WO2004042064A2 - Disorders of lipid metabolism - Google Patents

Abstract

The present invention provides isolated or recombinant nucleic acid molecules having one or more mutations linked to disorders of lipid metabolism such as Chylomicron Retention Disease (CMRD), Anderson's Disease (AD), Chylomicron Retention Disease with Marinesco-Sjogren Syndrome (CMRD with MSS), obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia; and the polypeptides encoded by the mutated nucleic acid molecule(s). The present invention further provides for the diagnosis, prophylaxis and/or treatment of disorders of lipid metabolism.

Description

Disorders of lipid metabolism

The present invention relates to isolated or recombinant nucleic acid molecules having one or more mutations linked to disorders of lipid metabolism such as Chylomicron Retention Disease (CMRD), Anderson's Disease (AD), Chylomicron

Retention Disease with Marinesco-Sjogren Syndrome (CMRD with MSS), obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia; and the polypeptides encoded by the mutated nucleic acid molecule(s). The present invention further provides for the diagnosis, prophylaxis and/or treatment of disorders of lipid metabolism, compositions comprising the nucleic acid molecule(s), the ρolyρeptide(s) or at least one antibody that binds to the polypeptide(s), as well as vaccines and antibodies which are immunospecific for the polypeptides. The invention further provides for a polynucleotide encoding short interfering RNA (siRNA) for inhibiting the expression of the nucleic acid molecule(s).

The small intestine is the site of absorption of dietary lipid (triglyceride, cholesteryl esters, phospholipids) and fat-soluble vitamins. The lipids enter the luminal surface of the absorptive cell as lipolytic products. Chylomicrons (Cms) are assembled in the secretary pathways, and emerge from the abluminal surface for transport in intestinal lymphatics to the blood. Cms are among the largest particles assembled and secreted from eukaryotic cells. Each of these particles carries a large neutral lipid load, surrounded by a single molecule of apolipoprotem (apo)B48, and a surface monolayer of phospholipids interspersed with free cholesterol. At the periphery, the lipid provides an important source of energy, which can be stored as fat depots in time of nutritional abundance. The remnants of peripheral Cm metabolism are cleared from the circulation by the liver, and further metabolized or re-packaged and secreted as triglyceride-rich, apoB-100 containing, very low density lipoproteins (VLDL). Elevated blood levels of apoB -containing lipoproteins are major risk factors for atheroscelerosis. Excess dietary lipid intake and/or abnormal metabolism of Cm and

NLDL may associate with the metabolic syndrome of type II diabetes, insulin resistance, visceral obesity and dyslipidemia. Apolipoprotein (apo)B is the major structural protein of both Cm and NLDL. In man, the small intestine produces the edited form of apoB mRΝA that encodes apoB48, while the liver produces apoBlOO. ApoB48 comprises the first 2152 amino acid residues of the 4536 amino acid apoBlOO protein. In the currently accepted model of Cm and NLDL assembly, apoB acquires a neutral lipid load in two steps. The first involves the formation of an apoB-free lipid droplet and a lightly lipidated apoB- containing particle. In the second, the apoB-free lipid droplet(s) and the first-step apoB-containing particles fuse to produce a triglyceride-rich, apoB-containing lipoprotein, which may undergo further lipidation in the Golgi apparatus.

The production of Cms is blocked in five inherited disorders of severe fat malabsorption, all associated with a failure to thrive in infancy, growth retardation and fat soluble vitamin deficiency. Co-dominant hypobetalipoproteinemia and recessive abetalipoproteinernia are caused by mutations of apolipoprotein (apo)B, and the microsomal triglyceride transfer protein (MTP), respectively. Affected individuals produce no Cms or NLDL, and blood cholesterol and triglyceride levels are markedly reduced. Chylomicron Retention Disease (CMRD), Anderson's Disease (AD), and CMRD with the neuromuscular disorder Marinesco-Sjogren Syndrome (CMRD with MSS) are three recessive disorders characterised by reduced cholesterol levels, and a selective absence of Cms from blood. In these conditions, Cm-like particles accumulate within enterocytes in membrane-bound compartments and in the cytoplasm, and fail to enter the intestinal lymphatics.

The small GTPase Sarlb is obligatory for the transport of Cm through the secretory pathway of enterocytes. Sarlb is defective in pure CMRD, AD, and CMRD with MSS. All three conditions are histologically characterised by an accumulation of Cm- like particles within large membrane-bound compartments of enterocytes. Sarlb shares 90% amino acid sequence identity with Sari a, compared to 60% sequence identity with the yeast Sari homologue, Sarlp. The small intestine and liver contain comparable levels of Sari a and Sarlb mRΝA. Sari proteins form an obligatory component of COPII-coated vesicles, which transport secretory and cell surface proteins (i.e. cargo), out of the endoplasmic reticulum (ER). COPII-coated vesicles bud from the transitional ER and fuse to form • the ER-Golgi intermediate compartment (ERGIC), where the COPII-coat is replaced by a COPI vesicle coat. To reach the Golgi apparatus ERGIC-clusters travel along microtubules.

The integral membrane protein Secl2 initiates the assembly of COPLT vesicles on the ER membranes by exchanging GDP on Sari for GTP. This exchange promotes the interaction of Sari with ER membrane lipids, the formation of a pre-budding complex through the recruitment of Sec23 complexed to Sec24 (Fig. 18F), and the capture of Secl3/31 heterodimers. The five subunits (i.e. Sari, Sec23/24 and Secl3/31) form the basic machinery required for budding and sorting of cargo into COPII-coated vesicles.

The biochemical basis for the requirement for Sarlb in the transport of Cm through the secretory pathway of enterocytes is unknown but genetic and biochemical studies in yeast and mammalian cells suggest three possibilities. First, Sarlb may promote the formation of large COPII vesicles to accommodate Cm, which vastly exceed the size of typical COPII-coated vesicles. Second, Sarlb situated on the cytosolic side of the ER membrane may interact with a putative adaptor molecule to recruit Cm/apoB48-containing lipoproteins, situated on the luminal side of the ER, into a COPII-coated vesicle. Third, specific amino acids on the surface of Sarlb (Fig 18E) may provide the signal(s) for exporting Cm from the ER in COPII-coated vesicles.

The transport of nascent Cm from the ER may be independent of COPII function. However, these conclusions were based on studies that used an antibody that may not have recognised Sarlb as well as Sari a and included experimental data in which only recombinant Sari a, and not Sarlb, was added to their assay system. Thus, while these studies may ultimately illuminate the role of Sari a in the intracellular trafficking of nascent Cm, they provide no insights into the role of Sarlb in this process.

The inventors have reasoned that CMRD, AD and CMRD with MSS have a common genetic basis with phenotypic variation arising from mutational heterogeneity, and evaluated this by performing a genome-side screen in ten patients from six families.

This evaluation led to the identification of a number of mutations in the gene (SARA2) encoding Sarlb GTPase. These mutations could be associated with CMRD, AD, CMRD with MSS. In addition, the identification of the defective SARA2 gene in CMRD, AD, CMRD with MSS leads to the suggestion that other disorders of lipid metabolism, such as obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia and hyperlipidemia, can be prevented and/or treated by blocking and/or reducing normal Sarlb activity.

According to a first aspect of the present invention there is provided an isolated or recombinant nucleic acid molecule comprising: a) the DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent; b) a fragment of the sequence of a), the fragment comprising at least one of the mutations defined in Table 1 ; c) a sequence which is complementary to the sequence of a) or b); or d) a sequence which codes for the same polypeptide as the sequence a), b) or c).

In a second aspect, the present invention provides an isolated or recombinant nucleic acid molecule comprising: a) a fragment of the DNA sequence shown in Figure 12a, or its RNA equivalent, the fragment including the mutation in bold and as defined in Table 1 ; b) a sequence which is complementary to the sequence of a); or c) a sequence which codes for the same polypeptide as the sequence a) or b). In a third aspect, the invention provides a method for screening for and/or diagnosis of a disorder of lipid metabolism in a subject, the method comprising the step of detecting and/or quantifying the amount of a nucleic acid molecule as defined in the first or second aspect in a biological sample obtained from said subject.

The mutation may be a missense mutation, such as G to A at position 109, G to A at position 409, T to A at position 537, G to A at position 536, or T to C at position 542 of the nucleic acid sequence as shown in Table 1. Alternatively the mutation may be a splice site mutation 349-1 G to C. This mutation can result in Exon 6 being skipped, or the failure to remove intron 5 such that GTAAGTTAA is added to Exon 5.

Alternatively the mutation may be duplication of TTAC at 555-558.

The nucleic acid molecule of a) is mutated SARA2 gene.

In the present invention, the disorders of lipid metabolism may be CMRD, AD,

CMRD with MSS, obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and/or hyperlipidemia.

The term 'RNA equivalent' when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA 'IP replaces 'T' in the genetic code. The nucleic acid molecule of the present invention may be in isolated, recombinant or chemically synthetic form.

As used herein with respect to nucleic acid molecules, "isolated or "recombinant" means any of a) amplified in vitro by, for example, polymerase chain reaction (PCR), b) recombinantly produced by cloning, c) purified by, for example, gel separation, or d) synthesised, such as by chemical synthesis.

The nucleic acid molecules of the present invention, including DNA and RNA, may be synthesised using methods known in the art, such as using conventional chemical approaches or polymerase chain reaction (PCR) amplification. The nucleic acid molecules of the present invention also permit the identification and cloning of mutated S ARA2 gene, for instance by screening cDNA libraries, genomic libraries or expression libraries.

The fragment of the sequence of a) above may comprise at least 15 nucleotides, at least

20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 nucleotides. The fragment may comprise in the range of 18 to 20 nucleotides.

With regard to such fragments, for the DNA sequences shown in Figures 12b and 12c or the RNA equivalent, the fragment comprises a sequence that includes or spans the change in sequence resulting from the mutation.

