WO2011090947A2 - Procédés de diagnostic et de traitement de la maladie de parkinson - Google Patents

Procédés de diagnostic et de traitement de la maladie de parkinson Download PDF

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WO2011090947A2
WO2011090947A2 PCT/US2011/021581 US2011021581W WO2011090947A2 WO 2011090947 A2 WO2011090947 A2 WO 2011090947A2 US 2011021581 W US2011021581 W US 2011021581W WO 2011090947 A2 WO2011090947 A2 WO 2011090947A2
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otx2
cells
vector
neurons
gene
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PCT/US2011/021581
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WO2011090947A3 (fr
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Chee Yeun Chung
Ole Isacson
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The Mclean Hospital Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present technology relates generally to the diagnosis and treatment of neurodegenerative diseases, including Parkinson's Disease.
  • Parkinson's disease is a progressive neurodegenerative disease characterized clinically by bradykinesia, rigidity, and resting tremor. Selective degeneration of specific neuronal populations is a universal feature of PD that contributes to the clinical
  • DA midbrain dopaminergic
  • a similar pattern of differential vulnerability is observed in rodent and primate models of PD, including toxic models utilizing 6-hydroxydopamine (6- OHDA) 5 and 1 -methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), indicating that such differential vulnerability between A9 and A10 DA neuronal populations may be conserved between species.
  • 6-hydroxydopamine 6-hydroxydopamine
  • MPTP 4-phenyl 1,2,3,6-tetrahydropyridine
  • This disclosure provides methods for treating or preventing Parkinson's Disease (PD) in a patient by increasing the level of Otx2 or a biologically active fragment thereof, in the midbrain of that patient.
  • the level of Otx2 is increased in the midbrain dopaminergic neurons including, for example, the A9 (substantia nigra) and/or A10 (ventral tegmental area) dopaminergic neurons.
  • the Otx2 levels may be increased by administering a vector comprising a polynucleotide encoding the Otx2 protein or biologically active fragment thereof, operably linked to at least a regulatory element, wherein the vector is taken up by the target cell ⁇ e.g., neuron or pluripotent stem cell) and the polynucleotide (and Otx2 protein) is expressed.
  • the vector is a viral vector including, for example, an adenovirus, adeno-associated virus, retrovirus, or lentivirus.
  • the vector may be delivered to the midbrain in vivo using any suitable technique including, for example, stereotactic microinjection of the vector into or near the substantia nigra. The delivery method is designed to promote uptake and expression of the vector by the dopaminergic neurons.
  • the invention features a method for treating or preventing PD in a patient by administering, to the brain of a patient, an Otx2 protein or biologically active fragment thereof.
  • the Otx2 protein is administered by intravenous or intraventricular injection.
  • the Otx2 protein may be soluble or may be encapsulated within a liposome.
  • the Otx2 protein is taken up by neurons (e.g., dopaminergic neurons, adrenergic neurons, serotonergic neurons, and/or cholinergic neurons).
  • the Otx2 protein is administered to the brain of the patient by implanting cells capable of expressing a recombinant Otx2 protein.
  • the cells are autologous and are transplanted directly into the midbrain of the patient.
  • the cells are derived from pluripotent stem cells, including umbilical cord blood stem cells, neuronal progenitor cells, fetal mesencephalic cells, embryonic stem cells, and postpartum derived cells (U.S. Patent No. 5,487,739).
  • the transplanted cells are encapsulated in a permeable capsule.
  • the invention provides an isolated nucleic acid comprising a nucleotide sequence that encodes an Otx2 protein or biologically active fragment thereof and at least a regulatory element.
  • the invention provides vectors comprising such isolated nucleic acids.
  • the Otx2-encoding nucleic acid may be operably linked to a promoter.
  • the promoter is a neuron-specific promoter including, for example, a neuron-specific enolase promoter or a synapsin-I promoter.
  • the vectors may be a naked DNA or a viral vector including, for example, those selected from the group of adenovirus, adeno-associated virus, retrovirus, lentivirus, and herpes simplex virus.
  • the vectors are typically contained in a pharmaceutically acceptable formulation including, for example, a formulation suitable for intravenous, intramuscular, intracerberoventricular, or intranigral injection.
  • the invention provides cells containing such isolated nucleic acids or vectors.
  • the cells include, for example, pluripotent stem cells, umbilical cord blood stem cells, neuronal progenitor cells, fetal mesencephalic cells, embryonic stem cells, and postpartum derived cells.
  • the invention provides a method of identifying a compound that treats or prevents Parkinson's disease in a human, involving the steps of: (a) providing cells that express Otx2; (b) contacting the cells with a candidate compound; and (c) assessing the expression level of the genes relative to the expression level of the genes in the absence of the candidate compound, in which a candidate compound that increases the expression of said Otx2 is identified as a compound useful for treating Parkinson's disease.
  • the invention provides a method of identifying a compound for treating or preventing Parkinson's disease involving the steps of: (a) providing cells that express a reporter gene under the control of a Otx2 regulatory element; (b) contacting the cell with a candidate compound; and (c) assessing the level of expression of the reporter gene in the presence and/or absence of the candidate compound, in which a candidate compound that increases the level of expression of the reporter gene is identified as a compound that is useful for the treatment or prevention of Parkinson's disease. Any suitable reporter gene may be used.
  • Exemplary useful reporter genes include but not limited to: glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase, and ⁇ -galactosidase.
  • GUS glucuronidase
  • CAT chloramphenicol transacetylase
  • GFP green fluorescent protein
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucuronidase
  • contacting further includes contacting the cell with a neurotoxic compound.
  • a neurotoxic compound includes but not limited to l-methyl-4-phenylpyridinium (MPP+), rotenone, isoquinoline, tetrahydroisoquinoline and 6-hydroxydopamine.
  • the cells are mammalian cells such as human cells or rodent cells (e.g., rat and mouse) cells, or non-human primate cells.
  • the cells may be neuronal cells.
  • the cells may be immortalized cells or they may be derived from cultured primary cells (e.g., cultured embryonic ventral mesencephalon cells). Useful immortalized cells include, for example, PC 12 cells.
  • the PC12 cells also recombinantly express Otx2.
  • the assessing step (c) includes measuring the level of Otx2 RNA.
  • the invention provides a method for diagnosing PD or susceptibility to PD comprising detecting the single nucleotide polymorphism rsl 123285 in the Otx2 gene in a sample from a subject, and diagnosing the subject as having or being susceptible to PD when the rsl 123285 SNP is present.
  • the subject is indicated for treatment for PD and/or treatment for PD is initiated when the rsl 123285 SNP is present.
  • the detecting comprises PCR, RT-PCR, or nucleic acid hybridization.
  • the detecting comprises an allele discrimination method selected from the group consisting of: allele-specific hybridization, allele-specific primer extension including allele-specific PCR, allele-specific oligonucleotide ligation, allele- specific cleavage of a flap probe, and allele-specific cleavage using a restriction
  • the detecting comprises amplifying a fragment of the Otx2 gene comprising the rsl 123285 SNP, if present.
  • the amplifying is accomplished by polymerase chain reaction (PCR), using e.g., a detectably labeled primer.
  • the detecting is accomplished with electrophoresis.
  • the detecting is accomplished using the TaqMan® PCR detection system.
  • the sample is selected from the group consisting of: plasma, serum, and buccal smears.
  • the invention provides a method of counseling an individual on the likelihood of developing Parkinson's disease, comprising determining if nucleic acid from the individual has a rsl 123285 SNP in the Otx2 gene and, counseling the individual on the likelihood of having an offspring afflicted with a neurodevelopmental disorder based on the presence or absence of the mutation, wherein the presence of the SNP is indicative of an increased likelihood.
  • the invention provides a kit for detecting rsl 123285 SNP in the Otx gene in a sample comprising a primer pair for amplifying a fragment of a nucleic acid sample which contains the c. rsl 123285 SNP, if present.
  • the primer pair is selected from the group consisting of: SEQ ID NOs: 3/5, SEQ ID NOs: 4/5, and SEQ ID NOs: 7/8.
  • FIG. 1 presents results of experiments demonstrating that Otx2 is elevated in A10 DA neurons in adult mouse, primate and human. Otx2 mRNA levels were compared between A9 and A10 DA neurons in mouse and human using laser capture microdissection and quantitative PCR.
  • A-C LCM of the mouse midbrain DA neurons. Selection of DA neurons was guided by quick TH immunostaining.
  • A TH-positive neurons in SN before laser capture.
  • B The TH-positive cells were targeted for laser capture with a 7.5 ⁇ laser diameter.
  • C Captured cells on the thermoplastic film were visualized before processing for RNA extraction.
  • A9 DA neurons in the SN showed very low or undetectable Otx2 immunoreactivity (P-R), whereas A10 DA neurons in the VTA showed strong Otx2 immunoreactivity (T-V, W; z-stack image of perforated square in V). Otx2 expression was not exclusive to DA neurons.
