WO2007008933A2 - Compositions and methods comprising complex alternative splice variants of myelin/oligodendrocyte genes (mogs) and antibodies directed thereto - Google Patents

Abstract

Provided are isolated MOG isoforms generated in vivo by exon splicing and that contribute to phenotypic diversity in the peripheral and central nervous systems. Presented herein are transcriptomic analyses for a gene expressed only in mammalian nervous system, myelin oligodendrocyte glycoprotein (MOG). Complex splicing patterns were exclusively found in primates and not in mice. Primate-specific transcripts predicted the existence of multiple MOG isoforms including truncated intra-membranous and/or alternate cytoplasmic domains, and soluble secreted proteins with AIu motifs. The multitude of MOG isoforms is predictive of functions unique to primates, including humans, with regards to maintenance of myelin structure, intracellular signaling, and/or expression of CNS autoimmunity via exposure of specific epitope determinants. Differential expression of MOG isoforms along the neuroaxis is correlative of the broad variability of phenotypes that characteristic of the CNS demyelinating disorder multiple sclerosis. MOG splice variants, isoforms, antibodies, and methods presented herein may be suitably employed for the identification, characterization, and treatment of diseases including, but not limited to, demyelinating and other neurodegenerative, infectious, inflammatory, inherited, and metabolic disorders such as multiple sclerosis (MS) and Alexander's disease.

Description

COMPOSITIONS AND METHODS COMPRISING COMPLEX ALTERNATIVE SPLICE VARIANTS OF MYELIN/OLIGODENDROCYTE GENES (MOGs) AND

ANTIBODIES DIRECTED THERETO

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of provisional patent application U.S. Serial No. 60/698,519, filed My 11, 2005, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION Technical Field of the Invention

The present invention relates generally to diseases of the central and peripheral nervous systems and other systemic diseases. More specifically, provided herein are alternative splice variants of myelin oligodendrocyte genes (MOGs), isoforms of MOG proteins encoded by such splice variants, antibodies that specifically bind to MOG isoforms, as well as therapeutic and diagnostic methods that employ MOG splice variants, isoforms, and/or antibodies specific thereto. Such MOG splice variants, isoforms, antibodies, and methods presented herein may be suitably employed for the identification, characterization, and treatment of diseases including, but not limited to, demyelinating and other neurodegenerative, infectious, inflammatory, inherited, and metabolic disorders such as multiple sclerosis (MS) and Alexander's disease.

Description of the Related Art

Multiple Sclerosis (MS) designates a group of heterogeneous, immune-mediated chronic demyelinating disorders of the central nervous system (CNS) affecting 350,000 Americans and over 1 million individuals worldwide. MS affects women twice as often as men, and thus also represents a significant women's health issue. Pathologically, MS is characterized by plaques of perivascular infiltration comprised of mononuclear cells and macrophages, accompanied by concentric destruction of the myelin sheaths (demyelination), death of oligodendrocytes, proliferation of astrocytes, and axonal damage. Lassmann, Multiple Sclerosis 4:93-98 (1998); Raine, Multiple Sclerosis and Chronic Relapsing EAE: Comparative Ultrastructural Neuropathology, in Multiple Sclerosis: Pathology, Diagnosis and Management 413-460 (Hallpike et al. eds., 1983); and Trapp et al. , The New England Journal of Medicine 338:278-285 (1998).

The etiology of MS is unknown; however, strong circumstantial evidence suggests that MS is an autoimmune disorder arising in a genetically susceptible host under the pressure of environmental triggers. Hohlfeld, Brain 120:865-916 (1997) and Oksenberg et al, Pathogenesis of Multiple Sclerosis: Relationship to Therapeutic Strategies, in Multiple Sclerosis: Advances in Clinical Trial Design, Treatment and Future Perspectives 14-46 (Goodkin et al. eds., 1996). To a large extent, our current knowledge of the factors that may participate in the pathogenesis of MS lesions is based on observations of experimental allergic encephalomyelitis (EAE), an autoimmune disorder that is produced in laboratory animals by sensitization with antigens of CNS myelin. Martin et al, Ann. Rev. Immunol. 10:153-187 (1992); Miller et al., Immunol. Today 15:356-361 (1994); and Wekerle et al., Ann Neurol 36:S47-S53 (1994).

In contrast to the often-stereotyped illnesses encountered in the many models of EAE₃ the clinical phenotype of human MS can be benign or rapidly disabling, with variable courses including relapsing, remitting, or progressive forms. This heterogeneity of clinical presentation most likely reflects complex influences of environment and/or inherited genetic factors, and may correlate with distinct neuropathological subtypes as suggested by recent analyses of biopsy and autopsy material that showed specific patterns of lesions with various proportions of inflammation, demyelination, and oligodendrocyte and axonal pathology. Lassmann, Multiple Sclerosis 4:93-98 (1998); Lucchinetti et al, Ann. Neurol. 47:707-717 (2000); and Storch et al, Ann Neurol 43:465-471 (1998). Effector mechanisms of tissue damage in CNS autoimmunity include direct toxicity of infiltrating T cells, secretion of proinflammatory cytokines, antibody-mediated toxicity, and complement and macrophage activation (reviewed in Brosnan et al, Brain Pathol 6:243-257 (1996)). Immunization of Callithrix jacchus (C. jacchus) marmosets with whole human white matter, and myelin/oligodendrocyte glycoprotein (MOG) in adjuvant produce chronic, relapsing-remitting disorders of mild to moderate clinical severity which are reminiscent of typical forms of human MS. The neuropathology of acute C. jacchus EAE consists of large concentric areas of primary demyelination, macrophage infiltration, astrogliosis, and death of oligodendrocytes. Massacesi et al, Ann. Neurol. 37:519-530 (1995); Genain et al, Immunol. Reviews 183:159-172 (2001); and Brok et al, Immunol Rev 183:173-185 (2001). Ultrastructural features of myelin breakdown are similar in marmoset EAE and human MS₅ suggesting common mechanisms of myelin destruction. Genain et al, Immunol. Reviews 183:159-172 (2001); and Raine et al, Ann Neurol 46:144-160 (1999). Remyelination occurs in chronic EAE. C. jacchus marmosets are small animals (350-400 gm), yet serial paraclinical and laboratory studies, such as peripheral blood reactivity to myelin antigens, CSF sampling, and in vivo magnetic resonance imaging (MRI) can be obtained. Genain et al, Proc. Natl. Acad. Sci. USA 92:3601-3605 (1995); Genain et al, Methods: a Companion to Methods in Enzymology 10:420-434 (1996); Jordan et al, AJNR Am. J. Neuroradiol. 20:965-976 (1999); and Hart et al, Am. J. Pathol. 153:649-663 (1998). As an outbred species marmosets exhibit a very broad immunologic repertoire against myelin antigens, which is similar to humans. In addition to whole myelin and MOG, susceptibility to myelin basic protein (MBP₅) MBP- derived peptides, and proteo lipid protein (PLP) has been demonstrated. Genain et al, Immunol. Reviews 183:159-172 (2001). Diverse epitope recognition and T cell receptor D chain utilization are seen in the encephalitogenic repertoires against myelin proteins. Genain et al, J. Clin. Invest. 94:1339-1345 (1994); Uccelli et al, Eur. J. Immunol. 3J_:474~479 (2001); Villoslada et al, Eur. J. Immunol. 3J_:2942-2950 (2001); and Mesleh et al, Neurobiol Dis 9:160-172 (2002). C. jacchus are unique primates for studies of autoimmunity because these monkeys are born as naturally occurring bone marrow chimeras. While sibling pairs or triplets are genetically distinct, they share, and are tolerant to, each other's bone marrow- derived cell populations, which permits adoptive transfer of T cell clones. Genain et al, J. Clin. Invest. 94:1339-1345 (1994); Villoslada et al, Eur. J. Immunol. 31:2942-2950 (2001); and Watkins et al, Journal of Immunology 144:3726-3735 (1990).

The MS-like lesion in C. jacchus is mediated by a complex interplay between cellular and humoral responses to myelin. MOG has been shown to be a target for demyelinating antibodies. Genain et al, J. Clin. Invest. 96:2966-2974 (1995). Importantly, pathogenicity of MOG-specific autoantibodies has also been demonstrated in selected cases of human MS. Genain et al, Nat Med 5_: 170-175 (1999). C. jacchus share a very high degree of homology with humans for myelin and immune system genes. The recent cloning of MOG-specific marmoset immunoglobulin genes has revealed similarity of gene usage and epitope recognition between marmosets and humans, von Bϋdingen et al, Immunogenetics 53:557- 563 (2001); and von Budingen et al, Proc. Natl. Acad.Sci. USA 99:8207-8212 (2002).

Bioinformatic analysis of expressed sequence tag and mRNA sequences has revealed an unexpectedly high frequency and complexity of alternative splicing. Moreover, whereas most gene structures and constitutive exons in major transcript isoforms are highly similar, alternatively spliced forms frequently lack conservation across species; this phenomenon may be one evolutionary pathway to generate species-specific transcriptome diversity. Modrek et al, Nat Genet 34, 177-80 (2003) and Nurtdinov et al, Hum MoI Genet Yl, 1313-20 (2003). This hypothesis can be tested by assessing alternative splicing patterns by sequencing full- length mRNAs as well as directed RT-PCR-based analyses using primers encompassing alternative splicing sites.

Myelin is a relatively recent invention of evolution that appeared about 400 million years ago, and is a unique feature of vertebrates that is essential for fast conduction of nerve potentials along axons of the peripheral and central nervous systems. Reviewed in Colman et al, "Glial ceil development: basic principles and clinical relevance" 85-100 (BIOS Scientific Publishers Ltd, Oxford, UK, 1996). It is a complex multi-lamellar structure synthesized by specialized cells, and comprises various cytoplasmic or transmembrane proteins with highly organized interactions with lipids and glycolipids responsible for integrity and maintenance of its structure and function. Most myelin proteins exist as protein families comprising several isoforms that result from alternative splicing from a unique gene. Reviewed in Campagnoni et al, Brain Pathol ϋ:74-91 (2001) and Baumann et al, Physiol Rev. 8J_.:871-927 (2001). In CNS myelin, 90% of the protein content belongs to 2 major protein families:, the myelin basic proteins (MBP), and proteolipid proteins (PLP). Quantitatively minor components include glycoproteins, such as myelin associated glycoprotein and myelin oligodendrocyte glycoprotein (MOG), a 26-28 kDa glycoprotein only present in mammals (Birling et al, Neurochem. Res. I8j937-45 (1993)) in contrast to the more ancestral MBP and PLP. Campagnoni et al, Brain Pathol.

