WO2004053128A1 - Mutations in gaba-b receptor 1 associated with epilepsy - Google Patents

Abstract

An isolated mammalian nucleic acid encoding a mutant GABABR1 protein wherein mutation event has occurred so as to produce an epilepsy phenotype.

Description

MUTATIONS IN GABA-B RECEPTOR 1 ASSOCIATED WITH EPILEPSY

Technical Field

The present invention is concerned with mutations associated with epilepsy.

Background Art

Epilepsies constitute a diverse collection of brain disorders that affect about 3% of the population at some time in their lives. An epileptic seizure can be defined as an episodic change in behaviour caused by the disordered firing of populations of neurons in the central nervous system. This results in varying degrees of involuntary muscle contraction and often a loss of consciousness. Epilepsy syndromes have been classified into more than 40 distinct types based upon characteristic symptoms, types of seizure, cause, age of onset and EEG patterns (Commission on Classification and Terminology of the International League Against Epilepsy, 1989) . However the single feature that is common to all syndromes is the persistent increase in neuronal excitability that is both occasionally and unpredictably expressed as a seizure.

A genetic contribution to the aetiology of epilepsy has been estimated to be present in approximately 40% of affected individuals (Gardiner, 2000) . As epileptic seizures may be the end-point of a number of molecular aberrations that ultimately disturb neuronal synchrony, the genetic basis for epilepsy is likely to be heterogeneous. There are over 200 endelian diseases which include epilepsy as part of the phenotype. In these diseases, seizures are symptomatic of underlying neurological involvement such as disturbances in brain structure or function. In contrast, there are also a number of "pure" epilepsy syndromes in which epilepsy is the sole manifestation in the affected individuals. These are termed idiopathic and account for over 60% of all epilepsy cases. Idiopathic epilepsies have been further divided into partial and generalized sub-types. Partial (focal or local) epileptic fits arise from localized cortical discharges, so that only certain groups of muscles are involved and consciousness may be retained. However, in generalized epilepsy, EEG discharge shows no focus such that all subcortical regions of the brain are involved. Although the observation that generalized epilepsies are frequently inherited is understandable, the mechanism by which genetic defects, presumably expressed constitutively in the brain, give rise to partial seizures is less clear.

Two broad groups of IGE are now known - the classical idiopathic generalized epilepsies (Commission on Classification and Terminology of the International League Against Epilepsy, 1989) and the newly recognized genetic syndrome of generalized epilepsy with febrile seizures plus (GEFS⁺) (Scheffer and Berkovic, 1997; Singh et al., 1999) .

The classical GEs are divided into a number of clinically recognizable but overlapping sub-syndromes including childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy etc (Commission on Classification and Terminology of the International League Against Epilepsy, 1989; Roger et al., 1992). The sub- syndromes are identified by age of onset and the pattern of seizure types (absence, myoclonus and tonic-clonic) .

GEFS⁺ was originally recognized through large multi- generation families and comprises a variety of sub- syndromes. Febrile seizures plus (FS⁺) is a sub-syndrome where children have febrile seizures occurring outside the age range of 3 months to 6 years, or have associated febrile tonic-clonic seizures. Many family members have a phenotype indistinguishable from the classical febrile convulsion syndrome and some have FS⁺ with additional absence, myoclonic, atonic, or complex partial seizures. The severe end of the GEFS⁺ spectrum includes myoclonic- astatic epilepsy. In terms of the molecular genetics of classical IGΞs, a number of linkage analysis studies have been performed on classical remitting childhood absence epilepsy. In a family with persisting absence evolving into a juvenile myoclonic epilepsy phenotype, linkage to chromosome lp has been claimed. An Indian pedigree with persisting absence and tonic-clonic seizures may link to 8q24. Linkage to this region was also suggested by a non-parametric analysis in IGE, irrespective of subsyndrome, but was not confirmed in another study. Other loci for IGEs that have been reported in single studies include 3pl4, 8p, 18 and possibly 5p. However to date, genes responsible for epilepsy in these families and mapping to these regions have not been found. In so far as GEFS⁺ is concerned, linkage analysis on rare multi-generation large families with clinical evidence of a major autosomal dominant gene have demonstrated loci on chromosomes 19q and 2q. Both the 19q and 2q GEFS⁺ loci have been confirmed in independently ascertained large families, and genetic defects at these loci have been identified. Families linked to 19q are known and a mutation in the gene for the βl subunit of the neuronal sodium channel (SCN1B) has been identified (Wallace et al., 1998). Families linked to 2q are also known and mutations in the pore-forming α subunit of the neuronal sodium channel (SCN1A) have been identified (PCT/AU01/01648; Wallace et al., 2001J; Escayg et al., 2000) . Studies on the more common small families with GEFS⁺ have not revealed these or other mutations to date. In addition to the SCNlB and SCNlA mutations in GEFS⁺, other gene defects have been discovered for human idiopathic epilepsies through the study of large families. Mutations in the alpha-4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) occur in the focal epilepsy syndrome of autosomal dominant nocturnal frontal lobe epilepsy (Australian patent AU-B-56247/96; Steinlein et al., 1995) and mutations in two potassium channel genes (KCNQ2 and KCNQ3) were identified in benign familial neonatal convulsions (Singh et al., 1998; Biervert et al., 1998; Charlier et al., 1998). Although initially regarded as a special form of IGΞ, this unusual syndrome is probably a form of inherited focal epilepsy.

Further to these studies, mutations in the beta-2 subunit (CHRNB2) of the neuronal nicotinic acetylcholine receptor have been identified (PCT/AU01/00541; Phillips et al., 2001). In combination, these studies support the proposition that the idiopathic epilepsies comprise a family of channelopathies with mutations in ion channel subunits of voltage-gated or ligand-gated types.

Further evidence for this comes from the identification of mutations in subunits of the ligand- gated GABA-A receptor. Mutations in the gamma-2 (GABRG2), delta (GABRD) , alpha-6 (GABRA6), pi (GABRPi) and epsilon (GABRΞ) subunits of the GABA-A receptor have been identified in childhood absence epilepsy, febrile seizures (including febrile seizures plus) and myoclonic epilepsy (PCT/AU01/00729; PCT/AU02/00910; Wallace et al., 2001a; Harkin et al . , 2002).

Gamma-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the central nervous system. GABA-ergic inhibition is mediated by two major classes of receptors, type A and type B. Type B (GABA-B) receptors are members of the class of receptors coupled to G- proteins and mediate a variety of inhibitory effects via secondary messenger cascades. Class A (GABA-A) receptors are ligand-gated chloride channels that mediate rapid inhibition.

There are approximately 16 separate, but related, genes which encode GABA-A receptor subunits. These are grouped on the basis of sequence identity into α, β, γ, δ, ε and p subunits and there are six α subunits (designated αl, 0C2 etc), three β subunits, three γ subunits and three p subunits. Each GABA-A receptor comprises five subunits which may each, at least in theory, be selected from any one of these subunits .

GABA-B receptors were discriminated from GABA-A receptors based on their sensitivity to baclofen (Bowery, 1993) and their dependence on G-proteins for effector coupling. The molecular targets of GABA-B receptor activation are Ca++ and K+ channels whose gating is directly modulated by liberation of G-protein following binding of GABA to the receptor.

Stimulation of the GABA-B receptor inhibits release of neurotransmitters including glutamate, GABA and acetylcholine through modulation of the Ca++ and K+ channels at presynaptic nerve terminals. GABA-B receptors also mediate a postsynaptic hyperpolarisation of neuronal cell bodies via the opening of G-protein-gated inwardly rectifying potassium channels (GIRKs) .

