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Title:  Method for diagnosing a person having multiple sclerosis
United States Patent: 
7,208,270
Issued: 
April 24, 2007

Inventors:  
Jansen; Burkhard (1080 Vienna, AT), Lucas; Trevor (1160 Vienna, AT)
Assignee: 
Jansen; Burkhard (Vienna, AT), Lucas; Trevor (Vienna, AT)
Appl. No.: 
10/176,372
Filed: 
June 21, 2002


 

Covidien Pharmaceuticals Outsourcing


Abstract

Described is a method for diagnosing a person having multiple sclerosis (MS) or being at risk of developing MS, comprising the following steps: providing a sample of a body fluid or tissue from said person, said sample containing at least one of the wild type SCF-Apoptosis-Response Gene- (wt-SARG-1-) protein and nucleic acids encoding wt-SARG-1, if taken from a person not having MS or a risk of aquiring MS, detecting the presence of wt-SARG-1-protein or nucleic acids encoding wt-SARG-1 in said sample and diagnosing MS or a risk of aquiring MS, if wt-SARG-1-protein or nucleic acids encoding wt-SARG-1 are not present in said sample.

Description of the Invention

The invention relates to a method for diagnosing a person having multiple sclerosis (MS) or being at risk of developing MS. Further, the invention relates to a method for diagnosing a person having cancer or being at risk of acquiring cancer.

Multiple sclerosis (MS) is a common demyelinating disease of the central nervous system (CNS) affecting up to 0.1% of the north European caucasian population and is considered an auto-immune syndrome directed against unidentified central nervous tissue antigens. The determination of susceptibility to MS development is complex and governed by both environmental and genetic factors (Ebers et al, 1995; Sawcer and Goodfellow, 1998; Sadovnick et al, 2000) with approximately 20% of patients having one or more affected relatives (Chataway et al, 1998). Although thought to be a polygenetic disease, candidate gene approaches have been adopted to isolate genes linked to MS (Weinshenker and Kantarci, 2000). Association with the Caucasian haplotype DRB*1501-DQA*0102-DQB1*0602 (Haines et al, 1998) and a point mutation in the protein tyrosine phosphatase receptor-type C (Jacobsen et al, 2000) have been linked to some cases. Recently, the importance of apoptosis in both T cell elimination and damage to neurons and oligodendrocytes in MS have been recognised (reviewed in Zipp, 2000).

However, to date no clear marker has been reported, although at least a portion of MS cases are clearly familial inherited. A mere recognition of MS or even providing a risk association would be very beneficial for early onset of therapy or preventive measures.

It is therefore an object of the present invention to provide an efficient diagnosis system for MS giving a clear indication and a clear correlation to this disease.

The subject matter of the invention is therefore a method for diagnosing a person having multiple sclerosis (MS) or being at risk of developing MS, characterised by the following steps: providing a sample of a body fluid or a tissue from said person, said sample containing at least one of the wild type SCF-Apoptosis-Response Gene 1- (wt-SARG-1-) protein and nucleic acids encoding wt-SARG-1, if taken from a person not having MS or a risk of aquiring MS, detecting the presence of wt-SARG-1-protein or nucleic acids encoding wt-SARG-1 in said sample and diagnosing MS or a risk of aquiring MS, if wt-SARG-1-protein or nucleic acids encoding wt-SARG-1 are not present in said sample.

