EP1556484A2 - Genes de la chaine lourde 1 de dyneine cytoplasmiques, produits d'expression, modele animal non humain : utilisation pour les maladies neurologiques humaines - Google Patents

Genes de la chaine lourde 1 de dyneine cytoplasmiques, produits d'expression, modele animal non humain : utilisation pour les maladies neurologiques humaines

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Publication number
EP1556484A2
EP1556484A2 EP03807821A EP03807821A EP1556484A2 EP 1556484 A2 EP1556484 A2 EP 1556484A2 EP 03807821 A EP03807821 A EP 03807821A EP 03807821 A EP03807821 A EP 03807821A EP 1556484 A2 EP1556484 A2 EP 1556484A2
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Prior art keywords
cytoplasmic dynein
amino acid
seq
dynein heavy
heavy chainl
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Rainer Klocke
Andreas Marquardt
Gisela Peraus
Gabriele Stumm
Philipp Wabnitz
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Ingenium Pharmaceuticals GmbH
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Ingenium Pharmaceuticals GmbH
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Publication of EP1556484A2 publication Critical patent/EP1556484A2/fr
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
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    • A—HUMAN NECESSITIES
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    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/03—Animal model, e.g. for test or diseases
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    • A01K2267/03—Animal model, e.g. for test or diseases
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    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2799/00—Uses of viruses
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    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • Cytoplasmic dynein heavy chainl genes, expression products, non-human animal model uses in human neurological diseases
  • the present invention ter alia relates to a non-human animal model for movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitoxicity disorders, and neurodegeneration.
  • This animal model bears a mutation in the cytoplasmic dynein heavy chainl gene.
  • the invention also relates to modified peptides and the conesponding nucleic acid sequences of the modified mouse and human cytoplasmic cytoplasmic dynein heavy chainl.
  • the invention relates to the use of these peptides and nucleic acids for manufacturing therapeutics suitable for the treatment of diseases, such as Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotropic lateral sclerosis (ALS), as well as other diseases associated with overexpression, over-activity, or undesirable activity of cytoplasmic dynein heavy chainl .
  • diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotropic lateral sclerosis (ALS), as well as other diseases associated with overexpression, over-activity, or undesirable activity of cytoplasmic dynein heavy chainl .
  • Eukaryotic cells are characterized by biochemical and physiological processes which under normal conditions are extremely highly balanced to achieve the preservation and propagation of the cells. hen such cells are components of multicellular organisms such as vertebrates, or more particularly organisms such as mammals, the regulation of the biochemical and physiological processes involves intricate signaling pathways. Frequently, such signaling pathways consist of extracellular signaling proteins, cellular receptors that bind the signaling proteins and signal transducing components located within the cells.
  • Signaling prqcesses may elicit a variety of effects on cells and tissues, including by way of nonlimiting example induction of cell or tissue proliferation, suppression of growth or proliferation and induction or suppression of differentiation or maturation of a cell or tissue.
  • Many pathological conditions involve dysregulation of expression of important effector proteins. In certain classes of pathologies the dysregulation is manifested as a diminished or suppressed level of synthesis and secretion of protein effectors.
  • a subject may be suspected of suffering from a condition brought on by diminished or suppressed levels of a protein effector of interest. Therefore, there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject and to compare the level with that characteristic of a nonpathological condition.
  • Degenerative disorders of motor neurons include a range of progressive diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the most common genetic cause of death of children (Nicole, S. et al.,J. Muscle Nerve 26, 4-13, 2002).
  • ALS amyotrophic lateral sclerosis
  • SMA spinal muscular atrophy
  • ALS In ALS, commonly known as Lou Gehrig's Disease in the US, the motor neurons (nerve cells in the brain, brain stem, and spinal cord that control movement of the skeletal muscles) gradually degenerate, resulting in progressive weakness and functional loss of involved muscles. Diseases that cause selective progressive death of motor neurons are surprisingly common « ALS is the third most frequent neurodegenerative cause of adult death, after Alzheimer disease and Parkinson disease, and is significantly more common than multiple sclerosis (Motor Neurone Disease Association Information Sheet Number 9, Motor Neurone Disease Association, 1998). Targeted at adults in their prime years of life, the average age of onset is the mid-fifties, although adults of all ages can be affected. Men get the disease slightly more often than women.
  • ALS familial
  • SOD1 encodes the ubiquitously expressed enzyme superoxide dismutase 1 which takes on an unknown dominant gain of function in ALS that results in the selective death of motor neurons. Mutations have also been found in alsin, probably a GTPase regulatory protein, in rare juvenile recessive forms of motor neuron disease (Hadano, S. et al. Nat. Genet. 29, 166-173, 2001; Yang, Y. et al.. Nat. Genet. 29, 160-165, 2001).
  • NFH neuroofilament heavy chain
  • diagnosing ALS is a combination of medical history and physical and neurological examinations performed by a clinical neurologist (Brooks, Benjamin R., El Escorial J Neurol Sci 124 (Suppl.), pages 96-107, 1994; Karitzky J, Ludolph AC. J Neurol Sci, Oct 15;191(l-2):35-41, 2001; Ludolph AC, and Knirsch U, J Neurol Sci, Jun;165 Suppl l:S14-20, 1999).
  • An electromyogram is a diagnostic test which is done to determine abnormal nerve and muscle activity. The clinical examination and tests also rule out other conditions that might mimic motor neuron disease.
  • the certainty ofthe diagnosis is determined by the clinical evidence of upper and lower motor neuron signs, according to the criteria established by the World Federation of Neurology. Therefore, a definitive diagnosis of ALS is performed when clearly detectable classical clinical signs such as muscular fibrillation of distal muscle groups or tongue, difficulty to swallow, and predominantly distal muscular atrophy with the typical histological picture of grouped angular shaped atrophic muscle fibres indicative of muscle denervation are present which means that the disease course is already proceeded.
  • PBP Progressive bulbar palsy
  • PMA Progressive muscular atrophy
  • PLS Primary lateral sclerosis
  • SMA spinal muscular atrophy
  • SBMA spinobulbar muscular atrophy
  • Glutamate also may lead to the production of detrimental calcium, which can churn out its own supply of DNA-harming free radicals.
  • the free radicals also may injure neurofilaments, proteins that serve as the skeleton ofthe cell.
  • the immune system Hofmann, Y. and Wirth, B. Hum. Mol. Genet. 11, 2037-2049, 2002
  • Abnormalities can lead to an accumulation of the toxic calcium.
  • Glutamate-induced excitotoxicity is a potential contributor to ALS pathogenesis.
  • Depolarization of the neuronal membrane after activation of neuronal glutamate receptors activates voltage-dependent Ca 2+ channels, allowing Ca 2+ entry into the cell.
  • excess activation of neuronal glutamate receptors can cause cell death via alterations in cytosolic free Ca 2+ homeostasis.
  • spinal motor neurons rapid recovery of synaptic glutamate is accomplished by the glutamate transporter EAAT2 present in astrocytes. Loss of EAAT2 transporter could lead to increased extracellular concentrations of glutamate and excitotoxic degeneration of motor neurons (Julien, Review; Cell. 2001 Feb 23;104(4):581-91).
  • mice have recently enabled mouse ALS models to make an excess of glutamate transport proteins in both brains and spinal cords. Preliminary studies of these mice show huge increases in their survival time, which led to the development of an "EAAT2 replacement therapy" for ALS patients at the John Hopkins University (The Robert Packard Center for ALS Research at John Hopkins, www.alscenter.org, 2002).
  • Riluzole retards nerve cells' release of glutamate. Although the effects of Riluzole are clearly modest, it has been the only drug that reliably shows clinical efficacy compared to the dozens of drugs studied by clinicans around the world in thousands of ALS patiens (Nervous Breakdown, A detailed analysis of the neurology market, UBS
  • defects in axonal transport may be an underlying common pathway that leads to the degeneration of motor neurons in ALS patients and in relevant spontaneous and transgenic mouse models such as wobbler, nmd or SOD1
  • Motor neurons of the brain and spinal cord are characterized by the length of their axons, which can reach a meter in length in an adult human.
  • the significant length of these neuronal projections makes active axonal transport essential for normal cellular function.
  • the axonal transport is microtubule dependant and includes both, an anterograde transport of organelles to the axonal synapse and the retrograde transport of multivesicular bodies and trophic factors back to the neuron cell body.
  • the retrograde transport is mediated by the dynein-dynactin complex.
  • Dyneins are cytoskeletal motor proteins. These can be defined as molecules that convert chemical energy, originating from nucleotide hydrolysis, into the mechanical force necessary for them to move along cytoskeletal polymers (c/Nallee and Howard (1990) Annu. Rev. Biochem 59: 909-932). Dyneins and kinesins constitute the superfamily of microtubule-dependent motor proteins, (Hirokawa (1998) Science 279, 519-526).
  • dyneins Although the superfamily of dyneins is probably less diverse than that of kinesins, the family, which contributes to the structure and function of flagellar and cilliary axonems (axonemal dyneins), comprises more than a dozen dynein heavy chain isoforms (Milisav (1998) Cell Motil Cytoskeleton 39, 261- 272). Only four dynein heavy chain isoforms, contributing to different forms of the protein complex, called cytoplasmic dynein, are known in mammals.
  • cytoplasmic dynein heavy chainl The most abundant form of cytoplasmic dynein, whose identity is defined by the cytoplasmic dynein heavy chainl, is involved in a wide range of cellular functions. In mouse and human, the homologous proteins are refened to as cytoplasmic dynein heavy chainl. These proteins conespond, respectively, to the translation product of the transcript ofthe mouse gene Dnchcl (Genbank Accession No. AY004877) and the translation product of the full-length cDNA of the human gene DNCH1. The full- length human cytoplasmic dynein heavy chainl cDNA is disclosed herein (SEQ ID NO: 17).
  • Cytoplasmic dynein was initially identified in nervous tissue (Paschal et al. (1987) J Cell Biol 105, 1273-1282) although it is expressed in several tissues (Mikami et al. (1993) Neuron 10, 787-796).
  • cDHC-l- preimplantation embryos were microscopically indistinguishable from cDHC+/- and cDHC+/+ littermates, by 8.5 days p.c, no cDHC-l- embryo were found.
  • the cytoplasmic dynein complex consists of at least four classes of subunits: heavy, intermediate, light intermediate and light dynein chains.
  • the dynein intermediate chain, dynein light intermediate chain, and dynein light chain specify the
  • the architecture ofthe complex is dominated by two heavy chains of 530 kDA each, which form a stem by the interaction of a large portion (amino acids 300 - 1140) of their N-termini.
  • This stem also binds the dynein chains of lower molecular weight, e.g. two 74 kDa intermediate chains (between amino acids 446 and 701 of the heavy chains) and four 53-59 kDa light intermediate chains (between amino acids 649 and 800 of the heavy chains) (Tynan et al. (2000) J Biol Chem. 275, 32769-32774).
  • Several 8-22 kDa light chains bind to the complex via interaction with the intermediate or light intermediate chains (c * Makoka et al.
  • the binding of dynactin to dynein is essential for dynein-dependent intracellular movements (Gill et al. (1991) J Cell Biol 115, 1639-1650; Schroer and Sheetz (1991) J Cell Biol 115, 1309-1318) including those required for neuronal function. It has been shown that antibodies which specifically disrupt the binding of dynactin to dynein block vesicle motility along micrombules in extruded squid axoplasm (Waterman-Storer et al. (1997) Proc Natl Acad Sci USA 94, 12180-12185).
  • Dynein-mediated intracellular movements are directed along micrombules towards their proximal (-) ends. This process is accomplished by ATP hydrolysis, which is predominantly catalysed by the first of four (counted from the one nearest to the N-terminus) P-loop motifs of each of two C-terminal globular heads which are formed by approximately 3000 amino acids of each of the two dynein heavy chains. Both globular heads also contain a microtubule binding domain, which is thought to be located at the tip of a stalk (Asai and Koonce (2001) Trends Cell Biol 11, 196- 202).
  • the present invention provides an animal model for conelating mutations or defects in the cytoplasmic dynein heavy chainl gene with human disorders, including movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders, and neurodegeneration, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, and specifically motor neuron degeneration, such as ALS (Amyotrophic Lateral Sclerosis), SMA (Spinal Muscular Atrophy), SBMA (Bulbo-Spinal Muscular Atrophy), PBP (progressive Bulbar Palpsy), PMA (Progressive Muscular Atrophy), and PLS (Primary Lateral Sclerosis), resulting in whole or in part from these mutations or defects in the cytoplasmic dynein heavy chainl gene or the protein it encodes.
