WO2006094673A2 - Synergistic mixtures of c6- to c12-alkanedi0ls and tropolone (derivatives) - Google Patents

Abstract

The present invention relates to a polynucleotide encoding a specific novel sorting nexin, SNX-B8/30 and is also related to a polypeptide encoded by said polynucleotides. The invention also relates to specific medical, pharmaceutical and scientific uses of said new member of a herein described sorting nexin (SNX) subgroup consisting of SNX-9, SNX- 18 and the herein described SNX-B8/30. Also provided are specific screening methods for agonists or antagonists influencing the function and/or expression of the SNX-B 8/30 and/or the further members of the herein identified subfamily of sorting nexins. Accordingly, the present invention also relates to novel pharmaceutical compositions. These pharmaceutical compositions may, inter alia, be employed in the treatment of diseases/disorders related to (pathological) APP metabolism and/or the insulin metabolism, like Alzheimer's disease or diabetes. Also provided are non-human transgenic animals comprising a modified and/or altered SNX-B8/30 or expressing a heterologous SNX-B8/30.

Description

Sorting Nexins in the medical intervention of neurological and/or metabolic disorders

The present invention relates to a polynucleotide encoding a specific novel sorting nexin, namely SNX-B 8/30 and is also related to a polypeptide encoded by said polynucleotides. The invention also relates to specific medical, pharmaceutical and scientific uses of said new member of a herein described sorting nexin (SNX) subgroup consisting of SNX-9, SNX- 18 and the herein described SNX-B8/30. Also provided are specific screening methods for agonists or antagonists influencing the function and/or expression of the SNX-B8/30 and/or the further members of the herein identified subfamily of sorting nexins. Accordingly, the present invention also relates to novel pharmaceutical compositions. These pharmaceutical compositions may, inter alia, be employed in the treatment of diseases/disorders related to (pathological) amyloid precursor protein APP metabolism and/or the insulin metabolism, like Alzheimer's disease or diabetes. Also provided are non-human transgenic animals comprising a modified and/or altered SNX-B8/30 or expressing a heterologous SNX-B8/30.

The Sorting Nexins (SNXs) are a large family of proteins that are defined by the presence of a SNX phox homology (PX) domain (SNX-PX), a subgroup of the PX domain superfamily. In all of those SNXs tested, the SNX-PX domains act as phosphoinositide-binding motifs that aid in the targeting of the SNX protein to phosphoinositide-enriched membranes. The SNXs are a family of oligomeric proteins found distributed between membranes and cytosol, and they contain a variety of protein-protein and protein-lipid interaction domains in addition to their SNX-PX domain. To date, 29 mammalian SNXs and 10 yeast SNX, or SNX-like proteins have been identified, although for the majority of these, little is known of their function, as also summarized in Carlton (2005), Traffic 6, 75-82.

The binding specificity of the SNXs for phospholipids vary and binding occurs to different Ptdlns phosphates. It is difficult to determine the exact specificity as demonstrated by the fact that it depends on the assay used for the determination of binding to said PtdIns(P) (reviewed in Worby (2002), Nature reviews 3, 919-931). Mammalian SNXl, the original family member, was identified as a yeast two-hybrid partner for the core kinase domain and the lysosomal targeting sequence of the EGF receptor. SNXl was found to associate with the sorting endosome, from where it was proposed to enhance the degradative sorting of the EGFR through an unknown mechanism.

Overexpression of SNXl markedly downregulated the amount of activated EGFR on the cell surface, moreover this effect was highly specific as overexpression of SNXl did not alter

ERBB2 or PDGFR levels (Kurten (1996), Science 272, 1008-1110).

The association of SNXl with the endosomale membrane requires PI3K activity, which indicates that D3 phosphate is essential for Ptdlns recognition.

SNX2 shares 63% of sequence identity with SNXl. Both proteins oligomerize as homo- oligomers or hetero-oligomers.

Mice that lack SNXl and/or SNX2 indicate that the SNXs are functionally redundant, due to the finding that the single-knockouts are viable and fertile, whereas the embryogenesis is arrested at midgestation in the double-knockout, providing a necessary function of SNXl and

SNX2 in embryogenesis (Schwarz (2002), MoI. Biol. Cell 13, 3588-3600).

Unlike SNXl, SNX2 and SNX4 do not interact with the transferrin-receptor (TfR), which indicates different binding specifities and may be a hint on different cellular functions (Zhong

(2002), Proc. Natl. Acad. Sci. USA 99, 6767-6772).

SNX3 lacks the C-terminal interaction domain, it is not able to interact with the other SNXs. Therefore it may restrict the function of other SNXs by binding to Ptdlns at crucial membrane sites (Haft (2000), MoI. Biol. Cell 11, 4105-4116).

SNX6 is involved in TGF-β signalling. At a functional level, overexpression of SNX6 inhibits TGF-β signalling. SNX6 hetero-oligomerizes with SNXl, SNX2 and SNX4 (Parks (2001), J. Biol. Chem. 276, 19332-19339).

SNX13 is so far the only SNX that interferes with signal transduction that is triggered by G- Protein-coupled-receptors (GPCRs). SNX13 stimulates the GTP-hydrolysis the Gas subunit, thereby modulating its activity.

Overexpression of SNXl 3 inhibits the degradation of the EGFR, which is the opposite effect to that seen for the overexpression of SNXl (Zheng (2001), Science 294, 1939-1942). SNXl 5 was isolated as a result of a database search using the PX-domain consensus sequence that was obtained from SNXl, SNX2, SNX3 and SNX4. Overexpression of SNXl 5 alters the morphology of several endosomal compartments, therefore it may be involved in the endocytotic pathway (Barr (2000), Traffic 1, 904-916).

SNX 17 was isolated on the basis of its ability to interact with P-selectin a cell-adhesion molecule. The function of that interaction remains elusive.

Functional studies showed that overexpression of SNXl 7 enhances the rate of endocytosis of the low-density lipoprotein receptor (LDLR) (Stockinger (2002), EMBO J. 21, 4259-4267).

The membrane localization of SNXs ist not only a result of PX domain function. The interaction of several SNXs (SNXl, -2, -4, -5, -6, -7, -8, -9 and -18) with membranes is also due to the Bin/Amphiphysin/Rvs (BAR) domain. The domain has been described as a sensor for membrane curvature and dimerization module.

A further function of the BAR domain the may be a potential binding to small G-proteins (Habermann, (2004), EMBO Rep. 5, 250-255).

The SNX family of proteins is involved in intracellular trafficking and protein sorting along the endocytotic pathway and may integrate into cellular signalling pathways (Worby (2002), loc. cit).

As documented herein above, several SNXs have been chemically/biochemically and/or genetically identified, however, there is hardly any physiological function known or attributed to known SNXs. Furthermore, SNX-15 was merely identified via database search as discussed above. Accordingly, the problem remains that means and methods have to be provided, where sorting nexins can be used for the medical and/or pharmaceutical benefit in particular in the intervention of human disorders.

This technical problem has been solved by the provision of the embodiments and claims provided herein. Accordingly, the present invention relates to a polynucleotide selected from the group consisting of (a) a polynucleotide having a nucleotide sequence encoding the polypeptide having the deduced amino acid sequence as shown in SEQ ID NO: 2;

(b) a polynucleotide having the coding sequence as shown in SEQ ID NO: 1 encoding the polypeptide as shown in SEQ ID NO: 2;

(c) a polynucleotide having a nucleotide sequence encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any one of (a) or (b), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide and wherein said fragment and derivative is capable of functioning as an SNX-B8;

(d) a polynucleotide having a nucleotide sequence which is at least 60% identical to a polynucleotide as defined in any one of (a) to (c) and which encodes a functional SNX-B8 protein;

(e) a polynucleotide encoding a polypeptide which is at least 45% identical to a polypeptide encoded by a polynucleotide as defined in any one of (a) to (c) and which encodes a functional SNX-B8 protein;

(f) a polynucleotide having a nucleotide sequence the complementary strand of which hybridizes to a polynucleotide as defined in any one of (a) to (e) and which encodes a functional SNX-B 8 protein;

(g) a polynucleotide having a nucleotide sequence being degenerate to the nucleotide sequence of the polynucleotide of any one of (a) to (f); or the complementary strand of such a polynucleotide.

As documented in the appended examples the present invention provides for the identification of a novel member of a subgroup of the sorting nexin family, namely the herein identified SNX-B8 also denoted as SNX-30. In the present invention this novel attributed sorting nexin activity is termed SNX-B8, SNX-30 and/or SNX-B8/3O. As documented in the appended examples it was surprisingly found that the herein identified sorting nexin SNX-B 8/30 modified the α- and β-secretase cleavage of amyloid precursor protein (APP).

The β-secretase cleaves APP at the N-terminus of the Aβ-peptide domain, thereby catalyzing the first step in Aβ-peptide generation. In contrast, the α-secretase cleaves within the Aβ- sequence, and thus precludes the generation of the pathogenic Aβ-peptide. As shown in the examples, in order to identify and mechanistically characterize proteins regulating α- and β-secretase cleavage of APP and thus Aβ -generation, a genome-wide expression cloning screen was carried out using a human brain cDNA library and identified a novel member of the sorting nexin family of proteins (SNX), the herein described SNX-B8. As discussed above, SNXs are a large family of cytoplasmic and membrane bound proteins assumed to be involved in protein trafficking from and to the endosomes. Western Blot analysis using cleavage site-specific antibodies revealed that transfection of SNX-B8 into HEK293 cells strongly stimulated the secretion of (soluble) APP, increasing mainly the α- secretase cleavage and only to a lower extent β-secretase cleavage. Surprisingly, SNX-B8 reduced the rate of APP endocytosis and increased the amount of mature APP in the cell lysate. SNX-B8 is a phospho-protein. Thus and without being bound by theory, it seems possible that phosphorylation of SNX-B8 controls APP trafficking and shedding. Importantly, the shedding-stimulating effect of SNX-B8 is specific for APP. SNX-B8 has little or no effect on the shedding of other membrane proteins undergoing an α-secretase like cleavage, such as TNF-receptor2 and L-selectin. Together, these results document that SNX-B8 is a novel modifier of the endocytic trafficking of APP, thereby controlling the amount of APP available for α- and β-secretase cleavage. Furthermore, the appended examples document the surprising finding that SNX-B 8/30 is also involved in insulin signaling. It is shown SNX- B8/30 is essential in insulin signal transduction in vivo.

Accordingly, the present invention provides with SNX-B8/30 a potent modifier and/or modulator of the APP metabolism as well as the insulin signal transduction. Accordingly, the present invention provides for novel medical and/or pharmaceutical means and methods for the treatment of APP related disorders and/or disorders of the insulin-signaling pathway. Besides further examples given herein below, these disorders comprise, but are not limited to Alzheimer's disease and diabetes.

Therefore, the present invention provides for a new biological function of a sorting nexin identified herein, namely the sorting nexin SNX-B8/30. The function of said SNX-B8/SNX- 30 defined and characterized herein is basically that it effects the endocytotic machinery of a given cell and is, accordingly, involved in endocytotic processes, namely and even specifically of amyloid precursor proteins (APP), of insulin receptor (and accordingly of insulin) and of the transferrin receptor. In particular, and as documented in the appended examples, SNX-B8/30 influences the proteolytic cleavage of amyloid precursor protein and is essential in signal transduction provided by insulin. Most importantly, the proteolytic cleavage of amyloid precursor protein is defined in an enhanced cleavage by α-secretase (α- cleavage) whereas the detrimental β-cleavage (mediated by a β-secretase) is merely weakly affected. Accordingly, SNX-B8/30 may be used to stimulate in an organism, preferably in a patient, more preferably in a human patient the α-cleavage of APP and thereby influencing positively detrimental depositions of amyloid plaques. Moreover, it is clearly documented in the appended examples that SNX-B8/30 is also involved in the regulation of endocytotic processes of APP itself. It is, furthermore, documented that SNX-B8/30 leads to a lower endocytotic rate. In summary, the SNX-B8/30 identified herein influences the proteolytic cleavage of amyloid precursor protein, it is furthermore essential in insulin signal transduction in vivo (whereby it is of note that insulin signaling is disturbed in particular in type 2 diabetes; "insulin resistance") and, in addition, SNX-B8/30 effects the endocytosis of APP as well as of the transferrin receptor. Accordingly, the polynucleotide as identified herein as well as the polypeptide denoted as SNX-B 8/30 (as well as its homologs) are particularly useful in the generation of host cell and/or non-human transgenic animals which may function in screening assays for Alzheimer medicaments as well as diabetes medicaments or for medicaments related to an impaired transferrin receptor system. Details are given herein below and are described in the appended examples.

It is of particular note that the herein identified SNX-B 8/30 forms a novel subgroup of sorting nexin molecules. This subgroup comprises sorting nexin 9 (SNX-9), sorting nexin 18 (SNX- 18) as well as the herein described SNX-B8/30. Accordingly, means and methods and in particular uses described herein below apply, mutatis mutandis, to the use of SNX-9 and SNX-18. Corresponding SNX-9 and SNX- 18 sequences are provided herein below and are characterized as SEQ ID NOS: 3 and 4 and 5 and 6, respectively.

Due to its sequence homology SNXl 8 (also called SNAG-I) has been described as a homologue of SNX9 (Worby and Dixon, 2002). However, at present, there are no publications showing experiments using SNXl 8. Thus, at present it is unclear, if and where SNXl 8 is expressed and what its function is. SNX9 has been described in several publications. Initially, it was identified under the name SH3PX1, since it contains a SH3 and a PX domain (Howard et al., 1999). It is ubiquitously expressed and was proposed to have a function in endocytosis (Lundmark and Carlsson, 2002; Lundmark and Carlsson, 2003; Lundmark and Carlsson, 2004), as it binds to the endocytic GTPase dynamin (Lundmark and Carlsson, 2004). Additionally, overexpression experiments showed that SNX9 binds to the immature forms of two proteases, ADAM9 and ADAMl 5 (Howard et al., 1999), suggesting that it may function in the intracellular trafficking of both proteases. This point is particularly interesting, since ADAM9 has been proposed to be an α- secretase for APP. However, the previous study (Howard et al., 1999) did not disclose whether the binding of SNX9 to ADAM9 had any effect on the proteolytic activity of these proteases or on APP shedding. Additionally, SNX9 binds the insulin receptor, but that study (Macaulay et al., 2003) did not analyze whether SNX9 binding altered insulin receptor signaling.

SNX9 may be phosphorylated by ACK or by an unknown kinase, but it remains controversial whether the phosphorylation occurs within the SH3 or the LC domain. The phosphorylation may alter the interaction of the SNX with binding partners (Clemens et al., 2000; Lin et al., 2002; Lundmark and Carlsson, 2004; Worby et al., 2002). hi contrast to vertebrates, C. elegans has only one homologue of the herein defined SNX9/18/B8 group. The C. elegans homologous is teremed "lst-4" which has been shown to be involved in vulva development (Yoo et al., 2004). Moreover, it is down-regulated upon EGFR signaling and upregulated upon Notch signal transduction. The underlying molecular mechanisms have not yet been established.

Neither SNX- 18 nor SNX-9 have been provided in the prior art as targets for the medical intervention in neurological or metabolic disorders, like Alzheimer's disease or diabetes.

hi accordance with the present invention, the term "nucleic acid sequence" means the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. In a preferred embodiment, the "polynucleotide" as defined herein is an RNA molecule and may, additionally, comprise further nucleotides, like poly- A stretches and/or 5 '-regulating sequences. Accordingly, the polynucleotide as shown in SEQ ED NO: 1 also relates to a RNA molecule, whereby the "T" (Thymidine) is replaced by an "U" (Uracile).

When used herein, the term "polypeptide" means a peptide, a protein, or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine (Se-Cys). Peptides, oligopeptides and proteins may be termed polypeptides. The terms polypeptide and protein are often used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

In order to determine whether a nucleic acid sequence has a certain degree of identity to the nucleic acid sequence encoding SNX-B8/SNX30, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as those mentioned further down below in connection with the definition of the term "hybridization" and degrees of homology.

For example, BLAST2.0, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. MoI. Evol. 36 (1993), 290-300; Altschul, J. MoI. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

%sequence identity x % maximum BLAST score

100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.

The present invention also relates to nucleic acid molecules which hybridize to the SNX- B8/SNX30 polynucleotide as defined herein. Identities as well as homologies are, accordingly, easily deducible by the person skilled in the art and corresponding examples are also provided in the experimental part.

The term "hybridizes" as used in accordance with the present invention may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N. Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames .(Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65°C. Non- stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecule. Such fragments may represent nucleic acid sequences which code for SNX-B8/SNX30 and which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementartity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95%, 97% or 98% and most preferably at least 99% identity with a nucleic acid sequence as described above encoding SNX-B 8/SNX30 as described herein above.

In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95%, 97%, 98% or 99% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

Moreover, the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.

The nucleic acid molecule according to the invention may be any type of nucleic acid, e.g. DNA, RNA or PNA (peptide nucleic acid).

For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomelic units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254:1497 (1991); and Egholm et al, Nature 365:666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

The DNA may, for example, be cDNA. In a preferred embodiment it is a genomic DNA. The RNA may be, e.g., mRNA. The nucleic acid molecule may be natural, synthetic or semisynthetic or it may be a derivative, such as peptide nucleic acid (Nielsen, Science 254 (1991), 1497-1500) or phosphorothioates. Furthermore, the nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.

The invention also provides for a polynucleotide as defined above, coding for the SNX-B8/30 defined herein, whereby said polynucleotide is fused to a heterologous polynucleotide, preferably encoding a heterologous polypeptide. This heterologous polypeptide may, inter alia, be a marker, like a green fluorescent protein or HA, as shown in the appended examples. Preferably, the nucleic acid molecule(s) of the present invention is part of a vector. Said vector may be a gene targeting vector or a gene expression vector. These are particularly useful in the generation of a non-human transgenic animal or a host cell expressing the SNX- B8/30 described herein. Therefore, the present invention relates in another embodiment to a vector comprising the nucleic acid molecule of this invention. Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.

The nucleic acid molecules of the present invention may be inserted into several commercially available vectors. Nonlimiting examples include plasmid vectors compatible with mammalian cells, such as pUC, pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCl neo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-ρSV2neo, ρBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag, pIZD35, pLXIN and pSIR (Clontech) and plRES-EGFP (Clontech). Baculovirus vectors such as pBlueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBacTM Baculovirus Expression System, insect cells and protocols (Invitrogen) are available commercially and may also be used to produce high yields of biologically active protein, (see also, Miller (1993), Curr. Op. Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: A Laboratory Manual, p. 127). hi addition, prokaryotic vectors such as pcDNA2; and yeast vectors such as pYes2 are nonlimiting examples of other vectors suitable for use with the present invention. For vector modification techniques, see Sambrook and Russel (2001), loc. cit. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection hi the host, e. g., antibiotic resistance, and one or more expression cassettes. The coding sequences inserted in the vector can be synthesized by standard, methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e. g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods. Furthermore, the vectors may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site or internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV- promoter (Rous sarcome virus), human elongation factor lα-promoter, CMV enhancer, CaM- kinase promoter or SV40-enhancer.

For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter, the lacUV5 or the trp promoter, has been described. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide, hi this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pRc/CMV, pcDNAl, ρcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTl (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gtl 1.

An expression vector according to this invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids and protein of this invention. Suitable origins of replication include, for example, the Col El, the SV40 viral and the M 13 origins of replication. Suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the lacZ promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhidrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedral polyadenylation signals. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like. Specifically-designed vectors allow the shuttling of DNA between different host cells, such as bacteria-yeast, or bacteria-animal cells, or bacteria- fungal cells, or bacteria-invertebrate cells.

Beside the nucleic acid molecules of the present invention, the vector may further comprise nucleic acid sequences encoding secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, -and preferably, a leader sequence capable of directing secretion of translated protein, or a part thereof, into, inter alia, the extracellular membrane. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the proteins, antigenic fragments or fusion proteins of the invention may follow. Of course, the vector can also comprise regulatory regions from pathogenic organisms.

For gene therapy, various viral vectors which can be utilized, for example, adenovirus (like Ad5), herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can also incorporate multiple genes. AU of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.

Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences, for example polynucleotide sequences encoding an antibody of the present invention, which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the inserted polynucleotide sequence.

Since recombinant retroviruses are preferably defective, they require assistance in order to produce infectious viral particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include, but are not limited to w2, PA317 and PAl 2, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced. Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for polynucleotides encoding an antibody of the present invention is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 pm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. ScL, 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988). The composition of the liposome is usually a combination of phospholipids, particularly high- phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell- specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries.

The present invention in addition relates to a host transformed with a vector of the present invention or to a host comprising the nucleic acid molecule of the invention. Said host may be produced by introducing said vector or nucleotide sequence into a host cell which upon its presence in the cell mediates the expression of a protein encoded by the nucleotide sequence of the invention or comprising a nucleotide sequence or a vector according to the invention wherein the nucleotide sequence and/or the encoded polypeptide is foreign to the host cell. By "foreign" it is meant that the nucleotide sequence and/or the encoded polypeptide is either heterologous with respect to the host, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host but located in a different genomic environment than the naturally occurring counterpart of said nucleotide sequence. This means that, if the nucleotide sequence is homologous with respect to the host, it is not located in its natural location in the genome of said host, in particular it is surrounded by different genes. In this case the nucleotide sequence may be either under the control of its own promoter or under the control of a heterologous promoter. The location of the introduced nucleic acid molecule or the vector can be determined by the skilled person by using methods well-known to the person skilled in the art, e.g., Southern Blotting. The vector or nucleotide sequence according to the invention which is present in the host may either be integrated into the genome of the host or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleotide sequence of the invention can be used to restore or create a mutant gene via homologous recombination.

