WO1995026363A1 - Neurotrophin-6: a new member of the neurotrophin family - Google Patents

Abstract

The present invention relates to nucleic acid molecules encoding a new neurotrophic factor termed neurotrophin-6 or parts thereof, recombinant DNA molecules comprising said nucleic acid molecules and to hosts transformed with said recombinant DNA molecules. Furthermore, the present invention relates to (poly)peptides having the biological and/or antigenic properties of neurotrophin-6, to antibodies directed to said factor, to pharmaceutical compositions and kits comprising said factor and/or said antibody as well as to methods for the isolation of said nucleic acid molecules and the production of said factor. The present invention also comprises in vitro methods for the detection of diseases or disorders of the nervous system.

Description

Neurotrophin-6: A new member of the neurotrophin family

The present invention relates to nucleic acid molecules encoding a new neurotrophic factor termed neurotrophin-6 or parts thereof, recombinant DNA molecules comprising said nucleic acid molecules and to hosts transformed with said recombinant DNA molecules. Furthermore, the present invention relates to (poly)peptides having the biological and/or antigenic properties of neurotrophin-6, to antibodies directed to said factor, to pharmaceutical compositions and kits comprising said factor and/or said antibody as well as to methods for the isolation of said nucleic acid molecules and the production of said factor. The present invention also comprises .in vitro methods for the detection of diseases or disorders of the nervous system.

The development of the vertebrate nervous system is regulated by both intrinsic and epigenetic factors. Around the time of target field innervation, substantial numbers of postmitotic neurons (approximately 40-60%) are eliminated - a phenomenon known -as "naturally occurring cell death". Surgical manipulation of target tissue influences the extent of neuronal cell death: removal of target reduces the number of surviving neurons without affecting the numbers of neurons initially generated; conversely, grafting extra target tissue enhances neuronal survival. (Cowan et al.; 1984; Hamburger, 1993; Oppenheim, 1991).

The occurrence of dying cells has been noted as a general feature of the normal embryonal development in vertebrates (Ernst, 1926) . One view is that a cell not only needs a signal from another cell to proliferate but also a signal for survival (Raff, 1992) . The finding that neuronal cell death or apoptosis can be prevented by inhibition of RNA or protein synthesis (Martin et al. , 1988; Scott and Davies, 1990) suggests that apoptotic neuronal cell death is an active process necessitating gene expression, i.e. the activation of a "suicide" programme. Thus, neurotrophic molecules seem to suppress the activation of this suicide programme. Neurotrophic factors - a family of similar proteins released by target cells in limited amounts - provide trophic support for which neurons must "compete" in order to survive. (Barde, 1989; Thoenen, 1991).

The prototypical neurotrophin, nerve growth factor (NGF) was the first peptide growth factor to be molecularly characterized and it became the paradigm of a target-derived neurotrophic molecule supporting the survival and differentiation of specific populations of peripheral and central neurons. Its importance has been established for sensory and sympathetic neurons by the demonstration that administration of exogeneous NGF can almost completely prevent natural nerve cell death; conversely function- blocking anti-NGF antibodies enhance neuronal cell death (Levi-Montalcini and Angeletti, 1968; Johnson et al. , 1986). Moreover, it has been demonstrated that the regionally differential expression of NGF reflects the extent of survival and the density of innervation of responsive neurons (Korεching and Thoenen, 1983; Davies et al., 1987).

The concept of a neurotrophin protein family was established when a survival factor for sensory neurons from mammalian brain, named brain-derived neurotrophic factor (BDNF) was purified from mammalian brain (Barde et al. , 1982) and upon molecular cloning was found to be about 50% identical to NGF (Leibrock et al., 1989). In addition to NGF and BDNF, this family at present includes neurotrophin-3 (NT-3; (Ernfors et al., 1990; Hohn et al., 1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al. , 1990; Rosenthal et al., 1990)) and neurotrophin-4 (NT-4; (Hallbδδk et al., 1991; Ip et al. , 1992)). A molecule homologous to NT-4 has been cloned from rat and human libraries and termed neurotrophin-5 (NT-5; (Berkemeier et al., 1991)). All neurotrophins display both overlapping and distinct survival activities on peripheral and central neurons (Barde, 1990; Thoenen, 1991) .

Small aquarium teleost fish provide unique experimental systems for studying developmental and neurobiological phenomena: Fish show a high degree of evolutionary conservation compared to other vertebrates with respect to gene expression patterns in developing brain and the establishment of early axonal pathways (Kimmel, 1993) .

Additionally, recent studies have established that the genes for neurotrophic factors exist in fish (Hallbδδk et al., 1991; Gδtz et al., 1992b). Furthermore the proteins encoded by the fish NGF and BDNF genes show survival activity on embryonic chick neurons implying that the interaction with the neuronal cell surface receptors has been conserved during evolution (Gδtz et al., 1992b).

On the other hand, neurogenesis in fish continues well into adult life and the capacity of regeneration in the central nervous system (CNS) distinguishes fish from higher vertebrate species where neurogenesis in most structures is completed at birth and axons in the CNS fail to regrow (Holder and Clarke, 1988) .

The complex interaction of neuron development and neurotrophic factors cannot completely be explained by the biological activity of the neurotrophic factors which have been so far identified. Consequently, there is a need to identify and molecularly clone additional neurotrophic factors necessary to understand said interactions. Such factors are also of paramount importance in diagnostic and pharmaceutical applications.

Accordingly, the technical problem underlying the present invention was to provide an additional factor which contributes to the accomplishment of the above-identified needs. The solution to said technical problem is provided by the embodiments characterized in the claims.

Accordingly, the present, invention relates to a DNA sequence encoding a (poly)peptide having the biological properties of neurotrophin-6 (NT-6) , selected from the group consisting of

(a) the DNA sequence of SEQ. LIST. No. 1 (cDNA) ;

(b) the DNA sequence of SEQ. LIST. No. 2 (genomic DNA) ;

(c) a DNA sequence hybridizing to a DNA sequence of (a) or (b)

(d) a DNA sequence being degenerate with respect to a DNA sequence of (a) , (b) or (c) ; and

(e) a subsequence of a DNA sequence of (a) to (d) of at least ten nucleotides encoding a (poly)peptide having the biological and/or antigenic properties of neurotrophin-6.

The DNA sequence of the present invention may be of natural, synthetic or semi-synthetic origin.

The term "hybridizing to a DNA sequence of (a) or (b) " as used herein refers to hybridizations at any stringency suitable to allow the successful hybridization and optionally the subsequent identification of a hybridizing DNA sequence. Preferably, the hybridization conditions are as follows: hybridization in 5x SSC (lx SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2), 40% formamide at 42°C with washes in 2x SSC at 60°C. Examples of such hybridizing sequences are DNA sequences which differ from the DNA sequence of the invention by substitutions, insertions and/or deletions.

The term "degenerate with respect to a DNA sequence of (a), (b) or (c)" refers to DNA sequences which code for the same amino acid sequence but have a variant DNA sequence due to the degeneracy of the genetic code.

The subsequence of feature (e) refers to any DNA sequence which deviates from the DNA sequence of (a) to (d) by its length but wherein the (poly)peptide encoded still has the biological properties and/or activities of neurotrophin-6. Also, but not necessarily, the antigenic or immunogenic properties of neurotrophin-6 may be retained.

In a preferred embodiment of the present invention, said DNA sequence is a vertebrate DNA sequence.

In a more preferred embodiment of the present invention, said DNA sequence is a fish DNA sequence and includes bony and cartilagous fish.

In a further more preferred embodiment, said DNA sequence is a mammalian DNA sequence.

In a particularly preferred embodiment, said mammalian DNA sequence is a human sequence.

A further object of the invention is to provide a RNA sequence having the following properties:

(a) it is complementary to a DNA sequence of the invention; and/or

(b) it hybridizes to a DNA sequence of the invention. Said RNA sequence may be of natural, synthetic or semi- synthetic origin. The hybridization conditions necessary to allow a successful hybridization are essentially the same as detailed above for the DNA hybridizations and are well-known to a person skilled in the art; see, e.g. Sambrook et al. , "Molecular Cloning, A Laboratory Manual", 2^nd edition 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor.

The invention further relates to a primer and a pair of primers capable of hybridizing to either strand of the DNA sequence or the RNA sequence of the invention, respectively, under suitable conditions. In the case of a pair of primers, one of the primers hybridizes to a part of the DNA or RNA sequence of the invention whereas the second primer hybridizes to a part complementary to the DNA or RNA sequence of the invention under the proviso that said two parts of said DNA or RNA strands are non-overlapping and not directly adjacent when said DNA or RNA strands are hybridized to each other and that the 5' termini of said primers when hybridized to said DNA or RNA strands are distal to each other whereas their 3 ' ends are proximal to each other.

In a preferred embodiment, said primers have a length of 15 nucleotides. In particularly preferred embodiments, these primers have a length of 17 and 21 nucleotides. Said primers may be of natural (e.g. as a fragment of a larger DNA molecule) , synthetic or semisynthetic origin. The production of the primers from said origins is well understood by those skilled in the art.

Suitable conditions for the hybridization of the primers to said DNA strands are any conditions under which a stable DNA/DNA or RNA/RNA hybrid is formed. Preferably, these conditions are stringent conditions. The term "stringent conditions" is well-known in the art and is described in standard textbooks of molecular biology such as Sambrook et al. , loc. cit.

Said primers may be used, for example, to function as primers for the enzymatic or synthetic elongation of a DNA sequence after hybridizing to said DNA strands. Provided that an enzymatic elongation is carried out, appropriate enzymes that may be used are DNA polymerase I or Taq DNA polymerase or any functional parts or derivatives thereof.

Furthermore, said primer(s) may be used in any process for diagnosing the DNA or RNA sequence of the invention and may be contained in any kit useful for said diagnosis. Accordingly, said kit is also comprised by the present invention. Said kit comprises at least one of said primers and, if an enzymatic elongation or amplification is intended, additionally (a) suitable buffer(s). The term "suitable buffer(s) " is well understood and construed by those skilled in the art.

In a preferred embodiment of the invention, said primers are used for the enzymatic amplification ("polymerase chain reaction", abbreviated as "PCR") of the DNA or RNA sequence of the invention and its complementary sequence. Suitable conditions, technical details and applications of the PCR technique and the products obtained therewith have extensively been described e.g. in Sambrook et al., loc. cit., and H. Erlich et al., Nature 331, (1988), 461, and the references cited therein.

Additionally, the invention relates to a recombinant DNA molecule comprising the DNA sequence of the invention. Said recombinant DNA molecule conveniently comprises a vector sequence in addition to the DNA sequence of the invention. Further DNA sequences may be comprised in the recombinant DNA molecule of the invention. An example of such a further DNA sequence is a gene encoding a different neurotrophin factor such as NGF, NT-3 , BDNF or NT-4/5.

The recombinant DNA molecule of the invention may be used for the propagation and/or the expression of the DNA sequence of the invention. The person skilled in the art is well aware of methodes for the construction of recombinant DNA molecules for either purpose; see, e.g. Sambrook et al., ibid.

Additionally, the recombinant DNA molecule of the invention may express a fusion protein comprising the (poly)peptide of the invention.

According to a preferred embodiment of the invention said hybridization conditions of step (a) are 30% formamide and 1 M NaCl at 42°C followed by washing steps in 300 mM NaCl at 60°C.

In a particularly preferred embodiment said recombinant DNA molecule is the recombinant vaccinia vector WNT-6. This vector is replication-competent and contains a sub-genomic fragment coding for NT-6 from Xiphophorus maculatus under the control of the strong Pll vaccinia promoter; see Bertholet et al. , Proc. Natl. Acad. Sci. USA 82, 2096-2100, (1985) . It was obtained by homologous recombination after transfection of the RK1₃ cell line with plasmid 1593 and subsequent selection for mycophenolic acid resistance as described, e.g., Falkner and Moss, J. Virol. 62, 1849-1854, (1988). Upon infection of a mammalian cell line, e.g. χ₃, this virus vector directs the production and secretion of recombinant NT-6.