Surprisingly, SARG-1 protein turned out to be a very specific marker for MS. Persons having either mutated SARG-1 protein or not expressing any SARG-1 protein due to mutations in SARG-1, have a clearly enhanced risk of MS. Investigations on the immuno-histochemical localisation of SARG-1 protein indicated that this protein is located in the grey and white matter of the CNS. The role of SARG-1 in apoptotic induction prompted a candidate gene approach to analyse the mutational status of SARG-1 in cases of familial MS. Indeed, DNA from 20 unrelated familial MS patients was examined by PCR amplification and DNA-sequencing of the SARG-1 locus and compared to SARG-1 sequences from healthy controls. It was found that all control samples demonstrated wild-type SARG-1 genomic sequences whereas in DNA from MS patients only 6 from 20 DNA samples were even able to be amplified by PCR, i.e. 14 from 20 did not show any detectable SARG-1 signals. In 4 of the 6 other patients genetic alterations were seen. A T.fwdarw.C point mutation at nucleotide 67 resulting in a substitution of phenylalanine with leucine at amino acid 23 (numbering of amino acids and nucleotides according to FIGS. 4 8); a C.fwdarw.T point mutation at nucleotide 359 resulting in the substitution of phenylalanine for serine at amino acid 120 (FIG. 16), A.fwdarw.G point mutation a nucleotide 89 resulting in the substitution of glycine for glutamic acid at amino acid 30 (FIG. 17), deletion of a codon between amino acid 116 and 121 resulting in the loss of a serine residue (FIG. 18). Sequencing of only 20 control DNA samples revealed only wild-type sequence.

Surprisingly, it was also observed that changes in wild type SARG-1 or SARG-1 protein was seen in several cancer cells. In sequence analysis of over 30 cancer cells, a G residue is always present at position 280 resulting in a leucine residue at position 94 instead of a valine (A nucleotide) or methionine (T nucleotide). In a human melanoma cell line two mutations in SARG-1 are found: A.fwdarw.G point mutation at nucleotide 74 resulting in the substitution of aspartic acid for glycine and a C.fwdarw.T point mutation at nucleotide 289 resulting in the substitution of histidine for thyrosine at amino acid 97.

There is a number of restriction sites involved in these mutations: T.fwdarw.C at nucleotide 67 creates a number of restriction sites: Eco88I, XhoI, PaeR7I, Sfr274I, Ama781I, BcoI, BsoBI, AvaI; C.fwdarw.T at nucleotide 359 creates an additional BseRI restriction-site.

Therefore, a further object of the present invention relates to a method for diagnosing a person having cancer or being at risk of aquiring cancer, characterised by the following steps: providing a sample of a body fluid or tissue from said person, said sample containing at least one of the wt SARG-1 protein and nucleic acids encoding wt-SARG-1, if taken from a person not having cancer or being at risk of aquiring cancer, detecting the presence of wt-SARG-1 protein or nucleic acids encoding wt-SARG-1 in said sample and diagnosing cancer or a risk of aquiring cancer, if wt-SARG-1 protein or nucleic acids encoding wt-SARG-1 are not present in said sample.

The source of the sample is always dependent on the nature of the cancer or disease to be diagnosed. Especially preferred samples according to the present invention are derived from human blood, plasma, serum, lymph, nerve-cell.containing tissue, cerebrospinal fluid, all biopsy-material, including tumor tissue, bone marrow, nervous tissue, skin, hair, tears, fetal material including amniocentesis material, uterine tissue, saliva, faeces, sperm, etc.

In principle, any method for detecting the presence of wt-SARQ-1 protein or nucleic acid encoding wt-SARG-1 in the sample may be applied according to the present invention. Preferably methods are applied which allow also a characterisation of specific SARG-1 mutants if present, either by giving the information that a mutant is present or by analysing the nature of the mutant form in detail.

Especially detecting the presence of point mutations may be preferred within the present invention, i.e. non-wt-forms of SARG-1 differing from wt-SARG-1 or wt-SARG-1 protein in one nucleic acid residue, or one amino acid residue, respectively. The method according to the present invention may be designed to identify those point mutations, especially point mutations leading to the different amino acid sequence, e.g. exchange of one amino acid residue from the wild type SARG-1 protein.

Suitable methods for detecting the presence of wt-SARG-1 protein or nucleic acids encoding are known in the art, preferably nucleic acids encoding wt-SARG-1 are detected by nucleic acid amplification methods, especially polymerase chain reaction methods, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, proteomics, etc. These methods have been shown as being fast, highly reliable and easily conductable on a high throughput basis. Those tests could be performed on standard tissue or body fluid samples, such as blood, hair or saliva.