  • ALS Amyotrophic Lateral Sclerosis
  • SMA Spinal Muscular Atrophy
  • SBMA Bulbo-Spinal Muscular Atrophy
  • PBP progress
  • the Cral (cramping 1) mutation in cytoplasmic dynein itself results in progressive motor neuron degeneration in heterozygous mice.
  • this invention provides an animal model that demonstrates the role of cytoplasmic dynein heavy chainl in movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders and neurodegeneration, specifically ⁇ -motor neuron degeneration, resulting from a mutation or defect in cytoplasmic dynein heavy chainl.
  • the Cral animal model described herein demonstrates a germline missense mutation that causes subtle changes in the cytoplasmic dynein heavy chain 1, and is sufficient to specifically induce age dependent motor neuron degeneration without grossly affecting the housekeeping function of the dynein-dynactin complex in other cell types.
  • the Cral mutation in cytoplasmic dynein heavy chain 1 allows normal development and normal function ofthe organism in early adulthood.
  • the animal model provided herein meets the cunently unfulfilled need for a validated model to study the causes and precise physiological effects of such diseases associated with abenant cytoplasmic dynein heavy chainl function.
  • this invention provides a means for identifying antagonists and inhibitors for cytoplasmic dynein heavy chainl in such disorders.
  • the present invention also provides significant data useful in neurobiology and clinical research, as it describes in an animal model a new pathway involved in neurodegeneration, and also suggests a possible genetic cause for ALS/Lou Gehrig's disease.
  • the present invention provides diagnostic tests for latent or progressive diseases, or a propensity to develope a disease or passing on a contributing or causatory recessive genetic mutation to progeny, wherein the disease is selected from, but not limited to, the group of human disorders, including movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders, and neurodegeneration, in particular Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, spinal muscular atrophy, and fiber type grouping in Musculus tibialis anterior.
  • the disease is selected from, but not limited to, the group of human disorders, including movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders, and neurodegeneration, in particular Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, spinal muscular atrophy, and fiber type grouping in Musculus tibi
  • the present invention provides diagnostic tests for latent or progressive diseases, or a propensity to develope a disease or passing on a contributing or causatory recessive genetic mutation to progeny, wherein the disease is selected from, but not limited to, the group of human motor neuron degeneration disorders, including ALS (Amyotrophic Lateral Sclerosis), SMA (Spinal Muscular Atrophy), SBMA (Bulbo-Spinal Muscular Atrophy), PBP (progressive Bulbar Palpsy), PMA (Progressive Muscular Atrophy), and PLS (Primary Lateral Sclerosis).
  • ALS Amyotrophic Lateral Sclerosis
  • SMA Spinal Muscular Atrophy
  • SBMA Bulbo-Spinal Muscular Atrophy
  • PBP progressive Bulbar Palpsy
  • PMA Progressive Muscular Atrophy
  • PLS Primary Lateral Sclerosis
  • This invention provides inter alia a non-human animal useful as a model of dynein heavy chain disorders in humans, particularly cytoplasmic dynein heavy chainl disorders.
  • the invention provides an animal model which carries a mutated cytoplasmic dynein heavy chainl gene encoding a cytoplasmic dynein heavy chainl protein with a modified amino acid sequence compared to the wild type sequence.
  • the invention also provides cell lines derived from the animal model ofthe present invention.
  • the present invention also relates to the use of the animal model of the invention for the study of disorders associated with deficiencies or malfunctions in cytoplasmic dynein heavy chainl.
  • the invention provides methods of diagnosis for deficiencies or malfunction in cytoplasmic dynein heavy chainl or the gene encoding it.
  • the invention provides a method for screening of preventive or therapeutic agents of disorders and symptoms associated with movement hyperactivity, hyperexcitability disorders (e.g.
  • the present invention provides mutated cytoplasmic dynein heavy chainl nucleic acids and amino acids having modified sequences compared to the wild type sequence, as well as vectors and cell lines for expressing the muteins recombinantly. These mutated nucleic acids and amino acids may also be used in the diagnostic and therapeutic methods contemplated herein.
  • the cytoplasmic dynein heaVy chainl mutein has an amino acid substitution at a position that conesponds to a conserved amino acid of mouse or human cytoplasmic dynein heavy chain 1 as described in an individualized manner in connection with the prefened muteins of the invention below.
  • the cytoplasmic dynein heavy chainl mutein has an amino acid substitution, e.g., at position 1055 resulting from a point mutation at nucleotide +3169 of the coding sequence, conesponding to position 3328 of SEQ ID NO:l (Genbank Ace. No. AY004877; murine Dnchcl mRNA). Also provided are recombinantly generated cytoplasmic dynein heavy chainl muteins of the invention (both the murine and human othologs), as well as antibodies binding to these muteins, and chimeric protein derivatives of these cytoplasmic dynein heavy chainl muteins.
  • compositions comprising the cytoplasmic dynein heavy chainl muteins of this invention with or without a pharmaceutically acceptable carrier are also contemplated.
  • the invention includes the use ofthe cytoplasmic dynein heavy chainl muteins of the present invention, the polynucleotides encoding them, and vectors bearing said polynucleotides, for the prevention, treatment or amelioration of a medical condition in a mammalian subject, particularly a human subject.
  • the invention provides the use of these muteins for the manufacture of a medicament for prevention, treatment or amelioration of any medical conditions characterized by movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders and neurodegeneration.
  • hyperexcitability disorders e.g. myoclonic cramping, epilepsy
  • excitotoxicity disorders e.g. neurodegeneration.
  • the invention also includes a method of gene delivery and expression in a target cell of a mammal, comprising the step of introducing a viral vector into the target cell, wherein the viral vector is derived from a virus that has a low replicative efficiency in the target cell and has at least one insertion site containing a wild type or mutant cytoplasmic dynein heavy chainl nucleic acid, wherein the nucleic acid is operably linked to a promoter capable of expression in the host.
  • the viral vector is a non-lytic viral vector.
  • the invention also provides a method of gene delivery and expression in a target cell of a mammal comprising the steps of: (a) providing an isolated nucleic acid fragment of wild type or mutant cytoplasmic dynein heavy chainl ofthe invention; (b) selecting a viral vector derived from a virus that has a low replicative efficiency in the target cell, wherein the vector has at least one insertion site for insertion of said isolated nucleic acid fragment operably linked to a promoter capable of expression in the target cells; (c) inserting the isolated nucleic acid fragment into the insertion site, and (d) introducing the vector into said target cell wherein said gene is expressed at detectable levels.
  • the virus is selected from the group consisting of retrovirus, adeno virus, and pox virus.
  • the virus is a strain that has been genetically modified or selected to be non-virulent in a host.
  • the pox virus is vaccinia.
  • the virus is selected from the group consisting of retrovirus, adenovirus, iridoviruses, coronaviruses, togaviruses, caliciviruses and picornaviruses.
  • ALS Amyotrophic Lateral Sclerosis
  • SMA Spinal Muscular Atrophy
  • SBMA Bobo-Spinal Muscular Atrophy
  • PBP progressive Bulbar Palpsy
  • PMA Progressive Muscular Atrophy
  • PLS Primary Lateral Sclerosis
  • Neurodegenerative diseases are likewise disease targets contemplated e.g. in connection with the methods of screening for a predisposition for a neurodegenerative disease according to the present invention.
  • the present invention provides in a further embodiment a method of identifying a protein or nucleic acid marker indicative of an increased risk of a mammalian subject, particularly a human subject, or a mouse, a rat, a rabbit, a cow or a hamster, preferably a mouse or a rat, of developing a neurodegenerative disease, said method comprising the step of analyzing a test sample derived from said subject for the presence of a difference compared to a similar test sample if derived from a subject of the same species unaffected by or known not to be at risk of developing said disease, wherein said difference is indicative of the presence of a mutation in an allele of a gene coding for a protein, which is a subunit of the dynactin/dynein complex.
  • the present invention provides a method of identifying a protein or nucleic acid marker indicative of an association of a neurodegenerative disease in a mammalian subject, particularly a human subject, or a mouse, a rat, a rabbit, a cow or a hamster, preferably a mouse or a rat, with a mutation in an allele of a gene coding for a protein, which is a subunit ofthe dynactin/dynein complex.
  • a method for identifying a predisposition of a mammalian subject, particularly a human subject, or a mouse, a rat, a rabbit, a cow or a hamster, preferably a mouse or a rat, for developing a neurodegenerative disease, said method comprising the step of determining whether a test sample derived from said subject indicates the presence of a mutation in an allele of a gene coding for a protein, which is a subunit of the dynactin/dynein complex, indicative of an increased risk of said subject of developing said neurodegenerative disease.
  • oligonucleotides suitable for identifying the above-mentioned mutations are provided.
  • kits containing such oligonucleotides, and solid supports, such as DNA chips, to which said oligonucleotides are bound are provided.
  • Figure 1 presents a heterozygous Cral animal (right) compared to a wildtype mouse (left) demonstrating cramping ofthe hindlimbs and forelimbs ofthe mutant animal.
  • Figure 2 is a chart diagram, indicating movement as ambulatory activity expressed as beam breaks (average counts per hour) detected by a Cage Rack Photobeam Activity Cage System (San Diego Instruments, Inc., San Diego, CA, USA) of heterozygous Cral (Cral/+) animals and wildtype individuals during four 6-hour periods of an artificial 24 hour diurnal cycle, in which darkness lasted from 6pm to 6am, and the period from 6 am to 6 pm was light.
  • Cage Rack Photobeam Activity Cage System San Diego Instruments, Inc., San Diego, CA, USA
  • Figure 3 is a listing ofthe latency to fall values (seconds) of heterozygous Cral mice (Cral/+) and control individuals (+/+) in a semi-quantitative hanging wire assay. Muscle endurance of adult heterozygous Cral individuals (Cral/+) was measured in comparison to wildtype individuals by measuring the mouse's latency to fall from an inverted wire grid, as described in Example 3.
  • Figure 4 depicts photomicrographs of hematophilin and eosin (H&E) stained histological sections of a Cral/+ and a wildtype mouse hippocampus. Neurons characterized by reversible excitatory neuronal damage can be identified by the dark staining ("dark neurons"; indicated by anows). (See Example 5)
  • Figure 5 depicts photomicrographs of cross sections of the lumbar area of spinal cords from a wild type (left panel) and a homozygous Cral /Cral individual (right panel).
  • the top row photomicrographs depicts a drastic reduction of the number of neurons in the anterior horn area of the Cral/Cral individual (right), as compared to the wildtype (left).
  • TUNEL stains of anterior horn are shown (see anow), demonstrating significantly enhanced levels of apoptosis in the anterior horns of the Cral/Cral individuals (right).
  • a reduction is seen in the number of spinal ganglia neurons of Cral/Cral individuals
  • Figure 5.2 shows that progressive impairment of muscle function and motor coordination is associated with decreasing numbers of ⁇ -motor neurons and altered composition of muscle fibre types.
  • Loss of spinal ⁇ -motor neurons in 16 months old Cral/+ (h) mice is accompanied by altered composition of muscle fibre types, with a predominance of large type 1 fibres in Periodic acid-Schiff (PAS) staining, compared to +/+ littermates
  • PAS Periodic acid-Schiff
  • Figure 6 represents a list of selected genes expressed in fetal brain of individual
  • Cral/Cral mice shown to be transcriptionally deregulated by comparison of transcript levels to those detected in wildtype individuals. The direction of deregulation (up or down) and the biological significance of those genes is indicated.
  • Figure 7 schematically depicts an exon structure comparison of the human cytoplasmic dynein heavy chainl (HsDNCHl) and the mouse cytoplasmic dynein heavy chainl (MmDnchcl) genes.
  • Exonic sequences were aligned with sequencher software (version 4.0.5, GeneCodes Corp., Ann Arbor Mi, USA). Assembly of the human exons and the mouse exons, respectively, results in a full-length cDNA of human DNCHl and a full-length cDNA of mouse Dnchcl (representing the protein coding sequence within the mRNA, defined under GenBank Accession No. AY004877) as indicated by the anows at the top of the figure. Equivalent exons are paired, with the human exon positioned above the conesponding mouse exon.