The GABA-B receptor exists as a heterodimer made up of the GABABRl and GABABR2 proteins. The coupling of these two proteins enables the receptor to be fully expressed in the plasma membrane of cells allowing the recording of GABA-B mediated changes in K+ channel conductance (Jones et al., 1998; White et al., 1998; Kaupmann et al., 1998).

The gene encoding the GABABRl protein has been mapped to chromosome 6p21.3 (Peters et al., 1998). From linkage analysis studies of classical IGEs such as juvenile myoclonic epilepsy (EJM) , a locus for these IGEs has been identified in proximity or within the HLA region on chromosome 6p (Greenberg et al., 1988J) . This finding was supported by two collaborating laboratories, in separate patient samples, and subsequently three groups provided further evidence for a 6p locus for juvenile myoclonic epilepsy in some, but not all, of their families. The functional properties of the GABA-B receptor together with localisation of the GABABRl gene to the candidate region of the EJM locus suggests that this gene is a plausible candidate gene for common IGE syndromes. However subsequent studies have failed to identify disease-causing mutations in this gene. The inventors of the current invention have analysed the GABABRl gene for mutations in IGE affected individuals and have identified a disease-causing mutation. This novel finding will be important for further applications such as the screening for drugs which interact with GABA-B receptors containing the mutant subunit and their subsequent application for the treatment of individuals with epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

Disclosure of the Invention

The present inventors have determined that the GABABRl gene of the GABA-B receptor is associated with epilepsy through the identification of a mutation in the GABABRl gene.

According to one aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mutant GABABRl protein wherein a mutation event has occurred so as to produce an epilepsy phenotype. The mutation could disrupt the functioning of an assembled GABA-B receptor but, equally well, the mutation could disrupt the assembly of a GABA-B receptor so as to produce an epilepsy phenotype or produce an epilepsy phenotype through some other mode of action. In one embodiment of the invention, the mutation lies in the large extracellular loop of the amino terminal domain of GABABRl.

In one form of the invention, the mutation is in exon 6 of the large extracellular loop of the amino terminal domain of GABABRl and results in the replacement of an aspartic acid residue with an asparagine residue at amino acid position 208 (based on the numbering of GABABRl isoform la) . The D208N mutation occurs as a result of a G to A nucleotide substitution at position 622 of the GABABRl coding sequence (based on the numbering of GABABRl isoform la) as illustrated in SEQ ID NO: 1. Preferably the mutation creates a phenotype of febrile seizures . In a further form of the invention, the mutation is in exon 1 of the large extracellular loop of the amino terminal domain of GABABRl and results in the replacement of an alanine residue with an aspartic acid residue at amino acid position 5 (based on the numbering of GABABRl isoform lb) or in the replacement of a proline residue with a serine residue at amino acid 46 (based on the numbering of GABABRl isoform lb) . The A5D mutation occurs as a result of a C to A nucleotide substitution at position 14 of the GABABRl coding sequence (based on the numbering of GABABRl isoform lb) as illustrated in SEQ ID NO: 3. The P46S mutation occurs as a result of a C to T nucleotide substitution at position 136 of the GABABRl coding sequence (based on the numbering of GABABRl isoform lb) as illustrated in SEQ ID NO: 5. Preferably the A5D mutation creates a phenotype of idiopathic generalised epilepsy or photosensitive idiopathic generalised epilepsy. Preferably the P46S mutation creates a phenotype of idiopathic generalised epilepsy, juvenile myoclonic epilepsy, or photosensitive juvenile myoclonic epilepsy.

In a further form of the invention, the mutation is in exon 8 of the large extracellular loop of the amino terminal domain of GABABRl and results in the replacement of a C nucleotide with a G nucleotide at position 945 of the GABABRl coding sequence (based on the numbering of

GABABRl isoform la) as illustrated in SEQ ID NO: 7. Preferably this mutation creates a phenotype of idiopathic generalised epilepsy.

In addition, the polymorphisms identified in Table 1 form part of the invention (SEQ ID Numbers: 8-20). These polymorphisms may reflect changes in GABABRl which result in subtle changes of function of the GABA-B receptor. These subtle changes may predispose individuals to epilepsy and when expressed in combination with other gene mutations, such as those in ion channel genes, may lead to specific sub-types of the disease (as described in PCT/AU01/00872, the contents of which are incorporated herein by reference.

In another aspect of the present invention there is provided an isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7-20.

In another aspect of the present invention there is provided an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7-20. The nucleotide sequences of the present invention can be engineered using methods accepted in the art for a variety of purposes. These include, but are not limited to, modification of the cloning, processing, and/or expression of the gene product. PCR reassembly of gene fragments and the use of synthetic oligonucleotides allow the engineering of the nucleotide sequences of the present invention. For example, oligonucleotide-mediated site- directed utagenesis can introduce further mutations that create new restriction sites, alter expression patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number of polynucleotide sequences, some that may have minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention includes each and every possible variation of a polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequences of the present invention, and all such variations are to be considered as being specifically disclosed.

The nucleic acid molecules of this invention are typically DNA molecules, and include cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified, or may contain non-natural or derivatised nucleotide bases as will be appreciated by those skilled in the art. Such modifications include labels, methylation, intercalators, alkylators and modified linkages . In some instances it may be advantageous to produce nucleotide sequences possessing a substantially different codon usage than that of the polynucleotide sequences of the present invention. For example, codons may be selected to increase the rate of expression of the peptide in a particular prokaryotic or eukaryotic host corresponding with the frequency that particular codons are utilized by the host . Other reasons to alter the nucleotide sequence without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half- life, than transcripts produced from the naturally occurring mutated sequence.

The invention also encompasses production of nucleic acid sequences of the present invention entirely by synthetic chemistry. Synthetic sequences may be inserted into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements may include regulatory sequences, promoters, 5" and 3* untranslated regions and specific initiation signals (such as an ATG initiation codon and

Kozak consensus sequence) which allow more efficient translation of sequences encoding the polypeptides of the present invention. In cases where the complete coding sequence, including the initiation codon and upstream regulatory sequences, are inserted into the appropriate expression vector, additional control signals may not be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals as described above should be provided by the vector. Such signals may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf et al., 1994).

The invention also includes nucleic acid molecules that are the complements of the sequences described herein.

The present invention allows for the preparation of purified polypeptide or protein from the polynucleotides of the present invention, or variants thereof. In order to do this, host cells may be transformed with a novel nucleic acid molecule as described above. Typically said host cells are transfected with an expression vector comprising a DNA molecule according to the invention. A variety of expression vector/host systems may be utilized to contain and express sequences encoding polypeptides of the invention. These include, but are not limited to, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus) ; or mouse or other animal or human tissue cell systems.

Mammalian cells can also be used to express a protein using a vaccinia virus expression system. The invention is not limited by the host cell or vector employed.

The polynucleotide sequences, or variants thereof, of the present invention can be stably expressed in cell lines to allow long term production of recombinant proteins in mammalian systems. Sequences encoding the polypeptides of the present invention can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. The selectable marker confers resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode a protein may be designed to contain signal sequences which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, glycosylation, phosphorylation, and acylation. Post-translational cleavage of a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells having specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO or HeLa cells), are available from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the foreign protein.

When large quantities of the protein product of the gene are needed, such as for antibody production, vectors which direct high levels of expression of this protein may be used, such as those containing the T5 or T7 inducible bacteriophage promoter. The present invention also includes the use of the expression systems described above in generating and isolating fusion proteins which contain important functional domains of the protein. These fusion proteins are used for binding, structural and functional studies as well as for the generation of appropriate antibodies .