  • FIG. 2 is a series of micrographs demonstrating that Otx2 is elevated in A10 DA neurons in the embryonic mouse midbrain. Immunostaining of Otx2 in El 7 mouse development also demonstrated that Otx2 immunoreactivity was higher in A10 DA neurons. The SN and the VTA areas are defined in a lower magnification view (A-C). Otx2 expression was absent in A9 DA neurons (D-G) whereas strong Otx2 immunoreactivity was detected in A10 DA neurons (H-K).
  • FIG. 3 is a series of graphs showing that Otx2 regulates expression of known A10- elevated genes in MN9D cells and primary VM cultures.
  • MN9D cells were transduced with lentivirus encoding YFP-NLS (nuclear localization signal) or Otx2 and harvested 4 days after transduction for mRNA isolation and quantitative PCR analysis.
  • YFP-NLS nuclear localization signal
  • FIG. 4 is a series of micrographs demonstrating that adult Otx2 conditional knockout mice lack the A10 DA neuronal projection (A-F).
  • A-F A10 DA neuronal projection
  • TH Immunostaining reveals that Enl cre/+ , Otx2 flox/flox mice show dramatic reduction of A10 neuronal projections including prefrontal cortex (PFC; A and B), nucleus accumbens (NAc; A and C), olfactory tubercle (OT; C), septum (D), amygdala (amg; E and F).
  • PFC prefrontal cortex
  • NAc nucleus accumbens
  • OT olfactory tubercle
  • D septum
  • amygdala amygdala
  • FIG. 5 presents results of experiments demonstrating that Otx2 overexpression protects DA neurons from MPP + toxicity in primary VM cultures.
  • Primary VM cultures were transduced with lentivirus encoding YFP-NLS, Otx2, control shRNA or shRNA against Otx2 on DIV 1 and 10 ⁇ MPP + was applied to the culture at DIV 8. Cultures were fixed at DIV 10 and stained with TH for TH positive cells counting.
  • Otx2 was overexpressed by lentiviral delivery of Otx2 with multiplicities of infection (MOI) of 5 and 10 (A-C)
  • MOI multiplicities of infection
  • A-C multiplicities of infection
  • FIG. 6 is a series of micrographs showing Otx2 immunoreactivity in postmitotic mDA neurons at E12 and E15 of mouse development.
  • Postmitotic DA neurons in the marginal zone of VM exhibit very low Otx2 immunoreactivity compared to intermediate zone (A-C. D; z-stack image of perforated square in C).
  • A-C. D z-stack image of perforated square in C.
  • E15 Otx2 immunoreactivity becomes stronger and distinction between Otx2 -positive and -negative neurons can be clearly made at this stage (E-G, H; z-stack image of perforated square in G).
  • nucleic acid includes a combination of two or more nucleic acids, and the like.
  • amplification means one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an "amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR"), numerous other methods are known in the art for amplification of nucleic acids ⁇ e.g., isothermal methods, rolling circle methods, etc). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods.
  • PCR polymerase chain reaction
  • allele and "allelic variant” are used interchangeably herein.
  • An allele is any one of a number of alternative forms or sequences of the same gene occupying a given locus or position on a chromosome. A single allele for each locus is inherited separately from each parent, resulting in two alleles for each gene. An individual having two copies of the same allele of a particular gene is homozygous at that locus, whereas an individual having two different alleles of a particular gene is heterozygous.
  • complement as used herein means the complementary sequence to a nucleic acid according to standard Watson/Crick base pairing rules.
  • a complement sequence can also be a sequence of R A complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
  • substantially complementary means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target or marker sequence, positioned 3 ' or 5' to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target or marker sequence.
  • diagnosis means determining a disease state or condition in a patient in such a way as to inform a health care provider as to the necessity or suitability of a treatment for the patient.
  • genomic DNA refers to some or all of the DNA from the nucleus of a cell. Genomic DNA may be intact or fragmented. In some embodiments, genomic DNA may include sequence from all or a portion of a single gene or from multiple genes, sequence from one or more chromosomes, or sequence from all chromosomes of a cell. In contrast, the term “total genomic nucleic acid” is used herein to refer to the full complement of DNA contained in the genome of a cell. As is well known, genomic nucleic acid includes gene coding regions, introns, 5' and 3' untranslated regions, 5' and 3' flanking DNA and structural segments such as telomeric and centromeric DNA, replication origins, and intergenic DNA. Genomic nucleic acid may be obtained from the nucleus of a cell, or recombinantly produced. Amplification techniques may also be used.
  • hybridize or “specifically hybridize” refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Hybridizations are typically conducted with probe-length nucleic acid molecules. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et ah, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower
  • nucleic acid refers broadly to segments of a chromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acid may be derived or obtained from an originally isolated nucleic acid sample from any source ⁇ e.g., isolated from, purified from, amplified from, cloned from, or reverse transcribed from sample DNA or RNA).
  • oligonucleotide refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any combination thereof. Oligonucleotides are generally between about 10 and about 100 nucleotides in length. Oligonucleotides are preferably 15 to 70 nucleotides long, with 20 to 26 nucleotides being the most common. The single letter code for nucleotides is as described in the U.S. Patent Office Manual of Patent Examining Procedure, section 2422, table 1. An oligonucleotide may be used as a primer or as a probe.
  • An oligonucleotide is "specific" for a nucleic acid if the oligonucleotide has at least 50% sequence identity with a portion of the nucleic acid when the oligonucleotide and the nucleic acid are aligned.
  • An oligonucleotide that is specific for a nucleic acid is one that, under the appropriate hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%>, at least 85%o, at least 90%>, at least 95% and more preferably at least 98%> sequence identity.
  • a "primer” for amplification is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence.
  • the 3' nucleotide of the primer should be identical to the target or marker sequence at a corresponding nucleotide position for optimal primer extension by a polymerase.
  • a "forward primer” is a primer that anneals to the anti-sense strand of dsDNA.
  • a “reverse primer” anneals to the sense-strand of dsDNA.
  • operably linked refers to that a nucleic acid molecule and one or more regulatory sequences ⁇ e.g., a promoter) are connected in such a way as to permit expression and/or translation of the product ⁇ e.g., a protein) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • promoter refers to a nucleic acid sequence sufficient to direct transcription of a gene. Also included in the invention are those promoter elements which are sufficient to render promoter dependent gene expression controllable for cell type specific, tissue specific or inducible by external signals or agents.
  • neuronal promoter refers to a promoter that results in a higher level of transcription of a gene in cells of neuronal lineage compared to the transcription level observed in cells of a non-neuronal lineage.
  • regulatory element refers to a nucleic acid sequence capable of modulating the transcription of a gene.
  • Non-limiting examples of regulatory element include promoter, enhancer, silencer, poly-adenylation signal, transcription termination sequence. Regulatory element may be present 5 Or 3' regions of the native gene, or within an intron.
  • Otx2 refers to a protein having an amino acid sequence substantially identical to the Otx2 sequence of SEQ ID NO: 1 (Table 1).
  • a suitable cDNA encoding Otx2 is provided at GenBank Accession No. AF093138 (Table 2; SEQ ID NO: 2).
  • the Otx2 proteins are not limited to a human-derived Otx2 protein having an amino acid sequence represented by SEQ ID NO: 1, but may have an amino acid sequence derived from other animals, particularly, a warm-blooded animal (e.g., rat, guinea pig, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.).
  • Table 1 Amino Acid Sequence of Otx2 (SEQ ID NO: 1)
  • biologically active Otx2 fragment is meant any protein or polypeptide that is substantially identical to a portion of SEQ ID NO: 1 and possesses at least one biological activity of Otx2.
  • the Otx2 fragment contains the DNA binding domain corresponding to amino acids 39-94 of SEQ ID NO: 1.
  • some specific biologically active Otx2 fragments include, for example, polypeptides containing amino acids 1-94, 39-94, 39-114, 39-150, 39, 39-170, 39-200, and 39-289 of SEQ ID NO: 1.
  • the Otx2 fragment is about 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or more amino acids in length.
  • Otx2 biological activity refers to any biological activity associated with the full length native Otx2 protein.
  • Otx2 biological activity refers to transcriptional activation of genes that relate to axon guidance cues, including, but not limited to neuropilin 1, neuropilin 2, slit 2, and adenylyl cyclase activating peptide.
  • the Otx2 biological activity refers to the action of protecting dopaminergic neurons from various insults, including MPP + toxicity.
  • the Otx2 biological activity is equivalent to the activity of a protein having an amino acid sequence represented by SEQ ID NO: 1 (e.g. about 0.01 to 100 fold, preferably about 0.5 to 20 fold, more preferably about 0.5 to 2 fold).
  • Measurement of Otx2 biological activity such as inducing transcription of genes that relate to axon guidance cues or protecting dopaminergic neurons from various insults can be performed according to the assays described in the Examples.