Said host may be any prokaryotic or eukaryotic cell. Suitable prokaryotic/bacterial cells are those generally used for cloning like E. coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis. Said eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell. Said prokaryotic cell may be bacterial cell (e.g., E coli strains HBlOl, DH5a, XLl Blue, Y1090 and JMlOl). Eukaryotic recombinant host cells are preferred. Examples of eukaryotic host cells include, but are not limited to, yeast, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichia pastoris cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells as well as zebra fish cells. Mammalian species-derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-I cells, COS-I cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-I (ATCC CCL 26), CHO cells (ATCC CRL1859, ATCC CRL 1866) and MRC-5 (ATCC CCL 171). As documented in the examples, also HEK293 cells may be employed and are useful as host cells in accordance with this invention.

hi a more preferred embodiment, the host according to the invention is a non-human transgenic organism. Accordingly, the present invention also provides for a non-human transgenic animal, whereas the here described SNX-B8/30 is expressed heterologously (for example the human ortholog is expressed in a mouse) or wherein the endogenous SNX-B8/30 is down regulated or not expressed (SNX-B8/3O - "knock out"). Said non-human organism may be a mammal, an amphibian, a fish, an insect, a fungus or a plant. Particularly preferred non-human transgenic animals are Drosophila species, Caenorhabditis elegans, Xenopus species, zebra fish, Spodoptera frugiperda, Autographa californica, mice and rats. Transgenic plants comprise, but are not limited to, wheat, tobacco, parsley and Arabidopsis. Transgenic fungi are also well known in the art and comprise, inter alia, yeasts, like S. pombe or S. cerevisae, or Aspergillus spec, Neurospora or Ustilago species or Pichia species.

In a preferred embodiment of the invention, a non-human transgenic animal is provided which comprises a mutation in the ortholog of SNX-B8 as defined herein. Preferably, said ortholog comprises a mutation which leads to a non-functional SNX-B8 expression, function or activity. Such a non-human transgenic animal may be considered as a "knockout" or a "knock-down" animal. Also provided is a non-human transgenic animal expressing in its somatic and/or its germ cells an expression product which is capable of interfering with the expression, function or activity of SNX-B8. Such an animal may, inter alia, comprises an expression product (as transgene) which is an inhibiting RNA or siRNA. Corresponding examples are known in the art, see, inter alia, Hasuwa (2002), FEBS Lett. 532, 227, Kunath (2003), Nat. Biotech. 21, 559 and are reviewed in Prawitt (2004) Cytogen. Res. 105, 412.

As a preferred embodiment of the herein described transgenic animal, the SNX-B8 ortholog mutation is a knock-out mutation and said expression product capable of interfering with the expression, function or activity of SNX-B8 leads to a "knock-out" or a "knock-down" of SNX-B8.

hi another embodiment, the present invention relates to a method or a process for producing the polypeptide encoded by the SNX-B8/3O nucleic acid molecule of the invention comprising culturing/raising the host of the invention and isolating the produced polypeptide. Accordingly, a process is provided for the production of a functional SNX-B8/30 molecule. A large number of suitable methods exist in the art to produce polypeptides in appropriate hosts. If the host is a unicellular organism or a mammalian or insect cell, the person skilled in the art can revert to a variety of culture conditions that can be further optimized without an undue burden of work. Conveniently, the produced protein is harvested from the culture medium or from isolated (biological) membranes by established techniques. Furthermore, the produced polypeptide may be directly isolated from the host cell. Said host cell may be part of or derived from a part of a host organism. Additionally, the produced polypeptide may be isolated from fluids derived from said host.

The polypeptide of the invention may accordingly be produced by microbiological methods or by transgenic non-human mammals. It is also envisaged that the polypeptide of the invention is recovered from transgenic plants. Alternatively, the polypeptide of the invention may be produced synthetically or semi-synthetically.

For example, chemical synthesis, such as the solid phase procedure described by Houghton Proc. Natl. Acad. Sci. USA (82) (1985), 5131-5135, can be used. Another method is in vitro translation of mRNA. A preferred method involves the recombinant production of protein in host cells as described above. For example, nucleotide acid sequences comprising all or a portion of any one of the nucleotide sequences according to the invention can be synthesized by PCR, inserted into an expression vector, and a host cell transformed with the expression vector. Thereafter, the host cell is cultured to produce the desired polypeptide, which is isolated and purified. Protein isolation and purification can be achieved by any one of several known techniques; for example and without limitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, preparative disc gel electrophoresis. In addition, cell-free translation systems can be used to produce the polypeptides of the present invention. Suitable cell-free expression systems for use in accordance with the present invention include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega). These systems allow the expression of recombinant polypeptides or peptides upon the addition of cloning vectors, DNA fragments, or RNA sequences containing coding regions and appropriate promoter elements. As mentioned supra, protein isolation/purification techniques may require modification of the proteins of the present invention using conventional methods. For example, a histidine tag can be added to the protein to allow purification on a (immobilized) nickel column (IMAC). Other modifications may cause higher or lower activity, permit higher levels of protein production, or simplify purification of the protein. As documented in the examples, "fusion" proteins are also provided in context of this invention, for example, a SNX-B 8/30-HA fusion polypeptide. Furthermore an antibody specifically binding to the polypeptide SNX-B 8/SNX30 is within the scope of the present invention.

In another aspect the present invention relates to an antibody or aptamer specifically recognizing SNX-B8/SNX3O which is described herein. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sides (Gold, Ann. Rev. Biochem. 64 (1995), 763-797). The term "specifically" in this context means that the antibody reacts with SNX-B8/SNX30, such as the polypeptides of the present invention encoded by the polynucleotides of the present invention. Preferably, this term also means that such an antibody does not bind to other polypeptides which, may be, related to said polypeptides of the present invention. Whether the antibody specifically reacts as defined herein above can easily be tested, inter alia, by methods known in the art to determine the specificity of an antibody, such as ELISA, etc..

The antibody of the present invention can be, for example, polyclonal or monoclonal. The term "antibody" also comprises derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the polypeptides of the invention as well as for the monitoring of the presence of such polypeptides, for example, in recombinant organisms or in diagnosis. They can also be used for the identification of compounds interacting with the proteins according to the invention (as mentioned herein below). For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7- 13).

The present invention furthermore includes chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab')2, Fv or scFv fragments; see, for example, Harlow and Lane, loc. cit. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, the (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies to polypeptide(s) of this invention. Also, transgenic animals may be used to express humanized antibodies to polypeptides of this invention. Most preferably, the antibody of this invention is a monoclonal antibody. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kohler and Milstein Nature 256 (1975), 495- 497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., US Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptides as described above. Furthermore, transgenic mice may be used to express humanized antibodies directed against said immunogenic polypeptides. It is in particular preferred that the antibodies/antibody constructs as well as antibody fragments or derivatives to be employed in accordance with this invention or capable to be expressed in a cell. This may, inter alia, be achieved by direct injection of the corresponding proteineous molecules or by injection of nucleic acid molecules encoding the same. Furthermore, gene therapy approaches are envisaged.

Accordingly, in context of the present invention, the term "antibody molecule" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies. The term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term "antibody molecule" also comprises bifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins. It is also envisaged in context of this invention that the term "antibody" comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, inter alia, viruses or vectors.. It is also envisaged in context of this invention that the term "antibody" comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, inter alia, viruses or vectors. It is particularly envisaged that such antibody constructs specifically recognize SNX-B8/SNX30 as described herein, such as the polypeptides of the present invention.

Accordingly, it is, jEurthermore, envisaged that said antibody construct is employed in gene therapy approaches for treating and/or preventing the diseases associated with SNX- B8/SNX30 which are described herein. Therefore, not only the antibodies provided herein and directed against the herein identified SNX-B8/SNX30 may be medically used, but also nucleic acid molecules encoding the same.

In a further embodiment an antibody specifically directed against SNX-B8/30 is provided. Such an antibody molecule may function as an inhibitor of SNX-B 8/30 and may, accordingly, be clinically useful. The antibody of the invention is preferably an antibody specifically binding to/interacting with an epitope comprising a polypeptide as set forth in SEQ ID NOS: 10 or 11.

As pointed out above, the antibody molecule of the present invention may be detectably labelled, for example with a toxin, a radioisotope or a fluorescent label.

The invention also provides for an antagonist of SNX-B8/30. Such an antagonist (of expression) may be an inhibiting RNA, RNAi, siRNA, shRNA or a ribozyme binding to or inhibiting the translation of a SNX-B 8/30 polynucleotide as defined herein. Recently the therapeutic use of, in particular, siRNAs in medical settings and in the treatment of human patients has been proven; see Soutschek (2004), Nature 432, 173-178. Accordingly, the inhibiting RNA molecules, RNAi, siRNA, shRNA or a ribozyme provided herein are particularly useful in a medical setting.

However, also other "antagonist" and "inhibitors" of SNX-B 8/30 are envisaged and comprise, inter alia, an antibody, an aptamer or an anticalin or an "antisense molecule".

A potentially useful inhibiting RNA of the present invention is preferably selected from an antisense construct hybridizing to a SNX-B 8/30 polynucleotide defined herein, RNAi, siRNA, shRNA or a ribozyme.

Accordingly, potential "antagonist(s)/inhibitor(s)" or partial inhibitors(s) for SNX-B 8/SNX30 may be selected from aptamers (Gold, Ann. Rev. Biochem. 64 (1995), 763-797)), aptazymes, RNAi, shRNA, RNAzymes, ribozymes (see e.g., EP-Bl 0 291 533, EP-Al 0 321 201, EP-Bl 0 360 257), antisense DNA, antisense oligonucleotides, antisense RNA, siRNA, antibodies (Harlow and Lane " Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988), affibodies (Hansson, Immunotechnology 4 (1999), 237-252; Herming, Hum Gene Ther. 13 (2000), 1427-1439), trinectins (Phylos Inc., Lexington, Massachusetts, USA; Xu, Cliem. Biol. 9 (2002), 933), anticalins, or the like. These compounds are, for example, described in EP 1 017 814. Said European patent also describes the process of preparing such anticalins with the ability to bind a specific target. Other potential inhibitors which can be used as a pharmaceutical composition are identified by the methods as described herein. In accordance with the present invention, the term "aptamer" means nucleic acid molecules that can bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995), Ann. Rev. Biochem 64 , 763-797).

Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, whereby the inhibitory effect is based on specific binding of a nucleic acid molecule to DNA or RNA. For example, the 5' coding portion of a nucleic acid molecule encoding SNX-B8/SNX30 and/or fragments thereof to be inhibited can be used to design an antisense oligonucleotide, e.g., of at least 10 nucleotides in length. The antisense DNA or RNA oligonucleotide hybridises to the niRNA in vivo and blocks translation of said mRNA and/or leads to destabilization of the mRNA molecule (Okano, J. Neurochem. 56 (1991), 560; Oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Boca Raton, FL, USA (1988).

The antisense molecule may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxyhnethyl) uracil, 5- carboxymethylarninomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galaetosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-manαosylqueosine, 5 '-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- tliiouracil, 5-methyluracil, uracil 5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5- methyl-2-thiouracil, 3- (3-amin3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. The antisense molecule may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose. In yet another embodiment, the antisense molecule comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense molecule is an a-anomeric oligonucleotide. An a- anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual-units, the strands run parallel to each other (Gautier, 1987, Nucl. Acids Res. 15: 6625-6641). The oligonucleotide is a 2'-O-methylribonucleotide (Inoue, 1987, Nucl. Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analogue (Inoue, 1987, FEBS Lett. 215: 327-330).

Antisense molecules of the invention (and to be employed as "inhibitors" of SNX-B 8/SNX30) may be synthesized by standard methods known in the art, e. g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin,1988, Proc. Natl. Acad. Sci. U. S. A. 85: 7448-7451), etc.

For applying a triple-helix approach, a DNA oligonucleotide can be designed to be complementary to a region of the gene encoding SNX-B 8/SNX30 and/or fragments thereof to be inhibited according to the principles laid down in the prior art (see for example Lee, Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241 (1988), 456; and Dervan, Science 251 (1991), 1360). Such a triple helix forming oligonucleotide can then be used to prevent transcription of the specific gene, and is, accordingly, an inhibition in the sense of this invention. The oligonucleotides described above can also be delivered to target cells via a gene delivery vector as described above in order to express such molecules in vivo to inhibit gene expression of the respective protein.

Examples for antisense molecules are oligonucleotides specifically hybridising to a polynucleotide encoding SNX-B 8/SNX30 and/or fragments thereof. Such oligonucleotides have a length, of preferably at least 10, in particular at least 15, and particularly preferably of at least 50 nucleotides. They are characterized in that they specifically hybridise to said polynucleotide, that is to say that they do not or only to a very minor extent hybridise to other nucleic acid sequences.

Another suitable approach is the use of nucleic acid molecules mediating an RNA interference (RNAi) effect. RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to specifically target a gene's product, resulting in null or hypomorphic phenotypes. Introduction of dsRNA into a eukaryotic cell results in the loss of the function of SNX- B8/SNX3O and/or fragments thereof. Because RNAi is also remarkably potent (i.e., only a few dsRNA molecules per cell are required to produce effective interference), the dsRNA must be either replicated and/or work catalytically . Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in RNAi constructs, a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures.

Likewise, RNA molecules with ribozyme activity which specifically cleave transcripts of a gene encoding SNX-B8/SNX30 and/or fragments thereof can be used as "antagonist/inhibitor" as provided. Said ribozymes may also target DNA molecules encoding the corresponding RNAs. Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of two different groups of ribozymes. The first group is made up of ribozymes which belong to the group I intron ribozyme type. The second group consists of ribozymes which as a characteristic structural feature exhibit the so-called "hammerhead" motif. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule. In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding SNX-B8/SNX30 and/or fragments thereof, for example a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are homologous to sequences encoding the target protein. The expression of ribozymes in order to decrease the activity in certain proteins is also known to the person skilled in the art and is, for example, described in EP-Bl 0 321 201 or EP-Bl 0 360 257.

In another preferred embodiment, the inhibiting nucleic acid molecule is siRNA as dislosed in Elbashir (2001), Nature 411, 494-498.

The shRNA approach for gene silencing is well known in the art and may comprise the use of st (small temporal) RNAs; see, inter alia, Paddison (2002) Genes Dev. 16, 948-958. As mentioned above, approaches for gene silencing are known in the art and comprise "RNA"- approaches like RNAi or siRNA. Successful use of such approaches has been shown in Paddison (2002) loc. cit, Elbashir (2002) Methods 26, 199-213; Novina (2002) Mat. Med. June 3, 2002; Donze (2002) Nucl. Acids Res. 30, e46; Paul (2002) Nat. Biotech 20, 505-508; Lee (2002) Nat. Biotech. 20, 500-505; Miyagashi (2002) Nat. Biotech. 20, 497-500; Yu (2002) PNAS 99, 6047-6052 or Brummelkamp (2002), Science 296, 550-553. These approaches may be vector-based, e.g. the pSUPER vector, or RNA pol III vectors may be employed as illustrated, inter alia, in Yu (2002) loc. cit.; Miyagishi (2002) loc. cit. or Brummelkamp (2002) loc. cit.

It is envisaged that said siRNA is targeted to deplete SNX-B 8/SNX30 and/or fragments thereof. In accordance with the present invention the term "targeted" means that (an) siRNA duplex(es) is/are specifically targeted to a coding sequence of SNX-B8/SNX30 and/or fragments thereof, to cause gene silencing by RNA interference (RNAi) since said siRNA duplex(es) is/are homologous in sequence to a gene desired to be silenced, for example, SNX- B8/SNX30 and/or fragments thereof. "Homologous in sequence" in the context of the present invention means that said siRNA duplex(es) is/are homologous in the sequence to a gene, for example the SNX-B8/SNX30 and/or fragments thereof desired to be silenced by the mechanism/pathway of RNA interference (RNAi). It is envisaged that the degree of homology between the siRNA duplex(es) and the sequence of the gene desired to be silenced is sufficient that said siRNA duplex(es) is/are capable to cause gene silencing of said desired gene initiated by double-stranded RNA (dsRNA), for example, (an) siRNA duplex(es). The person skilled in the art is readily in a position to determine whether the degree of homology is sufficient to deplete SNX-B 8/SNX30 and/or fragments thereof.

When using the term "to deplete" in the context of the present invention, it means that due to a process of sequence-specific, post-transcriptional gene silencing (PTGS) expression of a desired gene, for example, SNX-B8/SNX30 is suppressed. Accordingly, the RNA encoding, for example SNX-B 8/SNX30 and/or fragments thereof may be partially or completely degraded by the mechanism/pathway of RNAi and, thus, may not be translated or only translated in insufficient amounts which causes a phenotype almost resembling or resembling that of a knock-out of the respective gene. Consequently, for example, no or at least to less of SNX-B8/SNX30 will be produced.

20- to 50-nucleotide RNAs, preferably 15, 18, 20, 21, 25, 30, 35, 40, 45 and 50-nucleotide RNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Most conveniently, siRNAs and the like are obtained from commercial RNA oligo synthesis suppliers, which sell RNA- synthesis products of different quality and costs. In general, 20 to 50-nucleotide RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. However, specific gene silcencing may also be obtained by longer RNA, for example long dsRNA which may comprise even 500 nt; see, inter alia, Paddison (2002), PNAS 99, 1443-1448. The preferred targeted region is selected from a given nucleic acid sequence beginning, inter alia, 50 to 100 nt downstream of the start codon.

As documented herein, preferred "inhibiting RNAs", "RNAi", "siRNA" or "shRNA" target a SNX-B8/30 nucleotide sequence, which, e.g., comprises or is a sequence as shown in SEQ ID NOS: 7, 8 or 9. These may also be the target for antisense molecules and ribozymes. For particular uses and methods provided herein, also inliibiting molecules directed against SNX- 9 or SNX-18 are envisaged. The appended SEQ ID NOS: 20 to 27 provide for corresponding target molecules and non-limiting examples of corresponding RNAi-molecules are also provided. The above cited SEQ ID NOS. comprise the target sequences for the inhibiting nucleic acid molecules. Under corresponding sections of said SEQ ID NOS, the relevant "strand" and "antistrand" in form of RNA molecules are provided. Accordingly, a target sequence (of SNX-B8/30) may, inter alia, be targeted by RNAi using duplex sequences as given in siRNAs of SEQ ID NOS: 28 and 29, 30 and 31, 32 and 33 and/or 50 and 51. Illustrative target sequence for inhibiting molecules for SNX-9 are given in SEQ ID NOS: 20, 21, 22, 23 and 24. Corresponding duplex sequences for RNAi approaches are given in SEQ ID NOS: 34 and 35, 36 and 37, 38 and 39, 40 and 41 and/or 42 and 43. Illustrative target sequences for SNX-18 inhibition are provided with SEQ ID NOS: 25, 26 and 27 and corresponding (duplex) sequences for RNAi approaches are given in SEQ ID NOS: 44 and 45, 46 and 47 and/or 48 and 49.

As documented herein, it is desired to enhance in certain disorders the expression or function of SNX-B 8/30. Accordingly, the invention also provides for an agonist/enhancer of the SNX- B8/30 polypeptide of the invention. Said agonist/enhancer may be a transcription factor capable of enhancing expression of any one of the SNX-B 8/30 polynucleotides as defined above.

In a further embodiment of the invention a composition comprising the polynucleotide, the vector, the host cell, the polypeptide, the antagonist/inhibitor, the RNAi or siRNA, the antibody or the agonist/enhancer of the invention is provided.

Preferably, said composition is a pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier and/or a diluent and/or an excipient and/or a microcapsulated host cell. Yet, also a diagnostic composition, optionally further comprising suitable means for detection is envisaged.

Dosage, pharmaceutical preparation and delivery of the compounds of the present invention as described herein for use in accordance with the present invention may be formulated in conventional manner according to methods found in the art, using one or more physiological carriers or excipients, see, for example Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7^th edition, Lippincott Williams & Wilkins Publishers, 1999. Thus, the compounds of the invention acceptable salts and solvates may be formulated for administration by inhalation, insufflation (either through the mouth, or nose), oral, buccal, parenteral, or rectal administration.

The pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the inhibitor described herein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In another embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. In a preferred embodiment, the pharmaceutical compositions are in a water- soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. The administration of the candidate agents of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, inrranasally, transdermally, intranodally, peritumourally, intratumourally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. hi addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

It is preferred that for oral administration, the pharmaceutical composition of the for example SNX-B 8/SNX30 inhibitors or enhancers may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutical acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulphate). Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparation may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol, syrup, cellulose derivatives, hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxycarbonates, soric acids). The preparations may also contain buffer salts, flavouring, coloring and sweetening agents as deemed appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the SNX-B8/SNX3O inhibitors, SNX-B8/30 enhancers, or the other medically useful compounds of the present invention.

Preferably, for administration by inhalation, the compounds of the present invention for use according to the present invention is conveniently delivered in the form of an aerosol spray presentation from a pressurised pack or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatine, for use in an inhaler or insufflator may be formulated containing a powder mix of the compounds of the invention and a suitable powder base such as lactose or starch.

It is also preferred that a compounds of the invention may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Site of injections include intravenous, intraperitoneal or sub-cutaneous. Formulations for injection may be presented in units dosage form (e.g., in phial, in multi-dose container), and with an added preservative. The compounds of the invention may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, or dispersing agents. Alternatively, the agent may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Compounds of the invention may, if desired, be presented in a pack, or dispenser device which may contain one or more unit dosage forms containing the said agent. The pack may for example comprise metal or plastic foil, such as blister pack. The pack or dispenser device may be accompanied with instruction for administration.

In the context of the present invention the term "subject" means an individual in need of a treatment of an affective disorder. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human.

The term "administered" means administration of a therapeutically effective dose of the aforementioned inhibitor to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents maybe administered alone or in combination with other treatments.

The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intraarterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, mtrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapuhnonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution dry spray.

The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

The dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily. In a preferred case the immune response is monitored using herein described methods and further methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Dosages will vary but a preferred dosage for intravenous administration of DNA encoding SNX-B8/SNX3O as described herein is from approximately 10⁶ to 10¹² copies of the DNA molecule. Similar ranges are envisaged for e.g. inhibitory molecules, like RNAi, siRNAs and the like. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The pharmaceutical composition of the invention may be administered locally or systemically. Administration will preferably be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium ion solution, Ringer's dextrose, dextrose and sodium ion, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The above described diagnostic composition may optionally comprises suitable means for detection. The nucleic acid molecule(s), vectors, host(s), antibody(ies), and polypeptide(s) described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of well-known carriers include glass, polystyrene, polyvinyl ion, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention.

Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acid molecule(s), vector(s) host(s), antibody(ies), aptamer(s), polypeptide(s), etc. on solid phases include but are not limited to ionic, hydrophogic, covalent interactions or (chemical) crosslinking and the like. Examples of immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Northern or Southern blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme rmmuno Assay), ELISA (Enzyme Linked Imrnuno Assay), FIA (Fluorescent hnmuno Assay), and CLIA (Chemioluminescent Immune Assay). Furthermore, the diagnostic compounds of the present invention may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays.