The present invention includes nucleic acid molecules comprising a sequence complementary to the sequence as defined in (a) and (b) above. Thus, for example, both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another). Also included are mRNA molecules and complementary DNA molecules (e.g. cDNA molecules).

Manipulation of the DNA encoding a protein is a particularly powerful technique for both modifying proteins and for generating large quantities of protein for purification purposes. This may involve the use of PCR techniques to amplify a desired nucleic acid sequence. Thus the sequence data provided herein can be used to design primers for use in PCR so that a desired sequence can be targeted and then amplified to a high degree.

A fourth aspect of the present invention provides a pair of nucleic acid primers, one of which can hybridise to the sense strand of the DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent upstream of one of the mutations defined in Table 1, and the other of which can hybridise to the antisense strand of the DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent respectively downstream of said mutation.

Typically the or each primer will be at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases, primers of at least thirty or at least thirty-five nucleotides in length may be used.

Chemical synthesis may be used to generate such primers. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.

If desired, a nucleic acid molecule as defined herein can also be used in hybridisation assays. A nucleic acid molecule as defined herein, or subsequences thereof comprising at least 8 nucleotides, can be used as a hybridisation probe. Hybridisation assays can be used for detection, prognosis, diagnosis, or monitoring of disorders of lipid metabolism. In particular, such a hybridisation assay can be carried out by a method comprising contacting a patient sample containing nucleic acid with a nucleic acid probe capable of hybridising to a nucleic acid molecule of the first aspect, under conditions such that hybridisation can occur, and detecting or measuring any resulting hybridisation.

Desirably such hybridising molecules are at least 10 nucleotides in length, are at least 25 or at least 50 nucleotides in length.

Desirably the hybridising molecules will hybridise to such nucleic acid molecules under stringent hybridisation conditions. One example of stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution which is about 0.9 molar. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes, such as low salt or high temperature conditions. As used herein, "highly stringent conditions" means hybridisation to filter-bound DNA in 0.5 M NaHPO , 7% sodium dodecyl sulphate (SDS), 1 mM EDTA at 65 °C, and washing in O.lxSSC/0.1% SDS at 68 °C (Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p.

2.10.3). For some applications, less stringent conditions for duplex formation are required. As used herein "moderately stringent conditions" means washing in 0.2xSSC/0.1% SDS at 42 °C (Ausubel et al., 1989, supra). Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex. Thus, particular hybridisation conditions can be readily manipulated, and will generally be chosen depending on the desired results. In general, convenient hybridisation temperatures in the presence of 50% formamide are: 42 °C for a probe which is 95 to 100% identical to the portion of the nucleic acid molecule as defined herein, 37°C for 90 to 95% identity and 32 °C for 70 to 90% identity. In the preparation of genomic libraries, DNA fragments are generated, some of which will encode parts or the whole of a polypeptide encoded by the nucleic acid molecule of the first aspect. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The DNA fragments can then be separated according to size by standard techniques, including but not limited to agarose and polyacrylamide gel electrophoresis, column chromatography and sucrose gradient centrifugation. The DNA fragments can then be inserted into suitable vectors, including but not limited to plasmids, cosmids, bacteriophages lambda or T₄, and yeast artificial chromosomes (YACs). (See, for example, Sambrook et al., 1989,

Molecular Cloning, A Laboratory Manual, ID Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II; Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York). The genomic library may be screened by nucleic acid hybridisation to labelled probe (Benton & Davis, 1977, Science 196:180; Grunstein & Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). h a fifth aspect, the present invention provides the nucleic acid molecule of the first and or second aspect and/or the nucleic acid primers of the third aspect in the manufacture of a diagnostic for the diagnosis of a disorder of lipid metabolism.

In a sixth aspect there is provided a method for screening and/or diagnosis of a disorder of lipid metabolism in a subject, the method comprising the steps of: a) a<-lministering a nucleic acid molecule of the first and/or second aspect to a biological sample obtained from a subject; and b) detecting and/or quantifying hybridisation of the nucleic acid molecule of the first and/or second aspect in the biological sample.

In a seventh aspect there is provided a method for screening for and or diagnosis of a disorder of lipid metabolism in a subject, the method comprising the steps of: a) administering nucleic acid primers of the fourth aspect to a biological sample obtained from a subject; b) hybridising the nucleic acid primers to a nucleic acid molecule in the biological sample; c) amplifying the nucleic acid molecule with the nucleic acid primers; and d) comparing the amplified nucleic acid molecule with a nucleic acid molecule of the first aspect or the second aspect

In addition to being used as primers and/or probes, hybridising nucleic acid molecules of the present invention can be used as anti-sense molecules to alter the expression of Sarlb by binding to complementary nucleic acid molecules. This technique can be used in anti-sense therapy.

In a specific embodiment, expression of mutant Sarlb polypeptide is inhibited by use of antisense nucleic acids.

In an eighth aspect, the present invention provides a nucleic acid molecule comprising at least six nucleotides that is antisense to a portion of a nucleic acid molecule of the first aspect or to a portion of a nucleic acid molecule of the second aspect for use in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of lipid metabolism.

The nucleic acid may comprise at least 10 nucleotides, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 nucleotides. The nucleic acid may comprise in the range of 18 to 20 nucleotides.

The nucleic acid molecule preferably includes at least one mutation as defined in Table 1.

In a ninth aspect, the present invention provides a method for the prophylaxis and/or treatment of a disorder of lipid metabolism comprising administering a nucleic acid molecule comprising at least six nucleotides that is antisense to a portion of a nucleic acid molecule of the first aspect or to a portion of a nucleic acid molecule of the second aspect to a subject.

Preferably the nucleic acid molecule includes at least one mutation as defined in Table 1.

As used herein, an "antisense" nucleic acid molecule refers to a nucleic acid capable of hybridising by virtue of some sequence complementarity to a portion of an RNA (preferably mRNA) derived from a nucleic acid molecule of the first aspect or of the second aspect. The antisense nucleic acid may be complementary to a coding and/or non-coding region of a mRNA derived from a nucleic acid molecule of the first aspect. Such antisense nucleic acids have utility as compounds that inhibit expression, and can be used in the treatment and/or prevention of disorders of lipid metabolism.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of (a)- (d) in the first aspect and (a) -(c) of the second aspect above (e.g. at least 50%, at least 75% or at least 90% or 95% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the hkelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.

The "percent identity" of two nucleic acid sequences can be or is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in either sequences for best alignment with the other sequence) and comparing the nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical nucleotides in the sequences being compared (i.e., % identity = # of identical positions/total # of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

In view of the foregoing description, the skilled person will appreciate that a large number of nucleic acids are within the scope of the present invention. Unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1) they may be DNA or RNA;

2) they may be single or double stranded; 3) they may be provided in recombinant form, e.g. covalently linked to a 5' and/or a

3' flariking sequence to provide a molecule which does not occur in nature;

4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature;

5) they may be provided in substantially pure form. Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids; and

6) they may be provided with introns or without introns (e.g. as cDNA).

The DNA or RNA molecules may be in the form of aptamers

Disorders of lipid metabolism can be treated and/or prevented using RNA interference (RNAi) to suppress expression of the mutated SARA2 gene. RNA interference is a process by which double stranded RNA can induce sequence specific post- transcriptional gene silencing or inhibition (WO01/75164).

In a tenth aspect, the present invention provides a RNA molecule, or a polynucleotide encoding such a molecule, comprising a double stranded structure which has a nucleotide sequence which is identical to a portion of the sequence of Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or which includes the respective mutation defined in Table 1.

In an eleventh aspect, the present invention provides the use of a RNA molecule, or polynucleotide encoding such a molecule as defined in the tenth aspect, in the manufacture of a medicament for the prophylaxis and or treatment of a disorder of lipid metabolism.

In a twelfth aspect, the present invention provides a method for the prophylaxis and/or treatment of a disorder of lipid metabolism, comprising administering to a subject a RNA molecule or polynucleotide encoding such a molecule of the tenth aspect.

The RNA molecule may have a length of from 19 to 25 nucleotides or 19 to 23 nucleotides, or 21 nucleotides. At least one strand may have a 3 ' overlap from 1 to 5 nucleotides, 1 to 3 nucleotides or 2 nucleotides. At least one of the RNA strands may be blunt ended.

One strand of the RNA molecule may have a 3' overhang and the other strand can be blunt-ended or have an overhang. If both strands comprise an overhang, the length of the overhangs may be the same or different for each strand. The RNA may comprise 21 nucleotide strands which are paired and which have overhangs of from about 1 to 3, particularly 2, nucleotides on both 3' ends of the RNA. In order to further enhance the stability of the RNA, the 3' overhangs can be stabilised against degradation through the inclusion of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, pyrimidine nucleotides may be substituted by modified analogues, e.g., substitution of uridine 2 nucleotides 3' overhangs by 2'- deoxythymidine is tolerated and does not affect the efficiency of RNAi.

The RNA molecules of this aspect of the present invention can be obtained using a number of techniques known to those of skill in the art. For example, the RNA can be chemically synthesised or recombinantly produced using methods known in the art. In the present invention, the RNA is useful as a sequence-specific mediator of RNA degradation and thus, for inhibiting mRNA of the mutated SARA2 gene associated with or causative of disorders of lipid metabolism. 21-23 nt RNAs can be produced and tested for their ability to mediate RNAi in a cell, such as a human or other primate cell. Those 21-23 nt human RNA molecules shown to mediate RNAi can be tested, if desired, in an appropriate animal model to further assess their in vivo effectiveness. Additional copies of 21-23 nt RNAs shown to mediate RNAi can then be produced.

Any dsRNA can be used in the methods of the present invention, provided that it has sufficient homology to the targeted portion to which it is identical in sequence to mediate RNAi. The dsRNA for use in the present invention corresponds to a nucleic acid molecule as defined above.