  • Measurement of transcriptional activity can be performed using any known method, such as a reporter assay or RT-PCR.
  • the term "preventing” is meant identifying a subject (i.e., a patient) having an increased susceptibility to PD but not yet exhibiting symptoms of the disease, and administering a therapy according to the principles of this disclosure.
  • the preventive therapy is designed to reduce the likelihood that the susceptible subject will later become
  • test sample refers to any liquid or solid material containing nucleic acids.
  • a test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture or a tissue sample from an animal, most preferably, a human.
  • biological sample such as cells in culture or a tissue sample from an animal, most preferably, a human.
  • sample tissues include, but are not limited to, blood, bone marrow, body fluids, cerebrospinal fluid, plasma, serum, or tissue (e.g. biopsy material).
  • Target nucleic acid refers to segments of a chromosome, a complete gene with or without intergenic sequence, segments or portions a gene with our without intergenic sequence, or sequence of nucleic acids to which probes or primers are designed.
  • Target nucleic acids may include wild type sequences, nucleic acid sequences containing mutations, deletions or duplications, tandem repeat regions, a gene of interest, a region of a gene of interest or any upstream or downstream region thereof.
  • Target nucleic acids may represent alternative sequences or alleles of a particular gene.
  • Target nucleic acids may be derived from genomic DNA, cDNA, or RNA.
  • target nucleic acid may be native DNA or a PCR amplified product.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With high stringency conditions, nucleic acid base pairing will occur only between nucleic acids that have sufficiently long segment with a high frequency of complementary base sequences. Exemplary hybridization conditions are as follows. High stringency generally refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C.
  • High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhardt's solution, 5X SSC (saline sodium citrate) 0.2%> SDS (sodium dodecyl sulphate) at 42°C, followed by washing in 0. IX SSC, and 0.1% SDS at 65°C.
  • Moderate stringency refers to conditions equivalent to hybridization in 50% formamide, 5X Denhardt's solution, 5X SSC, 0.2% SDS at 42°C, followed by washing in 0.2X SSC, 0.2% SDS, at 65°C.
  • Low stringency refers to conditions equivalent to hybridization in 10% formamide, 5X Denhardt's solution, 6X SSC, 0.2% SDS, followed by washing in IX SSC, 0.2% SDS, at 50°C.
  • the term "substantially identical", when referring to a protein or polypeptide, is meant one that has at least 80%>, 85%, 90%>, 95%, or 99% sequence identity to a reference amino acid sequence.
  • the length of comparison is preferably the full length of the polypeptide or protein, but is generally at least 10, 15, 20, 25, 30, 40, 50, 60, 80, or 100 or more contiguous amino acids.
  • a "substantially identical" nucleic acid is one that has at least 80%, 85%, 90%), 95%o, or 99% sequence identity to a reference nucleic acid sequence.
  • the length of comparison is preferably the full length of the nucleic acid, but is generally at least 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, or more.
  • treating is meant administering a pharmaceutical composition for the purpose of improving the condition of a patient by reducing, alleviating, or reversing at least one adverse effect or symptom.
  • a therapeutically effective amount refers to a quantity of compound (e.g., a Otx2 protein or biologically active fragment thereof) delivered with sufficient frequency to provide a medical benefit to the patient.
  • a therapeutically effective amount of a protein is an amount sufficient to treat or ameliorate a symptom of PD.
  • vector refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a cell, for example by a process of transformation.
  • Vectors may be viral or non-viral.
  • Viral vectors include retroviruses, adenoviruses, herpesvirus, papovirus, or otherwise modified naturally occurring viruses.
  • Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defmed-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
  • Non-viral vector may include plasmid that comprises a heterologous polynucleotide capable of being delivered to a target cell, either in vitro, in vivo or ex-vivo.
  • the heterologous polynucleotide can comprise a sequence of interest and can be operably linked to one or more regulatory elements and may control the transcription of the nucleic acid sequence of interest.
  • a vector need not be capable of replication in the ultimate target cell or subject.
  • the term vector may include expression vector and cloning vector.
  • Suitable expression vectors are well-known in the art, and include vectors capable of expressing a polynucleotide operatively linked to a regulatory element, such as a promoter region and/or an enhancer that is capable of regulating expression of such DNA.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA.
  • Appropriate expression vectors include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • the methods and compositions of this invention are based on the discovery that an elevated Otx2 level is neuroprotective of midbrain dopaminergic neurons.
  • the mDA are involved in many neurological diseases and psychiatric diseases. Considerable progress has been made on biology of mDA neurons and their development in an effort to understand how to treat these devastating diseases. For example, many transcription factors that determine mDA neuron phenotype have been identified, including Nurrl, Pitx3, Lmxla, Lmxlb, Enl, and Otx2. Even within mDA neurons, there are two major subgroups; A9 DA neurons in the SN and A10 DA neurons in the VTA.
  • A9 DA neurons to the dorsolateral striatum and A10 DA neurons to nucleus accumbens, cortex, septum, amygdala and olfactory tubercle.
  • A9 DA neurons in the SN preferentially degenerate, whereas A10 DA neurons in the adjacent VTA are relatively spared.
  • Otx2 is a homeodomain transcription factor, whose expression is restricted to the forebrain and midbrain in the nervous system during development. Otx2 plays an important role in proliferation and differentiation of mDA neuronal progenitor during mitosis, demonstrated by studies using various Otx2 mutant mice. The present inventors discovered that Otx2 is elevated in postmitotic A10 DA neurons in mice and humans and the levels of Otx2 affected the pattern of A10 axonal projection and vulnerability of mDA neurons to a PD-relevant toxin.
  • Otx2 Overexpression of Otx2 increased levels of known A10 elevated genes, including neuropilin 1, neuropilin 2, slit2 and adenylyl cyclase activating peptide in both MN9D cells and ventral mesencephalic (VM) cultures, whereas knockdown of endogenous Otx2 levels via shRNA reduced expression of these genes in VM cultures. Lack of Otx2 in the VM of Otx2 conditional knockout mice, caused reduction of mDA neurons and selective loss of A10 DA projections. Otx2 overexpression protected DA neurons in VM cultures from MPP+, whereas downregulation of Otx2 using shR A increased their susceptibility.
  • Otx2 is important for establishing subgroup phenotypes of postmitotic mDA neurons, and may alter disease susceptibility.
  • Parkinson's Disease a human disease characterized primarily by a loss of dopaminergic neurons particularly in the A9 midbrain region, can be treated or prevented by increasing the expression or activity of Otx2, in DA cells of human subjects.
  • This invention features methods and compositions for treating or preventing PD.
  • the invention features methods of gene therapy to express Otx2 in the midbrain, suitably in the dopaminergic neurons of the midbrain, of a patient.
  • Gene therapy including the use of viral vectors as described herein, seeks to transfer new genetic material (e.g., polynucleotides encoding Otx2 or a biologically active fragment thereof) to the cells of a patient with resulting therapeutic benefit to the patient.
  • expression vectors encoding the gene of interest is administered directly to the patient. The vectors are taken up by the target cells (e.g. , neurons or pluripotent stem cells) and the Otx2 gene expressed.
  • Adenoviruses are able to transfect a wide variety of cell types, including non- dividing cells. There are more than 50 serotypes of adenoviruses that are known in the art, but the most commonly used serotypes for gene therapy are type 2 and type 5. Typically, these viruses are replication-defective; and genetically-modified to prevent unintended spread of the virus. This is normally achieved through the deletion of the El region, deletion of the El region along with deletion of either the E2 or E4 region, or deletion of the entire adenovirus genome except the cis-acting inverted terminal repeats and a packaging signal (Gardlik et al., Med Sci Monit. 11 : RAl 10-121, 2005).
  • Retroviruses are also useful as gene therapy vectors and usually (with the exception of lentiviruses) are not capable of transfecting non-dividing cells. Accordingly, any appropriate type of retrovirus that is known in the art may be used, including, but not limited to, HIV, SIV, FIV, EIAV, and Moloney Murine Leukaemia Virus (MoMLV). Typically, therapeutically useful retroviruses including deletions of the gag, pol, or env genes.
  • the invention features the methods of gene therapy that utilize a lentivirus vectors to express Otx2 in a patient.
  • Lentiviruses are a type of retroviruses with the ability to infect both proliferating and quiescent cells.
  • An exemplary lentivirus vector for use in gene therapy is the HIV-1 lentivirus.
  • Previously constructed genetic modifications of lentiviruses include the deletion of all protein encoding genes except those of the gag, pol, and rev genes (Moreau-Gaudry et al., Blood. 98: 2664-2672, 2001).
  • Adeno-associated virus (AAV) vectors can achieve latent infection of a broad range of cell types, exhibiting the desired characteristic of persistent expression of a therapeutic gene in a patient.
  • the invention includes the use of any appropriate type of adeno-associated virus known in the art including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6 (Lee et al, Biochem J. 387: 1-15, 2005; U.S. Patent Publication 2006/0204519).