Furthermore, the present invention relates to a method for the isolation of a mammalian, preferably a human DNA sequence of the invention comprising the following steps: (a) hybridizing a vertebrate, preferably a fish, a mammalian or most preferably a human DNA library with a suitable probe under reduced stringency conditions; and

(b) isolating the hybridizing clones and determining the (partial) DNA sequence thereof according to standard procedures.

The term "human DNA library" refers to a genomic or a cDNA library obtained from a suitable human tissue or from suitable human cells. An example of such a suitable tissue is total brain, or specific brain aereas such as cortex, cerebellum, or hippo campus. In other less preferred embodiments of the invention, said DNA libraries are derived from other vertebrates, preferably from other mammals or fish.

The term "suitable probe" refers to any probe which may be employed for the successful identification of the DNA sequence of the invention. Said probe preferably hybridizes to the coding region of NT-6 (amino acid residues 1-286) .

The DNA sequences isolated from said human DNA library may then be subjected to DNA sequencing in order to confirm that the desired sequence has indeed been obtained. Various DNA sequencing protocols are well-known in the art.

A further object of the invention is to provide a method for the isolation of a DNA or RNA sequence of the invention comprising the following steps:

(a) electrophoresing vertebrate, preferably fish, mammalian, most preferably human DNA restricted with

(an) appropriate restriction enzyme(s) or RNA derived from any of said organisms through an appropriate matrix, preferably an agarose gel;

(b) transfering the electrophoresed DNA or RNA to a second matrix, preferably a nitrocellulose filter; (c) detecting the location of the DNA sequence or the RNA sequence of the invention using an appropriate probe;

(d) isolating the DNA or RNA sequence of the invention electrophoresed through a preparative gel on the basis of the data obtained in steps (a) to (c) ; and

(e) cloning the isolated DNA or RNA sequence in an appropriate vector.

The term "appropriate matrix" as used herein refers to ^• any matrix which is capable of separating DNA or RNA molecules according to their molecular weight. Examples of said matrices are polyacrylamide or agarose.

The term "second matrix" refers to any matrix onto which said DNA or RNA can be transferred and maintained. The transfer and maintenance of said DNA or RNA to said matrix is well-known in the art (and referred to herein as e.g. Southern, Northern or Western blotting, see Sambrook et al. , loc. cit.). Examples of said matrices are nitrocellulose filters and DBM paper.

As an appropriate probe any probe may be used which is capable of detecting the DNA or RNA sequence of the invention. Examples of such probes are DNA or RNA probes which hybridize to the DNA or RNA sequences of the invention under appropriate conditions. Preferably, the probe used for hybridization is the 389 bp Nsil-EcoRI fragment referred to in figure IA which is used under low stringency hybridization conditions as defined herein. Preferably said probes are labelled with a detectable marker such as a radioactive marker. Alternatively, the probe may be an antibody directed to said DNA or RNA sequence of the invention. However, the person skilled in the art is capable of designing other detection systems which work equally well. Once the person skilled in the art has determined the size of the DNA or RNA sequence of interest on the basis of the data obtained from steps (a) to (c) , he is in the position to isolate said DNA or RNA sequence from preparative gels using standard molecular weight markers. Methods for isolating DNAs or RNAs from preparative gels are known in the art and have been described e.g. by Sambrook et al. , loc. cit.

If the DNA sequence isolated does not comprise the complete (poly)peptide encoding region, it may be used to screen cDNA or genomic libraries from which the complete sequence may then be isolated.

The cloning of the DNA or RNA of the invention can then be carried out according to standard procedures.

In a preferred embodiment the probe used for identifying the desired DNA sequence in the methods referred to above is a fish probe.

In a particularly preferred embodiment, said fish probe is the 389 bp Nsil-EcoRI fragment derived from the platyfish Xiphophorus maculatus (genomic sequence) or the swordtail Xiphophorus helleri (cDNA sequence) and detailed in Figure IA.

Another object of the invention is to provide a method for the isolation of a DNA sequence of the invention, preferably a vertebrate, more preferably a mammalian or a fish and most preferably a human DNA sequence comprising the following steps:

(a) amplification of said DNA sequence with a set of primers of the invention; and

(b) cloning the amplified DNA sequence and determining its (partial) sequence according to standard procedures. Said amplification is carried out according to a technique well-known in the art as "polymerase chain reaction" (PCR) , see above. The person skilled in the art is well aware of details and applications of the PCR technique which is described in detail e.g. in Sambrook et al. , loc. cit. or Ehrlich et al., loc. cit.

In a preferred embodiment, at least one of the primers hybridizes to one of the conserved neurotrophin domains. Said domains are determined by aligning the amino acid sequences of the various members of the neurotrophin family. In the neurotrophin-6 molecule, said conserved domains are located in the following parts of the molecule: amino acid 50-57 (Q-L-F-Y-E-T-T-C) and 122-127 (W-R-F-I-R-I) .

The second primer must hybridize to an oligonucleotide corresponding to the sequence V-S-A-L-T.

The present invention also relates to a host transformed with a recombinant DNA molecule of the invention.

In a preferred embodiment, said host is a bacterial, fungal, yeast or mammalian cell or a transgenic animal.

An example of a bacterial cell which may be used for the transformation is an E. coli cell. However, the person skilled in the art is in the position to choose any other bacterial cell suitable to be transformed by the recombinant DNA molecule of the invention.

An example of a mammalian cell is the cell line K13 (ATTC number CCL 37) .

Transgenic animals carrying the NT-6 gene can be generated by techniques well-known in the art. The various techniques presently available have been summarized by Hanahan, D. Science 246, 1265-1275, (1989) and comprises e.g. micro-injection into fertilized eggs, into blastocysts and aggravation. Said transgenic animals may carry either additional NT-6 genes or a construct which disrupts the endogenous NT-6 genes. Both types of transgenic animals allow the study of NT-6 gene function as well as the interaction of its gene product with other neurotrophins and their receptors.

A further object of the invention is to provide a (poly)peptide encoded by a DNA sequence of the invention. Said (poly)peptide may be obtained e.g. by translation of an mRNA sequence transcribed from a DNA sequence of the invention. Alternatively, the (poly)peptide may be produced by chemical procedures well known in the art based upon the knowledge of the DNA sequence of the invention. The polypeptide of the invention may also be a semisynthetic product obtainable by combining e.g. biological translation and chemical synthesis.

Also comprised by the present invention are (poly)peptides which have been posttranslationally modified as well as those wherein amino acids have been replaced by different ones which do not essentially interfere with the biological and/or antigenic properties of NT-6.

Said (poly)peptides may furthermore be fusion proteins comprising the (poly)peptide of the invention.

In a preferred embodiment, the (poly)peptide of the invention has at least one of the following properties:

(a) its precursor molecule consists of 286 amino acids;

(b) it has an amino acid sequence at the carboxy terminal half of the precursor starting at lysin 143 which encodes the mature neurotrophin-6 of 143 amino acids;

(c) a predicted M_r of 15,968; (d) a hydrophobic domain at the N-terminus (amino acids 1 to 19) with the characteristics of a signal peptide;

(e) a pro-region (amino acids 20 to 142) containing two multiple basic motifs (R-X-K/R-R at residues 63-66 and 139-142);

(f) a pi value of 10.8;

(g) a stretch of 40 amino acids extending from amino acid position 151 to amino acid position 275 conserved in all neurotrophins known so far;

(h) biological activity on sympathetic and sensory dorsal root ganglia neurons, but not on parasympathetic ciliary and nodose neurons;

(i) it is a heparin binding molecule; and

(j) it acts as a survival factor for chick sensory neurons of dorsal root ganglia.

The conserved stretch of 40 amino acids of feature (g) has been found in the following neurotrophins: NGF, NT-3, NT-4/5 BDNF and NT-6. It is one of the characteristics which assigns a polypeptide to the neurotrophin family.

The biological activity referred to in feature (h) may be described as follows. NT-6 is a survival molecule for sensory neurons prepared from eight day old chick embryos. It is furthermore a survival molecule for sympathetic neurons prepared from 10 or 11 days old chick embryos.

In a further preferred embodiment, the (poly)peptide of the invention is glycosylated. The glycosylation may be obtained by expressing the recombinant DNA molecule of the invention e.g. in a mammalian or a yeast cell. Depending on the origin of the host cell, the glycosylation pattern obtained will vary. The preferred glycosylation pattern is that obtained by expressing the recombinant DNA molecule of the invention in a human cell. Glycosylation sites for asparagin-linked glycosylation are the motifs N-H-S (amino acids 32-34) , N-R-T (amino acids 76-78) .

In another preferred embodiment, the (poly)peptide of the invention is not glycosylated. A non-glycosylated molecule can be obtained by expressing the recombinant DNA molecule of the invention e.g. in bacterial cells such as E. coli cells. Also, a glycosylated product may be treated with appropriate enzymes to remove the sugar chains.

Additionally, the present invention relates to a molecule having the same three-dimensional structure at least with respect to the biologically functional domains as the (poly)peptide encoded by the DNA or RNA sequence of the invention.

Said molecule may be of proteinaceous matter, but may also comprise carbohydrate or nucleic acid matter. It is feasible that said molecule consists of either of these compound classes alone or a mixture thereof as long as the three-dimensional structure at least with respect to the biologically functional domains of NT-6 is retained. Preferably the whole structure of NT-6 is retained by said molecule. Alternatively or additionally, at least one of the antigenic properties specific for NT-6 is retained.

The molecule of the invention is useful e.g. in mimicking the biological and/or antigenic properties of the (poly)peptide of the invention and may therefore be employed in the methods, kits and pharmaceutical compositions of the invention.

The present invention relates further to a method for the production of the (poly)peptide comprising the following steps: (a) culturing a host cell of the invention in a suitable medium under conditions which allow the expression of said (poly)peptide;

(b) adding heparin to the medium at the onset of the culturing or at least 0.5 hours before harvesting; and

(c) purifying the protein from the medium.

The medium used for the culturing of the host cell of the invention is any medium which allows the expression of the (poly)peptide of the invention. As is well known to the person skilled in the art, the composition of said medium depends on the particulars and the origin of the host cell employed.- For example, if an E. coli host cell is used, Luria Broth (Sambrook et al. "Molecular Cloning, ...) . If, alternatively, a mammalian host cell is employed, Dulbecco's modified Eagle's medium is used.

The addition of heparin in the expression system employed is essential to release NT-6 bound to cell surface and/or extracellular matrix components into the medium.

The produced (poly)peptide of the invention may then be purified from the culture medium by any method or combination of methods known in the art.

In a preferred embodiment of the invention, said purification comprises the following steps:

(ca) loading the medium on a reverse phase column;

(cb) washing the column with 0.1% trifluoroacetic acid;

(cc) eluting the column with trifluoroacetic acid containing 60% acetonitrile; and

(cd) lyophilisation of the eluate.

In a further preferred embodiment of the invention, said purification comprises the following steps: (ca¹) centrifugation of the medium for 10 min at 400xg and 4°C; (cb¹) adjustment of the medium to a concentration of 0.5 M acetic acid; (cc¹) precipitation of the cells with methanol/chloroform.

The above described embodiments are preferably used if smaller amounts of NT-6 are purified.

In another preferred embodiment of the invention, said purification process comprises the following steps:

(ca¹') chromatography of the medium over a controlled- pore glass column; (cb'') chromatography of the product of step (ca'') over a heparin column; and (cc¹') chromatography of the product of step (cb'') over a reversed-phase MPLC-column.

For example, if the protein of the invention is recovered from a 2000 ml culture, the following steps may be carried out.