  • Figure 8 is a comparative listing of all exons of the mouse (MmDnchcl) and human (Hs DNCHl) dynein heavy chainl genes. The size of each exon is indicated by its length in basepairs (bp).
  • the human gene consists of 78 exons, and the mouse gene consists of 77 exons. The difference in exon number between these two species is caused by an additional RNA splice event in human, separating exon 67 (61 bp) and exon 68 (124 bp); mouse exon 67 having 185 bp.
  • Figure 9 depicts the amino acid sequence alignment between the human (Hs DNCHl) and the mouse (Mm Dnchcl) cytoplasmic dynein heavy chainl proteins. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 97%.
  • Figure 10 depicts the amino acid sequence alignment between the human (Hs DNCHl), the mouse (Mm Dnchcl), and the rat cytoplasmic dynein heavy chainl proteins, indicating highly conserved amino acid residues between species. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 96%.
  • Figure 11 depicts the amino acid sequence alignment between the human and the mouse cytoplasmic dynein intermediate chain 1 proteins. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree ofidentity is 88%.
  • Figure 12 depicts the amino acid sequence alignment between the human, the mouse, and the rat cytoplasmic dynein intermediate chain 1 proteins, indicating highly conserved amino acid residues between species. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 87%.
  • Figure 13 depicts the amino acid sequence alignment between the human and the mouse cytoplasmic dynein intermediate chain 2 proteins. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree ofidentity is 94%.
  • Figure 14 depicts the amino acid sequence alignment between the human, the mouse, and the rat cytoplasmic dynein intermediate chain 2 proteins, indicating highly conserved amino acid residues between species. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 93.5%.
  • Figure 15 depicts the amino acid sequence alignment between the human and the mouse DCTN1 proteins. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 97%.
  • Figure 16 depicts the amino acid sequence alignment between the human, the mouse, and the rat DCTN 1 proteins, indicating highly conserved amino acid residues between species. Black boxes indicate amino acid identity, and grey boxes indicate conserved amino acid substitutions. The degree of identity is 95%.
  • This invention provides a non-human animal useful as a model of dynein heavy chain disorders in humans, particularly cytoplasmic dynein heavy chainl disorders.
  • the Cral mutation, disclosed herein was generated in offspring of male C3HeB/FeJ mice (Cral, Institute of Mammalian Genetics at the GSF - National Research Center for Environment and Health) mutagenized with N-ethyl-N- nitrosourea (E ⁇ U). The mutation was detected by Cral mice displaying an unusual twisting of the body and clenching of the hindlimbs when suspended by the tail.
  • Dyneins are implicated in a number of different diseases and disorders.
  • the mouse mutant of the present invention is suitable to investigate the role of cytoplasmic dynein heavy chainl not only in transport processes in neurons but in all cell types expressing cytoplasmic dynein heavy chainl. Additionally, the mouse mutant of the present invention enables the analysis of cytoplasmic dynein malfunction during development whereas the transgenic mouse described by LaMonte et al. ((2002) Neuron 34, 715- 727) does not express the transgene before birth and, therefore, it is characterized by a late onset of symptoms.
  • dynein can associate with its cargo also through interactions with proteins other than dynactin (for review: Karcher et al. (2002) Trends in Cell Biology 12, 21-27). Since the mutation found in the cytoplasmic dynein heavy chain 1 gene of the mouse mutant of the present invention is located outside the binding site for the dynein intermediate chains which impart the binding of dynactin, the mouse mutant of the present invention will prove useful for studying dynactin-independant dynein functions.
  • HAPl a neuronal cytoplasmic protein
  • huntingtin the protein which carries a polyglutamine repeat that is expanded in Huntington's disease (>36 units). It has been shown that the affinity of HAPl for huntingtin is increased if the latter bears an expanded polyglutamine repeat (cf Li et al. (2002) J Biol Chem, in press). HAPl has been found on synaptic vesicles (Gutebuch et al. (1998) J Neurosci 18, 7674-7686) and it binds to P150 (Li et al.
  • APP amyloid precursor protein
  • the mouse mutant of the present invention provides a means to analyze whether three proteins (huntingtin, HAPl and APP) involved in the pathogenesis of the neurodegenerative diseases, Huntington's disease and Alzheimer's dementia are linked by the same intracellular transport pathways. This connection will help reveal the pathogenetic mechanisms of both diseases.
  • Lissencephaly syndromes are a familiy of diseases which involve incomplete development of the human brain (Dobyns et al. (1993) JAMA 270, 2838-2842) characterized by extensive disorganization of cortical neurons. This defect is thought to be due to a failure ofthe capability of neuronal cell body migration during the early stage of brain development. As a consequence of this, the convolutions characteristic of the normal brain surface are reduced in extent and number leading to a smooth appearence of wide portions ofthe brain.
  • lissencephaly include Norman-Roberts Syndrome, Fukuyama Syndrome, Walker- Warberg Syndrome and Miller-Dieker Syndrome. Some cases of isolated lissencephaly have been shown to arise from mutations in the LIS-1 gene (e.g. Lo Nigro et al. (1997) Hum Mol Genet 6, 157-164).
  • mice for the Lisl gene die early in embryogenesis whereas mice bearing mutations which cause different degrees of reduction of the function of Lisl exhibit conesponding degrees of brain disorganization as well as cerebellar defects (Hirotsune et al. (1998) Nature Genetics 19, 333-339).
  • the mouse mutant of the present invention provides a mean to comparatively analyze the overlap and the differences of cytoplasmic dynein and Lis 1 function in a mammalian model organism.
  • the homozygous knockout of the gene encoding cytoplasmic dynein heavy chainl in the mouse results in embryonic lethality before 8.5 days post coitus. Heterozygous individuals have no obvious phenotype (Harada et al. (1998) J Cell Biol 141, 51-59).
  • the transgenic dynamitin-overexpressing mouse described by LaMonte et al. ((2002) Neuron 34, 715-727) is characterized by a motomeuron specific inhibition ofthe functions of cytoplasmic dynein, which is mediated by the accessory protein complex dynactin. Due to the developmental regulation of the transgene by the Thy2 promotor of the transgene construct, inhibition is restricted to postnatal stages.
  • the characterization of such an animal model i.e. one that reflects the consequences of malfunction of cytoplasmic dynein heavy chainl in vivo in all types of cells and during embryonic and fetal development
  • the present invention provides a non-human animal model which expresses a modified cytoplasmic dynein heavy chainl protein compared to the amino acid sequence of the wild type protein.
  • the expressed cytoplasmic dynein heavy chainl may have similarity in sequence and secondary structure to a vertebrate cytoplasmic dynein heavy chainl.
  • the vertebrate is preferably from a genus selected from the group consisting of Homo sapiens, Mus musculus (e.g. mice), Rattus (e.g. rats), Oryctologus (e.g. rabbits) and Mesocricetus (e.g. hamsters).
  • a genus selected from the group consisting of Homo sapiens, Mus musculus (e.g. mice), Rattus (e.g. rats), Oryctologus (e.g. rabbits) and Mesocricetus (e.g. hamsters).
  • Animals carrying a mutated cytoplasmic dynein heavy chainl allele expressing the modified cytoplasmic dynein heavy chainl exhibit a variety of phenotypical features including: myoclonic cramps (which are especially pronounced in the hindlimbs), movement hyperactivity, reduced muscle endurance, excitatory neuronal damage ("dark neurons") in the hippocampus (gyrus dentatus, CA4, CA3) as well as in the upper layer ofthe cortex and in the Purkinje cell layer ofthe cerebellum. These animals also exhibit decreasing numbers of ⁇ -motor neurons and altered composition of musculus vastus fibre types.
  • homozygous individuals Compared with heterozygous animals, homozygous individuals exhibit an elevated incidence of perinatal lethality (100% in homozygous animals, versus approximately 20% amongst heterozygous animals), accelerated neurondegeneration in the anterior horns of the spinal cord, and accelerated neurodegeneration in the dorsal root ganglia. Neurodegeneration in heterozygous animals is apparent at a much later stage, i.e. late adulthood.
  • the observed neurological phenotype of myoclonic cramping and epilepsy in combination with the morphological conelate of reversible excitatory neuronal cell stress within the hippocampal layers CA4 and CA3 are indications for a hyperexcitability status in Cral animals. This in turn is consistent with an ambulatory locomotor hyperactivity of Cral animals. Hyperexcitation, if occuring repeatedly or continuously over longer periods of time, could result in excitotoxicity by mechanisms such as enhanced generation of reactive oxygen species (ROS) (see Haberny et al. (2002) Toxicol Sci, 68, 9-17; Bondy and LeBel, (1993) Free Radic Biol Med 14, 633-642; Tapia et al.
  • ROS reactive oxygen species
  • ROS ROS are suggested to be involved in the development of several neurodegenerative disorders, e.g. ALS, Parkinson's disease and Alzheimer's disease as well as aging. Therefore there is a possible link between the hyperexcitation status in Cral animals and the development of neurodegeneration at a later age.
  • modified refers to an alteration compared to the wild type.
  • phenotype refers to a collection of morphological, physiological, behavioral and biochemical traits possessed by a cell or organism that results from the interaction of the genotype and the environment.
  • the animal model of the present invention displays readily observable abnormalities. In a prefened embodiment the animal of the invention shows at least 2, preferably at least 4, more preferably 6 and most preferably all ofthe above listed phenotypical features.
  • the present invention provides nucleic acid sequences encoding wild type and mutated murine and human cytoplasmic dynein heavy chainl proteins, i.e., muteins as described in an individualized manner in connection with the prefened muteins ofthe invention below.
  • this invention provides mutated nucleic acid sequences for wild type murine cytoplasmic dynein heavy chainl mRNA (GenBank Accession Number AY004877.1, SEQ ID NO:l).
  • SEQ ID NO:l wild type murine cytoplasmic dynein heavy chainl mRNA
  • SEQ ID NO:l a mutated version of SEQ ID NO:l, containing a point mutation at position 3328 (which is position +3164 of the polypeptide coding sequence) is shown in SEQ ID NO:3.
  • this invention provides a mutated coding nucleic acid sequence for human cytoplasmic dynein heavy chainl containing the open reading frame for the human ortholog nucleic acid sequence, with a point mutation at the conesponding position (position +3170) in the sequence, as shown in SEQ ID NO:5.
  • this invention provides the wild type coding nucleic acid sequence for human cytoplasmic dynein heavy chainl, as shown in SEQ ID NO: 17.
  • nucleic acid sequences that are contemplated as within the scope of this invention include sequences that hybridize to the nucleic acid sequences encoding cytoplasmic dynein heavy chainl shown in SEQ ID NOS:l, 3, 5, and 17, other than those known in the prior art (as described above), with at least 75%, preferably at least 80%, more preferably at least 90%, even more preferably 95% and most preferably at least 99% sequence homology to the sequence of SEQ ID NOS: 1, 3, 5, or 17. Additionally, nucleic acid sequences that are degenerate with respect to the foregoing sequences are contemplated.
  • nucleic acid sequences encoding cytoplasmic dynein heavy chainl or mutant cytoplasmic dynein heavy chainl of the invention may exist alone or in combination with other nucleic acids as, for example, vector molecules, such as plasmids, including expression or cloning vectors.
  • nucleic acid sequence refers to any contiguous sequence series of nucleotide bases, e.g., a polynucleotide, and may be ribonucleic acid (RNA) or deoxy-ribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxy-ribonucleic acid
  • nucleic acid sequence is cDNA.
  • isolated nucleic acid molecule refers to a nucleic acid molecule that is separated from other nucleic acid molecules ordinarily present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (e.g., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism that is the natural (wild type) source of the DNA.
  • Cytoplasmic dynein heavy chainl molecules can be isolated using standard hybridization and cloning techniques, as described, for instance, in Sambrook et al.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or, 5 alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides conesponding to cytoplasmic dynein heavy chainl nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an 10 automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or
  • oligonucleotide is used to refer to a series of continguous nucleotides (a polynucleotide) of about 100 nucleotdies (nt) or less, e.g., portions of a nucleic acid sequence of about 100 nt, 50 nt, or 20 nt in length,
  • nucleotide sequences of about 15 nt to 30 nt in length.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof.
  • homologous nucleic acid sequence or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level, respectively.
  • homologous nucleotide sequences encode those sequences coding for isoforms of cytoplasmic dynein heavy chainl
  • Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel et al. (eds.), Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, USA, 1989) at 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • the present invention also provides murine and human cytoplasmic dynein heavy chainl amino acid sequences (wild type polypeptides and muteins).