On the other hand, preferred methods for detecting the presence of wt-SARG-1 protein encompass the application of a wt-SARG-1 protein antibody, especially a monoclonal antibody, e.g. in a ELISA-format. Such antibodies may be easily produced on an industrial scale with a high degree of standardisation potential.

The method according to the present invention is especially suited to be applied within a screening test format.

The SARG-1 intron/exon structure is given in FIG. 19. The transcription start site initiator consensus YYCARR is underlined. Donor (GU) and acceptor (AG) splice sites are underlined in italics; exons are in bold type. Coding exon sequences are in italic. SARG-1 is located on human chromosome 20q12-13.12.

The present invention also relates to a further aspect, to a nucleic acid molecule comprising a sequence according to Seq.ID.No. 1 (FIG. 4) encoding human wild-type SARG-1. Such nucleic acids may be used for diagnosis but also for therapeutic aspects by providing therapeutic molecules or gene sequences for gene therapy aspects, e.g. by antisense strategies, design of small molecule drugs.

The present invention also encompasses nucleic acid molecules comprising a sequence according to Seq.ID.No. 1, wherein one nucleic acid residue is exchanged by a different nucleic acid residue (e.g. T is replaced by C, G or A) wherein said exchange preferably results in a different SARG-1-protein amino acid sequence.

Especially preferred exchanges are selected from a T to C exchange at position 67 of Seq.ID.No. 1, an A to G exchange at position 74 of Seq.ID.No. 1, an A to G exchange at position 89 of Seq.ID.No. 1, a C to T exchange at position 289 of Seq.ID.No. 1 and a C to T exchange at position 359 of Seq.ID.No. 1. These exchanges relate to exchanges already observed in MS patients or cancer cells. Further exchanges resulting in a viable phenotype are also preferred.

Other preferred mutations in the nucleic acid molecule according to the present invention comprises a deletion in the coding region, preferably a deletion of one or more codons (e.g. 3 nucleic acids, or 6, 9, 12, etc.). One of these mutations leads to the deletion of a codon between amino acids 116 and 121, resulting in the loss of a serine residue (FIG. 19). Mutations leading to non-functional SARG-1 on SARG-1 protein may also be located in the controlling regions (5' or 3') and/or in the sequences, especially at critical positions for correct splicing.

When the nucleic acid molecule according to the present invention is used for a diagnostic purpose, it is not necessary to use the whole sequence. For use as a probe or performing a method according to the present invention a fragment of Seq.ID.No. 1, preferably having a length of at least 12, more preferred at least 15, especially at least 20, nucleic acid residues is suitable for performing various tests, especially diagnostic tests with these probes, e.g. as a probe to identify or isolate nucleic acid samples or even chromosomal samples or as PCR primers, etc.

The nucleic acid molecules according to the present invention are not restricted to the coding sequence according to Seq.ID.No. 1, but also relate to the genomic counterparts including the whole-exon/intron-structure of this gene, especially also imitations in the non-coding-region resulting in non-wild type forms of the protein (or non-translated forms of the protein) are encompassed by the present invention.

The present invention also relates to a polypeptide being encoded by this nucleic acid molecule, e.g. comprising an amino acid sequence according to Seq.ID.No. 2. There is single potential N-gly-cosylation site with consensus Asn-X-Ser/Thr (amino acid residues 131-133) -- see Original Patent.

All threonine and serine residues may be O-glycosylated. Computer predictions indicate high likelihood of glycosylation of serine residues 91, 108, 113, 117, 118, 119, 120, 121, 123, 124 and 133 and the threonine residues 88, 107, 111, 112, 115 and 125.

Similarly, all threonine and serine residues may be phosphorylated. Computer predictions indicate high likelihood of phosphorylation of serine residues 54, 92, 108, 113, 116, 117, 118, 119, 120, 121, 123, 124, 129, 133 and 144 and the threonine residues 61, 88, 107, 112 and 139.

No signal sequences, characteristic domains or other 3-dimensional structures have been detected other than potential protein kinase recognition sites. Of course, also amino acid sequences also having an amino acid residue exchange or a deletion are also encompassed by the present invention.