Appropriate labels and methods for labeling are known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like P, P, S or ¹²⁵I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).

A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immunoassays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymology", Academic Press, Inc. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

A diagnostic application in which the kit or the diagnostic composition of the present invention is used comprises any amplification technique. The term "amplification technique" refers to any method that allows the generation of a multitude of identical or essentially identical (i.e. at least 95% more preferred at least 98%, even more preferred at least 99% and most preferred at least 99.5% such as 99.9% identical) nucleic acid molecules or parts thereof. Such methods are well established in the art; see Sambrook et al. "Molecular Cloning, A Laboratory Manual", 2^nd edition 1989, CSH Press, Cold Spring Harbor. Various PCR techniques, including real-time PCR are reviewed, for example, by Ding, J. Biochem. MoI. Biol. 37 (2004), 1-10.

PCR is an example of an amplification technique. PCR is a powerful technique used to amplify DNA millions of fold, by repeated replication of a template, in a short period of time. The process utilizes sets of specific in vitro synthesized oligonucleotides to prime DNA synthesis. The design of the primers is dependent upon the sequences of the DNA that is desired to be analyzed. It is known that the length of a primer results from different parameters (Gillam (1979), Gene 8, 81-97; Innis (1990), PCR Protocols: A guide to methods and applications, Academic Press, San Diego, USA). Preferably, the primer should only hybridize or bind to a specific region of a target nucleotide sequence. The length of a primer that statistically hybridizes only to one region of a target nucleotide sequence can be calculated by the following formula: (Vi) ^x (whereby x is the length of the primer). For example a hepta- or octanucleotide would be sufficient to bind statistically only once on a sequence of 37 kb. However, it is known that a primer exactly matching to a complementary template strand must be at least 9 base pairs in length, otherwise no stable-double strand can be generated (Goulian (1973), Biochemistry 12, 2893-2901). It is also envisaged that computer-based algorithms can be used to design primers capable of amplifying the nucleic acid molecules of the invention. Preferably, the primers of the invention are at least 10 nucleotides in length, more preferred at least 12 nucleotides in length, even more preferred at least 15 nucleotides in length, particularly preferred at least 18 nucleotides in length, even more particularly preferred at least 20 nucleotides in length and most preferably at least 25 nucleotides in length. The invention, however, can also be carried out with primers which are shorter or longer. The person skilled in the art can readily design primers to be used in the diagnostic method of the invention, particular on basis of the nucleic acid molecules provided herein and homologous molecules as defined herein above. In a diagnostic method also the appended examples provide for means and methods how specific primers (or probes) may be generated. "Primers" and "probes" are particularly useful in the diagnostic methods provided herein.

The PCR technique is carried out through many cycles (usually 20 - 50) of melting the template at high temperature, allowing the primers to anneal to complimentary sequences within the template and then replicating the template with DNA polymerase. The process has been automated with the use of thermostable DNA polymerases isolated from bacteria that grow in thermal vents in the ocean or hot springs. During the first round of replication a single copy of DNA is converted to two copies and so on resulting in an exponential increase in the number of copies of the sequences targeted by the primers. After just 20 cycles a single copy of DNA is amplified over 2,000,000 fold.

The invention as provided herein is particularly useful in the medical intervention of different diseases like, for example, diseases related to APP metabolism, insulin pathway, transferrin receptor pathway and the like. As documented in the appended examples SNX-B 8/30 as well as the other members of the herein defined SNX subgroup, namely SNX-9 and SNX-18 are useful in the prevention, amelioration and/or treatment of disorders linked to the physiological pathways and metabolisms. Accordingly, SNX-B8/30, SNX-9 as well as SNX-18 (in their polynucleotide as well as their polypeptide form) may be employed in the medical intervention of these disorders. These disorders comprise, in particular neurological, neurodegenerative disorders, diabetes and obesity, whereby a particular referred disorder to be treated are Alzheimer's disease and diabetes. For these medical interventions also enhancers/agonists of SNX-B8/30 (or of the other members of the family, namely SNX-9 and SNX-18) may be employed. Accordingly, the present invention also relates to the use of the SNX-B8/30 polynucleotide of the invention, the vector, the host cell, the SNX-B8/30 polypeptide or the agonist/enhancer described herein above for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating neurological, neurodegenerative disorders, diabetes or obesity. Preferably, said neurological and/or neurodegenerative disorders are selected from the group consisting of Alzheimer's disease, Parkinson's disease or a prion-disease. Prion disorders comprise Creutzfeld- Jacob-disease; Kuru-Ruru, Gerstmann-Straussler-Scheinker syndrome, BSE Bovine Spongiform Encephalopathy "mad-cow disease", Scrapie in sheep, TME (transmissible mink encephalopathy) in mink or CWD (chronic wasting disease) in muledeer, elk. Also envisaged is that the above-mentioned compounds, leading to an enhanced level or an enhanced function of SNX-B8/30 are useful for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating a disorder, wherein α-cleavage of the Amyloid Precursor Protein (APP) is inhibited, wherein α-secretase is inhibited and/or malfunctioning or wherein it is desired that α-secretase activity is enhanced.

Alzheimer's disease (AD) is the most frequent cause of death in the industrialized countries besides cardiovascular diseases and cancer. AD affects roughly one million people in

Germany and about 4 million in the US.

According to our current knowledge, the pathogenesis of AD starts with the generation of the amyloid β peptide (Aβ). Two proteases cleave Aβ out of the much larger amyloid precursor protein (APP) (Fig. IA). Subsequently, Aβ forms aggregates, which are neurotoxic and lead to nerve cell loss in the brain and finally to death (Selkoe and Schenk, 2003).

Currently, there are only drugs ameliorating the symptoms of AD but no drugs interfering with the disease-causing process. Thus, much research effort is put into developing ways of stopping the first steps in the process, namely the generation and aggregation of Aβ. The Alzheimer protein APP is a membrane protein and consists of a large extracellular domain, a transmembrane and a cytoplasmic domain (Fig. IA). The two proteases, which cleave APP and generate Aβ, are referred to as β- and γ-secretase (Fig. IA). β-secretase cleaves first, γ-secretase cleaves second.

As an alternative to the β-secretase cleavage, APP may be cleaved by α-secretase, which cleaves within the Aβ domain (Fig. IA). Thus, α-cleavage precludes Aβ peptide generation, since the resulting APP fragments do not contain the full Aβ sequence anymore. Additionally, α- but not β-cleavage generates a secreted form of APP (sAPPα), which is neuroprotective. Therapeutically, it would therefore be desirable to enhance α-cleavage and to reduce β- cleavage and thus, reduce Aβ generation and increase sAPPα secretion (Lichtenthaler and Haass, 2004). To this aim, it is necessary to understand the molecular and cellular processes that regulate to which extent APP is cleaved by α- or β-secretase. At present, little is known about these processes, but it is clear that the stimulation of certain cellular signal transduction pathways, such as the insulin signaling pathway, the MAP kinase pathway or protein kinase A (PKA) can stimulate α-cleavage of APP (Fig. IB) (Allinson et al., 2003). To elucidate the mechanisms controlling APP cleavage we used a functional genomics screening system (expression cloning) and identified proteins that stimulate APP cleavage by α- or β-secretase. The α- or β-cleavage of APP are also referred to as shedding or ectodomain shedding, which in general stands for the proteolytic conversion of membrane proteins to their soluble counterparts (Fig. IB). Besides APP, a large number of membrane-anchored proteins can be proteolytically cleaved in an α-secretase like fashion (Blobel, 2002).

Type 2 diabetes affects about 150 million people world-wide and is responsible for 95% of all cases of diabetes. In type 2 diabetes cells become insulin-resistant. Under normal conditions, insulin binds to its receptor and induces a signal transduction cascade. As one of the consequences, sugar transporters move to the cell surface and allow sugar entry into the cell, which leads to a reduced blood sugar level. In insulin-resistant cells, the insulin signal transduction is not working properly and is reduced. One of the therapeutic aims for diabetes is therefore to stimulate insulin signal transduction in insulin resistant cells (Musi and Goodyear, 2002). Thus, genes affecting insulin signal transduction may be novel drug targets for the development of anti-diabetes drugs. Diabetes is a risk factor for AD (Arvanitakis et al., 2004), and decreased insulin signaling has been observed in AD brain. Reduced insulin signaling may have several consequences, which could contribute to the pathogenesis of AD and exacerbate the symptoms. The complex consequences of reduced insulin signaling are mechanistically not fully elucidated but include reduced expression of IDE (an enzyme which can cleave and thereby neutralize Aβ) (Zhao et al., 2004), reduced secretion of the neurotrophic and neuroprotective, α-secretase cleaved APP (sAPPα) and increased phosphorylation of the protein tau (reviewed in Gasparini et al., 2002). Upon hyperphosphorylation tau can form the so-called neurofibrillary tangles (NFTs). Besides the amyloid aggregates the NFTs are a second hallmark in AD brains. Taken together, it is assumed that increasing insulin signaling may be therapeutically helpful for AD (discussed in Zhao et al., 2004). This increase of insulin signaling may be obtained by the agonists/enhancers of SNX-B 8/30 function/expression as provided herein.

Obesity is a complex disorder of appetite regulation and/or energy metabolism controlled by specific biological factors. Besides severe risks of illness such as diabetes, hypertension and heart disease, individuals suffering from obesity are often isolated socially. Human obesity is strongly influenced by environmental and genetic factors, whereby the environmental influence is often a hurdle for the identification of (human) obesity genes.

Obesity is defined as a Body Mass Index (BMI) of 30 kg/m² or more. BMI is calculated by dividing the weight in kg by the height in metres squared. "Overweight" is defined as a BMI between 25 and 30 kg/m². A person is considered obese if he or she has 20 percent (or more) extra body fat for his/her age, height, sex, and bone structure.

Major advances have recently been made in identifying components of the homeostatic system(s) that regulate body weight/mass. Several candidate genes have been associated with mammalian/human obesity or its metabolic complications (Kopelman, Nature 404 (2000), 634-643). The "human obesity gene map" contains entries for more than 40 genes and 15 chromosomal regions in which published studies indicate a possible relationship to adiposity or a related phenotpye (Barsh (2000), loc. cit, Perusse, Obes. Res. 7 (1999), 111-129). Said "obesity gene map" comprises, however, mainly large chromosomal areas and does not provide for distinct genes involved in obesity. Lately, Snyder (2003) has published an extended version of the "obesity gene map" and more than 430 genes, markers, chromosomal regions have been associated or linked with human obesity phenotypes; Snyder, Obes. Res. 12 (2004), 369-439. Accordingly, obesity is not to be considered as a single disorder but a heterogeneous group of conditions with (potential) multiple causes. Most importantly, obesity is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Kolterman, J. Clin. Invest 65 (1980), 1272-1284) and a clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman (2000), loc. cit; Colditz, Arch. hit. Med. 122 (1995), 481-486). Due to the clear involvement of SNX-B8/30 in the insulin(msulin receptor pathway (as documented in the appended examples), the compounds of the present invention leading to an enhanced SNX-B8/30 function (or expression) are also useful in the treatment of obesity.

However, it is also envisaged that SNX-B8/30 (or the other two members of the herein defined subgroup, namely SNX-9 and SNX-18) are down-regulated or their expression and function is inhibited in order to achieve a positive medical intervention. Accordingly, also the use of antagonists or inhibitors of the herein defined SNX-B8/30 (or of SNX-9 or of SNX-18) for the preparation of a pharmaceutical composition for the treatment of, for example, cancer or a transferrin receptor related disorders are described. Accordingly, the invention also relates to the use of the antagonist/inhibitor of particular SNX-B8/30 (or SNX-9; SNX-18), the inhibiting RNA, the shRNA, RNAi or siRNA described herein and directed against the expression of SNX-B8/30 or the (inhibitory) antibody described above for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating cancer or transferrin- receptor-related disorders. Said transferrin-receptor-related disorder may be cancer or an apoptosis-related disorder. Said cancer is preferably selected from the group of cancers, where insulin receptor expression is high, like cancers of prostate, breast or colon.

Transferrin (Tf) has several polymorphisms with > 30 different species detected to date. Three major isotypes known as B, C and D are found, whereas the majority of people carry the C allele in particular Cl .

Several studies have shown a link between Tf polymorphism and susceptibility to diseases e.g. cardiovascular disease (CVD) and Alzheimer's disease (AD). Individuals possessing the Tf C2 allele in combination with the C282Y allele of the haemochromatosis (HFE) gene have a higher risk of developing AD. This risk is further increased in individuals carrying the allele apolipoproein E epsilon 4 (Apo E4).

Tf is synthesized in a variety of cells including predominantely hepatocytes, but also in Sertoli, ependymal, oligodendroglial, metastatic melanoma cell lines and human breast cancer cell lines. TF has been detected in various body fluids including plasma, bile, amniotic, cerebrospinal, lymph and breast milk.

The Transferrin-Receptor exists at least in two distinct types, designated TfRl and TfR2. TfRl is expressed on a range of cells, including blood cells, erythroid cells, hepatocytes, monocytes and cells of the blood-brain barrier. TfR2 is expressed as two transcripts (alphaTfR2 and betaTfR2) whereas alphaTfR2 is predominantly expressed on liver cells and betaTfR2 at low levels on a variety of cell types. Non-dividing cells can have extremely low levels of TfR expression, whereas rapidly proliferating cells (e.g. carcinoma cells) can express up to 100.000 copies per cell.

The antimicrobial activity of Tf is mainly linked to the reduction of free iron via Tf but also apo-Tf has an antimicrobial effect. Apo-Tf is . being capable of reducing the adhesion of bacteria to surfaces.

Tf has been implicated in growth and differentiation activities including myotrophic, embryo- morphogenic, proliferative, mitogenic, neurotrophic, chemotactic and angiogenic activities. These activities seem to be at least partially iron-binding independent since apo-Tf can have growth promoting effects.

Tf has been suggested to have paracrine and autocrine roles e.g. the proliferation of brain melanoma metastasis is promoted by Tf produced by brain cells.

Cell response to Tf signal can change throughout the life cycle of the cell. E.g. intracranial injection of apo-Tf in two-to-seven day old rats results in rapid differentiation of oligodendroglial cells whereas the same treatment has no effect in ten-day old rats. Iron-bound Tf has been shown to inhibit apoptosis in ovarian cancer cell lines. The apoptotic pathway acts by up regulating ferritin which results in reduced levels of intracellular iron. The presence of iron-bound Tf can restore intracellular iron levels, thereby preventing cell death. Tf can modulate different cellular events. An example is the antiproliferative and anti- apoptotic effect of the binding of Tf to insulin-like growth factor-binding protein 3 (IGFBP- 3). The complex of Tf and IGFBP-3 prevents the proliferative effect of IGFBP-3 on bladder smooth muscle cells and the apoptotic effect in prostate cancer cells. Accordingly, the antagonist/inhibitor of SNX-B8/30 is particularly useful in the treatment of, inter alia, prostate cancers or breast or colon cancers.

In diabetes patients there is an increased risk of developing a cardiovascular disease (CVD).

Oxidative damage and neuropathy observed in some diabetes patients lead to an increased loss of Tf and thereby to increased levels of unbound iron. The negative effect of unbound iron can be treated by infusion with apo-Tf to scavenge the iron.

In radiotherapeutic treatments Tf decreases and therefore free iron is increased. Infusion with apo-Tf has been useful.

The antimicrobial effect of infusion with apo-Tf is thought to be caused by the reduction of free iron which is necessary for the growth of pathogens.

A main field of therapeutic interest is targeted drug delivery via the Tf-TfR transport system.

Tf can bind a variety of metals which are useful in treatment and/or diagnostics e.g. ⁶⁷Ga³⁺ and ¹¹¹In³⁺ radioisotopes.

Conjugation of Tf with small molecules, peptides, proteins or genes can be used. An example is the delivery of a Tf-conjugated diphtheria toxin to malignant brain tumors (phase III is under way for glioblastoma).

Tf in combination with other factors (e.g. IGF-I and IL-2) can promote cytotoxicity and proliferation in lymphokine activated killer cells (LAK) and natural killer cells (NK).

The iron-binding ability of Tf has been used in conjunction with the anti-malaria drug, artemisinin (ART), a substance which breaks down into a toxic compound in the presence of

Fe²⁺.

The effects of targeted drug delivery are even more efficient in cells overexpressing the TfR e.g. leukemic cells (45-95 %) compared with normal mononuclear blood cells (0,4 - 1,3 %).

The delivery systems could be tailored by altering the metal binding site or by inserting peptide sequences for specific delivery of drugs to rapidly dividing cells.

A further aspect of the present invention is the use of a modulator of SNX-B 8/SNX3O activity or expression for the preparation of a pharmaceutical composition for treating a disorder. In the context of the present invention the term "modulator" means (a) compound(s), a complex of compounds, (a) substance(s) or complex of substances which can modify, i.e. modulate the activity of SNX-B8/SNX30 or the expression of SNX-B 8/SNX3O either directly or indirectly. The modulation can, for example, occur at the protein level.

Therefore and in accordance with this invention, also methods for screening activators as well as inhibitors of SNX-B8/30 function or expression are provided. Such "activators" and "inhibitors" are considered as "modulators".

Therefore, the invention also provides for a method for identifying an antagonist/inhibitor of an SNX-B 8 molecule comprising the steps of:

(a) contacting the SNX-B8/30 polynucleotide, the vector, the host cell, the SNX-B8/30 polypeptide or a non-human transgenic animal expressing SNX-B8/30 of the invention with an inliibitor/antagonist to be screened;

(b) determining whether the inhibitor/antagonist to be screened effects the expression of the SNX-B 8/30 polynucleotide as defined herein; and

(c) determining whether said inhibitor/antagonist to be screened is capable of down- regulating and/or inhibiting the expression of the polynucleotide coding for SNX- B8/30.

The invention also provides for a method for screening of an inhibitor/antagonist for SNX-B8 function comprising the steps of: (a) contacting a cell expressing SNX-B 8 with a compound to be tested; (b) determining whether in said cell SNX-B 8 is functional in the presence of the compound to be tested when compared to a cell not contacted with said compound; and (c) identifying the compound which inhibits SNX-B8 function and/or expression.

The above recited methods may comprise an additional step (V), wherein steps (a) and (b) are carried out in a control experiment in the absence of an inhibitor/antagonist to be screened.

The hereby identified "antagonists"/"inhibitors" of SNX-B8/30 expression or function are particularly useful in the treatment of cancer.

hi accordance with the present invention, the term "inhibitor", "antagonist" denotes molecules or substances or compounds or compositions or agents or any combination thereof described herein below, which are capable of inhibiting and/or reducing SNX-B8/SNX30 expression and/or function. The term "inhibitor" when used in the present application is interchangeable with the term "antagonist". The term "inhibitor" comprises competitive, non-competitive, functional and chemical antagonists as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. The term "partial inhibitor" in accordance with the present invention means a molecule or substance or compound or composition or agent or any combination thereof that is capable of incompletely blocking the action of agonists through, inter alia, a noncompetitive mechanism. It is preferred that said inhibitor alters, interacts and modulates SNX- B8/SNX30 expression and/or function.

The person skilled in the art can easily employ the compounds and the methods of this invention in order to elucidate the inhibitory effects and/or characteristics of a test compound to be identified and/or characterized in accordance with any of the methods described herein and which is an inhibitor of SNX-B8/SNX30 expression or function.

However, also provided are methods for the identification or verification of "positive modulators of SNX-B8/30". Therefore, the invention also provides for a method for identifying an agonist/enhancer of SNX-B 8 molecule expression comprising the steps of:

(a) contacting the SNX-B8/30 polynucleotide, the vector, the host cell, the SNX-B8/30 polypeptide or the non-human transgenic animal as described above with an agonist/enhancer to be screened;

(b) determining whether the agonist/enhancer to be screened effects the expression of the SNX-B8/30 polynucleotide; and

(c) determining whether said agonist/enhancer to be screened is capable of up-regulating and/or enhancing the expression of the polynucleotide coding for SNX-B8.

The invention also provides for a method for screening of an agonist/enhancer for SNX-B 8 function comprising the steps of:

(a) contacting a cell expressing SNX-B8 with a compound to be tested;

(b) determining whether in said cell SNX-B 8 function is altered in the presence of the compound to be tested when compared to a cell not contacted with said compound; and

(c) identifying the compound which alters SNX-B8 function and/or expression. The methods provided above may also comprise an additional step Qo'), wherein steps (a) and (b) are carried out in a control experiment in the absence of an agonist/enhancer to be screened.

Therefore, the present invention also relates to a method for identifying a compound which is capable of enhancing or reducing the expression of the SNX-B 8/SNX30 gene comprising the steps of contacting a cell which expresses the SNX-B8/30 gene from its natural promoter or a reporter gene driven by the SNX-B 8/SNX30 promoter and determining whether the expression of the gene is increased or reduced when compared to conditions in which the compound is not present.

Potential candidate molecules or candidate mixtures of molecules may be, inter alia, substances, compounds or compositions which are of chemical or biological origin, which are naturally occurring and/or which are synthetically, recombinantly and/or chemically produced or compounds or compositions described hereinabove. Thus, candidate molecules may be proteins, protein-fragments, peptides, amino acids and/or derivatives thereof or other compounds, such as ions, which bind to and/or interact with SNX-B8/SNX30. Such binding and/or interacting candidate compounds may be found employing, inter alia, yeast two-hybrid systems or modified yeast two-hybrid systems as described, for example in Fields, Nature 340 (1989), 245-246; Gyuris, Cell 75 (1993), 791-801; or Zervos, Cell 72 (1993), 223-232. As "agonist", in accordance with this invention, molecules/substances are denoted which have an affinity as well as an intrinsic activity. Mostly, said intrinsic activity (α) is defined as being proportional to the quotient of the effect, triggered by said agonist (EA) and the effect which can be maximally obtained in a given biological system (Emax): therefore, the intrinsic activity can be defined as

The highest relative intrinsic activity results from EA/Emax=l. Agonists with an intrinsic activity of 1 are full agonists, whereas substances/molecules with an intrinsic activity of >0 and <1 are partial agonists. Partial agonists show a dualistic effect, i.e. they comprise agonistic as well as antagonistic effects. Preferably, in the context of the present invention, an agonist (or full agonist) is an endogenous substance or a drug that can interact with SNX-B8/SNX30 and initiate a maximal or complete physiological or a pharmacological response characteristic of SNX-B8/SNX30. A partial agonist is an endogenous substance or a drug that also provokes physiological or a pharmacological response but, the maximum response is less than the maximum response to a full agonist, regardless of the amount of drug applied.