The RNA can be introduced into human cells or a human in order to mediate RNA interference in the cells or in cells in the individual, such as to prevent or treat a disorder of lipid metabolism. In this method, the mutated SARA2 gene is targeted, and the corresponding mRNA is degraded by RNAi. In this embodiment, an RNA of about 21 to about 23 nucleotides that targets the corresponding mRNA (the mRNA of the targeted gene) for degradation is introduced into the cell or organism. The cell or organism is maintained under conditions under which degradation of the corresponding mRNA occurs, thereby mediating RNA interference of the mRNA of the gene in the cell or organism. In the event that the RNA is introduced into a cell in which RNAi, does not normally occur, the factors needed to mediate RNAi are introduced into such a cell or the expression of the factors is induced in such a cell.

Alternatively, an ex vivo method may be used to treat cells from an individual to degrade mutated SARA2 gene that causes or is associated with a disorder of lipid metabolism. In this embodiment, cells to be treated are obtained from the individual using known methods (e.g., phlebotomy or collection of bone marrow) and RNAs that mediate degradation of the corresponding mRNA(s) are introduced into the cells, which are then re-introduced into the individual. If necessary, biochemical components needed for RNAi to occur can also be introduced into the cells. In an thirteenth aspect, the present invention provides an isolated or recombinant polypeptide comprising: a) the amino acid sequence shown in Figure 3, 5, 7, 9, 11, 13a, 13b or 15; or b) a fragment of a polypeptide as defined in a), which is at least 5 amino acids long and includes at least one of the mutations defined in Table 1.

The polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well known degeneracy of the genetic code. All of these molecules are within the scope of the present invention. They can be inserted into vectors and cloned to provide large amounts of DNA or RNA for further study. Suitable vectors may be introduced into host cells to enable the expression of polypeptides used in the present invention using techniques known to the person skilled in the art.

The polypeptides or fragments thereof of the present invention may be provided in isolated or recombinant form, and may be fused to other moieties. The polypeptides or fragments thereof may be provided in substantially pure form, that is to say free, to a substantial extent, from other proteins. Thus, a polypeptide may be provided in a composition in which it is the predominant component present (i.e. it is present at a level of at least 50%; preferably at least 75%, at least 90%, or at least 95%; when determined on a weight/weight basis excluding solvents or carriers).

In order to more fully appreciate the present invention, polypeptides within the scope of a)-b) above will now be discussed in greater detail.

Polypeptides within the scope of a)

A polypeptide within the scope of a), may consist of the particular amino acid sequence given in Figure 3, 5, 7, 9, 11, 13a, 13b or 15 or may have an additional N-terrninal and/or an additional C-teπninal amino acid sequence. Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-teπninus to part of the β galactosidase enzyme (Itakwa etal, Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification. For example, a fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobihsed antibodies or immobihsed antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a polypeptide and need not provide any particular advantageous characteristic to the polypeptide. Such polypeptides are within the scope of the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it is preferred that the resultant polypeptide should exhibit the immunological or biological activity of the polypeptide having the amino acid sequence shown in Figure 3, 5, 7, 9, 11, 13a, 13b or 15.

Polypeptides within the scope of b)

As discussed supra, it is often advantageous to reduce the length of a polypeptide, provided that the resultant reduced length polypeptide still has a desired activity or can give rise to useful antibodies. Feature b) of the thirteenth aspect of the present invention therefore covers fragments of polypeptides a) above, the fragments including at least one of the mutations defined in Table 1.

The skilled person can determine whether or not a particular fragment has activity using the techniques disclosed above.

"Fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a). The fragment may or may not possess a functional activity of the polypeptide defined in a).

A polypeptide as defined herein may be useful as antigenic material, and may be used in the production of vaccines for treatment or prophylaxis disorders of lipid metabolism.

Such material can be "antigenic" and/or "immunogenic". Generally, "antigenic" is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. "Immunogenic" is taken to mean that the protein is capable of ehciting a protective immune response in a subject. Thus, in the latter case, the protein may be capable of not only generating an antibody response but, in addition, non-antibody based immune responses.

It is well known that it is possible to screen an antigenic protein or polypeptide to identify epitopic regions, i.e. those regions which are responsible for the protein or polypeptide's antigenicity or immunogenicity. Methods well known to the skilled person can be used to test fragments for antigenicity. Thus, the fragments of the present invention may include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein. The key issue may be that the fragment retains the antigenic/immunogenic properties of the polypeptide from which it is derived.

Fragments may possess at least a degree of the antigenicity/immunogenicity of the polypeptide from which they are derived.

In a fourteenth aspect, the present invention provides the use of a polypeptide as defined in the thirteenth aspect in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of lipid metabolism, wherein the medicament is a vaccine. The vaccine optionally comprises one or more suitable adjuvants. Examples of adjuvants well-known in the art include inorganic gels, such as aluminium hydroxide, and water-in-oil emulsions, such as incomplete Freund's adjuvant. Other useful adjuvants will be well known to the skilled person.

In yet further aspects, the present invention provides:

(a) the use of a polypeptide as defined herein in the preparation of an immunogenic composition, preferably a vaccine;

(b) the use of such an immunogenic composition in inducing an immune response in a subject; and

(c) a method for the treatment and/or prophylaxis of a disorder of lipid metabolism in a subject, or of vaccinating a subject against a disorder of lipid metabolism , which comprises the step of administering to the subject an effective amount of a polypeptide as defined herein, preferably as a vaccine.

A further aspect provides a method of diagnosis of a disorder of lipid metabolism in a subject, the method comprising detecting and/or quantifying the amount of a polypeptide as defined above in a biological sample obtained from said subject. Preferably, an antibody is used for detecting and/or quantifying the amount of a polypeptide as defined in the thirteenth aspect of the invention in a biological sample obtained from said subject.

In one embodiment, binding of antibody in tissue sections can be used to detect aberrant polypeptide localisation or an aberrant level of polypeptide. In a specific embodiment, antibody to a polypeptide as defined herein can be used to assay a patient tissue for the level of the polypeptide where an aberrant level of polypeptide is indicative of a disorder of lipid metabolism. As used herein, an "aberrant level" means a level that is increased or decreased compared with the level in a subject free from the disorder of lipid metabolism or a reference level. If desired, the comparison can be performed with a matched sample from the same subject, taken from a portion of the body not affected by the disorder of lipid metabolism.

In one embodiment, tissue from a subject is analysed for quantitative detection of a polypeptide as defined in the thirteenth aspect, wherein a change in abundance of the polypeptide in the tissue from the subject relative to tissue from a subject or subjects free from a disorder of lipid metabolism (e.g., a control sample or a previously determined reference range) indicates the presence of a disorder of lipid metabolism.

Suitable immunoassays include, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.

In a further aspect, the present invention provides an antibody which binds to at least one polypeptide as defined in the thirteenth aspect. Preferably the antibody binds specifically to a polypeptide as defined in the thirteenth aspect, for example by recognising a region encoding the mutation.

In a further aspect, the present invention provides the use of an antibody of the invention for screening for and/or diagnosis of a disorder of lipid metabolism.

In a further aspect, the present invention provides a method for the screening for and/or diagnosis of a disorder of lipid metabolism in a subject, which comprises detecting and/or quantifying the amount of a polypeptide as defined in the thirteenth aspect in a biological sample obtained from said subject using an antibody of the invention.

In a further aspect, the present invention provides a method for the prophylaxis and/or treatment of a disorder of lipid metabolism in a subject, which comprises administering to said subject a therapeutically effective amount of an antibody of the invention.

hi a yet further aspect, the present invention provides the use of an antibody of the invention in the preparation of a medicament for use in the prophylaxis and/or treatment of a disorder of lipid metabolism.

Preferred antibodies bind specifically to polypeptides of the present invention so that they can be used to purify and or inhibit the activity of such polypeptides. The antibodies maybe monoclonal or polyclonal.

Thus, the polypeptide of the present invention may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The term "antibody" as used herein refers to immunoglobulin molecules and inununologically-active portions of immunoglobuhn molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobuhn molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognise a specific domain of a polypeptide used in the invention, one may assay generated hybridomas for a product which binds to a polypeptide fragment containing such domain. For selection of an antibody that specifically binds a first polypeptide homologue but which does not specifically bind to (or binds less avidly to) a second polypeptide homologue, one can select on the basis of positive binding to the first polypeptide homologue and a lack of binding to (or reduced binding to) the second polypeptide homologue.

For preparation of monoclonal antibodies (mAbs) directed toward a polypeptide used in the invention, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature

256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

The hybridoma producing the mAbs used in the invention may be cultivated in vitro or in vivo. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilising known technology (PCT/US90/02545).

The monoclonal antibodies include but are not limited to human monoclonal antibodies and chimeric monoclonal antibodies (e.g., human-mouse chimeras). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb. (See, e.g., U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816397) Humanised antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Patent No. 5,585,089).

Chimeric and humanised monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP-A-184,187; EP-A-171,496; EP-A-173,494; WO 86/01533; U.S. Patent No. 4,816,567; EP-A-125,023; Better et al, 1988, Science 240:1041-1043; Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al, 1987, J. Immunol. 139:3521-3526; Sun et al, 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al, 1987, Cane. Res. 47:999-1005; Wood et al, 1985, Nature 314:446-449; Shaw et al, 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science

229:1202-1207; Oi et al, 1986, Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al, 1986, Nature 321:552-525; Nerhoeyan et al. (1988) Science 239:1534; and Beidler et al, 1988, J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunised in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide used in the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harboured by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg & Huszar (1995), Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, ie. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognise a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognising the same epitope. (Jespers et al. (1994)

Bio/technology 12:899-903).

The antibodies used in the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilised to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labelled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulphide stabilised Fv antibody domains recombinantly fused to either the phage gene HI or gene NIII protein. Phage display methods that can be used to make the antibodies used in the present invention include those disclosed in Brinkman et al, J. Immunol Methods

182: 41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al, Gene 1879- 18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994);. PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Νos. 5,698,426; 5,223,409;

5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinanfly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992); and Sawai et al, AJRI 34:26-3A (1995); and Better et al, Science 240:1041-1043 (1988).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988).

