFC (amino acids 57-70 in

SEQ ID NO: 9);

peptide R29: CRRRARSPHDLSL (amino acids 74-85 of SEQ ID NO: 9);

peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO: 9); and

peptide R31: STWRTVDRLSATAC (amino acids 123-136 i

SEQ ID NO: 9).

Of this group, only peptides R30 and R31, relatively close to the C terminus, recognized the denatured protein under reducing conditions on a western blot.

We have also identified additional neublastin-derived peptides derived from the mature protein, as discussed below, which are predicted surface-exposed loops based on the known GDNF structure (Eigenbrot and Gerber, Nat. Struct. Biol., 1997, 4, 435-438 ), and thus can be used for antibody generation: region 1: CRLRSQLVPVRALGLGHRSDELVRFRFC (AA43-70 in

SEQ ID NO: 9);

region 2: CRRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AA74-107 in SEQ ID NO: 9);

region 3: CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AA108-136 i

SEQ ID NO: 9).

In another aspect of the invention, antibodies that specifically bind neublastin or neublastin-derived peptides can be used to detect the presence of such neurotrophic neublastin factors in various media, and in particular to diagnose conditions or diseases that are associated with the neublastin molecules according to the invention. Many different methods for such detection, including ELISA, RIA and FACS, are known in the art.

The antibodies according to this invention can also be used to block the effect of the neurotrophic factor, and can in particular be neutralizing antibodies.

Methods for producing the polypeptides

A cell comprising a DNA sequence encoding a neublastin polypeptide according to the invention is cultured under conditions which enable the production of the polypeptide, followed by extraction of the polypeptide from the culture medium, as described in more detail below. When cells are to be genetically modified for the purpose of producing a neublastin polypeptide, the cells can be modified by conventional methods or by gene activation.

According to conventional methods, a DNA molecule containing a neublastin cDNA or genomic DNA sequence can be contained in an expression construct and transfected into cells using standard methods, including, but not limited to, liposome, polybrene, or DEAE -dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection or velocity-driven microprojectiles ("biolistics"). Alternatively, a system can be used that emits DNA using a viral vector. Viruses that are known to be able to be used for gene transfer include adenovirus, adeno-associated virus, lentivirus, herpes virus, measles virus, poliovirus, retrovirus, Sindbis virus and vaccinia virus, such as canary pox virus, as well as baculovirus infection of insect cells, especially SfP9 insect cells.

Alternatively, the cells can be modified using a gene activation ("GA") method, as described in US Patent Nos. 5,733,761 and 5,750,376.

Accordingly, the term "genetically modified", as used herein in reference to cells, is intended to include cells that express a particular gene product after the introduction of a DNA molecule encoding the gene product, and/or regulatory elements that control expression of a coding sequence for the gene product. The DNA molecule can be introduced by means of gene targeting, which makes it possible to incorporate the DNA molecule at a specific genomic location.

Recombinant expression vectors

In a further aspect, the invention provides a recombinant expression vector comprising the polynucleotide according to the invention. The recombinant expression vector according to the invention can be any suitable eukaryotic expression vector. Preferred recombinant expression vectors are the ubiquitin promoter containing vector pTEJ-8 (Johansen, T.E., Schoeller, M.S., Tolstoy, S., Schwartz, T., FEBS Lett., 1990, 257, 289-294) and derivatives thereof, e.g. pUbilz. A preferred, commercially available eukaryotic expression vector is e.g. the viral promoter containing vector pcDNA-3 (available from Invitrogen). Another preferred expression vector makes use of the SV40 early and adenovirus major late promoters (derived from plasmid pAD2beta; Norton and Coffin, Mol. Cell. Biol., 1985, 5, 281).

This invention also provides prokaryotic expression vectors and synthetic genes (syngenes) with codon optimization for prokaryotic expression. Syngenes were constructed with lower GC content and preferred bacterial codons (eg E. coli). The syngene is cloned into two vectors, pET19b and pMJB164, a derivative of pET19b. The construction with pET19b is shown in Figure 14. In this construction, the sequence coding for the mature neublast domain is fused directly to an initiating methionine. The construction with pMJB164 is shown in Figure 15.

Production cells

In yet another aspect, the invention provides a production cell that is genetically manipulated to comprise the isolated polynucleotide sequence according to the invention and/or a recombinant expression vector according to the invention. The cell according to the invention can in particular be genetically manipulated to transiently or stably express, overexpress or co-express the polypeptide according to the invention. Methods for generating transient and stable expression are known in the art.

The polynucleotide according to the invention can be inserted into an expression vector, e.g. a plasmid, virus or other expression carrier, and is operably linked to expression control sequences by ligation in such a way that expression of the coding sequence is achieved under conditions comparable to the expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns and stop codons, all held in the correct reading frame of the polynucleotide of the invention to enable correct translation of mRNA. Expression control sequences may also include additional components, such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or an inducible promoter. When cloned in bacterial systems, the

such inducible promoters as pL from bacteriophage X, plac, ptrp, ptac (ptrp-lac hybrid promoter) are used. When cloning in mammalian systems, promoters derived from the genome in mammalian cells, e.g. the ubiquitin promoter, the TK promoter or the metallothionein promoter, or from mammalian viruses, e.g. the retrovirus long end repeat, the adenovirus late promoter or the vaccinia virus 7.5K promoter are used. Promoters obtained using recombinant DNA or synthetic

techniques can also be used to provide transcription of the polynucleotide according to the invention.

Suitable expression vectors usually comprise an expression origin site, a promoter and specific genes enabling phenotypic selection of the transformed cells, and include vectors such as the 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 the oocyte expression vector PTLN [Lorenz, C., Pusch M. & Jentsch, T.J., Heteromultimeric CLC chloride channels with novel properties, Proe. Nati. Acad. Pollock. USA, 1996, 93, 13362-13366].

In a preferred embodiment, the cell according to the invention is a eukaryotic cell, e.g. a mammalian cell, e.g. a human cell, an oocyte or a yeast cell. The cell according to the invention can without limitation be a human embryonic kidney cell (HEK), e.g. a HEK293 cell, a BHK21 cell, a Chinese Hamster Ovary (CHO) cell, a Xenopus laevis oocyte cell (XLO). In another embodiment, the cell according to the invention is a fungal cell, e.g. a cell of filamentous fungi. In another preferred embodiment, the cell is an insect cell, most preferably the Sf9 cell. Further preferred mammalian cells according to the invention are PC12, HiB5, RN33b cell lines and human neural progenitor cells. Most preferred are human cells.

Examples of primary or secondary cells include fibroblasts, epithelial cells, including breast and intestinal epithelial cells, endothelial cells, formed elements from the blood, including lymphocytes and bone marrow cells, glial cells, hepatocytes, keratinocytes, muscle cells, nerve cells or the precursors of these cell types. Examples of immortalized human cell lines that can be used in the present methods include, but are not limited to, Bowes melanoma cells (ATCC accession number CRL 9607), Daudi cells (ATCC accession number CCL 213), HeLa cells and derivatives of HeLa cells (ATCC accession number CCL 2, CCL 2.1 and CCL 2.2), HL-60 cells (ATCC accession number CCL 240), HT-1080 cells (ATCC accession number CCL 121), Jurkat cells (ATCC accession number TIB 152), KB- carcinoma cells (ATCC accession number CCL 17), K-562 leukemia cells (ATCC accession number CCL 243), MCF-7 breast cancer cells (ATCC accession number BTH 22), MOLT-4 cells (ATCC accession number 1582), Namalwa cells (ATCC accession number CRL 1432), Raji cells (ATCC accession number CCL 86), RPMI-8226 cells (ATCC accession number CCL 155), U-937 cells (ATCC accession number CRL 1593), WI-38VA13 sub-line 2R4 cells (ATCC accession number

CLL 75.1) and 2780AD ovarian carcinoma cells (Van der Blick et al., Cancer Res., 1988, 48, 5927-5932, as well as heterohybridoma cells produced by fusion of human cells and cells of other species. Secondary human fibroblast strains, such as WI-38 (ATCC deposit number CCL 75) and MRC-5 (ATCC deposit number CCL 171), can also be used.

When the cell according to the invention is a eukaryotic cell, incorporation of the heterologous polynucleotide according to the invention can in particular be carried out by infection (using a viral vector), by transfection (using a plasmid vector), using calcium phosphate precipitation, micro-injection, electroporation, lipofection or other physico-chemical methods known in the art.

In a more preferred embodiment, the isolated polynucleotide sequence according to the invention and/or a recombinant expression vector according to the invention is transfected into a mammalian host cell, a nerve precursor cell, an astrocyte cell, a T cell, a hematopoietic stem cell, a non-dividing cell or a cerebral endothelial cell comprising at least one DNA molecule capable of mediating cellular immortalization and/or transformation.

Activation of an endogenous gene in a host cell can be carried out by means of the introduction of regulatory elements, in particular by the introduction of a promoter capable of effecting transcription of an endogenous gene which codes for the neublastin polypeptide according to the invention.

Pharmaceutical preparations

In another aspect, new pharmaceutical preparations are provided which comprise a therapeutically effective amount of the polypeptide according to the invention.

For use in therapy, the polypeptide of the invention may be administered in any suitable form. In a preferred embodiment, the polypeptide according to the invention is incorporated into a pharmaceutical preparation together with one or more adjuvants, or several excipients, carriers and/or diluents, and the pharmaceutical preparation is prepared by experts in the art by using common methods known in the field.

Such pharmaceutical preparations may comprise the polypeptide according to the invention or antibodies thereof. The preparation can be administered alone or in combination with one or more other agents, drugs or hormones.

The pharmaceutical composition may be administered by any suitable route, including, but not limited to, oral, intravenous, intramuscular, interarterial, intra-medullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, anteral, topical, sublingual, or rectal application, buccal, vaginal, intraorbital, intra-cerebral, intracranial, intraspinal, intraventricular, intra-cisternal, intracapsular, intrapulmonary, transmucosal application or via inhalation.

Intrapulmonary delivery methods, apparatus and drug preparation are described e.g. in US Patent Nos. 5,785,049, 5,780,019 and 5,775,320. Administration may be by means of periodic injections of a bolus of the preparation, or may be done more continuously by means of intra-venous or intraperitoneal administration from a reservoir which is externally (eg, an IV bag) or internally (eg, a biodegradable implant, a biological artificial organ, or a colony of implanted neublastin-producing cells). See e.g. US Patent Nos. 4,407,957, 5,798,113 and 5,800,828. Intrapulmonary delivery methods and apparatus are described e.g. in US Patent Nos. 5,654,007, 5,780,014 and 5,814,607.

In particular, administration of a neublastin of this invention may be accomplished using any suitable delivery means, including: (a) pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer, 41:1270 (1993) ; Cancer Research, 44:1698 (1984), (b) microencapsulation (see, e.g., US Patent Nos. 4,352,883, 4,353,888 and 5,084,350), (c) continuous release polymer implants (see, e.g., Sabel , US Patent No. 4,883,666, (d) macroencapsulation (see, e.g., US Patent Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733, and published PCT Patent Application No. WO 92/19195 and WO 95/05452, (e) naked or unencapsulated cell implants for the CNS (see, e.g., US Patent Nos. 5,082,670 and 5,618,531, (f) injection, either subcutaneous, intravenous, intra-arterial, intramuscular or to another suitable site, and (g) oral administration in a capsule, as a liquid,

tablet, pill or prolonged-release preparation.