(2001) and Baumann et al, Physiol. Rev. 81_.: 871- 927 (2001).

MOG is encoded by a gene located in the major histocompatibility complex region (MHC; Pham-Dinh et al, Proc. Natl Acad. ScL U.S.A. 90:7990-4 (1993)) and is the smallest representative in the immunoglobulin superfamily of adhesion proteins. MOG is the focus of considerable interest due to the highly immunogenic properties of its surface exposed, IgV- like extracellular domain, and its potential role as target for pathogenic {e.g., disease- inducing) antibody responses in CNS autoimmune demyelination. Pham-Dinh et al, Proc. Natl. Acad. Sci. U.S.A. 90:7990-4 (1993); Bernard et al, JMoI Med 75:77-88 (1997); Iglesias et al, GUa 36:220-34 (2001); Delarasse et al J Clin Invest 112:544-53 (2003); and Pham- Dinh et al, Oligodendrocyte Glycoprotein Gene, in, Myelin Biology and Disorders 1, 469- 497 (R. A. Lazzarini ed., 2004). The sequence of MOG is highly conserved between species, both at the nucleotide and amino acid levels as observed for other myelin genes. A preliminary study indicates, however, that alternative splicing patterns of the MOG gene are likely to be more complex in humans compared to mice. Pham-Dinh et al, Genomics 29:345- 52 (1995) and Ballenthin et al, J. Neurosci. Res. 46:271-81 (1996). Exon 2 that for MOG encodes the IgV-like domain appears to have undergone adaptative "shuffling" processes during evolution as it is shared with a number of other genes of the BT/B7 family. Henry et al, Immunol Today 20:285-8 (1999) and Linsley Protein Set 3:1341-3 (1994). These evolutionary events themselves provide the potential for differential expression of MOG and BT/B7 isoforms as an additional source of phenotypic diversity among species. The expression of MOG isoforms, similar to substitutions in gene nucleotide sequences that may result in functionally different proteins, can underlie different biological roles for this protein in different species and, within a single species in health and disease.

Because structural features of the expressed MOG proteins and their topographical expression control the exposure of major immunogenic determinants within CNS (von Budingen et al, Eur. J. Immunol. 34:2072-83 (2004); Breithaupt et al, Proc. Natl Acad. ScI U.S.A. 100:9446-51 (2003); Clements et al, Proc. Natl. Acad. Sci. U.S.A. 100:11059-64 (2003); Mesleh et al, Neurobiol. Dis. 9:160-72 (2002)), the findings disclosed herein undescore the complex splicing patterns of MOG as novel factors critical to phenotypic expression of CNS-directed autoimmunity in higher mammals.

SUMMARY OF THE INVENTION

Thus, the present invention provides, for the first time, a complete analysis of the alternative splicing of the MOG gene in five representative species from rodent to human and discloses that the most complex splicing patterns are exclusive to higher mammals including human and non-human primates, unlike for other myelin proteins. As discussed in further detail herein, the present invention relates, generally, to diseases of the central and peripheral nervous systems and other systemic diseases. More specifically, the present invention provides alternative splice variants of myelin oligodendrocyte genes (MOGs), isoforms of MOG proteins encoded by such splice variants, antibodies that specifically bind to MOG isoforms, as well as therapeutic and diagnostic methods that employ MOG splice variants, isoforms, and/or antibodies specific thereto. Such MOG splice variants, isoforms, and antibodies presented herein may be suitably employed in compositions and methods for the identification, characterization, and treatment of autoimmune diseases including, but not limited to, demyelinating and other neurodegenerative, infectious, inflammatory, inherited, and metabolic disorders such as multiple sclerosis (MS) and Alexander's disease. The invention provides the following:

1. An isolated alternative splice variant of a myelin oligodendrocyte genes (MOGs) selected from the group consisting of the nucleotide sequence presented in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70.

2. An isoform of a MOG protein selected from the group consisting of the amino acid sequences presented in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.

3. A protein epitope encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 107-184.

4. An antibody that specifically binds to one or more of the MOG isoforms selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.

5. An antibody that specifically binds to one or more peptide epitope encoded by a nucleotide sequences selected from the group consisting of any one of SEQ ID NOs: 107-

184.

6. A method for detecting a MOG isoform comprising the steps of: obtaining a sample suspected of containing a MOG isoform; contacting said sample with an antibody described herein; removing unbound antibody; and detecting bound antibody.

The invention also provides an isolated polynucleotide encoding an alternative splice variant of a myelin oligodendrocyte glycoprotein (MOG), said polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70.

The invention also provides an isolated polynucleotide comprising at least about 10 contiguous nucleotides spanning the junction between exon 2 and exon 4, between exon 2 and AIuB, or between exon 2 and exon 2' of an isoform of MOG. In some embodiments, the polynucleotide comprises at least about 15, about 20, about 25, or about 30 nucleotides, or longer than 30 nucleotides.

The invention also provides an isolated isoform of a MOG protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69. The invention also provides an MOG protein epitope encoded by any of the polynucleotides described herein. The invention also provides an antibody specifically binds to the MOG protein epitope. The invention provides an antibody that specifically binds to one or more of the MOG isoforms selected from the group consisting of α4Cj, α5, α6, α7Cj, If-AlulOB, AIuIOA, AIuIOB, and Ig-2'. In some embodiments, the antibody does not bind to one or more of the MOG isoforms selected from the group consisting of αl, α2, α3, α4, βl, β2, β3, β4, and lg-AlulOA.

The invention also provides a method for detecting a MOG isoform comprising detecting a MOG isoform described herein in a sample. In some embodiments, the detection is performed by contacting said sample with an antibody described herein, and detecting specific bound antibody. In some embodiments, the detection is performed by detecting the presence of an RNA encoding the MOG isoform in the sample.

The invention also provides a method for detecting autoreactive antibodies to one or more MOG isoforms described herein, comprising contacting a biological sample which is suspected to contain autoreactive antibodies with one or more MOG isoforms, and detecting binding of antibodies to the one or more MOG isoforms. The invention also provides a cell or a living organism transfected with a polynucleotide described herein. In some embodiments, the cell is a CHO cells. In some embodiments, the living organism is nematodes, drosophila, fish (e.g., zebrafish) and mouse.

The invention also provides a method of generating an animal model (e.g., for multiple sclerosis), comprising administering a polypeptide comprising one or more of the MOG isoforms described herein or a polynucleotide comprising a sequence encoding one or more of the MOG isoforms in an amount effective to induce anti-myelin response in the animal. The invention also provides a animal model generated by the method. In some embodiments, the animal is a mammal, such as, a mouse, a rat, a rabbit, a bovine, a pig, and a primate. In some embodiments, a primate is a marmoset or a macaque. The invention also provides a method for inducing tolerance to an MOG in an animal comprising administering a polypeptide comprising one or more of the MOG isoforms described herein or a polynucleotide comprising a sequence encoding one or more of the MOG isoforms in an amount effective to induce tolerance. In some embodiments, T cell tolerance is induced. In some embodiments, B cell tolerance is induced. In some embodiments, both T cell tolerance and B cell tolerance are induced.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE IDENTIFIERS

Figure 1 Comparative sequences of the mature MOG protein between mammalian species. Note high conservation in the extracellular domain, the second hydrophobic transmembrane domain (HR2) and the carboxy-terminal cytoplasmic tail. MOG domains are encoded as follows: exon 2, aa 1-116 (most of the Ig-like domain); exon 3, aa 117-154 (HRl; della Gaspera et al, Eur J Biochem 258:478-84 (1998)); exons 4 and 5, aa 155-161 and aa

162-168, respectively (intracellular loop); exon 6B, aa 169-207 (HR2); exons 7 and 8 A encode aa 208-214, and 215-218, respectively (intracellular C-terminal tail). *: Asparagine 31 as a glycosylation site.

Figure 2 Diversity of MOG alternate splicing. Splicing variants were RT-PCR amplified from CNS of different mammalian species. The thick band in each lane corresponds to the αl MOG major isoform (about 830 nucleotides), according to the position of the species-specific primers used (primer sequences in Table Sl; primer position Fig. 3A). Minor forms seen under the major form are not found in mouse. Figure 3 PCR amplifications of human MOG cDNAs. All nested PCR experiments are from a first PCR with primer set 1-03AT (primer 1, located in exon 1; primer 03AT, in the non-coding part). PCR products are described by exon composition. Primers pairs are positioned below the schematic exon composition in A and indicated above each gel in B, C, D. Exon sizes is indicated above exons when they are described for the first time. The size of 5' and 3' exons is reduced due to the use of internal primers. M, molecular weight marker.

Figure 3 A the MOG gene with its encoded domains: Exon 1 ; leader peptide sequence

(L); exon 2, extracellular IgV-like domain (Ig-EC); exon 3, hydrophobic transmembrane domain (HRl); exon 4, 5, 7, and 8, intracellular cytoplasmic regions (IC); exon 6 (6B according to the new designation issued from alternative splicing in Pham-Dinh et al, Genomics 29:345-52 (1995) encodes a second hydrophobic region (HR2).