The invention further provides for the use of bispecific antibodies, which can be made by methods known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, 1991, EMBO J. 10:3655-3659.

According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details for generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 1986, 121:210.

The invention provides for the use of functionally- active fragments, derivatives or analogues of the anti-polypeptide immunoglobulin molecules. "Functionally-active" means that the fragment, derivative or analogue is able to elicit anti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognise the same antigen that is recognised by the antibody from which the fragment, derivative or analogue is derived. Specifically, in a preferred embodiment, the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognises the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.

The present invention provides antibody fragments such as, but not limited to, F(ab')₂ fragments and Fab fragments. Antibody fragments which recognise specific epitopes may be generated by known techniques. F(ab')₂ fragments consist of the variable region, the light chain constant region and the CHI domain of the heavy chain and are generated by pepsin digestion of the antibody molecule. Fab fragments are generated by reducing the disulphide bridges of the F(ab')₂ fragments. The invention also provides heavy chain and light chain dimmers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al, 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody of the invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al, 1988, Science 242:1038-1041).

In other embodiments, the invention provides fusion proteins of the immunoglobulins of the invention (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N- terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the immunoglobulin. Preferably the immunoglobulin, or fragment thereof, is covalently linked to the other protein at the N-terminus of the constant domain. As stated above, such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.

The immunoglobulins used in the invention include analogues and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding. For example, but not by way of limitation, the derivatives and analogues of the immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analogue or derivative may contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the art relating to the localisation and activity of the polypeptides of the invention, e.g., for imaging or radioimaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. and for radiotherapy.

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives or analogues thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesised oligonucleotides (e.g., as described in Kutmeier et al, 199 A, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

If an antibody molecule that specifically recognises a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an antibody), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunising an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies. Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al, 1991, Nature 352:624; Hane et al, 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of the antibody molecule is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., WO 86/05807; WO 89/01036; and U.S. Patent No. 5,122,464). Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulphide bond with an amino acid residue that does not contain a sulphydryl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253:6551), PCR based methods, etc. In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al, 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al, 1984, Nαtwre 312:604-608; Takeda et al, 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human antibody constant region, e.g., humanised antibodies.

Once a nucleic acid encoding an antibody molecule has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DΝA technology using techniques well known in the art. Thus, methods for preparing the polypeptides used in the invention by expressing nucleic acid containing the antibody molecule sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DΝA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, ΝY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, ΝY).

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.

The host cells used to express a recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalo virus is an effective expression system for antibodies (Foecking et al, 198, Gene 45:101; Cockett et al, 1990, Bio/Technology 8:2).

A variety of host-expression vector systems may be utilised to express an antibody molecule of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculo virus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMN) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions comprising an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al, 1983, EMBOJ. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pJJSf vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Nan Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa calif ornica nuclear polyhedrosis virus (AcΝPN) is used as a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example, the polyhedrm gene) of the virus and placed under control of an AcΝPN promoter (for example, the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems (e.g., an adeno virus expression system) may be utilised.

As discussed above, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, cells lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g., neomycin or hygromycin), and selecting for expression of the selectable marker. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

The expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Nol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule used in the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Alternatively, any fusion protein may be readily purified by utilising an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al, 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

In a preferred embodiment, antibodies of the invention or fragments thereof are conjugated to a diagnostic or therapeutic moiety. The antibodies can be used for diagnosis or to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Patent No.4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin;

125 131 ill 99 and suitable radioactive nuclides include I, I, In and Tc.

An antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. Patent No. 4,676,980.

An antibody can be used as a therapeutic that is administered alone.

As discussed herein, certain polypeptides, nucleic acid molecules and antibodies find use in the treatment and/or prophylaxis of disorders of lipid metabolism.

Thus, in a further aspect, the present invention provides a pharmaceutical formulation comprising at least one polypeptide, nucleic acid molecule or antibody of the invention, optionally together with one or more pharmaceutically acceptable excipients, carriers or diluents. Preferably, the pharmaceutical formulation is for use as a vaccine and so any additional components will be acceptable for vaccine use. In addition, the skilled person will appreciate that one or more suitable adjuvants may be added to such vaccine preparations.

The medicament will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition maybe in any suitable form (depending upon the desired method of administering it to a patient).

It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (mcluding subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.

Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal adrninistration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration maybe formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient maybe formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal adrninistration may be presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators.

Pharmaceutical compositions adapted for vaginal adrninistration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted forparenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

Dosages of the polypeptide, nucleic acid or antibody used in of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice. In the context of the present invention, the biological sample can be obtained from any source, such as a serum sample or a tissue sample. The biological sample may be obtained using a small intestinal biopsy.

The invention also provides diagnostic kits, comprising an antibody against a polypeptide as defined in the thirteenth aspect. In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the antibody for diagnosis, prognosis, therapeutic monitoring or any combination of these applications; (2) a labelled binding partner to the antibody; (3) a solid phase (such as a reagent strip) upon which the antibody is immobilised; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any combination thereof. If no labelled binding partner to the antibody is provided, the anti- polypeptide antibody itself can be labelled with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

The invention also provides a kit comprising a nucleic acid probe capable of hybridising to RNA as defined in the first aspect. In a further aspect there is provided a kit comprising in one or more containers a pair of primers as defined in the fourth aspect (e.g., each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides) that under appropriate reaction conditions can prime amplification of at least the mutated portion of a nucleic acid molecule as defined in the first aspect, such as by polymerase chain reaction (see e.g., Innis et al, 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Qβ replicase, cyclic probe reaction, or other methods known in the art.

The invention provides methods for identifying agents, candidate compounds or test compounds that bind to a polypeptide as defined in the thirteenth aspect or have a stimulatory or inhibitory effect on the expression or activity of a polypeptide as defined herein. The compounds of the invention include but are not limited to any compound, e.g., a small organic molecule, protein, peptide, antibody, nucleic acid, etc. that restores the profile towards normal with the proviso that such compounds or treatments include, but are not limited to, taxol, cyclophosphamide, tamoxifen, and doxorubacin.

In another embodiment, symptoms of a disorder of lipid metabolism, may be ameliorated by decreasing the level or activity of a polypeptide as defined in the thirteenth aspect by using nucleic acid sequences of the first aspect in conjunction with well-known gene "knock-out," ribozyme or triple helix methods to decrease expression of the polypeptide. In this approach, ribozyme or triple helix molecules are used to modulate the activity, expression or synthesis of the nucleic acid sequence, and thus to ameliorate the symptoms of the disorder of lipid metabolism. Such molecules may be designed to reduce or inhibit expression of a mutant SARA2 gene. Techniques for the production and use of such molecules are well known to those of skill in the art.

Endogenous polypeptide expression can also be reduced by inactivating or "knocking out" a mutated SARA2 gene as defined herein, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317:230-

234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al, 1989, Cell 5:313- 321; and Zijlstra et al, 1989, Nature 342:435-438). For example, a mutant SARA2 gene encoding a non-functional polypeptide (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene encoding the polypeptide) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas & Capecchi, 1987 and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly admimstered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, and as discussed above, RNA interference may be used to silence or inhibit expression of a nucleic acid molecule of the invention.

A further aspect provides a method for identifying agents (e.g. drug candidates or test compounds) that have an inhibitory effect on the expression of a nucleic acid molecule of the first or second aspect, or activity of a polypeptide of the thirteenth aspect, comprising contacting a nucleic acid molecule of the first or second aspect, or a polypeptide of the thirteenth aspect with a candidate agent, and determining if the agent inhibits expression of the nucleic acid molecule or the activity of the polypeptide

In a further aspect, the present invention provides a method of screening for an agent that interacts with Sarlb, the method comprising identifying an agent that interacts with a surface of Sarlb away from the Sec23-Sarl binding surface, the surface comprising the amino acid residues Proline 139, Glutamic acid 140, Arginine 146, Methionine 150, Serine 162, Isoleucine 163, Serine 164, Leucine 109, Alanine 125, Aspartic acid 116, Glutamic acid 113, Serine 117 and/or Isoleucine 80, the method comprising contacting Sarlb polypeptide with a candidate agent and determining whether the agent interacts with the surface.

In a further aspect, the present invention provides a method of screening for an agent that interacts with a polypeptide of the present invention, the method comprising contacting a polypeptide of the invention with a candidate agent and determining whether the agent interacts with the polypeptide. Preferably the method identifies an agent that interacts with the surface of the polypeptide away from the Sec23-Sarl binding surface. In a further aspect, the present invention provides the use of an agent identified using the above methods of screening in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of lipid metabolism.

In CMRD, AD, and CMRD with MSS there is a failure to absorb lipid from the intestine. The consequences of this can be considered to be the reverse of the problems that lead to obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia. Therefore, the identification of defective SARA2 gene in CMRD, AD, and CMRD with MSS leads to the suggestion that other disorders of lipid metabolism, such as obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia can be prevented and/or treated by blocking and/or reducing normal Sarlb activity. By blocking Sarlb activity, CMRD may be mimicked and furthermore lipid absorption can be reduced.

In a further aspect of the present invention there is provided a method for the prophylaxis and/or treatment of disorders of lipid metabolism, comprising blocking and/or reducing Sarlb activity in a subject.

The disorders of lipid metabolism may be obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and/or hyperlipidemia

Blocking and/or reducing of Sarlb activity may be carried out by: i) Decreasing transcription of Sarlb gene (SARA2); ii) Reducing mRNA levels; iii) Decreasing GTPase activity of Sarlb protein; or iv) Interfering with the interaction of Sarlb protein with other proteins that augment GTP hydrolysis, and chylomicron and very low density lipoprotein export from the intestinal absorption cell and hepatocyte, respectively.