In one embodiment, a neublastin is delivered directly into the CNS, preferably to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS site, most preferably intrathecally.

In another preferred embodiment, we think of systemic delivery by means of subcutaneous injection, intravenous administration or intravenous injection.

Other applicable parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable injection systems, pump delivery, encapsulated cell delivery, liposomal delivery, needle delivery injection, needleless injection, nebulizer, aerosol device, electroporation, and transdermal patch.

Further details regarding formulation and administration techniques can be found in the latest edition of

Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).

The active ingredient can be administered in one or more doses per day. Currently assumed appropriate dosages are between 0.5 ng neublastin/kg body weight and approx. 50 ug/kg per administration, and from approx. 1.0 ng/kg to approx. 100 ug/kg daily. The pharmaceutical neublastin preparation should provide a local concentration of neurotrophic factor from approx. 5 ng/ml cerebrospinal fluid ("CSF") to 25 ng/ml CSF.

The dose that is administered must of course be carefully adjusted according to the age, weight and condition of the individual being treated, as well as the route of administration, the dosage form and regimen and the desired result, and the exact dosage should of course be determined by the doctor.

In further embodiments, the neublastin polypeptide according to the invention can be administered by means of genetic delivery, by using cell lines and vectors as described below under treatment methods. To produce such therapeutic cell lines, the polynucleotide according to the invention can be introduced into an expression vector, e.g. a plasmid, virus or other expression carrier, and is operably linked to expression control sequences by ligation in such a way that expression of the coding sequence is achieved under conditions 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 held in the correct reading frame of the polynucleotide of the invention to enable correct translation of mRNA. Expression control sequences may also include additional components, such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or an inducible promoter. Constitutive promoters can be synthetic, viral or derived from the genome of mammalian cells, e.g. the human ubiquitin promoter. In a preferred embodiment, the therapeutic cell line will be a human, immortalized nerve cell line that expresses the polypeptide according to the invention. For implantation, we are thinking of implanting between approx. IO<5> and 10<10> cells, more preferably IO<6> to approx. IO<8> cells.

Treatment methods

Polynucleotides and proteins, polypeptides, peptide fragments or derivatives produced therefrom, as well as antibodies directed against such proteins, peptides or derivatives as described herein, can be used for the treatment or alleviation of a disorder or disease in a living animal body, including a human, where the disorder or the disease responds to the activity of neurotrophic agents.

The polypeptides as described herein can be used directly via e.g. injected, implanted or ingested pharmaceutical preparations for the treatment of a pathological process responsive to the neublastin polypeptides.

The polynucleotide as described herein, including the complementary sequences thereof, can be used for the expression of the neurotrophic factor as described herein. This can be achieved by means of cell lines that express such proteins, peptides or derivatives according to the invention, or by means of virus vectors that code for such proteins, peptides or derivatives according to the invention, or by means of host cells that express such proteins, peptides or derivatives. These cells, vectors and preparations can be administered to treatment target areas to affect a disease process that responds to the neublastin polypeptides.

Suitable expression vectors can be derived from lentivirus, retrovirus, adenovirus, herpes or vaccinia virus, or from various bacterially produced plasmids which can be used for in vivo delivery of nucleotide sequences to a whole organism or a 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 delivery via slow release systems. In yet another aspect of the invention, "antisense" nucleotide sequences that are complementary to the neublastin gene or parts thereof can be used to inhibit or promote neublastin expression.

In particular, the disorder or disease may be damage to the nervous system caused by trauma, surgery, ischaemia, infection, metabolic diseases, nutrient deficiency, malignancy or toxic agents, and genetic or idiopathic processes.

In particular, the damage may have occurred to sensory neurons or retinal ganglion cells, including neurons in the dorsal root ganglion or in any of the following tissues: the geniculate, petrosal, and nodose ganglia; the vestibuloacoustic complex of the eighth cranial nerve; the ventrolateral pole of the maxillo-mandibular lobe of the trigeminal ganglion; and the mesencephalic-trigeminal nucleus.

In a preferred embodiment of the method as described herein, the disease or disorder is a neurodegenerative disease that includes damaged and traumatic neurons, such as traumatic lesions in peripheral nerves, the medulla and/or spinal cord, cerebral ischemic nerve damage, neuropathy and especially peripheral neuropathy, peripheral nerve trauma or peripheral nerve injury, ischemic stroke, acute brain injury, acute spinal cord injury, nervous system tumors, multiple sclerosis, exposure to neurotoxins, such metabolic diseases as diabetes or renal dysfunctions and damage caused by infectious agents, neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, Parkinson's disease, Parkinson's -Plus syndromes, progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome), olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome (multiple system atrophy), Guamanian parkinsonism-dementia complex, amyotrophic lateral sclerosis or any other congenital or is a neurodegenerative disease and memory disorder associated with dementia.

In a preferred embodiment, we are thinking of treating sensory and/or autonomic system neurons. In another preferred embodiment, we contemplate the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis ("ALS") and spinal muscular atrophy. In yet another preferred embodiment, we are thinking of using the neublastin molecules as described herein to improve nerve healing after traumatic injury. In one embodiment, we are thinking of using a nerve conduction channel with a matrix containing neublastin polypeptides. Such nerve conduction channels are described e.g. in US Patent No. 5,834,029.

In a preferred embodiment, the polypeptides and nucleic acids as described herein (and pharmaceutical preparations containing the same) are used in the treatment of peripheral neuropathies. Among the peripheral neuropathies contemplated for treatment with the molecules described herein are trauma-induced neuropathies, e.g. those caused by physical injury or disease state, physical injury to the brain, physical injury to the spinal cord, stroke associated with brain injury and neurological disorders related to neuro-degeneration.

We also contemplate the treatment of chemotherapy-induced neuropathies (such as those caused by the delivery of chemotherapeutic agents, eg taxol or cisplatin); toxin-induced neuropathies, drug-induced neuropathies, vitamin deficiency-induced neuropathies; idiopathic neuropathies; and diabetic neuropathies. See e.g. US Patent Nos. 5,496,804 and 5,916,555.

We also contemplate the treatment of non-neuropathies, monomultiplex neuropathies and polyneuropathies, including axonal and demyelinating neuropathies, using the neu-blastin nucleotides and polypeptides as described herein.

In another preferred embodiment, the polypeptides and nucleic acids (and pharmaceutical preparations containing the same) are used in the treatment of various disorders of the eye, including photoreceptor loss in the retina in patients affected by macular degeneration, retinitis pigmentosa, glaucoma and similar diseases.

Another object described herein is to provide a method for preventing the degenerative changes associated with the above-mentioned diseases and disorders by implanting in the mammalian brain, including humans, vectors or cells capable of producing a biologically active form of neublastin or a precursor of neublastin, i.e. a molecule that can easily be converted into a biologically active form of neublastin by the body, or additional cells that secrete neublastin can be encapsulated, e.g. in semipermeable membranes.

Cells can be grown in vitro for use in transplantation or inoculation into mammalian brain, including humans.

In a preferred embodiment, the gene encoding the polypeptide as described herein is transfected into a suitable cell line, e.g. in an immortalized rat neural stem cell line such as HiB5 and RN33b or in a human immortalized neural progenitor cell line, and the resulting cell line is implanted into the brain of a living body, including a human, to secrete the therapeutic polypeptide of the invention into the CNS, e.g. by using the expression vectors described in International Patent Application WO 98/32869.

Methods for diagnosis and screening

A neublastin nucleic acid can be used to determine whether an individual is predisposed to developing a neurological disorder that results from a defect in the neublastin gene, e.g. a defect in a neublastin allele, which has been acquired e.g. by genetic inheritance, by abnormal embryonic development or by acquired DNA damage. The analysis can be e.g. by demonstrating one or more deletions or one or more point mutations in the neublast gene, or by demonstrating the inheritance of such predisposition to such genetic defects with specific restriction fragment length polymorphisms (RFLPs), by demonstrating the presence or absence of a normal neublast gene by hybridization of a nucleic acid sample from the patient with one or more nucleic acid probes specific for the neublast gene, and by determining the ability of the probe to hybridize to the nucleic acid.

A neublastin nucleic acid can in particular be used as a hybridization probe. Such hybridization analyzes can be used to detect, provide prognosis and diagnosis or monitor the various conditions, disorders or disease states that are associated with abnormal levels of the mRNAs that code for the neublastin protein. A neublastin nucleic acid can be engineered as a "marker" for neublastin-neurotrophic factor-dependent physiological processes. These processes include, but are not limited to, "normal" physiological processes (eg, nerve function) and pathological processes

(e.g. neurodegenerative disease). The characterization of one or more specific patient subpopulations with aberrant (that is, elevated or absent) levels of the neublastin protein and/or neublastin-encoding mRNA may lead to new disease classifications. By "abnormal levels" as defined herein is meant an increased or decreased level relative to the level in a control sample or in an individual who has not had the disorder determined by quantitative or qualitative means.

The neublastin nucleic acids and polypeptides of this invention can also be used to screen to identify neublastin analogs, including small molecule mimics of neublastin. In one contemplated embodiment, there is provided a method of identifying a candidate compound that induces a neublastin-mediated biological effect, the method comprising the steps of providing a test cell which, when contacted with neublastin, is induced to express a detectable product, exposing the cell to the candidate compound and detecting the detectable product. The expression of the detectable product provides an indication of the candidate compound's ability to induce the neublastin-mediated biological effect.

The neublastin nucleic acids and polypeptides of this invention can further be used on DNA chips or protein chips, or in computer programs to identify related novel gene sequences and proteins encoded by them, including allelic variants and single nucleotide polymorphisms ("SNPs"). Such methods are described e.g. in US Patent Nos. 5,795,716, 5,754,524, 5,733,729, 5,800,992, 5,445,934 and 5,525,464.

Examples

Example 1

Methods for the isolation of neublastin nucleic acids

Method 1: Rapid screening of human fetal brain cDNA with respect to the neublast gene

A 290 bp fragment was identified in two high-speed genome sequences (HGTS) submitted to GenBank (accession numbers AC005038 and AC005051) by its homology to human persephin. From the nucleic acid sequence of the 290 bp fragment, two neublastin-specific primers were synthesized. The neublastin top-strand primer ("NBNint.sense") had the sequence 5'-CCT GGC CAG CCT ACT GGG-3<1>[SEQ ID NO: 17]. The neublastin bottom strand primer ("NBNint.antisense") had the sequence 5'-AAG GAG ACC GCT TCG TAG CG-3' [SEQ ID NO: 18]. With these primers, 96-well PCR reactions were performed.

A 96-well master plate containing plasmid DNA from 500,000 cDNA clones was loaded with approximately 5,000 clones per well. well. A 96-well subplate was used with E. coli DH10B glycerol standard containing 50 clones per well.

A neublastin nucleic acid was identified by three rounds of amplification using polymerase chain reaction ("PCR") techniques; amplification increased the number of copies of the nucleic acid in the sample.

Master plate screening: Using the 96-well PCR screening technique described above, a human fetal brain cDNA master plate was screened with the gene-specific primers to isolate the human neublastin cDNA.

Thirty nanograms (30 ng) human fetal brain cDNA

(6 ng/ul, Origene Technologies) was obtained from the corresponding well of the master plate and placed in a total volume of 25 μl containing the following reagents: 0.2 mM each of the two previously mentioned gene-specific primers (ie NBNint.sense and NBNint.antisense), 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and 0, 5 units of Taq DNA polymerase (5 U/µl, Advanced Biotechnologies, UK).

