EP1214597A1 - Diagnosis, prognosis and treatment of trinucleotide repeat-associated diseases and intranuclear inclusions-associated diseases - Google Patents
Diagnosis, prognosis and treatment of trinucleotide repeat-associated diseases and intranuclear inclusions-associated diseasesInfo
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- EP1214597A1 EP1214597A1 EP00960253A EP00960253A EP1214597A1 EP 1214597 A1 EP1214597 A1 EP 1214597A1 EP 00960253 A EP00960253 A EP 00960253A EP 00960253 A EP00960253 A EP 00960253A EP 1214597 A1 EP1214597 A1 EP 1214597A1
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- Prior art keywords
- protein
- cag
- cell
- cells
- stretch
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to neurodegenerative disorders. More particularly, the invention relates to Machado-Joseph disease (MJD).
- the present invention also relates to trinucleotide repeat expansions and more particularly to CAG repeats, also termed expansions of a coding CAG repeat (exp-CAG), and GCG repeats. More particularly, the invention relates to: (1) exp-CAG associated diseases; (2) intranuclear inclusions (INI) in patients and cellular models of exp-CAG associated diseases; and (3) the elucidation of the mechanism responsible for the toxic effects of such repeats.
- the present invention therefore relates to the diagnosis, prognosis and treatment of repeat- associated diseases and INI-associated diseases as well as to assays for the identification of agents which could be used for the treatment of such diseases or disorders.
- Coding CAG triplet repeat expansions cause several neurodegenerative disorders, including Machado-Joseph disease (MJD) 1"5 .
- MJD Machado-Joseph disease
- INI intranuclear filamentous inclusions
- exp-CAG expanded CAG repeat disorders
- Similar INI are found in oculopharyngeal muscular dystrophy, which is caused by a short expansion of analanine encoding GCG repeat.
- transcriptional or translational frameshifts occurring within expanded CAG tracts result in the production and accumulation of polyalanine-containing mutant proteins.
- alanine polymers might deposit in cells forming INI and lead to nuclear toxicity Support for this disease model is provided using lymphoblast cells from MJD patients, as well as inpontine neurons of MJD brain and in in vitro cell culture models of the disease Evidence that alanine polymers alone are toxic to cells is also provided and strongly suggests that a similar pathogenic mechanism underlies the other CAG repeat disorders
- CAG repeats code for polyglutamine in the protein containing same It is commonly believed that these polyglutamine stretches in proteins are toxic to cells, and these repeats are also termed CAG/polyglutamine repeats (Iver et al 1999, Nature Medicine 5 383-384) These diseases, also termed polyglutamine diseases, are thought to occur by "a gain function mechanism" Unfortunately, the mechanism explaining toxicity of the polyglutamine diseases, apparently through an aggregation in nuclear inclusions, has yet to be provided, although transgenic mice bearing a polyglutamine repeat in a recombinant protein were shown to display intranuclear polyglutamine inclusions (Hardy et al 1998, Science 282 1076-1079) Of relevance, although the pathogenic effect of these inclusion bodies is not clearly understood, it is recognized that numerous types of genes can contain these so-called CAG
- INI are also found in oculopharyngeal muscular dystrophy (OPMD) 10 , which is caused by short expansions of a poiyalanine (polyAla) encoding GCG tract in the PABP2 gene 11 (also see PCT/CA98/01133)
- OPMD oculopharyngeal muscular dystrophy
- polyAla poiyalanine
- PABP2 PABP2 gene 11
- exp-GCG very small GCG expansions
- poiyalanine tracts are prone to aggregation and may be very toxic 11
- This contention is supported by the observation that polyAla peptides containing more than 9 alanines (Ala) in a row form ⁇ -pleated sheet fib ⁇ llar macromolecules spontaneously in vitro , which in turn are extremely resistant to chemical and enzymatic degradation 13
- Short tnnucleotide repeat expansions causing a human disease have been first described in PCT application number PCT/CA98/
- the invention therefore concerns the identification of the mechanistic action of expanded CAG tracts in cell pathogenesis, cell death or disease More specifically, the invention relates to the identification that mutational events within these CAG enable them to encode poiyalanine stretches which accumulate in intranuclear filamentous inclusions and somehow trigger toxicity in cells
- the present invention relates totranslational and/or transc ⁇ ptional frameshift events occurring within the CAG tracts, thereby resulting in the production and accumulation of polyalanine-containing mutant proteins
- the present invention in addition, relates to a formal identification of the toxic effects of poiyalanine stretches on nuclear toxicity
- the Applicant is thus the first to have demonstrated that CAG expansions can give rise to production of mutant proteins containing poiyalanine stretches
- the Applicant is also the first to have demonstrated that poiyalanine stretches in proteins are indeed toxic to cells
- the instant invention also relates to gene replacement technologies aimed at deleting a repeat-containing protein giving rise to a mutant protein by a normal corresponding protein (i e lacking the repeat or having a smaller repeat)
- the present invention also provides the means to determine a predisposition to developing a disease or condition associated with the expression of a polyamino acid-containing protein such as polyalanine-containing proteins This determination could thus enable a better prognosis of the disease and condition and enable a determination of the best treatment or prevention of the disease or condition
- Another aim of the present invention is thus to provide means to screen humans (and more broadly animals) to identify those that might have a predisposition to developing a disease associated with the expression of genes that lead to polyalanine-containing proteins such as CAG, GCG-repeat containing genes.