In order to express and purify the protein as a fusion protein, the appropriate cDNA sequence is inserted into a vector which contains a nucleotide sequence encoding another peptide (for example, glutathionine succinyl transferase) . The fusion protein is expressed and recovered from prokaryotic or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence. The desired protein is then obtained by enzymatic cleavage of the fusion protein.

Fragments of the polypeptides of the present invention may also be produced by direct peptide synthesis using solid-phase techniques. Automated synthesis may be achieved by using the ABI 431A Peptide Synthesizer (Perkin-Elmer) . Various fragments of this protein may be synthesized separately and then combined to produce the full-length molecule.

According to still another aspect, the present invention provides an isolated mammalian polypeptide, said polypeptide being a mutant GABABRl protein wherein a mutation event has occurred so as to produce an epilepsy phenotype .

In one form of the invention the mutation event is a substitution in which an aspartic acid residue is replaced with an asparagine residue in the large extracellular loop of the amino terminal domain of GABABRl. Preferably the substitution is a D208N transition (based on the numbering of GABABRl isoform la) as illustrated in SEQ ID NO:2.

In a further form of the invention the mutation event is a substitution in which an alanine residue is replaced with an aspartic acid residue, or in which a proline residue is replaced with a serine residue in the large extracellular loop of the amino terminal domain of GABABRl. Preferably the substitution is an A5D or P46S transition (based on the numbering of GABABRl isoform lb) as illustrated in SEQ ID Numbers: 4 and 6.

In another aspect of the present invention there is provided an isolated polypeptide comprising the amino acid sequence set forth in any one of SEQ ID Numbers:2, 4 or 6. In another aspect of the present invention there is provided an isolated polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NO:2, 4 or 6. According to still another aspect of the invention, there is provided a GABA-B receptor that incorporates a GABABRl protein as described above. Preferably, there is a mutation at amino acid position 208 of the GABABRl protein (isoform la) of the isolated receptor complex, amino acid position 5 of the GABABRl protein (isoform lb) of the isolated receptor complex, or amino acid position 46 of the GABABRl protein (isoform lb) of the isolated receptor complex. Typically the mutation is a D208N mutation in GABABRl (isoform la), an A5D mutation in GABABRl (isoform lb) or a P46S mutation in GABABRl (isoform lb) .

According to still another aspect of the present invention there is provided an expression vector comprising a nucleic acid molecule as described above.

According to still another aspect of the present invention there is provided a cell comprising a nucleic acid molecule as described above.

According to still another aspect of the present invention there is provided a method of preparing a polypeptide, said polypeptide being a mutant GABABRl protein of a GABA-B receptor, comprising the steps of:

(1) culturing a cell as described above under conditions effective for polypeptide production; and

(2) harvesting the polypeptide.

The mutant GABABRl protein may be allowed to assemble with other subunits constituting the GABA-B receptor that are co-expressed by the cell (such as the GABABR2 protein), whereby the assembled mutant GABA-B receptor complex is harvested.

According to still another aspect of the invention there is provided a polypeptide which is the product of the process described above . Substantially purified protein or fragments thereof can then be used in further biochemical analyses to establish secondary and tertiary structure. Such methodology is known in the art and includes, but is not restricted to, X-ray crystallography of crystals of the proteins or of the assembled ion channel incorporating the proteins or by nuclear magnetic resonance (NMR) . Determination of structure allows for the rational design of pharmaceuticals to interact with the mutated GABA-B receptor as a whole or through interaction with the mutant GABABRl protein of the mutant receptor (see drug screening below) , alter the overall GABA-B receptor charge configuration or charge interaction with other proteins, or to alter its function in the cell .

It will be appreciated that having identified for the first time a mutation in the GABABRl gene responsible for epilepsy, the mutant GABABRl protein will enable therapeutic methods for the treatment of epilepsy as well as other disorders associated with GABA-B receptor dysfunction and also enables methods for the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

Therapeutic Applications

According to still another aspect of the invention there is provided a method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction, comprising administering a selective antagonist, agonist or modulator of a polypeptide as described above to a subject in need of such treatment.

In still another aspect of the invention there is provided the use of a selective antagonist, agonist or modulator of a polypeptide as described above in the manufacture of a medicament for the treatment of the disorder.

In one aspect, a suitable antagonist, agonist or modulator will restore wild-type function to GABA-B receptors containing GABABRl mutations that form part of this invention, or will negate the effects the mutant receptor has on cell function. Using methods well known in the art, a mutant GABA-B receptor, or GABABRl protein of the receptor, that is causative of the disease may be used to produce antibodies specific for the mutant receptor or GABABRl protein of the receptor or to screen libraries of pharmaceutical agents to identify those that bind the mutant receptor or GABABRl protein of the receptor.

In one aspect, an antibody, which specifica,lly binds to a mutant GABA-B receptor or mutant GABABRl protein of the invention, may be used directly as an agonist, antagonist or modulator, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues that express the mutant receptor.

In a still further aspect of the invention there is provided an antibody which is immunologically reactive with a polypeptide as described above, but not with a wild-type GABA-B receptor or GABABRl protein thereof.

In particular, there is provided an antibody to an assembled GABA-B receptor containing a mutation in the GABABRl protein that forms part of the receptor, which is causative of epilepsy or another disorder associated with GABA-B receptor dysfunction. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies as would be understood by the person skilled in the art.

For the production of antibodies, various hosts including rabbits, rats, goats, mice, humans, and others may be immunized by injection with a polypeptide as described above or with any fragment or oligopeptide thereof which has immunogenic properties . Various adjuvants may be used to increase immunological response and include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface-active substances such as lysolecithin. Adjuvants used in humans include BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to the mutant GABA-B receptor, or mutant GABABRl protein thereof, have an amino acid sequence consisting of at least 5 amino acids, and, more preferably, of at least 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of GABABRl amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to a mutant GABA-B receptor, or mutant GABABRl protein thereof, may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique. (For example, see Kohler et al., 1975; Kozbor et al., 1985; Cote et al., 1983; Cole et al., 1984) .

Monoclonal antibodies produced may include, but are not limited to, mouse-derived antibodies, humanised antibodies and fully human antibodies. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (For example, see Orlandi et al., 1989; Winter and Milstein, 1991).

Antibody fragments which contain specific binding sites for a mutant mutant GABA-B receptor, or mutant GABABRl protein thereof, may also be generated. For example, such fragments include, F(ab*)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (For example, see Huse et al., 1989). Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between an ion channel and its specific antibody. A two-site, monoclonal- based imunoassay utilizing antibodies reactive to two non-interfering GABA-B receptor epitopes is preferred, but a competitive binding assay may also be employed. In a further aspect of the invention there is provided a method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction, comprising administering an isolated nucleic acid molecule which is the complement (antisense) of any one of the nucleic acid molecules described above and which encodes an RNA molecule that hybridizes with the mRNA encoding a mutant GABABRl of the invention, to a subject in need of such treatment .

In a still further aspect of the invention there is provided the use of an isolated nucleic acid molecule which is the complement (antisense) of a nucleic acid molecule of the invention and which encodes an RNA molecule that hybridizes with the mRNA encoding a mutant GABABRl of the invention, in the manufacture of a medicament for the treatment of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

Typically, a vector expressing the complement (antisense) of the polynucleotides of the invention may be administered to a subject in need of such treatment. Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (For example, see Goldman et al . , 1997).