PCR thermal cycling reactions were performed using the following procedure and conditions. DNA was first denatured at 94 °C for 3 min and then followed by 35 cycles of denaturation at 94 °C for 1 min each, annealing at 55 °C for 1 min, an initial extension at 72 °C for 90 s and a final extension at 72 °C for 5 minutes. The products of 96 individual PCR reactions were analyzed by gel electrophoresis using a 2% agarose gel containing ethidium bromide dye. The 102 bp positive PCR product seen in one well was found to correspond to a unique 96-well subplate.

The 102 bp nucleic acid fragment had the following sequence [SEQ ID NO: 13]:

Subplate screening: The 96-well human fetal brain subplate was screened using PCR-mediated amplification by placing 1 µl of the glycerol standard from the corresponding subplate well in a total volume of 25 µl containing: 0.2 mM each of the two gene-specific primers, 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and 0.5 units of Tag-DNA- polymerase (5 U/µl, Advanced Biotechnologies, UK).

The same PCR thermal cycling conditions as described for the master plate screening were used. The 96 individual PCR reaction mixtures were analyzed on a 2% agarose gel containing ethidium bromide, and a positive well was identified which gave the 102 bp PCR fragment.

Colony PCR: 1 ml of the glycerol standard from the positive subplate well was diluted 1:100 in Luria nutrient broth (LB). 1 ml and 10 ml of the above dilution were then plated onto two separate agar plates containing Luria nutrient broth ("LB") and 100 µg/ml carbenicillin. The LB plates were then incubated overnight at 30°C. From these plates, 96 colonies were picked into a new 96-well PCR plate containing: 0.2 mM of each of the two previously mentioned gene-specific primers, 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and 0.5 units of Tag-DNA polymerase (5 U/ul, Advanced Biotechnologies, UK) in a final volume of 25 ul.

The same PCR thermal cycling conditions as described for the master plate screening were used. The 96 individual PCR reaction mixtures were then analyzed on a 2% agarose gel containing ethidium bromide. A positive colony containing the 102 bp fragment was then identified.

Sequencing of the plasmid DNA prepared from this positive colony revealed a full-length cDNA of 861 bp [SEQ ID NO: 8]. The cDNA encoded a pre-pro-neublastin [SEQ ID

NO: 9]. Automated DNA sequencing was performed using the "BigDye" terminator cycle sequencing kit (PE Applied Biosystems, USA). The sequencing gels were run on an ABI Prism 377 (PE Applied Biosystems, USA).

Method 2: Cloning of neublastin cDNA from human brain

An additional method for amplifying the full-length neublastin cDNA or the cDNA fragment can be performed using RACE ("Rapid Amplification of DNA ends") and the neublastin-specific primers NBNint.sense and NBNint.antisense described above, combined with vector-specific or adapter specific -got primers, e.g. by using the Marathon cDNA amplification kit (Clontech Laboratories, USA, cat. no. K1802-1).

Whole human brain Marathon-Ready cDNA (Clontech Laboratories, USA, catalog no. 7400-1) can be used to amplify the full-length neublastin cDNA. Useful primers for amplification include a neublastin top strand primer, 5'-ATGGAACTTGGACTTGG-3' [SEQ ID NO: 19] ("NBNext.sense"), and a neublastin bottom strand primer, 5'-TCCATCACCCACCGGC-3' [SEQ ID

NO: 20] ("NBNext.antisense"), combined with the adapter primer API included with the Marathon-Ready cDNA. An alternative top-strand primer has also been used, 5'-CTAGGAGCCCATGCCC-3'

[SEQ ID NO: 28]. A further alternative bottom strand primer, 5'-GAGCGAGCCCTCAGCC-3' [SEQ ID NO: 33] can also be used. Likewise, alternative bottom strand primers, SEQ ID NOS: 24 and 25, can also be used.

Method 3: Cloning of neublastin cDNA from human brain

Another method for cloning neublastin cDNA is by screening adult or fetal brain libraries with one or more neublastin probes described here (and as exemplified in figure 1). These libraries include: A,gtll human brain (Clontech Laboratories, USA, cat. no. HL3002b) or Å.gtll human fetal oster brains (Clontech Laboratories, USA, cat. no. HL-3002b).

Method 4: Rapid screening of mouse fetal cDNA with regard to the neublast gene

A rapid screening procedure for the neublast gene was performed as follows. A 96-well master plate containing plasmid DNA from 500,000 cDNA clones was filled with approximately 5,000 clones per well. well. A 96-well subplate was used with E. Coli glycerol standard containing 50 clones per well. Three rounds of PCR-mediated amplification were performed to identify a gene of interest (ie, neublastin).

Master plate screening: A mouse fetal cDNA master plate was screened by 96-well PCR using gene-specific primers to isolate the mouse neublastin cDNA. The following two primers were synthesized:

(1) neublastin-C2 primer (NBNint.sense): 5'-GGCCACCGCTCCGACGAG-3' [SEO ID NO: 21]; and (2) neublastin-C2as primer (NBNint.antisense): 5'-GGCGGTCCACGGTTCTCCAG-3' [SEQ ID NO: 22]. By using these two gene-specific primers, a 220 bp positive PCR product was identified. The 220 bp nucleic acid had the following sequence [SEQ ID NO: 14]:

96-well PCR reactions were then performed as follows. 30 ng of mouse fetal brain cDNA (6 ng/µl, Origene Technologies) was obtained from the corresponding well of the master plate and placed in a total volume of 25 µl which also contained: 0.2 mM of each of the two previously mentioned gene-specific primers (i.e. C2 -primer (NBNint.sense) and neublastin-C2as primer

(NBNint.antisense)), 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and

0.5 units Ta g-DNA polymerase (5 U/pl, Advanced Biotechnologies, UK).

The following PCR heat cycling conditions were used: An initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min each, annealing at 55 °C for 1 min, extension at 72 °C for 90 seconds and a final extension at 72 °C for 5 minutes. The

The 96 individual PCR reaction mixtures were analyzed on a

2% agarose gel containing ethidium bromide dye. The 220 bp

large positive PCR product seen from one well was found to correspond to a unique 96-well subplate. The 96 individual PCR reaction mixtures were then analyzed by gel electrophoresis on a 2% agarose gel containing ethidium bromide dye. The 220 bp positive PCR product that had been identified corresponded to a unique well in a 96-well subplate.

Subplate screening: The 96-well mouse embryo subplate was screened by PCR-mediated amplification by placing 1 µl of the glycerol standard from the corresponding subplate well in a final total volume of 25 µl containing: 0.2 mM of each of the two previously mentioned gene-specific primers, 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and 0.5 units Taq DNA polymerase (5 U/µl, Advanced Biotechnologies, UK). The PCR heat cycle was performed according to the conditions described above for the master plate screening.

The individual 96 PCR reaction mixtures were then analyzed on a 2% agarose gel containing ethidium bromide, and a positive well that produced the 220 bp fragment was identified.

Colony PCR: 1 ml of the glycerol standard from the positive subplate well was diluted 1:100 in Luria nutrient broth (LB). 1 ml and 10 ml of the aforementioned dilution were then plated on two separate LB plates, containing 100 µg/ml carbenicillin, and incubated at 30 °C overnight. A total of 96 colonies were isolated and transferred to a 96-well PCR plate containing: 0.2 mM of each of the two previously mentioned gene-specific primers, 1 x standard PCR buffer (buffer V, Advanced Biotechnologies, UK), 0, 2 mM dNTPs (Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA) and

0.5 units of Tag-DNA polymerase (5 U/ul; Advanced Biotechnologies, UK) in a final volume of 25 µl.

PCR thermal cycling was performed according to the conditions described above (see “master plate screening”). The 96 individual PCR reaction mixtures were analyzed by gel electrophoresis on a 2% agarose gel containing ethidium bromide. A positive colony was identified by the presence of the 220 bp fragment. Plasmid DNA was prepared from this positive colony. The clone was sequenced by automated DNA sequencing using the "BigDye" terminator cycle sequencing kit with AmpliTaq DNA polymerase. The sequencing gels were run on ABI Prism 377

(PE Applied Biosystems). The resulting sequence from this clone revealed a full-length cDNA of 2136 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 a mouse pre-pro-neublastin polypeptide.

Example 2

Cloning of genomic neublastin

As discussed above, applicants identified a 290 bp nucleic acid fragment in two human BAC clones deposited in GenBank (with accession numbers AC005038 and AC005051) that had regions of homology to persephin and to the flanking sequences of persephin. Applicants used the 861 bp predicted sequence described above to design additional primers, with the goal of cloning a nucleic acid encoding additional neublastin nucleic acids using Lasergene Software (DNAStar, Inc.). Two pairs of primers were used to clone the neublast gene by applying PCR reactions to genomic DNA. The two pairs of primers are illustrated below.

Primer pair #1

5' CCA AgC CCA CCT ggg TgC CCT CTT TCT CC 3' (sense) [SEQ ID NO: 23].

5' CAT CAC CCA CCg gCA ggg gCC TCT CAg 3<*>(antisense) [SEQ ID NO: 24].

Primer pair #2

Using primer pair no. 1, an 887 bp DNA fragment was amplified from a preparation of human genomic DNA purchased from Clontech Laboratories (cat. no. 6550A-1).

PCR procedure: PCR was performed using the Expand High Fidelity PCR system (Boehringer Mannheim) with buffer 1. The PCR reaction mixture was supplemented with 5% dimethyl sulfoxide (DMSO) and 17.5 pmol of each dNTP in a total volume of 50 pl. Thermal cycling was performed with a pre-denaturation step at 94°C for 2 minutes, followed by 35 two-step cycles at 94°C for 10 seconds and 68°C for 1 minute, respectively. Heat cycling was terminated by incubation at 68°C for 5 minutes. Thermal cycling was performed in a PTC-225 DNA Engine Tetrad thermocycler (MJ Research, MA). The PCR products were analyzed by gel electrophoresis on 2% agarose (FMC) and then photographed.

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

Fragments obtained by PCR amplification of human genomic DNA with the second pair of primers (primer pair #1 above) were sequenced, and an additional 42 bp region at the 3'-prime end of the open reading frame was revealed. The full-length sequence was analyzed by comparing it to the sequences of nucleic acids of other neurotrophic factors, as well as by mapping exon-intron boundaries using computer programs to find genes identifying likely splice junctions and regions of high-coding potential using Netgene and Gene Mark software (Brunak et al., J. Mol. Biol., 1991, 220, 49-65); Borodovsky et al., Nucl. Acids Res., 1995, 23, 3554-62). The exon-intron boundaries were confirmed using the cDNA obtained from the Rapid Screen described above.

As illustrated in Figure 7, the resulting neublast has two exons separated by a 70 bp intron. Together, the exons have a predicted amino acid sequence of a full-length neublastin polypeptide. The predicted cDNA [SEQ ID

NO: 3] contains an open reading frame (ORF) which codes for

238 amino acid residues [SEQ ID NO: 4]. The neublastin-2 clone contained the complete coding sequence for pro-neublastin. The amino acid sequence encoded by the gene showed high homology to three proteins, persephin, neurturin and GDNF.