  • Herpes simplex virus replicates in epithelial cells, but is able to stay in a latent state in non-dividing cells such as the midbrain dopaminergic neurons.
  • the gene of interest may be inserted into the LAT region of HSV, which is expressed during latency.
  • Other viruses that have been shown to be useful in gene therapy include parainfluenza viruses, poxviruses, and alphaviruses, including Semliki forest virus, Sinbis virus, and Venezuelan equine encephalitis virus (Kennedy, Brain. 120: 1245-1259, 1997).
  • Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defmed-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
  • naked DNA may be administered using an injection, a gene gun, or electroporation.
  • DNA-mediated gene transfer has also been characterized in liver, heart, lung, brain and endothelial cells. See Zhu, et al., Science, 261 : 209-211, 1993; Nabel, et al., Science, 2 : ⁇ M2- ⁇ M , 1989.
  • DNA for gene transfer also may be used in association with various cationic lipids, polycations and other conjugating substances. See Przybylska et al., J. Gene Med., 6: 85-92, 2004; Svahn, et al. , J. Gene Med., 6: S36-S44, 2004.
  • cationic liposomes for use in this invention are DOTMA, DOPE, DOSPA, DOTAP, DC-Choi, Lipid GL-67.TM., and EDMPC. These liposomes may be used in vivo or ex vivo to encapsulate a Otx2 vector for delivery into target cells ⁇ e.g. , neurons or pluripotent stem cells).
  • vectors made in accordance with the principles of this disclosure will contain regulatory elements that will cause constitutive expression of the Otx2 coding sequence.
  • neuron-specific regulatory elements such as neuron-specific promoters are used in order to limit or eliminate ectopic Otx2 expression in the event that the vector is incorporated into cells outside of the target region.
  • Several regulatory elements are well known in the art to direct neuronal specific gene expression including, for example, the neural-specific enolase (NSE), and synapsin-1 promoters (Morelli et al. J. Gen. Virol. 80: 571-583, 1999).
  • ex vivo gene therapy is used to effect Otx2 expression in the midbrain of a patient.
  • this therapeutic strategy involves using the expression vectors and techniques described above to transfect cultured cells in vitro prior to
  • autologous cells are isolated, transfected, and implanted into the patient.
  • the use of autologous cells minimizes the likelihood of rejection or other deleterious immunological host reaction.
  • Other useful cell types include, for example, pluripotent stem cells, including umbilical cord blood stem cells, neuronal progenitor cells, fetal mesencephalic cells, embryonic stem cells, and postpartum derived cells (U.S. Patent Application 2006/0233766).
  • pluripotent stem cells including umbilical cord blood stem cells, neuronal progenitor cells, fetal mesencephalic cells, embryonic stem cells, and postpartum derived cells (U.S. Patent Application 2006/0233766).
  • cells are encapsulated in a
  • the encapsulated cells are modified to express a secreted version of Otx2 which provides therapeutic benefit to the surrounding brain regions.
  • Cell transplantation therapies typically involve grafting the Otx2-expressing replacement cell populations into the lesioned region of the nervous system (e.g., the A9 region of the substantia nigra), or at a site adjacent to the site of injury. Most commonly, the therapeutic cells are delivered to a specific site by stereotaxic injection. Conventional techniques for grafting are described, for example, in Bjorklund et al. (Neural Grafting in the Mammalian CNS, eds. Elsevier, pp 169-178, 1985), Leksell et al. (Acta Neurochir., 52: 1-7, 1980) and Leksell et al. (J. Neurosurg., 66:626-629, 1987). Identification and localization of the injection target regions will generally be done using a non-invasive brain imaging technique (e.g., MRI) prior to implantation (see, for example, Leksell et al, J. Neurol.
  • MRI magnetic resonance imaging
  • administration of cells into selected regions of a patient's brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted.
  • the cells can be injected into the brain ventricles or intrathecally into a spinal cord region.
  • the cell preparation permits grafting of the cells to any predetermined site in the brain or spinal cord. It also is possible to effect multiple grafting concurrently, at several sites, using the same cell suspension, as well as mixtures of cells.
  • the cells are prepared for implantation.
  • the cells are suspended in a physiologically compatible carrier, such as cell culture medium (e.g., Eagle's minimal essential media), phosphate buffered saline, or artificial cerebrospinal fluid (aCSF).
  • Cell density is generally about 50 to about 500 cells/ml (preferably about 100-150 cells/ml).
  • the volume of cell suspension to be implanted will vary depending on the site of implantation, treatment goal, and cell density in the solution.
  • the Otx2-expressing cells are encapsulated within permeable membranes prior to implantation. Encapsulation provides a barrier to the host's immune system and inhibits graft rejection and inflammation. Several methods of cell encapsulation may be employed. In some instances, cells will be individually encapsulated. In other instances, many cells will be encapsulated within the same membrane. Several methods of cell encapsulation are well known in the art, such as described in European Patent Publication No. 301 ,777, or U.S. Patents 4,353,888, 4,744,933, 4,749,620, 4,814,274, 5,084,350, and 5,089,272.
  • the isolated cells are mixed with sodium alginate and extruded into calcium chloride so as to form gel beads or droplets.
  • the gel beads are incubated with a high molecular weight (e.g., MW 60-500 kDa) concentration (0.03-0.1% w/v) polyamino acid (e.g., poly-L-lysine) to form a membrane.
  • a high molecular weight e.g., MW 60-500 kDa
  • polyamino acid e.g., poly-L-lysine
  • the interior of the formed capsule is re-liquefied using sodium citrate. This creates a single membrane around the cells that is highly permeable to relatively large molecules (MW -200-400 kDa), but retains the cells inside.
  • the capsules are incubated in physiologically compatible carrier for several hours in order that the entrapped sodium alginate diffuses out and the capsules expand to an equilibrium state.
  • the resulting alginate-depleted capsules is reacted with a low molecular weight polyamino acid which reduces the membrane permeability (MW cut-off -40-80 kDa).
  • Nucleic acids that encode a Otx2 protein or fragment thereof may be introduced into various cell types or cell-free systems for expression, thereby allowing purification of the Otx2 protein for large-scale production and patient therapy.
  • Eukaryotic and prokaryotic Otx2 expression systems may be generated in which a Otx2 gene sequence is introduced into a plasmid or other vector, which is then used to transform living cells. Constructs in which the Otx2 cDNA contains the entire open reading frame inserted in the correct orientation into an expression plasmid may be used for protein expression. Prokaryotic and eukaryotic expression systems allow for the Otx2 protein to be recovered, if desired, as fusion proteins or further containing a label useful for detection and/or purification of the Otx2 protein.
  • Typical expression vectors contain regulatory elements that direct the synthesis of large amounts of mRNA corresponding to the inserted Otx2 nucleic acid in the plasmid-bearing cells. They may also include a eukaryotic or prokaryotic origin of replication sequence allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector-containing cells to be selected for in the presence of otherwise toxic drugs, and sequences that increase the efficiency with which the synthesized mR A is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome). Cell lines may also be produced that have integrated the vector into the genomic DNA, and in this manner the gene product is produced on a continuous basis.
  • viruses e.g., the OriP sequences from the Epstein Barr Virus genome
  • Expression of foreign sequences in bacteria requires the insertion of the Otx2 nucleic acid sequence into a bacterial expression vector.
  • plasmid vectors contain several elements required for the propagation of the plasmid in bacteria, and for expression of the DNA inserted into the plasmid. Propagation of only plasmid-bearing bacteria is achieved by introducing, into the plasmid, selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs.
  • the plasmid also contains a transcriptional promoter capable of producing large amounts of mRNA from the cloned gene. Such promoters may be (but are not necessarily) inducible promoters that initiate transcription upon induction.
  • the plasmid also preferably contains a polylinker to simplify insertion of the gene in the correct orientation within the vector.
  • Stable or transient cell line clones of mammalian cells can also be used to express a Otx2 protein.
  • Appropriate cell lines include, for example, COS, HEK293T, CHO, or NIH cell lines.
  • the appropriate expression vectors containing a Otx2 gene, fragment, fusion, or mutant are constructed, they are introduced into an appropriate host cell by transformation techniques, such as, but not limited to, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection.
  • the host cells that are transfected with the vectors of this invention may include (but are not limited to) E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression in SF9 insect cells), or cells derived from mice, humans, or other animals.
  • a recombinant protein Once a recombinant protein is expressed, it can be isolated from cell lysates using protein purification techniques such as affinity chromatography. Once isolated, the recombinant protein can, if desired, be purified further by e.g. , by high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).
  • HPLC high performance liquid chromatography
  • the present invention includes the administration of Otx2, and biologically active fragments thereof, for the treatment or prevention of PD.
  • the administration of Otx2, regardless of its method of manufacture, will be in an amount, frequency, and duration sufficient to ameliorate at least one symptom of PD.