  • mutein applies to a protein arising as a result of a mutation.
  • the wild type murine cytoplasmic dynein heavy chainl amino acid sequence is shown in SEQ ID NO:2.
  • a mutated version wherein Tyr at position 1055 has been mutated to a Cys is shown in SEQ ID NO:4.
  • a prefened embodiment is of a human mutein with the same amino acid change at the conesponding position (native Tyr residue to mutant Cys residue at position 1057) in the sequence is shown in SEQ ID NO:6.
  • Another prefened embodiment is the wild type human dynein heavy chein 1 amino acid sequence, as shown in SEQ ID NO: 18.
  • the wild type residue of the modified human cytoplasmic dynein heavy chainl protein is replaced by an amino acid with different size and/or polarity, e.g., a non-conservative amino acid substitution.
  • residue 1055 of murine cytoplasmic dynein heavy chainl and residue 1057 of human cytoplasmic dynein heavy chainl according to the present invention is replaced by an amino acid other than Trp or Phe and preferably is replaced by an amino acid selected from Ala, Ser, Thr, Pro, Gly, Met, Leu, He, Nal, or Cys, more preferably by Met, Leu, He, Nal, or Cys, and most preferably by Cys.
  • the murine cytoplasmic dynein heavy chainl mutein of the present invention has the amino acid sequence shown in SEQ ID ⁇ O:4.
  • the human cytoplasmic dynein heavy chainl mutein ofthe present invention has the amino acid sequence shown in SEQ ID NO:6.
  • the present invention is not limited to muteins with a mutation of the residue at, e.g., position 1055 of the amino acid sequence shown in SEQ ID NO: 4, and it is not limited to the mutation of the residue at, e.g., position 1057 of the amino acid sequence shown in SEQ ID NO: 6 or at a conesponding position in other dynein heavy chain proteins (especially cytoplasmic dynein heavy chainl proteins from different species). Rather it encompasses additional muteins with modifications in the amino acid sequence of the mouse or human cytoplasmic dynein heavy chainl proteins as long as they impart a phenotype as described herein in connection with the animals. Such muteins, may comprise mutations, such as single or multiple further amino acid substitutions, deletions and insertions.
  • One embodiment of the invention relates to muteins wherein the amino acid residue affected by the mutation, e.g., a substimtion, deletion, or insertion, conesponds to a residue selected from the group of conserved amino acid residues between Leu302 (302L) and Phe 1140 (1140F) of the cytoplasmic dynein heavy chainl amino acid sequence, e.g., SEQ ID NO:2 (Mus musculus) specified in Table 19.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 302L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 303K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 305G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 306K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 307R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 308F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 309H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 310A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 31 IT.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 312N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 314F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 315D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 317D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 319G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 320L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 321K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 322Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 324L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 327N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 329D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 330 Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 33 IN.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 333L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 334M.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 335K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 337F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 338P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 342L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 344S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 345 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 346T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 35 OK.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 354 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 3581.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 359F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 361H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 362L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 363R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 364K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 366R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 368T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 369K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 370Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 371P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 373Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 374R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 376L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 378L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 380E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 381 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 3821.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 383S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 384R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 385D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 386L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 389Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 390L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 39 IL.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 392K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 393N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 394L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 399L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 400M.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 406E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 407F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 41 IM.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 414C.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 418F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 421W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 422D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 423D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 424E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 425Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 427K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 43 IL.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 432L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 433R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 434D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 4351.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 437K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 439K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 440R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 445K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 448W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 454H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 457L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 460R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 467F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 468R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 470Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 471H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 472E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 473 Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 475R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 477N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 4781.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 480R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 48 IN.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 482L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 483R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 484P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 519E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 52 IN.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 524A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 525 Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 526E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 529K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 531V.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 532D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 534L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 535D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 543A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 544W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 545E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 547A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 549K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 55 OR.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 551Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 5551.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 558N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 559E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 560T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 5621.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 563T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 566L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 569Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 570L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 575N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 577N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 578E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 579M.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 580F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 5821.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 583F.
  • a prefened mutein of the invention is one wherein the conserved amino acid . affected by said mutation is the amino acid 584S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 585R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 587N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 588 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 589L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 590F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 592R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 593P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 5951.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 596R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 597G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 598 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 5991.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 60 IE.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 602Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 603 Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 604T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 606L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 6071.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 609R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 6 ION.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 61 IK.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by saidm ⁇ tatioh ⁇ s the amino acid 613D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 6141.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 617L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 62 IF.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 639L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 640P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 641P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 646W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 655Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 656L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 659Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 66 IK.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 662R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 663N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 664E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 665D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 666N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 667L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 668G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 669K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 671W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 672E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 674H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 677G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 680L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 68 IK.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 683D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 684G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 685D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 687F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 690F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 69 IL.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 697F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 700W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 704N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 705 ⁇ .
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 7151.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 717T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 73 IL.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 733L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 736N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 744L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 746K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 747E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 748N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 752K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 755 G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 756F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 757R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 758N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 759P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 760L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 7621.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 763N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 764 ⁇ .
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 766A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 767H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 768Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 769A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 770N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 771Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 774P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 776A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 778S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 779L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 7801.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 78 IE.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 784R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 785T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 802L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 804 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 808K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 816E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 817G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 820L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 822 W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 824S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 825 Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 826K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 828D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 830Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 834L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 836E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 838N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 842Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 843E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 848L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 849L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 862L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 865C.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 867Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 87 IT.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 876L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 880Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 883N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 884D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 886L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 888L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 891 Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 892S.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 893 ⁇ .
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 894L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 897W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 898N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 899N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 90 IL.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 902D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 906E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 909L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 912R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 920W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 92 IT.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 95 IP.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 955N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 957V.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 963T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 965Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 968Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 971P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 975E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 977R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 987W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1001R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1003Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1017Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1019N.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1021L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1025P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1027G
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1031L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1032E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1035Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1048Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1052W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1053L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1055Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1056Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1058L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1059W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1062Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1063 A.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1070L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1071 G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1077W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1081L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 10841.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1088R.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1091F.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1092D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1096T.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1102P.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1107Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1109K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1111Q.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1113K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1117K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1118Y.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1119D.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1121 W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1122H.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1123K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1124E.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1126L.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1128K.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1129F
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1130G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1134G.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1137W.
  • a prefened mutein of the invention is one wherein the conserved amino acid affected by said mutation is the amino acid 1140F.
  • human muteins of the cytoplasmic dynein heavy chainl having mutations as described herein, e.g., canying a substitution, deletion, or insertion at a position within the human amino acid sequence conesponding to any of the above individualized conserved amino acids of the mouse amino acid sequence are likewise within the scope of the invention.
  • the conesponding human amino acids will, as they are conserved between, e.g. mouse and human, be identical while their relative position within the amino acid sequence may vary.
  • the skilled person will be readily able to identify the conesponding human amino acids, e.g., within SEQ ID NO: 18, or from Figures 9 or 10.
  • mutant proteins described by SEQ ID NO:4, and SEQ ID NO: 6 the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence encoding a dynein heavy chain polypeptide (e.g., the sequences of SEQ ID NO:3 and SEQ ID NO:5), thereby leading to changes in the amino acid sequence of the encoded dynein heavy chain proteins without altering properties of the mutant dynein heavy chain proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 3 and SEQ ID NO:5.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of a dynein heavy chain polypeptide without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the cytoplasmic dynein heavy chainl polypeptide or polypeptide fragments of the present invention are predicted to be particularly unamenable to alteration.
  • such alterations replace an amino acid with one of similar size and polarity (e.g., conservative substitution).
  • the polypeptide has at least 75 %, preferably at least 80 %, more preferably at least 90 %, even more preferably 95% and most preferably at least 99 % sequence identity with the wild type cytoplasmic dynein heavy chainl sequence.
  • the polypeptide is identical with the wild type sequence except for a replacement of the Tyr residue at position 1055 ofthe amino acid sequence, as shown in SEQ ID NO: 4.
  • the polypeptide is identical to the wild type sequence except for a replacement ofthe Tyr residue at position 1057 ofthe amino acid sequence, as shown in SEQ ID NO: 6 or at the conesponding position in other cytoplasmic dynein heavy chainl sequences.
  • Prefened modifications of the amino acid sequence in addition to the replacement at, e.g., position 1055 of the amino acid sequence shown in SEQ ID NO: 4, and at, e.g., position 1057 ofthe amino acid sequence shown in SEQ ID NO: 6, or at a conesponding position in other dynein heavy chains are at any of the positions which are conserved among the vertebrate dynein heavy chains as described in individualized manner in connection with the prefened muteins above.
  • Prefened modifications of the amino acid sequence in addition to the replacement at position 1055 of the amino acid sequence shown in SEQ ID NO: 4, and at position 1057 of the amino acid sequence shown in SEQ ID NO: 6, or at a conesponding position in other dynein heavy chains are at positions which are not conserved among the vertebrate dynein heavy chains.
  • Amino acids in the cytoplasmic dynein heavy chain 1 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085, 1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro proliferat ⁇ ve activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904, 1992; de Nos et al., Science 255:306-312, 1992).
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide or protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of dynein heavy chain protein in which the protein is separated from cellular components of the cells from which it the protein is isolated or in which it is recombinantly produced.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
  • additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second comparison amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at conesponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the conesponding position in the second sequence, then the molecules are homologous at that position (e.g., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch (1970) J Mol Biol 48, 443-453.
  • GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3
  • the coding region of the analogous nucleic acid sequences refened to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NOS:l, 3, 5, and 17.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc., Madison WI, USA).
  • the MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method (see, for example, Higgins and Sharp (1988) Gene 73:237-244.
  • the clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low homology or of no homology between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the method described in Hein (1990) Methods Enzymol 183, 626-645. Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • the comparison of two or more amino acid or nucleic acid sequences to determine sequence identity can be performed between ortholog sequences, preferably between mouse and human, more preferably between mouse, rat, and human sequences.
  • ortholog sequences preferably between mouse and human, more preferably between mouse, rat, and human sequences.
  • this amino acid or nucleotide is "evolutionary conserved" for the purpose of this invention.
  • the term "evolutionary conserved" also comprises amino acid substitutions, where an amino acid is replaced by another (i.e., different) amino acid that represents a conservative substitution as defined below.
  • Non-conservative substitutions are defined as exchanges of an amino acid by another amino acid listed in a different group of the five standard amino acid groups shown below:
  • Conservative substitutions are defined as exchanges of an amino acid by another amino acid listed within the same group of the five standard amino acid groups shown above. Three residues are parenthesized because of their special role in protein architecture. Gly is the only residue without a sidechain and therefore imparts flexibility to the chain. Pro has an unusual geometry which tightly constrains the chain. Cys can participate in disulfide bonds.
  • the cytoplasmic dynein heavy chainl expressed in the animal model of the present invention canies a mutation, which affects the conserved amino acids of the muteins described in individualized manner in connection with the prefened muteins ofthe invention above.
  • the cytoplasmic dynein heavy chainl expressed in the animal model of the present invention has the amino acid sequence shown in SEQ ID ⁇ O:4.
  • the cytoplasmic dynein heavy chainl expressed in the animal model of the present invention has an amino acid sequence selected from the sequences shown in SEQ ID NO:6 and SEQ ID NO: 18.
  • the invention also provides novel chimeric or fusion proteins as used herein, a novel "chimeric protein" or "fusion protein” comprises a novel cytoplasmic dynein heavy chainl polypeptide linked to a non-cytoplasmic dynein heavy chainl polypeptide (e.g., a polypeptide that does not comprise cytoplasmic dynein heavy chainl or a fragment therof).
  • the fusion protein is a GST-cytoplasmic dynein heavy chainl fusion protein in which the cytoplasmic dynein heavy chainl sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) sequences.
  • GST glutthione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant cytoplasmic dynein heavy chainl polypeptides.
  • the fusion protein is a cytoplasmic dynein heavy chainl -immunoglobulin fusion protein in which the cytoplasmic dynein heavy chainl sequences are fused to sequences derived from a member of the immunoglobulin protein family, especially Fc region polypeptides. Also contemplated are fusions of cytoplasmic dynein heavy chainl sequences (mutant or wild type or functional fragments) fused to amino acid sequences that are commonly used to facilitate purification or labeling, e.g. polyhistidine tails (especially hexahistidine segments), FLAG tags, streptavidin.