Example 3

Expression of neublastin nucleic acids

Expression of neublastin RNA was detected in both neural and non-neural tissues of rodents and humans, and at various developmental immature and adult stages, using the techniques described below.

Method for detection of neublastin RNA expression using RT-PCR: Based on the neublastin DNA sequence identified as SEQ ID NO: 1, the following primers were synthesized: (1) a neublastin C2 primer, 5'-GGCCACCGCTCCGACGAG -3'

[SEQ ID NO:21], and (2) a neublastin-C2as primer, 5'-GGCGGTCCACGGTTCTCCAG-3' [SEQ ID NO: 22]. This primer set was used to RT-PCR amplify a DNA fragment from adult and fetal human whole brain mRNA. Among the DNA fragments produced by this reaction was one of 220 bp. Identification of this 220 bp DNA fragment confirmed that the neublast gene is expressed in adult and fetal brain tissue. A 220 bp DNA fragment was also amplified from genomic DNA using these primers.

Method for detection of neublastin RNA expression using northern blot hybridization: Northern blots with polyA<+->RNA from adult human tissue were purchased from a commercial supplier (Clontech Laboratories, USA) and probed with a<32>P-label neublastin cDNA. The labeled neublastin cDNA was prepared according to the methods described in Example 1 above.

Preparation of Probes: A neublastin nucleic acid DNA fragment (nucleotides 296-819 of SEQ ID NO: 8) was labeled using the Rediprime II Labeling Kit (Amersham, Cat. No. RPN1633) for use as a hybridization probe, as recommended by the manufacturer . Briefly, the DNA sample was diluted to a concentration of 2.5-25 ng in 45 µl 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 boiling water bath, rapidly cooling the sample by placing it on ice for 5 minutes and then briefly centrifuging it to bring the contents to the bottom of the the reaction tube. The total amount of denatured DNA was added together with 5 µl of Redivue [<32>P]-dCTP (Amersham Pharmacia Biotech Ltd.) in the reaction tube containing buffered solution of dATP, dGTP, dTTP, exonuclease-free Klenow enzyme and arbitrary primer in the dried stabilized form. The solution was mixed by pipetting up and down twice, moving the pipette tip around in the solution, and the reaction mixture was incubated at 37°C for 10 minutes. The labeling reaction was stopped by adding 5 µl of 0.2 M EDTA. For use as a hybridization probe, the labeled DNA was denatured into single strands by heating the DNA sample to 95-100°C for 5 minutes and then rapidly cooling the DNA sample on ice for 5 minutes. The tube was centrifuged and its contents mixed well. Finally, the single-stranded DNA probe was purified using the nucleotide removal kit (Qiagen).

Hybridization techniques: Prepared northern blots were purchased from a commercial supplier ("Multiple Tissue Northern Blots, Clontech Laboratories, USA, catalog nos. 7760-1 and 7769-1) and were hybridized according to the manufacturer's instructions using the neublastin-<32> P-labeled probe prepared above. For hybridization, ExpressHyb solution (Clontech Laboratories, USA) was used, and a concentration of about 3 ng/ml of the labeled probe was used. The ExpressHyb solution was heated to 68 °C and then stirred to dissolve any precipitate. Each northern blot membrane (10 x 10 cm) was prehybridized in at least 5 ml of ExpressHyb solution at 68°C for 30 minutes in a Hybaid hybridization oven according to the manufacturer's instructions. The neublastin -32P-labeled probe was denatured at 95-100 °C for 2 min and then rapidly cooled on ice. Fourteen microliters (14 µl) of the labeled probe was added to 5 ml of fresh ExpressHyb and thoroughly mixed. The ExpressHyb solution which was used in the pre-hybridization, was replaced by evenly distributing over the blots the 5 ml of new ExpressHyb solution containing the labeled DNA probe. The blots were incubated at 68°C for 1 hour in a Hybaid hybridization oven. After incubation, the blots were rinsed and washed several times at low stringency (2 x SSC buffer containing 0.05% SDS at room temperature), followed by a high stringency wash (0.1 x SSC containing 0.1% SDS at 50 C) [20 x SSC is 0.3 M NaC/0.3 M Na citrate, pH 7.0]. Blots were exposed to a Hyperfilm MP (Amersham Pharmacia Biotech, Ltd.) at -80°C using intensifying screens.

The results of the northern blot hybridization experiments are shown in Figure 1. Figure IA (left) and Figure IB (right) are northern blots of polyA<+->RNA probed with <32>P-labeled neublastin cDNA, as described in Example 3. The markers represent polynucleotides of 1.35 kilobase pairs ("kb"), 2.4 kb, 4.4 kb, 7.5 kb and 9.5 kb in size. The membrane in Figure IA was prepared with mRNA extracted from various adult human tissues: From the results of the northern blot hybridization analysis, the applicants conclude that neublastin mRNA is expressed in many adult human tissues. The highest level of neublastin expression is detected in the heart, skeletal muscle and pancreas. The membrane in Figure 1B was prepared with RNA extracted from various regions of the adult human brain. In the adult brain, the highest expression level is seen in the caudate nucleus and in the thalamus. An mRNA transcript of approximately 5 kb was the predominant form of neublastin mRNA expressed in the brain.

Method for detection of neublastin RNA expression using in situ hybridization in tissue

The following techniques are used to measure the expression of neublastin RNA in animal tissue, e.g. rodent tissue, with a neublastin antisense probe.

Expression in mice

Preparation of tissue samples: Time-pregnant mice (B&K Universal, Stockholm, Sweden) were euthanized by cervical dislocation on day 13.5 or 18.5 after onset of pregnancy. Embryos were removed by dissection under sterile conditions and immediately immersed in a solution of 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde ("PFA") for 24-30 hours and then removed from PFA and stored in PBS. The tissue was prepared for sectioning by immersing the tissue in a solution of 30% sucrose and then encapsulating it in TissueTech (O.C.T. Compound, Sakura Finetek USA, Torrance, CA). Six series of coronal or sagittal sections (12 µm each) were cut on a cryostat and thaw-mounted on positively charged slides. Neonatal heads/brains (P1, P7) were fixed following the same procedure as for the embryonic stages, and adult brain tissue was dissected, immediately frozen on dry ice and cut on a cryostat without any prior packaging.

Preparation of neublastin riboprobes: An antisense neublastin RNA probe (hereafter a "neublastin riboprobe") was made as follows. Nucleotides 1109-1863 of the mouse neublastin cDNA sequence [SEQ ID NO: 15] were subcloned into the BlueScript vector (Stratagene). The resulting plasmid was cut into a linear DNA using EcoRI restriction endonuclease. The £coRI DNA fragment was transcribed in vitro with T3 RNA polymerase and digoxigenin ("DIG") RNA labeling kit according to the manufacturer's instructions (Boehringer Mannheim).

Hybridization: Cryostat sections were fixed for 10 min in 4% PFA, treated for 5 min with 10 mg/ml proteinase K, dehydrated sequentially in 70% and 95% ethanol for 5 and 2 min, respectively, and then allowed to air dry. Hybridization buffer (50% deionized formamide, 10% of a 50% dextran/sulfate solution, 1% Denhardt's solution, 250 µg/ml yeast tRNA, 0.3 M NaCl, 20 mM Tris-HCl (pH 8), 5 mM EDTA, 10 mM NaPO 4 , 1% sarkosyl) containing 1 µg/ml of the DIG-labeled probe was heated to 80 °C for 2 min and applied to the sections. The sections were then covered with parafilm and incubated at 55°C for 16-18 hours.

The next day, the sections were washed at high stringency (2 x SSC containing 50% formamide) at 55°C for 30 minutes and then washed in RNase buffer and incubated with 20 µg/ml RNaseA for 30 minutes at 37°C. To detect the DIG-labeled probe, sections were preincubated in 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 a 1:5000 dilution of alkaline phosphatase-coupled anti-DIG antibody (Boehringer Mannheim). The following day, each section was given four 2-hour washes in PBS containing 0.1% Tween-20 and then given two 10-minute washes in NTMT buffer (100 mM NaCl,

100 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 0.1% Tween-20). The sections were then incubated in BM violet substrate containing 0.5 mg/ml levamisole for 48 hours. The color reaction was stopped by washing in PBS. The sections were air-dried and covered with coverslips with DPX (KEBO-lab, Sweden).

The results of the in situ hybridization reactions are shown in Table 1.

As shown in Table 1, at embryonic day 13.5 ("E13.5") neublastin was expressed in the spinal cord and hindbrain, and weakly in the forebrain. Neublastin expression was also detected in the developing retina and in the sensory ganglia (dorsal root ganglia and trigeminal ganglia (V)). Outside the nervous system, a weak signal was found in the kidney, lungs and small intestine, indicating that neublastin is also expressed in those tissues.

At embryonic day 18.5 ("E18.5"), neublastin was expressed most prominently in the trigeminal ganglion (V). Neublastin expression was also detected in the retina, striatum and cerebral cortex. In addition, expression was seen in dentition.

Again referring to Table 1, increased neublastin expression from the E18.5 time point to postnatal days 1 and 7 was seen in the cerebral cortex, striatum and trigeminal ganglion (V). Neublastin expression was more prominent in the outer layers of the cerebral cortex than in the inner layers of the cerebral cortex. At P7, expression was found in the same structures as on day 1, but in addition, neublastin expression was found in the hippocampus, especially in the dentate gyrus and in the cerebellum. In the adult murine brain, neublastin was highly expressed in serrated convolutions, with very low or undetectable levels of neublastin expression detected in other tissues tested.

Expression in the rat

The following experiment describes the hybridization of rat tissue with an alkaline phosphatase-labeled oligodeoxynucleotide neublastin antisense probe.

Preparation of tissue samples: Rat embryos (E14) were obtained from pregnant Wistar rats (Mollegaard Breeding, Denmark) after pentobarbital anesthesia. Postnatal rats (PO, P7, adults) were euthanized by decapitation. Dissected brains and whole heads were immediately immersed in cold 0.9% NaCl, frozen and sectioned at 20 µm on a cryostat (coronal and sagittal sections, 10 series).

In Situ Hybridization: Two series of sections were hybridized using an antisense alkaline phosphatase-(AP)-conjugated oligodeoxynucleotide probe (5'-NCA GGT GGT CCG TGG GGG GCG CCA AGA CCG G-3' [SEQ ID NO: 27] , oligo no.

164675, DNA Technology, Denmark). This probe is complementary to bases 1140-1169 of the mouse neublastin cDNA according to SEQ ID NO: 15.

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

Post-hybridization treatment consisted of four

30 min rinses in 1 x SSC (0.15 M NaCl, 0.015 M Na citrate) at 55 °C, followed by three 10 min rinses in Tris-HCl, pH 9.5, at room temperature before addition of AP developer. AP developer was prepared immediately before use and contained nitro blue tetrazolium (NBT, Sigma), 5-bromo, 4-chloro, 3-indolyl phosphate (BCIP, Sigma) and Tris-HCl-MgCl2 buffer, pH 9.5 (Finsen et al., Neurosci., 1992, 47, 105-113). AP development took place in the dark at room temperature for 48 h. The color reaction was stopped by rinsing the sections in distilled water. The sections were dehydrated in graded acetone, softened in xylene-phenol-creosote (Allchem, UK), cleared in xylene and placed under glass coverslips using Eukitt (Bie&Berntsen, Denmark).