Additional antisense or gene-targeted silencing strategies may include, but are not limited to, the use of antisense oligonucleotides, injection of antisense RNA, transfection of antisense RNA expression vectors, and the use of RNA interference (RNAi) or short interfering RNAs (siRNA) . Still further, catalytic nucleic acid molecules such as DNAzymes and ribozymes may be used for gene silencing (Breaker and Joyce, 1994; Haseloff and Gerlach, 1988) . These molecules function by cleaving their target mRNA molecule rather than merely binding to it as in traditional antisense approaches.

In a further aspect, a suitable agonist, antagonist or modulator may include peptides, phosphopeptides or small organic or inorganic compounds that can restore wild-type activity of GABA-B receptors containing mutations in GABABRl protein of the receptor as described above.

Peptides, phosphopeptides or small organic or inorganic compounds suitable for therapeutic applications may be identified using nucleic acids and peptides of the invention in drug screening applications as described below. Molecules identified from these screens may also be of therapeutic application in affected individuals carrying other GABA-B receptor mutations, or individuals carrying mutations in genes other than those comprising the GABA-B receptor, if the molecule is able to correct the common underlying functional deficit imposed by these mutations and those of the invention. There is therefore provided a method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising administering a compound that is a suitable agonist, antagonist or modulator of a GABA-B receptor and that has been identified using mutant GABABRl of the invention.

In some instances, an appropriate approach for treatment may be combination therapy. This may involve the administering an antibody or complement (antisense) to a mutant GABA-B receptor, or mutant GABABRl protein thereof, of the invention to inhibit its functional effect, combined with administration of wild-type GABABRl which may restore levels of wild-type GABA-B receptor formation to normal levels. Wild-type GABABRl can be administered using gene therapy approaches as described above for complement administration.

There is therefore provided a method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising administration of an antibody or complement to a mutant GABA-B receptor, or mutant GABABRl protein thereof, of the invention in combination with administration of wild-type GABABRl. In still another aspect of the invention there is provided the use of an antibody or complement to a mutant GABA-B receptor, or mutant GABABRl protein thereof, of the invention in combination with the use of wild-type GABABRl, in the manufacture of a medicament for the treatment of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

In further embodiments, any of the agonists, antagonists, modulators, antibodies, complementary sequences or vectors of the invention may be administered alone or in combination with other appropriate therapeutic agents. Selection of the appropriate agents may be made by those skilled in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficacy with lower dosages of each agent may be possible, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Drug Screening

According to still another aspect of the invention, nucleic acid molecules of the invention as well as peptides of the invention, particularly purified mutant GABABRl protein and cells expressing these, are useful for the screening of candidate pharmaceutical compounds for the treatment of epilepsy as well as other as other disorders associated with GABA-B receptor dysfunction.

Still further, it provides the use of a mutant GABA-B receptor polypeptide complex for the screening of candidate pharmaceutical compounds.

Still further, it provides the use wherein high throughput screening techniques are employed.

Compounds that can be screened in accordance with the invention include, but are not limited to peptides (such as soluble peptides), phosphopeptides and small organic or inorganic molecules (such as natural product or synthetic chemical libraries and peptidomimetics) .

In one embodiment, a screening assay may include a cell-based assay utilising eukaryotic or prokaryotic host cells that are stably transformed with recombinant molecules expressing the polypeptides or fragments of the invention, in competitive binding assays. Binding assays will measure the formation of complexes between a mutant GABA-B receptor, or mutant GABABRl protein thereof, and the compound being tested, or will measure the degree to which a compound being tested will inhibit or restore the formation of a complex between a mutant GABA-B receptor, or mutant GABABRl protein thereof, and its interactor or ligand. The invention is particularly useful for screening compounds by using the polypeptides of the invention in transformed cells, transfected or injected oocytes, or animal models bearing mutated GABABRl such as transgenic animals or gene targeted (knock-in) animals (see transformed hosts) . Drug candidates can be added to cultured cells that express a mutant GABABRl protein (appropriate wild-type GABA-B receptor subunits such as GABABR2 should also be expressed for correct receptor assembly) , can be added to oocytes transfected or injected with a mutant GABABRl protein (appropriate wild-type GABA- B receptor subunits such as GABABR2 must also be injected for correct receptor assembly) , or can be administered to an animal model expressing a mutant GABABRl protein. Determining the ability of the test compound to modulate mutant GABA-B receptor activity can be accomplished by a number of techniques known in the art. These include for example measuring the effect on the flow of potassium ions through G-protein-gated inwardly rectifying potassium channels that are activated by the receptor as compared to the current of a cell or animal containing only wild-type GABA-B receptors. In order for this type of electrophysiological assay to be performed, the GABA-B receptor effectors (for example GIRK1 or GIRK2) wpuld also need to be expressed in the transformed ςells or transfected oocytes .

Current in cells can be measured by a number of approaches including the patch-clamp technique (methods described in Hamill et al, 1981) or using fluorescence based assays as are known in the art (see Gonzalez et al.

1999) . Drug candidates that alter the current to a more normal level are useful for treating or preventing epilepsy as well as other disorders associated with GABA-B receptor dysfunction. Non cell-based assays may also be used for identifying compounds that can inhibit or restore binding between the mutant GABA-B receptors, or mutant GABABRl protein thereof, of the invention, and their interactors. Such assays are known in the art and include for example AlphaScreen technology (PerkinElmer Life Sciences, MA, USA) . This application relies on the use of beads such that each interaction partner is bound to a separate bead via an antibody. Interaction of each partner will bring the beads into proximity, such that laser excitation initiates a number of chemical reactions ultimately leading to fluorophores emitting a light signal. Candidate compounds that inhibit the binding of the mutant GABA-B receptor, or mutant GABABRl protein thereof, with its interactor will result in loss of light emission, while candidate compounds that restore the binding of the mutant GABA-B receptor, or mutant GABABRl protein thereof, with its interactor will result in positive light emission. These assays ultimately enable identification and isolation of the candidate compounds.

High-throughput drug screening techniques may also employ methods as described in WO84/03564. Small peptide test compounds synthesised on a solid substrate can be assayed for mutant GABABRl protein or mutant GABA-B receptor binding. Bound mutant GABA-B receptor or mutant GABABRl polypeptide is then detected by methods well known in the art. In a variation of this technique, purified polypeptides of the invention can be coated directly onto plates to identify interacting test compounds.

The invention also contemplates the use of competition drug screening assays in which neutralizing antibodies capable of specifically binding the mutant GABA-B receptor compete with a test compound for binding thereto. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants of the mutant receptor.

The polypeptides of the present invention may also be used for screening compounds developed as a result of combinatorial library technology. This provides a way to test a large number of different substances for their ability to modulate activity of a polypeptide. A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical applications. In addition, a mimic or mimetic of the substance may be designed for pharmaceutical use. The design of mimetics based on a known pharmaceutically active compound ("lead" compound) is a common approach to the development of novel pharmaceuticals. This is often desirable where the original active compound is difficult or expensive to synthesise or where it provides an unsuitable method of administration. In the design of a mimetic, particular parts of the original active compound that are important in determining the target property are identified. These parts or residues constituting the active region of the compound are known as its pharmacophore. Once found, the pharmacophore structure is modelled according to its physical properties using data from a range of sources including x-ray diffraction data and NMR. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be added. The selection can be made such that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, does not degrade in vivo and retains the biological activity of the lead compound. Further optimisation or modification can be carried out to select one or more final mimetics useful for in vivo or clinical testing.