- CAG repeat expansions also known as CAG repeats or CAG/polyglutamine stretches
- CAG repeats were recognized as a common denominator in numerous neurological diseases and were thought to code for polyglutamine stretches in the mutant protein
- These polyglutamine stretches were thought to confer "a gain of toxic property to these proteins" (Iver et al. supra) by a mechanism that was not understood (Hardy et al. supra)
- CAG tract toxicity was also referred to as polyglutamine diseases
- the present invention demonstrates that these CAG repeats actually encode poiyalanine stretches
- the present invention opens the way to numerous methods of diagnosing, prognosing or treating CAG repeat-dependent diseases or conditions
- it provides means to diagnose, prognose and treat diseases or conditions associated with polyalanine-containing proteins (i.e. GCG repeats)
- Non-limiting examples thereof comprise methods using ligands (i.e. polyclonal and monoclonal antibodies), nucleic acid sequences, restriction length polymorphisms (RFLPs) and the like
- MJD-Ala frameshifted ataxin-3 protein
- a method for the diagnosis of a disease associated with protein accumulation in intranuclear inclusions which comprises obtaining a sample of a patient and determining a presence of the protein accumulation in the intranuclear inclusions, wherein this protein accumulation is indicative of a disease related thereto
- (3) toxicity to cells which comprises a) incubating a cell harboring an expression vector of the present invention, comprising a repeat domain which can give rise to a polyamino acid-containing protein associated with a disease or condition in an animal, with a compound, and b) assessing one of (1) an expression of the polyamino acid-containing protein, (2) accumulation of the polyamino acid- containing protein, and (3) toxicity to cells, whereby a modulator is selected when the agent significantly modulates one of the expression, accumulation and toxicity, as compared to a control agent
- the instant invention also relates to GCG repeats encoding poiyalanine stretches and their association with protein accumulation in a cell nucleus, swallowing difficulty and/or ptosis in a patient
- a method for the diagnosis or prognosis of a disease associated with protein accumulation in a cell nucleus, and/or swallowing difficulty and/or ptosis in a human patient which comprises a) obtaining a sample of a patient, and b) determining the extract of the poiyalanine stretch in an alanine stretch-containing protein having the ammo acid sequence
- a human PAB II protein comprising a polymorphic GCG repeat encoding a poiyalanine stretch having the sequence Met (Ala) 6+n Ala, wherein n is 0, and wherein the sequence is indicative of a non-disease phenotype associated with protein accumulation in a cell nucleus, swallowing difficulty, and/or ptosis in a human patient
- Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art Such standard techniques can be found in reference manuals such as for example Sambrook et al.
- rDNA recombmant DNA
- nucleic acid molecule refers to a polymer of nucleotides
- Non-limiting examples thereof include DNA (i e. genomic DNA, cDNA) and RNA molecules (i e mRNA)
- the nucleic acid molecule can be obtained by cloning techniques or synthesized.
- DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
- DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of ammo acids which can be referred to as a polypeptide, protein, protein fragment and the like.
- amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
- amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
- theo gos are designed to bind to a complementary sequence under selected conditions.
- the nucleic acid i e. DNA or RNA
- the nucleic acid may be obtained according to well known methods.