Figure 3B Hexl-HexO8A primer set revealed the previously unknown MOG isoform α4 (479 bp), the major MOG isoform αl (827 bp, all constitutive exons), and the isoform lacking exon 6B, previously designated as α2 (713 bp). Pham-Dinh et al, Genomics 29:345- 52 (1995). Δ: denotes deleted exon. Figure 3 C Hexl-HexO5 primer set: additional minor transcripts besides the expected major isoform αl at 592 bp: a 244 bp band corresponding to splicing variants where the 348 bp sequence of exon 2 is skipped, as found in α4, α5 and α6 transcripts; and a 730-bp band containing a previously published sequence of 138-bp Ballenthin, P. A. & Gardinier, M. V. Myelin/oligodendrocyte glycoprotein is alternatively spliced in humans but not mice (J Neurosci Res 46, 271-81 (1996)) located in intron 2 (position 8583-8720 according to numbering in Roth et al, Genomics 28:241-50 (1995). The latter sequence belongs to the AIu repeat family and has been designated the AIu- 10 exon.

Figure 3D antisense primer derived from the AIu- 10 sequence (HexOAlu). Two bands and 4 different sequences were obtained: a 574-bp isoform comprising exons 1, 2 and AIu- 10 (MOG Ig-AIu- 10); a 226-bp sequence lacking exon 2 (MOG AIu- 10); and 2 similar forms with a deletion of 20 bp in the 5' end of exon AIu- 10. The full AIu- 10 exon was designated AIu- 1OA, and the partly excised one, AIu- 1OB.

Figure 4A presents a schematic representation of the exon composition of MOG splice variants across species. Exon numbering is according to Pham-Dinh et al, Genomics 29:345- 52 (1995). Open boxes, non-coding regions. Black boxes, constitutive coding exons; alternative exons: yellow, 2'; red, AIu A; blue, AIu B; green, 6 A. Previously published human isoforms: MOG αl, α2, α3, βl, β2, β3, β4, Ig-Alu-IOA, in black type; newly discovered isoforms: MOG α4, α5, α6, Ig-AlulOB, AIu-IOA, AIu-IOB, Ig-2'_s in red type and framed; **: α4Cj and α7Cj, the 2 marmoset-specific MOG isoforms. Bovine and mouse MOG transcripts are not represented here as these isoforms do not differ from any of those found in primates (please refer to Table 1 for mouse/bovine transcript composition). Three families of mRNAs are defined, based on use of one of two alternative acceptor splice sites in exon 8 in the 3' part of the gene (α and β), or the presence of a premature stop codon introduced by exonisation (spliced-mediated) of AIu- 10 or 2' sequences from intron 2 (AIu forms). *: indicate alternative in-frame stop codons. Figure 4B presents a schematic topological model of the MOG protein isoforms of the present invention. Exon 1 (leader peptide) is not represented. White boxes, constitutive exons contained in the major MOG protein. Striped boxes, C-terminal protein domain encoded by the alternative 8B exon, defining the β isoform family. Colored boxes indicate domains encoded by alternative exons. The highly hydrophobic exon 6B-encoded domain is represented as semi-embedded in the membrane in the forms containing exon 3 (encoding the transmembrane domain), whereas it is tentatively drawn spanning the membrane when exon 3 is skipped as in oc7Cj. Exons 4 and 5 in α7Cj are tentatively represented as extracellular. The N-glycosylation site is indicated by an asterisk above asparagine 31 (N 31).

Figure 5 demonstrates a developmental analysis of MOG transcript expression in the human brain. Transcripts containing exon 8B which is characteristic of, and specific for, the 3 'end of the MOG β isoforms, were amplified with Hexl/08B primer pairs and hybridized with labelled Hex3 primer. Transcripts containing exon 8 A, coding for the 3' end of the MOG α isoforms, were amplified with Hexl/08A primer pairs and hybridized with labelled Hex3 primer. Transcripts containing exon AIu were amplified with Hexl/HexOAlu primer pairs and hybridized with labelled AIu primer. All the relatively major MOG isoforms were detectable using RT-PCR without re-amplification. Only the α 1 MOG isoform was detectable in all samples at all times tested, whereas splicing variants were detectable in only the 2 year old child and adult brain tissue samples.

SEQ ID NO: 1 is the amino acid sequence of human MOG αl ; SEQ ID NO: 2 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 1;

SEQ ID NO: 3 is the amino acid sequence of human MOG α2;

SEQ ID NO: 4 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 3; SEQ ID NO: 5 is the amino acid sequence of human MOG α3;

SEQ ID NO: 6 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 5;

SEQ ID NO: 7 is the amino acid sequence of human MOG α4 NCBI Accession No. AY786325; SEQ ID NO: 8 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 7;

SEQ ID NO: 9 is the amino acid sequence of human MOG α5 NCBI Accession No. AY566846;

SEQ ID NO: 10 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 9;

SEQ ID NO: 11 is the amino acid sequence of human MOG α6 NCBI Accession No. AY566847;

SEQ ID NO: 12 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 11; SEQ ID NO: 13, is the amino acid sequence of human MOG Ig-Alu-IOB NCBI

Accession No. AY566848;

SEQ ID NO: 14 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 13;

SEQ ID NO: 15 is the amino acid sequence of human MOG AIu- 1OA NCBI Accession No. AY566849;

SEQ ID NO: 16 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 15;

SEQ ID NO: 17 is the amino acid sequence of human MOG AIu- 1OB NCBI Accession No. AY566850; SEQ ID NO: 18 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 17;

SEQ ID NO: 19 is the amino acid sequence of human MOG Ig-2' NCBI Accession No. AY566851;

SEQ ID NO: 20 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 19;

SEQ ID NO: 21 is the amino acid sequence of human MOG βl NCBI Accession No. AY566852;

SEQ ID NO: 22 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 21 ; SEQ ID NO: 23 is the amino acid sequence of human MOG β2 NCBI Accession No.

AY566853;

SEQ ID NO: 24 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 23;

SEQ ID NO: 25 is the amino acid sequence of human MOG β3 NCBI Accession No. AY566854;

SEQ ID NO: 26 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 25;

SEQ ID NO: 27 is the amino acid sequence of human MOG β4; SEQ ID NO: 28 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 27;

SEQ ID NO: 29 is the amino acid sequence of human MOG Ig-AIu- 1OA; SEQ ID NO: 30 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 29;

SEQ ID NO: 31 is the amino acid sequence of murine MOG αl; SEQ ID NO: 32 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 42;

SEQ ID NO: 33 is the amino acid sequence of murine MOG α2 NCBI Accession No. AY566830;

SEQ ID NO: 34 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 33; SEQ ID NO: 35 is the amino acid sequence of bovine MOG αl ;

SEQ ID NO: 36 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 35;

SEQ ID NO: 37 is the amino acid sequence of bovine MOG α2 NCBI Accession No. AY566831; SEQ ID NO: 38 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 37;

SEQ ID NO: 39 is the amino acid sequence of bovine MOG α4 NCBI Accession No. AY566832;

SEQ ID NO: 40 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 39.

SEQ ID NO: 41 is the amino acid sequence of bovine MOG α5 NCBI Accession No. AY566833;

SEQ ID NO: 42 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 41; SEQ ID NO: 43 is the amino acid sequence of macaque MOG αl NCBI Accession

No. AF399846;

SEQ ID NO: 44 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 43;

SEQ ID NO: 45 is the amino acid sequence of macaque MOG α2 NCBI Accession No. AY566838;

SEQ ID NO: 46 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 45;

SEQ ID NO: 47 is the amino acid sequence of macaque MOG α3 NCBI Accession No. AY566839; SEQ ID NO: 48 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 47;

SEQ ID NO: 49 is the amino acid sequence of macaque MOG α4 NCBI Accession No. AY566840;

SEQ ID NO: 50 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 49; SEQ ID NO: 51 is the amino acid sequence of macaque MOG α5 NCBI Accession

No. AY566841;

SEQ ID NO: 52 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 51;

SEQ ID NO: 53 is the amino acid sequence of macaque MOG aβ NCBI Accession No. AY566842;

SEQ ID NO: 54 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 53;

SEQ ID NO: 55 is the amino acid sequence of macaque MOG βl NCBI Accession No. AY566843; SEQ ID NO: 56 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 55;

SEQ ID NO: 57 is the amino acid sequence of macaque MOG β2 NCBI Accession No. AY566844;

SEQ ID NO: 58 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 57;

SEQ ID NO: 59 is the amino acid sequence of macaque MOG β3 NCBI Accession No. AY566845;

SEQ ID NO: 60 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 59; SEQ ID NO: 61 is the amino acid sequence of macaque MOG Ig-2' NCBI Accession

No. AY786326;

SEQ ID NO: 62 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 61.

SEQ ID NO: 63 is the amino acid sequence of marmoset MOG αl NCBI Accession No. AY566834;

SEQ ID NO: 64 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 63;

SEQ ID NO: 65 is the amino acid sequence of marmoset MOG α4 NCBI Accession No. AY566835; SEQ ID NO: 66 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 65;

SEQ ID NO: 67 is the amino acid sequence of marmoset MOG α4Cj NCBI Accession No. AY566836;

SEQ ID NO: 68 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 67;

SEQ ID NO: 69 is the amino acid sequence of marmoset MOG oc7Cj NCBI Accession No. AY566837;

SEQ ID NO: 70 is the nucleotide sequence of a cDNA encoding the amino acid sequence as depicted in SEQ ID NO: 69; SEQ ID NO: 71 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MsI (5' ATGGCCTGTTTGTGGAGCTTC 3');

SEQ ID NO: 72 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Ms2 (5' TTCTTCAGAGACCACTCTTACC 3');

SEQ ID NO: 73 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MsO3 (5' ACAAGGCCTACAGAGACCTG 3 ');

SEQ ID NO: 74 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MsO6B (5' CCAGTCTTCGGTGCAGCCAG 3');

SEQ ID NO: 75 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MsO8A (5 'CCTTCCGCTCC AGGAAGAC A 3'); SEQ ID NO: 76 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MsO8B (5' GGAAGAAATATCCACCATTTG 3 ');

SEQ ID NO: 77 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Bexl (5' G AC AGTGGAG ATGGC CAGT 3');

SEQ ID NO: 78 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Bex2 (5' CAGTTCAGAGTAATAGGACCAGG 3');

SEQ ID NO: 79 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as BexO3 (5' ACGAGGCCCACGGTGATCTG 3');