It is also possible to isolate a target-specific antibody and then solve its crystal structure. In principle, this approach yields a pharmacophore upon which subsequent drug design can be based as described above. It may be possible to avoid protein crystallography altogether by generating anti-idiotypic antibodies (anti- ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analogue of the original receptor. The anti-id could then be used to isolate peptides from chemically or biologically produced peptide banks.

Another alternative method for drug screening relies on structure-based rational drug design. Determination of the three dimensional structure of the polypeptides of the invention, or the three dimensional structure of the GABA- B receptors which incorporate these polypeptides allows for structure-based drug design to identify biologically active lead compounds.

Three dimensional structural models can be generated by a number of applications, some of which include experimental models such as x-ray crystallography and NMR and/or from in silico studies of structural databases such as the Protein Databank (PDB) . In addition, three dimensional structural models can be determined using a number of known protein structure prediction techniques based on the primary sequences of the polypeptides (e.g. SYBYL - Tripos Associated, St. Louis, MO), de novo protein structure design programs (e.g. MODELER - MSI Inc., San Diego, CA, or MOE - Chemical Computing Group, Montreal, Canada) or ab initio methods as described, for example, in US Patent Numbers 5331573 and 5579250, the contents of which are incorporated herein by reference . Once the three dimensional structure of a polypeptide or polypeptide complex has been determined, structure- based drug discovery techniques can be employed to design biologically-active compounds based on these three dimensional structures. Such techniques are known in the art and include examples such as DOCK (University of

California, San Francisco) or AUTODOCK (Scripps Research Institute, La Jolla, California) . A computational docking protocol will identify the active site or sites that are deemed important for protein activity based on a predicted protein model. Molecular databases, such as the Available Chemicals Directory (ACD) are then screened for molecules that complement the protein model . Using methods such as these, potential clinical drug candidates can be identified and computationally ranked in order to reduce the time and expense associated with typical ^Λwet lab' drug screening methodologies. Compounds identified through screening procedures as described above, and which are based on the use of the mutant nucleic acid and polypeptides of the invention, can also be tested for their effect on correcting the functional deficit imposed by other gene mutations in affected individuals including other GABABRl mutations.

Such compounds form a part of the present invention, as do pharmaceutical compositions containing these and a pharmaceutically acceptable carrier.

Pharmaceutical Preparations

Compounds identified from screening assays and shown to restore GABA-B receptor wild-type activity can be administered to a patient at a therapeutically effective dose to treat or ameliorate epilepsy as well as other disorders associated with GABA-B receptor dysfunction, as described above. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorder.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from these studies can then be used in the formulation of a range of dosages for use in humans .

Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiological acceptable carriers, excipients or stabilisers which are well known. Acceptable carriers, excipients or stabilizers are non-toxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including absorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; binding agents including hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming σounterions such as sodium; and/or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG) .

The formulation of pharmaceutical compositions for use in accordance with the present invention will be based on the proposed route of administration. Routes of administration may include, but are not limited to, inhalation, insufflation (either through the mouth or nose) , oral, buccal, rectal or parental administration.

Diagnostic Applications

Polynucleotide sequences of the invention may be used for the diagnosis of epilepsy, as well as other as other disorders associated with GABA-B receptor dysfunction, and the use of the nucleic acid molecules incorporated as part of the invention in diagnosis of these disorders, or a predisposition to these disorders, is therefore contemplated.

According to one aspect of the present invention there is provided the use of a nucleic acid molecule as described above in the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

The polynucleotides that may be used for diagnostic purposes include oligonucleotide , sequences, genomic DNA and complementary RNA and DNA molecules . The polynucleotides may be used to detect and quantitate gene expression in biological samples. Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may also be used, with or without PCR amplification. To detect a specific nucleic acid sequence, hybridisation using specific oligonucleotides, restriction enzyme digest and mapping, PCR mapping, RNAse protection, and various other methods may be employed. Oligonucleotides specific to particular sequences can be chemically synthesized and labelled radioactively or nonradioactively and hybridised to individual samples immobilized on membranes or other solid-supports or in solution. The presence, absence or excess expression of mutant GABABRl may then be visualized using methods such as au oradiography, fluorometry, or colorimetry.

In a further diagnostic approach, the nucleotide sequences of the invention may be useful in assays that detect the presence of associated disorders, particularly those mentioned previously. The nucleotide sequences may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridisation complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

In order to provide a basis for the diagnosis or prognosis of epilepsy and other disorders as described above, which are associated with GABABRl mutations or variants, the nucleotide sequence of the gene can be compared between normal tissue and diseased tissue in order to establish whether the patient expresses a mutant gene.

Accordingly, in a further aspect of the present invention there is provided a method for the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising the steps of:

(1) obtaining DNA from a subject suspected of epilepsy or another disorder associated with GABA-B receptor dysfunction; and

(2) comparing the GABABRl gene of said DNA to the DNA of wild-type GABABRl.

In order to provide a basis for the diagnosis of a disorder associated with abnormal expression of GABABRl due to GABABRl gene mutations, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding GABABRl, under conditions suitable for hybridisation or amplification.

Standard hybridisation may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Another method to identify a normal or standard profile for GABABRl expression is through quantitative RT-PCR studies. RNA isolated from body cells of a normal individual is reverse transcribed and real-time PCR using oligonucleotides specific for the relevant gene is conducted to establish a normal level of expression of the gene. Standard values obtained in both these examples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder. Once the presence of a disorder is established and a treatment protocol is initiated, hybridisation assays or quantitative RT-PCR studies may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

According to a further aspect of the invention there is provided the use of a polypeptide as described above in the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction. According to a still further aspect of the invention there is provided a method for the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising the steps of:

(1) obtaining the GABA-B receptor from a subject suspected of epilepsy or another disorder associated with

GABA-B receptor dysfunction; and

(2) comparing the GABABRl subunit of said receptor with the corresponding GABABRl subunit of the wild-type GABA-B receptor. When a diagnostic assay is to be based upon mutant GABABRl proteins constituting a GABA-B receptor, a variety of approaches are possible. For example, diagnosis can be achieved by monitoring differences in the electrophoretic mobility of normal and mutant GABABRl proteins that form the GABA-B receptor. Such an approach will be particularly useful in identifying mutants in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the electrophoretic migration of the resultant protein. Alternatively, diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the various amino acid residues, or by functional assays demonstrating altered function of the gene products. In another aspect, antibodies that specifically bind mutant GABA-B receptors may be used for the diagnosis of a disorder, or in assays to monitor patients being treated with agonists, antagonists or modulators of the mutant GABA-B receptor. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for mutant GABA-B receptors include methods that utilize the antibody and a label to detect a mutant GABA-B receptor in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by covalent or non-covalent attachment of a reporter molecule.

A variety of protocols for measuring the presence of mutant GABA-B receptors, including but not restricted to, ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing a disorder. The expression of a mutant GABA-B receptor is established by combining body fluids or cell extracts taken from test mammalian subjects, preferably human, with antibody to the mutant receptor under conditions suitable for complex formation. The amount of complex formation may be quantitated by various methods, preferably by photometric means.

Antibodies specific for the mutant receptor will only bind to individuals expressing the said mutant receptor and not to individuals expressing only wild-type receptor (i.e. normal individuals) . This establishes the basis for diagnosing the disorder.

Once an individual has been diagnosed with a disorder, effective treatments can be initiated as described above.

Microarray

In further embodiments, complete cDNAs, oligonucleotides or longer fragments derived from any of the GABABRl polynucleotide sequences described herein may be used as probes in a microarray. The microarray can be used to diagnose epilepsy, as well as other disorders associated with GABA-B receptor dysfunction, through the identification of genetic variants, mutations, and polymorphisms, to understand the genetic basis of a disorder, or can be used to develop and monitor the activities of therapeutic agents.