Control reactions consisted of (1) pretreatment of the sections with RNase A (50 ug/ml, Pharmacia, Sweden) before hybridization; (2) hybridization of the sections with a 100-fold excess of unlabeled probe; and (3) hybridization of the sections with hybridization buffer alone. The results of the hybridization reactions are shown in Table 2.

On embryonic day 14 (E14), neublastin was weakly expressed in rat embryos in the forebrain, hindbrain and spinal cord. Neublastin mRNA was also detected in the eye (retina), dorsal root ganglia, trigeminal ganglia (V) and in the kidneys, lungs, heart, liver and intestines. In newborn (PO) rats, there was marked neublastin expression in the spinal cord and in the striatum. Neublastin expression was also detected in the olfactory bulb and in the hippocampus. In 7-day-old (P7) rats, neublastin was expressed in the cerebral cortex, striatum, olfactory bulb and in the cerebellum. A marked signal could be seen in the hippocampus. In adult rats, very low or undetectable levels of neublastin expression were detected in most regions of the brain. Weak signals were detected in the thalamic nucleus, and marked neublastin expression was detected in the hippocampus.

Example 4

Neublastin polypeptides

The open reading frame or coding region (CDS) identified in SEQ ID NO: 8 encodes the pre-pro polypeptide (designated "pre-pro-neublastin"). The amino acid sequence predicted from this open reading frame is shown in SEQ ID NO: 9. Based on SEQ ID NO: 9, three variants of neublastin polypeptides were identified. These variants include:

(i) the polypeptide designated herein as NBN140, having the amino acid sequence designated as SEQ ID NO: 10; (ii) the polypeptide designated herein as NBN116, having the amino acid sequence designated as SEQ ID NO: 11; and (iii) the polypeptide designated herein as NBN113, having the amino acid sequence designated as SEQ ID NO: 12.

Based on the coding region (CDS), as identified in SEQ ID NO: 3, which encodes the pre-pro polypeptide having the amino acid sequence designated as SEQ ID NO: 4, three variants of neublastin were also identified. These variants include: (i) the polypeptide having the amino acid sequence designated as SEQ ID NO: 5; (ii) the polypeptide having the amino acid sequence designated as SEQ ID NO: 6; and (iii) the polypeptide having the amino acid sequence designated as SEQ ID NO: 7.

Based on clustal W (1.75) based multiple sequence alignment, SEQ ID NO: 9 was aligned with the amino acid sequences of GDNF, persephin and neurturin. This composition is illustrated in table 3.

From the amino acid sequence assembly shown in Table 3, it can be seen that neublastin has seven conserved cysteine residues at sites that are conserved within the TGF-β superfamily. In one embodiment, the preferred neublastin polypeptide contains seven cysteines conserved as in SEQ ID NO: 2 at positions 8, 35, 39, 72, 73, 101 and 103, or as in SEQ ID NOS: 4 and 9 at positions 43, 70, 74 , 107, 108, 136 and 138. These seven conserved cysteine residues are known within the TGF-β superfamily to form three intramonomer disulfide bonds (as can be seen, for example, in SEQ ID NO: 2 between cysteine residues 8-73, 35-101 and 39-103, and e.g. in SEQ ID NOS: 4 and 9 between cysteine residues 43-108, 70-136 and 74-138) and one intermonomer disulfide bond (which can be seen e.g. in SEQ ID NO: 2 between cysteine residues 72-72, and for example in SEQ ID NOS: 4 and 9 between cysteine residues 107-107), which together with the extended beta-strand region constitute the conserved structural motif of the TGF-β superfamily. See e.g. Daopin et al., Proteins, 1993, 17, 176-192.

Based on this sequence assembly, neublastin was shown to be a member of the GDNF subfamily of neurotrophic factors (LGLG - FR(Y/F)CSGSC - QxCCRP - SAxxCGC, the GDNF subfamily fingerprint, underlined in Table 3).

The homology between neublastin and other members of the GDNF family was calculated and the results are shown in Table 4 below.

Example 5

Production of neublastin

We have produced neublastin in both eukaryotic and prokaryotic cells, as described below.

Expression vectors. The full-length cDNA encoding neublastin was inserted into the eukaryotic expression vector pUbilz. This vector was generated by cloning the human UbC promoter into a modified version of pcDNA3.1/Zeo. The unmodified pcDNA3.1/Zeo is commercially available (Invitrogen). The modified pcDNA3.1/Zeo is smaller than the parent vector because the ampicillin gene (from position 3933 to 5015) and a sequence from position 2838 to 3134 were removed. In this modified version of pcDNA3.1/Zeo, the CMV promoter was replaced with the UbC promoter from pTEJ-8 (Johansen et al., FEBS Lett, 1990, 257, 289-294), resulting in pUbilz.

Mammalian cell expression. The pUbilz vector, which contained neublastin coding sequences, was then transfected into the mammalian cell line HiB5, which is an immortalized rat nerve cell line (Renfranz et al., Cell, 1991, 66, 713-729). Several HiB5 cell lines stably expressing neublastin (as determined by RT-PCR) have been established. In one of these stable cell lines, HiB5pUbilzNBN22 expression was confirmed by hybridization of 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 a neublastin source for studies of neurotrophic neublastin activity.

Figure 2 shows the expression of neublastin cDNA in the HiB5pUbilzNBN22 clone (ie northern blot probed with 32 P-labelled neublastin cDNA according to the present invention, as described below). The blot was prepared using total RNA extracted from untransfected HiB5 cells, HiB5pUbilzNBN22 cells and HiB5pUbilzGDNF14, respectively, as indicated). The positions of the 28S and 18S rRNA bands corresponding to 4.1 kb and 1.9 kb, respectively, are indicated on the blot.

As shown in Figure 3, antibodies raised against neublastin-derived polypeptides also recognized an approximately 13 kilodalton ("kD") protein in conditioned medium from the HiB5pUbilz-NBN22 clone, but not from non-transfected HiB5 cells (see Example 6).

The predicted molecular weights of the unmodified (ie, lacking 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, respectively. .7 kilodaltons ("kD"), 12.4 kD and 12.1 kD.

Methods: A northern blot of total RNA (10 µg) from untransfected HiB5 cells and the HiB5pUbilzNBN22 clone was prepared by electrophoresis on a 0.8% formaldehyde-agarose gel 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 arbitrary labeling covering SEQ ID NO: 8 and additional nucleotides from the 5'UTR and 3<1>UTR in the neublastin cDNA. The blot was exposed to a Hyperfilm MP (Amersham) at -80°C using intensifying screens.

Conditioned medium from HiB5pUbilzNBN22 or untransfected HiB5 cells incubated overnight in serum-free medium supplemented with N2 supplement (Life Technologies, cat. no. 17502-048) was concentrated and separated on 15% polyacrylamide gels (Amersham Pharmacia Biotech, cat. no. 80 -1262-01). Proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech, cat. no. RPN-303F), and nonspecific protein binding sites were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20. Membranes were incubated overnight with a polyclonal neublastin antibody (1:1000), followed by incubation with a secondary antirabbit IgG antibody (Amersham Pharmacia Biotech, cat. no. NA 934) conjugated to horseradish peroxidase (1:2000). Immunostaining was visualized using enhanced chemiluminescence (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 illustrations of the expression of neublastin protein in transfected HiB5 cells. Overnight medium from untransfected HiB5 cells [lane 1] or from a HiB5 clone stably transfected with neublastin cDNA

[lane 2], was concentrated as described below. The medium was then analyzed by western blotting using two different polyclonal antibodies, Ab-1 and Ab-2 described in Example 10, which are specific for neublastin. In the medium derived from transfected cells, both antibodies were found to recognize a protein with a molecular weight of approximately 15 kDa. This protein was not seen in non-transfected HiB5 cells.

The cloned cDNA encoding neublastin can also be inserted into another eukaryotic expression vector, e.g. the eukaryotic expression vector TEJ-8 (Johansen et al., FEBS Lett., 1990, 267, 289-294) or pcDNA-3 (Invitrogen), and the resulting expression plasmid is transfected into an alternative mammalian cell line, e.g. Chinese hamster ovary ("CHO") cells, the HEK293, COS, PC12, or RN33b cell lines, or human neural stem cells. Stable cell lines expressing neublastin are used, e.g. to produce the neublastin protein.

Expression in CHO cells

Construction of plasmid pJC070. 14. To express

the neublastin cDNA in Chinese hamster ovary cells, a cDNA fragment encoding the pre-pro form of human neublastin was inserted into the mammalian expression vector pEAG347 to generate plasmid pJC070.14. pEAG347 contains the tandem SV40 early and adenovirus major late promoters (derived from plasmid pAD2beta, Norton and Coffin, Mol. Cell. Biol., 1985, 5, 281), a unique NotI cloning site, followed by SV40 late transcription- termination and polyA signals (derived from plasmid pCMVbeta, MacGregor and Caskey, Nucl. Acids. Res., 1989, 17, 2365). In addition, pEAG347 contains a pUC19-derived plasmid backbone and a pSV2dhfr-derived dhfr for MTX selection and amplification in transfected CHO cells.

Plasmid pJC070.14 was generated in two steps. First, a fragment encoding the pre-pro form of human neublastin was isolated from plasmid pUbilz-NBN using the polymerase chain reaction with the oligonucleotides KD2-824 5<1>AAGGAAAAAA GCGGCCGCCA TGGAACTTGG ACTTGGAGG3' [SEQ ID NO: 31], KD2- 825

5'TTTTTTCCTT GGCGGCGCT CAGCCCAGGC AGCCGCAGG3' [SEQ ID NO: 32]

and PFU polymerase. The fragment was cloned into the Srf-1 site in pPCR-Script Amp SK(+) to generate plasmid pJC069. In the second step, a partial Not-1 digestion was performed on plasmid pJC069 to generate a 685 bp fragment (containing the neublast gene) which was cloned into the Not-1 site of plasmid pEAG347 to generate plasmid pJC070.14. Transcription of the neublast gene in plasmid pJC070.14 is controlled by the adenovirus major late promoter.

Generation of CHO cell lines expressing neublastin. 200 µg of pJC070.14 was linearized by digestion with the restriction endonuclease Mlu-1. The DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated. The linearized DNA was resuspended in 20 mM Hepes,

pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 6 mM dextrose (HEBS) and introduced into ~4E7 CHO dukx Bl(dhfr-) cells (p23) by electroporation (280 V and 960 uF) . After electroporation, cells were returned to culture in a+ modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) for two days. The cells were then trypsinized and replated in 100 mm dishes (100,000 cells/plate) in α-MEM (lacking ribo- and deoxyribonucleosides), supplemented with 10% dialyzed FBS, for 5 days. The cells were then split at a density of

100,000 cells/100 mm plate and selected in 200 nM methotrexate. Resistant colonies were picked and scaled up to 6-well plates; conditioned medium from each clone was screened using a specific assay for neublastin, described below. The 12 clones expressing the highest level of neublastin were scaled up into T162 flasks and then re-assayed. As shown in Figure 10, the CHO cell lines produced neublastin in the range of 25-50 ng/ml.

Ternary complex assay for neublastin. We assayed for the presence of neublastin in the medium of CHO cell line supernatants using a modified form of a ternary complex assay described by Sanicola et al (Proe Nati Acad Sei USA, 1997, 94, 6238).