SEQ ID NO: 80 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as BexO6B (5' CCGAGAACCGGC ACGATGAC 3'); SEQ ID NO: 81 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as BexO8A (5' GCAACTCAATGGATGTGACATC 3');

SEQ ID NO: 82 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as BexO8B (5' TGCAGCACAGGTGTCCTCTG 3');

SEQ ID NO: 83 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Hexl (5 ' GGAACAGTAGAGATGGC AAGC 3 ' );

SEQ ID NO: 84 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Mr2 (5' GAGCTGCTGAAAGACGATATT 3 ');

SEQ ID NO: 85 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MrO3 (5' TGCGAGGAGAACCAGCACT 3'); SEQ ID NO: 86 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MrO6B (5' GGTT ATCTTCC AGCAGGGCAC 3');

SEQ ID NO: 87 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MrO8A (5' GCAACTTGATGGATGAGACATC 3');

SEQ ID NO: 88 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as MrO8B (5 ' TCCAGCACAGATGTCCTCTG 3 ');

SEQ ID NO: 89 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Hexl (5' GGAACAGTAGAGATGGCAAGC 3');

SEQ ID NO: 90 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Hex2 (5' CAGTTCAGAGTGATAGGACCAAG 3'); SEQ ID NO: 91 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as McO3 (5' ACGAGGCCGAC AGTGATCTG 3');

SEQ ID NO: 92 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as McO6A (5' CCTAGAACACCAAAGGATT 3');

SEQ ID NO: 93 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO6B (5' CCAAGAACCGGCACAATTAC 3');

SEQ ID NO: 94 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO8A (5' GCAACTTGATGGATGAGATGTC 3');

SEQ ID NO: 95 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as McO8B (5' TCCAGCACAGGTGTCCTCTG 3'); SEQ ID NO: 96 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Hexl (5 ' GGAACAGTAGAGATGGCAAGC 3 ');

SEQ ID NO: 97 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as Hex2 (5 ' CAGTTCAGAGTGATAGGACCAAG 3 ');

SEQ ID NO: 98 ■ is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO2 (5 ' TGATCTCGGAAGAAGC AGGT 3 ' );

SEQ ID NO: 99 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO3 (5' ACGAGGCCAACAGTGATCTG 3');

SEQ ID NO: 100 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO5 (5' CAAAAGTCCGGTGGAGATTC 3'); SEQ ID NO: 101 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexOόA (5' CCTAGAACACCAAAGGACT 3');

SEQ ID NO: 102 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO6B (5' CCAAGAACCGGCACAATTAC 3');

SEQ ID NO: 103 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO8 A (5 ' GCAACTTGATGGATGAGATGTC 3 ');

SEQ ID NO: 104 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO8B (5' TCCAGTACAGGTGTCCTCTG 3');

SEQ ID NO: 105 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexOAlu (5' CATGGTGACACAAGCCTGTG 3'); SEQ ID NO: 106 is the nucleotide sequence of an MOG oligonucleotide primer designated herein as HexO3 AT (5 ' AGAAGTGTGCTTATGAAAACTTGT 3 ');

SEQ ID NO: 107 is the nucleotide sequence of the 5' splice site for human MOG gene exonl - exon2 (5' CAG GTAAGACA 3');

SEQ ID NO: 108 is the nucleotide sequence of the 3' splice site for human MOG gene exonl - exon2 (5' TGTCTTGGACAG GG 3');

SEQ ID NO: 109 is the nucleotide sequence of the 5' splice site for human MOG gene exon2 -- exon2' (5' AAG GTGAGTAG 3');

SEQ ID NO: 110 is the nucleotide sequence of the 3' splice site for human MOG gene exon2 ~ exon2' (5' ATCCTTACTTAG CT 3'); SEQ ID NO: 111 is the nucleotide sequence of the 5' splice site for human MOG gene exon2 - AIuA (5' AAG GTGAGTAG 3');

SEQ ID NO: 112 is the nucleotide sequence of the 3' splice site for human MOG gene exorώ - AIuA (5' TTTTTGAGACAG TG 3');

SEQ ID NO: 113 is the nucleotide sequence of the 5' splice site for human MOG gene exon2 - AIuB (5 ' AAG GTGAGTAG 3 ');

SEQ ID NO: 114 is the nucleotide sequence of the 3' splice site for human MOG gene exorώ - AIuB (5' TCTGTCACCCAG GA 3');

SEQ ID NO: 115 is the nucleotide sequence of the 5' splice site for human MOG gene exorώ - exon3 (5' AAG GTCTGCAC 3'); SEQ ID NO: 116 is the nucleotide sequence of the 3' splice site for human MOG gene exorώ - exon3 (5' CCTGCCTTTCAG AT 3');

SEQ ID NO: 117 is the nucleotide sequence of the 5' splice site for human MOG gene exon3 - exon4 (5' GAG GTACAGGG 3');

SEQ ID NO: 118 is the nucleotide sequence of the 3' splice site for human MOG gene exon3 - exon4 (5' TTTCTATTTTAG GA 3');

SEQ ID NO: 119 is the nucleotide sequence of the 5' splice site for human MOG gene exon4 - exon5 (5' TAG GTGAGTTC 3');

SEQ ID NO: 120 is the nucleotide sequence of the 3' splice site for human MOG gene exon4 - exon5 (5' TTTCTCTTTCAG AG 3'); SEQ ID NO: 121 is the nucleotide sequence of the 5' splice site for human MOG gene exon5 - exon6A (5' TTG GTGCAGTG 3');

SEQ ID NO: 122 is the nucleotide sequence of the 3' splice site for human MOG gene exon5 - exon6A (5' TTTTGTCCCCAG AG 3');

SEQ ID NO: 123 is the nucleotide sequence of the 5' splice site for human MOG gene exon5 - exonόB (5 ' TTG CACCTGGG 3 ');

SEQ ID NO: 124 is the nucleotide sequence of the 3' splice site for human MOG gene exon5 - exonόB (5' TGAACAATTCAG AT 3');

SEQ ID NO: 125 is the nucleotide sequence of the 5' splice site for human MOG gene exon5 -- exon7 (5' TTG CACCTGGG 3'); SEQ ID NO: 126 is the nucleotide sequence of the 3' splice site for human MOG gene exon5 ~ exon7 (5' TTTTGTTTTCAG GG 3');

SEQ ID NO: 127 is the nucleotide sequence of the 5' splice site for human MOG gene exon5 ^» exon8B (5' TTG CACCTGGG 3');

SEQ ID NO: 128 is the nucleotide sequence of the 3' splice site for human MOG gene exon5 - exon8B (5' TTCTCTCTCCAG TC 3');

SEQ ID NO: 129 is the nucleotide sequence of the 5' splice site for human MOG gene exonθA - exon7 (5' CAG GTGGGTGG 3');

SEQ ID NO: 130 is the nucleotide sequence of the 3' splice site for human MOG gene exonβA - exon7 (5' TTTTGTTTTCAG GG 3'); SEQ ID NO: 131 is the nucleotide sequence of the 5' splice site for human MOG gene exonόB - exon7 (5' CAG GTGCAGTG 3');

SEQ ID NO: 132 is the nucleotide sequence of the 5' splice site for human MOG gene exonόB -- exon7 (5' TTTTGTTTTCAG GG 3');

SEQ ID NO: 133 is the nucleotide sequence of the 5' splice site for human MOG gene exon7 ~ exon8A (5' TAC GTAAGTTC 3');

SEQ ID NO: 134 is the nucleotide sequence of the 3' splice site for human MOG gene exon7 - exonSA (5' TCCTTCTTCTAG GA 3');

SEQ ID NO: 135 is the nucleotide sequence of the 5' splice site for human MOG gene exon7 ~ exon8B (5' TAC GTAAGTTC 3'); SEQ ID NO: 136 is the nucleotide sequence of the 3' splice site for human MOG gene exon7 ~ exon8B (5' TTCTCTCTCCAG TC 3');

SEQ ID NO: 137 is the nucleotide sequence for human MOG gene exonl (5' ATGGCAAGCTTATCGAGACCCTCTCTGCCCAGCTGCCTCTGCTCCTTCCTCCTCCT CCTCCTCCTCCAAGTGTCTTCCAGCTATGCAG); SEQ ID NO: 138 is the nucleotide sequence for human MOG gene exon2 (5'

GGCAGTTCAGAGTGATAGGACCAAGACACCCTATCCGGGCTCTGGTCGGGGATG AAGTGGAATTGCCATGTCGCATATCTCCTGGGAAGAACGCTACAGGCATGGAGGT GGGGTGGTACCGCCCCCCCTTCTCTAGGGTGGTTCATCTCTACAGAAATGGCAAG GACCAAGATGGAGACCAGGCACCTGAATATCGGGGCCGGACAGAGCTGCTGAAA GATGCTATTGGTGAGGGAAAGGTGACTCTCAGGATCCGGAATGTAAGGTTCTCAG ATGAAGGAGGTTTCACCTGCTTCTTCCGAGATCATTCTTACCAAGAGGAGGCAGC AATGGAATTGAAAGTAGAAG);

SEQ ID NO: 139 is the nucleotide sequence for human MOG gene exon2' (5' CTCCCTTAAAGCAGAGATCAAGATGA);

SEQ ID NO: 140 is the nucleotide sequence for human MOG gene exonAluA (5' TGTCTCACTCTGTCACCCAGGATTGGTTGCAGTGGCACGATCATGGCTCATTGCA GCCTCCACCTCCCAGGCTCAAGTGA);

SEQ ID NO: 141 is the nucleotide sequence for human MOG gene exonAluB (5' GATTGGTTGCAGTGGCACGATCATGGCTCATTGCAGCCTCCACCTCCCAGGCTCA AGTGATCCTCCTGACTCAGCCTCTTGAATAG); SEQ ID NO: 142 is the nucleotide sequence for human MOG gene exon3 (5'