The person skilled in the art can, therefore, easily employ the compounds and the methods of this invention in order, to elucidate the agonistic and/or antagonistic effects and/or characteristics of a compound/molecule/substance to be identified and/or characterized in accordance with any of the above described methods.

The methods for screening "inhibitors" or "activators" of SNX-B8/30 may also be employed for the screening of medically useful activators of SNX-9 or SNX- 18 (for example for the intervention in Alzheimer's disease or diabetes) or medically useful inhibitors of SNX-9 or SNX- 18 expression or function. These inhibitors of other subgroup members SNX-9/SNX-18 may also be employed in the treatment of, inter alia, cancer as described for SNX-B8/30 inhibitors/antagonists. The person skilled in the art may easily modify the above recited screening methods by employing, inter alia, SNX-9 or SNX- 18 polypeptides, or nucleic acid molecules encoding the same.

The term "test compound" or "compound to be tested" refers to a molecule or substance or compound or composition or agent or any combination thereof to be tested by one or more screening method(s) of the invention as a putative "inhibitor" or "activator'V'enhancer" of SNX-B8/SNX3O. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof or any of the compounds, compositions or agents described herein. It is to be understood that the term "test compound" when used in the context of the present invention is interchangeable with the terms "test molecule", "test substance", "potential candidate", "candidate" or the terms mentioned hereinabove.

Accordingly, small peptides or peptide-like molecules as described hereinbelow are envisaged to be used in the screening methods for "inhibitor(s)" or "activators" SNX-B8/SNX3O. Such small peptides or peptide-like molecules bind to and occupy the active site of a protein thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. Moreover, any biological or chemical composition(s) or substance(s) may be envisaged as SNX-B 8/SNX30 inhibitor or activator. The inhibitory function of the inhibitor can be measured by methods known in the art and by methods described herein. Such methods comprise interaction assays, like immunoprecipitation assays, ELISAs, RIAs as well as specific inhibition assays, like the assays provided in the appended Examples. It is also envisaged that elements of the SNX-B8/SNX30 pathway may be used, e.g., enzymes. Said enzymes may be present in whole cell extracts of cells expressing SNX- B8/SNX30 or said enzymes may be purified, partially purified or recombinantly expressed as described hereinbelow.

Also preferred potential candidate molecules or candidate mixtures of molecules to be used when contacting a cell expressing SNX-B 8/SNX30 (or a member of the herein defined subgroup SNX-9/SNX-18) may be, inter alia, substances, compounds or compositions which are of chemical or biological origin, which are naturally occurring and/or which are synthetically, recombinantly and/or chemically produced. Thus, candidate molecules may be proteins, protein-fragments, peptides, amino acids and/or derivatives thereof or other compounds, such as ions, metabolites, intermediates or enzymes.

Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

In addition, the generation of chemical libraries is well known in the art. For example, combinatorial chemistry is used to generate a library of compounds to be screened in the assays described herein. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in every possible combination to yield peptides of a given length. Millions of chemical compounds can theoretically be synthesized through such combinatorial mixings of chemical building blocks. For example, one commentator observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. (Gallop, Journal of Medicinal Chemistry, Vol. 37, No. 9,1233-1250 (1994)). Other chemical libraries known to those in the art may also be used, including natural product libraries. Once generated, combinatorial libraries are screened for compounds that possess desirable biological properties. For example, compounds which may be useful as drugs or to develop drugs would likely have the ability to bind to or interact with the SNX-B8/30 target protein identified, expressed and purified as described herein.

In the context of the present invention, libraries of compounds are screened to identify compounds that function as inhibitors or activators of the target gene product, here SNX- B8/SNX30 (or the gene target product of the other member of the subgroup defined herein, namely SNX-9 or SNX-18). First, a library of small molecules is generated using methods of combinatorial library formation well known in the art. U. S. Patent Nos. 5,463,564 and 5,574,656 are two such teachings. Then the library compounds are screened to identify those compounds that possess desired structural and functional properties. U. S. Patent No. 5,684, 711, discusses a method for screening libraries. To illustrate the screening process, the target cell or gene product and chemical compounds of the library are combined and permitted to interact with one another. A labeled substrate is added to the incubation. The label on the substrate is such that a detectable signal is emitted from metabolized substrate molecules. The emission of this signal permits one to measure the effect of the combinatorial library compounds on the enzymatic activity of target enzymes by comparing it to the signal emitted in the absence of combinatorial library compounds. The characteristics of each library compound are encoded so that compounds demonstrating activity against the cell/enzyme can be analyzed and features common to the various compounds identified can be isolated and combined into future iterations of libraries. Once a library of compounds is screened, subsequent libraries are generated using those chemical building blocks that possess the features shown in the first round of screen to have activity against the target cell/enzyme. Using this method, subsequent iterations of candidate compounds will possess more and more of those structural and functional features required to inhibit (or to enhance) the function of the target cell/enzyme, until a group of (enzyme)inhibitors or activators with high specificity for the enzyme can be found. These compounds can then be further tested for their safety and efficacy as antibiotics for use in animals, such as mammals. It will be readily appreciated that this particular screening methodology is exemplary only. Other methods are well known to those skilled in the art. For example, a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known. For example, some techniques involve the generation and use of small peptides to probe and analyze target proteins both biochemically and genetically in order to identify and develop drug leads. Such techniques include the methods described in PCT publications No. WO 99/35494, WO 98/19162, WO 99/54728.

Preferably, candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons, preferably less than about 750, more preferably less than about 350 daltons.

Candidate agents may also comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

Exemplary classes of candidate agents may include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

As mentioned above, candidate agents are also found among biomolecules including peptides, amino acids, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alleviation, esterification, amidification, etc. to produce structural analogs.

Other candidate compounds to be used as a starting point for the screening of inhibitors of SNX-B8/SNX30 are aptamers, aptazymes, RNAi, shRNA, RNAzymes, ribozymes, antisense DNA, antisense oligonucleotides, antisense RNA, antibodies, affybodies, trinectins, anticalins, or the like compounds which are described in detail hereinbelow. Accordingly, the person skilled in the art is readily in a position to have candidate compounds at his disposal which can be used in the screening methods for inhibitors of SNX-B8/SNX30 biosynthesis as a basis to, inter alia, improve or further develop the capability of such compounds to inhibit or activate SNX-B8/SNX30 biosynthesis. Accordingly, the person skilled in the art can readily modify such compounds by methods known in the art to improve their capability of acting as an inhibitor in the sense of the present invention. The capability of one or more of the aforementioned compounds to inhibit SNX-B8/SNX30, preferably in a eukaryotic cell, more preferably in mammalian cells is tested as described hereinabove.

In one embodiment of the present invention, the SNX-B8/SNX30 as described herein are isolated and expressed. These recombinant proteins are then used as targets in assays to screen libraries of compounds for potential drug candidates.

Current cell-based assays used to identify or to characterize compounds for drug discovery and development frequently depend on detecting the ability of a test compound to modulate the activity of a target molecule located within a cell or located on the surface of a cell. Most often such target molecules are proteins such as enzymes, receptors and the like. Corresponding examples are given, inter alia, in the experimental part, providing for an assay based on APP shedding, endocytosis, insulin signalling as well as endocytosis of the transferrin receptor.

A number of highly sensitive cell-based assay methods are available to those of skill in the art to detect binding and interaction of test compounds with specified- target molecules. However, these methods are generally not highly effective when the test compound binds to or otherwise interacts with its target molecule with moderate or low affinity, hi addition, the target molecule may not be readily accessible to a test compound in solution, such as when the target molecule is located inside the cell or within a cellular compartment such as the periplasm of a bacterial cell. Thus, current cell-based assay methods are limited in that they are not effective in identifying or characterizing compounds that interact with their targets with moderate to low affinity or compounds that interact with targets that are not readily accessible. The cell-based assay methods of the present invention have substantial advantages over current cell-based assays.

Current methods employed in the arts of medicinal and combinatorial chemistry are able to make use of structure-activity relationship information derived from testing compounds in various biological assays including direct binding assays and cellbased assays. Occasionally compounds are directly identified in such assays that are sufficiently potent to be developed as drugs. More often, initial hit compounds exhibit moderate or low potency. Once a hit compound is identified with low or moderate potency, directed libraries of compounds are synthesized and tested in order to identify more potent leads. Generally these directed libraries are combinatorial chemical libraries consisting of compounds with structures related to the hit compound but containing systematic variations including additions, subtractions and substitutions of various structural features. When tested for activity against the target molecule, structural features are identified that either alone or in combination with other features enhance or reduce activity. This information is used to design subsequent directed libraries containing compounds with enhanced activity against the target molecule. After one or several iterations of this process, compounds with substantially increased activity against the target molecule are identified and may be further developed as drugs. This process is facilitated by use of the sensitized cells of the present invention since compounds acting at the selected targets exhibit increased potency in such cell-based assays, thus, more compounds can now be characterized providing more useful information than would be obtained otherwise.

Moreover, in a preferred embodiment the method for screening inhibitors or activators SNX- B8/SNX30 (or the other members of the herein defined subgroup, namely SNX-9 or SNX- 18) function or expression are screened in a high through put screening assay. High-throughput screening methods are described in U.S. Pat. Nos. 5,585,277 and 5,679,582, in U.S. Ser. No. 08/547,889, and in the published PCT application PCT/US96/19698, and may be used for identifying an inhibitor or activator of SNX-B8/SNX30 function or expression as described herein. High-throughput screening methods and similar approaches which are known in the art (Spencer, Biotechnol. Bioeng. 61 (1998), 61-67; Oldenburg, Annu. Rev. Med. Chem. 33 (1998), 301-311) carried out using 96-well, 384-well, 1536-well (and other) commercially available plates. In this method, large numbers of different small test compounds, e.g. aptamers, peptides, low-molecular weight compounds as described herein, are provided or synthesized on a solid substrate, such as plastic pins or some other surface. Further methods to be employed in accordance with the present invention comprise, but are not limited to, homogenous fluorescence readouts in high-throughput screenings (as described, inter alia, in Pope, Drug Discovery Today 4 (1999), 350-362).

After a "modulator" of SNX-B8/30 (or the other members of the herein defined subgroup, namely SNX-9 or SNX- 18) is screened, and identified by the methods provided herein, said "modulator" can in addition be formulated as pharmaceutical composition. Accordingly, the invention also provides for a method for the production of a pharmaceutical composition comprising the screening methods of the invention and, optionally, further comprising a pharmaceutically acceptable carrier and/or a diluent and/or an excipient and/or a microcapsulated host cell.

In context of the invention also a kit is provided, comprising a SNX-B8/30 polynucleotide, the SNX-B 8/30 polypeptide, the antagonist/inhibitor or the agonist/enhancer of the invention.

Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific or diagnostic assays or the like. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia, for detecting one or more of the nucleic acid molecules described herein which encode (a) polypeptide(s) involved endocytotic pathways as described herein. Thus, said kit could be, for example, employed in a variety of applications, e.g., as diagnostic kit, as research tool or therapeutic tool. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.

hi a further embodiment of the invention, a method is provided for the preparation of a non- human double transgenic animal comprising the steps of

(a) mating with a non-human transgenic animal described herein above (the "SNX- B 8/30" (over)expressing animals or knock-out/knock-down animals) with a further non-human transgenic animal, said further animal comprising a modification of/in an a gene which leads to an altered APP metabolism or which is related to APP processing and/or an altered insulin metabolism or which is related to the insulin pathway;

(b) selecting the off-spring of said mating which carries at least one copy of a heterologous polynucleotide of claim 1 to 4 or at least one copy of a gene comprising a mutation in an ortholog os SNX-B8 and at least one further copy of a gene leading to an altered APP metabolism or which is related to APP processing and/or an altered insulin metabolism or which is related to the insulin pathway.

It is also envisaged that the above method for the preparation of a "double transgenic animal" comprises a further step, i.e. a step (c), wherein said offspring is again mated and whereby generating double- homozygous non-human transgenic animals. However, also heterozygous non-human animals are useful in particular in drug screening assays, however also as scientific, medical and pharmacological research tools. Accordingly, the invention also relates to double-transgenic animals obtained by the method described herein. It is evident for the person skilled in the art that also other double-transgenics may be obtained and produced by mating a given non-human transgenic animals with a "SNX-B 8/30 transgenic" as provided herein. Accordingly, the invention also provides for any double-transgenic non-human animal wherein at least one copy of the SNX-B 8/30 ortholog is modified/mutated or wherein at least one copy of a heterologous SNX8/30 (for example the human SNX8/30 described herein expressed in a mouse) is expressed.

As used herein above, the term "comprising a modification of/in a gene" may relate to the expression of an additional gene, for example a heterologous gene from a different species or to the expression of a(n) additional copy (copies) of said gene, e.g. also genes of the same species (so-called "over"-expressors). Said term also relates to genes which are mutated, silenced and/or down-regulated. The same applies mutatis mutandis for the "SNX-B8/3O" transgenics defined herein above.

The term "gene which is related to APP processing" comprises, inter alia, wildtype genes, like APP, presenilins, (presenilin 1 or presenilin 2) or BACE. Said wildtype genes may be overexpressed or may relate to heterologously expressed wildtype genes like the expression of corresponding human genes in a non-human transgenic animal. It is evident for the skilled artisan that the term "a gene which leads to an altered APP metabolism" may not only comprise the expression of a (heterologous) wildtype gene or additional copies of a homologous wildtype gene but also to gene(s) comprising a mutation. For example may APP expressed in said non-human transgenic animal which comprises a mutation like, e.g. the Swedish-, Arctic-, Indiana- or London mutation.

Several corresponding and useful non-human transgenic models, in a particular mouse models have been described and reviewed in Price (1998) Annual Review of Genetics Vol. 32: 461- 493, Khachaturian (2002); Alzheimer Disease & Associated Disorders. 16 Supp 1:S9-S12 as well as in German (2004) Rev Neurosci. 15(5):353-369. In particular German (2004) loc cit, states that mice over-expressing mutant Alzheimer's disease (AD)-related proteins exhibit many of the neuropathological and behavioral features of the human disease. Transgenic animals have been created that express mutations in the amyloid precursor protein (APP), presenilin (PS)I, and PS2, and also animals expressing more than one of these mutations. For example, in APP mouse models, there are age-related accumulations of amyloid-beta (Abeta)- containing neuritic plaques in the hippocampus and cerebral cortex, activation of astrocytes and microglial cells in regions containing plaques, and degeneration of cholinergic nerve terminals in brain regions that eventually become plaque containing. Missing in the APP and PS mouse models are neurofibrillary tangles and robust neuronal loss in cerebral cortical and subcortical regions such as the basal forebrain cholinergic andiocus coeruleus noradrenergic nuclei. Neurofibrillary tangles can be produced in mice expressing mutant tau protein, and the tangle formation is further enhanced in animals that also express mutant APP. Studies in APP mouse models indicate that, like AD, there are abnormalities in adult hippocarnpal neurogenesis. The animal models of AD are employed to develop and test treatments that reduce brain levels of the Abeta42 protein, neuritic plaque load and glial activation. The mating with the non-human transgenic animals of this invention lead to double-transgneic animals which are particularly useful in drug screening approaches for medicaments to stop the neurodegenerative process and restore hippocampal neurogenesis, damaged brain circuits may be replaceable in patients with AD.

Stalder (Am J Pathol 1999, 154:1673-1684) characterized transgenic mice (APP23) generated by Sommer from animals that carried the human APP gene with a double mutation of Swedish familial AD under a neuron-specific promoter, whereas Frautschy (Proc Natl Acad Sci USA 1997, 94:13287-13292) characterized mice originally generated by Hsiao (Science 1996, 2774:99-102) from animals that carried the same mutation in APP under a different promoter, the PRP promoter, which is expressed in both neurons and glia. The Swedish mutation is associated with elevated levels of production of AB in brain, plasma, and fibroblasts of affected and at-risk individuals and in a variety of cells transfected with such constructs; see Suzuki in Science 1994, 264:1336-1340. Both studies document aggregation and phenotypic activation of microglia associated with dense amyloid deposits, but limited or no association of microglia with diffuse amyloid deposits.

Non-human transgenic animals comprising a gene mutation or a gene insertion leading to a modified APP metabolism may, e.g., be selected from the group consisting of animals expressing human APP or a variant/mutation thereof, like the "Swedish mutation", "Indiana mutation" and/or the "London mutation; V642I". It is also envisaged and non-limiting that animals comprising a gene encoding the "Flemish" or "Arctic" mutation are employed in context of this invention.

Such animals are well known in the art, see, inter alia, Dominguez-del-Toro (2004) Eur J Neurosci. 20(7): 1945- 1952; Wenk (2004) Neuroscience.;125(3):769-776 or Jin (2004) Proc Natl Acad Sci U S A. 101(36):13363-13367. It is also envisaged to mate the non-human transgenic animals of this invention ("SNX-B 8/30 animals") with animal models lacking APP, like mice described by Wang (2005) J Neurosci. 2005 Feb 2;25(5):1219-25.

More than 100 different mutations in presenilin 1 (PSl) have been associated with inherited early onset Alzheimer's disease (AD) and changes in the presenilin metabolism and fucnetion have been associated with modified APP-metabolism. Accordingly, in context with this invention, also double-mutated non-human transgenic animals are to be produced in accordance with this invention, wherein a first mutation relates to SNX-B 8/30 and the second mutation influences presenilin expression and/or function. These animals may have an alteration in their presenilin 1 or 2 or may express presenilin form another sprecies, like PSl or PS2 from human. Therefore, also presenilin (in a particular 1) animals (in particular mice) are useful in the embodiment provided above. Such presenilin transgenic animals are known in the art, see, inter alsi, Cataldo 2004; J Neuropathol Exp Neurol. 63(8):821-30; Jankowsky (2004) Neurobiol Aging. 25(7):885-92.

BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the beta- secretase cleavage site and is involved in Alzheimer's disease. Accordingly, it is also envisaged that animals with BACE modifications be used in the method provided herein and that double-transgenic animals be produced by mating the "SNX-B8/30" non-human transgenic animals with these BACE-animals. BACE-"knock outs" are, inter alia described in Pastorino (2004) MoI Cell Neurosci. 25(4):642-649 and BACE- over-expressing mice are known form Lee (2005) J Cell Biol. 168(2):291-302. In these overexpressing mice it was shown that the subcellular processing of APP is altered and that Abeta deposition is inhibited.

In this context, it is also of note that in one embodiment of the invention, also already "doubly-modified" non-human transgenic animals may be mated with the non-human transgenic animals provided in this invention, i.e. the SNX-B8/30 over-expressing animals or animals where the corresponding ortholog comprises a mutation, like, inter alia, SNX-B8/30 knock-outs. A corresponding, non-limiting example may be, the double transgenic for amyloid precursor protein (AA substitution K670N,M671L) and presenilin-1 (AA substitution M146V) where both synaptic and cognitive deficits have been described, see, e.g. Gong, 2004, J Clin Invest. 114:1624-1634. Also further "double-transgenic" non-human animals may be obtained by the method provided above, whereby as mating partners non-human transgenic animals are employed which comprise a modification in genes relating to metabolic pathways, in particular to the insulin pathway. Accordingly, the non-human transgenic animals provided herein and comprising a modified gene relating to the herin described SNX B8/30 gene (or an orthologue thereof) are, in accordance with this invention amted with such animals comprising gene modifications in a metabolic pathway. Non-limiting examples of such non-human transgenic animals, having modifications in a metabolic pathway, like the insulin pathway are provided below.

Also in this context, it is evident for the skilled artisan that the terms "gene which alters insulin metabolism" or " a gene which is related to the insulin pathway" may comprise heterologously wildtype genes, additional copies of the relevant (homologous) wildtype genes as well as mutated versions of said genes. Also comprised are in the context (as well as in the context relating to APP processing or APP metabolism) "knock-out" or "knock-down" non- human transgenic animals to be mated with the herein defined "SNX-B8/30" non-human transgenics. The corresponding exemplified genes in context of "insulin metabolism" or "insulin pathway" may comprise, but are not limited to insulin, insulin receptor(s), insulin- like growth factor(s), insulin receptor substrate(s), lipoprotein lipase(s), PD kinase, Akt3 and the like.

Binding of insulin and insulin-like growth factor I (IGF-I) to their corresponding receptors (insulin receptor (IR) and IGF-I receptor (IGF-IR)) induces a signaling cascade involving multiple protein-protein interactions. Proteins called insulin receptor substrates 1-4 (IRS 1-4) bind to IR and IGF-IR and can in turn activate proteins such PI3-Kinase. Fernandez et al. ((2001) Genes Dev. 75, 1926) generated a mouse model of diabetes which over-expresses a dominant-negative form of IGF-IR specifically in muscle. Expression of the mutant IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance.

IRS 1-4 are all involved in insulin signaling, but their knock-out has differential effects on insulin signaling and - in particular - on glucose metabolism and insulin resistance (for a review see Le Roith (2002) Curr Opin Clin Nutr Metab Care 5:371). Knock-out of IRSl and 2 revealed a role in insulin responsiveness in major tissues, IRS4 had a role in glucose tolerance, whereas IRS3 knock-out mice did not seem to be essential in these processes. Thus, IRS-I, -2 and -4 transgenic animals may be used for mating with the non-human "SNX-B8" transgenics provided herein.

Another genetic (mouse) model for diabetes has a transgenic muscle- and liver-specific overexpression of lipoprotein lipase (Kim et al. (2001) Proc. Natl Acad Sci 98,7522). Muscle- lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate- 1 -associated phosphatidylinositol 3-kinase activity. In contrast, liver-lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. Accordingly, also this non-human transgenic is to be mated with the SNX-B8/30 animals provided herein.

Also envisaged is the mating of the inventive tansgenic ,,SNX-B8/30" animals with animals which show a reduced expression of , e.g. PI-3 kinase or Akt-2. Such a "reduced expression" may, inter alia, be obtained by employing corresponding RNAi technology, i.e. the generation of non-human tansgenic animals expressing a corresponding inhibitory molecule, like an RNAi.