Amino acid residue exchanges of the polypeptide according to the present invention are preferably selected from amino acid residues Phe23, Asp25, Glu30, His97 and Ser120, especially Phe23 to Leu 23, Asp25, to Gly25, Glu30 to Gly30, His97 to Tyr97 and Ser120 to Phe120 exchanges.

The present invention provides SARG-1 mutant forms as specific markers for (acute) myeloid leukaemia or other leukaemia subtypes as described hereinafter. Deletions of the SARG-1 gene may be partial or full to serve as marker. Diagnostic tests for screening for the presence or absence of such a marker are easily conceivable and reduced to practice by the skilled man in the art.

Preferred polypeptides according to the present invention are re-combinantly produced which exhibit structural differences compared to wt-SARG-1 protein, e.g. differential glycosylation, especially non-homogeneous glycosylation.

The present invention also relates to a method for making an antibody preparation comprising administering a polypeptide according to the present invention to an animal, allowing said animal to generate antibodies against said polypeptide, extracting antibody-containing body fluids or tissue from said animal and preparing an antibody preparation against said polypeptide from said body fluids or tissue. This method is especially applicable for making polyclonal antibodies.

For making monoclonal antibodies a method for making such an antibody preparation is preferred, comprising administering a polypeptide according to the present invention to an animal, allowing said animal to generate antibodies against said polypeptide, removing the spleen of said animal, preparing fusion cells of said spleen cells with suitable hybridoma generating cells, generating hybridoma cells producing monoclonal antibodies against said polypeptide, cloning and culturing said hybridoma cells, thereby expressing moncolonal antibodies, and preparation of said monoclonal antibodies. The skilled man in the art thereby relies on methods readily available for such purposes and e.g. described in "Antibodies: A laboratory manual" by Ed Harlow, Cold Spring Harbor Laboratory; David Lane, Imperial Cancer Research Fund Laboratories, 1988. Of course, also phage display peptides may also easily be generated.

The present also relates to a kit for performing the in vitro diagnosing method according to the present invention which comprises at least means for detecting the presence of wt-SARG-1 protein or nucleic acids encoding wt-SARG-1. The skilled man in the art can envisage the basis of the disclosure of the present application a wide number of suitable alternatives, e.g. anti-wt-SARG-1 protein antibodies, nucleic acid probes selectively binding to wt-SARG-1, nucleic acid primers defining a region being selective for a wt-SARG-1, a chip comprising said nucleic acid probes or said nucleic acid primers. Other preferred means or assays are assays in which proteins bind to SARG-1 such as antibodies or peptides including mutation specific antibodies, ELISAS, Western Blotting assays, flow cytometry assays and assays using immunohistochemical techniques including confocal microscopy.

A further aspect of the present invention relates to a transgenic non-human animal model of the present invention, especially an animal wherein the SARG-1 gene has been mutated or knocked out. A SARG-1 knock out mouse is especially preferred. Methods for providing such models, especially the mouse models, are readily available to the skilled man in the art. Such an animal model is extremely useful in studying genetic variations and mutations of SARG-1, especially with respect to its MS-related disorders.

The term "transgenic" is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a mammalian cell, particularly a mammalian cell of a living animal. The transgene is used to transform a cell, meaning that a permanent or transient genetic change, preferably a permanent genetic change, is induced in a cell following incorporation of exogenous DNA. A permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.

Transgenic animals comprise an exogenous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially in germ cells. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the germline sequence. During the initial construction of the animal, "chimeras" or "chimeric animals" are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals.

Transgenic animals fall into two groups, colloquially termed "knockouts" and "knockins". In the present invention, knockouts have a partial or complete loss of function in one or both alleles of the endogenous SARG-1. Knockins have an introduced transgene with altered genetic sequence and function from the endogenous gene. The two may be combined, such that the naturally occurring gene is disabled, and an altered form introduced.