- physiologically relevant is meant to describe a frameshrfting event which can result in the production of a toxic protein in vivo
- Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted sequences employed
- the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system
- the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hyd ⁇ zidation thereof with its targeted sequence (see below and in Sambrook et al , 1989, Molecular Cloning - A Laboratory Manual, 2nd
- oligonucleotide or "DNA” molecule or sequence refers to a molecule comprised of thedeoxynbonucleotides adenme (A), guanine
- oligonucleotide or "DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA As used herein, particular DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction
- Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure Examples of hybridization conditions can be found in the two laboratory manuals referred above ⁇ ambrook et al , 1989, supra and Ausubel et al , 1989, supra) and are commonly known in the art
- a hybridization to a nitrocellulose filter as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50%formam ⁇ de, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 ⁇ g/ml denatured carrier DNA (i e salmon sperm DNA) The non-specifically binding probe can then be washed off the filter by several washes in 0 2 x SSC/0 1% SDS
- Probes or primers of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann Reports Med Chem 23 295 and Moran et al , 1987, Nucleic acid molecule Acids Res , 14 5019 Probes or primers of the invention can be constructed of either ⁇ bonucleic acid (RNA) or deoxy ⁇ bonucleic acid (DNA), and preferably of DNA
- probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and
- RNA detection Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds Other detection methods include kits containing probes on a dipstick setup and the like
- Probes can be labeled according to numerous well known methods (Sambrook et al , 1989, supra)
- detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies
- Other detectable markers for use with probes include biotin and radionucleotides It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe
- radioactive nucleotides can be incorporated into probes of the invention by several methods Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kmase, using the Klenow fragment of Pol I of E coll in the presence of radioactive dNTP (i e uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
- oligonucleotides or “oligos” define a molecule having two or more nucleotides ( ⁇ bo or deoxy ⁇ bonucleotides) The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill
- An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods
- a "primer” defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions
- Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ rep case system and NASBA (Kwoh et al , 1989, Proc Natl Acad Sci USA 86, 1173-1177, ⁇ zardi et al , 1988, BioTechnology 6 1197-1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra) Preferably, amplification will be carried out using PCR
- PCR Polymerase chain reaction
- a nucleic acid sample e g , in the presence of a heat stable DNA polymerase
- An extension product of eachp ⁇ mer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith
- the extension product synthesized from each pnmer can also serve as a template for further synthesis of extension products using the same primers Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess whether the sequence or sequences to be detected are present Detection of the amplified sequence may be carried
- Ligase chain reaction is carried out in accordance with known techniques (Weiss, 1991 , Science 254 1292) Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al , 1992, Proc Natl Acad Sci USA 89 392-396, and ibid , 1992, Nucleic Acids Res 20 1691-1696)
- SDA Strand displacement amplification
- the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
- a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific ammo acid sequence thereby giving rise the a specific polypeptide or protein It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art
- heterologous i e a heterologous gene region of a DNA molecule is a subsegment segment of DNA within a larger segment that is not found in association therewith in nature
- the term 'heterologous can be similarly used to define two polypeptidic segments not joined together in nature
- Non- limiting examples of heterologous genes include reporter genes such as green fluorescence protein, luciferase, chloramphenicoi acetyl transferase, ⁇ - galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides
- vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned Numerous types of vectors exist and are well known in the art
- expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more
- expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host
- the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
- control element sequences such as promoter sequences
- Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript
- two sequences, such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence
- two sequences such as a promoter and a "reporter sequence"
- Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcnptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
- Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest
- This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (i e SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography )
- the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies The purified protein can be used for diagnostic or therapeutic applications
- the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule
- Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence
- the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
- a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
- Eukaryotic promoters will often, but not always, contain "TATA" boses and "CCAT” boxes Prokaryotic promoters contain Shme-
- variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention
- the functional derivatives of the present invention can be synthesized chemically or produced through recombmant DNA technology All these methods are well known in the art
- chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention
- moieties could affect the physico-chemical characteristic of the derivative (i e solubility, absorption, half life and the like, decrease of toxicity)
- Such moieties are examphfied in Remington's Pharmaceutical Sciences (1980) Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art
- allele defines an alternative form of a gene which occupies a given locus on a chromosome
- a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell
- a mutation can be, for example, a detectable change in one or more deoxy ⁇ bonucleotide
- nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutations and experimentally induced mutations exist
- a mutant polypeptide can be encoded from a mutant nucleic acid molecule
- mutant proteins can be produced through aberrant events during replication, transcription and/or translation Frameshifting (the switching from a particular reading frame to another) is such a mechanism that can modify the sequence of the translated protein
- purified refers to a molecule having been separated from a cellular component
- a purified protein has been purified to a level not found in nature
- a “substantially pure” molecule is a molecule that is lacking in all other cellular components
- molecule As used herein, the terms “molecule”, “compound”, “agent”, or “ligand” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds
- molecule therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like
- Non limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceuticaMherapeutical agents
- the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling
- the terms “rationally selected” or “rationally designed” are meant to define, for example, compounds which have been chosen based on the configuration of the poiyalanine domains of the present invention
- macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule”
- peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by
- the present invention also provides antisense nucleic acid molecules which can be used for example to modulate the expression of the mutant proteins of the present invention
- An antisense nucleic acid molecule according to the present invention refers to a molecule capable of forming a stable duplex or triplex with a portion of its targeted nucleic acid sequence (DNA or RNA)
- the use of antisense nucleic acid molecules and the design and modification of such molecules is well known in the art as described for example in WO 96/32966, WO 96/11266, WO 94/15646, WO 93/08845 and USP 5,593,974
- Antisense nucleic acid molecules according to the present invention can be derived from the nucleic acid sequences and modified in accordance to well known methods For example, some antisense molecules can be designed to be more resistant to degradation to increase their affinity to their targeted sequence, to affect their transport to chosen cell types or cell compartments, and/or to enhance their lipid solubility bu using nucleotide analogs and/
- indicator cells refers to, for example, cells that express a fusion protein comprising a poiyalanine segment (e g a "CAG" repeat) and an identifiable or selectable phenotype or characteristic which enables an assessment of the level of fusion protein expression (e g a reporter protein)
- Such indicator cells can be used in the screening assays of the present invention
- the indicator cells have been engineered so as to express a chosen derivative fragment, homolog, or mutant of a repeat
- the repeats should not be limited to CAG repeats Indeed, GCG repeats can also be used
- the invention should not be limited to poiyalanine repeats, since the present invention provides for the testing of polyserme fragments and other polyamino acids which could be expressed from a frameshifting event
- the cells can be yeast cells or higher eukaryotic cells such as mammalian cells (WO 96/41169)
- the indicator ceils are higher eukaryotic
- a poiyalanine polypeptide segment of the present invention is provided as a fusion protein
- the design of constructs therefor and the expression and production of fusion proteins are exemplified herein and are well known in the art (Sambrook et al , 1989, supra, and Ausubel et al , 1994, supra)
- Non limiting examples of such fusion proteins include a hemaglutinin fusions (HA) and Gluthione-S-transferase (GST) fusions
- HA hemaglutinin
- GST Gluthione-S-transferase
- fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like
- sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) repeat domain It will be clear to the person of ordinary skill that whether a repeat domain of the present invention, variant, derivative, or fragment thereof retains its function in enabling a concentration of the protein containing same in INI or triggering toxicity in cells or animals, can be readily determined by using the teachings and assays of the present invention and the general teachings of the art
- the repeat domains of the present invention can be modified, for example by/ ⁇ vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds
- some derivative or analogs having lost their biological function may still find utility, for example for raising antibodies
- these antibodies could be used for detection or purification purposes
- these antibodies could also act as competitive or non-com petitive inhibitor and be found to be modulators of the biological activity of the repeat domain
- a host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e g a DNA construct) when such DNA has been introduced inside the cell
- the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
- thetransfecting DNA may be maintained on a episomal element such as a plasmid
- a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA Transfection methods are well known in the art (Sambrook et al , 1989, supra, Ausubel et al , 1994 supra).
- the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents
- the therapeutic agent according to the present invention can be introduced into individuals in a number of ways
- the therapeutic agent can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes Having shown that a poiyalan
- the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (i e DNA construct, protein, cells), the response and condition of the patient as well as the severity of the disease
- composition within the scope of the present invention should contain the active agent (i e fusion protein, nucleic acid, and molecule) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects
- the nucleic acids, fusion proteins and molecules in accordance with the present invention can be administered to mammals (i e humans) in doses ranging from 0 005 to 1 mg per kg of body weight per day of the mammal which is treated
- Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed , Mack Ed )
- the amount administered should be chosen so as to avoid adverse side effects
- the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
- the present invention also relates to a kit for diagnosing a disease or condition associated with the expression of a repeat domain, encoding for example a poiyalanine stretch, or a predisposition to contracting same comprising a nucleic acid, a protein or a ligand in accordance with the present invention.