ATCCTTTCTACTGGGTGAGCCCTGGAGTGCTGGTTCTCCTCGCGGTGCTGCCTGTG CTCCTCCTGCAGATCACTGTTGGCCTCGTCTTCCTCTGCCTGCAGTACAGACTGAG AG);

SEQ ID NO: 143 is the nucleotide sequence for human MOG gene exon4 (5' GAAAACTTCGAGCAGAGATAG);

SEQ ID NO: 144 is the nucleotide sequence for human MOG gene exon5 (5' AGAATCTCCACCGGACTTTTG);

SEQ ID NO: 145 is the nucleotide sequence for human MOG gene exonόA (5' AGTCCTTTGGTGTTCTAGGACCCCAGGTTAAGGAACCAAAAAAGACAG); SEQ ID NO: 146 is the nucleotide sequence for human MOG gene exonόB (5'

ATCCCCACTTTCTGAGGGTGCCCTGCTGGAAGATAACCCTGTTTGTAATTGTGCCG GTTCTTGGACCCTTGGTTGCCTTGATCATCTGCTACAACTGGCTACATCGAAGACT AGCAG);

SEQ ID NO: 147 is the nucleotide sequence for human MOG gene exon7 (5' GGCAATTCCTTGAAGAGCTAC);

SEQ ID NO: 148 is the nucleotide sequence for human MOG gene exonδA (5' GAAATCCCTTCTGA);

SEQ ID NO: 149 is the nucleotide sequence for human MOG gene exon8B (5' TCTTCCACCTGGAAGCCCTCTCTGGCTAA); SEQ ID NO: 150 is the nucleotide sequence for murine MOG gene exonl (5'

ATGGCCTGTTTGTGGAGCTTCTCTTGGCCCAGCTGCTTCCTCTCCCTTCTCCTCCTC CTTCTCCTCCAGTT GTCATGCAGC TATGCAG);

SEQ ID NO: 151 is the nucleotide sequence for murine MOG gene exon2 (5' GACAATTCAGAGTGATAGGACCAGGGTATCCCATCCGGGCTTTAGTTGGGGATGA AGCAGAGCTGCCGTGCCGCATCTCTCCTGGGAAAAATGCCACGGGCATGGAGGT GGGTTGGTACCGTTCTCCCTTCTCAAGAGTGGTTCACCTCTACCGAAATGGCAAG GACCAAGATGCAGAGCAAGCACCTGAATACCGGGGACGCACAGAGCTTCTGAAA GAGACTATCAGTGAGGGAAAGGTTACCCTTAGGATTCAGAACGTGAGATTCTCAG ATGAAGGAGGCTACACCTGCTTCTTCAGAGACCACTCTTACCAAGAAGAGGCAGC AATGGAGTTGAAAGTGGAAG);

SEQ ID NO: 152 is the nucleotide sequence for murine MOG gene exon3 (5' ATCCCTTCTATTGGGTCAACCCCGGTGTGCTGACTCTCATCGCACTTGTGCCTACG ATCCTCCTGCAGGTCTCTGTAGGCCTTGTATTCCTCTTCCTGCAGCACAGACTGAG AG); SEQ ID NO: 153 is the nucleotide sequence for murine MOG gene exon4 (5'

GAAAACTTCGTGCAGAAGTAG);

SEQ ID NO: 154 is the nucleotide sequence for murine MOG gene exon5 (5' AGAATCTCCATCGGACTTTTG);

SEQ ID NO: 155 is the nucleotide sequence for murine MOG gene exonόB (5' ATCCTCACTTCCTGAGGGTGCCCTGCTGGAAGATAACACTGTTTGTTATTGTGCCT GTTCTTGGACCCCTGGTTGCCTTGATCATCTGCTACAACTGGCTGCACCGAAGACT GGCAG);

SEQ ID NO: 156 is the nucleotide sequence for murine MOG gene exon7 (5' GACAGTTTCTTGAAGAGCTAA); SEQ ID NO: 157 is the nucleotide sequence for murine MOG gene exonδ (5'

GAAACCCCTTTTGA);

SEQ ID NO: 158 is the nucleotide sequence for bovine MOG gene exonl (5' ATGGCCAGTTTATTGAGCTCCTCTCTGCCCAGCTGTCTCCCCTCCCTCCTCTTCCTC CTCCTCCAGTTGACTTCCAGCTC TGCAG); SEQ ID NO: 159 is the nucleotide sequence for bovine MOG gene exon2 (5'

GACAGTTCAGAGTAATAGGACCAGGGCACCCCATCCGGGCGCTGGTAGGGGATG AAGTGGAATTGCCCTGTCGCATATCTCCAGGAAAGAACGCTACAGGCATGGAGG TGGGATGGTATCGGCCCCCCTTCTCCAGGGTGGTTCATCTCTACCGAAATGGCAA GGACCAAGACGAAGAGCAGGCACCTGAATACCGGGGCCGCACACAGCTGCTAAA AGAGACCATTGGGGAAGGGAAGGTGACCCTCAGGATCCGGAATGTGAGGTTCTC AGATGAAGGAGGTTTTACCTGCTTCTTCCGAGATCACTCTTACCAAGAGGAGGCA GCGATGGAATTGAAAGTGGAAG);

SEQ ID NO: 160 is the nucleotide sequence for bovine MOG gene exon3 (5' ATCCCTTCTACTGGATCAACCCCGGCGTGCTGGTGCTCATCGCGGTCCTGCCAGTG CTCCTCCTACAGATCACCGTGGGCCTCGTCTTCCTGTGCCTGCAGCGCAGACTCCG AG);

SEQ ID NO: 161 is the nucleotide sequence for bovine MOG gene exon4 (5' GAAAACTCTGGGCAGAGATAG);

SEQ ID NO: 162 is the nucleotide sequence for bovine MOG gene exon5 (5' AGAATCTCCACCGGACTTTTG);

SEQ ID NO: 163 is the nucleotide sequence for bovine MOG gene exon6B (5' ATCCCCACTTCCTGATGGTGCCCTGCTGGAAGATAACCCTATTTGTCATCGTGCCG GTTCTCGGACCCCTGGTGGCCTTGATCATCTGCTATAACTGGCTACACCGCAGACT AGCAG); SEQ ID NO: 164 is the nucleotide sequence for bovine MOG gene exon7 (5'

GGCAATTTCTTGAAGAGCTAA);

SEQ ID NO: 165 is the nucleotide sequence for bovine MOG gene exonδA (5' GAAACCCCTTCTGA);

SEQ ID NO: 166 is the nucleotide sequence for macaque MOG gene exonl (5' ATGGCAAGCTTATCAAGACCCTCTCTGCCCAGCTGCCTCTGCTCCTTCCTCCTCCT CCTGCTCCTCCAAGTGTCTTCCAGCTACGCAG);

SEQ ID NO: 167 is the nucleotide sequence for macaque MOG gene exon2 (5' GACAGTTCAGAGTGATAGGACCAAGACAACCTATCCGGGCTCTGGTCGGTGATG AAGTGGAATTGCCATGTCGCATATCTCCTGGGAAGAACGCTACAGGCATGGAAGT GGGATGGTACCGGCCCCCCTTCTCTAGGGTGGTTCATCTCTACAGAAATGGCAGG GACCAAGATGGAGAGCAAGCACCTGAATATCGGGGCCGGACAGAGCTGCTGAAA GACGCTATTGGTGAGGGAAAGGTGACTCTCAGGATCCGGAATGTAAGGTTCTCA GATGAAGGAGGTTTCACCTGCTTCTTCCGAGATCATTCTTACCAAGAGGAGGCA GCAATAGAATTGAAAGTGGAAG);

SEQ ID NO: 168 is the nucleotide sequence for macaque MOG gene exon2' (5' AGATCAAGATGAAAAGGGCTTCAGCTGCGGCATGGCACATGGAGATTAGAGCGG GGCTTTTGGATGCTGAGGAGCAGACCTAG);

SEQ ID NO: 169 is the nucleotide sequence for macaque MOG gene exon3 (5' ATCCCTTCTACTGGGTCAGCCCTGCAGTGCTGGTTCTCCTCGCGGTGCTGCCTGTG CTCCTTCTGCAGATCACTGTCGGCCTCGTCTTCCTCTGCCTGCAGTATAGACTGAG AG);

SEQ ID NO: 170 is the nucleotide sequence for macaque MOG gene exon4 (5' GAAAACTTCGAGCAGAGATAG);

SEQ ID NO: 171 is the nucleotide sequence for macaque MOG gene exon5 (5' AGAATCTCCACCGGACTTTTG); SEQ ID NO: 172 is the nucleotide sequence for macaque MOG gene exon6A (5'

AATCCTTTGGTGTTCTAGGATCCCAGGTTAAGGAACCAAAAAAGAAAG);

SEQ ID NO: 173 is the nucleotide sequence for macaque MOG gene exon6B (5' ATCCCCACTTTCTGAGGGTGCCCTGCTGGAAGATAACCCTGTTTGTAATTGTGCCG GTTCTTGGACCCCTGGTTGCCTTGATCATCTGCTACAACTGGCTACATCGAAGACT AGCAG);

SEQ ID NO: 174 is the nucleotide sequence for macaque MOG gene exon7 (5' GGCAATTCCTTGAAGAGCTAA);

SEQ ID NO: 175 is the nucleotide sequence for macaque MOG gene exon8A (5' GAAACCCTTTCTGA); SEQ ID NO: 176 is the nucleotide sequence for macaque MOG gene exon8B (5'

TCTTCCACTTGGAAACCCTTCCTGGCTAA);

SEQ ID NO: 177 is the nucleotide sequence for marmoset MOG gene exonl (5' ATGGCAAGCTTATCGAAACCCTCCCTGCCCAGCTACCTCTGCTTCCTCCTGCTCCT CCTCCATGTGTCTTCCAGCTACGGAG); SEQ ID NO: 178 is the nucleotide sequence for marmoset MOG gene exon2 (5'