  • amino acid sequences of the present invention may be made by using peptide synthesis techniques well known in the art, such as solid phase peptide synthesis (see, for example, Fields et al. "Principles and Practice of Solid Phase Synthesis” in Synthetic Peptides, A Users Guide, Grant GA (ed.), (W.H. Freeman Co. New York NY, USA, 1992) at Chapter 3, pp. 77-183; Barlos K and Gatos D "Convergent Peptide Synthesis” in FMOC Solid Phase Peptide Synthesis, Chan WC and White PD (eds.), (Oxford University Press, New York NY, USA, 2000) at Chapter 9, pp. 215-228) or by recombinant DNA manipulations and recombinant expression.
  • solid phase peptide synthesis see, for example, Fields et al. "Principles and Practice of Solid Phase Synthesis” in Synthetic Peptides, A Users Guide, Grant GA (ed.), (W.H. Freeman Co. New York
  • Antibodies Also included in the invention are antibodies to fragments of cytoplasmic dynein heavy chainl polypeptides and muteins as described in individualized manner in connection with the prefened muteins of the invention above (including amino terminal fragments), as well as antibodies to fusion proteins containing cytoplasmic dynein heavy chainl polypeptides or fragments of cytoplasmic dynein heavy chainl polypeptides or muteins as described in individualized manner in connection with the prefened muteins of the invention above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, e.g., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Ig immunoglobulin
  • Such antibodies include, e.g., polyclonal, monoclonal, chimeric, single chain, F ab , F ab - and F( a b ' ) 2 fragments, and an F a expression library.
  • an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • a cytoplasmic dynein heavy chainl polypeptide 1 of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • Antigenic peptide fragments of the antigen for use as immunogens includes, e.g., at least 7 amino acid residues ofthe amino acid sequence ofthe mutated region such as an amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 afhino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Prefened epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of cytoplasmic dynein heavy chainl polypeptide that is located on the surface of the protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis of a cytoplasmic dynein heavy chainl polypeptide will indicate which regions of a cytoplasmic dynein heavy chainl protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. (See, for example, Hopp and Woods (1981) Proc Nat Acad Sci USA 78, 3824-3828; Kyte and Doolittle (1982) J Mol Biol 157, 105-142.)
  • Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • a protein of the invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the protein of the invention, a synthetic variant thereof, or a derivative of the foregoing.
  • An appropriate immunogenic preparation can contain, for example, the naturally occuning immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by Wilkinson (Wilkinson (2000) The Scientist, 14, 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen, the antigen binding site being characterized by its unique binding affinity for this epitope.
  • the epitope may be characterized by its binding affinity for antigen binding sites of one or more particular MAbs having binding sites directed towards that epitope.
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256, 495.
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice (Academic Press / Elsevier Science Sidcup, Kent, UK, 1986) at pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT- deficient cells.
  • Prefened immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More prefened immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor (1984) J Immunol, 133, 3001; Brön et al., Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc., New York NY, USA, 1987) at pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem., 107:220.
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a prefened source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison (1994) Nature 368, 812-13) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens of the mvention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321, 522-525; Riechmann et al.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions conespond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature 321, 522-525; Riechmann et al. (1988) Nature 332, 323-327; and Presta (1992) Cun Op Struct Biol 2, 593-596).
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor et al. (1983) Immunol Today 4, 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole et al. in: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., New York NY, USA, 1985) at pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote et al. (1983) Proc Natl Acad Sci USA 80, 2026-2030) or by transforming human B-cells with Epstein Ban Virus in vitro (see Cole et ⁇ /.in: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., New York NY, USA, 1985) at pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter (1992) J Mol Biol 227, 381-388; Marks et al. (1991) J Mol Biol 222, 581-597).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene reanangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen (see PCT publication WO94/02602).
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • the prefened embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO-A-96/33735 and WO-A-96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • a method of producing a nonhuman host exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598.
  • Such a host can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent reanangement of the locus and to prevent formation of a transcript of a reananged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest is disclosed in U.S. Patent No. 5,916,771.
  • the method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • Fgh Fragments and Single Chain Antibodies can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see for example U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F ab expression libraries (see for example Huse et al. (1989) Science 246, 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F( ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F( ab')2 fragment;
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for an antigenic protein of the invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello (1983) Nature 305, 537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the conect bispecific structure. The purification of the conect molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO-A-93/08829 and in Traunecker et al. (1991) EMBO J 10, 3655- 3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is prefened to have the first heavy chain constant region (CHI) containing the site necessary for light- chain binding present in at least one of the fusions.
  • CHI first heavy chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the prefened interface comprises at least a part ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. ((1985) Science 229, 81) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments can be recovered directly from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al. ((1992) J Exp Med 175, 217-225) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V L domains of one fragment are forced to pair with the complementary V and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH heavy-chain variable domain
  • V L domains of one fragment are forced to pair with the complementary V and VH domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared (Tutt et al. (1991) J Immunol 147, 60-69).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CDl, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO- A-91/00360; WO-A-92/20373; EP-A3-0308936).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • the antibody of the invention can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al. (1992) J Exp Med 176; 1191-1195 and Shopes (1992) J Immunol 148, 2918- 2922).
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al. (1989) Anti-Cancer Drug Design 3, 219-230).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha-
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 1, 131 In, 90 Y, and 186 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluor
  • a ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science 238, 1098-1104.
  • Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see WO-A-94/11026).
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein die antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • vectors preferably expression vectors, containing a nucleic acid encoding a cytoplasmic dynein heavy chainl protein or a mutein as described in individualized manner in connection with the prefened muteins of the invention above, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded circular DNA molecule into which additional DNA segments can be ligated.
  • viral vector Another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are refened to herein as "expression vectors”.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (e.g., express) cytoplasmic dynein heavy chainl protein or mutein as described in individualized manner in connection with the prefened muteins ofthe invention above. Accordingly, the invention further provides methods for producing cytoplasmic dynein heavy chainl protein or mutein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding cytoplasmic dynein heavy chainl protein has been introduced) in a suitable medium such that cytoplasmic dynein heavy chainl protein or mutein is produced.
  • the method further comprises isolating cytoplasmic dynein heavy chainl protein or mutein from the medium or the host cell.
  • a host cell of the invention can also be used to produce non-human transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which cytoplasmic dynein heavy chainl protein- or cytoplasmic dynein heavy chainl mutein-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous cytoplasmic dynein heavy chainl sequences have been introduced into their genome or homologous recombinant animals in which endogenous cytoplasmic dynein . heavy chainl sequences have been altered.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, which contains one or more cells bearing genetic information (a transgene), received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or transfection with recombinant DNA, or infection with a recombinant virus.
  • transgenic animals include non-human primates; ungulates such as cows, horses, goats, sheep; dogs, cats, and also chickens, amphibians, etc. Standard methods are known in the art that may be used in conjunction with the polynucleotides and of the invention and methods described herein to produce a transgenic animal expressing a modified cytoplasmic dynein e heavy chainl ofthe invention.
  • transgene is a foreign gene that has been artificially introduced into the genome of of an non human organism.
  • a transgene present in germ cells is inherited by offspring. If such offspring in fact possesses the transgene, they too are transgenic animal.s A transgene present only in somatic cells is nor passed on to the gametes.
  • transgenic describes an organism whose genome incorporates and expresses genes from another species.
  • the present invention provides a non-human animal model which expresses a cytoplasmic dynein heavy chainl protein modified as described herein, e.g. for the muteins described in individualized manner in connection with the prefened muteins above, as compared to the amino acid sequence of the wild type protein.
  • the cytoplasmic dynein heavy chainl expressed may have similarity in sequence and secondary structure to a vertebrate dynein heavy chain, including, but not limited to mammalian dynein heavy chain proteins, preferably of bovine, rat, and preferably mouse origin.
  • the animal is preferably from a genus selected from the group consisting of Mus (e.g., mice), Rattus (e.g., rats), Oryctologus (e.g., rabbits) and Mesocricetus (e.g., hamsters).
  • Mus e.g., mice
  • Rattus e.g., rats
  • Oryctologus e.g., rabbits
  • Mesocricetus e.g., hamsters
  • the animal is a mouse.
  • Animals canying a mutated cytoplasmic dynein heavy chainl allele expressing the modified cytoplasmic dynein heavy chainl proteins ofthe invention exhibit a variety of phenotypical features including: myoclonic cramps (which are especially pronounced in the hindlimbs), movement hyperactivity, reduced muscle endurance, excitatory neuronal damage ("dark neurons") in the hippocampus (gyrus dentatus, CA4, CA3) as well as in the upper layer ofthe cortex and in the Purkinje cell layer ofthe cerebellum.
  • homozygous individuals Compared with heterozygous animals, homozygous individuals exhibit an elevated incidence of perinatal lethality (100% in homozygous animals, versus approximately 20% amongst heterozygous animals), accelerated neurondegeneration in the anterior horns of the spinal cord, and accelerated neurodegeneration in the dorsal root ganglia. Neurodegeneration in heterozygous animals is apparent at a much later stage, i.e. late adulthood.
  • phenotype refers to a collection of morphological, physiological, behavioral and biochemical traits possessed by a cell or organism that results from the interaction ofthe genotype and the environment.
  • the animal model of the present invention displays readily observable abnormalities.
  • the animal of the invention shows at least 2, preferably at least 4, more preferably 6 and most preferably all of the above listed phenotypical features.
  • mice were generated carrying a point mutation in the thirteenth exon (exon 13) ofthe mouse cytoplasmic dynein heavy chainl gene, which is illustrated in SEQ ID NO:l, thereby replacing the tyrosine residue at position 1055 in the N-terminal domain of the protein.
  • This modification results in the above mentioned phenotypical features.
  • Amino acid position 1055 according to the present invention refers to the non-mature cytoplasmic dynein heavy chainl protein, as numbered in SEQ ID NO:4.
  • the mature protein is encompassed by the present invention and may be expressed in the animal model of the present invention.
  • a mature protein may result from a cleavage of several amino acids, and therefore may alter the numbering of the amino acids in the mature protein relative to the numbering of an unprocessed protein.
  • the tyrosine residue conesponding to the Tyr located at position 1055 in the amino acid sequence of SEQ ID NO:2 is conserved among the cytoplasmic dynein heavy chain proteins of different species as can be seen in the alignment in Table 9.
  • the non-human animal model of the present invention canies, e.g., a nucleic acid sequence encoding cytoplasmic dynein heavy chainl, whereby the codon for the amino acid at position 1055 of the amino acid sequence shown in SEQ ID NO:2 or the codon conesponding to said position in other dynein heavy chain proteins (especially cytoplasmic dynein heavy chainl proteins), like position 1057 of the amino acid sequence shown in SEQ ID NO: 18, which encodes a Tyr in the wild type, is mutated to encode a different amino acid.
  • the animal model of the present invention expresses the amino acid sequence shown in SEQ ID NO:4.
  • the animal model of the invention canies a modified cytoplasmic dynein heavy chainl nucleic acid sequence derived from a vertebrate, preferably from a mammal, in particular from mouse, for example, a cytoplasmic dynein heavy chainl nucleic acid sequence encoding a cytoplasmic dynein heavy chainl mutein as described in individualized manner in connection with the prefened muteins of the invention above.
  • the nucleic acid sequence is derived from the nucleic acid sequence of SEQ ID NO:l, 3, 5, or 17.
  • the animal model of the invention preferably expresses a modified cytoplasmic dynein heavy chainl protein of the invention (e.g. as shown in SEQ ID NO:4, or SEQ ID NO:6 or a mutein as described in individualized manner in connection with the prefened muteins above) in all of its cells, and particularly in the brain and spinal cord.
  • a modified cytoplasmic dynein heavy chainl protein of the invention e.g. as shown in SEQ ID NO:4, or SEQ ID NO:6 or a mutein as described in individualized manner in connection with the prefened muteins above
  • animals which express the modified cytoplasmic dynein heavy chainl protein in some, but not all cells which are termed cellular mosaic animals, are also contemplated.
  • the present invention further provides for inbred successive lines of animals carrying the mutant cytoplasmic dynein heavy chainl nucleic acid of the present invention that offer the advantage of providing a virtually homogenous genetic background.