Therefore, in a further embodiment of the invention, a "double-transgenic" non-human animal is provided which has/comprises at least one modified/altered or at least one additional SNX- B8/30 allele or gene copy and at least one further modified or altered allele of a further genetic locus (or an additional, further heterologous gene/allele). These double-transgenic non-human animals are particularly useful in screening methods as provided herein as well as research tools in, inter alia, neurological research, obesity research, diabetes research or cancer research.

The above defined uses of the polynucleotides encoding SNX-B8/30, of SNX-B8/30 polypeptides (or functional fragments thereof), of the vector provided herein, of the inventive host cells as well as of the agonists/enhancers of SNX-B8/30 defined herein are particular relevant in a medical setting, in particular in the treatment, prevention and/or amelioration of neurological neurodegenerative disorders as well as in metabolic disorders, in particular diabetes and/or obesity. The corresponding method of preventing, ameliorating and/or treating said disorders comprises the administration of any of the above recited compounds in a pharmaceutically acceptable form and in a therapeutically active amount to a subject in need of such a treatment, prevention and amelioration.

Most preferably, the neurodegenerative disorder to be treated is Alzheimer's disease. Further corresponding disorders are provided herein above. Similarly, the invention provides for a method of treating, ameliorating and/or preventing cancer (or a tumorous disease) and/or transferrin receptor related disorders by administering to a subject in need of such a treatment, amelioration and/or prevention a pharmaceutically active form and in a therapeutically active amount of an antagonist/inhibitor of SNX-B8/30 (or of SNX-9 and/or SNX-18), of an inhibiting RNA,shRNA, RNAi or siRNA of the invention or of (inhibiting or antagonizing) antibody as defined above.

The subject to be treated by the methods provided herein is preferably a human subject. In the context of the present invention the term "subject" means an individual in need of a treatment of an affective disorder. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human. The subject will receive a "therapeutically effective amount" or a "pharmaceutically effective amount" of the inventive substances disclosed herein.

The term "administered" means administration of a therapeutically effective dose of the aforementioned inventive compounds to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents maybe administered alone or in combination with other treatments.

The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intraarterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermal^, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution dry spray.

The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

The figures show:

Figure 1: Proteolytic cleavage of APP

A. . Cleavage of APP by α-, β- and γ-secretase.

B. The molecular machinery receives incoming signals and reacts by increasing α- cleavage of APP.

Figure 2: The novel sorting nexin SNX-B8: sequence alignment, domain structure, homologues

A. Shown is the amino acid alignment of the novel SNX-B 8/SNX30 and its two homologues SNX9 and 18 as well as the C. elegans homologue. In contrast to vertebrates, C. elegans has only one member of the SNX9/18/B8 group, which is named lst-4. The SH3 domain is underlined with a box, the PX domain with a black bar and the BAR domain with a dotted line.

B. Overview of the domain structure of several of the known SNXs. The SNXs are assembled in different subgroups depending on the nature of the functional domains. The novel SNX-B8 is a homologue of SNX9 and SNXl 8 and consists of a SH3 domain, a PX domain and a BAR domain. The domain between the SH3 and the PX domain is now termed low-complexity domain, but is not indicated in the figure, (figure adapted from Habermann (2004) EMBO Rep. 5, 250)

Figure 3: Dephosphorylation of SNX-B8

SNXB8-HA was immunoprecipitated with HA antibodies (1:200) from 293E cell lysates transiently overexpressing SNXB8-HA. Immunoprecipitated SNXB8-HA coupled to Protein A Sepharose (PAS) was incubated with (+) or without (-) shrimp alkaline phosphatase (SAP at a dilution of 1:100) in SAP Buffer containing protease inhibitors (protease inhibitor mix, Sigma) overnight at 37°C. After centifugation PAS beads were incubated with protein loading buffer at 95°C and loaded on a 8 % SDS gel, blotted and analyzed with an HA antibody.

Figure 4; SNX-B8 stimulates the ectodomain shedding of APP

Schematic drawing of the AP-APP fusion protein. The ectodomain of secretory alkaline phosphatase (AUc. Phos.) was fused to the N-terminus of full-length APP695. The Aβ peptide domain is indicated as a white box. Horizontal bars indicate the epitopes recognized by the antibodies 22Cl 1, 192Wt (specific for β- secretase cleaved APP), W02 (specific for α-secretase cleaved APP) and 6687. Arrows indicate the proteolytic cleavage sites of α-, β- and γ-secretase. β- secretase has a major and a minor (small arrow) cleavage site. M: membrane.

B. Measurement of alkaline phosphatase activity in AP-APP expressing cells Using lipofectamine, HEK293 cells stably expressing the alkaline phosphatase (AP)-APP fusion protein were transiently transfected with empty control (Con) vector, with the known APP α-secretase ADAMlO (ADlO), SNX-B8, wild-type dynamin (Dyn), a dominant-negative dynamin mutant (DynK44A) or a dominant-negative dynamin mutant lacking its N-terminus (Dyn ΛNT). One day after transfection the medium was replaced by fresh medium. 24 h later, the conditioned medium was collected and the cells were lysed in solubilization buffer (see below). The phosphatase activity measured in the conditioned medium was corrected for the protein concentration in the cell lysate (relative AP activity) and represents the mean of at least two independent experiments, each one carried out in duplicate. For the alkaline phosphatase measurements, aliquots of the conditioned medium were treated for 30 min at 65 ⁰C to heat- inactivate the endogenous alkaline phosphatase activity.

C. Measurement of alkaline phosphatase activity in AP-APP expressing cells.

The experiment was carried out as in Fig. 4B. The indicated plasmids were used for transfection. All four proteins carrying the HA epitope tag were expressed in the cell lysate as determined by immunoblot analysis using an antibody against the HA-tag. To this aim, aliquots of the lysates (corrected for protein concentration in the lysates) were directiy loaded onto the electrophoresis gels. Antibody HA.11 was purchased from Covance.

D. Measurement of SNX-B 8 effect on TNFR2- and L-selectin shedding Plasmids encoding the indicated proteins were transiently transfected into HEK293 cells stably expressing AP fusion proteins of L-selectin or TNFR2. The phosphatase activity in the conditioned medium was determined as in Fig. 4B. MEKK2 is a kinase that served as a positive control to demonstrate that the shedding of TNFR2 and L-Selectin can be stimulated.

All cDNAs were cloned into the peak 12 vector carrying the EFl alpha promoter.

Figure 5: SNX-B8 stimulates the a- and β-secretase cleavage of APP HEK293 cells expressing endogenous APP (left panels) and COS7 cells stably transfected with APP (right panel) were transiently transfected with SNX-B 8 carrying the HA epitope tag or with empty control vector. Experiments were carried out in duplicate. Secreted APP was detected in the supernatant with antibodies specific for α- (W02) or β-secretase cleaved APP (192 wt). Full- length (fl.) APP in the cell lysate was detected with antibody 6687 against the C- terminus of APP (epitopes of the antibodies are indicated in Fig. 4A). SNX-B8 expressed in the cell lysate was detected with an antibody against the HA-tag. Aβ was immunoprecipitated from the conditioned medium with a polyclonal antiserum against Aβ and detected with monoclonal antibody 6E10.

Figure 6: APP uptake assay

The APP uptake assay was essentially carried out as described previously (Kaether et al., J. Cell Biol. 158, 551). In brief: one day after transfection, COS cells transiently transfected with ρCEP-APP695 and GFP or pCEP-APPwt and SNXB8-GFP grown on glass coverslips were incubated on ice with anti-APP antibody 5313 (polyclonal, against ectodomain of APP) diluted 1:200 in PBS supplemented with 0.5 niM MgCl₂ and 1 mM CaCl₂ for 20 min, washed with cold PBS and returned to standard medium at 37°C . After 0, 7, 25 or 35 min cells were fixed in 4% paraformaldehyd, lysed with Triton X-100, blocked with gelatine, stained with a fluorescently labeled secondary antibody and analyzed by Immunofluorescence. Cells were scored for the analysis depending on whether they showed or not endocytosis APP at the indicated time points.

Figure 7: Expression analysis of SNX-B8

A. HEK293 cell lysates were precipitated with a polyclonal antibody against SNX-B8. The same antibody was used for detection in the immunoblot and revealed two to four protein bands of a similar apparent molecular weight of around 75 kDa (labeled SNX-B8), which correspond to the protein bands of HA-epitope tagged SNX-B8 expressed in HEK293 cells (direct loading of cell lysates). Additionally, the heavy chain of the SNX-B8 antibody used for the immunoprecipitation is detected in the left lane (labeled IgG). The vertical line between the two lanes indicates that both lanes were next to each other on the same blot, but that a longer exposure of the blot was used for the right lane (no immunoprecipitation).

B. Multiple tissue Northern blot analysis of SNX-B8 expression. A 5 '-probe of SNX-B8 was used for the detection and revealed two mRNA species of around 4,8 and 8 kb. To control for the amount of mRNA loaded in every lane, the blot was reprobed for actin (lower panel), the level of which was particularly low in brain. Compared to the actin levels, SNX-B8 shows a ubiquitous expression, which is particularly strong in the pancreas.

C. C-terminally GFP-tagged SNX-B8 was transiently transfected into COS cells.

GFP fluorescence was detected by standard procedures using a fluorescence microscope.

Figure 8: SNX-B8 inhibits endocytosis of transferrin (Tf) in a dose-dependent manner

COS cells transiently transfected with GFP as a control or GFP-tagged SNX- B8 were incubated on ice with Alexa-fluorophore labeled Tf. After removing the non-bound Tf the cells were moved back to 37⁰C, allowing endocytosis of the bound Tf. After 7 or 20 min respectively, the cells were fixed and analyzed by fluorescence microscopy for internalized fluorescent Tf. At 7 min, most GFP expressing cells showed Tf endocytosis. At the same time most cells weakly expressing SNX-B8 showed endocytosis, whereas cells with a middle or strong expression of SNX-B8 showed strongly reduced or no endocytosis, respectively. At 20 min, SNX expressing cells showed increased rates of endocytosis, revealing that SNX-B8 expression does not completely abolish but reduce the rate of Tf endocytosis.

Figure 9: RNAi experiment A. Reduction of SNX-B8/30 expression by RNAinterference. HEK293 cells were transiently co-transfected with HA-epitope tagged SNX-B 8/30 and either empty pSuper vector (control) or pSuper RNAi vector containing the indicated short hairpin sequences of the SNX-B8 sequence. Aliquots of cell lysates were directly loaded onto electrophoresis gels. Immunoblot detection was carried out with an anti-HA-tag antibody.

Short hairpin sequences encoding bases 13-31 (ggccgagccctctatgact; SEQ ID NO: 7; see RNAi I) or 1629-1647 (gcacatgatgcagaactac; SEQ ID NO: 8; see RNAi II) of SNX-B8/30 were cloned into the pSuper vector (Brurnmelkamp et al. Science 2002, 296, 550). HEK293 cells were transiently co-transfected with HA-epitope tagged SNX-B8/30 and either empty pSuper vector (control) or pSuper RNAi vector containing the indicated short hairpin sequences of the SNX-B8 sequence. Aliquots of cell lysates were directly loaded onto electrophoresis gels. Immunoblot detection was carried out with an anti-HA- tag antibody.

B. Knock-down of endogenous SNX-B8 in HEK293E cells. Oligo siRNA SNXB8a (CCUCAACCGUUUCUCAUGCdTdT and the corresponding antisense oligo; the desoxyribonucleotide sequence "dTdT" at the 3 '-end_. enhances the oligo 's stability) was transfected into HEK293E cells. SNX-B8 was immunoprecipitated from the cell lysate using a monoclonal antibody and was detected with a polyclonal antibody. Accordingly it can be documented that also endogenous SNX-B8/30 can successfully be down regulated by means and methods provided herein.

The Examples illustrate the invention.

Example 1: Expression cloning screen for activators of APP shedding

hi order to identify proteins stimulating APP α- or β-cleavage, an expression cloning approach was used. Pools of cDNAs from a human brain cDNA library were transfected into a reporter cell line, which allows to easily monitor APP shedding. This reporter cell line is already published (Lichtenthaler, 2003). Among the proteins/cDNAs stimulating APP shedding, were BACEl and the catalytic subunit of PKA, both of which are known regulators of APP shedding. Additionally, a N-terminally truncated form of dynamin-1 was identified, which acts as a dominant-negative mutant of dynamin function, thereby inhibiting endocytosis (data not shown). A dominant-negative dynamin mutant has previously been shown to inhibit APP endocytosis and to stimulate APP secretion, presumably by making more APP available for α-secretase cleavage at the cell surface. Together, these cDNAs validate the screening approach since that physiologically relevant cDNAs were obtained.

Example 2: Identification of a novel sorting nexin

A novel protein has been identified. Based on the predicted protein-domain structure the novel protein can be assigned to the sorting nexin (SNX) protein family. Sorting nexins are a large family of proteins (Fig. 2B). Their biological function is only partly understood. SNXs are thought to be involved in intracellular transport from and to the endosomes and may be involved in regulating stability and degradation of cell surface receptors (Worby and Dixon, 2002). Currently, 29 SNXs have been described.

SNXs are characterized by the presence of a well-conserved phosphoinositide-binding domain (PX domain, Fig. 2B) but otherwise do not share a high degree of similarity (Fig. 2A). In fact, some SNXs may not even function in protein trafficking and therefore be falsely assigned to the SNX group (Worby and Dixon, 2002). SNXs can be divided into subgroups depending on what other domains are present besides the PX domain (Fig. 2B). The inventive SNX- B8/SNX30 together with SNX9 and SNXl 8 form such a subgroup (which we refer to as the SNX9/18/B8 subgroup), said subgroup being characterized by a SH3 domain, a low- complexity domain (which is not shown in Fig. 2B, but is the domain between the SH3 and the PX domain), a PX domain and a BAR domain (Fig. 2B). The BAR domain was originally referred to as coiled coil domain (Lundmark and Carlsson, 2004). The herein illustrated SNX- B8/30 sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 and relate to the human protein. Further homologous orthologs are shown in SEQ ID NOS: 12 and 13 (mouse), 14 and 15 (rat) and 16 and 17 (C. elegans). It is of note that C. elegans comprises only one SNX of the herein defined SNX-B8/30, SNX-9 and SNX-18 family.

Genbank entries NM_153271 (hypothetical protein MGC32065), BC018775, AL833039 and ACl 05020 appear to be identical to the cDNA provided herein. The cDNA obtained in the examples provided here has however a longer 5' untranslated region (5' UTR) than most submitted sequences, but has the same 5'UTR as ACl 05020. The molecule provided herein is surprisingly be shown as being a functional member of the SNX-family and is denoted in context of the invention as "SNX30" or "SNX-B8". The above receited Genebank entries are not annotated.

SNX-B8/SNX30 encoded protein has a homology of 50-60% on protein-level to its homologues SNX9 and 18, with the homology being higher within the SH3, the PX and parts of the BAR domain.

SNX9 and SNX-18 sequences are provided in SEQ ID NOS: 3 and 4 and SEQ DD NOS: 5 and

6, respectively.

Nucleotide alignment

The alignment values (%identity) were calculated by blasting the SNX-B8/SNX30 nucleotide sequence against the Genbank database. The only SNX homologue obtained is SNXl 8, which shows in three short stretches (72, 56, 31 bp) the following homology- values:

72 bp (95% identity), 56 bp (91% identity), 31 bp (93 % identity).

The overall identity between the nucleotide sequence of SNX-B8 and its homologues was calculated using the EMBL-EBI Emboss program (in this case the values for identity and similarity are identical):

Protein alignment

The alignment values (%identity or similarity) were calculated using the EMBL-EBI Emboss program:

The C.elegans SNX (lst-4) is more homologous to the SNX-B8/SNX30 described herein than to SNX9 and SNX18.

Furthermore, the following similarities/identities between SNX-B8/30 orthologues has been deduced:

Protein sequence

The alignment values (% identity and similarity) were calculated by blasting the human SNX- B8 protein sequence against the Genbank database.

The protein alignment values for the human vs. C.elegans protein are already shown in the table above (Example 2). Nucleotide sequence (identity=homology)

The alignment values (%identity or similarity) were calculated using the EMBL-EBI Emboss program:

Example 3: Expression and phosphorylation of SNX-B8/SNX30

Transfection studies were carried out using lipofectamine 2000 according to the manufacturers instructions (Invitrogen). Transfection of SNX-B 8/SNX30 into human embryonic kidney HEK293 cells, followed by its detection in the cell lysate by Western blotting reveals a protein with an apparent molecular weight of around 70-75 kDa (Fig. 3), which is in the range of its calculated molecular weight (65 IcDa). The protein shows two to four bands of slightly different molecular weight, which can be converted to a single band upon dephosphorylation (Fig. 3). This result reveals that SNX-B8/SNX30 may be phosphorylated at multiple sites. SNX-B8/SNX30 is also expressed endogenously in HEK293 cells, where it shows the same pattern in the immunoblot as the transfected protein (as shown in Figure 7A). Moreover, SNX-B8/SNX30 is ubiquitously expressed, as analyzed by Northern blot (as shown in Figure 7B). Immunofluorescence of C-terrninally GFP -tagged SNX-B8/30 shows a partly cytoplasmic and partly membrane-associated and vesicular staining (as shown in Figure 7C), which is consistent with the function of the SNX in membrane and protein trafficking processes.

Example 4: Effect of SNX-B8/SNX30 expression on APP shedding

Compared to control transfected cells, overexpression of SNX-B8/SNX30 stimulated APP shedding using an alkaline phosphatase-APP fusion protein (Fig. 4A and B). Using lipofectamine, HEK293 cells stably expressing the alkaline phosphatase (AP)-APP fusion protein (Lichtenthaler et al. (2003), J. Biol. Chem. 278, 487113) were transiently transfected with empty control (Con) vector, or the same vector encoding HA epitope tagged SNX-B 8. One day after transfection the medium was replaced by fresh medium. 24 h later, the conditioned medium was collected and the cells were lysed. The phosphatase activity measured in the conditioned medium was corrected for the protein concentration in the cell lysate (relative AP activity) and represents the mean of at least two independent experiments, each one carried out in duplicate. For the alkaline phosphatase measurements, aliquots of the conditioned medium were treated for 30 min at 65 °C to heat-inactivate the endogenous alkaline phosphatase activity.

The two homologues, SNX9 and SNXl 8, had the same effect (Fig. 4C), suggesting that all three proteins may have similar functions in APP shedding.

Example 5: Specificity of the APP shedding effect

Importantly, another member of the SNX family, SNXl, which does not belong to the same subgroup as SNX9/18/B8 defined herein, did not have any effect on APP shedding (Fig. 4C). This suggests that the observed effect is specific for the SNX-B8/SNX3O and corresponding homologue subgroup. Moreover, SNX-B8/SNX30 and SNX9 had no effect on the shedding of L-selectin and only a minor effect on the shedding of TNF receptor 2 (TNFR2), as measured by alkaline phosphatase fusions of both proteins (Fig. 4D). L-selectin and TNFR2 - like APP — belong to a large and diverse group of membrane proteins undergoing ectodomain shedding (Blobel, 2002). Thus, results provided herein show that the SNX9/18/B8 subgroup is not a general stimulator of protein shedding but specifically act on APP (or a subset of proteins) undergoing shedding. Accordingly, it was surprisingly found that the SNX9/18/30 subgroup provided herein is specifically involved in APP metabolism.

Example 6: Effect of SNX-B8/SNX30 expression on APP α- and β-cleavage

It was also tested whether SNX-B8/SNX3O specifically stimulates α- or β-secretase cleavage of APP or both of them. To this aim SNX-B8/SNX30 was transiently transfected into HEK293 cells expressing endogenous APP. SNX-B8/SNX30 strongly stimulated α-secretase cleavage of endogenous APP but only had a minor stimulatory effect on β-secretase cleavage (Fig. 5). Similar effects on α-secretase cleavage were observed in COS7 cells overexpressing APP. β-secretase cleavage in the COS7 was not increased, but rather slightly reduced (Fig. 5). No major changes of Aβ secreted from the APP overexpressing HEK293 cells were observed upon SNX-B8/SNX30 transfection. Taken together, transfection of SNX-B 8/SNX3O activates the α-cleavage of APP in different cell lines and has a mild stimulatory effect (HEK293 cells) or not effect (COS7 cells) on β-cleavage. The observed increase in the α-cleavage of APP is certainly of therapeutic benefit for Alzheimer's disease, since the α-cleaved APP has neurotrophic and neuroprotective properties.

Example 7: Functional characterization of SNX-B8/SNX30 in endocytosis in regard of APP

Given, that both the dynamin K44A mutant and the SNX-B8/SNX30 stimulate APP shedding (Fig. 4B) it was also tested whether SNX-B8/SNX30 expression inhibits APP endocytosis similar to the dynamin K44A mutant. To this aim COS cells were transiently transfected with APP and either empty vector (as a control) or SNX-B8/SNX30. APP endocytosis was measured with a previously published antibody uptake assay (Kaether et al., 2002), which revealed that SNX-B8/SNX30 expression indeed inhibited APP endocytosis (Fig. 6). A similar inhibitory effect was also observed for the endocytosis of the transferrin receptor (as shown in Figure 8) using a published protocol (Leprince et al., 2003), suggesting that SNX- B8/SNX3O may affect the intracellular trafficking of different cell surface proteins. As already documented above, a direct interaction between SNX-B 8/SNX30 and APP could not be observed in co-immunoprecipitation experiments. It is likely that SNX-B8/SNX3O acts on the endocytic or intracellular trafficking machinery and thereby affects APP endocytosis. Taken together, these experiments indicate that SNX-B8/SNX30 may be involved in the endocytic process of cell surface proteins.

Example 8: Functional characterization of the sorting nexins with regard to insulin receptor signal transduction

Since not only the dynamin K44A mutant but also insulin signaling stimulate APP shedding, we tested a possible role of SNX-B8/SNX30 in insulin signaling, because this SNX also stimulated APP shedding, as documented above. To this aim we used the model organism Caenorhabditis elegans, where loss-of-function phenotypes in insulin signaling are well described. C. elegans offers the additional advantage of being easily amenable to gene expression knock-down experiments using RNA interference (RNAi) (Kamath et al., 2001). Moreover, C. elegans has only one member of the SNX9/18/B8 group, termed lst-4 (Genbank accession number for the protein is CAD56253: C. elegans hypothetical protein Y37A1B.2 or lst-4), such that a loss-of-function phenotype of SNX-B8/SNX30 or its homologues should be more readily visible. The C. elegans SNX-B8/SNX30 orthologue lst-4 has been identified by computational search of Notch receptor target genes and has been implicated in vulva development in C. elegans. Other functions or mechanistic details of its functions have not been described in that paper (Yoo et al., 2004). No other in vivo functional studies have been published about the SNX9/18/B8 group.