In this assay, the ability of GDNF-like molecules can be evaluated with respect to their ability to mediate binding between the extracellular domain of RET and the various coreceptors, GFRa1, GFRa2 and GFRa3. Soluble forms of RET and the coreceptors were generated as fusion proteins. A fusion protein between the extracellular domain of rat RET and uterine alkaline phosphatase (RET-AP) and a fusion protein between the extracellular domain of rat GFRα1 (described in published application WO 97/44356, November 27, 1997) and the Fc domain in human IgG1 (rGFRα-Ig) has been described (Sanicola et al., Proe Nati Acad Sei USA, 1997, 94, 6238).

To generate a fusion protein between the extracellular domain of murine GFRα3 and the Fc domain of human IgGl (mGFRα3-Ig), a DNA fragment encoding amino acids 1-359 of murine RETL3 was ligated to a fragment containing the Fc domain of human IgG1 and cloned into the expression vector pEAG347 to generate plasmid pGJ144. Plasmid pGJ144 was transfected into Chinese hamster ovary (CHO) cells to generate a stable cell line producing the fusion protein, which was purified on a protein A-Sepharose immunoaffinity column using standard methods. It can be summarized that if the GDNF-like molecule can mediate binding of the co-receptor to RET in this assay, then the RET-AP fusion protein will be retained on the plate, and the amount retained can be measured by using a chemiluminescent substrate for alkaline phosphatase.

Dynex Microlite-1 ELISA plates (Dynex Technologies) were coated with 1 mg/ml goat antibody specific for human Fc in 50 mM bicarbonate/carbonate, pH 9.6, for 16 h. The plates were emptied and filled with 300 µl of 1% 1-block (Tropix) in TBS/0.5% Tween-20 (TBST) for 1 hour. After washing three times with TBST, the wells were filled with 100 µl of 1 µg/ml rGFRα1-Ig or mGFRα3-Ig diluted in conditioned medium from 293-EBNA cells expressing the RET-AP fusion gene. 100 µl of conditioned medium from the CHO neublastin clones was then added to the top well of a column of wells, and two-fold serial dilutions were made with each row of wells, and incubated for 1.5 hours at room temperature. The plates were then washed three times with TBST and twice with 200 mM Tris, pH 9.8, 10 mM MgCl 2 (CSPD buffer). The wash solution was then replaced with 425 µM CSPD (Tropix) in CSPD buffer containing 1 mg/ml Sapphire chemiluminescence enhancer (Tropix) and incubated for 30 minutes at room temperature. The chemiluminescence result was measured using a Dynatech luminometer.

In the initial experiments, we investigated whether neublastin produced using the CHO cell lines could mediate the binding of GFRa1 or GFRa3 to the extracellular domain of RET. As shown in Figure 11, conditioned medium from CHO cell clone #53 gave a robust signal in the ternary complex assay when the mGFRα3-Ig fusion protein was included, but no signal when the rGFRα1-Ig fusion protein was included, indicating that neublastin binds to GFRα3, but not to GFRα1. This behavior clearly distinguishes neublastin from GDNF; as shown in Figure 11, GDNF binds to GFRa1 but not to GFRa3. No coreceptor fusion protein signal was observed when conditioned medium from the parent CHO cell line or pure medium was analyzed.

To quantify the expression levels of neublastin in the CHO cell lines, a standard curve was prepared using rGFRα1-Ig and GDNF, starting at a concentration of 1 ng/ml. Neublastin concentrations for the various CHO cell lines were then calculated using this standard curve; the levels produced using five CHO cell lines are shown in Figure 10. As this calculation depends on the untested assumption that the binding affinity between GDNF and GFRα1 is equal to the binding affinity between neublastin and GFRα3, these levels are only approximate.

Analysis of neublastin from CHO cell line supernatants

To further analyze the neublastin produced by the CHO cell lines, the protein was extracted from the medium, using the GFRα3-Ig fusion protein and analyzed by western blots with polyclonal antibodies raised against neublastin peptides.

In the first experiment, the neublastin was extracted with mGFRα3-Ig attached to Sepharose beads. mGFRa3-Ig was attached to Sepharose beads using the conditions suggested by the manufacturer, Pharmacia Inc. 100 µl of mGFRa3-Ig-Sepharose was added to 1.0 ml samples of conditioned medium from a negative control CHO cell line or from the neublastin-producing CHO cell line #16. The suspensions were incubated for 2 hours on a rocking platform. Each suspension was centrifuged and the supernatant removed, followed by three 1.0 ml washes with 100 mM HEPES, 100 mM NaCl, pH 7.5. Each resin was resuspended in 100 µl of 2x reducing sample buffer and heated to 100°C for 5 minutes. 20 µl of the sample buffer supernatant and 10 µl of a molecular weight standard (FMC) were added to each well in a 10-20% precast SDS-PAGE gel (Owl Scientific). The gel was electrophoresed at 40 mA constant current for 72 minutes.

For western blot analysis, the protein was electroblotted to nitrocellulose (Schleicher and Schuell) in a Hofer Scientific apparatus in a buffer system of 10 mM CAPS, 10% methanol, 0.05% SDS, pH 11.2 (45 min at 400 mA constant current-strength) . After the 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 parts, and the excess dye was removed by gentle shaking in distilled water. The membranes were blocked in 2% nonfat dry milk in TBS overnight at 4 °C. The membranes were incubated individually with two of the affinity-purified antineublastin peptide antibodies (R30 and R31) at a concentration of 1.0 µg/ml in 2% nonfat dry milk in TBS). The membranes were washed with three 10 minute washes in TBS-Tween and incubated in a 1:5000 dilution of goat antirabbit IgG-HRP conjugate (Biorad) for 30 minutes. The membranes were washed with three 10 minute washes of TBS-Tween and developed with ECL substrate (Amersham). As shown in Figure 12, specific bands were detected in the proteins extracted from the neublastin-producing CHO cell line with both antibodies (lanes 2 and 4) when compared to the bands observed in the extracted proteins from the negative control cell line (lanes 1 and 3).

The molecular weight of the lower species is approx. 13 kD and probably constitutes the mature domain of neublastin, generated after cleavage of the pro-domain. This cleavage can occur after any of the three Arg (eg, -RXXRJ'-) residues in the pre-pro-neublastin protein to produce either the 140AA, 116AA, or 113AA forms, as indicated in SEQ ID NO, respectively NO: 10, 11 or 12. The predicted molecular weights of the unmodified (ie, lacking 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, respectively. Further analysis will be necessary to confirm the structure of these species as well as the other neublastin-specific bands.

In the second experiment, the neublastin was extracted with hGFRα3-Ig captured on an ELISA plate. To produce a fusion protein between the extracellular domain of human GFRα3 (described in published application WO 97/44356, November 27, 1997) and the Fc domain of human IgGl (hGFRα3-Ig), a DNA fragment encoding the amino acids 1-364 in human GFRα3, ligated to a fragment containing the Fc domain from human IgG1 and cloned into the expression vector CH269 described by Sanicola et al. (Proe Nati Acad Sei USA, 1997, 94, 6238). The fusion protein encoded by this plasmid was transiently expressed in 293 Epstein-Barr virus-encoded nuclear antigen (EBNA) cells and purified on a protein A-Sepharose immunoaffinity column using standard methods.

Six wells in a 96-well plate were coated overnight at 4°C with goat anti-human IgG (Fcy fragment specific, Jackson Immunologics) at a concentration of 25 mg/ml in PBS (300 ml/well). The wells were blocked for 1 hour at room temperature with 400 ml of 1% BSA in PBS. After three washes with PBST (PBS + 0.05% Tween 20), 300 ml hGFRα3-Ig (10 mg/ml in PBS containing 0.1% BSA) was added to each well. The plate was incubated for 1 hour at room temperature and shaken gently (200 beats/minute) to maximize binding. The wells were then emptied and washed again three times with PBST. 250 ml of conditioned medium from a negative control CHO cell line or from the neublastin-producing CHO cell line #25 was added to each of three wells. The plate was incubated for 3 hours at room temperature and gently shaken (300 strokes/minute). The wells were then washed twice with PBST. 25 ml of non-reducing Laemmli loading buffer was added to the first well and the plate was rapidly shaken for 5 minutes to elute the bound proteins (1300 beats/minute). The contents were transferred to the next well, and the procedure was repeated to elute the proteins bound in the second and third wells. After addition of (3-mercaptoethanol (5% final concentration) the samples were boiled for 5 minutes and analyzed by SDS-PAGE on a 10-20% polyacrylamide gel.

For western blot analysis, the proteins were transferred to nitrocellulose. The membrane was blocked and probed in 5% nonfat dry milk, PBST and washed in PBST. Neublastin was detected by electrochemiluminescence after reaction with polyclonal antibodies (R30 and R31) raised against two neublastin peptides (at 1 µg/ml), followed by reaction with HRP-conjugated goat anti-rabbit antibodies (BioRad). As shown in Figure 13, five neublastin-specific bands were detected in the extracted proteins from the neublastin-producing CHO cell line (lane 2). The lower two bands are very similar to the bands observed in Figure 12; again, the lower band probably represents the mature domain of neublastin produced after cleavage of the pro-domain.

Subsequent analysis (data not shown) of the bands in Figure 13 shows that deglycosylation with PGNase F of the approx. 18 kD band reduces that band to a size equivalent to the bottom band in the gel in Figure 13. This suggests that neublastin can be produced as a glycosylated protein in mammalian cells.

Expression of neublastin in E . coli

To express the neublast gene in E. coli, syngenes with lower GC content and preferred E. coli codons were constructed. The syngene is cloned into two vectors, pET19b and pMJB164, a derivative of pET19b. The construct with pET19b is shown in Figure 14. In this construct, the sequence encoding the mature domain of neublastin (NBN113) is directly fused to an initiating methionine. The pMJB164 construct is shown in Figure 15. In this construct, the mature domain of neublastin is fused to a histidine tag (ie, 10 histidines) separated by an enterokinase cleavage site. The initiating methionine precedes the histidine tag.

Nucleotide sequence coding for neublastin in Figure 14

Nucleotide sequence that codes for his-tagged neublastin in Figure 15

Example 6

Effect of neublastin on the survival of rat embryonic dopaminergic neurons and ChAT activity

In this series of experiments, the effect of conditioned medium from neublastin-producing HiB5pUbilzNBN22 cells above was determined.

Preparation of cultures: The ventral midbrain or spinal cord was dissected from rat E14 embryos in cold Hank's buffered saline (HBSS). Tissue pieces were incubated in sterile filtered 0.1% trypsin (Worthington) and 0.05% DNase (Sigma) in HBSS at 37°C for 20 min. Tissue pieces were then rinsed four times in HBSS + 0.05% DNase and dissociated using a 1 ml automatic pipette. The suspension was then centrifuged at 600 rpm for 5 minutes, and the pellet was re-suspended in serum-conditioned medium (SCM, DMEM with 10% fetal calf serum). The total number of cells was determined by the tryphan blue dye exclusion method and plated at a density of 100,000 cells/cm<2>in poly-L-lysine-coated 8-well chamber slides (Nunc) for determination of dopaminergic neuron survival, or at 200,000 cells/cm<2> in 48-well plates (Nunc) for ChAT activity measurements. Cells were incubated in SCM at 5% CO2/95% O2 and 95% humidity at 37°C for 24-48 hours before changing to serum-free medium (SFM) supplemented with neurotrophic factors.

For determination of dopaminergic neuron survival, cells were left for 5 days in SFM + trophic factor supplements and then fixed for 5 minutes in 4% PFA and stained for tyrosine hydroxylase by immunohistochemistry.