- a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper.
- Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another
- Such containers will include a container which will accept the test sample (DNA protein or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products.
- Figure 1 shows the Western blot analysis of lymphoblastoid cells from controls and MJD patients, a, Schematic representation of the MJD- Ala protein that results from a frameshift in the CAG tract showing the new C- termmus (italicized, used to raise the FS1 and FS2 antibodies).
- FIG. 3 shows the immunohistochemical detection of INI in MJD pontine neurons Immunoprobing with FS1 antiserum in MJD pons (a) and control pons (b), immunoprobing with anti-ubiquitm in MJD pons (c) and control pons (d) INI in pontine neurons are indicated by arrowheads Double labeling immunofluorescence analysis of MJD pons showing ubiquitm-labeled INI (e, h) and FS1 -labeled INI (f, i), and the composite image of bothlabelmgs (g, j) For all panels, the magnification before publication is 1000x, before reproduction
- Figure 4 shows the constructs used in the transfection experiments All constructs, with the exception of pM JD11 , represent full-length MJD-1 Solid black boxes indicate the repeat portion of the constructs Staggered ends indicate that EGFP will only be expressed if aframeshift occurs Encircled blown up detail of pMJD1 is also present in pMJD2 and pMJD3, details of pMJD5 are present in pMJD6, details of pMJD7 are present in pMJD8 and details of pMJD9 are the same as pMJDIO
- Figure 5 shows the transfection experiments with different M JD/EGFP constructs a, DNA sequence of the clones with EGFP out of frame (pMJD1 , pMJD2 and pMJD3) and b, with EGFP inglutamme frame (pMJD4), both (a, b) showing the predicted ammo acid sequence c-e, Fluorescence at 72 hours of COS-7 cells transfected with pMJD1 (c), pMJD2 (d) and pMJD3 (e) where the M JD(CAG) n /EGFP fusion protein is translated only when frameshifts to GCA- polyAla occur f, COS-7 cells transfected with pMJD4 where EGFP is in frame with CAG-Gln g, COS-7 cells transfected with the vector pEGFP-N1 h, pe ⁇ nuclear fluorescent aggregates observed in cells transfected with pMJD3 Pictures of sections c, d, e, f and
- Figure 6 shows the time-course immunocytochemical analysis of COS-7 cells transfected with constructs encoding atax ⁇ n-3 with either a polyAla or a polyGln tract Immunoprobing with anti-HA antibody at time-points 8 hours (a) pMJD7, (b) pMJD9, (c) pMJD8, (d) pMJDIO and (e) vector pEGFP-N1 alone, 20 hours (f) pMJD7, (g) pMJD9, (h) pMJD8, ⁇ ) pMJDIO and 0) vector pEGFP-N1 alone, 24 hours (k) pMJD7, (I) pMJD9, (m) pMJD8, (n) pMJDIO and (o) vector pEGFP-N1 alone, 48 hours (p) pMJD7, (q) pMJD9, (r) pMJD ⁇ , (s) pMJDIO and (t) vector
- Figure 7 shows the western analysis of transfected COS-7 cells (in figure 5) Blots were probed with (a) anti-HA, (b) 1C2 Arrow indicates threshold between stacking and resolving portions of the gel Cells were harvested at 72 hours after transfection
- Figure 8 shows the immunocytochemical analysis of COS-7 cells transfected with a truncated polyAla-encodmg construct Immunoprobing with anti-HA antibody in (a) cells transfected with 42A and (b) mock-transfected cells For all panels pictures were taken at 1000x magnification, before reproduction
- the pMJD1 and pMJD3 constructs were modified by adding epitopes for FS1 and HA in the alanine frame (pMJD5 and pMJD6, Fig. 4). Western blots of cellstransfected with these constructs were probed with anti-HA. No signal for the 14 CAG-bearing pMJD5 construct was detected, but a band corresponding to aggregated protein was detected at the top of the gel for pMJD ⁇ , showing that with 82 repeats, frameshifts are occurring. The absence of a band corresponding to the predicted size of the ataxin-3/HA protein (Fig. 5K) indicates that all frameshifted protein is accumulating as insoluble aggregates. These experiments further demonstrate thatframeshifts do occur and that their frequency increases with the size of the CAG repeat.