Down-regulation of SNX-B8/SNX30 expression in C. elegans via RNAi led to phenotypic changes in said animals which closely resembled phenotypes found in worms with a reduced insulin signal transduction (Burgering and Kops, 2002; Cassada and Russell, 1975): they showed an increased tendency for dauer formation. Dauer is a particular stage during larval development (Burgering and Kops, 2002) and occurs if TGFβ or insulin signaling do not occur properly. The results shown in table I are described in detail below. Since they rule out an involvement of the SNX in TGFβ signaling, they clearly position the C. elegans SNX- B8/SNX30 in the insulin signaling pathway and show that this SNX is required for normal insulin signal transduction.

Wild-type C. elegans as well as mutant strains carrying known gain-of-function or loss-of- function mutations in genes in the insulin signaling pathway were incubated at 15°C or 26°C. At 15⁰C (permissive temperature), the mutants behave as wild-type worms or show a mild phenotype. At 26°C (non-permissive temperature) the mutant phenotype becomes fully visible. For example, at 15⁰C worms with loss-of-function mutations in the insulin receptor (daf-2(el370)) or the PDK-I kinase (pdk-l(sa680)) show no or only mild phenotypes compared to wild-type worms (upper part of table I). However, at the non-permissive temperature of 26°C the animals show complete dauer formation (lower part of table I). In contrast, gain-of-function mutations in PDK-I (mgl42) or the PI3-kinase AGE-I (sa315), do not show any dauer formation, not even at 26⁰C. Likewise, the loss of function mutation of the transcription factor DAF-16 (m26) does not show dauer formation at either temperature. These results using known mutant worms were expected and served as controls. A knockdown of SNX-B8/SNX30 (lst-4) led to partial dauer formation already at the permissive temperature of 15°C (32.3% versus none for the wild-type). At the higher non-permissive temperature of 26 ⁰C, 71% of the worms show partial dauer formation and ~2% complete dauer formation, indicative of a severe impairment of insulin signaling. The most dramatic effects were seen when the insulin receptor mutation and the SNX knock-down were combined (daf~2(el370); lst-4(KNAϊ)). 99.6% of the worms showed dauer formation at 15°C, showing that insulin signaling is abolished. Thus, the daf-2(el370); lst-4(RNAϊ) worms show a synergistic effect, which is much stronger than the individual effects of the insulin receptor mutation (daf-2(el370)) or the SNX knock-down (lst-4(KNAi)) alone. This synergistic effect indicates that both insulin receptor and the SNX act in the same pathway. A similar synergistic effect is observed for the SNX knock-down in the PDK-I mutant worm (Ist- ¥(RNAi); pdk-l(sa680), again pointing to an essential role of the SNX in insulin signaling. Not only the insulin receptor but also the TGFβ signaling pathway and the cGMP pathway control dauer formation. However, in contrast to the insulin signaling mutants no synergistic effects were observed for the SNX knock-down combined with a loss of function mutation in the C. elegans TGFβ ligand (DAF-7) and the C. elegans guanylyl cyclase (DAF-Il). These results clearly show an essential role for the SNX-B8/SNX30 in insulin signal transduction but not in TGFβ or cGMP signaling.

Table 1. Effects of Ce sorting nexin homolog lst-4 on dauer formation

1A. Phenotype of progeny at 15°C (%)

L4 larvae partial strain and adult dauer dauer L1 arrest no.^a wild-type 100 0 0 0 831 rrf-3(pk1426) 100 0 0 0 792 lst-4^b 67.4 0 32.3^d 0.3 597 lst-4° 98.9 0 0 1.1 627 daf-2(e1370) 100 0 0 0 856 daf-2(e1370); lst-4^b 0 99.6 0 0.4 889 pdk-1(sa680) 95.0 4.4 0.6 0 792 lst-4^b; pdk-1(sa680) 0.2 99.6 0 0.2 1090 pdk-1(mg142) 99.3 0 0 0.7 1069 lst-4^b; pdk-1(mg142) 100 0 0 0 978 age-1(sa315) 99.8 0 0 0.2 1076 age-1(sa315); lst-4^b 99.7 0 0 0.3 981 daf-16(m26) 100 0 0 0 764 daf-16(m26); lst-4^b 99.9 . 0 0 0.1 906 daf-7(e1372) 32.2 67.8 0 0 11 15 daf-7(e1372); lst-4^b 24.0 76.0 0 0 1085 daf-11(m47) 61.6 38.2 0 0.2 1065 lst-4^b; daf-11 (m47) 98.7 0.2 1.1 0 1230 dyn-1(ky51) 100^e 0 0 0 1142 lst-4^b; dyn-1(ky51) . 99.8^f 0 0.2 0 940

1 B. Phenotype of prog eny at 26°C (%) wild-type 99.7 0 0 0.3 865 rrf-3(pk1426) 99.7 0 0 0.3 814 lst-4^b 25.5 1.7 71.9 0.9 619

Ist-4^C 87.4 0.2 12.3 . 0.1 682 daf-2(e1370) 0 100 0 0 778 daf-2(e1370); lst-4^b 0 100 0 0 730 pdk-1(sa680) 3.7 96.3 0 0 767 lst-4^b; pdk-1(sa680) 0 100 0 0 878 pdk-1(mg142) 99.9 0 0 0.1 828 lst-4^b; pdk-1(mg142) 100 0 0 0 677 age-1(sa315) 99.9 0 0 0.1 668 age-1(sa315); lst-4^b 100 0 0 0 719 daf-16(m26) 100 0 0 0 687 daf-16(m26); lst-4^b 99.9 0 0 0.1 850 daf-7(e1372j 0 99.9 0 0.1 717 daf-7(e1372); lst-4^b 0 99.9 0 0.1 808 daf-11 (rr>47) 1.8 97.8 0 0.4 666 lst-4^b; daf-11(m47) 6.0 93.5 0.5 0 850 dyn-1(ky51) 17.4 0 0 85.5 620 lst-4^b; dyn-1(ky51) 0 0 0 100^g 702 ^a Total number of animals scored. ^b All data with lst-4^b were done by RNAi by feeding. ^c rrf-3(pk1426) was used for RNAi by feeding. ^d Similarity to partial dauers. ^e 0.9% of dyn-1(ky51) are phenotypically degenerated at 15⁰C. ^f 0.4% of lst-4^b; dyn-1(kyδ1) are phenotypically degenerated at 15°C.

⁹ Most of the animals were dead at the timepoint of scoring. The experiments provided above and the phenotype analysis are carried out in accordance with Hertweck et al. (2004), Dev. Cell, 6, 577-588. RNAi sequences directed against the C. elegans ortholog (Ist-4) are shown in SEQ ID NOS: 18 and 19, appended below.

Example 9: Co-immunoprecipitation of SNX9 and dynamin /SNX-B8/SNX30 and dynamin

HEK293 cells were transiently transfected with plasmids encoding SNX9 or SNX-B8/SNX3O. Both proteins carried a C-terminal HA-epitope tag. Transfection was carried out as described above. Two days after transfection cell lysates were prepared using the following buffer: 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40 and Sigma protease inhibitor mix. Cell lysates were incubated with anti-HA-tag antibody Ha.11 (Covance; dilution 1:100) and Protein A- Sepharose beads (30 μl). After rotating incubation at 4°C for 2 h, samples were precipitated by centrifugation. The beads were washed 3 times with the above solubilization buffer or the buffer with an increased NaCl concentration (500 mM). Samples were boiled in protein sample buffer containing mercaptoethanol and loaded onto electrophoresis gels. For the detection of dynamin binding antibody dynamin I/II from Cell Signaling was used. The coimmunoprecipitation was also carried out under identical conditions using the dynamin antibody for immunoprecipitation and the HA antibody for detection.

Since we confirmed that SNX9 binds to dynamin (Lundmark and Carlsson, 2003), it is possible (without being bound by theory) that overexpression of the SNX9/18/B8 proteins sequesters dynamin within the cell, thus reducing the amount of dynamin available for general endocytosis — and not only for the endocytosis of those proteins that normally require the SNX (such as the insulin receptor). However, an interaction of SNX-B8/SNX30 with dynamin could not be detected. Accordingly, SNX-B8/SNX30 does not appear to simply sequester dynamin.

Example 10: Screen for interaction partners for SNX9 and SNX-B8/SNX30 using split ubiquitin assay

The split ubiquitin assay (Fetchko (2004) Methods, 32, 349) which is similar to the Yeast- Two-Hybrid assay was used to screen for interaction partners. Screening with SNX9 reveals Nucleoporin, COG5, SEC6 and 14-3-3eta as potential binding partners. Screening with SNX- B8/SNX30 reveals RACKl as a potential binding partner. These results show that both SNXs have different binding partners and, thus, potentially different functions.

Example 11: Experimental set-up for testing the involvement of SNX-B8/SNX30 in insulin signaling in human cell lines:

A plasmid encoding SNX-B8/SNX3O is stably or transiently transfected into HEK293 cells. These cells are serum-starved for 3 hours and then stimulated for a few minutes with insulin or buffer (as a control). Cell lysates are prepared and tested by Western blotting for the amount of phosphorylated (and thus activated) ERK kinase and Akt kinase. Both kinases are activated by the insulin receptor. If increased amounts of phosphorylated kinases are detected upon insulin stimulation, this indicates that expression of the SNX stimulates insulin signaling. Additionally the opposite strategy is to be followed. Upon down-regulation of SNX-B8/SNX30 expression by RNAi, the effects on insulin signaling is to be measured.

Example 12: Functional differences between the known SNX9 and the inventive SNX- B8/SNX30 and functional analysis

The invention provides for a novel subgroup of sorting nexins comprising the members SNX- 9, SNX-18 as well as the herein identified SNX-B8/30. However, even if said members of the novel subgroup share functional and structural features, there are some differences as documented in this experimental part and as listed below.

than SNX-B8/SNX30, but has lower effect on has a stronger effect on APP shedding

APP shedding

Interaction partners in split ubiquitin assay Interaction partners in split ubiquitin assay

(similar to yeast two hybrid assay): (similar to yeast two hybrid assay):

Nucleoporin RACKl

COG5

SEC6 These results show that both SNXs have

14-3-3 eta different binding partners and thus, potentially different functions

Upon transfection does not inhibit TfR Upon transfection does inhibit TfR endocytosis (data from literature) endocytosis

How can the novel SNX9/18/B8 subgroup of sorting nexins be involved in three cellular processes - namely, in insulin signaling, APP shedding and endocytosis?

Without being bound by theory, the following functional explanations are provided.

Insulin signaling and SNX9/18/B8

The experiments provided herein and relating to the SNX-B 8/SNX30 knock-down (via RNAi approaches) in C. elegans document that said SNX is required for insulin signaling. RNAi approach for "knock-down" are discussed and shown in Hertweck et al. (2004), loc. cit. Since insulin signaling is disturbed in diabetes and in Alzheimer's disease, the SNX-B 8/SNX30 has an essential role in the pathogenesis of both diseases. The exact molecular step in insulin signaling is up-stream of the PI3-kinase. The SNX-B8/SNX30 (and its homologues SNX9 and SNXl 8) modulate the intracellular trafficking of the insulin receptor, its interaction with cytoplasmic binding partners, as well as the endocytosis of the insulin receptor. Without being bound by theory it is speculated that the SNX binds directly to the insulin receptor. This is very likely, since the cytoplasmic domain of the insulin receptor comprises a proline-rich region, potentially binding to SH3 domains. Importantly, SNX-B8/SNX30 and its homologues have a SH3 domain.

SNX9/18/B8 and endocvtosis Experiments provided herein document that SNX-B8/SNX30 expression interfered with the endocytosis of APP and the transferrin receptor. However, it is unlikely that SNX-B 8/SNX30 simply sequesters dynamin and thereby inhibits endocytosis. Although it was found SNX9 binds to dynamin (Lundmark and Carlsson, 2003), no interaction with dynamin was detected for SNX-B8/SNX30. There are additional reasons speaking against SNX-B8/SNX30 being part of the general cellular endocytic machinery (such as it is the case for dynamin). The dynamin loss-of-function mutation led to early developmental arrest in C. elegans and thus to a much more severe phenotype than the RNAi of the C. elegans SNX (lst-4). The C. elegans SNX lst-4 knock-down did not show a synergistic effect with the dynamin mutant (in contrast to the insulin signaling mutants), making it unlikely that SNX-B 8/SNX30 is required for exactly the same functions as dynamin. Additionally, it is noteworthy that SNX-B8/SNX30 is only expressed in multicellular organisms but not in yeast, where endocytosis occurs in the absence of SNX.

SNX9/18/B8 and APP shedding

Given the function of SNX-B8/SNX30 in insulin signaling documented herein and in the endocytic process, there may be two mutually not excluding possibilities how the SNX may influence APP shedding. Since SNX-B 8/SNX30 is required for insulin signaling and since insulin signaling stimulates APP shedding, expression of SNX-B8/SNX30 stimulates APP shedding, potentially by stimulating insulin signaling.

Without being bound by theory in another possibility, expression of SNX-B8/SNX30 influence the intracellular trafficking of APP, such as the endocytosis (as documented herein).

As a result, more APP is found at the cell surface and undergoes an increased cleavage by the secretases (which is what the inventors found). Reduced APP endocytosis is known to increase APP shedding (as discussed above), which is consistent with findings provided herein.

Altering the SNX expression level would be expected to alter APP shedding through one of the above mechanisms. In fact, it is known that the SNX expression level can undergo dramatic changes in vivo. For example, vulval precursor cells in C. elegans can have a high expression or a nearly completely suppressed expression level of the SNX-B8/SNX3O (Yoo et al., 2004).

Without being bound to theory, an additional way how SNX-B 8/SNX30 may control the amount of APP shedding is through phosphorylation. This invention also documents for the SNX-B8/SNX30. Since phosphorylation often modifies protein function, SNX-B8/SNX30 may be used as a molecular switch, which - depending on its phosphorylation status - could increase or decrease APP shedding and insulin signal transduction. Thus, SNX-B8/SNX30 may be used to influence the molecular processes underlying disorders or pathological conditions, like Alzheimer's disease and diabetes. Given, that SNX9 and SNXl 8 have similar effects on APP shedding as SNX-B8/SNX30 (Fig. 4C), they may be medically employed as SNX-B8/SNX3O. As documented above, also SNX-9 and SNX-18 are comprised in a novel, functionally are structurally defined subgroup of sorting nexins. Accordingly, the embodiments provided herein for SNX-B8/30 apply, mutatis mutandis, for SNX-9 and SNX- 18. Therefore, the present invention also provides for screening methods for antagonists or agonists of SNX-9 and/or SNX- 18 function and/or expression. The antagonists/agonists provided by the methods disclosed herein may specifically influence the function/expression of each SNX of the herein defined "SNX-9/18/B8"-subgroup but may also be agonists or antagonists for each member.

hi particular, the agonists/enhancers of SNX-B 8 (as well as agonists/enhancers of SNX- 18 and/or SNX-9) are particularly useful in the treatment of neurodegenerative disorders, like Alzheimer's disease.

Furthermore, agonist/enhancers (as well as the proteins themselves or nucleic acid molecules encoding the same) of SNX-B8 and/or SNX-18 may be used in medical intervention of metabolic disorders, like diabetes or obesity. Also the corresponding nucleic acid molecules may be employed, for example in gene therapy approaches. Corresponding medical means and methods are provided in the specification above as well as in the appended claims. .

The invention relates to and/or provides for the following sequences:

SEQ ID NO: 1: SNX-B8/30 (DNA, Homo sapiens)

atggcactgaaaggccgagccctctatgactttcacagtgagaacaaggaggaaatcagcatccagcaggatgaggacctggtcatct ttagcgagacctcactggatggctggctgcagggccagaacagccgtggggagacagggctctttcctgcctcttatgtggagatcgt ccgttctggcatcagcaccaaccatgctgactactccagcagccctgcaggctctcccggagcccaggtgagcttgtacaacagcccc agtgtggccagcccagctaggagtggtgggggcagtggcttcctctcaaaccagggtagctttgaggaggatgatgatgatgactgg gatgactgggacgacggatgcacagtggtggaggagccacgggctggtgggctgggcaccaacgggcaccctcccctcaacctct cctaccctggtgcctaccccagccagcacatggccttccggcccaagccaccactggagcggcaggacagcctggcatctgccaag cgaggcagtgtggtgggccgtaacctcaaccgtttctcatgctttgtgcgttctggagtggaggccttcatcctgggtgatgtgcccatga tggccaagatcgctgagacatactccattgaaatgggccctcgtggcccccagtggaaggccaatccccacccatttgcctgctctgtg gaggaccccacaaaacagaccaaattcaagggcatcaaaagctacatctcctacaagctcacacccacccatgctgcctcacccgtct accggcgctacaaacactttgactggctctataaccgcctgctacacaagttcactgtcatctcggtgccccacctgcctgagaagcag gccactggccgcttcgaggaggacttcatcgaaaagcggaagcggagactcatcctctggatggaccacatgaccagccaccctgtg ctctcccagtacgaaggcttccagcatttcctcagctgcctggatgacaagcagtggaagatgggcaaacgccgggcggagaaggat gagatggtgggtgccagcttcctgctcaccttccagatccccaccgagcaccaggacttgcaggacgtggaagatcgcgtggacactt tcaaggccttcagtaagaagatggacgacagcgtcctgcagctcagcactgtggcatcagagctggtgcgtaaacatgtggggggctt ccgcaaggaattccagaagctgggcagtgccttccaggccatcagtcattccttccagatggacccccccttttgctctgaggccctcaa cagtgccatttctcacacgggccgtacctatgaagccatcggggagatgtttgctgagcagcccaagaatgacctcttccagatgctgg acacactgtctctctaccagggcctgctctccaacttccctgacatcatccatctacaaaaaggcgccttcgccaaggtgaaggagagc caacgcatgagtgacgagggccgcatggtgcaggacgaggcagacggcattcgcaggcgctgccgcgtggtgggtttcgccctgc aggccgagatgaaccacttccaccagcgccgtgagctcgacttcaagcacatgatgcagaactacttgcgccagcagatcctcttctac cagcgggtgggccagcagctggagaagaccctgcgcatgtatgacaacctc

SEQ ID NO: 2 SNX-B8/30 (Protein; Homo sapiens)

MALKGRALYDFHSENKEEISIQQDEDLVIFSETSLDGWLQGQNSRGETGLFPASYVEI

VRSGISTNHADYSSSPAGSPGAQVSLYNSPSVASPARSGGGSGFLSNQGSFEEDDDDD

WDDWDDGCTWEEPRAGGLGTNGHPPLNLSYPGAYPSQHMAFRPKPPLERQDSLAS

AKRGSVVGRNLNRFSCFVRSGVEAFILGDVPMMAKIAETYSIEMGPRGPQWKANPHP

FACSVEDPTKQTKFKGIKSYISYKLTPTHAASPVYRRYKHFDWLYNRLLHKFTVISVP

HLPEKQATGRFEEDFIEKRKRRLILWMDHMTSHPVLSQYEGFQHFLSCLDDKQWKM

GKRRAEKDEMVGASFLLTFQIPTEHQDLQDVEDRVDTFKAFSKKMDDSVLQLSTVA

SELVRKHVGGFRKEFQKLGSAFQAISHSFQMDPPFCSEALNSAISHTGRTYEAIGEMF

AEQPKNDLFQMLDTLSLYQGLLSNFPDIIHLQKGAFAKVKESQRMSDEGRMVQDEA

DGIRRRCRWGFALQAEMNHFHQRRELDFKHMMQNYLRQQILFYQRVGQQLEKTL

RMYDNL

SEQ ID NO: 3: SNX9 (DNA; Homo sapiens) atggccaccaaggctcgggttatgtatgattttgctgctgaacctggaaataatgaactgacggttaatgaaggagaaatcatcacaatc acaaatccggatgtaggtggaggatggctggaaggaagaaacatcaaaggagaacgagggctggttcccacagactacgttgaaatt ttacccagtgatggaaaagatcaattttcttgtggaaattcagtggctgaccaagccttccttgattctctctcagccagcacagctcaggc cagttcgtcggctgccagcaacaatcaccaggttggcagtggcaatgacccctggtcagcctggagtgcctccaaatctgggaactgg gaaagctcagaaggctggggggcccagccagagggggctggagcccaaagaaacacaaacactcccaacaactgggacactgcc ttcggccacccccaggcctaccaaggaccagcaactggtgatgatgatgactgggatgaagactgggatgggcccaaatcctcttcct actttaaggattcagagtcagctgatgcaggcggcgctcagcgaggaaacagtcgtgctagttcctcatccatgaaaattccccttaaca aatttcctggatttgcgaaacctggcacggaacagtatttgttggccaaacaactagcaaaacccaaagagaaaattcccatcattgttgg agattatggcccaatgtgggtttatcctacctctacttttgactgtgtggtagcagatcccaggaaaggctccaaaatgtatggtctaaaga gctacatcgaatatcagctaacacctactaacactaatcgatctgtaaaccacaggtataagcactttgactggttatatgagcgtctcctg gttaagtttgggtcagccattccaatcccttctcttccagacaaacaagtcacaggccgctttgaagaggaatttatcaaaatgcgcatgg agagacttcaggcctggatgaccaggatgtgtcgccatccagtaatctcagaaagtgaagttttccagcagttcctaaatttccgagatg. agaaggaatggaaaactggaaagaggaaggccgagagagatgagctggcgggagtcatgatattttccaccatggaaccagaggc acctgacttggacttagtagaaatagagcagaagtgcgaggctgtggggaagttcaccaaggccatggatgacggcgtgaaggagct gctgacggtggggcaggagcactggaagcgctgcacgggcccattacccaaggaatatcagaagataggaaaggccttgcagagtt tggccacagtgttcagttccagtggctatcaaggtgaaacagatctcaatgatgcaataacagaagcaggaaagacttatgaagaaatt gccagtctcgtggcagaacagccaaagaaagatctccatttcctgatggaatgtaatcacgagtataaaggttttcttggctgcttccctg acatcattggcactcacaagggagcaatagaaaaagtgaaagaaagtgacaaactagttgcaacaagtaaaatcaccctacaagacaa acagaacatggtgaagagagtcagcatcatgtcttacgcgttgcaagctgagatgaatcactttcacagtaaccggatctatgattacaa cagtgtcatccgcctgtacctggagcagcaagtgcaattttacgaaacgattgcagaaaagctgaggcaggccctcagccgctttcca gtgatg