GGCAGTTCAGAGTGATAGGACCAAGTCACCCCATCCAGGCTCTGGTCGGGGATGC AGCAGAATTGCCATGTCGCATATCTCCTGGGAAGAATGCTACAGGCATGGAGGTG GGGTGGTACCGCTCCCCCTTCTCTAGGGTGGTTCATCTCTACAGAAATGGCAAGG ATCAAGATGGAGAGCAGGCACCTGAATATCGGGGTCGAACAGAGCTGCTGAAAG ACGATATTGGTGAGGGAAAGGTGACTCTCAAGATCCGGAATGTAAGATTCCCAG ATGAAGGAGGTTTCACCTGCTTCTTCCGAGATCATTCTTACCAAGAGGAGGCCGC AATGCAATTGAAAGTGGAAG);

SEQ ID NO: 179 is the nucleotide sequence for marmoset MOG gene exon3 (5' ATCCCTTCTACTGGGTCAGCCCTGGAGTGCTGGTTCTCCTCGCAGTGCTCCCTGTG CTCTTCCTGCAGATCACTGTGGGCCTCGTCTTCCTCTACCTGCAGCATAGACTGAG AG);

SEQ ID NO: 180 is the nucleotide sequence for marmoset MOG gene exon4 (5' GAAAACTTCGAGCAGAGATAG);

SEQ ID NO: 181 is the nucleotide sequence for marmoset MOG gene exon5 (5' AGAATCTCCACCGG ACTTTTG);

SEQ ID NO: 182 is the nucleotide sequence for marmoset MOG gene exonό (5' ATCCCCACTTTCTGAGGGTGCCCTGCTGGAAGATAACCCTGTTTGTAATTGTGCCG GTTCTTGGACCGCTGGTCGCCTTGATTATCTGTTACAACTGGCTACATCGAAGATT AGCAG); SEQ ID NO: 183 is the nucleotide sequence for marmoset MOG gene exon7 (5'

GGCAATTCCTTGAAGAGCTAA);

SEQ ID NO: 184 is the nucleotide sequence for marmoset MOG gene exonδ (5' GAAATCCCTTCTGA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, generally, to diseases of the central and peripheral nervous systems and other systemic diseases. As indicated above, the present invention is based upon the complete analysis of the alternative splicing of the MOG gene in five representative species from rodent to human and discloses that the most complex splicing patterns are exclusive to higher mammals including human and non-human primates, unlike for other myelin proteins. Such MOG splice variants, isoforms, antibodies, and methods presented herein may be suitably employed for the identification, characterization, and treatment of diseases including, but not limited to, demyelinating and other neurodegenerative, infectious, inflammatory, inherited, and metabolic disorders such as multiple sclerosis (MS) and Alexander's disease. More specifically, the present invention provides alternative splice variants of myelin oligodendrocyte genes (MOGs) (depicted in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 3O₅ 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 70); isoforms of MOG proteins encoded by such splice variants (depicted in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69); any fragments of the variant MOG proteins; antibodies that specifically bind to one or more of the MOG isoforms depicted in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69 and/or one or more of the peptide epitopes encoded by the nucleotide sequences depicted in SEQ ID NOs: 107-184); as well as therapeutic and diagnostic methods that employ MOG splice variants, isoforms, and/or antibodies specific thereto. Such MOG splice variants, isoforms, and antibodies presented herein may be suitably employed in compositions and methods for the identification, characterization, and treatment of autoimmune diseases including, but not limited to, multiple sclerosis.

An epitope that "specifically binds" (used interchangeably herein) to an antibody or a polypeptide is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an isoform of MOG protein is an antibody that binds this isoform or isoform epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other isoform or isoform epitopes or non-MOG epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')₂, Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example.

The polynucleotide encoding an alternative splice variant of MOG protein described herein may be used to transfect a cell, such as CHO cells, to generate a cell line expressing the variant. Such cell lines may be used in detecting autoreactive antibodies (such as human antibodies) to the MOG variant expressed.

One or more of the MOG isoforms or polynucleotides described herein may be administered to an animal (such as a mammal) to induce immune response to MOG protein to establish a disease model. In some embodiments, the polypeptide is administered with complete freund's adjuvant.

One or more of the MOG isoforms or polynucleotides described herein may be administered to an animal having autoimmune reaction to myelin oligodendrocyte glycoproteins. In some embodiments, the polypeptide is administered with incomplete freund's adjuvant, splenocyte-coupled. Administration can be by any means known in the art, including: orally (inducing tolerance), mucosally (lung spray for inducing tolerance), by eye drops, intrathecally, intracranially, intradermally, intranasally, intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, intravenous peripherally, and/or intravenous centrally.

Polynucleotides may be administered naked or in vectors, such as viral vector (adeno, AAV, vanninia, fowlpox, etc.).

The present invention will be best understood in conjunction with the following, non- limiting Examples, which set forth the methodology for identifying each of the MOG splice variants, sequences of MOG splice variants from a variety of rodent and primate species, including those disclosed in SEQ ID NO and MOG isoforms encoded by the presently disclosed MOG splice variants. It will be appreciated that antibodies, including fully-rodent antibodies, chimeric rodent-human antibodies, humanized antibodies, and fully-human antibodies that bind specifically to MOG isoforms are all encompassed by the scope of the present invention and will be prepared by those of skill in the art through routine experimentation following well established procedures including, but not limited to, those described in further detail herein. EXAMPLES

The following Examples are offered by way of illustration, not limitation.

Example 1 IDENTIFICATION OF MULTIPLE MOG ISOFORMS

Sequencing of macaque and marmoset MOG cDNAs (NCBI numbers: AF399846, AY566834; SEQ ID NOs: 43 and 63, respectively) confirmed the high degree of conservation previously observed for the predicted extracellular domain of MOG as compared to human and rodent sequences (the present invention and Pham-Dinh et al, Oligodendrocyte Glycoprotein Gene, in, Myelin Biology and Disorders 1, 469-497 (R.A. Lazzarini ed., 2004). See, Figure 1. By contrast, the full set of CNS MOG transcripts amplified using a single round of PCR with the most distal MOG primer sets revealed multiple transcripts with clear- cut size and quantitative differences. Figure 2B. In addition to the major transcript present in all species, several other bands were detected in human, bovine, C. jacchus, and M. fascicularis brain amplified transcripts.

Alternative Splicing of the Human MOG Gene

Characterizing the splicing sites used in the primary transcript of the MOG gene led to discovery of 15 MOG isoforms in the human brain (presented herein in Figures 3 and 4A). The corresponding encoded MOG protein isoforms are also schematically depicted in Figure 4B. Consensus motifs for splicing were detected at all intron-exon boundaries (see, Table 1, below), and the results of specific nested PCR experiments are presented in Figure 3. Primer sets encompassing potential splicing sites were used to specifically explore the 5' part of the gene. Figure 3A. Three isoforms lacking exon 2 (encoding the IgV-like domain) were identified (Figure

3A), each differing by exon composition in their respective 3' ends: the α4 transcript contained exon 6B, the α5 transcript was devoid of exon 6, and the α6 transcript contained exon 6A (Figure 4A; NCBI numbers: AY786325, AY566846, and AY566847; and presented herein as SEQ ID NOs: 7, 9, and 11₅ respectively). A previously published sequence of 138-bp (Ballenthin et al, J Neurosci Res 46:271-

81 (1996)) located in intron 2 (position 8583-8720 according to numbering in the human MOG gene was also identified (Fig. 3B); this sequence belongs to the AIu repeat family and was designated Alu-10 exon, as it is the tenth AIu sequence found within the gene. Roth et al. Genomics 28:241-50 (1995).

Table 1 Splice Sites in the Human MOG Gene

The presence of the AIu sequence in additional transcripts was confirmed by performing PCRs using an antisense primer derived from the Alu-10 sequence. Figure 3C. Two bands were obtained, cloned, and sequenced. Surprisingly, 4 different sequences were found: i) an isoform comprising exons 1, 2 and Alu-10, designated herein as Ig-AIu- 10; ii) a sequence lacking exon 2, designated herein as Alu-10; iii) and iv) two additional forms similar to i) and ii) but presenting a deletion of 20 bp in the 5' end of exon Alu-10, indicating the occurrence of a further alternative splicing event in this exon by use of an additional acceptor splicing site. Thus, the full AIu- 10 exon was designated AIu- 1OA, and the partly excised one, AIu- 1OB (NCBI numbers: AY566848 (SEQ ID NO: 13; Ig-AIu-IOB), AY566849 (SEQ ID NO: 15; AIu-I OA), AY566850 (SEQ ID NO: 17; AIu-IOB). Only one band corresponding to the splicing of exon 1 to exon 2 was found when this boundary was explored, indicating that no additional exons were encoded by sequences located within intron 1. By contrast, amplification of transcripts from exon 2 to 3 revealed an additional splicing variant comprising a 163-bp sequence at the junction between the classic exons 2 and 3. This sequence, which is located within intron 2 upstream to exon AIu-10 (position 7923 to 8092), was designated exon T (NCBI number: AY566851; SEQ ID NO: 19). Id. Identification of Alternative MOG Transcripts in Other Mammalian Species

The exon composition of alternative variants in non-human mammalian species was explored using species-specific 1-08B primer sets (Figure 2 and Table 2, below) that corresponded to the non-selective amplification of α and β isoforms (thus including the sequences encoding the major α-specific carboxy-terminus and the alternative β-carboxy- terminus, respectively).

Table 2 MOG Oligonucleotide Primers

In a second step, nested PCRs were performed with the 1-08A primer sets, which amplified only α forms, after Bell restriction cutting of exon 6B (contained in αl major isoforms), thus permitting selective amplification of minor α transcripts. AU of these mRNA variants were further cloned and sequenced to fully assess exon organization of the corresponding cDN As. See, Table 3.