According to a further aspect of the present invention, tissue material obtained from animal models generated as a result of the identification of specific GABABRl human mutations (see below), particularly those disclosed in the present invention, can be used in microarray experiments. These experiments can be conducted to identify the level of expression of GABABRl, or the level of expression of any cDNA clone from whole-tissue libraries, in diseased tissue as opposed to normal control tissue. Variations in the expression level of genes, including GABABRl, between the two tissues indicates their possible involvement in the disease process either as a cause or consequence of the original GABABRl mutation present in the animal model. These experiments may also be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known in the art. (For example, see Schena et al., 1996; Heller et al., 1997).

Transformed Hosts

The present invention also provides for genetically modified (knock-out, knock-in and transgenic), non-human animal models transformed with nucleic acid molecules of the invention. These animals are useful for the study of the function of a GABA-B receptor, to study the mechanisms of disease as related to a GABA-B receptor, for the screening of candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express mutant GABA-B receptor, and for the evaluation of potential therapeutic interventions.

Animal species which are suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates such as monkeys and chimpanzees. For initial studies, genetically modified mice and rats are highly desirable due to the relative ease in generating knock-in, knock-out or transgenics of these animals, their ease of maintenance and their shorter life spans. For certain studies, transgenic yeast or invertebrates may be suitable and preferred because they allow for rapid screening and provide for much easier handling. For longer term studies, non-human primates may be desired due to their similarity with humans.

To create an animal model for a mutated GABA-B receptor of the invention, several methods can be employed. These include, but are not limited to, generation of a specific mutation in a homologous animal gene, insertion of a wild type human gene and/or a humanized animal gene by homologous recombination, insertion of a mutant (single or multiple) human gene as genomic or minigene cDNA constructs using wild type or mutant or artificial promoter elements, or insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase.

To create transgenic mice in order to study gain of gene function in vivo, a GABABRl mutant of the invention can be inserted into a mouse germ line using standard techniques such as oocyte microinjection. Gain of gene function can mean the over-expression of a gene and its protein product, or the genetic complementation of a mutation of the gene under investigation. For oocyte injection, one or more copies of the mutant gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo- pregnant foster mother. The live-born mice can then be screened for integrants using analysis of tail DNA for the presence of the relevant human GABABRl gene sequence. The transgene can be either a complete genomic sequence injected as a YAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing all of the coding region and other elements found to be necessary for optimum expression.

To generate knock-out mice or knock-in mice, gene targeting through homologous recombination in mouse embryonic stem (ES) cells may be applied. Knock-out mice are generated to study loss of gene function in vivo while knock-in mice (which are preferred) allow the study of gain of function or to study the effect of specific gene mutations. Knock-in mice are similar to transgenic mice however the integration site and copy number are defined in the former.

For knock-out mouse generation, gene targeting vectors can be designed such that they delete (knock-out) the protein coding sequence of the GABABRl gene in the mouse genome. In contrast, knock-in mice can be produced whereby a gene targeting vector containing the relevant mutant GABABRl gene can integrate into a defined genetic locus in the mouse genome. For both applications, homologous recombination is catalysed by specific DNA repair enzymes that recognise homologous DNA sequences and exchange them via double crossover.

Gene targeting vectors are usually introduced into ES cells using electroporation. ES cell integrants are then isolated via an antibiotic resistance gene present on the targeting vector and are subsequently genotyped to identify those ES cell clones in which the gene under investigation has integrated into the locus of interest. The appropriate ES cells are then transmitted through the germline to produce a novel mouse strain.

In instances where gene ablation results in early embryonic lethality, conditional gene targeting may be employed. This allows genes to be deleted in a temporally and spatially controlled fashion. As above, appropriate ES cells are transmitted through the germline to produce a novel mouse strain, however the actual deletion of the gene is performed in the adult mouse in a tissue specific or time controlled manner. Conditional gene targeting is most commonly achieved by use of the cre/lox system. The enzyme cre is able to recognise the 34 base pair loxP sequence such that loxP flanked (or floxed) DNA is recognised and excised by cre. Tissue specific cre expression in transgenic mice enables the generation of tissue specific knock-out mice by mating gene targeted floxed mice with cre transgenic mice. Knock-out can be conducted in every tissue (Schwenk et al., 1995) using the *deleter' mouse or using transgenic mice with an inducible cre gene (such as those with tetracycline inducible cre genes), or knock-out can be tissue specific for example through the use of the CD19-cre mouse (Rickert et al., 1997) . According to still another aspect of the invention there is provided the use of genetically modified non- human animals as described above for the screening of candidate pharmaceutical compounds (see drug screening above) . These animals are also useful for the evaluation (eg therapeutic efficacy, toxicity, metabolism) of candidate pharmaceutical compounds, including those identified from the invention as described above, for the treatment of epilepsy as well as other as other disorders associated with GABA-B receptor dysfunction. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

Modes for Performing the Invention

Example 1: Epilepsy sample collection

A large collection of individuals affected with epilepsy have undergone careful clinical phenotyping and additional data regarding their family history has been collated. Informed consent was obtained from each individual for blood collection and its use in subsequent experimental procedures. Clinical phenotypes incorporated classical IGE cases including juvenile myoclonic epilepsy, as well as GEFS+ and febrile seizure cases. In addition idiopathic partial epilepsy cases (temporal lobe epilepsy) were included.

DNA was extracted from collected blood using the

QIAamp DNA Blood Maxi kit (Qiagen) according to manufacturers specifications or through procedures adapted from Wyman and White (1980). Stock DNA samples were kept at a concentration of 1 ug/ul.

In preparation for SSCP analysis, samples to be screened were formatted into 96-well plates at a concentration of 30 ng/ul. These master plates were subsequently used to prepare exon specific PCR reactions in the 96-well format.

Example 2 : Identification of sequence alterations in the GABABRl gene SSCP analysis of GABABRl exons followed by sequencing of SSCP bandshifts was performed on individuals constituting the 96-well plates to identify sequence alterations.

Primers used for SSCP were labelled at their 5' end with HEX and typical PCR reactions were performed in a total volume of 10 μl. All PCR reactions contained 67 mM Tris-HCl (pH 8.8); 16.5 mM (NH₄)₂Sθ₄; 6.5 μM EDTA; 1.5 mM MgCl₂; 200 μM each dNTP; 10% DMSO; 0.17 mg/ml BSA; 10 mM β- mercaptoethanol; 5 μg/ml each primer and 100 U/ml Taq DNA polymerase. PCR reactions were performed using 10 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds followed by 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. A final extension reaction for 10 minutes at 72°C followed.

Ten μl of loading dye comprising 50% (v/v) formamide, 12.5 mM EDTA and 0.02% (w/v) bromophenol blue were added to completed reactions which were subsequently run on non- denaturing 4% polyacrylamide gels with a cross-linking ratio of 35:1 (acrylamide:bis-acrylamide) and containing 2% glycerol. Gel thickness was lOOum, width 168mm and length 160mm. Gels were run at 1200 volts and approximately 20mA, at 18°C and analysed on the GelScan

2000 system (Corbett Research, Australia) according to manufacturers specifications.