  • a genetically homogenous line of animals provides a functionally reproducible model system for disorders or symptoms associated with movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders and neurodegeneration; over-activity or undesirable activity of endogenous cytoplasmic dynein heavy chainl ; over-expression, over-production or undesirable production of endogenous cytoplasmic dynein heavy chainl; excessive or undesirable condition shown to be modulated by endogenous cytoplasmic dynein heavy chainl .
  • the animal model of the present invention may use any of the cytoplasmic dynein heavy chainl muteins described herein, e.g., those described in an individualized manner in connection with the prefened muteins of the invention above, and is not limited to the modification of the residue at position 1055 of the amino acid sequence shown in SEQ ID NO:4 or at a conesponding position in other dynein heavy chain orthologs (e.g. position 1057 ofthe amino acid sequence shown in SEQ ID NO:6) so long as the mutein used imparts the desired mutant dynein phenotype to the animal ofthe present invention.
  • Suitable muteins include those with modifications in the amino acid sequence of the mouse or human cytoplasmic dynein heavy chainl proteins so long as they impart a phenotype as described herein in connection with the animals ofthe invention.
  • Such muteins may comprise mutations, such as single or multiple further amino acid substitutions, deletions and insertions, particularly mutations that affect the conserved amino acids of mouse or human cytoplasmic dynein heavy chainl, as described, e.g., in an individualized manner in connection with the prefened muteins ofthe invention above.
  • Amino acid insertional derivatives ofthe present invention include amino and or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • the cytoplasmic dynein heavy chainl expressed in the animal model of the invention has a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or most preferably 99%, with the wild type cytoplasmic dynein heavy chainl sequence from vertebrate, preferably from mammals, most preferably from bovine, and from rat and even most preferably from mouse (SEQ ID NO:2), excluding the wild type mouse cytoplasmic dynein heavy chainl sequence itself.
  • the cytoplasmic dynein heavy chainl expressed in the animal model of the invention is human cytoplasmic dynein heavy chainl (SEQ ID NO: 18) or has a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or most preferably 99%, with the human cytoplasmic dynein heavy chainl sequence in SEQ ID NO: 18 (e.g. SEQ ID NO:6).
  • the human wild type cytoplasmic dynein heavy chainl sequence is a further embodiment ofthe invention.
  • the animal model expresses a polypeptide as shown in SEQ ID NO:4.
  • Further prefened embodiments include the animal model ofthe invention, that expresses a polypeptide selected from those shown in SEQ ID NO:6 and SEQ ID NO: 18.
  • Prefened modifications of the cytoplasmic dynein heavy chainl amino acid sequence in the animal models of the present invention in addition to the modification at, e.g, position 1055 of the amino acid sequence shown in SEQ ID NO:4, (or in addition to the modification in the equivalent position 1057 of the amino acid sequence shown in SEQ ID NO:6) are at positions which are conserved among the vertebrate dynein heavy chain proteins (especially those conserved among vertebrate cytoplasmic dynein heavy chainl proteins), as described in individualized manner in connection with the prefened muteins above.
  • Another prefened modification ofthe cytoplasmic dynein heavy chainl amino acid sequence in the animal models of the present invention in addition to the modification at position 1055 ofthe amino acid sequence shown in SEQ ID NO:4, (or in addition to the modification in the equivalent position 1057 of the amino acid sequence shown in SEQ ID NO: 6) are at positions which are not conserved among the vertebrate dynein heavy chain proteins (especially those not conserved among vertebrate cytoplasmic dynein heavy chainl proteins), as described above.
  • the animal model of the invention may carry a mutated cytoplasmic dynein heavy chainl nucleic acid according to the present invention derived from the same species or from a different species.
  • the mutated cytoplasmic dynein heavy chainl nucleic acid of the present invention is homozygous in the animals of the present invention.
  • transcription of the mutated cytoplasmic dynein heavy chainl gene of the present invention is under the control of the promoter sequence controlling transcription of the endogenous wild type cytoplasmic dynein heavy chainl sequence of the animal, although a different promoter may be used.
  • the animals of the invention can be produced by using any technique known to the person skilled in the art; including but not limited to micro-injection, electroporation, cell gun, cell fusion, micro-injection into embryos of teratocarcinoma stem cells or functionally equivalent embryonic stem cells.
  • the animals ofthe present invention may be produced by the application of procedures which result in an animal with a genome that incorporates and/or integrates exogenous genetic material in such a manner as to modify or disrupt the function ofthe normal cytoplasmic dynein heavy chainl gene or protein. The prefened procedure for generating animal models of this invention as described in Example 1.
  • the procedure may involve obtaining genetic material, or a portion thereof, which encodes a cytoplasmic dynein heavy chainl.
  • the isolated native sequence is then genetically manipulated by the insertion of a mutation appropriate to replace the residue at position 1055 of the amino acid sequence shown in SEQ ID NO:2.
  • the manipulated construct may then be inserted into embryonic stem cells, e.g. by electroporation.
  • the cells subjected to said procedure are screened to find positive cells, e.g. cells which have integrated into their genome the desired construct encoding an altered cytoplasmic dynein heavy chainl .
  • the positive cells may be isolated, cloned (or expanded) and injected into blastocysts obtained from a host animal ofthe same species or a different species. For example, positive cells are injected into blastocysts from mice, the blastocysts are then transfened into a female host animal and allowed to grow to term, following which the offspring of the female are tested to determine which animals are transgenic, e.g. which animals have an inserted exogenous mutated DNA sequence.
  • One method involves the introduction of the recombinant gene at the fertilized oocyte stage ensuring that the gene sequence will be present in all ofthe germ cells and somatic cells ofthe "founder" animal.
  • the term "founder animal” as used herein means the animal into which the recombinant gene was introduced at the one cell embryo stage.
  • the animals ofthe invention can also be used as a source of primary cells from a variety of tissues, for cell culture experiment, including but not restricted to, the production of immortalized cell lines by any methods known in the art, such as retroviral transformation.
  • Cells from the animals may advantageously exhibit desirable properties of both normal and transformed cultured cells, e.g. they will be normal or nearly normal morphologically and physiologically, but can be cultured for long, and perhaps indefinite periods of time.
  • the present invention provides such primary cells and cell lines derived therefrom, obtained from the animals of the present invention. These primary cells or cell lines derived thereof may be used for the construction of an animal model ofthe present invention.
  • cell lines may be prepared by the insertion of a nucleic acid construct comprising the nucleic acid sequence of the invention or a fragment thereof comprising the codon imparting the above-described phenotype to the animal model of the invention (vide infra).
  • Suitable cells for the insertion include primary cells harvested from an animal as well as cells which are members of an immortalized cell line.
  • Recombinant nucleic acid constructs ofthe invention, described below, may be introduced into the cells by any method known in the art, including but not limited to, transfection, retroviral infection, micro-injection, electroporation, transduction or DEAE-dextran.
  • Cells which express the recombinant construct may be identified by, for example, using a second recombinant nucleic acid construct comprising a reporter gene which is used to produce selective expression.
  • Cells that express the nucleic acid sequence of the invention or a fragment thereof may be identified indirectly by the detection of reporter gene expression.
  • compositions containing mutein nucleic acids or proteins of the invention e.g. those muteins as described in individualized manner in connection with the prefened muteins of the invention above, as well as pharmaceutical compositions containing antibodies to them.
  • the compositions are preferably suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable earners.
  • the compounds are especially useful in that they have very low, if any toxicity.
  • mutated nucleic acid sequences and muteins of this invention, and antibodies thereto may be used in pharmaceutical compositions, when combined with a pharmaceutically acceptable canier.
  • pharmaceutically acceptable canier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable earners are described in the most recent edition of Remington's Pharmaceutical Sciences (e.g. currently the 18th edition), Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton PA, USA, 1990), a standard reference text in the field, which is incorporated herein by reference.
  • Prefened examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g. intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (e.g. topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ, U.S.A.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifiingal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylceUulose and/or polyvinylpynolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • diluents include, without limitation, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, ofthe active ingredient.
  • the compounds ofthe invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • Injectable compositions are preferably aqueous isotonic solutions or suspensions.
  • the compositions may be sterilized and or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • the compounds of the present invention can be administered in intravenous
  • Injectables can be prepared in conventional forms, either * as liquid solutions or suspensions.
  • Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained- released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.
  • prefened compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Other prefened topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1 % to 15 %, w/w or w/v.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the canier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual earners to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug earners.
  • Such polymers can include polyvinylpynolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or amphipathic block copolymers of hydrogels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity ofthe condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or anest the progress ofthe condition.
  • Oral dosages of the present invention when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally.
  • the compositions are preferably provided in the fonn of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient. Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.
  • Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compositions may contain 0.1-99%, preferably 1-70% ofthe cytoplasmic dynein heavy chainl polypeptide.
  • the pharmaceutical compositions can be provided with an adjuvant.
  • adjuvants are discussed above.
  • adjuvants can be used to increase the immunological response, depending on the host species, include Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • animals are injected with antigen using several injections in a series, preferably including at least three booster injections.
  • the animals ofthe present invention present a phenotype whose characteristics are representative of many symptoms associated with disorders associated with dynein heavy chain deficiency (especially cytoplasmic dynein heavy chainl deficiency), therefore making the animal model of the present invention a particularly suitable model for the study of these diseases including movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders and neurodegeneration; over-activity or undesirable activity of endogenous cytoplasmic dynein heavy chainl; over-expression, over-production or undesirable production of endogenous cytoplasmic dynein heavy chainl; excessive or undesirable condition shown to be modulated by endogenous cytoplasmic dynein heavy chainl .
  • diseases including movement hyperactivity, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders and neurodegeneration; over-activity or undesirable activity of endogenous cytoplasmic dynein heavy chainl; over
  • the animal model of the invention presents a phenotype characterized by cramping,movement hyperactivity; excitatory neuronal damage in the CA3 and CA4 sector of the hippocampus, as well as in the upper layer of the cortex and in the Purkinje cell layer ofthe cerebellum and neurondegeneration.
  • these are all phenotypical characteristics of human diseases related to hyperexcitability and neurodegeneration, such as epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. Therefore, the animals of the invention can be used to study diseases or symptoms associated with movement hyperactivity and impairment, hyperexcitability disorders (e.g. myoclonic cramping, epilepsy), excitotoxicity disorders, cognitive disorders and neurodegeneration in general.
  • the animals of the present invention can also be used to identify early diagnostic markers for diseases associated with cytoplasmic dynein heavy chainl deficiency.
  • Sunogate markers including but not limited to ribonucleic acids or proteins, can be identified by performing procedures of proteomics or gene expression analysis known in the art.
  • procedures of proteomics analysis include, but are not restricted to, ELISA, 2D-gel, protein microanays or mass spectrophotometric analysis of any organ or tissue samples, such as blood samples, or derivatives thereof, preferably plasma, at different age or stage of cytoplasmic dynein heavy chainl activity deficiency associated disease development, or symptom thereof.
  • gene expression analysis procedures include, but are not restricted to, differential display, cD A microanays, analysis of quality and quantity of ribonucleic acids species from any organ or tissue samples, such as blood samples, or derivatives thereof, at different age or stage of development of cytoplasmic dynein heavy chainl. activity deficiency associated disease, or symptom thereof.
  • the animal model of the present invention can be used to monitor the activity of agents useful in the prevention or treatment of the above-mentioned diseases and disorders.
  • the agent to be tested can be administered to an animal of the present invention and various phenotypic parameters can be measured or monitored.
  • the animals of the invention may be used to test therapeutics against any disorders or symptoms that have been shown to be associated with cytoplasmic dynein heavy chainl deficiency or overexpression.
  • the animals ofthe present invention can also be used as test model systems for materials, including but not restricted to chemicals and peptides, particularly medical drugs, suspected of promoting or aggravating the above-described diseases associated with cytoplasmic dynein heavy chainl deficiency.
  • the material can be tested by exposing the animal of the present invention to different time, doses and/or combinations of such materials and by monitoring the effects on the phenotype of the animal of the present invention, including but not restricted to movement activity; latency to fall in a hanging wire assay, motor coordination test assays, such as rotarod, stationary beam and coat hanger motor performance, excitatory neuronal damage in the hippocampus (CA3, CA4 and gyrus dentatus) and in the pyramidal area of the cortex, histopathological and biochemical parameters of motor neuron degeneration and spinal muscular atrophy, as well as tracer experiments to determine the functionality ofthe retrograd axonal transport.