Cells for ChAT activity were allowed to stand for 3 days with SFM and were then lysed in HBSS + 0.1% Triton X-100 and immediately frozen on dry ice until ChAT activity measurement.

Addition of trophic factor: Conditioned medium was collected from non-transfected HiB5 control or HiB5-producing neublastin (HiB5pUbilzNBN22) or GDNF (HiB5pUbilzGDNF-L17). HiB5pUbilzNBN22 produces approx. 20 ng GDNF/24 hours/10<5> cells, as determined by GDNF-ELISA on conditioned medium, collected from the cells. The respective cell lines were incubated overnight with DMEM + 1% FCS, and the supernatant was removed and stored at -20 °C until use. The supernatants were diluted 1:50 in SFM when added to the cells. Separate wells were treated with Hib5 control supernatant (1:50) + purified recombinant rat GDNF (from 0.03 to 10 ng/ml).

The results of these experiments are shown in Figure 4. Figures 4A-4C are illustrations of the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the survival of cultured rat embryonic dopaminergic, ventral midbrain neurons and ChAT activity in cholinergic cranial motor neurons in serum-free medium, as described below in example 5.1. Figure 4A is an illustration of the dose-response curve of recombinant GDNF on ChAT activity (dpm/hr) measured at DIV5 in serum-free cultures originally established 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 is an illustration of ChAT activity (dpm/hr) measured at DIV5 in serum-free cultures originally established from E14 ventral midbrain. Diluted conditioned medium from either neublastin-producing HiB5pUbilzNBN22 cells (neublastin) or GDNF-producing HiB5GDNFL-17-(GDNFL-17) cells was added as indicated in the figure [i.e. neublastin 1:10; neublastin 1:50; GDNFL-17 1:50].

Figure 4C is an illustration of the number of tyrosine hydroxylase immunoreactive cells per well [number of TH<+->cells/well] at DIV7 in serum-free cultures originally established from E14 rat ventral midbrain. Diluted conditioned medium from either non-transfected HiB5 cells (HiB5) or neublastin-producing HiB5pUbilzNBN22 cells (neublastin) or recombinant GDNF at various concentrations were added as indicated in the figure [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].

Conditioned medium from neublastin-transfected HiB5 cells diluted 1:40 significantly increased the number of TH-immunoreactive cells per well compared to control (untransfected) HiB5 cells at an equivalent and a lower dilution (1:10 and 1:40) (see, e.g., Figure 4B). The increase in TH-immunoreactive cells is comparable to the increase seen at a maximal GDNF concentration (10 ng/ml). This indicates that neublastin secreted into the medium has an effect on the survival of the dopaminergic neuron population from the rat embryonic ventral midbrain. In contrast, unlike GDNF secreted from transfected HiB5 cells, no effect of conditioned medium from neublastin-transfected HiB5 cells is seen on another neuronal population in the same culture, the cholinergic neurons (see, eg, Figure 4A).

Example 7

Effect of neublastin on the survival of slice cultures of qrisebryonic dopaminergic ventral midbrain neurons

In this experiment, the effect of co-culture of neublastin-producing HiB5pUbilzNBN22 cells with slice cultures of ventral midbrain from porcine embryos is shown.

Preparation of cultures: Ventral midbrain (VM) was isolated from porcine embryos (E28, n=12) under sterile conditions, cut into 400 µm pieces and placed in chilled Gey's balanced salt solution (GIBCO) with glucose (6.5 mg/ml). The tissue pieces were cultured using the interface culture method, originally developed by Stoppini et al. [L. stoppini,

ON. Buchs, D. Muller, J. Neurosci. Methods, 1991, 37, 173-182].

Briefly, pieces were placed on semiporous membranes (Millipore, 0.3 µm, 8 pieces/membrane corresponding to one VM) placed in inserts in 6-well plates (Costar) with serum-containing medium (Gibco BRL). Each well contained 1 ml of medium (50% Optimem, 25% horse serum, 25% Hank's balanced salt solution (all GIBCO)) supplemented with D-glucose to a final concentration of 25 mM. On day 0, 7000 transfected HiB5pUbilzNBN22 (neublastin) or 7000 non-transfected HiB5 cells (control) were inoculated onto each tissue piece. The co-cultures were first grown in an incubator at 33 °C for 48 hours, allowing the HiB5 cells immortalized with a temperature-sensitive oncogene to proliferate, and then placed in an incubator at 37 °C where the HiB5 cells differentiate. The medium was changed twice a week. Antimitotics and antibiotics were not used at any stage.

Determination of dopamine by HPLC: On days 12 and 21 in vitro the culture medium was collected and analyzed for dopamine using 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, the cultures were fixed in 4% paraformaldehyde in phosphate buffer for 60 min, dehydrated in a 20% sucrose solution for 24 h, frozen, cryostat sectioned at 20 µm (4 series) and mounted on gelatin-coated microscope slides. One series of sections was immunostained for tyrosine hydroxylase (TH). Briefly, sections were washed in 0.05 M Tris-buffered saline (TBS, pH 7.4) containing 1% Triton X-100 for 3 x 15 min and incubated with 10% intact bovine serum (FBS, Life Technologies ) in TBS for 30 min. The tissue was then incubated for 24 h at 4 °C with monoclonal mouse anti-TH antibody (Boehringer Marinheim) diluted 1:600 in TBS with

10% FBS. After rinsing in TBS with 1% Triton X-100 for 3x

After 15 minutes, the sections were incubated for 60 minutes with biotinylated anti-mouse IgG antibody (Amersham) diluted 1:200 in TBS with 10% FBS. The sections were then washed in TBS with 1% Triton X-100 (3 x 15 minutes) and incubated for 60 minutes with streptavidin peroxidase (Dako) diluted 1:200 in TBS with 10% FBS. After washing in TBS (3 x 15 minutes), bound antibody was visualized by treatment with 0.05% 3,3-di-aminobenzidine (Sigma) in TBS containing 0.01 H 2 O 2 . Finally, the sections were dehydrated in alcohol, cleared in xylene and coverslipped in Eukitt.

Cell counts and morphometric analysis: Quantification of immunoreactive TH-ir neurons was performed using bright field microscopy (Olympus). Only cells showing strong staining with a well-preserved cellular structure and a clear nucleus were counted. Calculation was based on cell counts in every fourth culture compartment using a x 20 objective. Cell counts were corrected for double counting according to Abercrombie's formula (M. Abercrombie, Anat. Ree, 1946, 94, 239-47) using the mean diameter of the nuclei of the TH-ir neurons (6.6 ± 0.2 µm, n = 30). The size of the nuclei was calculated using a neuron tracking system (Neurolucida, MicroBrightField, Inc.).

The results of these experiments are shown in Figure 5.

Figures 5A-5C are illustrations of the effect of neublastin secreted from HiB5pUbilzNBN22 cells on the function and survival of slice cultures of pig embryonic dopaminergic ventral midbrain neurons co-cultured with either HiB5pUbilzNBN22 cells (neublastin) or HiB5 cells (control), as described below. Figure 5A and Figure 5B illustrate dopamine released into the medium at DIV12 [dopamine (pmol/ml) - day 12] and DIV21 [dopamine (pmol/ml) - day 21], respectively. Figure 5C is an illustration of the number of tyrosine hydroxylase immunoreactive cells per culture [TH-ir cells per culture] at DIV21.

At day 12, HPLC analysis revealed that medium from HiB5-neublastin cocultures contained 84% more dopamine than medium from HiB5-C cocultures (Figure 5A). At day 21, the difference was 78% (Figure 5B), and cell counts showed that HiB5-neublastin cocultures contained 66% more tyrosine hydroxylase immunoreactive neurons than HiB5-C cocultures (P < 0.05) (Figure 5C). This indicates that neublastin secreted from the HiB5pUbilzNBN22 clone has a strong survival effect on dopaminergic embryo porcine neurons.

Example 8

Survival of dorsal root ganglion cells in serum-free medium

This example demonstrates the neurotrophic activity of a neublastin polypeptide compared to known neurotrophic factors.

Pregnant female mice were euthanized by cervical dislocation. The embryos were processed for culture as follows.

Electrolytically tipped tungsten needles were used to dissect dorsal root ganglia from indicated stages of C57/BI6 mice (Mollegaard Breeding, Denmark). Embryoganglia were incubated for 5 min at 37°C with 0.05% trypsin (Gibco/BRL) in calcium- and magnesium-free Hank's balanced salt solution. Postnatal ganglia were treated with collagenase/dispase 1 mg/ml for 30-45 minutes and then trypsin/DNAse 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 were gently triturated with a flame-cleaned Pasteur pipette to obtain a single cell suspension.

The cells were plated in 24-well plates (Nunc) precoated with polyornithine (0.5 mg/ml/overnight) and laminin (20 mg/ml for 4 hours, Gibco/BRL). The neurons were incubated at 37 °C in a humidified 5% CO2 incubator in a defined medium consisting of Ham's 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 neurons were clearly recognized by their bipolar morphology under phase-contrast optics. The percentage neuronal survival in the absence or presence of trophic factors (added to the culture medium before plating the neurons at 10 ng/ml) or of conditioned medium from the neublastin-producing HiB5pUbilzNBN22 cells was determined by counting the neurons in the wells after

4 8 hours.

The results of these experiments are shown in Figure 9, where: 0 is the control experiment (in the absence of factors); 1 is experiment in the presence of GDNF; 2 are experiments in the presence of neurturin; 3 are experiments in the presence of neublastin according to the invention;

E12 is data from experiments performed on DRG cells isolated from embryonic day 12;

E16 is data from experiments performed on DRG cells isolated from embryonic day 16;

PO is data from experiments performed on DRG cells isolated from the day of birth;

P7 is data from experiments performed on DRG cells isolated from postnatal day 7; and

P15 is data from experiments performed on DRG cells isolated from day 15 after birth.

These results clearly show that the neurotrophic factor according to the invention exhibits activities comparable to or even superior to those of the well-established neurotrophic factors.

Example 9

In vivo - effects of neublastin on nigradopamine neurons

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

Lentivirus production: To generate a lentivirus transfer vector encoding neublastin, pHR'-neublastin, a 1331 bp BamHI fragment from neublastin cDNA, was subcloned into the BamHI/BglII site of pSL301 (Invitrogen). From this construct, a 1519 bp BamHI/XhoI fragment was excised and ligated into the BamHI/XhoI site of pHR<1>carrying a woodchuck hepatitis virus post-translational fragment (Zufferey, R., Donello, J.E., Trono, D ., Hope, T.J., "Wood-chuck hepatitis virus posttranscriptional regulatory element enhances 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 into the BamHI/XhoI site of pHR'.

Preparation of the lentiviral vector has been described by e.g. Zufferey et al. (Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., Trono, D.: "Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo"; Nat. Bio-technol., 1997, 15 (9 ) 871-875). Briefly, the transfer constructs and the helper plasmids pR8.91 and pMDG were co-transfected into 293T cells. Virions released into the medium were collected at 48 and 72 h post-transfection. To concentrate the virus, the medium was centrifuged for 1.5 h at 141,000 g and the pellet was dissolved in DMEM. The titer of a control carrying the gene for Green Fluorescent Protein ("GFP") was determined to be 10<8>transforming units (TU)/ml by GFP fluorescence in 293T cells. An RNA "slot blot" technique (von Schwedler, U., Song, J. , Aiken, C, Trono, D., "Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells", J. Virol., 1993, 67 (8) 4945-4955) was used to determine virus particle titers. In the GDNF supernatant and the neublastin supernatant, there were 10-fold fewer particles compared to the GFP supernatant.