PCR products showing a conformational change were subsequently sequenced. This first involved re- amplification of the amplicon from the relevant individual (primers used in this instance did not contain 5' HEX labels) followed by purification of the PCR amplified templates for sequencing using QiaQuick PCR preps (Qiagen) based on manufacturers procedures . The primers used to sequence the purified amplicons were identical to those used for the initial amplification step. For each sequencing reaction, 25 ng of primer and 100 ng of purified PCR template were used. The BigDye sequencing kit (ABI) was used for all sequencing reactions according to the manufacturers specifications. The products were run on an ABI 377 Sequencer and analysed using the EditView program. Table 1 shows the results of the GABABRl SSCP screen. Analysis of exon 6 of the GABABRl gene (isoform la) identified a bandshift in an individual with febrile seizures. Sequencing of this bandshift identified a G to A nucleotide change which corresponds to nucleotide 622 of the coding sequence of the GABABRl gene (based on the numbering of isoform la of the gene) as represented by SEQ ID NO: 1. The nucleotide change results in the replacement of an aspartic acid amino acid residue with an asparagine residue at position 208 of the encoded protein (based on the numbering of isoform la of the gene) , as represented by SEQ ID NO: 2. This nucleotide and associated amino acid change was also seen in the affected individuals monozygotic twin brother who also had febrile seizures. This change was not present in the control population indicating that it is causative of the epilepsy seen in these individuals.

SSCP analysis of the lb isoform of GABABRl identified 2 bandshifts, one in an individual with idiopathic generalised epilepsy and one in an individual with juvenile myoclonic epilepsy. Both of these individuals were photosensitive. Sequencing of one of these bandshifts identified a C to A nucleotide change which corresponds to nucleotide 14 of the coding sequence of this lb isoform of the gene as represented by SEQ ID NO: 3. The nucleotide change results in the replacement of an alanine amino acid residue with an aspartic acid residue at position 5 of the encoded protein (based on the numbering of isoform lb of the gene), as represented by SEQ ID NO:4. This change was not present in the control population indicating that it is causative of the epilepsy seen in this individual. Sequencing of the second bandshift seen in the lb isoform identified a C to T nucleotide change which corresponds to nucleotide 136 of the coding sequence of this lb isoform of the gene as represented by SEQ ID NO: 5. The nucleotide change results in the replacement of a proline amino acid residue with a serine residue at position 46 of the encoded protein (based on the numbering of isoform lb of the gene), as represented by SEQ ID NO: 6. This change was not present in the control population indicating that it is causative of the epilepsy seen in this individual. In addition, a further bandshift was identified in an individual with idiopathic generalised epilepsy. Sequencing of this bandshift revealed a C to G nucleotide change which corresponds to nucleotide 765 of the coding sequence of the GABABRl gene (based on the numbering of isoform la of the gene) as represented by SEQ ID NO:7. This nucleotide change does not alter the amino acid sequence but interestingly was not detected in the normal population. While not wishing to be found by theory, it is believed that this polymorphism may impart a change in GABABRl characteristics (such as reducing the half-life of the GABABRl mRNA therefore potentially reducing the levels of GABABRl protein) leading to the epilepsy phenotype or may be causative of the IGE phenotype in this individual when expressed in combination with an as yet undetermined second gene alteration.

During the mutation screen of GABABRl 13 single nucleotide polymorphisms (SNPs) were identified (Table 1) . 10 of these were in intronic sequences of the gene, while 3 were in the coding region but did not alter the amino acid sequence. These polymorphisms may reflect changes in

GABABRl which result in subtle changes of function of the GABA-B receptor. These subtle changes may predispose individuals to epilepsy and when expressed in combination with other gene mutations, such as those in ion channel genes, may lead to specific sub-types of the disease (see

PCT/AU01/00872) .

Example 3 : Functional significance of the GABABRl mutations To test the effects of the GABABRl mutations on GABA- B receptor function a number of procedures can be adopted as are known in the art. These may include, but are not restricted to, electrophysiological analysis of Xenopus laevis oocytes. Firstly, cDNAs encoding the GABABRl subunit of the human GABA-B receptor can be subcloned into pcDNA3.1(+) and the relevant mutation (such as the c622G—>A mutation, the cl4C—»A mutation or the cl36C—>T mutation) can be introduced into GABABRl of the human GABA-B receptor using the QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA) . Successful mutagenesis can be confirmed through DNA sequencing. Secondly, in order for correct GABA-B receptor assembly to be achieved, co- expression of the GABABR2 gene in the oocytes is needed. Finally, in order for an electrophysiological assay to be performed, an effector of the GABA-B receptor is also needed to be co-expressed in the oocytes. Such an effector would include the G-protein-gated inwardly rectifying potassium channels such as GIRK1 or GIRK2. Once the appropriate genes have been cloned, wild-type and mutant cDNAs can be linearised and gel purified and cRNA made using the T7 RNA polymerase Message Machine in vitro RNA synthesis kit (Ambion) . Samples of cRNA can be run on denaturing agarose gels to ensure they are the correct size and are not degraded.

Xenopus laevis oocytes (stage V or VI) can be microinjected with ~25 ng of cRNA representing the mutant GABABRl, wild-type GABABR2 and representative wild-type GIRK genes. Oocytes can subsequently be stored in OR2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, pH 7.5, supplemented with 2% foetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, Life Technologies) and incubated at 18°C for three to five days to allow for adequate receptor expression. Current recordings from oocytes can be made using a two-electrode voltage clamp (AxoClamp 2B, Axon Instruments), with recordings made at a holding potential of -80 mV. Electrodes can be filled with 3 M KCl and are normally of 1-2 MΩ resistance. Oocytes can be continuously perfused with ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM BaCl and 5 mM HEPES, pH 7.5). Gamma-Aminobutyric acid (GABA; Sigma- Aldrich, Australia) can be applied by bath perfusion.

The electrophysiological responses of the GABA-B receptor incorporating the mutant GABABRl gene can then be compared to wild-type GABA-B receptor responses in order to determine the functional significance of the GABABRl mutation.

Example 4 : Analysis of receptors and receptor subunits The following methods are used to determine the function and structure of mutant GABABRl and GABA-B receptors containing the mutant GABABRl protein.

Molecular biological studies The ability of the mutant GABA-B receptor or mutant GABABRl protein to bind known and unknown proteins can be examined. Procedures such as the yeast two-hybrid system are used to discover and identify any functional partners. The principle behind the yeast two-hybrid procedure is that many eukaryotic transcriptional activators, including those in yeast, consist of two discrete modular domains. The first is a DNA-binding domain that binds to a specific promoter sequence and the second is an activation domain that directs the RNA polymerase II complex to transcribe the gene downstream of the DNA binding site. Both domains are required for transcriptional activation as neither domain can activate transcription on its own. In the yeast two-hybrid procedure, the gene of interest or parts thereof (BAIT) , is cloned in such a way that it is expressed as a fusion to a peptide that has a DNA binding domain. A second gene, or number of genes, such as those from a cDNA library (TARGET) , is cloned so that it is expressed as a fusion to an activation domain. Interaction of the protein of interest with its binding partner brings the DNA-binding peptide together with the activation domain and initiates transcription of the reporter genes. The first reporter gene will select for yeast cells that contain interacting proteins (this reporter is usually a nutritional gene required for growth on selective media) . The second reporter is used for confirmation and while being expressed in response to interacting proteins it is usually not required for growth.

Mutant GABABRl or GABA-B receptor interacting genes may also be targets for mutation in epilepsy as well as other disorders associated with GABA-B receptor dysfunction. The nature of the interacting genes and proteins can be studied such that these partners can also be targets for drug discovery.

Structural studies

Mutant GABABRl or GABA-B receptor recombinant proteins can be produced in bacterial, yeast, insect and/or mammalian cells and used in crystallographical and NMR studies. Together with molecular modelling of the mutant GABABRl protein or mutant GABA-B receptor, structure-driven drug design can be facilitated.