Table 3 MOG splicing variants across species

Designation Exon Composition (Deletion) g

Mouse: One band corresponded to the major isoform, αl, after amplification with the mouse-specific 1-08B primer set. Figure 2. Another form, however, corresponding to the minor human α2 (lacking exon 6B), was further detected using 1-08 A α isoform-specific nested amplifications. Table 1 (NCBI number: AY566830; SEQ ID NO: 33). Neither β nor other α forms were found after nested amplification following restriction cutting of PCR products using EcoRl or BamHΪ. Bovine: Sequencing of the lower MW band detected in agarose gels (Figure 2), showed that this variant lacked exon 2 and may correspond to the α4 human MOG isoform. Figure 4A. After restricted amplification of the major form by Bell restriction cutting, 2 additional bands were detected corresponding to human α2 and α5 isoforms. Figure 4A. Further sequencing identified 4 variants, αl, α2 (NCBI number AY566831; SEQ ID NO: 37), oc4 (NCBI number AY566832; SEQ ID NO: 39), α5 (NCBI number AY566833; SEQ ID NO: 41). See, Table 1. No β forms were found after cutting by EcoRl in bovine brain PCR products.

Marmoset: Sequencing of the band visible on agarose gels below the major form αl (Figure 2; NCBI number: AY566834; SEQ ID NO: 63) showed a α4 isoform (Figure 4A), and also an unexpected isoform deleted of 154 on 348 bp at the 5' end of exon 2. In this variant, exon 1 is spliced to nucleotide 273 in exon 2, due to a cryptic consensus 'ag' dinucleotϊde acceptor site at position 271-272. By analogy to the human α4 form, which lacks exon 2, this form was designated α4Cj. Using the exon-specifϊc restriction cutting strategy, another form lacking exon 3, designated α7Cj, was detected. Figure 4 A and Table 1. Neither α4Cj nor α7Cj forms were observed in the other species studied and thus appear to be unique to marmosets (NCBI numbers: AY566836, AY566837; SEQ ID NOs: 67 and 69, respectively).

Macaque: The complex expression pattern detected in this species comprised 4 faint bands in addition to the major form (Figure 2), and appeared virtually identical to that of humans. Sequencing revealed one form without exon 2 (the oc4 isoform), and another containing exon 8B in place of exon 8 A, thus corresponding to a macaque βl isoform. Using α-specific amplification, the following isoforms were detected: α2, α3, cc4, α5, and α6. In addition, using the nested amplification strategy, described herein above, followed by EcoRl cutting, β2 and β3 forms were also identified. Thus the human and M. fascicularis brains both exhibited αl, cc2, α3, α4, α5, α6, as well as βl, β2₅ and β3 MOG transcripts (presented in Table 1; NCBI numbers: AF399846, AY566838, AY566839, AY566840, AY566841, AY566842, AY566843, AY566844, and AY566845; SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, and 59, respectively).

The AIu and 2' exon sequences were searched in non-human primates, C. jacchus and M. fascicularis, using a short PCR amplification from exon 2 to 3. In contrast to humans, only exon 2' was found in the M. fascicularis (NCBI number: AY786326; SEQ ID NO: 61). Thus, by comparison with all other species disclosed herein, the closest to humans was macaque, which is also the only other mammal to express β forms of MOG.

In total, the data presented herein demonstrated the diversity generated by splicing of the primary transcript from the MOG gene during recent evolution. The mature MOG protein was encoded by the major MOG transcript αl . Although the pattern of transcription appeared similar to that of major structural myelin protein genes such as MBP and PLP (Campagnoni et al, Brain Pathol ϋ:74-91 (2001) and Baumann et al, Physiol Rev ϋ :871-927 (2001), MOG was unique in that: i) it was only found in mammalian genomes where it was located within the MHC (Pham-Dinh et al Proc, Natl. Acad. ScI U.S.A. 90:7990-4 (1993); Bernard et al. J MoI Med l '5:77-88 (1997); Iglesias et al, GHa 36:220-34 (2001); Delarasse et al J CHn Invest 112:544-53 (2003); and Pham-Dinh et al, Oligodendrocyte Glycoprotein Gene, in, Myelin Biology and Disorders 1, 469-497 (R. A. Lazzarini ed., 2004)) and ii) whereas other myelin genes tended to be ubiquitously expressed (Campagnoni et al, Brain Pathol ϋ: 74-91 (2001) and Baumann et al, Physiol Rev 81:871-927 (2001)) expression of the MOG protein was restricted to myelin (Colman et al, 85-100 (BIOS Scientific Publishers Ltd, Oxford, UK, 1996)); and iii) when comparing rodents to higher mammals, MOG was overall less conserved than other myelin proteins (Popot et al, Annu Rev Biophys Biophys Chem 19:369- 403 (1990)), especially within the transmembrane domain. Figure 1. Alternative splicing of major and minor transcripts of MOG exhibited a striking increase in complexity paralleling evolutionary events; the most complex patterns were found in human and non-human primates. Fifteen alternative splicing variants using combinations of 11 exons were identified in humans, 10 in Old World primates (M. fascicularis), and only 2 in mice (when including the major form α-1). The β variants were only found in macaques and humans, and soluble forms were not expressed in non-primate species. Table 1. This phenomenon is unique to MOG and has not been reported for another well- studied myelin gene, the GoIWMBP gene, as Golli/MBP isoforms are well conserved between human and mouse. Campagnoni et al, Brain Pathol JJL: 74-91 (2001) and Pribyl et al. Proc. Natl. Acad. Sd. U.S.A. 90, 10695-9 (1993). Moreover, contrary to both the MOG and Golli/MBP genes, the PLP gene encodes two mouse-specific isoforms, which have not yet been found in humans (Campagnoni et al, supra) in addition to the strictly conserved PLP/DM20 isoforms found in most vertebrate species.

In all species examined herein, several alternating variants showed splicing out of sequences encoding the second hydrophobic domain of MOG (exon 6B), or the carboxy- terminus (exon 8A). These sequences were replaced by either hydrophilic exon 6 A, or a new carboxy-terminus (exon 8B). Figure 4; Table 1.

Without wishing to be limited to any specific mechanistic theory, it is believed that the resulting variant proteins, especially those containing exon 8B (β forms) may underlie distinct MOG-cytoskeleton interactions and transmembrane signaling pathways. It has recently been reported that interaction between myelin proteins and the myelin membrane lipids, as opposed to proteins themselves, may be essential for membrane stability and integrity (Hu et al. Proc.

Natl. Acad. Set U.S.A. lOJj 13466-71 (2004)), thus both MOG/lipid interactions and signaling pathways through MOG could potentially differ between rodents and primates including man.

Similar to MOG, the evolutionarily-related butyrophilin and bg antigen members of the B7/BT family genes (Linsley et al., Protein Sci 3:1341-3 (1994), are also alternatively spliced in the short exons encoding heptads forming the cytoplasmic part of their molecule. Reviewed in Pham-Dinh et al, Myelin biology and disorders Vol. 1, supra and Henry et al, Immunol Today 20:285-8 (1999).

In addition to myelin maintenance and function, the intrinsic alternative splicing machinery may operate to regulate the expression of immunologically relevant domains of MOG in primates. In the 5' part of the gene, MOG splicing variants that lack exon 2 (exposed encephalitogenic IgV-like domain) are found in all species except mouse. Table 1. The immune system may ignore these isoforms during development, thus truncated MOG proteins may play a role in maintenance of central and peripheral tolerance and/or in inflammatory and demyelinating diseases. Bernard et al, J. MoI Med. 75:77-88 (1997) and Iglesias et al., GHa 36:220-34 (2001). The expression patterns of the MOG isoforms, disclosed herein may be utilized in the context of CNS in diagnostic methods in which physiological and pathological conditions are examined. Similarly, primate-specific exons encode Ig-like domain isoforms resulting from exonisation of premature stop codon-containing exons, such as AIu or 2' sequences, downstream of exon 2 sequences. These transcripts corresponded to species-specific protein motifs predicted to be soluble rather than membrane-bound. Figure 4B. It is comtemplated that expression of such soluble MOG proteins may have a profound impact on immune homeostasis or dysregulation.

Quite surprisingly, C. jacchus marmosets presented a pattern of MOG alternative splicing with only 4 isoforms, including the 2 variants unique to this species, α4Cj and α7Cj. Table 1 and Figure 4. These New World primates evolved from a split of the Anthropoidea into Platirrhini (New World primates) and Catarrhini (Old World primates) about 35-40 millions years ago, while M. fascicularis and H. sapiens diverged at 35 million years in the Old World primate tree. Both M. fascicularis and C. jacchus evolved with unique characteristics such as limited variability in MHC class I and II genes, and -for marmosets- bone marrow chimerism. Watkins et al., J Immunol. 144:3726-35 (1990); Uccelli et al., J. Immunol. 158_: 1201-7 (1997); Cadavid et al, Proc. Natl. Acad. ScL U.S.A. 94_:14536-41 (1997) ; Antunes et al, Proc. Natl Acad. Set U.S.A. 95:11745-50 (1998). Differentiation of other immune system genes is quite similar to man in complexity. Uccelli et al, J. Immunol. 158:1201-7 (1997) and von Budingen et al , J. Clin. Immunol. 2J_.:155-70 (2001). The marmoset isoform oc4Cj may be regrouped with isoforms where exon 2 is spliced out, resulting in truncated proteins lacking the IgV -like domain found in all species except mouse; α4Cj appears to be soluble. The α7Cj isoform lacked exon 3 (the first transmembrane domain) and, thus, encoded an isoform having an extracellular domain extended by the hydrophilic peptides encoded by exons 4 and 5. These proteins possessed a distinct tertiary structure and/or may be less tightly associated with the myelin membrane than the major MOG isoform. The most complex neuropathological patterns of experimental inflammatory deniyelination were reproduced in C. jacchus and appear to be correlative of human MS.

Unlike existing mouse models of MS, the demyelinating component of lesions in C. jacchus is produced by a subgroup of anti-MOG-antibodies that are pathogenic because they specifically bind to conformational determinants of the IgV-like domain but do not specifically bind to its linear domains located in the exposed random loops, von Budingen et al Eur. J. Immunol. 34:2072-83 (2004). The presently disclosed findings in the C. jacchus system, disclosed herein, demonstrate how the differential expression of the spliced variants of MOG, through presentation of specific antigenic determinants within the CNS, influences susceptibility to disease and the expression of phenotype in primates including man. Moreover, the topographical distribution and quantitative expression of MOG variants that contain determinants exposed, to pathogenic antibodies, influences the location, severity, and density of demyelinating lesions along the neuraxis in disorders like MS.