SEQ ID NO: 4: SNX9 (Protein; Homo sapiens]

MATKARVMYDFAAEPGNNELTVNEGEIITITNPDVGGGWLEGRNIKGERGLVPTDYV

EILPSDGKDQFSCGNSVADQAFLDSLSASTAQASSSAASNNHQVGSGNDPWSAWSAS

KSGNWESSEGWGAQPEGAGAQRNTNTPNNWDTAFGHPQAYQGPATGDDDDWDED

WDGPKSSSYFKDSESADAGGAQRGNSRASSSSMKIPLNKFPGFAKPGTEQYLLAKQL

AKPKEKIPHVGDYGPMWVYPTSTFDCWADPRKGSKMYGLKSYIEYQLTPTNTNRS

VNHRYKHFDWLYERLLVKFGSAIPIPSLPDKQVTGRFEEEFIKMRMERLQAWMTRM

CRHPVISESEVFQQFLNFRDEKEWKTGKRKAERDELAGVMIFSTMEPEAPDLDLVEIE

QKCEAVGKFTKAMDDGVKELLTVGQEHWKRCTGPLPKEYQKIGKALQSLATVFSSS

GYQGETDLND AITEAGKTYEEIASLVAEQPKKDLHFLMECNHEYKGFLGCFPDIIGTH KGAIEKVKESDKLVATSKITLQDKQNMVKRVSMSYALQ AEMNHFHSNRIYDYNSVI RLYLEQQVQFYETIAEKLRQALSRFPVM

SEQ ID NO: 5: SNX18 (DNA; Homo sapiens)

atggcgctgcgcgcccgggcgctgtacgacttcaggtcggagaacccaggagagatctcgctgcgagagcacgaggtgctgagcc tgtgcagcgagcaggacatcgagggctggctcgagggggtcaacagccgcggcgaccgcggcctcttcccggcctcctatgtgca ggtgatccgcgcccccgagcctggcccggcgggagacggcggcccgggcgccccggcccgctacgccaatgtgccccccgggg gcttcgagcccctgcccgtcgcgccccccgcctccttcaagccgccgcctgacgccttccaggcgctgctgcagccacagcaggcg ccgcctccgagcaccttccagccgcccggcgcgggcttcccgtacggcgggggcgccctgcagccgtcgcctcagcagctctacg gcggctaccaggccagccaaggcagcgatgatgactgggacgacgagtgggacgacagctccacggtggcggacgagccgggc gctctgggcagcggagcatacccggacctcgacggctcgtcttcggcgggtgtgggcgcagccggccgctaccgcctgtccacgc gctccgacctgtccctgggctcccgcggcggctcggtccccccgcagcaccacccgtcggggcccaagagctcggccaccgtgag ccgcaacctcaatcgcttctccaccttcgtcaagtccggcggggaggccttcgtgctgggggaggcgtcaggcttcgtgaaggacgg ggacaagctgtgcgtggtgctggggccctatggccccgagtggcaggagaacccctacccgttccagtgcaccatcgacgacccca ccaagcagaccaagttcaagggcatgaagagctacatctcctacaagctggtgcccacgcacacgcaggtgccggtgcatcggcgc tacaagcacttcgactggctgtacgcgcgcctggcggagaagttcccggtcatctccgtgccccacctgcccgagaagcaggccacc ggccgcttcgaggaggacttcatctctaagcgcaggaagggcctgatctggtggatgaaccacatggccagccacccagtgctggcg cagtgcgacgtcttccagcacttcctgacgtgccccagcagcaccgacgagaaagcctggaagcagggcaagaggaaggccgaga aggacgagatggtgggcgccaacttcttcctgacccttagcacgccccccgccgctgcccttgacctgcaggaggtggagagcaag atagacggcttcaagtgcttcaccaagaagatggacgacagcgcgctgcagctcaaccacacggccaacgagttcgcgcgcaagca ggtgaccggcttcaaaaaggagtatcagaaggtgggccagtccttccgcggcctcagccaggcctttgagctggaccagcaggcctt ctcggtgggcctgaaccaggctatcgccttcaccggagatgcctatgacgccattggcgagctcttcgcggagcagcccaggcagga cctggatGccgtcatggacctattagcgctgtatcaggggcatctggctaacttcccggacatcatccacgttcagaaaggtaaagcctg gcccttagagcaggtgatatggagtgtattgtgcaggctgaaaggggcgactttgacagcagtaccactgtgggtttcagactcatattc tacaggtgaggaagcgagcagagacgtggacgcctgggtcttttccctagagtgtaagttggattgctcgacaggcagcttcctcctcg agtatcttgcattagggaatgagtactctttctcgaaggttcaaagagtacctttgatgacagtgctatcattttag

SEQ ID NO: 6: SNX18 (Protein; Homo Sapiens)

MALRARALYDFRSENPGEISLREHEVLSLCSEQDIEGWLEGVNSRGDRGLFPASYVQ VIRAPEPGPAGDGGPGAPARYANVPPGGFEPLPVAPPASFKPPPDAFQALLQPQQAPP PSTFQPPGAGFPYGGGALQPSPQQLYGGYQASQGSDDDWDDEWDDSSTVADEPGAL GSGAYPDLDGSSSAGVGAAGRYRLSTRSDLSLGSRGGSVPPQHHPSGPKSSATVSRN

LNRFSTFVKSGGEAFVLGEASGFVKDGDKLCWLGPYGPEWQENP YPFQCTIDDPTK

QTKFKGMKSYISYKLVPTHTQVPVHRRYKHFDWLYARLAEKFPVISVPHLPEKQATG

RFEEDFISKRRKGLIWWMNHMASHPVLAQCDVFQHFLTCPSSTDEKAWKQGKRKAE

KDEMVGANFFLTLSTPPAAALDLQEVESKTDGFKCFTKKMDDSALQLNHTANEFARK

QVTGFKKEYQKVGQSFRGLSQAFELDQQAFSVGLNQAIAFTGDAYDAIGELFAEQPR

QDLDPVMDLLALYQGHLANFPDIIHVQKGKAWPLEQVIWSVLCRLKGATLTAVPLW

VSDSYSTGEEASRDVDAWVFSLECKLDCSTGSFLLEYLALGNEYSFSKVQRVPLMTV

LSF

SEQ ID NO: 7: target sequence RNAi-I directed against human SNX-B8/30 (nucleotide; Homo sapiens)

ggccgagccctctatgact

The corresponding strand and antisense sequences to be used are as follows: ggccgagcccucuaugacutt (SEQ ID NO: 28) and agucauagagggcucggcctt (SEQ ID NO: 29) The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 8: target sequence RNAi-2 directed against human SNX-B8/30 (nucleotide; Homo sapiens)

gcacatgatgcagaactac

The corresponding strand and antisense sequences to be used are as follows: gcacaugaugcagaacuactt (SEQ ID NO: 30) and guaguucugcaucaugugctt (SEQ ID NO: 31) The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector. SEQ ID NO: 9: target sequence RNAi-3 directed against human SNX-B8/30 (nucleotide; Homo sapiens)

cctcaaccgtttctcatgc

The corresponding strand and antisense sequences to be used are as follows: ccucaaccguuucucaugctt (SEQ E) NO: 32) and gcaugagaaacgguugaggtt (SEQ ID NO: 33) The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 10: Epitope of SNX-B8/30 (protein; Homo sapiens)

ISTNHADYSSSPAGSP

SEQ ID NO: 11; Epitope of SNX-B8/30 (protein; Homo sapiens)

FRPKPPLERQDSLAS

SEQ ID NO: 12: SNX-B8/30 (nucleic acid/mouse)

ATGGCACTGAAAGGCCGAGCCCTCTATGATTTCCACAGTGAGAACAAGGAGGAA

ATCAGTATCCAGCAGGACGAAGAGCTGGTTATCTTCAGTGAGACCTCACTGGACG

GCTGGCTGCAGGGACAGAACAGCCGTGGGGAGACAGGGCTCTTCCCTGCTTCTTA

CGTCGAGATTGTCCGTCCTGGCATCAGCACCAACCATGTGGACTATTCCAACAGC

CCTGCAGGCTCCCTGGGCACCCAGGGGAGTTTGTATAGCAGCCCGAGTATGGCCA

GTCCAGCCAGGAGTGGTGGGGGCAGCGGCTTCCTCTCAAACCCAGGAAGCTTCG

AGGATGATGATGATGATGATTGGGATGACTGGGATGATGGGTGCACAGTGGTAG

AAGAGCCACTAGCTGGCGGCCTAGGTACCAATGGGCACCCTCCACTCAACCTCTC

CTACCCGGGTGCCTACCCCAACCAGCATATGGCTTTCCGACCCAAGGCACCCCTG

GAAAGGCAGGACAGCCTGGCGTCTGCCAAGCGGGGCAGTGTGGTAGGACGGAAC

CTCAATCGTTTCTCGTGCTTCGTACGCTCTGGAGTGGAGGCCTTCATCCTTGGTGA

TGTGCCCATGATGGCCAAGATTGCTGAGACTTACTCCATTGAGATGGGCCCACGT GGCCCTCAGTGGAAGGCCAACCCCCACCCATTTGCCTGCTCCATAGAGGACCCCA

CCAAACAGACCAAGTTCAAAGGCATTAAAAGTTACATCTCTTACAAGCTTACACC

CACCCACGCAGGCTCGCCTGTTTACCGGCGCTACAAACACTTTGATTGGCTGTAC

AACCGCCTTCTACACAAGTTCACAGTGATCTCAGTGCCCCACCTGCCTGAGAAGC

AGGCCACAGGCCGCTTCGAAGAGGACTTCATCGAGAAGCGCAAGCGAAGGCTCA

TCCTCTGGATGGACCACATGACCAGCCACCCTGTGCTCTCCCAGTATGAGGGCTT

CCAGCACTTCCTCAGCTGCCTGGATGACAAGCAGTGGAAGATGGGTAAGCGCCG

GGCAGAGAAGGATGAGATGGTGGGCGCCAGTTTTCTGCTCACTTTCCAGATCCCC

ACAGAGCACCAGGATCTGCAGGATGTAGAGGACCGCGTGGATACATTCAAGGCT

TTCAGTAAGAAGATGGATGACAGCGTCCTACAGCTTAGCAACGTGGCGGCGGAG

CTGGTGCGGAAGCATGTGGGGGGCTTCCGCAAGGAATTCCAGAAGCTAGGAAGT

GCCTTCCAGGCCATTAGCCATGCCTTCCAGATGGACCCTCCCTTTAGGTCTGATGC

TCTCAACAATGCCATTTCTCACACTGGCCGGACCTATGAAACCGTTGGCGAAATG

TTTGCGGAACAGCCCAAGCACGACCTCTTCCAAATGCTTGACACACTGTCTCTCT

ACCAGGGCCTACTCTCCAACTTCCCTGACATTATCCACCTGCAGAAAGGTGCCTTT

GCCAAGGTGAAGGAAAGCCAGCGCATGAGTGATGAGGGCCGAATGGCTCAGGAA

GAGGCAGATGGCATTCGCAGGCGCTGCCGTGTGGTAGGCTTCGCCCTGCAGGCCG

AGATGAACCATTTCCACCAGCGCCGTGAGCTCGATTTTAAGCATATGATGCAAAG

.CTACCTGCGCCAGCAGATTCTTTTCTACCAGCGGGTAGGCCAGCAGCTGGAGAAG

ACTCTCCACATGTATGACCACCTC

SEQ ID NO: 13: SNX-B8/30 (amino acid; mouse)

MALKGRALYDFHSENKEEISIQQDEELVIFSETSLDGWLQGQNSRGETGLFPASYVEI

VRPGISTNHVDYSNSPAGSLGTQGSLYSSPSMASPARSGGGSGFLSNPGSFEDDDDDD

WDDWDDGCTWEEPLAGGLGTNGHPPLNLSYPGAYPNQHMAFRPKAPLERQDSLA

SAKRGSVVGRNLNRFSCFVRSGVEAFILGDVPMMAKIAETYSIEMGPRGPQWKANPH

PFACSIEDPTKQTKFKGIKSYISYKLTPTHAGSPVYRRYKHFDWLYNRLLHKFTVISVP

HLPEKQATGRFEEDFIEKRKRRLILWMDHMTSHPVLSQYEGFQHFLSCLDDKQWKM

GKRRAEKDEMVGASFLLTFQIPTEHQDLQDVEDRVDTFKAFSKKMDDSVLQLSNVA

AELVRKHVGGFRKEFQKLGSAFQAISHAFQMDPPFRSDALNNAISHTGRTYETVGEM

FAEQPKHDLFQMLDTLSLYQGLLSNFPDIIHLQKGAFAKVKESQRMSDEGRMAQEEA DGIRRRCRWGFALQAEMNHFHQRRELDFKHMMQSYLRQQILFYQRVGQQLEKTLH MYDHL

SEQ ID NO: 14: Rat orthologue of SNX-B8/30 (nucleic acid; rat)

atggcactgaaaggccgagccctctatgatttccacagcgagaacaaggaggaaatcagcatccagcaggacgaagacttggttatct tcagcgagacctcactggacggctggctgcagggacagaacagccgtggggagacagggctcttccctgcttcttacgttgagattgt tcgtcctggcatcagcaccaaccatggggactattccaacagccctgcaggctccctgggcacccaggtgagtttgtatagcagcccta gtacggccagtccagccaggagtagtgggggcagtggcttcctctcaaaccaaggaagctttgaggatgatgatgacgatgactggg atgactgggatgatggatgcacagtggtagaagagccactggctggtggcctaggtaccaatggacaccctccacttaacctctcctac ccgggtgcctaccccaaccagcatatggctttcagacccaaggcacccctggaaaggcaggacagtctagcgtctgccaagcgggg cagtgttgtaggacggaacctcaatcgtttctcatgctttgtacgctctggagtggaggccttcatccttggtgatgtgcccatgatggcca agattgctgagacctactccattgagatgggcccacgtgggcctcagtggaaggccaatccccacccatttgcctgctccatagagga ccccaccaagcagaccaagttcaaaggcattaaaagttacatctcctacaagcttacgcccacccacgctggctcacctgtttaccgcc gttacaaacactttgactggctatacaaccgccttctgcacaagttcacagtaatttcagtgccccacctgcctgagaagcaggccacag gccgctttgaggaggacttcattgagaaacgta^'agcgaaggctcatcctctggatggaccacatgaccagccaccctgtgctctccca gtatgagggcttccagcacttcctcagctgcctggatgacaaacagtggaagatggggaaacgccgagcagagaaggatgagatgg tgggtgccagtttcctgctcaccttccagatccccacagaacaccaggatctgcaggatgtagaggatcgggtggacgcattcaaggct ttcagtaagaagatggatgacagcgtcctacagcttagcactgtagcagcagaattggtgcggaaacatgtaggaggcttccgcaagg aattccagaagctcggaagtgccttccaggccattagccatgccttccagatggaccctccctttaggtctgaggctctcaacaatgcca tttctcacactggccggacctatgaaaccgttggagaaatgtttgctggacagcccaagcatgacctcttccaaatgctcgacacactgt ctctctaccagggcctactctccaacttccctgacattatccacctgcagaaaggtgQctttgccaaggtgaaggagagccagcgcatg agtgatgagggccgcatggctcaggaagaggcagacggtattcgcaggcgctgccgtgtggtaggctttgccctgcaggccgagat gaaccatttccaccagcgccgtgagctcgacttcaagcatatgatgcaaagctacctgcgccagcagatccttttctaccagcgggtgg gccagcagctggagaagacgctccgcatgtatgatcacctctga

SEQ ID NO: 15: Rat orthologue of SNX-B8/30 (amino acid; rat)

MALKGRALYDFHSENKEEISIQQDEDLVIFSETSLDGWLQGQNSRGETGLFPASYVEI

VRPGISTNHGDYSNSPAGSLGTQVSLYSSPSTASPARSSGGSGFLSNQGSFEDDDDDD

WDDWDDGCTWEEPLAGGLGTNGHPPLNLSYPGAYPNQHMAFRPKAPLERQDSLA

SAKRGSVVGRNLNRFSCFVRSGVEAFILGDVPMMAKIAETYSIEMGPRGPQWKANPH

PFACSIEDPTKQTKFKGIKSYISYKLTPTHAGSPVYRRYKHFDWLYNRLLHKFTVISVP HLPEKQATGRFEEDFIEKRKRRLILWMDHMTSHPVLSQYEGFQHFLSCLDDKQWKM

GKRRAEKDEMVGASFLLTFQIPTEHQDLQDVEDRVDAFKAFSKKMDDSVLQLSTVA

AELVRKHVGGFRKEFQKLGSAFQAISHAFQMDPPFRSEALNNAISHTGRTYETVGEM

FAGQPKHDLFQMLDTLSLYQGLLSNFPDIIHLQKGAFAKVKESQRMSDEGRMAQEEA

DGIRRRCRWGFALQAEMNHFHQRRELDFKHMMQSYLRQQILFYQRVGQQLEKTLR

MYDHL

SEQ ID NO: 16: C. elegans orthologue of SNX-B8/30 (nucleic acid)

atggctcaggtgaaagccgagtatgattttcaaagtcaaccaaacacaggcgaactgtcaatctcggctggcgaagttttgacggttatt cgagagaacatcgacggaggatggatagaaggtcgaaacgtgcgtggatccgtcggactctttcccgaatcctacgtaactccctatc aagcttctcgtccgccaccggtcctaccaccaccactcccaccaacttcaagcggtccaccagctgcctcatcgcgtccttttgatgattg gggaggcgcttcagaagttgctgctccaccatcctacggagcccaacatcatcatcagccaacgccatctgtaccggaagtcactaga tcttcatatccatcacagaatgatgattttgacgatgaatggacggatgaggatgatgagcaggaaccaactcggcccaacgttcaatcc tcgatcggctcgaactcccgtcgtgacctcagccgtagccactcggaacacggtggacctgatcgaggatccaataaggtcaacaag aacatcaatcgattctcgaattttgtaaagtccggagtcgaagcgtacgtgattggtgaatcaaagaccacctcacagattagtgaacga catgaagttgtgatgaacaatggaattattcaatggaaaccgattcaacagtactacacgtgtatagtggataaaccgaagaaagagtca aagctgaaagggctgaagagcttcatcgcatattcgattacgtcgagtttgactaatattcaggtatcccgtcgctacaagcatttcgattg gctccacgagcagctatcggctaaatatgtgctcattccgattccaccgttgccagagaaacaggtcgccggaagatacgaggaggat cttattgatcatcggaagcacattcttcagttatgggtcaacaagatttgtaggcatccagtgttgagtcagtcggaagtttggctccacttt atcagttgcaccgacgaaaaagattggaagaatgggaagcggcgagctgaaaaggatgaatacattggtggagctttcctgaactgta tcactgttccacatcaaccattggatccgaataatgtcgatatgcaagttgaacgattccaacggagcgtcaaaacatcggaagaagcg atgcgtgtgatgcaggagagaatgaacatgttccagaaggtcttcgctggaccggtaaaacaaaattggcaaaagatgggaagcgcg ttcaagacacttcagcaatcttttgagattgatgagaccgttgccagtcgccgcctgacagaagctctcgcctacactgcatcagaatatc atgaaattgggcaagtattcgatgcgcacacgaagaatgacatggaaccagtgcttgagaacctttattcgtataagggaacagtgcaa aatgtgccggatattattcaagtgcacaagcaagctgttcaaaagttccgtgacagcgaaggacgtctctcgtcggccgaggctgaaaa gatgaagcaaagaatcgatgcgatgagctacactgtaattgctgaagttcaacatcaaacagcagagaaagtggaggatatgaaatcg acaatgggcacatatttgaagaaacaagcaatgttttatcaggaagtggcgaccaagctgacttcattagccgctagatatgattga

SEQ ID NO: 17: C. elegans orthologue of SNX-B8/30 (amino acid)

MAQVKAEYDFQSQPNTGELSISAGEVLTVIRENIDGGWIEGRNVRGSVGLFPESYVTP YQASRPPPVLPPPLPPTSSGPPAASSRPFDDWGGASEVAAPPSYGAQHHHQPTPSVPE VTRSSYPSQNDDFDDEWTDEDDEQEPTRPNVQSSIGSNSRRDLSRSHSEHGGPDRGSN KVNI^INRFSNFVKSGVEAYVIGESKTTSQISERHEVVMNNGIIQWKPIQQYYTCΓVD KPKKESKLKGLKSFIAYSITSSLTNIQVSRRYKHFDWLHEQLSAKYVLFF IPPLPEKQV

AGRYEEDLIDHRKHILQLWVNKICRHPVLSQSEVWLHFISCTDEKD WKNGKRRAEK

DEYIGGAFLNCITWHQPLDPNNVDMQVERFQRSVKTSEEAMRVMQERMNMFQKVF AGPVKQNWQKMGSAFKTLQQSFEIDETVASRRLTEALAYTASEYHEIGQVFDAHTK NDMEPVLENLYSYKGTVQNVPDIIQVHKQAVQKFRDSEGRLSSAEAEKMKQRIDAM SYTVIAEVQHQTAEKVEDMKSTMGTYLKKQAMFYQEVATKLTSLAARYD

SEQ ID NO: 18: C. elegans SNX-B8/30 (Ist-4) RNAi sequence (1^st strand)

tcgctaccaaacttaatgaggtcgatcctcaaataggacaccgctggagactccacaaaagctcacaacagatttgttttgttattctacag taatcctacagtactcctaccgtaccatatcacaaaatagagctctagtagttggttacgggtctcaccacgctgacaacaagtttctgtta ctcgtgtcgatttacagttgtcagcgtggcgagacccattttcttgtgacgtatacgtaataactgtagctcccagagtgctccattccggct tgatctacgttgatctacaaaaaacgcgcgaattgagacgtagagttatccactgatttcgcacagtaccatttcgctttgatcttcgaaaat gcgggagaagagacgcagacatctcatctgatttcgcatggttaagagcgtgctgaagtcacaatttttcttgaaaagtatccccgcatttt tcgtagatcaaaccgcaatgagacagcctgacaccacgtgtgcacggttaaaaacgtgctgacgtcacattttgtcgggcaaaaaatcc cgcattttttgtagatcaatccgtgatgggaccacatgacacacagatcccattte^ aattccaggtcctaccaccaccactcccaccaacttcaagcggtccaccagctgcctcatcgcgtccttttgatgattggggaggcgctt cagaagttgctgctccaccatcctacggagcccaacatcatcatcagccaacgccatctgtaccggaagtcactagatcttcatatccat cacagaatgatgattttgacgatgaatggacggatgaggatgatgagcaggtatttttaa^ ccgtcctatttataccctctaactcctaaatctatatatcctaaaagaccaaacgaaaagtcca

SEQ ID NO: 19: C. elegans SNX-B8/30 (Ist-4) RNAi sequence (2^nd strand)

TGGACTTTTCGTTTGGTCTTTTAggatatatagatttaggagttagagggtataaataggacggacagaaactgga caatcaaaaaattaaaataaaattttaaaaatacctgctcatcatcctcatccgtccattcatcgtcaaaatcatcattctgtgatggatatga agatctagtgacttccggtacagatggcgttggctgatgatgatgttgggctccgtaggatggtggagcagcaacttctgaagcgcctc cccaatcatcaaaaggacgcgatgaggcagctggtggaccgcttgaagttggtgggagtggtggtggtaggacctggaatttttgagt atttatgaaattgtttaaaaagttagaaaattgaaatgggatctgtgtgtcatgtggtcccatcacggattgatctacaaaaaatgcgggattt tttgcccgacaaaatgtgacgtcagcacgtttttaaccgtgcacacgtggtgtcaggctgtctcattgcggtttgatctacgaaaaatgcg gggatacttttcaagaaaaattgtgacttcagcacgctcttaaccatgcgaaatcagatgagatgtctgcgtctcttctcccgcattttcgaa gatcaaagcgaaatggtactgtgcgaaatcagtggataactctacgtctcaattcgcgcgttttttgtagatcaacgtagatcaagccgga atggagcactctgggagctacagttattacgtatacgtcacaagaaaatgggtctcgccacgctgacaactgtaaatcgacacgagtaa cagaaacttgttgtcagcgtggtgagacccgtaaccaactactagagctctattttgtgatatggtacggtaggagtactgtaggattactg tagaataacaaaacaaatctgttgtgagcttttgtggagtctccagcggtgtcctatttgaggatcGACCTCATT AAGTTTG GTAGCGA

SEQ ID NO: 20: target sequence RNAi-4 directed against human SNX-9 (nucleotide; Homo sapiens)

AAGAGAGUCAGCAUCAUGUCU

The corresponding strand and antisense sequences to be used are as follows: GAGAGUCAGCAUCAUGUCUTT (SEQ ID NO: 34) and

AGACAUGAUGCUGACUCUCTT (SEQ ID NO: 35)

The "tt" sequence at the 3 ' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector. .