  • the symptoms of PD that may be ameliorated include, for example, phenotypic symptoms ⁇ e.g., resting tremor) or
  • neuroanatomical symptoms ⁇ e.g., protecting or restoring midbrain dopaminergic neurons.
  • the therapeutic molecules can be administered to a subject, e.g., a human, alone or in combination with any pharmaceutically acceptable carrier or salt known in the art.
  • Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • Exemplary pharmaceutically acceptable carriers include physiological saline and artificial cerebrospinal fluid (aCSF).
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (21st edition), 2005, Lippincott Williams & Wilkins Publishing.
  • compositions of a therapeutically effective amount of a compound of the invention, or pharmaceutically acceptable salt-thereof can be administered parenterally ⁇ e.g. intramuscular, intraperitoneal, intravenous, or subcutaneous injection), or by intrathecal or intracerebroventricular injection in an admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
  • parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate.
  • Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispersing agents.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for the proteins of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Liquid formulations can be sterilized by, for example, filtration through a bacteria- retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
  • the protein or therapeutic compound can be administered in a sustained release composition, such as those described in, for example, U.S. Patent No. 5,672,659 and U.S. Patent No. 5,595,760.
  • a sustained release composition depends on the type of condition being treated. If the condition consists of an acute or subacute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition. Alternatively, for preventative or long-term treatments, a sustained released composition will generally be preferred.
  • a candidate compound that is beneficial for treating or preventing PD can be identified using the methods described herein.
  • a candidate compound can be identified for its ability to increase the expression or biological activity of Otx2 gene.
  • Candidate compounds that modulate the expression level or biological activity of the polypeptide of the invention by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 100%, or more relative to an untreated control not contacted with the candidate compound are identified as compounds useful for treating and preventing PD.
  • a wide array of cell types may be used in the screening methods of this invention to identify candidate compounds for the treatment of PD by assessing the effects of the candidate compounds on the expression of Otx2.
  • Primary fetal dopaminergic neurons or cell lines exhibiting some characteristics of the dopaminergic neuronal phenotype may be used in the present invention.
  • Cell lines have the advantage of providing a homogeneous cell population, which allows for reproducibility and sufficient number of cells for experiments.
  • Primary dopaminergic cultures are derived from tissues harvested from developing ventral mesencephalon (VM) containing the substantia nigra. They have the advantage of containing authentic dopaminergic neurons cultured in a context of their naturally occurring neighboring cells.
  • VM ventral mesencephalon
  • DAN Human Dopaminergic Neuron Precursor
  • vectors comprising Otx2 coding sequence operably linked to at least one regulatory element may be introduced into the cells.
  • Vectors encoding Otx2 nucleic acid sequence may further comprise non-Otx2 nucleic acid sequence which may be co-expressed with Otx2 as a fusion product or as a co-transcript.
  • Nonlimiting examples of such non-Otx2 nucleic acid sequence includes His-tag (a stretch of poly histidines), FLAG-tag, and Green Fluorescent Protein (GFP). His-tag and FLAG-tag can be used in many different methods, such as purification of Otx2 protein.
  • the tags can also serve as an important site for antibody recognition.
  • Exemplary, eukaryotic vectors include the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, Nature, Vol. 277: 108-114 (1979)), the
  • Otx2 RNA may be assessed by microarray such as by Affymetrix GeneChip® technology, or by Northern blot analysis (Sambrook, et ah, Molecular Cloning: A Laboratory Manual (1989), Second Edition, Cold Spring Harbor Press, Plainview, NY).
  • the Otx2 polypeptide may be detected by immunological methods ⁇ e.g., ELISA, RIA, Western blot). Methods for screening a candidate compound can also be found in U.S. Application Publication 2006/0078890. The contents of which are incorporated herein to its entirety.
  • reporter gene that is operably linked to at least a Otx2 regulatory element can be used to identify a candidate compound for treating or preventing PD.
  • Assays employing the detection of reporter gene products are extremely sensitive and readily amenable to automation, hence making them ideal for the design of high-throughput screens.
  • Assays for reporter genes may employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products.
  • Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. Such vectors contain cassettes encoding reporter genes such as lacZ/p-galactosidase, green fluorescent protein, and luciferase, among others.
  • a genomic DNA fragment carrying at least a Otx2 regulatory element is first cloned using standard approaches (such as those described by Sambrook et al.).
  • the DNA carrying at least one Otx2 regulatory element is then inserted, by DNA subcloning, into a reporter vector, thereby placing a vector-encoded reporter gene under the control of Otx2 regulatory element.
  • the activity of at least a Otx2 regulatory element operably linked to the reporter gene can then be directly observed and quantified as a function of reporter gene activity in a reporter gene assay.
  • At least a Otx2 regulatory element could be cloned upstream from a luciferase reporter gene within a reporter vector. This could be introduced into the test cells, along with an internal control reporter vector (e.g. , a lacZ gene under the transcriptional regulation of the ⁇ -actin promoter). After the cells are exposed to the test compounds, reporter gene activity is measured and the reporter gene activity is normalized to internal control reporter gene activity.
  • an internal control reporter vector e.g. , a lacZ gene under the transcriptional regulation of the ⁇ -actin promoter
  • the present inventors discovered that a single nucleotide polymorphism in this Otx2 gene shows strong association with Parkinson's disease risk.
  • methods of detecting a particular nucleic acid segment of interest in a sample of nucleic acids include a single nucleotide polymorphism (SNP) in the Otx2 gene.
  • SNP single nucleotide polymorphism
  • the present invention provides variant nucleic acids or SNPs associated with PD.
  • a polymorphism in the promoter region of the Otx2 gene shows a strong association with PD risk in patients older than 60 years.
  • the PD- associated SNP is res 1123285.
  • the PD-associated SNPs disclosed herein are useful for diagnosing, screening for, and evaluating predisposition to PD and related pathologies in humans.
  • the present invention provides individual variants associated with PD and genomic sequences containing SNPs, methods of detecting these polymorphisms in a test sample, methods of determining the risk of an individual of having or developing a PD, methods of using the disclosed variants to select a treatment strategy, and methods of using the variants of the present invention for human identification.
  • nucleic acid molecules may be double-stranded molecules and that reference to a particular site on one strand refers, as well, to the corresponding site on a complementary strand.
  • reference to an adenine, a thymine (uridine), a cytosine, or a guanine at a particular site on one strand of a nucleic acid molecule also defines the thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule.
  • Probes and primers may be designed to hybridize to either strand and variant genotyping methods disclosed herein may generally target either strand.
  • identifying a variants position reference is generally made to the protein-encoding strand, only for the purpose of convenience.
  • variant peptides, polypeptides, or proteins of the present invention include peptides, polypeptides, proteins, or fragments thereof, that contain at least one nucleotide or amino acid residue that differs from the corresponding nucleotide or amino acid sequence of the art-known nucleotide/protein (the art-known protein may be interchangeably referred to as the "wild-type", "reference", or "normal” protein).
  • Such variant proteins can result from a codon change caused by a nonsynonymous nucleotide substitution at a protein- coding SNP position (i.e., a missense mutation) disclosed by the present invention.
  • Variant proteins can also result from a nonsense mutation, i.e., a SNP that creates a premature stop codon, a SNP that generates a read-through mutation by abolishing a stop codon, or a SNP that otherwise alters the structure, function/activity, or expression of a protein, such as a SNP in a regulatory region (e.g., a promoter or enhancer) or a SNP that leads to alternative or defective splicing, such as a SNP in an intron or a SNP at an exon/intron boundary.
  • a nonsense mutation i.e., a SNP that creates a premature stop codon, a SNP that generates a read-through mutation by abolishing a stop codon, or a SNP that otherwise alters the structure, function/activity, or expression of a protein, such as a SNP in a regulatory region (e.g., a promoter or enhancer) or a SNP that leads to alternative or defective splic
  • nucleotide numbering of the coding DNA and RNA reference sequence is as follows. Nucleotide numbering is in relation to the translation initiation codon, starting with number 1 at the A of the ATG. Standard mutation nomenclature based on coding DNA reference sequences and protein level amino acid sequences uses the prefixes "c.” and "p.,” respectively. Standard mutation nomenclature based on a genomic DNA reference sequence requires a prefix "g.” In one embodiment, the methods detect a variant in the promoter of the Otx2 gene. In one embodiment, the PD- associated SNP is rsl 123285 (NG_008204.1 :g.7666G>C).
  • Nucleic acid DNA or RNA
  • Examples include tissue samples or any cell-containing bodily fluid.
  • Biological samples may be obtained by standard procedures and may be used immediately or stored, under conditions appropriate for the type of biological sample, for later use.
  • test samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, drawing of blood or other fluids, surgical or needle biopsies, and the like.
  • the test sample may be obtained from a patient.
  • the test sample may contain cells, tissues or fluid obtained from a patient suspected being afflicted with or a carrier for an allele associated with increased suspectibility to PD.
  • the test sample may be a cell-containing liquid or a tissue.