The human MOG gene contains as many as 14 AIu sequences concentrated in 3 introns. Roth et al, Genomics 28:241-50 (1995). The Alu-10 exon belongs to the J-subclass of AIu element, one of the oldest AIu subfamilies, and is located in the antisense orientation (position 8568-8863; 296 bp length), the most prone to be exonised. The genomic sequence surrounding the acceptor site of the Alu-10 exon in the MOG gene (Table ?), was similar to that previously determined as necessary for exonisation for the AIu-I family subtype. Lev- Maor et al, Science 300:1288-91 (2003). The 4 MOG Alu-containing isoforms described herein were only found in human and provide additional examples wherein AIu elements enriched the transcriptome. Kreahling et al, Trends Genet. 20:1-4 (2004). It is believed that translation of these polyadenylated RNAs, resulting from the exonisation of AIu and 2' sequences specific to higher primates, further distinguishes the primate proteome from that of other mammals. Alu-10 and 2' sequences of the MOG gene are located in similar positions in the chimpanzee and human genomes (http://genome.ucsc.edu/cgi-bin/hgBlat).

The complexity of alternative splicing pattern of the MOG gene correlated with the position of mammals in the phylogenic evolutionary tree. This phenomenon appears to be unique to the MOG gene and has not been observed for other major myelin genes. It is believed that the MOG gene is located in the MHC, an unstable genomic region that, because of its prime importance in defining and regulating self-tolerance and autoimmunity, is rapidly evolving across species. The MOG gene appears to represent an important milestone in mammalian evolution in the context of its myelin-restricted expression, and provides a link between CNS biology and autoimmunity. The presence of soluble isoforms and β isoforms unique to primates further clarifies the roles of MOG as a structural myelin protein and as a receptor for signaling. Johns et al., J. Neurochem. 72:1-9 (1999). These soluble MOG isoforms and β isoforms provide antigenic targets for demyelinating antibodies with similar antigenic determinants in humans and primates.

Example 2 Expression of the MOG spliced variants during development of human CNS To study MOG alternative transcript regulation/expression during myelinogenesis,

RT-PCR analysis was performed on human brain tissues for the presence of the MOG mRNA variants. RNA from adult C57BL/6 mouse, bovine and human brain tissue was obtained from Clontech (Biosciences, France); additional samples of brain tissue (frontal lobe) were obtained from a 21 -week-old human foetus, a 40-day-old infant and a 2-year-old child. Brain tissue from adult macaques (M Fascicularis) was obtained from two animals bred at the INRA monkey colony (Jouy-en-Josas, France), and brain tissue from two adult common marmosets (C. jacchus) was obtained from the New England Regional Primate Research Center, Southborough, MA, USA.

Standard molecular biology techniques were performed as known to those skilled in the art. mRNA isolated from brain tissue was reverse-transcribed following a 3' RACE protocol (rapid amplification of cDNA ends) using a dT18-adapter primer as previously described. (Pham-Dinh et al., J Neurochem. (1994) 63:2353-2356). In order to complete a panel of MOG cDNAs, the major full-length MOG mRNAs from macaque and marmoset brains were amplified, cloned and sequenced, as previously described. (Pham-Dinh et al., PNAS (1993) 90:7990-7994). The number and relative abundance of MOG splicing variants were evaluated in samples from human, macaque, marmoset, bovine and mouse, after performing a first round of PCR using species-specific sense primers located in exon 1 and reverse primers located in exon 8 part B as described herein.

In order to clone the corresponding cDNAs, the representation of minor splicing variants was increased using a previously published strategy. (Pham-Dinh et al., Genomics (1995) 29:345-352). In brief, the 1-08B PCR products, containing potentially all splicing MOG variants (containing alternate α- or β- 3 '-ends), were successively digested with exon- specific restriction enzymes, then amplified with nested polymerase chain reactions. To amplify variants without exon 6B, restriction enzyme Bell was used (cleavage site at nucleotide 671 in exon 6B). To amplify variants without exon 2, restriction enzyme BamHI (cleavage site nucleotide 338 in exon 2) was used for all species except bovine, where restriction enzyme HaeII was used instead ofBamHI. To amplify only α - variants a second round of amplification was performed using 1-08 A primer sets. To selectively amplify the β -^-variants (in which exon 8 part A is spliced out), restriction enzyme EcoRI was used (cleavage site at nucleotide 886 in exon 8A), and further amplifications were performed using the 1-08B primer sets.

The final nested PCR products were sub-cloned into pGEM-T easy vector (Promega Biotech). Recombinant colonies were analyzed by PCR, and positive clones were then characterized by direct sequencing of the purified PCR products to assess exon composition and boundaries in the splicing variants (GenomeExpress, Meyran, France). In order to analyse the developmental expression of MOG splicing variants in human brains, RT-PCR were performed by standard procedures for 30 cycles using mRNA from foetal 21 -weeks, post-natal 40-days, 2-years old child, and adult brain (frontal lobes) samples. The PCR products were analyzed by Southern blot analysis using exon-specific primers. Transcripts containing exon 8 A were amplified with Hex 1 /Hex 08 A primer pairs and hybridized with labelled Hex3 primer. Transcripts containing exon 8B were amplified with Hexl/ Hex 08B primer pairs and hybridized with labelled Hex3 primer. Transcripts containing exon AIu were amplified with Hexl/ Hex OAIu primer pairs and hybridized with labelled AIu primer. To avoid heteroduplex formation during electrophoretic separation, RT- PCR products were run in denaturing conditions in NaOH buffer at 4⁰C, transferred onto nylon membranes and probed with a [(X-³²P] dCTP 3 'end-labelled exon-specific primer. All amplified isoforms detected by hybridization were assessed by cloning and sequencing.

As shown in Fig. 5 the major αl mRNA isoform is present at all time points tested, albeit weakly at 21 weeks (fetus) and at 40 days after birth, at which time the other variants were undetectable under the experimental conditions used. By contrast, a dramatic change appears in the brain tissue from a 2-year old child and an adult, in that the level of the major MOG αl mRNA isoform becomes more abundant and the other isoforms α4, α5, βl, Ig-AIu- 1OA and B and AIu-I OA and B become detectable.

Example 3 Induction of EAE with alpah-4 cj newly discovered form

(LLQVSSSYGEMARIKMESRHLNIGVEQSY) was conducted as described below:

Marmosets were immunized with the complete sequence of the alpha-4 marmoset MOG comprising the peptide unique to this species, and not found in human, bovine, macaque, or mouse. Peptide was synthesized by conventional, standard Moc chemistry, and purified >95% by HPLC, then dissolved in PBS and homogeneized with an equal volume of complete Freund's adjuvant supplemented with 3 mg/ml of H37Ra, as previously used for immunization of marmosets with other antigens (Genain and Hauser, Immunol Reviews, 2001). Animals received B. pertussis on the day of immunization and 48 hrs. Later. Imunization were performed using 1 mg of peptide per animal, inoculated in 4 sites on the hips and the shoulder blades, intra-dermally.

Two animals immunized in this fashion developed clinical signs of mild EAE, with a relapsing remitting course. The onset of symtoms was observed for both animals around 15 days after immunization (score of 2). Several remissions (score of 0), and relapses (n= 6 to 8 over the course of greater than 410 days) were oberved in both the animals. The maximal score observed were 4 and 6 on the marmoset grading scale, indicative of clear but mild disease. For comparison, EAE scores in animals induced with the alpha- 1 form of MOG has scores of 4 to 24, depending on severity. Signs in the animals immunized with marmoset peptides included paparesis with decreased tone, respectively (Villosalda et al., 200O₅ J Exp Med).

Claims

What is claimed is:

1. An isolated polynucleotide encoding an alternative splice variant of a myelin oligodendrocyte glycoprotein (MOG), said polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 10, 12, 14, 16, 18, 20, 34, 36, 38, 40, 42, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64₅ 66, 68, and 70.

2. An isolated polynucleotide comprising at least contiguous about 10 nucleotides spanning the junction between exon 2 and exon 4, between exon 2 and AIuB, or between exon 2 and exon 2' of an isoform of MOG.

3. An isolated isoform of a MOG comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 11, 13, 15, 17, 19, 33, 35, 37, 39, 41, 45, 47, 49,

51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.

4. An MOG protein epitope encoded by the polynucleotide of claim 2.

5. An antibody specifically binds to the MOG protein epitope of claim 4.

6. An antibody that specifically binds to one or more of the MOG isoforms selected from the group consisting of α4Cj, α5, α6, αVCj, If-AlulOB, AIuIOA, AIuIOB₅ and Ig-2'.

7. The antibody of claim 6, wherein the antibody does not bind to one or more of the MOG isoforms selected from the group consisting of αl, α2, α3, α4, βl, β2, β3, β4, and Ig-AIuIOA.

8. A method for detecting a MOG isoform comprising detecting a MOG isoform according to claim 3 in a sample.

9. The method of claim 8, wherein the detection is performed by contacting said sample with an antibody of claim 5 or claim 6, and detecting specific bound antibody.

10. The method of claim 8, wherein the detection is performed by detecting the presence of an RNA encoding the MOG isoform in the sample.

11. A method of generating an animal model for multiple sclerosis, comprising administering one or more of the MOG isoforms of claim 3 in an amount effective to induce anti-myelin response in the animal.

12. An animal model generated by the method of claim 11.

13. The method of claim 11, wherein the animal is selected from the group consisting of a mouse, a rat, a rabbit, a bovine, a pig, and a primate.

14. The method of claim 13, wherein the primate is a marmoset or a macaque.

15. A method for inducing tolerance to an MOG comprising administering one or more of the MOG isoform of claim 3 in an amount effective to induce tolerance.

16. The method of claim 15, wherein T cell tolerance is induced.

17. The method of claim 15, wherein B cell tolerance is induced.

18. A cell transfected with a polynucleotide according to claim 1.