SEQ ID NO: 21: target sequence RNAi-5 directed against human SNX-9 (nucleotide; Homo sapiens)

AACCUACUAACACUAAUCGAU

The corresponding strand and antisense sequences to be used are as follows: CCUACUAACACUAAUCGAUTT (SEQ ID NO: 36) and

AUCGAUUAGUGUUAGUAGGTT (SEQ ID NO: 37)

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 22: target sequence RNAi-6 directed against human SNX-9 (nucleotide; Homo sapiens)

AACAGTCGTGCTAGTTCCTCA The corresponding strand and antisense sequences to be used are as follows: CAGUCGUGCUAGUUCCUCATT (SEQ ID NO: 38) and

UGAGGAACUAGCACGACUGTT (SEQ ID NO: 39)

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 23: target sequence RNAi-7 directed against human SNX-9 (nucleotide; Homo sapiens)

t tea gtg get gac caa gcc

The corresponding strand and antisense sequences to be used are as follows: uucaguggcugaccaagcctt (SEQ ID NO: 40) and ggcuuggucagccacugaatt (SEQ ID NO: 41) The "tt" sequence at the 3 ' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 24: target sequence RNAi-8 directed against human SNX-9 (nucleotide; Homo sapiens)

a cct ggc acg gaa cag tat

The corresponding strand and antisense sequences to be used are as follows: accuggcacggaacaguautt (SEQ ID NO: 42) and auacuguuccgugccaggutt (SEQ ID NO: 43) The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides (dTdT), whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 25: target sequence RNAi-9 directed against human SNX-18 (nucleotide; Homo sapiens) CTGTGGGTTTCAGACTCAT

The corresponding strand, and antisense sequences to be used are as follows: CUGUGGGUUUCAGACUCAUTT (SEQ ID NO: 44) and

AUGAGUCUGAAACCCACAGTT (SEQ ID NO: 45)

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 26: target sequence RNAi-IO directed against human SNX-18 (nucleotide; Homo sapiens)

GCAGGTGATATGGAGTGTA

The corresponding strand and antisense sequences to be used are as follows: GCAGGUGAUAUGGAGUGUATT (SEQ ID NO: 46) and

UACACUCCAUAUCACCUGCTT (SEQ ID NO: 47)

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 27: target sequence RNAi-Il directed against human SNX-18 (nucleotide; Homo sapiens)

GGACCTATTAGCGCTGTAT

The corresponding strand and antisense sequences to be used are as follows: GGACCUAUUAGCGCUGUAUTT (SEQ ID NO: 48) and

AUACAGCGCUAAUAGGUCCTT (SEQ ID NO: 49)

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

SEQ ID NO: 28: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens) ggccgagcccucuaugacutt

SEQ ID NO: 29: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

agucauagagggcucggcctt

SEQ ID NO: 30: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

gcacaugaugcagaacuactt

SEQ ID NO: 31: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

guaguucugcaucaugugctt

SEQ ID NO: 32: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

ccucaaccguuucucaugctt

SEQ ID NO: 33: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

gcaugagaaacgguugaggtt

SEQ ID NO: 34: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

GAGAGUCAGCAUCAUGUCUTT

SEQ ID NO: 35: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

AGACAUGAUGCUGACUCUCTT

SEQ ID NO: 36: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens) CCUACUAACACUAAUCGAUTT

SEQ ID NO: 37: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

AUCGAUUAGUGUUAGUAGGTT

SEQ ID NO: 38: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

CAGUCGUGCUAGUUCCUCATT

SEQ ID NO: 39: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

UGAGGAACUAGCACGACUGTT

SEQ ID NO: 40: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

uucaguggcugaccaagcctt

SEQ ID NO: 41: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

ggcuuggucagccacugaatt

SEQ ID NO: 42: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

accuggcacggaacaguautt

SEQ ID NO: 43: inhibiting RNA for SNX-9 (nucleotide; Homo sapiens)

auacuguuccgugccaggutt

SEQ ID NO: 44: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

CUGUGGGUUUCAGACUCAUTT SEQ ID NO: 45: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

AUGAGUCUGAAACCCACAGTT

SEQ ID NO: 46: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

GCAGGUGAUAUGGAGUGUATT

SEQ ID NO: 47: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

UACACUCCAUAUCACCUGCTT

SEQ ID NO: 48: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

GGACCUAUUAGCGCUGUAUTT

SEQ ID NO: 49: inhibiting RNA for SNX-18 (nucleotide; Homo sapiens)

AUACAGCGCUAAUAGGUCCTT

SEQ ID NO: 50: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

CCUCAACCGUUUCUCAUGCTT

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides.

SEQ ID NO: 51: inhibiting RNA for SNX-B8/30 (nucleotide; Homo sapiens)

GCAUGAGAAACGGUUGAGGTT

The "tt" sequence at the 3' end of the nucleotides are desoxyribonucleotides, whereas the preceding nucleotides are ribonucleotides. In general, the "tt" sequence at the 3' end of the nucleotides in context of inhibiting RNA provided herein may be employed in form of desoxyribonucleotides (dTdT), whereas the preceding nucleotides are ribonucleotides. Additionally, the indicated target sequence (as desoxyribonucleotides) may directly be cloned into siRNA vectors such as the pSuper vector.

Additional references cited herein

Allinson, 2003, J Neurosci Res 74, 342-352.

Arvanitakis, 2004, Arch Neurol 61, 661-666.

Beall, 2002, Int J Parasitol 32, 399-404.

Blobel, 2002, Inflamm Res 51, 83-84.

Burgering, 2002, Trends Biochem Sci 27, 352-360.

Carlton, 2005, Traffic 6, 75-82.

Cassada, 1975, Dev Biol 46, 326-342.

Chyung, 2003, Inhibition of receptor mediated endocytosis demonstrates generation of amyloid beta -protein at the cell surface. J Biol Chem.

Clemens, 2000, Proc Natl Acad Sci U S A 97, 6499-6503.

Gasparini, 2001, J Neurosci 21, 2561-2570.

Gasparini, 2002, Trends Pharmacol Sci 23, 288-293.

Gomme, 2005, Drug Discovery Today 10, 267-273.

Habermann, 2004, EMBO Rep 5, 250-255.

Howard, 1999, J Biol Chem 274, 31693-31699.

Kaether, 2002, J Cell Biol 158, 551-561.

Kamath, 2001, Genome Biol 2, RESEARCH0002.

Leprince, 2003, J Cell Sci 116, 1937-1948.

Lichtenthaler, 2003, J Biol Chem 278, 48713-48719.

Lichtenthaler, 2004, J Clin Invest 113, 1384-1387.

Lin, 2002, J Biol Chem 277, 10134-10138.

Lundmark, 2002, Biochem J 362, 597-607.

Lundmark, 2003, J Biol Chem 278, 46772-46781.

Lundmark, 2004, J Biol Chem 279, 42694-42702.

Macaulay, 2003, Biochem J Pt.

Musi, 2002, Curr Drug Targets Immune Endocr Metabol Disord 2, 119-127.

Selkoe, 2003, Annu Rev Pharmacol Toxicol 43, 545-584.

Solano, 2000, Faseb J 14, 1015-1022.

Worby, 2002, Nat Rev MoI Cell Biol 3, 919-931.

Worby, 2002, J Biol Chem 277, 9422-9428.

Yoo, 2004, Science 303, 663-666.

Zhao, 2004, J Neurosci 24, 11120-11126.

Claims

Claims

1. A polynucleotide selected from the group consisting of

(a) a polynucleotide having a nucleotide sequence encoding the polypeptide having the deduced amino acid sequence as shown in SEQ ID NO: 2;

(b) a polynucleotide having the coding sequence as shown in SEQ ID NO: 1 encoding the polypeptide as shown in SEQ ID NO: 2;

(c) a polynucleotide having a nucleotide sequence encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any one of (a) or (b), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide and wherein said fragment and derivative is capable of functioning as an SNX-B 8;

(d) a polynucleotide having a nucleotide sequence which is at least 60% identical to a polynucleotide as defined in any one of (a) to (c) and which encodes a functional SNX-B 8 protein;

(e) a polynucleotide encoding a polypeptide which is at least 45% identical to a polypeptide encoded by a polynucleotide as defined in any one of (a) to (c) and which encodes a functional SNX-B 8 protein;

(f) a polynucleotide having a nucleotide sequence the complementary strand of which hybridizes to a polynucleotide as defined in any one of (a) to (e) and which encodes a functional SNX-B8 protein;

(g) a polynucleotide having a nucleotide sequence being degenerate to the nucleotide sequence of the polynucleotide of any one of (a) to (f); or the complementary strand of such a polynucleotide.

2. The polynucleotide of claim 1, wherein said polynucleotide is DNA, cDNA, genomic DNA, RNA or PNA.

3. The polynucleotide of claim 1 or 2, wherein said polynucleotide is fused with a heterologous polynucleotide.

4. The polynucleotide of claim 3, wherein said heterologous polynucleotide encodes a heterologous polypeptide.

5. A vector containing the polynucleotide of any one of claims 1 to 4.

6. The vector of claim 5, wherein said polynucleotide is operatively linked to expression control sequences allowing expression in archaeal, prokaryotic or eukaryotic host cells.

7. The vector of claim 5 or 6 which is a gene targeting vector or a gene expression vector.

8. A host cell genetically engineered with the polynucleotide of any one of claims 1 to 4 or the vector of claim 5, 6 or 7.

9. The host cell of claim 8, wherein said host cell is a cell derived from a non-human transgenic organism.

10. The host cell of claim 9, wherein said non-human organism is a mammal, an amphibian, an insect, a fungus or a plant.

11. A non-human transgenic animal transfected with the vector of claim7.

12. A non-human transgenic animal comprising/or heterologously expressing a polynucleotide of claim 1 to 4.

13. A non-human transgenic animal comprising a mutation in the ortholog of SNX-B 8 as defined in claim 1.

14. The non-human transgenic animal of claim 13, wherein said ortholog comprises a mutation which leads to a non-functional SNX-B 8 expression, function or activity.

15. A non-human transgenic animal expressing in its somatic and/or its germ cells an expression product which is capable of interfering with the expression, function or activity of SNX-B 8.

16. The non-human transgenic animal of claim 15, wherein said expression product is an inhibiting RNA or siRNA.

17. The non-human transgenic animal of any one of claims 13 to 16, wherein said SNX- B8 ortholog mutation is a knock-out mutation and wherein said expression product capable of interfering with the expression, function or activity of SNX-B 8 leads to a knock-out or a knock-down of SNX-B 8.

18. A process for producing a polypeptide which is a functional SNX-B8 protein comprising genetically engineered cells in vitro with the vector of claim 5, 6 or 7, wherein said polypeptide is encoded by a polynucleotide of any one of claims 1 to 4.

19. A process for producing cells capable of expressing a polypeptide which is a functional SNX-B 8 protein comprising genetically engineered cells in vitro with the vector of claim 5 or 6, wherein said polypeptide is encoded by a polynucleotide of any

^■ one of claims 1 to 4.

20. A polypeptide having the amino acid sequence encoded by a polynucleotide of any one of claims 1 to 4 or obtainable by the process of claim 18.

21. An antibody specifically binding to said polypeptide of claim 20.

22. An inhibiting RNA, RNAi, siRNA, shRNA or a ribozyme binding to or inhibiting the translation of a polynucleotide of any one of claims 1 to 4.

23. An antagonist/inhibitor of the polypeptide of claim 20, wherein said antagonist/inhibitor is an antibody, an aptamer, an anticalin or an inhibiting RNA.

24. The inhibiting RNA of claim 23, wherein said inhibiting RNA is an antisense construct hybridizing to a polynucleotide of any one of claims 1 to 4, RNAi, siRNA, shRNA or a ribozyme.

25. The inhibiting RNA, RNAi, siRNA or shRNA of any one of claims 22 to 24 targets a nucleotide sequence comprising a nucleotide sequence or being a sequence as shown in SEQ ID NOs: 7, 8 or 9.

26. The RNAi of claim 25, whereby the duplex sequences of siRNAs are selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 28 and 29, 30 and 31, 32 and 33 and/or 50 and 51.

27. The antibody of any one of claims 21 or 23, wherein said antibody specifically recognizes the polypeptide set forth in SEQ ID NOs: 10 or 11.

28. The antibody of any one of claims 21, 23 or 27, wherein said antibody is polyclonal, monoclonal, chimeric, single chain, single chain Fv, human antibody, humanized antibody, or Fab fragment.

29. ^' The antibody of any of claim 21, 23, 27 or 28, wherein said antibody is labeled.

30. The antibody of claim 29, wherein said label is a toxin, radioisotope or fluorescent label.

31. The antibody of any one of claims 21, 23, or 27 to 30, wherein said antibody is immobilized.

32. An agonist/enhancer of the polypeptide of claim 20, wherein said agonist/enhancer is a transcription factor capable of enhancing expression of any one of the polynucleotides of claims 1 to 4.

33. A composition comprising the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6, or 7, the host cell of claim of any one of claims 8 to 10, the polypeptide of claim 20, the antagonist/inhibitor of claim 23, the RNAi or siRNA of claim 22, 24 or 25, the antibody of any one of claims 21, 23, or 27 to 31 or the agonist/enhancer of claim 32.

34. A composition of claim 33, wherein said composition is a pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier and/or a diluent and/or an excipient and/or a microcapsulated host cell.

35. A composition of claim 33, wherein said composition is a diagnostic composition, optionally further comprising suitable means for detection.

36. Use of the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6 or 7, the host cell of claim 8, the polypeptide of claim 20 or the agonist/enhancer of claim 32 for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating neurological, neurodegenerative disorders, diabetes or obesity.

37. Use of claim 36, wherein said neurological and/or neurodegenerative disorders are selected from the group consisting of Alzheimer's disease, Parkinson's disease, Down syndrome or a prion-disease.

38. Use of the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6 or 7 , the host cell of claim 8, the polypeptide of claim 20 or the agonist/enhancer of claim 32 for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating a disorder, wherein α-cleavage of the Amyloid Precursor Protein (APP) is inhibited or wherein α-secretase is inhibited and/or malfunctioning.

39. Use of the antagonist/inhibitor of claim 23, the inhibiting RNA, shRNA, RNAi or siRNA of any one of claims 22, 24 or 25 or the antibody of any one of claims 21, 23 or 27 to 31 for the preparation of a pharmaceutical composition for preventing, ameliorating and/or treating cancer and/or transferrin-receptor-related disorders.

40. The use of claim 39, wherein said transferrin-receptor-related disorder is cancer or an apoptosis-related disorder.

41. Use of claim 40, wherein said cancer is selected from the group consisting of prostate, breast or colon.

42. Method for identifying an antagonist/inhibitor of an SNX-B8 molecule comprising the steps of:

(a) contacting the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6 or 7, the host cell of any one of claims 8 to 10, the polypeptide of claim 20 or a non-human transgenic animal of any one of claims 11 to 15 with an inhibitor/antagonist to be screened;

(b) determining whether the inhibitor/antagonist to be screened effects the expression of a polynucleotide as defined in claims 1 to 4; and

(c) determining whether said inhibitor/antagonist to be screened is capable of down-regulating and/or inhibiting the expression of the polynucleotide coding for SNX-B8.

43. A method for screening of an inliibitor/antagonist for SNX-B8 function comprising the steps of:

(a) contacting a cell expressing SNX-B8 with a compound to be tested;

(b) determining whether in said cell SNX-B8 is functional in the presence of the compound to be tested when compared to a cell not contacted with said compound; and

(c) identifying the compound which inhibits SNX-B8 function and/or expression.

44. The method of claims 42 and 43, wherein said method comprises an additional step (V), wherein steps (a) and (b) are carried out in a control experiment in the absence of an inhibitor/antagonist to be screened.

45. Method for identifying an agonist/enhancer of SNX-B 8 molecule expression comprising the steps of:

(a) contacting the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6 or 7, the host cell of any one of claims 8 to 10, the polypeptide of claim 20 or the non-human transgenic animal of any one of claims 11 to 16 with an agonist/enhancer to be screened;

(b) determining whether the agonist/enhancer to be screened effects the expression of a polynucleotide as defined in claims 1 to 4; and

(c) determining whether said agonist/enhancer to be screened is capable of up- regulating and/or enhancing the expression of the polynucleotide coding for SNX-B8.

46. A method for screening of an agonist/enhancer for SNX-B8 function comprising the steps of:

(a) contacting a cell expressing SNX-B8 with a compound to be tested;

(b) determining whether in said cell SNX-B8 function is altered in the presence of the compound to be tested when compared to a cell not contacted with said compound; and

(c) identifying the compound which alters SNX-B8 function and/or expression.

47. The method of claims 45 and 46, wherein said method comprises an additional step (V), wherein steps (a) and (b) are carried out in a control experiment in the absence of an agonist/enhancer to be screened.

48. A method for the production of a pharmaceutical composition comprising the method of any one of claims 42 to 47 and optionally further comprising a pharmaceutically acceptable carrier and/or a diluent and/or an excipient and/or a microcapsulated host cell.

49. A method for the production of a diagnostic composition comprising the method of any one of claims 42 to 47 and optionally further comprising suitable means for detection.

50. A kit comprising a polynucleotide of any one of claims 1 to 4, the polypeptide of claim 20, the antagonist/inhibitor of claim 23 or the agonist/enhancer of claim 32.

51. A method for the preparation of a non-human double transgenic animal comprising the steps of:

(a) mating with a non-human transgenic animal of any one of claims 11 to 17 with a further non-human transgenic animal, said further animal comprising a modification of/in an a gene which leads to an altered APP metabolism or which is related to APP processing and/or which leads to an altered insulin metabolism or which is related to the insulin pathway;

(b) selecting the off-spring of said mating of step (a) which carries at least one copy of a heterologous polynucleotide of claim 1 to 4 or at least one copy of a gene comprsing a mutation in an ortholog os SNX-B8 and at least one further copy of a gene leading to an altered APP metabolism or which is related to APP processing and/or an altered insulin metabolism or which is related to the insulin pathway.

52. The method of claim 51, further comprising a step (c), wherein said offspring is again mated thereby generating double- homozygous non-human transgenic animals.

53. The method of claim 51 or 52, wherein said a gene leading to an altered APP- metabolism or which is related to APP processing is selected from the group consisting of wild-type APP, APP comprising a mutation, presenilin 1, presenilin 2, and BACE.

54. The method of claim 51 or 52, wherein said gene leading to an altered insulin metabolism or which is related to the insulin pathway is selected from the group consisting of human insulin, insulin-like growth factor, mutated insulin, mutants of insulin-like growth factor, IRS-I, IRS-2, IRS-4, PD kinase and Akt-2.

55. A non-human transgenic animal obtained by the method of any one of claims 51 to 54.

56. A method of ameliorating, preventing and/or treating a neurological and/or neurodegenerative disorder, a metabolic disorder, diabetes or obesity comprising the step of administering to a patient in need of such an amelioration, prevention and/or treatment a pharmaceutically active amount of the polynucleotide of any one of claims 1 to 4, the vector of claim 5, 6 or 7, the host cell of claim 8, the polypeptide of claim 20 or the agonist/enhancer of claim 32.

57. A method of ameliorating, preventing and/or treating cancer and/or a transferrin- receptor related disorder comprising the step of administering to a patient in need of such an amelioration, prevention and/or treatment a pharmaceutically active amount of the antagonist/inhibitor of claim 23, the inhibiting RNA, shRNA, RNAi or siRNA of any one of claims 22, 24 or 25 or the antibody of any one of claims 21, 23 or 27 to 31.