Decreased transcription of SARA2 may be achieved by interfering with transcription by, for example, interfering with the activity of specific transcription factors thereby up-regulating or down-regulating Sarlb activity. Such methods are well known on the art.

Alternatively, in another embodiment, symptoms of a disorder of lipid metabolism and in particular obesity, insulin resistance, type II diabetes, atheroscelerosis, and hyperlipidemia, maybe ameliorated by decreasing the level or activity of a Sarlb polypeptide by using well-known gene "knock-out," ribozyme or triple helix methods to decrease expression. In this approach, ribozyme or triple helix molecules are used to modulate the activity, expression or synthesis of the nucleic acid sequence encoding Sarlb (SARA2), and thus to ameliorate the symptoms of the disorder of lipid metabolism. Such molecules may be designed to reduce or inhibit expression of SARA2. Techniques for the production and use of such molecules are well known to those of skill in the art.

Endogenous polypeptide expression can also be reduced by inactivating or "knocking out" SARA2, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al, 1989, Cell 5:313-321; and Zijlstra et al, 1989, Nature 342:435-438). For example, a Sarlb gene encoding a non- functional polypeptide (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene encoding the polypeptide) can be used, with or without a selectable marker and or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas & Capecchi, 1987 and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors. Alternatively, and as discussed above in relation to the nucleic acid molecules of the invention, RNA interference may be used to silence or inhibit expression of SARA2 gene.

mRNA levels may be reduced by, for example, using antisense nucleic acid molecules. For example, expression of Sarlb maybe inhibited by use of antisense nucleic acid molecules. The antisense nucleic acid molecule is capable of hybridising by virtue of some sequence complementarity to a portion of an RNA (preferably mRNA) derived from the nucleic acid sequence of Sarlb. The antisense nucleic acid may be complementary to a coding and/or non-coding region of a mRNA derived from the Sarlb. Such antisense nucleic acids have utility as compounds that inhibit expression, and can be used in the treatment and/or prevention of disorders of lipid metabolism, and particularly obesity, insulin resistance, type II diabetes, atheroscelerosis, and hyperlipidemia.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule for Sarlb (e.g. at least 50%, at least 75% or at least 90% or 95% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions. The "percent identity" discussed above in relation to the ninth aspect applies equally to this aspect.

In a further aspect, the present invention provides a method for identifying agents (e.g. drug candidates or test compounds) that have an inhibitory effect on the expression or activity of Sarlb polypeptide comprising contacting SARA2 gene or Sarlb polypeptide with candidate agents, and selecting those agents which inhibit expression of SARA2 gene or activity of Sarlb polypeptide. The invention also provides methods of identifying agents that bind to Sarlb polypeptide. The methods can be used in high throughput drug discovery. Examples of agents, candidate compounds or test compounds of the present invention include, but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145; U.S. Patent No. 5,738,996; and U.S. Patent No.5,807,683, each of which is incorporated herein in its entirety by reference).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.,

1994, Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al, 1994, J. Med. Chem.

37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int.

Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and

Gallop et al., 1994, J. Med. Chem. 37:1233, each of which is incorporated herein in its entirety by reference.

Libraries of compounds maybe presented, e.g., presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc.

Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310), each of which is incorporated herein in its entirety by reference.

In one embodiment agents are identified in a cell-based assay system. In accordance with this aspect of the present invention, cells expressing Sarlb are contacted with an agent, such as a drug candidate, or a control and the ability of the agent to inhibit Sarlb activity, e.g. by interacting with Sarlb, is studied. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate compounds. The cell, for example, can be of prokaryotic origin (e.g., E. coli) or eukaryotic origin (e.g., yeast or mammalian). In certain instances, the Sarlb or the candidate compound is labeled, for example with a radioactive label (such as ³²P, ³⁵S or ¹²⁵I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable detection of an interaction between Sarlb and an agent, such as a drug candidate. The ability of the candidate compound to interact directly or indirectly with Sarlb can be determined by methods known to those of skill in the art. For example, the interaction between an agent and Sarlb can be determined by flow cytometry, a scintillation assay, immunoprecipitation or western blot analysis.

Inhibition of Sarlb can be determined by contacting cells expressing Sarlb with an agent, such as a drug candidate; and deteirnining whether secretion of chylomicrons and/or very low density lipoproteins is decreased in the presence of the agent.

In another embodiment, agents are identified in a cell-free assay system.

In accordance with this embodiment, a native or recombinant Sarlb, is contacted with an agent or a control and the ability of the agent to inhibit Sarlb activity, e.g. by interacting with Sarlb is determined. If desired, this assay maybe used to screen a plurality (e.g. a library) of agents. Preferably, the Sarlb is first immobilized, by, for example, contacting the Sarlb with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of the Sarlb with a surface designed to bind proteins. The Sarlb may be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. Further, the Sarlb may be a fusion protein comprising the Sarlb or a biologically active portion thereof and a domain such as glutatMonine-S-transferase. Alternatively, the Sarlb can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL). The ability of the agent to interact with Sarlb can be determined by methods known to those of skill in the art.

In another embodiment, agents that modulate (i.e., upregulate or downregulate) the expression of Sarlb are identified by contacting cells (e.g., cells of prokaryotic origin or eukaryotic origin) expressing Sarlb gene (SARA2) with a candidate agent or a control (e.g., phosphate buffered saline (PBS)) and deteimining the expression of SARA2 gene or mRNA encoding Sarlb. The level of expression of SARA2 gene or mRNA encoding Sarlb in the presence of the candidate agent is compared to the level of expression of the S ARA2 gene or mRNA encoding the Sarlb in the absence of the candidate agent (e.g., in the presence of a control). The candidate agent can then be identified as a modulator of the expression of Sarlb. When expression of Sarlb or mRNA encoding Sarlb is significantly less in the presence of the candidate agent than in its absence, the candidate agent is identified as an inhibitor of the expression of Sarlb or mRNA encoding Sarlb. The level of expression of Sarlb or the mRNA that encodes it can be determined by methods known to those of skill in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR, and protein levels can be assessed by western blot analysis.

In a further aspect of the present invention there is provided a method of identifying agents that modulate the activity of Sarlb, the method comprising contacting a preparation containing Sarlb, or cells (e.g., prokaryotic or eukaryotic cells) expressing the Sarlb with a test agent or a control and determining the ability of the test agent to modulate (e.g., stimulate or inhibit) the activity of Sarlb.

The activity of Sarlb may be assessed by detecting GTP hydrolysis, the interaction of Sarlb with Sec23/24 and/or Sec 13/31, or by measuring vesicle fusion. Based on the present description, techniques known to those of skill in the art can be used for measuring these activities. Thus the method may comprise: i) contacting a preparation containing Sarlb, or cells (e.g., prokaryotic or eukaryotic cells) expressing Sarlb with a test agent or a control, and GTP; ii) measuring GTP hydrolysis; and iii) determining whether GTP hydrolysis is affected in the presence of the agent.

Preferably the GTP is labelled GTP. The GTP may be fluorescently or radioactively labelled. A radioactive label may be ³²P

Preferably the GTP hydrolysis is measured by detecting release of labelled phosphate from the GTP.

Preferably a control is provided to ensure that the agent(s) are specific to Sarlb. The control may comprise contacting the agent(s) with a further GTPase and measuring

GTP hydrolysis.

Alternatively, the method comprises: i) contacting an agent with Sarlb and Sec23/24 and or Sarlb and Seel 3/31; and ii) detecting whether the agent inhibits the interaction.

The interaction between Sarlb and Sec23/24 and/or Sarlb and Sec 13/31 can be measured using methods well known in the art. One such method is by studying vesicle fusion. This may be determined through the use of fluorescent probes to determine whether vesicles fuse in the presence of the agent(s).

The term "agent" is used herein to describe a wide variety of physical, chemical or biological factors. For example, physical agents include, without limitation, the diet of a subject, a change in temperature or humidity, exposure to ultraviolet radiation and the like. Biological and chemical agents include exogenous factors such as pharmaceutical compounds (including candidate compounds and test compounds), toxic compounds, proteins, peptides, chemical compositions, natural pathogens, such as microbial agents including bacteria, viruses and lower eukaryotic cells such as fungi, yeast and simple multicellular organisms, as well as endogenous factors which occur naturally in the body, including, without limitation, hormones, enzymes, receptors, ligands and the like, which may or may not be recombinant. In a further aspect, the present invention provides the use of an agent identified using the above methods in the manufacture of a medicament for the prophylaxis and/or treatment of a disorder of lipid metabolism. The disorder may be obesity, insulin resistance, type Ji diabetes, atheroscelerosis, dyslipidemia, and/or hyperlipidemia.

In a further aspect, the present invention provides a method of screening for an agent that interacts with a polypeptide of the present invention. Preferably the method identifies an agent that interacts with the surface of the polypeptide away from the Sec23-Sarl binding surface.

h a further aspect, the present invention provides the use of an agent identified using the above method of screening in the manufacture of a medicament for the prophylaxis and/or treatment of a disorder of lipid metabolism.