In a knockout, preferably the target gene expression is undetectable or insignificant. A knock-out of a SARG-1 means that function of the SARG-1 protein has been substantially decreased so that expression is not detectable or only present at insignificant levels or mutated according to the teachings according to the present invention to perform as suitable model for the situation in humans as described herein. This may be achieved by a variety of mechanisms, including introduction of a mutation or disruption of the coding sequence, e.g. insertion of one or more stop codons, insertion of a DNA fragment, etc., deletion of coding sequence, substitution of stop codons for coding sequence, etc. In some cases the exogenous transgene sequences are ultimately deleted from the genome, leaving a net change to the native sequence. Different approaches may be used to achieve the "knock-out". A chromosomal deletion of all or part of the native gene may be induced, including deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletions of genes that activate expression of SARG-1. A functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native genes (for example, see Li and Cohen (1996) Cell 85:319 329). "Knock-outs" also include conditional knock-outs, for example where alteration of the target gene occurs upon exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.

A "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression or function of the native SARG-1. Increased (including ectopic) or decreased expression may be achieved by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene. These changes may be constitutive or conditional, i.e. dependent on the presence of an activator or re-presser.

The exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence. The introduced gene may be a wild-type gene, naturally occurring polymorphism or mutation, or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions. The introduced sequence may encode wild-type human or animal SARG-1 protein or a mutation thereof, or may utilize the SARG-1 promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal. By "operably linked" is meant that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules, e.g. transcriptional activator proteins, are bound to the regulatory sequence s).

Specific constructs of interest, include, but are not limited to anti-sense SARG-1, which will block native SARG-1 expression, expression of dominant negative SARG-1 mutations, and over-expression of a SARG-1. A detectable marker, such as lac Z may be introduced into the locus, where upregulation of expression will result in an easily detected change in phenotype. Constructs utilizing the SARG-1 promoter region, in combination with a reporter gene or with the coding region are also of interest.

A series of small deletions and/or substitutions may be made in the SARG-1 to determine the role of different exons in DNA binding, transcriptional regulation, etc. By providing expression of SARG-1 protein in cells in which it is otherwise not normally produced, one can induce changes in cell behavior.

DNA constructs for homologous recombination will comprise at least a portion of the SARG-1 with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive-and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527 537.

For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF). When ES cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopreg-nant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.

Binding partners of human SARG-1 include protein-O-mannosyltrans-ferase 1 (POMT1), microtubule-associated protein 1 A (MAP1A), ATpase, Na+/K+ transporting beta 1 polypeptide (ATP1B1), SWI/SNF complex 60 KDa subunit (BAF60c) alpha-Actinin 2, exon 16, rab GDP dissociation inhibitor 1 (GDI1) and proteasome 26 S subunit, ATPase 3 (PSMC3).

Is is a further aspect of the present invention to use SARG-1 binding proteins for modulating activity of SARG-1 proteins (including mutants) and vice versa. Moreover, SARG-1 (wild type) or SARG-1 binding proteins may be used for treating MS or cancer, preferably myeloproliferative disorders, polycythaemia, myelodys-plasia and myeloid leukaemia, especially acute myeloid leukaemia, by administering an effective amount of SARG-1 or SARG-1 binding protein (or a complex thereof) to an MS or cancer patient. Instead of wt-SARG-1 protein, also fragments of SARG-1 may be used which are suitable for advantageous treatment of such patient (e.g. fragments binding to the SARG-1 binding proteins). Minimum requirements for such fragments are easily found by the skilled man in the art especially using the mouse models described herein.
 


Claim 1 of 11 Claims

1. A method for diagnosing in a patient multiple sclerosis (MS) or the increased risk for acquiring MS, wherein the method comprises the steps of providing a sample of a body fluid or tissue from the patient; and testing the sample to determine whether the sample contains a wild type SCF-Apoptosis-Response Gene-1 protein (SARG-1 protein) (SEQ ID NO. 2), whereby the absence of a wild type SARG-1 protein in the sample indicates that the patient has MS or an increased risk of acquiring MS.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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