Glass powder (PG-120-200, Sigma) is dissolved in trifluoroacetic acid, washed extensively with 0.15 M NaCl containing 10 mM Pj_, pH 7.5 (NaCl/P and packed into a chromatography column (4 ml bed volume) . The conditioned medium (1.6 1) is pumped over this column at a flow rate of 30 ml/h. Subsequently, the glass beads are washed with 500 ml NaCl/Pi and 30 ml 10 mM acetic acid, then NT-3 is eluted with 20 ml 30% acetonitrile, 100 mM NaCl and 100 mM acetic acid. After evaporation of the acetonitrile by vacuum concentration, the sample is applied to a heparin-Sepharose- affinity column (1 ml Heparin-Hitrap, Pharmacia) and eluted with 4 ml 700 mM NaCl/20 mM sodiumphosphate buffer, pH 7.0. The eluate is then applied to a reversed-phase CQ chromatography column (Aquapore RP-300 column, 2.1 mm x 220 mm, Applied Biosystemε) , equilibrated with 0.1% trifluoroacetic acid, and an acetonitrile gradient (7-70% acetonitrile in 90 min at 0.4 ml/min) is started; NT-6 elutes as a major peak at approximately 35% acetonitrile. After evaporation, proteins may be constituted with water and, optionally, further purified by gel filtration on a UltroPac TSK G3000SW column (7.5 mm x 600 mm) equilibrated with 0.1% trifluoroacetic acid and 30% acetonitrile.

The purified NT-6 may then be lyophilised and reconstituted with water.

The purification of NT-6 may be monitored by standard SDS-PAGE procedures or by biological assays as described e.g. in^" Example 5.

The above method is conveniently used for the large- scale preparation of NT-6.

A further object of the invention is to provide an antibody or antibody fragment or derivative thereof to an epitope of the (poly)peptide of the invention or of the molecule having the three-dimensional structure at least with respect to the biologically functional domains as the (poly)peptide encoded by the DNA or RNA sequence of the invention.

The term "antibody" relates to any antibody obtainable by immunization of an animal with the (poly)peptide of the invention as immunogen or hapten or by genetic engineering or by a combination thereof which is specific for NT-6. Genetically engineered antibodies may be obtained e.g. by expression in mammalian, bacterial, yeast or plant cells. Depending on the host cell, the glycosylation pattern of said antibody will vary. Genetically engineered antibodies also comprise humanized antibodies with at least a part of the variable region, preferably the CDR domains, derived from human genes and the remainder derived from one or different other organisms. The term "antibody fragment" relates to any fragment of an antibody which is capable of binding to an epitope specific for the (poly)peptide of the invention. Examples of said fragments are Fab and F(ab')2 fragments.

The term "derivative" relates to any not naturally occurring antibody or any antibody fragment still capable of binding to the (poly)peptide of the invention. Examples of such derivatives are posttranslationally modified antibodies, antibodies into which amino acids have been introduced, from which amino acids have been deleted or in which amino acids have been substituted, all without substantially changing the specificity of said antibody. Further examples are isolated L or H chains of said antibody as long as they determine its specificity as well as antibodies labelled with a detectable marker such as a radioactive marker or an enzyme.

In a preferred embodiment, the antibody or antibody fragment or derivative thereof of the invention is a monoclonal antibody or antibody fragment or derivative thereof. Methods for producing said monoclonal antibodies are well-known in the art and have been described in detail e.g. in Harlow and Lane, "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988.

A further preferred embodiment of the antibody or antibody fragment or derivative thereof of the invention relates to a polyclonal antibody or antibody fragment or derivative thereof. Methods for producing polyclonal antibodies are equally well-known in the art and have been described in detail e.g. in Harlow and Lane, loc. cit.

The present invention also relates to a method for the production of an antibody of the invention comprising the following steps: (a) immunizing a suitable mammal with a (poly)peptide of the invention or a molecule having the three- dimensional structure at least with respect to the biologically functional domains as the (poly)peptide encoded by the DNA or RNA sequence of the invention or an antigenic portion thereof;

(b) isolating the polyclonal antiserum specific to said (poly)peptide or molecule or said antigenic portion thereof according to standard procedures; or

(c) immortalizing cells secreting antibodies specific to said (poly)peptide or molecule or said antigenic portion thereof according to standard procedures.

As is understood by the person skilled in the art, the (poly)peptide or molecule of the invention or antigenic portion thereof is either immunogenic by itself or it acts as a hapten coupled to an immunogenic carrier. Examples of immunogenic carriers which may be used in the method of the invention are keyhole limpet hemocyanin and bovine serum albumine. Methods for coupling haptens to carriers as well as immunization strategies are known to the person skilled in the art and can be followed up, e.g. in Harlow and Lane, loc. cit. The same reference also details purification methods for antibodies.

In a preferred embodiment of the method of the invention, said antigenic portion comprises the amino acid sequence

K A V S H T M H R G E Y S V C.

The amino acid sequence referred to above may be used alone or contained in a longer (poly)peptide as immunogen or hapten as long as it retains its three-dimensional antigenic structure specific for neurotrophin-6. A further object of the invention relates to pharmaceutical composition comprising a substantially pure (poly)peptide or the molecule having the same three- dimensional structure at least with respect to the biologically functional domains as the (poly)peptide encoded by the DNA or RNA sequence of the invention, optionally in combination with a pharmaceutically acceptable carrier.

The term "substantially pure" is intended to mean that no further ingredient is comprised in the (poly) eptide or molecule preparation which interferes with the activity of the pharmaceutical composition or causes adverse side effects upon application.

The active pharmaceutical compositions of the invention, which comprises the (poly)peptide or molecule of the invention or derivatives produced therefrom or a combination of the (poly)peptide or molecule of the invention and a second agent such as NGF or skeletal muscle extract, may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

The amount of (poly)peptide or molecule of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the dose-response curve and the pharmaceutical compositions of the invention first in vitro, e.g. in appropriate bioassay systems, using neurons prepared from the central (such as retinal ganglion cells, cholinergic neurons, dopaminergic neurons, motoneurons) and/or peripheral (such as sympathetic neurons, sensory neurons) nervous system of chick or rat and then in useful animal model systems prior to testing in humans. Based on in vitro data, in a specific embodiment of the invention, a pharmaceutical composition effective in promoting the survival of sensory neurons may provide a local (poly)peptide concentration of between about 5 and 100 ng/ml. In an additional specific embodiment of the invention, a pharmaceutical composition effective in promoting the growth and survival of dopaminergic, cholinergic and other subpopulations of neurons may provide a local polypeptide concentration of between about 10 ng/ml and 100 ng/ml.

Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intrathecal, intraventricular and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

Further, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

The invention also provides for pharmaceutical compositions comprising (poly)peptides of the invention administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the (poly)peptide of the invention. It is envisioned that it may be possible to introduce cells actively producing the polypeptide of the invention, substances related thereto, antagonists thereof or antibodies thereto into areas in need of increased or decreased concentrations of NT-6.

In a preferred embodiment of the invention, said pharmaceutical composition may be used for the increase of neuron survival.

It is envisaged that said pharmaceutical composition will support in particular the survival of sensory neurons, e.g. the petrosal, geniculate, and ventrolateral trigeminal ganglia.

In a further preferred embodiment of the invention, said pharmaceutical composition is for the increase of neuron growth.

For example, said pharmaceutical composition may be used for the increase of neurite outgrowth, e.g. of sensory neurons.

An additional preferred embodiment of the invention relates to a pharmaceutical composition for the support of differentiated cell function.

Thus, said pharmaceutical composition may be used for the treatment of conditions where lack of differentiation of neural crest cells is involved.

A further preferred embodiment of the invention relates to a pharmaceutical composition for the treatment of a disease or disorder of the nervous system. In a more preferred embodiment the pharmaceutical composition of the invention may be used for the treatment of a disease or disorder of the nervous system which is a degenerative disease.

Thus, the (poly)peptide of the invention can be used in the treatment of hereditary spastic paraplegia with retinal degeneration (Kjellin and Barnard-Scholz syndromes) , retinitis pig entosa, Stargardt disease, Usher syndrome (retinitis pigmentosa with congenital hearing loss) , and Refsum syndrome (retinitis pigmentosa, hereditary hearing loss, and polyneuropathy) , to name but a few. It is possible that a defect in NT-6 synthesis or responsiveness may be the underlying etiology for syndromes characterized by a combination of retinal degeneration and other sensory dysfunction.

In a furthermore preferred embodiment of the invention, said disease or disorder of the nervous system comprises damage of the nervous system.

In particularly preferred embodiments of the invention, said damage is selected from the group consisting of trauma, surgery, infarction, infection, and malignancy.

Additionally, said pharmaceutical composition may be used for the treatment of ischemia, nutritional deficiency or metabolic disease.

Furthermore, said pharmaceutical composition may be used for the treatment of neuropathic side effects caused by the treatment of anti-static agents in cancer patients.

In a further preferred embodiment of the invention said pharmaceutical composition may be used for the treatment of a disease or disorder or damage of the nervous system which is caused by a toxic agent. In particular, the (poly)peptide of the invention can be locally administered to sensory neurons which have been severed by any of the above enumerated causatives, including, but not limited to, neurons in dorsal root ganglia or in any of the following tissues: the geniculate, petrosal, and nodose ganglia; the vestibuloacoustic complex of the Vlllth cranial nerve; the ventrolateral pole of the maxillomandibular lobe of the trigeminal ganglion; and the mesencephalic trigeminal nucleus. It may be desirable to administer the (poly)peptide of the invention by adsorption onto a membrane, e.g. a silastic membrane, that could be implanted in the proximity of the severed nerve. The present invention can also be used for example in hastening the recovery of patients suffering from diabetic neuropathies, e.g. mononeuropathy multiplex.

In a further particularly preferred embodiment of the invention, said pharmaceutical composition may be used for the treatment of a disorder of the nervous system comprising a congenital disorder of the retina.

An additional particularly preferred embodiment of the invention relates to a pharmaceutical composition for the treatment of a degenerative disease which is selected from the group consisting of Alzheimer's disease, Huntington's chorea, retinal disease, Parkinson's disease and "Parkinson- Plus" syndrome.

It may also be envisioned that the (poly)peptide or molecule of the invention can be used in conjunction with surgical implantation of tissue in the treatment of Alzheimer's disease and/or Parkinson's disease.

In a further particularly preferred embodiment of the present invention, said pharmaceutical composition is for the treatment of a "Parkinson-Plus" syndrome selected from the group consisting of Progressive Spranuclear Palsey (Steele-Richardson-Olszweski Syndrome) , Olivopontocerebellar Atrophy, Shy-Drager Syndrome (Multiple Systems Atrophy) , and Guamanian Parkinsonism-Dementia complex.

A further particularly preferred embodiment of the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder of the nervous system which comprises disease or disorder of the sensory neurons or a tumor.

In a further particularly preferred embodiment of the present invention said pharmaceutical composition is for the treatment of a tumor which is a neuroblastoma.

Further, the (poly)peptide of the invention may be used to promote the survival of dopaminergic substantia nigra neurons in a dose-dependent manner, supporting the use of the (poly)peptide of the invention in the treatment of disorders of CNS dopaminergic neurons, including, but not limited to, Parkinson's disease.

In addition, the (poly)peptide of the invention will sustain the survival of CNS cholinergic neurons and, in particular, basal forebrain cholinergic neurons, indicating that the (poly)peptide of the invention may be useful in the treatment of disorders involving cholinergic neurons, including, but not limited to, Alzheimer's disease. It has been shown that approximately 35 per cent of patients with Parkinson's disease suffer from Alzheimer-type dementia; NT- 6 related (poly)peptide produced according to the invention may prove to be useful single agent therapy for this disease complex. Similarly, NT-6 related (poly)peptide produced according to the invention may be used therapeutically to treat Alzheimer's disease in conjunction with Down's Syndrome. The (poly)peptide produced according to the invention can be used in the treatment of a variety of dementias as well as congenital learning disorders. It is also expected that the (poly)peptide of the invention suppresses the proliferation of astroglial cells, supporting the use of BDNF for diminishing scar formation in the CNS (for example, following surgery, trauma, or infarct) as well as the use of said (poly)peptide in the treatment of astroglial derived CNS tumors. Also the (poly)peptide of the invention may be used to upregulate the expression of NGF receptor, and thereby may be advantageously administered prior to or concurrently with NGF to a patient in need of such treatment.