The methods of screening above in relation to normal Sarlb apply equally to the polypeptides and nucleic acid molecules of the present invention, as appropriate.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

Examples

The invention will now be described with reference to the following examples, which should not in any way be construed as limiting the scope of the present invention. The examples refer to the figures in which:

Figure 1 provides a) the nucleic acid sequence (SARA2) encoding Sarlb, and b) the amino acid sequence for Sarlb;

Figure 2 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1; Figure 3 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 2;

Figure 4 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1 ;

Figure 5 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 4;

Figure 6 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1;

Figure 7 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 6;

Figure 8 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1;

Figure 9 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 8;

Figure 10 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1;

Figure 11 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 10;

Figure 12a provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1; Figure 12b provides the nucleic acid sequence resulting from the mutated sequence in Figure 12a;

Figure 12c provides an alternative nucleic acid sequence resulting from the mutated sequence in Figure 12a;

Figure 13a provides the amino acid sequence encoded by the nucleic acid sequence of Figure 12b;

Figure 13b provides the amino acid sequence encoded by the nucleic acid sequence of Figure 12c;

Figure 14 provides the nucleic acid sequence of mutated SARA2, the mutation being defined in Table 1;

Figure 15 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 14;

Figure 16 - Pedigrees, linkage analysis, and fine-mapping of the Anderson's Disease (AD), Chylomicron Retention Disease (CMRD) with Marinesco-Sjogren Syndrome

(CMRD/MSS) gene locus. Fasting plasma total cholesterol (TC) and apolipoprotein (apo)B levels are shown. Microsatellite loci spanning an interval of XcM are shown on the left. Polymorphic DNA markers D5S2110 and D5S816 delineate a 4cM region of homozygosity (affected sibs, families 1, 2, 5 and 6) and is boxed. Sarlb resides between D5S2115 and D5S816 on the physical map (http://www.ensembl.orR). The affected and unaffected chromosomes are represented by filled-in and blank bars, respectively. Parents of the affected children are depicted as obligate carriers. Squares, males; circles, females; double lines, consanguineous matings; filled squares and circles, affected individuals.

Figure 17 - Mutation analysis of the Sarlb gene in Chylomicron Retention Disease, Anderson's Disease (AD), Chylomicron Retention Disease (CMRD) with Marinesco- Sjogren Syndrome (CMRD/MSS). Sequence chromatographs showing the mutations and the translated amino acids of mutant alleles. Amino acids shown in magenta represent missense mutations. The Sarlb gene of patient 3 (family lb, AD) also contains the G37R missense mutation on both alleles (sequence trace not shown). The D 137N missense mutation was also present on one allele in patient H/l of family

3 (CMRD). The second allele contained a two base deletion (sequence not shown). The five missense mutations were not present in the Sarlb of gene of 86 controls.

Figure 18 - Sarlb gene organisation (A), sequence alignment (B), location of mutations in Chylomicron Retention Disease (CMRD), Anderson's Disease (AD), Chylomicron Retention Disease (CMRD) with Marinesco-Sjogren Syndrome (CMRD/MSS), (C, D), the molecular surface of Sari, with Sec23 (E), and the prebudding complex of Sari with Sec23 and Sec24 (F). (A) Exons, and associated exon-intron boundaries, of Sarlb (AF092130) were PCR-amplified, and sequenced. Filled rectangles, exons; lines, introns. (B) Alignment of human Sarlb (X9Y6B6) with human ADP-ribosylation factor ARF 1 (AAA3061.1), Sarla(Q9NR3), yeast Sacchromyces cerevisiae Sarlp (NP_015106) and hamster Sari (1F6B B). The amino acid residues mutated in CMRD and AD (Gly³⁷ (family 1,AD), Asp¹³⁷ (families 2 and 3, CMRD), Ser¹⁷⁹ (family 4, CMRD), Leu¹⁸¹ (family 7) are displayed in magenta. The residues mutated due to frame shift in family 5 (CMRD) are boxed. The outcome of the homozygous mutation in family 6 (CMRD and MSS) may produce a mRNA species that lacks exon 6 sequences, which encode amino acids 117-160 of Sarlb. Residues that differ between human Sarlb and Sarla are displayed in green. Residues that contribute to the Sarlp-Sec23 interface of the Sari- are shown in light blue. The two residues highlighted in red differ between human Sarlb and hamster Sari . The secondary structure elements are based on the X-ray crystal structure of hamster Sari (1F6B_B). Blue cylinders represent α-helices. Yellow arrows depict β-strands. Loops are shown in grey and residues with unassigned coordinates by a dashed line. (C). Ribbon representation of hamster Sarl-GDP. Sari has a structural core of six β-strands (yellow) that are sandwiched between three α- helices (blue) on either side. GDP and the four residues (Gly³⁷, Asp¹³⁷, Ser¹⁷⁹ and L¹⁸¹) mutated in CMRD and AD are depicted in a stick representation. GDP atoms: green, carbon; blue, nitrogen; oxygen, red and phosphorous, yellow. Side chains or mutated residues, magenta. The location of the magnesium ion is shown as a grey sphere. (D) Detailed view of the GDP binding site of Sari. A distance cut off of X A was used to identify side chains contributing to the guanine nucleotide binding site. The atoms of the bound GDP and of the four residues mutated in AD and CMRD

(Gly³⁷, Asp¹³⁷, Ser¹⁷⁹ and Leu¹⁸¹) are shown in: green, carbon; blue, nitrogen; oxygen, red and phosphorous, yellow. Gly³⁷ sits at the base of the nucleotide binding site in contact with the ribose ring of GDP. Asp¹³⁷ forms three hydrogen-bonds, two with

170 170 the guanine base, and one with the OG atom of Ser . The OG atom of Ser forms a second hydrogen bond with the main chain nitrogen atom of Lys¹⁸², an integral component of the local fold. The aliphatic side chains of Leu¹⁸¹ and Lys¹³⁵ form extensive van der Waals contacts the hydrophobic face of the purine ring. (E) The Sarlb-Sec23 interface was modelled on the coordinates of yeast Sarl-Sec23/24. (F) Sequences within Sari and Sec24 face towards the ER membrane. In C-E, the figures were prepared using PREPI.

Figure 19 - Expression of Sarlb and Sarla in human tissues. The coding sequences of Sarlb and Sarla were amplified from control cDNA (MTC panel I, BD Biosciences, ) using TITANIUM™ Taq DNA Polymerase, and radiolabelled (Rediprime II DNA labelling system, Amersham, Uppsala), and hybridised to the same multiple tissue northern blot (BD Biosciences,). B-actin was used as a loading control. The blot was stripped between hybridisations. Numbers indicate the sizes of Standard RNA in kilobases (kb).

Figure 20 - Effect of high levels of human Sarla and Sarlb expression on the secretion of apoB48 and apoBlOO - containing lipoproteins from stable McA- RH7777 cell lines.

Example 1

A genome side screen in ten patients from six families was performed. One family originated from Algeria, and comprised an affected sibling pair with AD. Three were white Canadians, and contributed four patients with CMRD (families 2-4, CMRD). The fifth was Turkish, and comprised two offspring with CMRD from a consanguineous union between first cousins. The sixth family, originating from a small village in southern Italy, contained an affected sibpair with CMRD and MSS.

We observed segregation of genetic markers D5S615 and D5S658, located on chromosome 5q31, with affected status in all six familires, with no recombinants (Fig. 18). Further refinement of the interval identified a region of homozygozity in four of the six families, one with AD (family 1), two with CMRD (families 2 and 5) and one with CMRD and MSS (family 6). The disease allele differed in these families, consistent with private-family mutations.

Analysis of the Ensembl database identified eight candidate genes for AD, CMRD and CMRD+MSS. These were non-kinase Cdc42 effector protein SPEC2, (AF189692); PDZ containing guanine nucleotide exchange factor I (AF117947);

Septin-like protein KIAA0202 (AK057797); HTGN29 (AF226055); Sec24a (AJ131244); Q96J42 (NM_024715); Rabkinesin-6, (AF070672), and Sarlb (AF092130). The Sarlb gene encodes an ADP-ribosylation factor (AFR) related small GTPase protein component of the cytoplasmic coat protein π complexes (COPH) vesicle coat, which is involved in endoplasmic reticulum (ER) to Golgi apparatus transport of proteins. This gene lies between D5S2115 and D5S816 on the physical map (Fig 16.). The coding sequences and consensus splice site sequences (Fig. 18 A) were amplified from genomic DNA by polymerase chain reaction (PCR) in all patients and family members.

We identified a mutation on both alleles of Sarlb in all patients (Table 2, Fig.16, Fig.lδA), and verified the genetic status of the heterozygote carriers (Fig. 16). In total, eight different mutations, including five missense mutations, one null allele, and one splice site mutation were identified in our cohort of families (Fig. 16, Table 2). The missense mutations were not present in a sample of 86 unrelated subjects, indicating that they are disease-causing mutations. A common polymorphism with an allele frequency of 0.125 in the control subjects (n=192) was identified in exon 6 of Sarlb (Fig. 18 A).

Table 1. Nucleotide and amino acid mutations associated with each of Figure 2 to 15.

In Figures 2 to 15 the mutations are indicated in bold. The start and stop codons are underlined.

Table 2. Molecular defects in Chylomicron Retention Disease (CMRD), Anderson's disease (AD) and chylomicron retention disease with Marinesco-Sjogren Syndrome (CMRD/MSS). The exons and flanking concensus splice sites were screened for mutations. Nucleotide and amino acids are numbered from A of the initiation codon (ATG). Abreviations: horn, homozygous; het, heterozygous; fs, frameshift; ins, insertion; del, delete; ref, reference; amino acids: G, glycine; R, arginine; D, aspartic acid; N, asparagine; S, serine; I, isoleucine; L, leucine; P, proline. N>

GO cr

I o w

Family Diagnosis Origin Status Mutation Amino-acid Protein effects no./individual

Het c.75_76delGT L28fsX34 Translation

r CD¹ <

arrested after 34 o residues o

4, π-i CMRD French Canadian Horn c.537T>A S179R GNP binding g 8.