- COS-7 ceils were transfected with these constructs, and with pMJD7 and pMJD8, which contained 14 CAG repeats and 82 CAG repeats respectively, as well as EGFP fused in frame with the CAG tracts.
- a HAepitope was also added at the COOH-terminus in frame with the GCA tracts, and so only detectable if frameshifts occurred (Fig. 4).
- a 12-mer peptide corresponding to the new predicted COOH- terminus of the atax ⁇ n-3 protein after frameshift occurs was used to raise antisera from two rabbits, FS1 and FS2 Injections were performed using 0 5 mg of peptide conjugated to KLH and sera were collected using standard protocols 42
- MJD and control lymphoblastoid cells were lysed in buffer containing NP-40 Equal amounts of protein were eiectrophoresed in ⁇ % SDS-polyacrylamide gels and transblotted to nitrocellulose membranes (as commonly known) Immunodetection was performed using FS1 (1 300), FS2 (1 300), ant ⁇ -atax ⁇ n-3 (1 1000), 1C2 (1 2000), anti-ubiquitm (1 400, DAKO) and FS1 and FS2 pre-immune serum (1 300) Results for FS1 and FS2 were always consistent, all experiments were repeated three times on different blots HRP conjugated secondary antibodies were used at a 1 10,000 dilution COS-7 cells transfected with various constructs were collected, washed and lysed in Sample Loading Buffer 100 ⁇ g of each sample was used to run on a 10% SDS-PAGE and transblotted onto nitrocellulose membranes Immunodetection was performed using antisera at following
- MJD and control lymphoblastoid cells were harvested and a total of 50x10 4 cells from each cell line were plated onto poly-D-lysine coated slides and fixed with acetone/methanol (1 :1). Immunodetection was performed using FS1 (1 :300), anti-ubiquitin (1 :300), anti-ataxin-3 (1 :500) and FS1 pre- immune serum (1 :300). Biotinylated secondary antibodies were used at a 1 :500 dilution and an amplification step was performed using the ABC kit (VECTOR). Reaction product was visualized using the VIP kit (VECTOR). Immunocyto- chemistry on COS-7 cells was performed on cover slips. At each time-point cells were fixed with 4% paraformaldehyde and immunodetection was performed using anti-HA probe (1 :500) and FS1 (1 :300). Secondary antibodies and subsequent amplification and detection procedures were carried out as described above.
- DNA amplification was performed using Pfu DNA polymerase (STRATAGENE) Primer MJD-5' TTTTAAGCTTAGACAAA-TAAACATGGAG (SEQ ID N0 1) was used in conjunction with MJD-3' CCGGTGGATCCCTCATCCTGATAGGTCCCGCTGCTG (SEQ ID NO 2) for pMJD1 , pMJD2 and pMJD3, or MJD-3'c CCGGTGGATCCCTCA- TTGATAGGTCCCGCTGCTG (SEQ ID NO 3) for pMJD4 (STRATAGENE) Primer MJD-5'(HIII) TTTAAGCTTCCCACCATGGAGT-CATCTTCCA (SEQ ID NO 4) was used in conjunction with MJD-3'(BI)
- CCGGTGGATCCCTCAGGGCGTAGTCGGGGACGTCGTAGGGGTACATGGAT GTGAACTCTGTCCTGATAGGTCCCGCTG (SEQ ID NO 5) for pMJD5 and pMJD6, or MJD-3'c(BI) CCGGTGGATCCCAGGGCGTAGTCGGGGACGTCG- TAGGGGTACATGGATGTGAACTCTGTCCTGATAGGTCCCGCTG (SEQ ID NO 6) for pMJD7 and pMJD ⁇ , or MJD-Ala
- the present invention thus shows that transcriptional or translational frameshifts occurring within expanded CAG tracts result in the production and accumulation of polyalanine-containing mutant proteins.
- These alanine polymers might deposit in cells forming INI and lead to nuclear toxicity. Support for this disease model is provided using lymphoblast cells from MJD patients, as well as in pontine neurons of MJD brain and in in vitro cell culture models of the disease.
- Evidence that alanine polymers aione are toxic to cells is also provided and strongly suggests that a similar pathogenic mechanism underlies the other CAG repeat disorders. How these accumulations lead to cell death still needs to be elucidated.
Abstract
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