  • Samples may include, but are not limited to, amniotic fluid, biopsies, blood, blood cells, bone marrow, fine needle biopsy samples, peritoneal fluid, amniotic fluid, plasma, pleural fluid, saliva, semen, serum, tissue or tissue homogenates, frozen or paraffin sections of tissue. Samples may also be processed, such as sectioning of tissues, fractionation, purification, or cellular organelle separation. [0096] The methods can be used to perform prenatal diagnosis using any type of embryonic or fetal cell or nucleic acid containing body fluid. Fetal cells can be obtained through the pregnant female, or from a sample of an embryo.
  • Fetal cells are present in amniotic fluid obtained by amniocentesis, chorionic villi aspirated by syringe, percutaneous umbilical blood, a fetal skin biopsy, a blastomere from a four-cell to eight-cell stage embryo (pre- implantation), or a trophectoderm sample from a blastocyst (pre -implantation or by uterine lavage).
  • the sample may be collected or concentrated by centrifugation and the like.
  • the cells of the sample may be subjected to lysis, such as by treatments with enzymes, heat, surfactants, ultrasonication, or a combination thereof.
  • the lysis treatment is performed in order to obtain a sufficient amount of nucleic acid derived from the individual's cells to detect using polymerase chain reaction.
  • variants in the Otx2 gene may be detected using an acellular bodily fluid.
  • Methods of plasma and serum preparation are well known in the art. Either "fresh" blood plasma or serum, or frozen (stored) and subsequently thawed plasma or serum may be used. Frozen (stored) plasma or serum should optimally be maintained at storage conditions of -20 to -70°C until thawed and used. "Fresh” plasma or serum should be refrigerated or maintained on ice until used, with nucleic acid (e.g., RNA, DNA or total nucleic acid) extraction being performed as soon as possible. Exemplary methods are described below.
  • nucleic acid e.g., RNA, DNA or total nucleic acid
  • Blood can be drawn by standard methods into a collection tube, typically siliconized glass, either without anticoagulant for preparation of serum, or with EDTA, sodium citrate, heparin, or similar anticoagulants for preparation of plasma. If preparing plasma or serum for storage, although not an absolute requirement, is that plasma or serum is first fractionated from whole blood prior to being frozen. This reduces the burden of extraneous intracellular RNA released from lysis of frozen and thawed cells which might reduce the sensitivity of the amplification assay or interfere with the amplification assay through release of inhibitors to PCR such as porphyrins and hematin.
  • Frsh plasma or serum may be fractionated from whole blood by centrifugation, using gentle centrifugation at 300-800 times gravity for five to ten minutes, or fractionated by other standard methods. High centrifugation rates capable of fractionating out apoptotic bodies should be avoided. Since heparin may interfere with RT- PCR, use of heparinized blood may require pretreatment with heparanase, followed by removal of calcium prior to reverse transcription. Imai, H., et al., J. Virol. Methods 36:181- 184, (1992). Thus, EDTA is a suitable anticoagulant for blood specimens in which PCR amplification is planned.
  • the nucleic acid to be amplified may be from a biological sample such as an organism, cell culture, tissue sample, and the like.
  • the biological sample can be from a subject which includes any animal, preferably a mammal.
  • a preferred subject is a human, which may be a patient presenting to a medical provider for diagnosis or treatment of a disease.
  • the biological sample may be obtained from a stage of life such as a fetus, young adult, adult, and the like.
  • Subjects may be humans being tested for the existence of an Otx2 polymorphism.
  • the volume of plasma or serum used in the extraction may be varied dependent upon clinical intent, but volumes of 100 to one milliliter of plasma or serum are usually sufficient.
  • RNA extraction Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al, Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 (1989).
  • RNA may be extracted from patient blood/bone marrow samples using MagNA Pure LC mRNA HS kit and Mag NA Pure LC Instrument (Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN).
  • Nucleic acid extracted from tissues, cells, plasma or serum can be amplified using nucleic acid amplification techniques well known in the art. Many of these amplification methods can also be used to detect the presence of mutations simply by designing oligonucleotide primers or probes to interact with or hybridize to a particular target sequence in a specific manner. By way of example, but not by way of limitation, these techniques can include the polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction. See Abravaya, K., et al, Nucleic Acids Research, 23:675-682, (1995), branched DNA signal amplification, Urdea, M.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • nested PCR ligase chain reaction
  • RNA reporters S., et al, AIDS, 7 (suppl 2):S11-S 14, (1993), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA).
  • NASBA isothermal nucleic acid sequence based amplification
  • RNA to cDNA Some methods employ reverse transcription of RNA to cDNA. As noted, the method of reverse transcription and amplification may be performed by previously published or recommended procedures. Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus Thermophilus .
  • one method, but not the only method, which may be used to convert RNA extracted from plasma or serum to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR Methods Applic, 4:S83-S91, (1994).
  • PCR is a technique for making many copies of a specific template DNA sequence.
  • the reaction consists of multiple amplification cycles and is initiated using a pair of primer sequences that hybridize to the 5' and 3' ends of the sequence to be copied.
  • the amplification cycle includes an initial denaturation, and typically up to 50 cycles of annealing, strand elongation and strand separation (denaturation).
  • the DNA sequence between the primers is copied.
  • Primers can bind to the copied DNA as well as the original template sequence, so the total number of copies increases exponentially with time.
  • PCR can be performed as according to Whelan, et ah, J of Clin Micro, 33(3):556-561(1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and lx PCR Buffer.
  • LCR is a method of DNA amplification similar to PCR, except that it uses four primers instead of two and uses the enzyme ligase to ligate or join two segments of DNA.
  • LCR can be performed as according to Moore et al., J Clin Micro, 36(4): 1028-1031 (1998). Briefly, an LCR reaction mixture contains two pair of primers, dNTP, DNA ligase and DNA polymerase representing about 90 ⁇ , to which is added 100 ⁇ of isolated nucleic acid from the target organism. Amplification is performed in a thermal cycler (e.g. , LCx of Abbott Labs, Chicago, IL).
  • a thermal cycler e.g. , LCx of Abbott Labs, Chicago, IL.
  • TAS is a system of nucleic acid amplification in which each cycle is comprised of a cDNA synthesis step and an RNA transcription step.
  • a sequence recognized by a DNA-dependent RNA polymerase i.e., a polymerase-binding sequence or PBS
  • PBS polymerase-binding sequence
  • polymerase is used to synthesize multiple copies of RNA from the cDNA template.
  • TAS DNA-dependent RNA transcription can result in 10-1000 copies for each copy of cDNA template.
  • TAS can be performed according to Kwoh et al., PNAS, 86: 1173-7 (1989). Briefly, extracted RNA is combined with TAS amplification buffer and bovine serum albumin, dNTPs, NTPs, and two oligonucleotide primers, one of which contains a PBS. The sample is heated to denature the RNA template and cooled to the primer annealing temperature. Reverse transcriptase (RT) is added the sample incubated at the appropriate temperature to allow cDNA elongation.
  • RT Reverse transcriptase
  • RNA polymerase is added and the sample is incubated at 37°C for approximately 25 minutes for the synthesis of RNA. The above steps are then repeated.
  • both RT and RNA polymerase are added following a 1 min 100°C denaturation followed by an RNA elongation of approximately 30 minutes at 37°C.
  • TAS can be also be performed on solid phase as according to Wylie et al., J Clin Micro, 36(12):3488-3491 (1998). In this method, nucleic acid targets are captured with magnetic beads containing specific capture primers.
  • the beads with captured targets are washed and pelleted before adding amplification reagents which contains amplification primers, dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase.
  • amplification reagents which contains amplification primers, dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase.
  • a 100 ⁇ TMA reaction mixture is placed in a tube, 200 ⁇ oil reagent is added and
  • amplification is accomplished by incubation at 42°C in a waterbath for one hour.
  • NASBA is a transcription-based amplification method which amplifies RNA from either an RNA or DNA target.
  • NASBA is a method used for the continuous amplification of nucleic acids in a single mixture at one temperature.
  • AMV avian myeloblastosis virus
  • RNase H RNase H
  • T7 RNA polymerase T7 RNA polymerase
  • an NASBA reaction mixture contains two specific primers, dNTP, NTP, 6.4 U of AMV reverse transcriptase, 0.08 U of Escherichia coli Rnase H, and 32 U of T7 RNA polymerase.
  • the amplification is carried out for 120 min at 41 °C in a total volume of 20 ⁇ .
  • self-sustained sequence-replication (3SR) reaction isothermal amplification of target DNA or RNA sequences in vitro using three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H.
  • This method may be modified from a 3 -enzyme system to a 2-enzyme system by using human immunodeficiency virus (HIV)-l reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H.
  • HAV human immunodeficiency virus
  • AMV avian myeloblastosis virus
  • the amplified RNA is obtained in a purer form compared with the 3 -enzyme 3SR (Gebinoga & Oehlenschlager Eur J Biochem, 235:256-261, 1996).