  • motor coordination test assays such as rotarod, stationary beam and coat hanger motor performance
  • excitatory neuronal damage in the hippocampus (CA3, CA4 and gyrus dentatus) and in the pyramidal area of the cortex histopathological and biochemical parameters of motor neuron degeneration and spinal muscular atrophy, as well as tracer
  • the animals of the present invention may be used for the dissection of the molecular mechanisms of the cytoplasmic dynein heavy chainl pathway, that is for the identification of downstream genes or proteins thereof regulated by cytoplasmic dynein heavy chainl activity and deregulated in cytoplasmic dynein heavy chainl activity deficiency associated disorders.
  • this can be done by performing differential proteomics analysis, using techniques including but not restricted to 2D gel analysis, protein chip microanays or mass spectrophotometry, on tissues of the animal of the present invention which express cytoplasmic dynein heavy chainl and which respond to cytoplasmic dynein heavy chainl stimuli.
  • the animal model of the present invention can be used to identify and clone so-called modifier genes which are able to modify, aggravate, reduce or inhibit the phenotype associated with a cytoplasmic dynein heavy chainl activity deficiency.
  • the animal model ofthe present invention can be mated to mice of different strains canying a different genetic background, which gives the possibility to map the genes modifying the phenotype.
  • the animal model of the present invention when produced in an C3HeB/FeJ inbred strain background can be bred to C57Bl/6Jico inbred mice.
  • the hybrid animals of this progeny are then further bred, either in back-cross strategy with C57Bl/6Jico inbred mice again, or in an intercross strategy between each other.
  • the modifier gene can be then mapped and cloned by using microsattelites or a single nucleotide polymorphism (SNP) strategy on the mice resulting from the backcross or intercross breeding that have been grouped with respect to their phenotype intensity.
  • SNP single nucleotide polymorphism
  • An exemplary method for detecting the presence or absence of cytoplasmic dynein heavy chainl in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting cytoplasmic dynein heavy chainl protein, mutein, or nucleic acid (e.g. mRNA, genomic DNA) that encodes cytoplasmic dynein heavy chainl protein or mutein such that the presence of cytoplasmic dynein heavy chainl is detected in the biological sample.
  • An agent for detecting cytoplasmic dynein heavy chainl mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to cytoplasmic dynein heavy chainl mRNA or genomic DNA.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with abenant cytoplasmic dynein heavy chainl expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with cytoplasmic dynein heavy chainl protein, nucleic acid expression or activity.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the invention provides a method for identifying a disease or disorder associated with abenant cytoplasmic dynein heavy chainl expression or activity in which a test sample is obtained from a subject and cytoplasmic dynein heavy chainl protein or nucleic acid (e.g. mRNA, genomic DNA) is detected, wherein the presence of cytoplasmic dynein heavy chainl protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant cytoplasmic dynein heavy chainl expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., blood, plasma, serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g. an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant cytoplasmic dynein heavy chainl expression or activity.
  • an agent e.g. an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g. an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • Agents, or modulators that have a stimulatory or inhibitory effect on cytoplasmic dynein heavy chainl activity can be administered to individuals to treat (prophylactically or therapeutically) cytoplasmic dynein heavy chainl -mediated disorders. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g. drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.
  • Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of cytoplasmic dynein heavy chainl protein, expression of cytoplasmic dynein heavy chainl nucleic acid, or mutation content of cytoplasmic dynein heavy chainl genes in an individual can be determined to thereby select one or more appropriate agents for therapeutic or prophylactic treatment ofthe individual.
  • the present invention also provides a diagnostic method for cytoplasmic dynein heavy chainl activity deficiency.
  • Patients' peptide material particularly that in or from blood, serum or plasma, is subjected to analysis for one or more of the amino acid sequences ofthe present invention.
  • the peptide material may be analyzed directly or after extraction, isolation and/or purification by standard methods.
  • the diagnostic method comprises the identification of the modified cytoplasmic dynein heavy chainl, e.g., modified as described in individualized manner in connection with the prefened muteins of the invention above, whereby the modification is, e.g., associated with the replacement of an amino acid at a position conesponding to position 1055 in the amino acid sequence shown in SEQ ID NO:2.
  • the diagnostic method comprises the identification of the modified cytoplasmic dynein heavy chainl , e.g., the mutein as described in individualized manner in connection with the prefened muteins of the invention above, whereby the modification is, e.g., associated with the replacement of an amino acid at a position conesponding to position 1057 in the amino acid sequence shown in SEQ ID NO: 18.
  • the diagnostic method comprises the identification of the modified cytoplasmic dynein heavy chainl,.
  • diagnostic methods include those employing detection of the modified cytoplasmic dynein heavy chainl by its failure to activate a biological pathway.
  • the diagnostic methods of the invention also include those employing detection of the modified cytoplasmic dynein heavy chainl by its activity in competing with and blocking the action of native dynein heavy chain.
  • Methods of identifying the modified cytoplasmic dynein heavy chainl include any methods known in the art which are able to identify altered conformational properties of the amino acid sequence of the present invention compared to those of the wild type cytoplasmic dynein heavy chainl. These include, without limitation, the specific recognition of the modified protein by other proteins, particularly antibodies; individual or combined patterns of amino acid sequence digestion by known proteases or chemicals.
  • the method exploits the failure of another protein to recognize the modified protein, examples being antibodies directed to an epitope of wild type cytoplasmic dynein heavy chainl that incorporates residue 1055 of SEQ ID NO:2, or SEQ ID NO: 18, and cytoplasmic dynein heavy chainl receptors in which this portion ofthe molecular surface of wild type cytoplasmic dynein heavy chainl is recognized or involved in cytoplasmic dynein heavy chainl activation.
  • the principle of the diagnostic method is the detection of a nucleic acid sequence encoding the modified cytoplasmic dynein heavy chainl ofthe invention.
  • the present invention is not only concerned with the identification of neurodegenerative disease relevant mutations in the dynein heavy chain 1 gene, but is also based on the recognition that other components (hereinafter also refened to as "subunits") of the dynactin/dynein complex may likewise be subject to mutations that are involved in, or affect, the pathogenesis of such diseases.
  • these subunits include in particular the cytoplasmic dynein heavy chain 1, cytoplasmic dynein intermediate chain 1, cytoplasmic dynein intermediate chain 2, cytoplasmic dynein light intermediate chain 1, cytoplasmic dynein light intermediate chain 2; cytoplasmic dynein 10 kDa light chain, cytoplasmic dynein light chain Tctex 1, cytoplasmic dynein light chain 2B, DCTN 1, DCTN 2, DCTN 3, DCTN 4, DCTN 5, DCTN 6, ARP1, ARP11, HAPl, and CLIP-170, preferably encoded by the genomic nucleic acid sequences identified in Table 25, or defined by the amino acid sequences identified in Tables 26, 27, and 29-35.
  • NCBI contigs are derived from genomic sequence data assembled by NCBI staff
  • the invention ter alia relates to the identification of a protein or a nucleic acid marker indicative of an increased risk of a mammalian subject, particularly a human subject, of developing a neurodegenerative disease or of an association of a neurodegenerative disease in a mammalian subject, particularly a human subject, with a mutation which leads to a neurodegenerative disease.
  • a neurodegenerative disease Preferably the above-mentioned neurodegenerative diseases are Alzheimer's Disease, Parkinson's Disease, or Huntington's Disease.
  • the neurodegenerative disease is a motomeuron degenerative disease.
  • the motomeuron degenerative disease is Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy, Bulbo-Spinal Muscular Atrophy, Progressive Bulbar Palsy, Progressive Muscular Atrophy, and Primary Lateral Sclerosis.
  • the mutation selectively affects cell types associated with or suspected to be involved in neurodegenerative diseases. In a more prefened embodiment, these cell types are motomeurons. In an even more prefened embodiment, the motomeurons are ⁇ -motorneurons. In another prefened embodiment, the mutation affects cellular processes, e.g., neuronal axonal transport, cellular transport, proliferation, differentiation, or apoptosis. In another prefened embodiment, the mutation leads to a deficiency or malfunction, e.g. an alteration in the functional interaction or a disruption of the dynactin/dynein complex in neurons, preferably in motomeurons.
  • a deficiency or malfunction e.g. an alteration in the functional interaction or a disruption of the dynactin/dynein complex in neurons, preferably in motomeurons.
  • said mutation results in a deletion of an amino acid or an insertion of an additional amino acid not normally present in the amino acid sequence of the protein encoded by said allele or in a substimtion by another amino acid of an amino acid encoded by said allele.
  • said deletion, substitution, or insertion is encoded by both alleles ofthe gene coding for said protein and/or occurs in an evolutionary conserved region of said protein.
  • the substitution of an amino acid which is identical between the conesponding mouse and human, preferably between the conesponding mouse, rat, and human protein encoded by said allele, by another amino acid, preferably by a non- conservative amino acid and/or the substitution of the amino acid occurs by a naturally occuning amino acid.
  • the amino acid affected by the mutation is encoded by a codon within the open reading frame of a nucleic acid sequence, preferably by an exon, as set forth in the following tables.
  • Table 36 Mouse cytoplasmic dynein intermediate chainl (GenBank Ace. No.: NM_0100631(SEQ ID NO.: 70)
  • Exon3 (nucleotide) TGCAGCCGCTGCATTTTTTAACATGGGATACCTGTTATTTTCATTATTTAGTCCCAACCCCTATGTCTC CCTCCTCGAAATCAGTGAGCACTCCCAGTGAAGCTGGAAGCCAAGACTCAGGCGATCTGGGGCCATTAA
  • Exon2 (nucleotide) ATCCAAGTATTTGAAGATGGAGCAGATACTACTTCCCCAGAGACTCCTGATTCTTCTGCTTCAAAGGTC CTCAAGAGAG
  • Exonl2 (nucleotide) GATGTCAATCGGGAGCTGACAAACCAGCAGGAAGCGTCTGTAGAGAGGCAGCAGCAGCCGCCGCCAGAG ACTTTTGATTTCAAAATCAAGTTTGCTGAGACCAAGGCTCATGCCAAG
  • Exonl9 (nucleotide) GTATCCGACACACTCCTGGACTGCAGGAAGCACTTGACGTGGGTGGTAGCTGTTCTGCAGGAGGTGGCA GCTGCAGCCGCCCAGCTTATTGCCCCCTTGGCAGAGAACGAGGGGCTGCCTGTGGCTGCACTGGAGGAG ' CTGGCCTTCAAAGCAAGCGAGCAG
  • Exon4 (nucleotide) AGGGAACTGATACAACTGCAAAGACTAGCAAACTG
  • Exonl 6 (nucleotide) TGCCCTCTCTCAGTGCAGTGTGGATGTGTATAAGAAAGTGGGCAGCCTGTACCCTGAGATGAGTGCCCA TGAGCGCTCCTTGGATTTCCTCATTGAACTGCTGCACAAGGATCAGCTGGATGAGACTGTCAATGTGGA GCCTCTCACCAAGGCCATCAAGTACTATCAG
  • the amino acid affected by the mutation is located in a domain of said protein, which is capable of binding to another subunit of the dynactin/dynein complex.
  • said domain comprises amino acids 147-157, amino acids 243-314, amino acids 140-157, or amino acids 1-123 of SEQ ID NO.: 61, in case the protein is mouse cytoplasmic dynein intermediate chain 1, or, in case the protein is human cytoplasmic dynein intermediate chain 1 amino acids 1-140, amino acids 157-174, amin acids 164-174, or amino acids 260-331 of SEQ ID NO.: 62.
  • said domain comprises amino acids 1-123, amino acids 122-139, amino acids 129-130, or amino acids 226-297 of SEQ ID NO.: 64, or, in case the protein is human cytoplasmic dynein intermediate chain 2 amino acids 1-149, amino acids 148-165, amino acids 155-165, or amino acids 252-323 of SEQ ID NO.: 65.
  • said domain comprises amino acids 39-150, amino acids 1006-1021, or amino acids 133-899 of SEQ ID NO.: 67, or, in case the protein is human DCTN 1 amino acids 39-150, amino acids 133-899, or amino acids 1006-1021 of SEQ ID NO.: 68.
  • the amino acid affected by the mutation is any one as described in individualized manner in connection with the prefened muteins ofthe invention above, e.g., those specified in Tables 19, 20, 21, 22 or 24.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is amino acid 39G.