Surgical procedures: All work involving animals was carried out in accordance with the rules established by the Ethical Committee for Use of Laboratory Animals at Lund University.

A total of 21 young adult female Sprague-Dawley rats (B&K Universal, Stockholm, Sweden) were used and housed under a 12-h light-dark cycle with free access to rat chow and water. Retrograde labeling and 6-OHDA lesions were performed 3 weeks before lesioning according to Sauer and Oertel (Sauer and Oertel, Neuroscience, 1994, 59, 401-415). Briefly, under Equithesin anesthesia (0.3 ml/100 g), rats were injected bilaterally with 0.2 µl of a 2% solution (dissolved in 0.9% NaCl) of the retrograde tracer Fluoro-Gold (FG, Fluorochrome, Inc., Englewood, CO). Injections were made using a 2 µl Hamilton syringe at the coordinates: AP = +0.5 mm; ML = +3.4 mm relative to bregma; DV = -5.0 mm in relation to the dura and the incisor piece set to 0.0 mm. In addition, 0.05 µl/minute was injected, allowing 5 minutes to pass before the needle was withdrawn again.

14 days after the FG injections, the animals received a total of five deposits (1 µl/deposit) of a lentiviral vector carrying the gene for green fluorescent protein (GFP), neublastin or GDNF. Four of the deposits were in the striatum along two needle areas at the following coordinates: AP = +1.0 mm, ML =

-2.6 mm, DVi = -5.0 mm, DV2= -4.5 mm and AP = 0.0 mm, ML = -3.7 mm, DVi= -5.0 mm, DV2= -4, 5 mm. The supranigral deposition was done at AP = -5.2 mm, ML = -2.0 mm, DVi= -6.3 mm. The tooth piece was set at -2.3 mm. 21 days after retrograde labeling and 7 days after lentivirus injections, the animals were again anesthetized, and with a 10 µl Hamilton syringe, a single deposit of 20 µg 6-OHDA (Sigma, calculated as free base and dissolved in 3 µl ice-cold saline solution supplemented with 0.02% ascorbic acid) injected into the right striatum at the same site as the FG deposits. The injection rate was 1 µl/minute, as a further 3 minutes were allowed to pass before the needle was withdrawn again. Tissue processing: 21 days after the 6-OHDA injection, the animals were deeply anesthetized with chloral hydrate and transcardially perfused with saline (pH 7.4, room temperature) for 1 minute, followed by 200 ml of ice-cold formaldehyde solution (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The brains were dissected and post-fixed in the same fixative for 3-4 hours and then transferred to 25% sucrose/0.01 M phosphate buffer for 48 hours. Five series of 40 µm sections through the striatum and substantia nigra (SN) were cut on a freezing microtome.

Quantification of dopaminergic neurons in the SN: The number of FG labeled in the SN "pars compacta" was determined by a blinded observer, as described previously (Sauer and Oertel, Neuroscience, 1994, 59, 401-415). Briefly, three consecutive sections centered around the level of the medial terminal nucleus of the accessory visual pathway (MTN, -5.3 in the atlas of Paxinos and Watson (1997)) were used, using and all labeled/stained neurons lateral to MTN were counted at 40x magnification (n = 6-7/group). FG-labeled neurons were included if they became clearly fluorescent under epiillumination at 330 nm, displayed a neuronal profile and produced at least one neuroinflammatory process.

On the lesion side of animals that received injections of lentivirus carrying GFP, the number of FG-positive nigral neurons was reduced to 18% of the number on the intact side. In contrast, animals injected with lentineublastin showed an almost complete protection of the number of FG-positive nigral neurons (89%). This was as effective as lenti-GDNF-treated animals, where 87% of the retrogradely labeled neurons remained on the lesion side. This shows that neublastin is a strong survival factor for injured adult nigral dopamine neurons, and that it is as strong as GDNF.

Figure 6 is an illustration of the in vivo effect of lentivirus-produced neublastin on nigral dopamine neurons. Neurons in the SN pars compacta of female Sprague-Dawley rats were retrogradely labeled with Fluorogold (FG) 3 weeks before a single injection of 6-hydroxydopamine (6-OHDA) into the right striatum. One week before the 6-OHDA injection, the animals received injections of lentiviral vectors expressing neublastin [neublastin], GDNF [GDNF] or Green Fluorescent Protein [GFP], as indicated in the figure. 21 days after the 6-OHDA injections, the number of FG-labeled neurons on both sides of the striatum was determined. The figure shows the percentage [% FG lesion/intact] of FG-labeled neurons in the lesioned (right) side versus the intact (left) side of the striatum in the three groups of animals.

Example 10

Production of antibodies

To prepare antibodies against neublastin, two rabbits were immunized with either 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) conjugated to carrier protein at 3 weekly intervals. Two rabbits for each peptide were immunized at weeks 0, 3, 6 and 10, and blood samples were taken at weeks 7 and 11. The other blood sample was affinity purified via a peptide affinity column. The antibodies were named Ab-1 and Ab-2 after the peptide.

Western blot: 2 x IO<6>HiB5 cells, stably transfected with cDNA for neublastin (Hib5pUbilzNBN22), or untransfected HiB5 cells were incubated overnight in serum-free medium with N2 supplement (GIBCO). The medium was concentrated on small concentrators with 5 kDa cutoff membranes (Millipore, Bedford, MA). Concentrated samples were added to 5x Laemmli sample buffer and heated to 95°C for 5 minutes. Samples were separated by SDS-polyacrylamide gel electrophoresis on 15% acrylamide gels and transferred to PVDF membranes. Residual protein binding sites were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20. Membranes were incubated overnight with neublastin antibody (1:1000), followed by incubation with a secondary antirabbit or antimouse IgG antibody conjugated to horseradish peroxidase (1:2000).

Immunostaining was visualized using enhanced chemiluminescence Plus (ECL+) according to the manufacturer's instructions (Amersham). The results of these experiments are shown in figure 3 and example 5.

Using standard techniques, we also raised rabbit polyclonal antibodies against the following peptides: peptide R27: GPGSRARAAGARGC (amino acids 30-43 of SEQ ID NO: 9); peptide R28: LGHRSDELVRFRFC (amino acids 57-70 of SEQ ID NO: 9); peptide R29: CRRRARSPHDLSL (amino acids 74-85 of SEQ ID NO: 9); peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID

NO: 9); and

peptide R31: STWRTVDRLSATAC (amino acids 123-136 of SEQ ID

NO: 9).

Only the peptides R30 and R31, relatively close to the C terminus, recognized the denatured protein under reducing conditions on a western blot.

Claims (31)

1. Isolated neublastin polynucleotide, characterized in that it is selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence SEQ ID NO: 1, b) a polynucleotide comprising an open reading frame which codes for expression of a neublastin polypeptide or a polypeptide derived therefrom , in which the mature part of the polypeptide has a degree of similarity with aai-aaiosi SEQ ID NO: 2 of at least 90%, c) a polynucleotide which, during hybridization under high stringency solution conditions, specifically hybridizes to the nucleic acid having the nucleotide sequence SEQ ID NO: 1, d) a polynucleotide comprising a nucleotide sequence that is at least 80% identical to the polynucleotide sequence set forth as SEQ ID NO: 1, wherein the encoded polypeptide has neurotrophic activity. 2. Polynucleotide according to claim 1, characterized in that the mature part of the coded polypeptide has a degree of similarity with aai-aaio5i SEQ ID NO: 2. of at least 95% 3. Polynucleotide according to claim 1, characterized in that the coded polypeptide consists of aai-aaiosi SEQ ID NO: 2. 4. Polynucleotide according to claim 1, characterized in that the coded polypeptide is SEQ ID NO: 5. 5. Polynucleotide according to claim 1, characterized in that the coded polypeptide is SEQ ID NO: 6. 6. Polynucleotide according to claim 1, characterized in that the coded polypeptide is SEQ ID NO: 7. 7. Polynucleotide according to claim 1, characterized in that it comprises SEQ ID NO: 3. 8. Polynucleotide according to claim 1, characterized in that it comprises a nucleotide sequence which is 80% similar to the polynucleotide sequence indicated in SEQ ID NO: 1. 9. Polynucleotide according to claim 1, characterized in that it comprises a nucleotide sequence which is 90% similar to the polynucleotide sequence indicated in SEQ ID NO: 1. 10. Expression vector, characterized in that it comprises the polynucleotide according to any one of claims 1-9. 11. Isolated host cell, characterized in that it comprises an expression vector according to claim 10. 12. Cell according to claim 11, characterized in that it is a eukaryotic cell. 13. Cell according to claim 12, characterized in that it is selected from the group consisting of mammalian cells, fungal cells and yeast cells. 14. Cell according to claim 13, characterized in that the mammalian cell is selected from the group consisting of a Chinese hamster ovary cell, HEK293, COS, PC12, HiB5, RN33b and human neural stem cells. 15. Polypeptide, characterized in that the mature part of the polypeptide has a degree of similarity with amino acids 1-105 in SEQ ID NO: 2 of at least 90%, in which the polypeptide has neurotrophic activity. 16. Polypeptide according to claim 15, characterized in that the mature part of the polypeptide has a degree of similarity with amino acids 1-105 in SEQ ID NO: 2 of at least 95%. 17. Polypeptide according to claim 15, characterized in that it is a neublastin neurotrophic factor polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 2. 18. Polypeptide according to claim 15, characterized in that it is a neublastin neurotrophic factor polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 4. 19. Polypeptide according to claim 15, characterized in that it is a neublastin neurotrophic factor polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 5. 20. Polypeptide according to claim 15, characterized in that it is a neublastin neurotrophic factor polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 6. 21. Polypeptide according to claim 15, characterized in that it is a neublastin neurotrophic factor polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 7. 22. Polypeptide according to any one of claims 15-21, characterized in that it is glycosylated. 23. Polypeptide according to claim 15, characterized in that it is coded for by a polynucleotide according to any of claims 1-9. 24. Method for producing the polypeptide according to claims 15-23, characterized in that the method comprises the step of expressing the polypeptide from a neublastin neurotrophic factor polynucleotide according to any one of claims 1-9. 25. Method according to claim 24, characterized in that it comprises the step of culturing a cell according to any one of claims 11 to 14 comprising said neublastin neurotrophic factor polynucleotide in a culture medium which enables the production of the polypeptide. 26. Method according to claim 25, characterized in that it further comprises the step of recovering the polypeptide from the culture medium. 27. Method according to claim 26, characterized in that the polypeptide is purified. 28. Preparation, characterized in that it comprises the polypeptide according to any one of claims 15-23, and a pharmaceutically acceptable carrier. 29. Polypeptide according to any one of claims 15-23, for use as a medicament. 30. Polypeptide according to any one of claims 15-23, for the therapeutic or prophylactic treatment of a neurodegenerative disease involving damaged and traumatic neurons. 31. Polypeptide according to claim 30, wherein the disease involves traumatic lesions of peripheral nerves, traumatic lesions of the marrow, traumatic lesions of the spine, cerebral, ischemic nerve injury, neuropathy, peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis or memory disorders associated with dementia.