Consequently, further particularly preferred embodiments of the invention relate to pharmaceutical compositions comprising the (poly)peptide or molecule of the invention for the promotion of the survival of dopaminergic neurons, of cholinergic neurons, in particular of basal forebrain cholinergic neurons or septal cholinergic neurons.

In addition, said pharmaceutical composition may be used for suppressing the proliferation of astroglial cells.

In a further preferred embodiment of the pharmaceutical composition of the present invention, said composition comprises at least one additional neurotrophic factor.

In a particularly preferred embodiment, said pharmaceutical composition additionally comprises NGF, NT-3, BDNF and/or NT-4/5.

It is envisioned that the (poly)peptide or molecule of the invention is used in conjunction with other cytokines to achieve a desired neurotrophic effect. For example, and not by way of limitation, the (poly)peptide or molecule of the invention can be used together with NGF or with skeletal muscle extract to achieve a synergistic stimulatory effect on growth of sensory neurons wherein synergistic is construed to mean that the effect of the combination of the (poly)peptide or molecule of the invention and a second agent achieves an effect greater than the same amount of either substance used individually. It is envisioned that said (poly)peptide or molecule may function synergiεtically with other CNS-derived peptide factors yet to be fully characterized, in the growth, development, and survival of a wide array of neuronal subpopulationε in the central nervouε system.

It is further envisioned that, based on the full characterization of the (poly)peptide of the invention, novel peptide fragments, derivatives, or mutants of the (poly)peptide of the invention may be developed which are capable of acting as antagonists of some, or all of the biological functions of NT-6. Such antagonists may be useful in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

A further object of the invention is to provide a pharmaceutical composition comprising an antibody or a fragment or a derivative thereof of the invention.

Doses and routes of administration may be the same or similar to the ones discussed above for the pharmaceutical compositions comprising the (poly)peptide or molecule of the invention and are selected by the physician in charge.

Said pharmaceutical composition can be administered to patients suffering from a variety of neurologic disorders and diseases and who are in need of such treatment. For example, patients who suffer from excessive production of NT-6 may be in need of such treatment. Anti-NT-6 antibodieε can be used in prevention of aberrant regeneration of senεory neuronε (e.g. post-operatively) , or in the treatment of chronic pain syndromes. In light of the high levels of NT-6 mRNA expected to be found in neuroblastoma tissue, it is poεεible that NT-6 εerveε as an autocrine tumor growth factor for neuroblastoma; accordingly, anti-NT-6 antibodieε can be adminiεtered therapeutically to achieve tumor regreεεion in a specific embodiment of the invention.

A further object of the invention is to provide an in vitro method for diagnosing a diεeaεe or disorder of the nervous system referred to herein above comprising

(a) contacting a tisεue probe with a detectably labelled DNA or RNA molecule of the invention or nucleic acid molecules complementary thereto under conditions which will allow hybridization to occur; and

(b) detecting any hybridization which has occurred.

Hybridization asεays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or diseaεe εtates asεociated with changeε in NT-6 expreεsion, including, in particular, conditions resulting in εenεory neuron damage and degeneration of retinal neurons. Such diseases and conditions include but are not limited to CNS trauma, infarction, infection, degenerative nerve diseaεe, malignancy, or post-operative changes including but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Chorea, and degenerative diseases of the retina. For example, total RNA in a tissue sample from a patient can be aεεayed for the presence of NT-6 mRNA, wherein the decrease in the amount of NT-6 mRNA is indicative of neuronal degeneration.

Another object of the invention relates to an n vitro method for diagnosing a disease or disorder of the nervous εyεtem referred to herein above compriεing

(a) exposing a tisεue probe to a detectably labelled antibody or a fragment thereof or a derivative thereof of the invention under conditions which will allow binding to occur; and

(b) detecting any binding which has occurred. Thus, said detectably labelled antibodies or fragments thereof or derivatives thereof can be used to diagnose diseases and disorders of the nervous syεtem, including, in particular, sensory disorders and degenerative diseaεeε of the retina, as well as those disorders and diseases listed supra. The antibodies of the invention can be used, for example, in m situ hybridization techniqueε using tissue sampleε obtained from a patient in need of such evaluation. In a further example, the antibodies or fragments or derivatives thereof of the invention can be used in ELISA procedures to detect and/or measure amounts of NT-6 present in tiεεue or fluid εamples; similarly, the antibodies or fragments or derivatives thereof of the invention can be used in Western blot analysis to detect and/or measure NT-6 present in tissue or fluid samples.

A further object of the present invention relates to an in vitro method for the diagnosis of diseaεes and/or disorders of the nervous system comprising the following steps:

(a) bringing the detectably labelled (poly)peptide or molecule having the three-dimensional structure at least with respect to the biologically functional domains as the (poly)peptide encoded by the DNA or RNA sequence of the invention into contact with cells or tisεue normally expressing the NT-6 receptors; and

(b) detecting any abnormalitieε in NT-6 receptor expression.

Such abnormalities may be, e.g., inherited or toxic damages of neurons.

Finally, the present invention relateε to a kit for the diagnoεiε of a diεeaεe or diεorder referred to herein above compriεing at leaεt (a) a DNA and/or a RNA molecule of the invention or a derivative thereof or nucleic acid molecules complementary thereto; and/or

(b) a primer or a pair of primers of the invention; and/or

(c) an antibody or a fragment or a derivative thereof according to the invention, optionally labelled with a detectable marker; and/or

(d) an antibody or a fragment thereof or a derivative thereof directed to a nucleic acid of the invention.

The kit of the invention iε conveniently uεed to provide the necessary tools for any in vitro method referred to above.

The DNA or RNA molecules of the invention or a derivative thereof, or complementary sequenceε thereto may themselves be labelled with a detectable marker such as an enzymatic or radioactive tag. Alternatively, if the DNA or RNA molecule of the invention iε used for the detection of the nucleic acid sequence of interest, a second nucleic acid sequence may be employed for the detection of the nucleic acid complex. Said second nucleic acid εequence iε labelled with a detectable marker and hybridizeε to a different portion of the DNA or RNA molecule of the invention or derivative thereof or complementary sequence thereto than the nucleic acid sequence of interest.

Further, the kit of the invention may comprise a primer or a pair of primers of the invention. Applications and uses of said primer or pair of primers have been discuεsed above.

Additionally or alternatively, the kit of the invention may comprise an antibody or antibody fragment or derivative thereof of the invention, optionally labelled with a detectable marker. Furthermore, the kit of the invention may comprise an antibody or a fragment or a derivative thereof which detects the nucleic acid of the invention, optionally labelled with a detectable marker. The detectable marker referred to above may be e.g. an enzyme capable of converting a substrate which results in a change of substrate color or a radioactive marker.

It is within the skill of the person skilled in the art to design various combinations of compounds comprised in the kit of the invention which may be used in the n vitro diagnostic methodε referred to above. Also, additional compounds not referred to above such as a second labelled antibody directed to an antibody comprised in the kit of the invention may be employed for detection purposes. Alternatively, such a εecond labelled antibody may be comprised in the kit of the invention. Furthermore, various buffers and diluents may be comprised in the kit of the invention.

It is also within the ordinary skill of the art to carry out various diagnostic tests referred to above such as

ELISAs or RIAs using the kit of the invention, as well as determining which buffers and/or diluents may also be contained in the kit of the invention.

The figures show:

Figure 1;

Structure and εequence of the fish NT-6 gene and its predicted protein. (A) Structure and restriction map depicting the inserts of overlapping genomic clones and a full length complementary DNA clone; the EcoRI sites are indicated. The hatched rectangle delineates the open reading frame of the predicted protein and the position of the intron is marked. (B) Southern blot of Eco-RI digested fish DNA hybridized under low stringency with the 400bp Nsil-EcoRI probe of NT-6 and washed at low (lane L) and high (lane H) stringency, respectively (see Example 1) . The sizeε of the hybridizing genomic bands are given in kbp; the 2.5kbp band representε NT-6, and the 2.9kbp fragment containε NGF. Size markers (lane M) are ³²P labeled Hindlll fragments of phage lambda. (C) Nucleotide and deduced amino acid sequence. The sequence of the cDNA is given; the genomic DNA encodeε a protein with identical εequence but divergeε 5' to nucleotide 132 of the cDNA due to an intron (arrow) . Stop codonε are indicated by aεterisks, the polyadenylation εignal is indicated with a dotted line. The precursor protein of 286 reεidueε iε proteolytically cleaved (arrowhead) to yield a εecreted protein of 143 residues. The cysteines are marked in εquareε and the inεertion not found in NGF iε' boxed. The two multi-baεic motifε for proteolytic cleavage are underlined.

Figure 2:

(A) Heparin requirement to release NT-6 from producing cells into the medium. Western blot analysis of medium and cell lysates from cells infected with a NT-6 recombinant vaccinia virus or a wildtyp virus (WT) vectors. Heparin (100 μg/ml) was added as indicated for a period of 20 hours or for 30 min at the end of the 20 hourε incubation period. A control blot was developed where the antiserum was preincubated in the presence of the competing NT-6 peptide (1 μg/ml) . (B) SDS-PAGE analysiε of purified NT-6 (2 μg of protein loaded) stained with Coomassie brillant blue.

Figure 3:

Expression of NT-6 transcripts in embryonal development and adult tisεueε of fiεh. Northern blot analysis of total RNA (10 μg/lane) from different stages of Xiphophorus embryos and adult tissues probed with a NT-6 riboprobe. Transcriptε of l,4kb and lkb are indicated. Figure 4 :

Localiεation of NT-6 mRNA by in εitu hybridization hiεtochemiεtry. Bright-field (A) and dark-field (B) micrographs showing a paraεagital section through the brain of a stage 21 platyfiεh embryo. Silver grainε are concentrated in the gray matter of the optic tectum. Control hybridization (C) of the adjacent εection hybridized with the εenεe probe. (D) High-power brightfield photograph of tectal εection εhowing localization of silver grains in neuronal cells. All sections were counterstained with toluidine blue. Scale bar, 100 μm in A-C; 10 μm in D.

Figure 5: Sequence alignment of NT-6 with other neurotrophins.

The deduced amino acid sequence of fish NT-6 is shown aligned to the other nerve growth factors. Conserved amino acids are highlighted by shading. The horizontal bars above the sequence delineate loop or turn segments exposed on the protein surface as determined by X-ray analysis of the mouse NGF structure (McDonald et al., 1991) . Of special interest is the strongly positively charged (+) insert in NT-6.

Figure 6: Effect of NT-6 on the survival of sensory neurons prepared from the DRG of eight days-old chick embryos.

Phase-contrast microgaphs of cultures in either the presence (A) or absence (B) of NT-6. After 2 days in culture, neurons with phase-bright cell bodieε and neurites were present in NT-6 treated wells; the same cultures without NT-6 show typical features ot apoptosiε.

(C) Dose-response curve of the survival effect of NT-6 on DRG neuronε plated onto plaεtic wells coated either with conditioned medium from a εchwannoma cell line (open circles) or with polyornithine plus laminin (filled circles) .

(D) Bar graph εhowing the effect on the εurvival of sensory neurons in the presence of NT-6 or NGF with or without the anti-NGF antibody (monoclonal clone 27/21, Korsching and Thoenen H., Proc. Natl. Acad. Sci. USA 80, (1983), 3513- 3516, available from Boehringer Mannheim, Cat. No. 1087754 (α-nerve growth factor mouse) ) .

The Examples illustrate the invention.