  • SDA is an isothermal nucleic acid amplification method.
  • a primer containing a restriction site is annealed to the template.
  • Amplification primers are then annealed to 5' adjacent sequences (forming a nick) and amplification is started at a fixed temperature.
  • Newly synthesized DNA strands are nicked by a restriction enzyme and the polymerase amplification begins again, displacing the newly synthesized strands.
  • SDA can be performed as according to Walker, et al, PNAS, 89:392-6 (1992). Briefly, an SDA reaction mixture contains four SDA primers, dGTP, dCTP, TTP, dATP, 150 U of Hinc II, and 5 U of exonuclease-deficient of the large fragment of E. coli DNA polymerase I (exo ⁇ Klenow polymerase). The sample mixture is heated 95°C for 4 minutes to denature target DNA prior to addition of the enzymes. After addition of the two enzymes, amplification is carried out for 120 min. at 37°C in a total volume of 50 ⁇ . Then, the reaction is terminated by heating for 2 min. at 95°C.
  • the Q-beta replication system uses RNA as a template.
  • RNA genome of the coliphage QP synthesizes the single-stranded RNA genome of the coliphage QP. Cleaving the RNA and ligating in a nucleic acid of interest allows the replication of that sequence when the RNA is replicated by Q-beta replicase (Kramer & Lizardi Trends Biotechnol. 1991 9(2):53-8, 1991).
  • a variety of amplification enzymes are well known in the art and include, for example, DNA polymerase, RNA polymerase, reverse transcriptase, Q-beta replicase, thermostable DNA and RNA polymerases. Because these and other amplification reactions are catalyzed by enzymes, in a single step assay the nucleic acid releasing reagents and the detection reagents should not be potential inhibitors of amplification enzymes if the ultimate detection is to be amplification based.
  • Amplification methods suitable for use with the present methods include, for example, strand displacement amplification, rolling circle amplification, primer extension preamplification, or degenerate oligonucleotide PCR (DOP). These methods of amplification are well known in the art and each described briefly below.
  • PCR is used to amplify a target or marker sequence of interest.
  • the skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target or marker sequence.
  • the length of the amplification primers depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.
  • oligonucleotides specific for alternative alleles Such oligonucleotides which detect single nucleotide variations in target sequences may be referred to by such terms as “allele-specific probes", or “allele-specific primers”.
  • allele-specific probes for analyzing polymorphisms is described in, e.g., Mutation Detection A Practical Approach, ed. Cotton et al. Oxford University Press, 1998; Saiki et al, Nature, 324: 163-166 (1986);
  • a probe or primer may be designed to hybridize to a segment of target DNA such that the SNP aligns with either the 5' most end or the 3' most end of the probe or primer.
  • the amplification may include a labeled primer, thereby allowing detection of the amplification product of that primer.
  • the amplification may include a multiplicity of labeled primers; typically, such primers are distinguishably labeled, allowing the simultaneous detection of multiple amplification products.
  • an allele-specific primer hybridizes to a region on a target nucleic acid molecule that overlaps a SNP position ⁇ e.g., NG-088204.1 :g.7666 G>T of FMRl) and only primes amplification of an allelic form to which the primer exhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res., 17:2427-2448).
  • the primer's 3'- most nucleotide is aligned with and complementary to the SNP position of the target nucleic acid molecule. This primer is used in conjunction with a second primer that hybridizes at a distal site.
  • Amplification proceeds from the two primers, producing a detectable product that indicates which allelic form is present in the test sample.
  • a control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site.
  • the single-base mismatch prevents amplification or substantially reduces amplification efficiency, so that either no detectable product is formed or it is formed in lower amounts or at a slower pace.
  • the method generally works most effectively when the mismatch is at the 3 '-most position of the oligonucleotide (i.e., the 3'-most position of the oligonucleotide aligns with the target SNP position) because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Exemplary allele-specific primer sequences for detecting the rsl 123285 (NG_008204.1 :g.7666G>C) SNP of the Otx2 gene are shown in Table 3 below.
  • a primer contains a sequence substantially
  • the primer has a mismatched nucleotide in one of the three nucleotide positions at the 3 '-most end of the primer, such that the mismatched nucleotide does not base pair with a particular allele at the SNP site.
  • the mismatched nucleotide in the primer is the second from the last nucleotide at the 3 '-most position of the primer. In another embodiment, the mismatched nucleotide in the primer is the last nucleotide at the 3'-most position of the primer.
  • primer or probe is labeled with a fluorogenic reporter dye that emits a detectable signal.
  • a fluorogenic reporter dye is a fluorescent dye
  • any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention.
  • Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.
  • Exemplary allele-specific probe sequences for detecting the rsl 123285 (NG 008204.1 :g.7666G>C) SNP of the Otx2 gene in a TaqMan® assay are shown in Table 4 below.
  • the detection reagent may be further labeled with a quencher dye such as Tamra, especially when the reagent is used as a self-quenching probe such as a TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al, 1995, PCR Method Appl, 4:357-362; Tyagi et al, 1996, Nature Biotechnology, 14:303- 308; Nazarenko et al, 1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635).
  • a quencher dye such as Tamra
  • the present invention also contemplates reagents that do not contain (or that are complementary to) a SNP nucleotide identified herein but that are used to assay one or more SNPs disclosed herein.
  • primers that flank, but do not hybridize directly to a target SNP position provided herein are useful in primer extension reactions in which the primers hybridize to a region adjacent to the target SNP position ⁇ i.e., within one or more nucleotides from the target SNP site).
  • a primer is typically not able to extend past a target SNP site if a particular nucleotide (allele) is present at that target SNP site, and the primer extension product can readily be detected in order to determine which SNP allele is present at the target SNP site.
  • particular ddNTPs are typically used in the primer extension reaction to terminate primer extension once a ddNTP is incorporated into the extension product.
  • Variant nucleic acids may be amplified prior to detection or may be detected directly during an amplification step ⁇ i.e., "real-time” methods).
  • the target sequence is amplified and the resulting amplicon is detected by electrophoresis.
  • the specific mutation or variant is detected by sequencing the amplified nucleic acid.
  • the target sequence is amplified using a labeled primer such that the resulting amplicon is detectably labeled.
  • the primer is fluorescently labeled.
  • detection of a variant nucleic acid is performed using the TaqMan® assay, which is also known as the 5' nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848).
  • the TaqMan® assay detects the accumulation of a specific amplified product during PCR.
  • the TaqMan® assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. The reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the excited reporter dye When attached to the probe, the excited reporter dye does not emit a signal.
  • the proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter.
  • the reporter dye and quencher dye may be at the 5' most and the 3' most ends, respectively or vice versa.
  • the reporter dye may be at the 5 ' or 3' most end while the quencher dye is attached to an internal nucleotide, or vice versa.
  • both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
  • DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present.
  • TaqMan® primer and probe sequences can readily be determined using the variant and associated nucleic acid sequence information provided herein.
  • a number of computer programs such as Primer Express (Applied Biosystems, Foster City, Calif), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the variants of the present invention are useful in diagnostic assays for neurodevelopmental disorders and related pathologies, and can be readily incorporated into a kit format.
  • the present invention also includes modifications of the TaqMan® assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).
  • real time PCR is performed using TaqMan® probes in combination with a suitable amplification/analyzer such as the ABI Prism® 7900HT Sequence Detection System.
  • the ABI PRISM® 7900HT Sequence Detection System is a high-throughput real-time PCR system that detects and quantitates nucleic acid sequences.
  • TaqMan® probes specific for the amplified target or marker sequence are included in the PCR amplification reaction. These probes contain a reporter dye at the 5' end and a quencher dye at the 3' end. Probes hybridizing to different target or marker sequences are conjugated with a different fluorescent reporter dye.
  • the fluorescently labeled probes bind specifically to their respective target or marker sequences; the 5' nuclease activity of Taq polymerase cleaves the reporter dye from the probe and a fluorescent signal is generated.
  • the increase in fluorescence signal is detected only if the target or marker sequence is complementary to the probe and is amplified during PCR.
  • a mismatch between probe and target greatly reduces the efficiency of probe hybridization and cleavage.
  • the ABI Prism 7700HT or 7900HT Sequence detection System measures the increase in fluorescence during PCR thermal cycling, providing "real time" detection of PCR product accumulation.
  • Real time detection on the ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescence and calculates Rn during each PCR cycle.
  • the threshold cycle, or Ct value is the cycle at which fluorescence intersects the threshold value.
  • the threshold value is determined by the sequence detection system software or manually.
  • MGB EclipseTM probes (Epoch Biosciences), which do not rely on a probe degradation can be used.
  • MGB EclipseTM probes work by a hybridization-triggered fluorescence mechanism.
  • MGB EclipseTM probes have the EclipseTM Dark Quencher and the MGB positioned at the 5'-end of the probe. The fluorophore is located on the 3'-end of the probe.