5, π-i CMRD Turkish Horn c.555- Gl 866X199 Membrane

558dupTTAC binding a

5, π-2 CMRD Turkish Horn c.555- G186fsX199 Membrane

558dupTTAC binding

6, π-i CMRD+MSS Italian Horn c.349-lG>C S117_K160del Skipping exon

6/non functional protein

6, π-2 CMRD+MSS Italian Horn c.349-lG>C S117_K160del Skipping exon

. 6/non functional protein

The COPπ machinery for transport of proteins between the endoplamic reticulum (E R) and the Golgi apparatus is highly conserved between yeast and mammals. Sari is involved in three critical steps in this process, which processively require GTP binding and hydrolysis. First, Sari possesses an amino terminal membrane anchor (in common with the ARFs) that is retracted in the GDP bound state and exposed in the GTP bound state. On GTP binding the release of this membrane anchor by Sari increases membrane binding, and enhances the affinity of binding of Sec23/24 to Sari (Fig 18F). The Sarl-Sec23/24 complex forms an extensive interaction with the ER membrane to create the prebudding complex. Second, Sec23 is the Sarl-GTPase activating protein (GAP). Sec23 inserts an arginine side chain "finger" into Sari to complete the active site. This mechanism is a feature of many proteins of the RAS family. GTP hydrolysis by the Sec23/24-Sarl GTP complex is a relatively extended reaction to allow prebudding complexes time to gather specific SNARE and cargo molecules. Third, the reaction is accelerated 10-fold by Sec 13/31 heterodimer recruitment, which triggers COPII coat disasembly, after cargo transfer to the Golgi apparatus. The crystal structure of a hamster Sari of the yeast Sec23/24-Sarl complex have been determined. Sequence alignment of the Sari family of proteins (Fig. 18B), and analysis of the hamster Sari (the hamster protein differs by only two amino acids in sequence from human Sarlb) shows that all five missense mutations of

Sarlb will have a marked effect in the region of guanine nucleotide binding on the local protein fold (Fig.l8C, D).

The mutation in three of the families (1-3) affects two highly conserved residues (Gly³⁷ and Asp¹³⁷) that are found in the guanine binding motifs (GxxxxGKT³⁹ and

NKxD¹³⁷) of all small GTP-binding proteins (Table 2, Fig. 17, Fig, 18B). Gly³⁷ sits at the base of the guanidine diphosphate (GTP) and guanidine triphosphate (GTP) binding cleft of the Sari family of GTPase, in contact with the ribose ring of the guanine base (Fig. 18C, D). An Arg residue at this position (family 1, AD) would almost certainly hinder the binding of guanine nucleotide by Sari, either through steric hindrance or more likely, changes in the geometry of the GDP/GTP binding cleft. In patients from family 1 the Golgi apparatus of the enterocytes and lacteals are completely devoid of CMs, despite a massive accumulation of Cm-like particles in membrane-bound ER related compartments. This observation is consistent with the results of an in vitro study which established that mutation of Asp³⁴ to Gly in yeast Sari interferes with GTP-Loading and destroys the ability of the protein to promote vesicle budding. In the Sari -GDP/GTP analogue structures, Asp¹³⁷ forms two hydrogen bonds with the Nl and N5 atoms of the guanine base (Fig. 18D). The Asp¹³⁷ to Asn mutation (families 2 and 3, CMRD), although conservative, results in the loss of one of these hydrogen bonds, which will almost certainly affect the affinity of Sarlb for guanine nucleotides (Fig.lδD). The Sari -GDP/GTP structures also indicate that three further missense mutations (Ser for Arg, S 179R; Ser for He, S 1791;

Leu for Pro, LI 8 IP) in CMRD and AD will profoundly affect the ability of Sarlb to bind guanine nucleotides (Table 2, Fig 17, Fig. 18B-D). Ser¹⁷⁹ forms a hydrogen bonding network between Asp¹³⁷ and the main chain amide of Lys¹⁸² (Fig. 18D). These interactions link two β-turns (amino acids 136-140 and 178-183) that are critical for the overall fold of the nucleotide binding cleft. Mutation of Ser¹⁷⁹ to either

Arg or lie would disrupt the hydrogen bond network and significantly affect the geometry of the nucleotide binding cleft. The Leu¹⁸¹ to Pro missense mutation would also perturb the local geometry of the β-turn formed by amino acids 178-183, and abrogate the hydrophobic packing interaction between Leu¹⁸¹ and the purine ring of GDP/GTP (Fig 18D).

In family 5 (individuals JJJJ and π/I) the mutant allele encodes a protein with twelve aberrant amino acids including three highly conserved residues (Gin¹⁹⁵, Tyr¹⁹⁶, He¹⁹⁷) at its C-terminal end (Fig. 18B).) which along with the N-terminal is involved in the initial interaction of Sari with the membrane. In this family there is very limited transport of Cms to the Golgi apparatus. A splice site mutation was identified on both alleles of the Sarlb gene in two patients (family 6, ITJ1 and JJI2) with CMRD and MSS (Fig. 17, Table 2). The mutation may produce a shorter transcript that encodes 119 amino acids (Table 2), or an mRNA species that lacks exon 6 sequences (amino acids 117-160) (Fig.18B) . This would have a massive impact on the structure and function of Sarlb, and since the gene is expressed in other tissues (Fig. 19a,b) including muscle and brain may well explain the musculo-skeletal and neurological abnormalites in CM with MSS.

The finding of Sarla and Sarlb mRNA at similar levels in the small intestine, and that only Sarlb mutations cause Cm retention, with no other secretary defect apparent, indicates that the difference in amino acid sequence between these proteins must confer the highly unusual selection of Cms as cargo by Sarlb. Whether this defect extends to the liver is unclear. However, patients with these diseases do have hypobetalipoproteinemia, suggesting at least a partial primary defect in VLDL secretion. Both Cm and VLDL are too large to travel in conventional 50-90(60)nm

COPπ vesicles. In the liver large lipid droplets accumulate in the terminal part of the smooth ER next to the Golgi apparatus, and in the Golgi apparatus NLDL is confined to the terminal saccules of the Golgi stack. Sari alone can initiate membrane cargo selection before recruitment of Sec23/24 and Secl3/31 through the formation of specialised export domains and the balance between vesicle and tubule formation.

For example procollagen is 200 nm in length. To help elucidate the specificity of Sarlb for lipoproteins we mapped the 20 variant amino acids between Sarla and Sarlb to the hamster Sari crystal structure. These variant residues mainly fall on one surface of the protein away from the Sec23-Sarl binding surface, which appear to be highly conserved in Sari as well as Sec23. These residues and this surface are not particularly hydrophobic as might be anticipated for a membrane associated or protein-protein interaction domain. However, this surface would appear to provide the constraints, which mediate the selection of this very unusual lipoprotein cargo. An understanding of the molecular basis for this process may indicate a novel therapeutic approach to obesity, and to postprandial hyperlipidemia, a condition that confers a high risk of atherosclerosis.

Both patients with CMRD and MSS had the diagnostic hallmarks of MSS including congenital cataracts, weakness and atrophy of proximal and distal muscles in the four limbs, skeletal anomalies, mental deficiency, cerebellar ataxia, and atrophy of the cerebellar hemispheres with a hypoplastic vermis. A diagnosis of spinocerebellar ataxia types 1-3, 6 and 7, hereditary denatatorubropallidoluysian atrophy, and/or Friedreich's ataxia were ruled by genetic studies. The brothers also presented with the less common features of the MSS, including optic neuropathy, hypergonadotropic hypogonadism, and moderately elevated serum concentrations of creatine kinase (CK). The affected individuals of families 2-4, and patient 11/2 of family 5 were also noted to have moderately elevated CK levels.

A whole-genome linkage analysis was performed using X polymorphic DNA markers at ~8cM intervals (Weber Human Screening Set Version 9, Research Genetics, Inc. Huntsville), followed by an additional 49 genetic markers to evaluate the X genomic regions that could not be excluded as containing the CMRD, AD and CMRD+MSS gene in the initial screen. D and D were the only markers to segregate with affected status in all families. Markers were genotyped in all family members from pedigrees 1-6.

Example 2

Caco-2 cells provide a well-characterised model for the in vitro study of Cm production. Caco-2 cells spontaneously differentiate in culture into enterocyte-like cells, with polarised apical and basolateral surfaces, brush-border microvilli and tight junctions. The differentiation process is accompanied by increased expression of apoB48 and 100, and increased secretion of VLDL and Cm. Importantly, differentiated Caco-2 cells emulate in vivo behaviour by increasing Cm secretion in response to oleic acid, and decreasing secretion in response to Pluronic L81. McA- RH7777 cells are a commonly used hepatoma cell line that produces apoB48- containing lipoproteins, as well as VLDL-apoB 100.

In the McA-RH7777 cell line, high levels of Sarlb expression increase the secretion of apoB48- and apoBlOO-containing lipoproteins by around 1.75-fold, whereas high levels of Sarla massively reduce the secretion of these lipoproteins (Fig. 20). The Sarlb result is particularly important as this suggests that Sarlb may promote the secretion of VLDL, as well as being obligatory for the secretion of Cm. This would accord with a report of fatty liver in two sibs with CMRD, which presumably relates to impaired trafficking of NLDL.

These studies indicate that: (1) endogenous Sarlb is limiting for the intracellular transport of nascent apoB-containing lipoproteins in McA-RH7777 cells; (2) Sarla may act through a dominant-negative mechanism to effectively reduce the availability of Sarlb for Sarlb-mediated transport of apoB-containing lipoproteins in COPπ- coated vesicles. If so, over-expression of Sarlb in cells over-expressing Sarla may over-come the observed transport defect.

The over-expression of Sarlb therefore promotes the secretion of hepatic apoB- containing lipoproteins (i.e. very low density lipoproteins), as well as chylomicrons. In lipid metabolism disorders, such as dyslipidemia and/or hyperlipidemia associated with obesity, insulin resistance, type H diabetes, and atheroscelerosis, there would therefore be a need to reduce Sarlb activity so that less lipid is absorbed, and released into blood in chylomicrons and very low density lipoproteins

Further, in CMRD, AD, and CMRD with MSS there is a failure to absorb lipid from the intestine. The consequences of this can be considered to be the reverse of the problems that lead to obesity, insulin resistance, type H diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia. Therefore, the identification of defective SARA2 gene in CMRD, AD, and CMRD with MSS leads to the suggestion that other disorders of lipid metabolism, such as obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia, and hyperlipidemia can be prevented and/or treated by blocking and/or reducing normal Sarlb activity. By blocking Sarlb activity, CMRD may be mimicked and furthermore lipid absorption can be reduced.