Example l: Cloning of NT-6

Neurotrophin-6 was cloned from a genomic library of the platyfish Xiphophorus maculatus (origin Rio Usumacinta, Mexico) using a radio labelled 360bp DNA probe corresponding to mature mouse NGF as follows: the probe was labeled with ³²P by nick-translation (Rigby et al., J. Mol. Biol. 113, 237-251, (1977). Hybridization of the filters with the plaque lifts was performed in 5x SSC (lx SSC iε 0.15 M NaCl, 0.015 M εodium citrate, pH 7.2), 40% formamide at 42°C with washes in 2x SSC at 55°C. In this screen, partially overlapping genomic clones (EcoRI and Xbal subfragments of two plaque purified cloneε, 3.5 and 5.5, Figure IA) were identified that hybridized to the mouse NGF probe under low-stringency hybridization conditions. DNA sequencing of the hybridizing region predicted a single long open reading frame of 858 base pairs encoding a polypeptide related to the nerve growth factors. The Southern blot was performed (as described by Southern, J. Mol. Biol. 98, 503-517, (1975)). The conditions of hybridization were aε followε: a 389bp Nsil-EcoRI probe was radiolabeled according to the method of Feinberg and Vogelεtein, Anal. Biochem. 312, 6-13, (1983) . The blot waε hybridized in 5x SSC/35% formamide at 42°C with washing at lx SSC at 60°C (low stringency) and 0.5x SSC at 65°C (high stringency). Two hybridizing bands were detected (Figure IB) ._^ The fragment of 2.5kbp corresponding to the cloned DNA hybridized strongly but there was also a weakly hybridizing band of 2.9kbp visible. Since this band disappeared upon increasing the wash stringency to 0.5x SSC at 65°C, it probably represented a sequence related to but different from the probe (Figure IB) .

Molecular cloning of the 2.9kbp EcoRI fragment was carried out in the following manner: Genomic DNA fragments in the size range 2.6-3.3kbp obtained by EcoRI digestion were separated by agarose gel electrophoresiε and purified according to the method of Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA 76, 615-619, (1979)) . They were ligated into the vector lambda gtlO, and packaged into phage particles as εpecified by the manufacturer of the packaging extract (Amerεham) . A positive clone with a 2.9kbp insert (clone A5.2) was isolated. Subsequent protein expression in RK 13 cells ATCC # CCL 37 and its biological characterization showed that the 2.9kbp band contained the bona fide, orthologous fish NGF gene. Fish NGF showed a εurvival activity for εympathetic and sensory neurons from the dorsal root ganglia from chick embryos, but not on nodose neurons. Sequence alignments of the neurotrophin encoded in the 2.5kbp EcoRI fragment to the other known neurotrophin sequenceε in fish, like the complete NGF and BDNF sequences of Xiphophorus (Gδtz et al. , 1992b), a partial NT-3 εequence of salmon (Hallbδδk et al. , 1991) and to the sequences of neurotrophins from other vertebrate species (Table I) suggeεted that it did not represent the true orthologue in Xiphophorus of any known neurotrophin but rather a paralogous novel member of the neurotrophin gene family termed neurotrophin-6.

The genomic organization of the NT-6 gene predicted a 22 amino acid inεertion in between the εecond and third cyεteine residues. In NGF this region is 9 amino acids long, in NT-6 the length is 31 amino acids. Studies on the NT-6 cDNA confirmed that this insertion iε indeed a coding sequence.

Example 2: Expression of Recombinant NT-6

Expression of the protein was carried out in the following manner:

A vaccinia virus expression vector with the NT-6 coding region inserted was constructed and used to infect the RK13 cell line. Rabbit kidney cells (RK13 cell line) grown as monolayers in a 10-tray cell factory (Nunc) were infected with VNNT-6 recombinant virus (multiplicity of infection greater 5). After 6h growth in Dulbecco's modified Eagle'ε medium containing 5% fetal calf serum, the medium was removed and εeru -free medium containing heparin (100 μg/ml, Sigma # H7005) waε added and incubation continued for 20h. At the end of the incubation period, the conditioned medium waε purified by chromatography on controlled-pore glaεs. Glasε powder (PG-120-200, Sigma) waε dissolved in trifluoroacetic acid, washed extensively with 0.15 M NaCl containing 10 mM sodiumphosphate buffer, pH 7.5 (NaCl/Pi) and packed into a chromatography column (4 ml bed volume) . The conditioned medium (1000 ml) was pumped over this column at a flow rate of 30 ml/h. Subsequently, the glass beads were washed with 500 ml NaCl/Pi and 30 ml 100 mM acetic acid, then NT-6 was eluted with 15 ml 30% acetonitrile, 100 mM NaCl and 100 mM acetic acid. Then the sample was applied to a heparin-Sepharose-affinity column (1 ml Heparin-Hitrap, Pharmacia) and eluted with 4 ml 700 mM NaCl/20 mM sodiumphosphate buffer, pH 7.0. The eluate is then applied to a reversed-phaεe Cg chromatography column (Auqapore RP-300 column, 2.1 mm x 220 mm, Applied Biosystems) , equilibrated with 0.1% trifluoroacetic acid, and an acetonitrile gradient (7-70% acetonitrile in 90 min at 0.4 ml/min) waε εtarted; NT-6 eluted aε a major peak at approximately 35% acetonitrile. After evaporation, proteins were constituted with water and, optionally, further purified by gel filtration on a UltroPac TSK G3000SW column (7.5 mm x 600 mm) equilibrated with 0.1% trifluoroacetic acid and 30% acetonitrile. Alternatively, conditioned serum-free medium was harvested 24h post infection after adding heparin (100 μg/ml; Sigma no. H7005) to the culture flask; see above the cell lysate was prepared by addition of Laemmli sample buffer Laemmli, U.K. Nature 227, 680-685, (1970) to the cell monolayer. To analyse the expression of NT-6, the medium following removal from the culture flask, was εpun to remove any detached cellε (10 min 400 g at 4°C) made 0.5 M with reεpect to acetic acid and precipitated. Alternatively, the medium (10 ml) was loaded onto a reversed phase cartridge (Sep-Pak C8 Plus, Millipore) , washed with 100 ml of 0,1% trifluoroacetic acid and eluted with 2 ml of 0,1% trifluoroacetic acid containing 60% acetonitrile. The samples were then lyophilized and resuεpended in water. After εeparation by 0.1% SDS - 15% polyacrylamide electrophoreεis (Laemmli, loc.cit.), proteins were transferred to nitrocellulose filter membranes (BA 83, 0.2 μm, Schleicher and Schϋll) by electroblotting as described (Kyhse-Andersen, 1984) . Antibodies were raised in rabbits against a peptide (KAVSHTMHRGEYSVC, see Figure 1C) which was syntheεized on a Applied Bioεystems 43 IA peptide synthesizer with Fmoc chemistry.

This peptide corresponds to the putative N-terminus of the mature NT-6 sequence. Primary antiseru : 500 μg peptide (500 μg) was dissolved in water and mixed with 1 ml of complete Freund'ε adjuvans to form an emulsion. Rabbits were immunized subcutanously. Four and five weeks later, 500 μg of peptide in incomplete Freund's adjuvans were used to boost the immune responεe and horseradish peroxidase conjugated goat anti-rabbit IgG (Jackεon Immunoreεearch no. 111-035-003) were uεed at dilutionε of 1:5.000; detection waε performed with chemilumineεcence (ECL-kit, Amerεham) . Conditioned medium obtained from cells infected with the NT-6 vaccinia virus was concentrated and analysed by western blotting (Figure 2) . In several experiments NT-6 was not detected in the medium after a one day expreεεion period (Figure 2, lane 1) . The cell lyεate however, contained a 36k antigen (Figure 2, lane 6) that waε not preεent in cells infected with wildtype virus (lane 7) and that could be eliminated by preincubation of the antibodies with the corresponding peptide (lane 8) . Furthermore, control experiments done in parallel with a mouse NGF vaccinia vector demonstrated that the infected cells secreted mature NGF ruling against a general problem of the expresεion system employed.

From the amino acid sequence it was deduced that NT-6 might not be a soluble factor as the other neurotrophins but rather contain a heparin binding domain probably formed by the positively charged amino acids in the insert. The three-dimensional structure of mouse NGF was recently determined at 2.3-A resolution by X-ray crystallography (McDonald et al, 1991) and it waε expected that the overall folding pattern and most elements of secondary structure of NT-6 are very similar. In mouse NGF, the segment in between cysteins 2 and 3 forms three consecutive reverse turns and projects in the solvent (McDonald et al., 1991). By homology to NGF the basic insert in NT-6 might be available for interaction with the heparin polyanion with the half-cystines involved in stabilizing the rigidity of the structure. Thus, it waε expected that recombinant NT-6 was sequestered at the cell surface and/or matrix but could possibly be released by the addition of heparin. When the expressing cells were grown in the presence of heparin, a specific band of 21kD was detected in the medium (Figure 2, lanes 2 and 3) . Only a small difference in the quantity of NT-6 released was noted when heparin was present only for 30 min at the end of the production period as compared to the whole period of incubation (20 hours) . Thuε, heparin doeε not seem to protect secreted soluble NT-6 from proteolytic degradation but acted via a release mechanism. On a per cell level, approximately 60-fold higher amounts of precursor versus mature NT-6 were present as was determined by Western Blot experiments; expresεing mouεe NGF in this system yields a ratio of precursor to mature protein close to 1 indicating that the release of NT-6 by heparin and/or its secretion were not optimized. NT-6 was purified to near homogeneity (Figure 2B) aε described in Example 2. The purification included the binding of NT-6 to heparin agarose and the subsequent elution with 700 mM NaCl supporting the above finding that NT-6 is a heparin binding molecule. N-terminal sequencing waε performed on a gas/liquid-phase sequencer 477A from Applied Biosystems equipped with an on-line 120A phenylthiohydantoin analyser using the conditions given by the manufacturer for 9 cycles yielded the sequence K-A-V-S-H-T-M-7-R demonstrating that proteolytic cleavage had occurred on the carboxyl side of the subtilisin-like serine protease PACE/furin cleavage motif (Figure 1C) .

Example 3: Developmental and Tissue-specific Expression of N -6 mRNA

Poly(A)+ RNA from fish embryos was iεolated by guanidinium thiocyanate lyεiε followed by ceεiumtrifluoroacetate centrifugation (Okayama et al., 1987) . Electrophoreεiε and blotting of RNA and hybridization of blots were performed as described by Krumlauf (Krumlauf, 1991) . Filterε were hybridized in 50% formamide at 65°C for 12-24 h and εubεequently washed at a final stringency of 0.2x SSC - 1% SDS at 80°C for lh before autoradiography. The εingle εtranded (antiεense) riboprobe was synthesized with RNA polymerase from a linearized plasmid template that contained the lkbp EcoRI subfragment of the cDNA (Figure IA) ; thiε template εerved alεo to produce a sense RNA probe for in situ hybridization. In situ hybridizations were performed on 7 or 10 μm sectionε of stage 21-23 Xiphophorus embryos that had been cut from paraffin-embedded specimen. Glaεε slides containing adjacent serial sections were hybridized with either sense or antisense NT-6 riboprobeε (see above) using the in situ hybridization protocol described by Wilkinson (Wilkinson and Green, 1990) . Riboprobes were labelled with 35S-UTP and used without alkaline hydrolysis.

The analysis of the NT-6 mRNA expression during embryonal development by Northern blotting revealed a transcript of 1.4kb from organogeneεiε onwardε (εtage 12) with an elevated expression in stages 18-22. The expression of this transcript continued in adult brain (Figure 3B) . NT-6 was also expresεed aε a 1.4kb RNA in adult gill, liver and eye but no expreεεion could be detected in εkin, spleen, heart and.muscle (Figure 3B) .

The small size of Xiphophorus embryos made the expresεion of NT-6 by in situ hybridization with consecutive serial sections throughout stage 21-23 embryos where NT-6 RNA level was highest possible. Sagital sections hybridized with a S-labelled NT-6 antisense riboprobe revealed expression in the gray matter of the optic tectum (Figure 4A and B) . Adjacent sections hybridized with the εenεe probe revealed no εpecific signal (Figure 4C) .