Industrial Applicability

The present invention allows for the diagnosis and treatment of diseases such as epilepsy and disorders associated with GABA-B receptor dysfunction.

TABLE 1

Note : Numbering is based on isoform la of GABABRl (see NCBI accession number NM_001470) except for the exon lb changes where numbering is based on isoform lb of GABABRl (see accession number NM_021903) . Exons 6 and 12 were split into two amplicons due to the large size of these exons .

10 References

References cited herein are listed on the following pages, and are incorporated herein by this reference.

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Claims

Claims :

1. An isolated nucleic acid molecule encoding a mutant GABABRl protein wherein a mutation event has occurred so as to produce an epilepsy phenotype.

2. An isolated nucleic acid molecule as claimed in claim

1 wherein a mutation event has occurred in the nucleotides encoding the large extracellular loop of the amino terminal domain of GABABRl .

3. An isolated nucleic acid molecule as claimed in claim

2 wherein said mutation event occurs in exon 6 of the GABABRl coding sequence.

4. An isolated nucleic acid molecule as claimed in claim

3 wherein said mutation event is a nucleotide substitution at position 622 of the GABABRl coding sequence based on the numbering of GABABRl isoform la.

5. An isolated nucleic acid molecule as claimed in claim

4 wherein said mutation event is a G-A nucleotide substitution.

6. An isolated nucleic acid molecule as claimed in claim 2 wherein said mutation event takes place in exon 1 of the GABABRl coding sequence.

7. An isolated nucleic acid molecule as claimed in claim

6 wherein said mutation event is a nucleotide substitution at position 14 of the GABABRl coding sequence based on the numbering of GABABRl isoform lb.

8. An isolated nucleic acid molecule as claimed in claim

7 wherein said mutation event is a C-A nucleotide substitution.

9. An isolated nucleic acid molecule as claimed in claim 6 wherein said mutation event is a nucleotide substitution at position 136 of the GABABRl coding sequence based on the numbering of GABABRl isoform lb.

10. An isolated nucleic acid molecule as claimed in claim 9 wherein said mutation event is a C-T nucleotide substitution.

11. An isolated nucleic acid molecule as claimed in claim 2 wherein said mutation event is in exon 8 of the GABABRl coding sequence.

12. An isolated nucleic acid molecule as claimed in claim

11 wherein said mutation event is a substitution at position 945 of the GABABRl coding sequence based on the numbering of GABABRl isoform la.

13. An isolated nucleic acid molecule as claimed in claim

12 wherein said mutation event is a C-G nucleotide substitution.

14. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOs : 1,

3, 5 and 7.

15. An isolated nucleic acid molecule consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 5 and 7.

16. An expression vector comprising a nucleic acid molecule as claimed in any one of claims 1 to 15.

17. A cell comprising a nucleic acid molecule as claimed in any one of claims 1 to 15.

18. A genetically modified non-human animal transformed with a nucleic acid molecule as claimed in any one of claims 1 to 15.

19. An isolated polypeptide, said polypeptide being a mutant GABABRl protein wherein a mutation event has occurred so as to produce an epilepsy phenotype.

20. An isolated polypeptide as claimed in claim 19 wherein a mutation event has occurred in the large extracellular loop of the amino terminal domain of GABABRl .

21. An isolated polypeptide as claimed in claim 20 wherein said mutation event is a substitution at position

208 based on the numbering of GABABRl isoform la.

22. An isolated polypeptide as claimed in claim 21 wherein the substitution is a D208N transition.

23. An isolated polypeptide as claimed in claim 20 wherein said mutation event is a substitution at position 5 based on the numbering of GABABRl isoform lb.

24. An isolated polypeptide as claimed in claim 23 wherein the substitution is an A5D transition.

25. An isolated polypeptide as claimed in claim 20 wherein said mutation event is a substitution at position 46 based on the numbering of GABABRl isoform lb.

26. An isolated polypeptide as claimed in claim 25 wherein the substitution is a P46S transition.

27. An isolated polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4 or 6.

28. An isolated polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4 or 6.

29. A GABA-B receptor that incorporates a GABABRl subunit as claimed in any one of claims 19 to 28.

30. A method of preparing a polypeptide comprising the steps of:

1) culturing a cell as claimed in claim 17 under conditions effective for polypeptide production; and

2) harvesting the polypeptide.

31. A polypeptide prepared by the method of claim 30.

32. An antibody which is immunologically reactive with a mutant polypeptide as claimed in any one of claims 19 to

28 or 31, or a GABA-B receptor as claimed in claim 29 but not with a wild-type GABA-B receptor.

33. The use of a polypeptide as claimed in any one of claims 19 to 28 or 31, a GABA-B receptor as claimed in claim 29, or an antibody as claimed in claim 36 for the screening of candidate pharmaceutical agents.

34. The use of genetically modified non-human animal as claimed in claim 18 or a cell as claimed in claim 17 in the screening of candidate pharmaceutical compounds.

35. A method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction, comprising administering a selective antagonist, agonist or modulator of a polypeptide as claimed in any one of claims 19 to 28, or a GABA-B receptor as claimed in claim

29 to a patient in need of such treatment.

36. The use of a selective antagonist, agonist or modulator of a polypeptide as claimed in any one of claims 19 to 28, or a GABA-B receptor as claimed in claim 29 in the manufacture of a medicament for the treatment of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

37. A method of treating epilepsy as well as other disorders associated with GABA-B receptor dysfunction, comprising administering an isolated nucleic acid molecule which is the complement (antisense) of a nucleic acid molecule as claimed in any one of claims 1 to 15 and which encodes an RNA molecule that hybridizes with the mRNA encoding a mutant GABABRl protein to a subject in need of such treatment .

38. The use of an isolated nucleic acid molecule which is the complement (antisense) of a nucleic acid molecule as defined in any one of claims 1 to 15 and which encodes an RNA molecule that hybridizes with the mRNA encoding a mutant GABABRl polypeptide in the manufacture of a medicament for the treatment of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

39. A method of treating epilepsy as well as other disorders associated with GABA-B dysfunction comprising administration of an antibody as claimed in claim 32.

40. The use of an antibody as claimed in claim 32 in the manufacture of a medicament for the treatment of epilepsy as well as other disorders associated with GABA-B dysfunction.

41. The use of a nucleic acid molecule as claimed in any one of claims 1 to 15 in the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

42. A method for the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising the steps of:

(1) obtaining DNA from a subject suspected of epilepsy or another disorder associated with GABA-B receptor dysfunction; and

(2) comparing the GABABRl gene of said DNA to the DNA of wild-type GABABRl.

43. A method as claimed in claim 42 wherein each DNA fragment is sequenced and the sequences compared.

44. A method as claimed in claim 42 wherein the DNA fragments are subjected to SSCP analysis.

45. The use of a polypeptide as claimed in any one of claims 19 to 28 or 31, a GABA-B receptor as claimed in claim 29, or an antibody as claimed in claim 32 in the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction.

46. A method for the diagnosis of epilepsy as well as other disorders associated with GABA-B receptor dysfunction comprising the steps of: (1) obtaining the GABA-B receptor from a subject suspected of epilepsy or another disorder associated with GABA-B receptor dysfunction; and

(2) comparing the GABABRl subunit of said receptor with the corresponding GABABRl subunit of the wild-type GABA-B receptor.

47. An isolated nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NOs: 8-20.

48. An isolated nucleic acid molecule consisting the sequence set forth in any one of SEQ ID NOs: 8-20.