The nucleic acid sequence encoding Sarlb (designated SARA2) is given in Figure la. The amino acid sequence for Sarlb is given in Figure lb.

It should be noted that Sarlb protein is a small GTPase and that the process of GTP hydrolysis is necessary for the movement of chylomicrons between the endoplasmic reticulum and the Golgi apparatus. In Table 2, all of the missense mutations in Sarlb interfere with GDP/GTP nucleotide (GNP) binding to the polypeptide and therefore by presumption with hydrolysis of this nucleotide. GTP hydrolysis leads to and is accelerated by the association of Sarlb with the other components of the so-called cytoplasmic coat II complexes (COPII) 2 vesicle lipid transport machinery. These include Sec23/24 and Seel 3/31 as noted above. Therefore the identification of agents that inhibit native Sarlb activity and especially GTP hydrolysis in native Sarlb polypeptide and the interaction with Sec23/24 and Seel 3/31 can lead to therapeutic approaches to obesity, insulin resistance, type II diabetes, atheroscelerosis, dyslipidemia and/or hyperlipidemia.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

Claims

Claims

1. An isolated or recombinant nucleic acid molecule comprising: a) the DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent; b) a fragment of the sequence of a), the fragment comprising at least one of the mutations defined in Table 1 ; c) a sequence which is complementary to the sequence of a) or b); or d) a sequence which codes for the same polypeptide as the sequence a), b) or c).

2. An isolated or recombinant nucleic acid molecule comprising: a) a fragment of the DNA sequence shown in Figure 12a, or its RNA equivalent, the fragment including the mutation in bold and as defined in Table 1; b) a sequence which is complementary to the sequence of a); or c) a sequence which codes for the same polypeptide as the sequence a) or b).

3. A method for screening for and/or diagnosis of a disorder of lipid metabolism in a subject, the method comprising the step of detecting and/or quantifying the amount of a nucleic acid molecule as defined in claim 1 or claim 2 in a biological sample obtained from said subject.

4. A pair of nucleic acid primers, one of which can hybridise to the sense strand of the DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent upstream of one of the mutations defined in Table 1 , and the other of which can hybridise to the antisense strand of the

DNA sequence shown in Figure 2, 4, 6, 8, 10, 12a, 12b, 12c or 14 with or without the nucleotides boxed, or its RNA equivalent respectively downstream of said mutation.

5. The nucleic acid molecule of claim 1 or 2 and/or the nucleic acid primers of claim 4 in the manufacture of a diagnostic for the diagnosis of a disorder of lipid metabolism.

6. A method for screening and/or diagnosis of a disorder of lipid metabolism in a subject, the method comprising the steps of: a) administering a nucleic acid molecule as claimed in claim 1 and/or claim 2 to a biological sample obtained from a subject; and b) detecting and/or quantifying hybridisation of the nucleic acid molecule as claimed in claim 1 and/or claim 2 in the biological sample.

7. A method for screening for and/or diagnosis of a disorder of lipid metabohsm in a subject, the method comprising the steps of: a) administering nucleic acid primers as claimed in claim 4 to a biological sample obtained from a subject; b) hybridising of the nucleic acid primers to a nucleic acid molecule in the biological sample; c) amplifying the nucleic acid molecule with the nucleic acid primers; and d) comparing the amplified nucleic acid molecule with a nucleic acid molecule as claimed in claim 1 or claim 2.

8. A nucleic acid molecule comprising at least six nucleotides that is antisense to a portion of a nucleic acid molecule as claimed in claim 1 or to a portion of a nucleic acid molecule as claimed in claim 2, for use in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of lipid metabolism.

9. A method for the prophylaxis and/or treatment of a disorder of lipid metabolism comprising administering a nucleic acid molecule comprising at least six nucleotides that is antisense to a portion of a nucleic acid molecule as defined in claim 1 or to a portion of a nucleic acid molecule as claimed in claim 2 to a subject.

10. A RNA molecule, or a polynucleotide encoding such a molecule, comprising a double stranded structure which has a nucleotide sequence which is identical to a portion of the sequence of Figure 2, 4, 6, 8, 10, 12a,12b, 12c or 14 with or without the nucleotides boxed, or which includes the respective mutation defined in Table 1.

11. The use of a RNA molecule, or polynucleotide encoding such a molecule as claimed in claim 10, in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of lipid metabohsm.

12. A method for the prophylaxis and/or treatment of a disorder of hpid metabohsm, comprising adtnimstering to a subject a RNA molecule or polynucleotide encoding such a molecule as claimed in claim 10.

13. An isolated or recombinant polypeptide comprising: a) the amino acid sequence shown in Figure 3, 5, 7, 9, 11, 13a, 13b or 15; or b) a fragment of a polypeptide as defined in a), which is at least 5 amino acids long and includes at least one of the mutations defined in Table 1.

14. The use of a polypeptide as claimed in claim 13 in the manufacture of a medicament for the treatment and/or prophylaxis of a disorder of hpid metabolism, wherein the medicament is a vaccine.

15. The use of a polypeptide as claimed in claim 13 in the preparation of an immunogenic composition, preferably a vaccine.

16. The use of an immunogenic composition as claimed in claim 15 in inducing an immune response in a subject.

17. A method for the treatment and/or prophylaxis of a disorder of lipid metabolism in a subject, or of vaccinating a subject against a disorder of lipid metabolism , which comprises the step of administering to the subject an effective amount of a polypeptide as claimed in claim 13, preferably as a vaccine.

18. A method of diagnosis of a disorder of lipid metabolism in a subj ect, the method comprising detecting and/or quantifying the amount of a polypeptide as claimed in claim 13 in a biological sample obtained from said subject.

19. A method as claimed in claim 18, wherein an antibody is used for detecting and/or quantifying the amount of a polypeptide.

20. An antibody which binds to at least one polypeptide as claimed in claim 13.

21. An antibody as claimed in claim 20, wherein the antibody binds specifically to a polypeptide as claimed in claim 13, for example by recognising a region encoding the mutation.

22. The use of an antibody as defined in claim 20 or 21 for screening for and/or diagnosis of a disorder of hpid metabolism.

23. A method for the screening for and/or diagnosis of a disorder of hpid metabohsm in a subject, which comprises detecting and/or quantifying the amount of a polypeptide as claimed in claim 13 in a biological sample obtained from said subject using an antibody as claimed in claim 20 or 21.

24. A method for the prophylaxis and/or treatment of a disorder of lipid metabolism in a subject, which comprises administering to said subject a therapeutically effective amount of an antibody as claimed in claim 20 or 21.

25. The use of an antibody as claimed in claims 20 or 21 in the preparation of a medicament for use in the prophylaxis and or treatment of a disorder of lipid metabolism.

26. A pharmaceutical formulation comprising at least one polypeptide as claimed in claim 13, nucleic acid molecule as claimed in claim 1 or claim 2, or antibody as claimed in claim 20 or 21, optionally together with one or more pharmaceutically acceptable excipients, carriers or diluents.

27. A method for identifying agents that have an inhibitory effect on the expression or activity of a polypeptide as claimed in claim 13, comprising contacting a nucleic acid molecule as claimed in claim 1 or 2, or a polypeptide as claimed in claim 13, with a candidate agent, and determining if the agent inhibits expression of the nucleic acid molecule or the activity of the polypeptide.

28. A method for the prophylaxis and/or treatment of a disorder of lipid metabolism, comprising blocking and/or reducing Sarlb activity in a subject.

29. The method as claimed in claim 28, wherein the method comprises decreasing transcription of Sarlb gene (SARA2).

30. The method as claimed in claim 28, wherein the method comprises reducing Sarlb mRNA levels.

31. The method as claimed in claim 28, wherein the method comprises decreasing GTPase activity of Sarlb protein.

32. The method as claimed in claim 28, wherein the method comprises interfering with the interaction of Sarlb protein with other proteins that augment GTP hydrolysis, and chylomicron and very low density hpoprotein export from the intestinal absorption cell and hepatocyte, respectively.

33. A method for identifying agents that have an inhibitory effect on the expression or activity of Sarlb polypeptide comprising contacting Sarlb gene (SARA2) or Sarlb polypeptide with a candidate agent, and determining if the agent inhibits expression of Sarlb gene (SARA2) or activity of Sarlb polypeptide.

34. A method as claimed in claim 33, comprising: i) contacting cells expressing Sarlb with an agent; and ii) determining whether secretion of chylomicrons and/or apoB is decreased in the presence of the agent.

35. A method as claimed in claim 33, wherein the activity of Sarlb is assessed by detecting GTP hydrolysis, the interaction of Sarlb with Sec23/24 and/or Secl3/31, or by measuring vesicle fusion.

36. A method as claimed in claim 35, wherein the method comprises: i) contacting a preparation containing Sarlb, or cells expressing the Sarlb with a test agent or a control and GTP; ii) measuring GTP hydrolysis; and iii) detertnining whether GTP hydrolysis is affected in the presence of the agent.

37. A method as claimed in claim 36, wherein the GTP is labelled.

38. A method as claimed in claim 37, wherein the GTP is fluorescently or radioactively labelled.

39. A method as claimed in claim 37 or 38, wherein the GTP hydrolysis is measured by detecting release of labelled phosphate from the GTP.

40. A method as claimed in claim 35, wherein the method comprises: i) contacting an agent with Sarlb and Sec23/24 and/or Sarlb and Secl3/31; and ii) detecting whether the agent inhibits the interaction.

41. A method for the prophylaxis and/or treatment of a disorder of lipid metabolism in a subject, comprising administering to said subject a therapeutically effective amount of an agent identified in a method as claimed in any one of claims 33 to 40.

2. The use of an agent identified in a method as claimed in any one of claims 33 to 40 in the manufacture of a medicament for the prophylaxis and/or treatment of a disorder of lipid metabolism.