Example : Determination of the Structure of NT-6

The deduced structure of the NT-6 precurεor of 286 amino acidε (predicted M_r of 31,424) εhowed all the features of a neurotrophin (Figure 1C) : (i) a hydrophobic domain at the N- terminuε with the characteristics of a signal peptide (amino acids 1-19) . (ii) a pro-region (amino acids 20- 142) containing two multiple basic motifs (R-X-K/R-R at reεidueε 63-66 and 139-142) , known to be necessary for intracellular proteolytic conversion of precursor proteins into their mature forms in the constitutive secretory pathway (Hosaka et al., 1991). (iii) an amino acid εequence at the carboxyl half of the precurεor εtarting at lyεine 143 which encodeε the mature NT-6 of 143 reεidueε (M_r 15,968, pi 10,8; see also below the data on the recombinant protein) . (iv) 40 residueε, including the εix cysteines that have been found to be conserved in all neurotrophins known so far that are alεo conεerved in NT-6 (Figure 5) indicating that NT-6 shares the same fold (that is, has the same secondary structure in the same orientation with the same chain topology) ; these residues are thought to be involved in the correct folding of the neurotrophins based on the X-ray diffraction εtructure of NGF (McDonald et al., 1991).

An intereεting feature of the predicted amino acid sequence is the existence of an insertion of 22 residues between cysteines 2 and 3 (Figures 1C and 5) . This segment containε six basic and eight bend-inducing residues. Although small insertions/deletions have been noted in sequence compariεonε of the different neurotrophins (see Figure 2) experimental evidence for the presence of this domain in the mRNA and therefore the translational product of the genomic clone waε obtained. A cDNA library from fish brain was prepared by reverse transcription with oligo(dT) -priming of poly(A)+ mRNA isolated from X.helleri (origin Rio Lancetilla) according to standard proceedures; εee Sambrook et al. loc. cit. The library waε εcreened with the ³²P-labelled 389bp Nsil-EcoRI fragment derived from the fish genomic clone (see Figure IA) . A complementary DNA clone iεolated from said cDNA library (abundance 1 clone per 500.000 recombinant phage clones screened) contained a l,4kbp coding length insert sequence. The encoded protein εequence iε identical to that deduced from the genomic clone. Three εilent nucleotide differences in the coding region are due to species difference: the genomic DNA was cloned from X. maculatus, the cDNA was cloned from X.helleri. It is noteworthy that in both the_ genomic and cDNA sequences in-frame stop codons upstream of the start codon are present (Figure 1C) . An intron is located 1 lbp upstream of the initiating ATG since the genomic and cDNA sequences diverge at this point and a splice site acceptor site is present in the genomic sequence (Figures IA and 1C) .

Example 5: Determination of the Biological Activity of NT-6

The purified recombinant NT-6 cell survival activity on embryonic chick sensory neurons prepared from dorsal root ganglia (DRG) was determined in the following manner:

Dorεal root ganglia were removed from chick embryos at embryonic day 8 as described previouεly (Barde et al. , 1980; Lindsay et al., 1985). Neuron-enriched cell suspensions were obtained by differential preplating to remove nonneuronal cells, and neurons were plated at a density of 1.500-2 000 cells per well (1 cm²) in the wells of 48-well plastic plates (Costar) that had been coated εequentially with polyornithine (500 μg/ml) pluε laminin (20 μg/ml) or conditioned medium from the εchwannoma cell line RN22 (Palm and Furcht, 1983) as indicated in Figure 6. The number of surviving neurite-bearing neurons was determined after 48h in the presence or absence of factors in microscopic fields covering 14% of each well. Results are the mean — standard deviation of the percent survival for 6 wells. All experimentε were performed at leaεt twice. NGF was purified from mouse salivary gland as deεcribed (Suda et al., 1978); mouse BDNF and NT-3 were expresεed and purified aε deεcribed (Gδtz et al., 1992a).

This ganglion contains several different types of neuronε which have been shown in previous experiments to be responsive to all the members of the nerve growth factor family (Lindsay et al., 1985; Davies et al., 1986; Leibrock et al, 1989; Hohn et al. , 1990; Maiεonpierre et al. , 1990; Hallbδδk et al., 1991) . The result with these dissociated cultures showed that NT-6 promotes the survival of sensory DRG neurons (Figure 6A, B) . Doεe-responεe experimentε with theεe cultures plated onto matrix components εecreted from the εchwannoma cell line RN22 demonstrated that the concentration of NT-6 to obtain half-maximal and optimal survival was 150 ng/ml and 500 ng/ml, respectively. 40% of the plated neurons survived in the presence of 500 ng/ml and increased concentration above 500 ng/ml did not produce more surviving neurons (Figure 6C) . Control purifications of medium from wildtype vaccinia virus-infected cells allowed only 9% of the plated neuronal cells to survive arguing against the possibility that the neurotrophic activity seen with the NT-6 preparation was due to trace amounts of a neurotrophin from the RK13 cells. Furthermore, when an antibody that inhibited the activity of NGF was included in the bioasεay, no inhibition of the εurvival activity of NT-6 was obεerved (Fig. 6D) . When the dose-responεe experimentε were performed on polyornithine/laminin εubstrate, 20% of the plated neuronε εurvived in the preεence of 1 μg/ml NT-6 indicating that the binding of NT-6 to a matrix component produced by εchwannoma cellε, presumably a heparin-binding molecule, potentiates its biological activity (Figure 6C) .

Table I

Amino acid identity in percent of fiεh NT-6 with other memberε of the nerve growth factor protein family (note that for εalmon NT-3 only 33 aminoacidε are known)

NGF, fiεh 61

NGF, man 56

BDNF, fiεh 48

BDNF, man 48

NT-3, fish 52

NT-3, man 46

NT-4, frog 39

NT-4/5, man 38

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Barde, Y.-A., Edgar, D. and Thoenen, H. (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1, 549-553.

Barde, Y.-A. (1989) Trophic factorε and neuronal survival. Neuron 2, 1525-1534.

Barde, Y.-A. (1990) The nerve growth factor family. Progr. Growth Factor Res. 2, 237-248.

Berkemeier, L.R., Winslow, J.W. , Kaplan, D.R. , Nikolicε, K. , Goeddel, D.V. and Roεenthal, A. (1991) Neurotrophin-5: A novel neurotrophic factor that activates trk and trkB. Neuron 7, 857-866.

Cowan, W.M. , Fawcett, J.W. , O'Leary, D.D.M. and Stanfield, B.B. (1984) Regressive events in neurogenesis. Science 225, 1258-1260.

Davies, A.M., Thoenen, H. and Barde, Y.-A. (1986) Different factors from the central nervous syεtem and periphery regulate the εurvival of εenεory neuronε. Nature 319, 497-499

Davies, A.M., Bandtlow, C. , Heumann, R. , Korsching, S., Rohrer, H. and Thoenen, H. (1987) Timing and site of nerve growth factor εyntheεiε in developing skin in relation to innervation and expression of the receptor. Nature 326, 353-358.

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Ernst, M. (1926) ϋber Untergang von Zellen wahrend der normalen Entwicklung bei Wirbeltieren. Zeitschr.Anat. u.Entw.geεch. 79, 228-262.

Gδtz, R. , Kolbeck, E , Lottspeich, F. and Barde, Y.-A. (1992a) Production and characterization of recombinant mouse neurotrophin-3. Eur.J.Biochem. 204 745-749.

Gδtz, R. , Raulf, F. and Schartl, M. (1992b) Brain-derived neurotrophic factor is more highly conserved in structure and function than nerve growth factor during vertebrate evolution. J.Neurochem. 59, 432-442. Hallbδδk, F. , Ibaήez, C.F.- and Perεεon, H. (1991)

Evolutionary εtudieε of the nerve growth factor family reveal a novel member abundantly expreεεed in Xenopuε ovary. Neuron 6, 845-858.

Hamburger, V. (1993) The hiεtory of the discovery of the nerve growth factor. J.Neurobiol. 24, 893-897.

Hohn, A., Leibrock, J. , Bailey, K. and Barde, Y.-A. (1990)

Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344, 339-341.

Holder, N. and Clarke, J.D.W. (1988) Is there a correlation between continuous neurogenesis and directed axon regeneration in the vertebrate nervous system? Trends Neurosci. 11, 94-99.

Hosaka, M. , Nagahama, M. , Kim, W.-S., Watanabe, T. , Hatεuzawa, K. , Ikemizu, J. , Murakami, K. and Nakayama, K. (1991) Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J.Biol.Chem. 266, 12127-12130.

Ip, N.Y., Ibanez, C.F., Nye, S.H., McClain, J. , Jones, P.F., Gies, D.R., Belluεcio, L. , Le Beau, M.M. , Espinosa, R.,III, Squinto, S.P., Perεεon, H. and Yancopoulos, G.D. (1992) Mammalian neurotrophin-4: Structure, chromosomal localization, tisεue diεtribution, and receptor εpecificity. Proc.Natl.Acad.Sci. USA 89, 3060-3064.

Johnεon, E.M. ,Jr, Rich, K.M. and Yip, H.K. (1986) The role of NGF in sensory neurons in vivo. TINS 9, 33-37.

Jones, K.R. and Reichardt, L.F. (1990) Molecular cloning of a human gene that is a member of the nerve growth factor family. Proc.Natl.Acad.Sci. USA 87, 8060-8064.

Kaisho, Y., Yoshimura, K. and Nakahama, K. (1990) Cloning and expression of a cDNA encoding a novel human neurotrophic factor. FEBS Lett. 266, 187-191.

Kimmel, CB. (1993) Patterning the brain of the zebrafish embryo. Annu.Rev.Neurosci. 16, 707-732.

Korsching, S. and Thoenen, H. (1983) Nerve growth factor in εympathetic ganglia and correεponding target organε of the rat: Correlation with denεity of sympathetic innervation. Proc.Natl.Acad.Sci. USA 80, 3513-3516.

Krumlauf, R. (1991) Northern Blot Analysiε of Gene Expreεεion. in Gene Tranεfer and Expression Protocols (E.J. Murray, ed) , pp. 307-323. Humana Press, Clifton. Kyhse-Andersen, J. (1984) Electroblotting of multiple gelε: a εimple apparatuε without buffer tank for rapid tranεfer of proteinε from polyacrylamide to nitrocelluloεe. Journal of Biochemical and Biophyεical Methodε 10, 203-209.

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SEQUENCE LISTING

( 1 ) GENERAL INFORMATION : „

( i ) APPLICANT :

(A) NAME: Max-Planck-Gesellschaft zur Foerderung der issenschaften e.V.

(B) STREET:

(C) CITY: Berlin

(E) COUNTRY: Federal Republic of Germany

(F) POSTAL CODE (ZIP) : none

(ii) TITLE OF INVENTION: Neurotrophin-6 : A new member of the neurotrophin family

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1429 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 143..1000

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAATTCGGGG ATTTTGATTT TGGGATAATT CTTCTGCAGC TCTGGGGGAA AACAAGAGAA 60

GAGGAATATA GCTGACTTAC TTTTCAACTT TTATCCAACA GTGGAGAGAG GAGAAGGAGC 120

ATTATAACGT GGACGTGTGC CC ATG AGG TCA TCA CTA CTG GTT CTG CTC CTC 172

Met Arg Ser Ser Leu Leu Val Leu Leu Leu 1 5 10

CTG ATC GGC GTC CAG GCT GTA CTG AAC ATG GGA GGT GGT TTG GCC CGG 220 Leu He Gly Val Gin Ala Val Leu Asn Met Gly Gly Gly Leu Ala Arg 15 20 25

AAC CCT GGA GCA GCC AAT CAC AGC GCA GGG CAA CAG GAG ACA GCA GCA 268 Asn Pro Gly Ala Ala Asn His Ser Ala Gly Gin Gin Glu Thr Ala Ala 30 35 40

GCC AGG GGA CAG CTT TCT CAG GAC CAA ACT TCA TAT CAG CAA CAC AGG 316 Ala Arg Gly Gin Leu Ser Gin Asp Gin Thr Ser Tyr Gin Gin His Arg 45 50 55 ACC ACC CAT CAT AGA ACC AAG AGG ACA CAG AGT GCA GCC TCA AAC ATG 364 Thr Thr His His Arg Thr Lys Arg Thr Gin Ser Ala Ala Ser Asn Met 60 65 70

CAG AAC AGA ACC CCT GTC ATT GGA CCC TCT CCA GCA GGT TCC TCT CCA 412 Gin Asn Arg Thr Pro Val He Gly Pro Ser Pro Ala Gly Ser Ser Pro 75 80 85 90