  • the probe is in solution and not hybridized, the three dimensional conformation brings the quencher into close proximity of the fluorophore, and the fluorescence is quenched.
  • the probe anneals to a target or marker sequence, the probe is unfolded, the quencher is moved from the fluorophore, and the resultant fluorescence can be detected.
  • Oligonucleotide probes can be designed which are between about 10 and about 100 nucleotides in length and hybridize to the amplified region. Oligonucleotides probes are preferably 12 to 70 nucleotides; more preferably 15-60 nucleotides in length; and most preferably 15-25 nucleotides in length. The probe may be labeled. Amplified fragments may be detected using standard gel electrophoresis methods. For example, in preferred embodiments, amplified fractions are separated on an agarose gel and stained with ethidium bromide by methods known in the art to detect amplified fragments.
  • Assay controls may be used in the assay for detecting carriers and individuals afflicted with or susecptible to a neurodegernative condition, such as PD. Positive controls for normal or wild type Otx2 gene may be used.
  • kits for diagnosing the PD or an increased risk of PD comprising a set of reagents for determining the presence or absence, or differential presence, of one or more markers (SNPs) indicative of a
  • the kit contains a set of nucleic acid primers for detecting one or more mutations in an Otx2 gene indicative of a suspectibility to PD.
  • the kit contains one or more of the primers or probes of SEQ ID NOs: 3-10.
  • Example 1 - Otx2 is elevated in A10 (VTA) DA neurons
  • Brains were cut using a cryostat with 10 ⁇ (for mouse) or 18 ⁇ (for human) thickness, mounted on LCM slides (Arcturus) and immediately stored at -70° C.
  • the tissue sections were fixed in cold acetone for 5 minutes, washed with PBS, incubated with rabbit anti-TH (Pel-Freez Biologicals, Rogers, AR; 1 :25) for 4 min, washed in PBS, and exposed to biotinylated anti-rabbit antibody (Vector Laboratories, Burlingame, CA; 1 :25) for 4 min.
  • Quantitative (Q) PCR was performed according to a previous protocol (Chung et ah, 2005). Briefly, RNA samples from A9 and A10 DA neurons were reverse- transcribed into cDNA using Sensiscript reverse transcriptase (Qiagen, Valencia, CA) and oligo dT as the primer. PCR reactions were set up in 25 ⁇ reaction volume using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) with 250 nM final concentrations of primers. For each primer pairs, triplicates of three to five independently collected A9 and A10 samples were compared to quantify relative gene expression differences between these cells using the 2 ⁇ AACT method. Beta-actin was used as an internal control gene.
  • coverslips were washed with PBS-T and blocked with donkey serum. Sections were then incubated with anti-TH (Pel-Freez), anti-Otx2 (Neuromics, 1 :500), or GFP (Invitrogen, 1 : 1000) antibodies overnight and subsequently incubated in the following Alexa secondary antibodies (Invitrogen, Carlsbad, CA). Covers lips were mounted onto glass slides and visualized using confocal microscopy.
  • Example 2 Otx2 regulates known AlO-elevated gene expression.
  • Otx2 is a transcription factor
  • Our previous microarray analysis on A9 and A10 DA neurons in adult mice identified two major categories of genes that were elevated in A10 DA neurons: 1) axon guidance cues and 2) neuropeptides (Chung et al., 2005).
  • MN9D cells to overexpress Otx2 via lentivirus.
  • MN9D cells normally express a very low baseline level of Otx2 expression.
  • Otx2 overexpression by lentivirus increased known AlO- elevated axon guidance cue genes including neuropilin 1, neuropilin 2 and slit2 (FIG. 3 A).
  • AlO-elevated neuropeptides such as vasoactive peptides (VIP) and adenylyl cyclase activating peptide (Adcyap) were also significantly increased by Otx2 overexpression in MN9D cells (FIG. 3A).
  • VIP vasoactive peptides
  • Adcyap adenylyl cyclase activating peptide
  • Otx2 overexpression resulted in an increase in levels of genes elevated in A10 DA neurons such as neuropilin 1, neuropilin 2, slit2 and Adcyap (FIG. 3B), whereas reduction of endogenous Otx2 by shRNA caused a decrease in levels of these genes (FIG. 3B).
  • the degree of Otx2-mediated changes in gene expression was smaller in primary VM cultures than in MN9D cells probably because unlike MN9D cells, DA neurons are only a small portion of primary VM cultures, consisting of heterogeneous cell populations.
  • results from primary VM cultures validated findings from MN9D cells, with the exception of VIP.
  • Lentivirus encoding shRNA production Lentiviruses were produced as described (Miyoshi et al, 1998; Dittgen et al, 2004). Briefly, 293T cells (Human Embryonic Kidney) were transfected by using the Perfectin transfection reagent (Genlantis, San Diego, CA) with three plasmids: expression vector and two helper, delta8.9 and vesicular stomatitis virus G protein plasmids at 4, 30, and 22 ⁇ g of DNA per 175-cm cell culture flask.
  • the supernatants were spun at 780 x g for 5 min, filtered at a 0.45- ⁇ pore size, spun at 83,000 x g for 1.5 h, and the pellet was resuspended in 100 ⁇ of PBS. Titers were adjusted to 1-2 x 106/ ⁇ 1.
  • shRNA targeting Otx2 The structure of the shRNA hairpins was sense-loop-antisense.
  • the oligonucleotides used for the construction of shRNA targeting Otx2 were:
  • A10 neuronal projection targets including prefrontal cortex (PFC), nucleus accumbens (NAc), septum, amygdala (Amg), and olfactory tubercle (OT).
  • PFC prefrontal cortex
  • NAc nucleus accumbens
  • Amg amygdala
  • OT olfactory tubercle
  • TH immunohistochemistry results revealed dramatic reduction of TH-positive projections in all of these targets in Enl cre/+ , Otx2 flox/flox mice, whereas projection to dorsolateral striatum, a target of A9 DA neuronal projection, was largely spared (FIG. 4A-F).
  • Otx2 may regulate the expression of these genes and that an axonal guidance cue map during development may be maintained in adult to some degree. It is most likely that the differential expression of Otx2 in mDA neurons observed during E15 and El 7 may regulate levels of these axonal guidance cues during this critical time for axonal projection, which in turn may contribute to the distinct projections pattern of A9 (SN) and A10 (VTA) DA neurons. Moreover, reduction of Otx2 in the knockout mice showed the dramatic reduction of A10 (VTA) projections and sparing of A9 (SN) projections despite the fact that DA neurons in both SN and VTA were reduced in these mice. These results suggest that Otx2 may be one of key transcription factors that influences DA projection potentially by elevating levels of certain guidance cues, including neuropilin 1, neuropilin 2 and slit2.
  • Example 4 - Otx2 makes DA neurons less vulnerable to MPP toxicity
  • VM cultures were challenged with MPP + after overexpressing or reducing Otx2 using the lentivirus encoding Otx2 or the lentivirus encoding shRNA against Otx2.
  • FIG. 5D shows that after reduction of endogenous Otx2 in VM cultures using lentivirus encoding shRNA against Otx2 caused -25% decrease in Otx2- positive TH-positive neurons in VM cultures (FIG. 5E-G) and made TH-positive neurons more susceptible to MPP + toxicity compared to control shRNA overexpressing conditions (FIG. 5H).
  • Primary VM cultures Primary cell cultures were performed using E12.5 mouse embryos. Briefly, ventral midbrains were isolated and tissue was dissociated using trypsine- EDTA (Invitrogen). The cells were seeded on Poly-L-ornithine/Laminin-coated coverslips at approximately 150,000 per coverslip, in medium, containing DMEM-F12, supplemented with 5% Fetal Calf Serum (FCS), 0.25% BSA (Sigma, Germany), 33 mM glucose, 50 U/ml penicillin, 50U/ml streptomycin, and 1% Fungizone (Invitrogen). The coverslips were incubated at 37°C for up to three weeks. The typical numbers of TH-positive cells per cover slip were between 1500 and 1800 cells. All cell counts were normalized against the controls in each experiment. Each of the experimental conditions is represented by at least 3 coverslips in each experiment.
  • Example 5 Otx2 polymorphism is associated with PD risk
  • SNP rsl 123285 located within the Otx2 gene, was chosen as a genetic marker because it is an informative variation, with a minor allele frequency of 0.28-0.50 among various populations based on the dbSNP Build 129.

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Abstract

La présente invention concerne des procédés de traitement ou de prévention de la maladie de Parkinson et des états associés en augmentant l'expression Otx2 dans les neurones dopaminergiques du mésencéphale du patient. La présente invention concerne en outre des procédés de diagnostic et d'évaluation de la maladie de Parkinson. Par exemple, des échantillons de test de patient sont analysés pour la présence d'une ou plusieurs séquences de variants spécifiques associées à la maladie de Parkinson. Dans certains modes de réalisation, le variant est une mutation promotrice dans le gène Otx2.
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