GAC CCC TCC AGC CCA GTG GTG GAC CCG AAG CTC TTC TCT AAG AGA CAT 460 Asp Pro Ser Ser Pro Val Val Asp Pro Lys Leu Phe Ser Lys Arg His 95 100 105

TAT CGC CCC TCA CCT CGT GTT GTC TTT AGC GAG GTA ATC CCT TCG CAT 508 Tyr Arg Pro Ser Pro Arg Val Val Phe Ser Glu Val He Pro Ser His 110 115 120

GAC GTT CTG GAT GGT GAG GGT TAT GAC TTT GAA AGG GTG AGG GGG CTG 556 Asp Val Leu Asp Gly Glu Gly Tyr Asp Phe Glu Arg Val Arg Gly Leu 125 . 130 135

AGG GTA AGG CGC AAA GCA GTA TCA CAC ACC ATG CAT CGA GGA GAG TAC 604 Arg Val Arg Arg Lys Ala Val Ser His Thr Met His Arg Gly Glu Tyr 140 145 150

TCT GTG TGT GAC AGT ATA AAT ACC TGG GTG AAC AAG ACA CGA GCC ACA 652 Ser Val Cys Asp Ser He Asn Thr Trp Val Asn Lys Thr Arg Ala Thr 155 160 165 170

GAC ATG TCT GGA AAT GAA GTG ACA GTA CTC TCC CAC GTT ACA GTC AAC 700 Asp Met Ser Gly Asn Glu Val Thr Val Leu Ser His Val Thr Val Asn 175 180 185

AAC AAG GTA AAG AAA CAG CTT TTT TAT GAG ACC ACC TGT AGA TCC CCG 748 Asn Lys Val Lys Lys Gin Leu Phe Tyr Glu Thr Thr Cys Arg Ser Pro 190 195 200

ACG CAC AGG AGT TCT GGA ATC GTA ATC GGG GGA CGA TCT GGA GGA CGA 796 Thr His Arg Ser Ser Gly He Val He Gly Gly Arg Ser Gly Gly Arg 205 210 215

GGT GGA AAG CAA GGC TCC AAG ACA GGC AAC TCG GGT TGT CGA GGC ATT 844 Gly Gly Lys Gin Gly Ser Lys Thr Gly Asn Ser Gly Cys Arg Gly He 220 225 230

GAC AGC CGC TAC TGG AAC TCC CAC TGC ACC AAC ACA GAC ATA TAT GTA 892 Asp Ser Arg Tyr Trp Asn Ser His Cys Thr Asn Thr Asp He Tyr Val 235 240 245 250

AGC GCC CTG ACC GTC TTC AAG GAA CAG ACA GCC TGG CGT TTC ATC CGC 940 Ser Ala Leu Thr Val Phe Lys Glu Gin Thr Ala Trp Arg Phe He Arg 255 260 265

ATC AAC GCT GCA TGT GTG TGT GTT CTC AGC CGG AAT TCT TGG TCA AGA 988 He Asn Ala Ala Cys Val Cys Val Leu Ser Arg Asn Ser Trp Ser Arg 270 275 280 AGA CCG GGA CAC TGACTGAGGT GTGGACCGCT CTTGACCACT GGACTATGAA 1040

Arg Pro Gly His 285

AGCAAACACA CATTTTTACA AAACAGCCAC TGCAGCTTCT ATGCCAATGC TCCCATCATA 1100

CACCCACTGA TAACAGCAGT CAATAAACAT ACACATGCAC ACACACACAC ACACACACAT 1160

ACACATTTCT TGATTCAGTC CCTTGTTCCC TGACCCTACC TCAGTCAAAG AAGTCCTAGT 1220

AGTTTTAAGT TATGAGGACC GCATGTTATA TTTATGGTTT ATACAGACAA ATATAAAAGT 1280

ATATGCAGTA TGTGTATATA TACAAAATTA TTTCAACTTT TTAAATGTTA TTAATGTTGT 1340

TATTGTTGTT GTGTTGTCCA TGCTGTTATT TATTAAACAC CTCAGTGCTT AAAAAAAAAA 1400

AAAAAAAAAA ACGGAGTTCC GCGGAATTC 1429

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 286 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Arg Ser Ser Leu Leu Val Leu Leu Leu Leu He Gly Val Gin Ala 1 5 10 15

Val Leu Asn Met Gly Gly Gly Leu Ala Arg Asn Pro Gly Ala Ala Asn 20 25 30

His Ser Ala Gly Gin Gin Glu Thr Ala Ala Ala Arg Gly Gin Leu Ser 35 40 45

Gin Asp Gin Thr Ser Tyr Gin Gin His Arg Thr Thr His His Arg Thr 50 55 60

Lys Arg Thr Gin Ser Ala Ala Ser Asn Met Gin Asn Arg Thr Pro Val 65 70 75 80

He Gly Pro Ser Pro Ala Gly Ser Ser Pro Asp Pro Ser Ser Pro Val 85 90 95

Val Asp Pro Lys Leu Phe Ser Lys Arg His Tyr Arg Pro Ser Pro Arg 100 105 110

Val Val Phe Ser Glu Val He Pro Ser His Asp Val Leu Asp Gly Glu 115 120 125

Gly Tyr Asp Phe Glu Arg Val Arg Gly Leu Arg Val Arg Arg Lys Ala 130 135 140 Val Ser His Thr Met His Arg Gly Glu Tyr Ser Val Cys Asp Ser He 145 150 155 160

Asn Thr Trp Val Asn Lys Thr Arg Ala Thr Asp Met Ser Gly Asn Glu 165 170 175

Val Thr Val Leu Ser His Val Thr Val Asn Asn Lys Val Lys Lys Gin 180 185 190

Leu Phe Tyr Glu Thr Thr Cys Arg Ser Pro Thr His Arg Ser Ser Gly 195 200 205

He Val He Gly Gly Arg Ser Gly Gly Arg Gly Gly Lys Gin Gly Ser 210 215 220

Lys Thr Gly Asn Ser Gly Cys Arg Gly He Asp Ser Arg Tyr Trp Asn 225 230 235 240

Ser His Cys Thr Asn Thr Asp He Tyr Val Ser Ala Leu Thr Val Phe 245 250 255

Lys Glu Gin Thr Ala Trp Arg Phe He Arg He Asn Ala Ala Cys Val 260 265 270

Cys Val Leu Ser Arg Asn Ser Trp Ser Arg Arg Pro Gly His 275 280 285

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2273 base pairs

(B) TYPE: nucleic acid

(C) STRA DEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TCTAGATTTA AAACACTCAA ATGAATGGCC CTTCCCATGA TTATTGCTTG ATATTTAGTT 60

TATTCTTGTG TTCTCCTCTT GGAGCATTTG GATATAGTTA TGTTAGACCT GTCTTAATTT 120

TTCCCCTTTG TGTAGTTTGT TTGAGTTTTA GTCACTAACA TCTACCGAGA ACGTCTGGGA 180

TGACTCCTAC CACCGTCACC CCAGACAGTC TCAGTAATTG TGTCCATCAG CAAGACAGTG 240

GTCAAAGGGT GCTATTTACA GTTTAGTTCA ACTCCCTTTG TGTCCGTCTT CCTCTCTCAG 300

TTTCCATTTT TCTCCCTGCC ACATCTGTAC CACATTTTCT CTGATTAGCT TCTTTGTCAT 360

TTCCTCAGCA TTAAGGCTAC TGGATTTTGT TTGTTTCTTC CTGGATTCTT TCGTTAGACT 420

GTCTGTCACA CTCACGCCAG TTCCATGTTC TGTTCCCTTA GTGACTACTG TATAAGTCTC 480

TATTATTATT ATTAAAGAAA CACTCCAAAC TCGGGTTCTG CCTGCTTTTA GTACAACTAG 540

AAACCAAATA TAACGATGCC TTCACAGAAC ACCTGTTTTA ATACTGACGA TAACGTACAC 600 ACAGGTCATT TATATTTACT AAATGAGTAA CTTGTGAAGG ACATTCTATG AACTTGATTT 660

TTTTTTTTAG GGTTATCAAA GTCTATCAAA AAATTAGCAA TGAAATACAT TATAGCTTGT 720

GGGCAGAACA TTGAGATAAA CTACATTAGA CTCAATTATA CATACATTCT TCTAAGCTAC 780

TCATTGGGCA ATTTCTCAAA CATAAAGCTG TTGTAATAGT GCTAACTAAG ATGATTTTCT 840

GTCTTTGACC CAGGACGTGT GCCCATGAGG TCATCACTAC TGGTTCTGCT CCTCCTGATC 900

GGCGTCCAGG CTGTACTGAA CATGGGAGGT GGTTTGGCCC GGAACCCTGG AGCAGCCAAT 960

CACAGCGCAG GGCAACAGGA GACAGCAGCA GCCAGGGGAC AGCTTTCTCA GGACCAAACT 1020

TCATATCAGC AACACAGGAC CACCCATCAT AGAACCAAGA GGACACAGAG TGCAGCCTCA 1080

AACATGCAGA ACAGAACCCC TGTCATTGGA CCCTCTCCAG CAGGTTCCTC TCCAGACCCC 1140

TCCAGCCCAG TGGTGGACCC GAAGCTCTTC TCTAAGAGAC ATTATCGCCC CTCACCTCGT 1200

GTTGTCTTTA GCGAGGTAAT CCCTTCGCAT GACGTTCTGG ATGGTGAGGG TTATGACTTT 1260

GAAAGGGTGA GGGGGCTGAG GGTAAGGCGC AAAGCAGTAT CACACACCAT GCATCGAGGA 1320

GAGTACTCTG TGTGTGACAG TATAAATACC TGGGTGAACA AGACACGAGC CACAGACATG 1380

TCTGGAAATG AAGTGACAGT ACTCTCCCAC GTTACAGTCA ACAACAAGGT AAAGAAACAG 1440

CTTTTTTATG AGACCACCTG TAGATCCCCG ACACACAGGA GTTCTGGAAT CGTAATCGGG 1500

GGACGATCTG GAGGACGAGG TGGAAAGCAA GGCTCCAAGA CAGGCAACTC AGGTTGTCGA 1560

GGCATTGACA GCCGCTACTG GAACTCCCAC TGCACCAACA CAGACATATA TGTAAGCGCT 1620

CTGACCGTCT TCAAGGAACA GACAGCCTGG CGTTTCATCC GCATCAACGC TGCATGTGTG 1680

TGTGTTCTCA GCCGGAATTC TTGGTCAAGA AGACCGGGAC ACTGACTGAG GTGTGGACCG 1740

CTCTTGACCA CTGGACTATG AAAGCAAACA CACATTTTTA CAAAACAGCC ACTGCAGCAT 1800

CTATGCCAAT GCTCCTATCA TACACCCACT GATAACAGCA GTCAATAAAC ATACACATGC 1860

ACACGCACAC ACACACACAT ACACATGTCT TCATTCAGTC CCTTGTTCCC TGACCCTACC 1920

TCAGTCAAAG AAGTCCTAGT AGTTTTAAGT TATGAGGACC GCATGTTATA TTTATGGTTT 1980

ATACAGACAA ATATAAAAGT ATATACAGTA TGTGTATATA TACAAAATTA TTTCAACTTT 2040

TTAAATGTTA TTAATGTTGT TATTGTTGTT GTGTTGTCCA TGCTGTTATT TATTAAACAC 2100

CTCAGTGCTT GCCTGCCTGG TTGTGCCGTT TTTATCCCAC TCAGACAGCG TTAAAGTGCT 2160

GTGGAAATGT TTTTGTTCCT CCCCTTTCTT GCTTTTTTGT CACACTGAAA AAAAATTACA 2220

CCAGAAAAAA AGCATATGGT GGTAAATAAA AATTAGTTTT TAACAATTAT TCC 2273

Claims

1. A DNA sequence encoding a (poly)peptide having the biological properties of neurotrophin-6, selected from the group consisting of

(a) the DNA sequence of SEQ. LIST. No. 1 (cDNA) ;

(b) the DNA sequence of SEQ. LIST. No. 2 (genomic DNA) ;

(c) a DNA sequence hybridizing to a DNA sequence of (a) or (b);

(d) a DNA sequence being degenerate with respect to a DNA sequence of (a) , (b) or (c) ; and

(e) a subsequence of a DNA sequence of (a) to (d) of at least ten nucleotides encoding a (poly)peptide having the biological and/or antigenic properties of neurotrophin-6.

2. A DNA εequence encoding a (poly)peptide having the same three-dimensional structure at leaεt with reεpect to the biologically functional domains as the (poly)peptide encoded by the DNA sequence of claim 1.

3. The DNA εequence of claim 1 or 2 which is a vertebrate DNA εequence.

4. The DNA sequence of any one of claims 1 to 3 which is a fish DNA sequence.

5. The DNA sequence of any one of claims 1 to 3 which iε a mammalian DNA sequence.

6. The DNA sequence of claim 5 which is a human DNA εequence.

7. A RNA sequence having the following propertieε:

(a) it iε complementary to a DNA εequence of any one of claimε 1 to 6; and/or

(b) it hybridizes to a DNA sequence of any one of claims 1 to 6.

8. A primer or a pair of primers hybridizing to the DNA sequence of any one of claims 1 to 6 or the RNA sequence of claim 7.

9. A recombinant DNA molecule comprising the DNA sequence of any one of claimε 1 to 6.

10. The recombinant DNA molecule of claim 9 which iε the vaccinia viruε expression vector WNT-6, the construction of which is described in Example X.

11. A method for the isolation of a DNA sequence of any one of claims 1 to 6 comprising the following stepε:

(a) hybridizing a vertebrate, preferably a fish or a mammalian, most preferably a human DNA library with a suitable probe under reduced stringency conditions; and

(b) isolating the hybridizing clones and determining the (partial) DNA sequence thereof according to standard procedures.

12. The method of claim 11 wherein the hybridization conditions of step (a) are 30% formamide and 1 M NaCl at 42°C followed by waεhing steps in 300 mM NaCl at 60°C.

13. A method for the isolation of a DNA or RNA sequence of any one of claims 1 to 6, 9 to 10 or a RNA sequence of claim 7 comprising the following stepε:

(a) electrophoreεing vertebrate, preferably fish, mammalian, most preferably human DNA restricted with (an) appropriate restriction enzyme(s) or RNA derived from any of εaid organiεmε through an appropriate matrix, preferably an agaroεe gel;

(b) transfering the electrophoresed DNA or RNA to a second matrix, preferably a nitrocellulose filter;

(c) detecting the location of the DNA εequence of any one of claimε 1 to 6, 9 or 10 or the RNA sequence of claim 7 using an appropriate probe;

(d) isolating the DNA sequence of any one of claims 1 to 6, 9 or 10 or the RNA εequence of claim 7 electrophoresed through a preparative gel on the basis of the data obtained in steps (a) to (c) ; and

(e) cloning the isolated DNA or RNA sequence in an appropriate vector.

14. The method of any one of claims 11 to 13 wherein the probe is a fish derived probe.

15. The method of claim 14 wherein the fish derived probe is the 389 bp Nsil-EcoRI fragment of Figure IA.

16. A method for the isolation of a DNA sequence of any one of claims 1 to 6 comprising the following steps:

(a) amplification of said DNA sequence with a set of primers of claim 8; and

(b) cloning the amplified DNA sequence and determining its (partial) sequence according to standard procedures.

17. The method of claim 16 wherein at least one of the primers hybridizes to one of the conserved neurotrophin domains.

18. A host transformed with a recombinant DNA molecule of claim 9 or 10.

19. The host of claim 18 which is a bacterial, fungal, yeast or mammalian cell or a transgenic animal.

20. A (poly)peptide encoded by a DNA sequence of any one of claimε 1 to 6, 9 or 10.

21. A (poly)peptide of claim 20 having at least one of the following properties:

(a) its precursor molecule consists of 286 amino acids;

(b) it has an amino acid εequence at the carboxy terminal half of the precurεor εtarting at lysin 143 which encodes the mature neurotrophin-6 of 143 amino acids;

(c) a predicted M_r of 15,968;

(d) a hydrophobic domain at the N-terminus (amino acidε 1 to 19) with the characteriεticε of a εignal peptide;

(e) a pro-region (amino acids 20 to 142) containing two multiple basic motifs (R-X-K/R-R at residues 63-66 and 139-142) ;

(f) a pi value of 10.8;

(g) a stretch of 40 amino acids extending from amino acid position 151 to amino acid position 275 conserved in all neurotrophins known so far;

(h) it diεplayε a biological activity on εymphatic or sensory, but not on ciliatory neurons; (i) it iε a heparin binding molecule; and (j) it acts as a survival factor for chick sensory neurons of dorsal root and nodose ganglia.

22. The (poly)peptide of claim 20 or 21 which is glycosylated.

23. The (poly)peptide of claim 20 or 21 which is not glycosylated.

24. A molecule having the same three-dimensional structure at least with respect ot the biologically functional domains as the (poly)peptide encoded by the DNA sequence of any one of claims 1 to 6, 9 or 10 or the RNA sequence of claim 7.

25. A method for the production of the (poly)peptide of any one of claims 20 to 23 comprising the following steps:

(a) culturing a host cell of claim 18 or 19 in a suitable medium under conditions which allow the expression of said (poly)peptide;

(b) adding heparin to the medium at the onset of the culturing or at least 0.5 hours before harvesting; and

(c) purifying the protein from the medium.

26. The method of claim 25 wherein step (c) comprises the following steps:

(ca) loading the medium on a reverse phase column;

(cb) washing the column with 0.1% trifluoroacetic acid;

(cc) eluting the column with trifluoroacetic acid containing 60% acetonitrile; and

(cd) lyophilisation of the eluate.

27. The method of claim 25 wherein step (c) comprises the following steps:

(ca') centrifugation of the medium for 10 min at 400xg and 4°C; (cb¹) adjustment of the medium to a concentration of 0.5

M acetic acid; (cc¹) precipitation of the cells with methanol/chloroform.

28. The method of claim 25 wherein step (c) comprises the following steps:

(ca' ¹) chromatography of the medium over a controlled- pore glass column ; (cb'") chromatography of the product of step (ca¹') over a heparin column; and (cc¹¹) chromatography of the product of step (cb'") over a reverεed-phaεe HPLC column.

29. An antibody or antibody fragment or a derivative thereof to an epitope of the protein of any one of claimε 20 to 23 or the molecule of claim 24.

30. The antibody or antibody fragment or a derivative thereof of claim 29 which iε a monoclonal antibody or antibody fragment or derivative thereof.

31. The antibody or antibody fragment or a derivative thereof of claim 29 which is a polyclonal antibody or antibody fragment or derivative thereof.

32. A method for the production of an antibody of any one of claims 29 to 31 comprising the following steps:

(a) immunizing a suitable mammal with a (poly)peptide of any one of claims 20 to 23 or a molecule of claim 24 or an antigenic portion thereof;

(b) isolating the polyclonal antiserum εpecific to εaid (poly)peptide or molecule or said antigenic portion thereof according to standard procedures; or

(c) immunizing cells secreting antibodieε εpecific to said (poly)peptide or molecule or εaid antigenic portion thereof according to εtandard procedures.

33. The method of claim 32 wherein said antigenic portion comprises the amino acid εequence

K A V S H T M H R G E Y S V C.

34. A pharmaceutical compoεition compriεing a εubεtantially pure (poly)peptide of any one of claimε 20 to 23, or a εubεtantially pure molecule of claim 24, optionally in combination with a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34 for the increase of neuron survival.

36. The pharmaceutical composition of claim 34 for the increase of neuron growth.

37. The pharmaceutical composition of claim 34 for the support of differentiated cell function.

38. The pharmaceutical composition of claim 34 for the treatment of a diεeaεe or diεorder of the nervouε system.

39. The pharmaceutical compoεition of claim 38 wherein said disease or disorder of the nervous system is a degenerative disease.

40. The pharmaceutical composition of claim 38 wherein said disease or disorder of the nervouε εyεtem compriεes damage of the nervous system.

41. The pharmaceutical composition of claim 40 wherein said damage is selected from the group consisting of trauma, surgery, infarction, infection, and malignancy.

42. The pharmaceutical composition of claim 38 or 41 wherein said diseaεe or disorder or said damage of the nervous system is caused by exposure to a toxic agent.

43. The pharmaceutical composition of claim 38 wherein said disorder of the nervouε εystem comprises a congenital disorder of the retina.

44. The pharmaceutical composition of claim 39 wherein said degenerative disease is selected from the group consisting of Alzheimers' diεeaεe, Huntington'ε chorea, retinal diεease, Parkinson'ε diεeaεe and "Parkinεon- Pluε" εyndrome.

45. The pharmaceutical composition according to claim 44, wherein the "Parkinεon-Plus" syndrome is selected from the group consisting of Progresεive Spranuclear Palsey (Steele-Richardson-Olszewski Syndrome) , Olivopontocerebellar Atrophy, Shy-Drager Syndrome (Multiple Systems Atrophy) , and Guamanian Parkinsonism- Dementia complex.

46. The pharmaceutical compoεition of claim 38 wherein εaid disease or disorder of the nervous syεtem comprises disease or disorder of the sensory neurons or a tumour.

47. The pharmaceutical composition of claim 46 wherein the tumour is a neuroblastoma.

48. The pharmaceutical compoεition of claim 38 or 46 for the promotion of the survival of dopaminergic neurons.

49. The pharmaceutical composition of claim 38 or 46 for the promotion of the εurvival of cholinergic neuronε.

50. The pharmaceutical composition of claim 49 in which the cholinergic neurons are basal forebrain cholinergic neurons or septal cholinergic neuronε.

51. The pharmaceutical compoεition of claim 34 for the treatment of neuropathic εide effectε cauεed by the treatment of anti-εtatic agents in cancer patients.

52. The pharmaceutical composition of claim 34 for suppressing the proliferation of astroglia cellε.

53. The pharmaceutical composition of any one of claims 34 to 51 furthermore compriεing at leaεt one additional neurotrophic factor.

54. The pharmaceutical compoεition of claim 52 wherein εaid additional factor is NGF, NT-3, BDNF or NT-4/5.

55. A pharmaceutical composition comprising an antibody or a fragment or derivative thereof of any one of claims 25 to 31.

56. An in vitro method for diagnosing a diseaεe or diεorder of the nervous system referred to in any one of claims 38 to 51 comprising

(a) contacting a tisεue probe with a detectably labelled DNA or RNA molecule of any one of claimε 1 to 10 under conditionε which will allow hybridization to occur; and

(b) detecting any hybridization which has occurred.

57. An m vitro method for diagnosing a disease or disorder of the nervous system referred to in any one of claims 38 to 51 comprising

(a) exposing a tissue probe to a detectably labelled antibody or fragment or derivative thereof of any one of claims 29 to 31 under conditions which will allow binding to occur; and

(b) detecting any binding which has occurred.

58. An m vitro method for the diagnosiε of diseaseε and/or disorders of the nervous syεtem referred to in any one of claimε 38 to 51 compriεing

(a) bringing the detectably labelled (poly)peptide of any one of claimε 20 to 23 or the detectably labelled molecule of claim 24 into contact with cellε or tissue normally expressing the NT-6 receptors; and

(b) detecting any abnormalities in NT-6 receptor expression.

59. A kit for the diagnoεiε of a diεease or disorder of the nervous system referred to in any one of claims 38 to 51 comprising at least

(a) a DNA and/or a RNA molecule of any one of claimε 1 to 7, 9 or 10 or a derivative thereof; and/or

(b) a primer or a pair of primers of claim 8; and/or

(c) an antibody or fragment or derivative thereof according to any one of claims 29 to 31;

(d) an antibody or a fragment thereof or a derivative thereof directed to a nucleic acid of any one of claims 1 to 10.