  • the amino acid affected by said mutation is amino acid 40H.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is amino acid 41R.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is amino acid 42G.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is amino acid 43 T.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is amino acid 44V.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 45 A.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 46Y.
  • the amino acid affected by said mutation is the amino acid 47V.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 48G.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 49 A.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 50T.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 51L.
  • the amino acid affected by said mutation is the amino acid 52F.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 53A.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 54T.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 55G.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 56K.
  • the amino acid affected by said mutation is the amino acid 57W.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 58V.
  • the amino acid affected by said mutation is the amino acid 59G.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 60V
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 611.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 62L.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 63D.
  • the amino acid affected by said mutation is the amino acid 64E.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 65 A.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 66K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 67G.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 68K.
  • the amino acid affected by said mutation is the amino acid 69N.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 70D.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 71G.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 72T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 73V.
  • the amino acid affected by said mutation is the amino acid 74Q.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 75 G.
  • the amino acid affected by said mutation is the amino acid 76R.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 77K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 78Y.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 79F.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 80T.
  • the amino acid affected by said mutation is the amino acid 81C.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 82D.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 83E.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 84G.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 85H.
  • the amino acid affected by said mutation is the amino acid 86G.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 871.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 88F.
  • another prefened embodiment is one wherem the amino acid affected by said mutation is the amino acid 89V.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 90R.
  • the amino acid affected by said mutation is the amino acid 91Q.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 92S.
  • the amino acid affected by said mutation is the amino acid 93 Q.
  • another prefened embodiment is one wherein the- amino acid affected by said mutation is the amino acid 941.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 95Q.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 96V.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 97F.
  • the amino acid affected by said mutation is the amino acid 98E.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 99D.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 100G.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 101 A.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 102D.
  • the amino acid affected by said mutation is the amino acid 103T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 104T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 105S.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 106P.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 107E.
  • the amino acid affected by said mutation is the amino acid 108T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 109P.
  • the amino acid affected by said mutation is the amino acid 110D.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 111 S.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 112S.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 113 A.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 114S.
  • the amino acid affected by said mutation is the amino acid 115K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 117L.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 118K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 119R.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 120E.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 12 IG.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 123D.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 126 A.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 127K.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 128T.
  • the amino acid affected by said mutation is the amino acid 129S.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 13 OK.
  • the amino acid affected by said mutation is the amino acid 13 IL.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 132R.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 133G.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 134L.
  • protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 135K.
  • the amino acid affected by said mutation is the amino acid 136P.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 137K.
  • the protein is human DCTN 1
  • another prefened embodiment. is one wherein the amino acid affected by said mutation is the amino acid 138K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 139A.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 140P.
  • the amino acid affected by said mutation is the amino acid 141T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 142 A.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 143R.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 144K.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 145T.
  • the amino acid affected by said mutation is the amino acid 146T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 147T.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 148R.
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 149R.
  • the protein is human DCTN 1
  • another prefened embodiment is one wherein the amino acid affected by said mutation is the amino acid 150P.
  • test sample derived from a mammalian subject may be directly obtained from said subject. It may, however, also be a sample that has been obtained previously.
  • test samples according to the invention are, for example, cDNA preparations that have been prepared from mRNA obtained from a tissue sample from a human subject at an earlier stage. It may also be cloned or PCR-amplif ⁇ ed DNA that originates from DNA contained in such tissue sample obtained at an earlier stage.
  • the test sample will be analyzed for a difference to a similar test sample derived from a human subject unaffected by or known not to be at risk of developing a neurological disease.
  • the method may include actually deriving or directly obtaining a test sample from such a mammalian subject, particularly a human subject, for comparative purposes, the necessary information regarding the relevant structural features and properties of such similar test sample to be used for comparison will often already be available.
  • it will often be sufficient for the purposes of the above methods ofthe invention to perform an analysis for a difference to a similar test sample as it would be observed if said similar test sample were in fact obtained from a subject of the same species, which subject is unaffected by or known not to be at risk of developing the above medical condition.
  • the test sample may be a nucleic acid sample, e.g., mRNA (or cDNA derived therefrom), or genomic DNA.
  • the nucleic acid sample or the protein sample comprises one or more of the nucleic acids coding for proteins or the proteins itself, which are subunits of the dynactin/dynein complex, preferably the cytoplasmic dynein heavy chain 1, e.g. according to SEQ ID NOS.: 28 or 29, the cytoplasmic dynein intermediate chain 1, e.g. according to SEQ ID NOS.: 30 or 31, the cytoplasmic dynein intermediate chain 2, e.g. according to SEQ ID NOS.: 32 or 33, the cytoplasmic dynein light intermediate chain 1, e.g.
  • the cytoplasmic dynein light intermediate chain 2 e.g. according to SEQ ID NOS.: 36 or 37
  • the cytoplasmic dynein 10 kDa light chain e.g. according to SEQ ID NOS.: 38 or 39
  • the cytoplasmic dynein light chain Tctex 1 e.g. according to SEQ ID NOS.: 40 or 41
  • the cytoplasmic dynein light chain 2B e.g. according to SEQ ID NO.: 42
  • DCTN 1 e.g. according to SEQ ID NOS.: 43 or 44
  • DCTN 2 e.g. according to SEQ ID NO.: 45
  • DCTN 3 e.g.
  • DCTN 4 e.g. according to SEQ ID NOS.: 48 or 49
  • DCTN 5 e.g. according to SEQ ID NO.: 50
  • DCTN 6 e.g. according to SEQ ID NOS.: 51 or 52
  • ARPl e.g. according to SEQ ID NOS. 53 or 54
  • ARPl l e.g. according to SEQ ID NOS. 55 or 56
  • HAPl e.g. according to SEQ ID NO. 57 or 58
  • CLIP-170 e.g.
  • the difference analyzed- may furthermore be one relating to the expression level of said nucleic acid or protein. Alternatively, it may be analyzed whether there is a difference in terms ofthe nucleotide or the amino acid sequence level.
  • the step of analysis for differences between the test samples may comprises the partial or complete determination of the sequence of the nucleic acid, or a PCR- amplified portion of the nucleic acid, of the test sample, and optionally also of the nucleic acid or at PCR-amplified portion ofthe nucleic acid ofthe similar test sample
  • another embodiment of the invention relates to oligonucleotides suitable for identifying a mutation in an allele coding for a protein, which is a subunit ofthe dynactin/dynein complex.
  • the oligonucleotide is suitable for hybridizing to the nucleic acid of said allele or a portion of its nucleic acid under stringent conditions.
  • the oligonucleotide is suitable as primer for amplifying the nucleic acid of said allele or a portion of its nucleic acid.
  • Suitable methods for the determination of partial or complete nucleic acid sequences, and thus, detection of the above-mentioned differences, are well known to the skilled artisan. They include, for example, Southern blotting, TGGE (temperature gradient gel electrophoresis), DGGE (denaturing gradient gel electrophoresis), SCCP (single chain conformation polymorphism) detection, and the like. High throughput sequence analysis methods such as those described by Kristensen et al. (Kristensen et al., BioTechniques 30 (2001), 318-332), which is incorporated herein by reference in its entirety, are likewise suitable, and hence, contemplated in connection with the present invention.
  • One embodiment of the present invention relates to the determination whether the above-mentioned differences are present in one or both alleles ofthe gene coding for the protein, which is a subunit of the dynactin/dynein complex, i.e., whether the mammalian subject (or certain cell samples derived therefrom) is (are) homozygous or heterozygous with respect to said mutation.
  • the portion of the allele to be tested is PCR-amplified and digested with a suitable restriction enzyme and the resulting restriction pattern obtained from the amplified portion is compared to the restriction pattern obtained from the conesponding amplified portion of the similar sample or the similar samples (see Example (8).
  • the above-mentioned determination whether differences are present in one or both alleles ofthe gene coding for said protein is TGCE, as explained below.
  • PCR-amplified portions of the allele via electrophoresis through a gradient of increasing temperature via TGCE (see Example 20). Differences in the amplified portion of the allele compared to amplified portions of the alleles contained in the similar test sample(s) are indicated by a difference in migration during electrophoresis.
  • the invention ter alia relates to high throughput screening methods.
  • the oligonucleotides according to the invention, used in these high throughput methods are fixed on a solid support to form an anay.
  • the solid support can comprise microplates, standard blotting membranes, or glass chips.
  • the solid support is a glass chip with a modified surface capable of covalently binding to said oligonucleotides.
  • Nucleic acid samples, preferably genomic DNA are tagged, e.g. with a fluorescent marker, and allowed to interact with said oligoncleotides bound to the solid support.
  • Hybridization will occur at complementary sequences between nucleic acid sample and oligonucleotide, which is indicative for the presence of a mutation in said nucleic acid sample. Hybridization is then determined by scanning with a laser beam and analyzing the signal on a computer.
  • Suitable methods for the determination of partial or complete amino acid sequences are likewise well known, and include, for example, detection of particular epitopes within a protein sample via specific antibodies in dot blot, slot blot, or Western blot assays, or via ELISAs or RIAs, or partial amino acid sequence determination on a sequencer via Edman degradation. Also, high-throughput methods may again be employed.
  • a further aspect of the present invention is represented by a method for identifying a predisposition of a mammalian subject, particularly a human subject, for developing a neurodegenerative disease, said method comprising the step of determining whether a test sample derived from said subject indicates the presence of a mutation in an allele of the gene coding for a protein, which is a subunit of the dynactin/dynein complex, indicative of an increased risk of said human subject of developing said neurodegnereative disease.
  • Also contemplated in connection with the present invention is a method for determining whether a neurodegenerative disease in a mammalian subject, particularly a human subject, which is associated with a mutation in an allele of a gene coding for a protein, which is asubunit of the dynactin/dynein complex, said method comprising the step of determining whether a test sample derived from said subject indicates the presence of a mutation in an allele ofthe gene coding for said protein.
  • test sample may also be a sample that has been obtained previously.
  • suitable test samples according to the invention are, for example, cDNA preparations that have been prepared from mRNA obtained from a tissue sample from a mammalian subject, particularly a human subject, at an earlier stage. It may also again be cloned or PCR- amplified DNA that originates from DNA contained in such tissue sample obtained at an earlier stage.
  • test sample which may be a nucleic -acid or protein test sample as previously defined
  • the test sample is analyzed for the presence of a mutation in an allele of a gene of a protein, which is a subunit of the dynactin/dynein complex, which mutation is indicative of an increased risk of developing such a neurodegenerative disease.
  • said methods would comprise analyzing said sample for the presence of amutation, which would indicate an altered expression or function of said protein.
  • mutations are ter alia those refened to herein in connection with the proteins and nucleic acids according to the invention, and that mutations of this kind may be readily identified, for example, by in vitro assays.
  • in vitro assays to determine whether the mutation in an allele of a gene coding for said protein affects the function of the complex may comprise the quantitative and/or qualitative determination of the binding of the subunits to each other within the complex.
  • Qualitative methods may comprise SDS-PAGE and subsequent Western blotting of homogenates of, e.g. the spinal cord, using antibodies specific for the dynactin/dynein subunits.
  • Disruption of the dynactin/dynein complex by a mutation in said proteins can be determined by, e.g. a difference in migration of the subunits compared to proteins derived from homogenates of similar tissue comprising the conesponding proteins without mutation (LaMonte et al, see above.).
  • disruption, or at least functional alteration of the dynactin/dynein complex due to mutations in its subunits may be analyzed by affinity chromatography as described in Karki et al. (J Biol Chem 270, 28806-28811, 1995) or co-precipitation experiments as described in Paschal et al. (J Biol Chem 268, 15318-23, 1993).
  • Quantitative methods to determine quantitative binding of the subunits within the complex to each other are, e.g. those using surface plasmon resonance (SPR), such as, for example, the Biacore technology (Biacore AB, Uppsala, Sweden) (see also Example 18).
  • SPR surface plasmon resonance
  • Further suitable in vitro assays comprise the step of determining whether a mutation in one or more subunits of the dynactin/dynein complex affects dynein motility.
  • Such tests comprise, e.g. immunohistology with appropiate antibodies.
  • antibodies directed against tubulin or synaptotagmin may be suitable (Martin et al, 1999, Mol Biol Cell 10, 3717-3728).
  • dynein motility may be determined by analysis of single-lipid-droplet motility as described by Welte et al. (Cell 92: 547-557, 1998) or a microtubule gliding assay (see Example 16).
  • the in. vitro assays may comprise tests to determine whether the mutation in an allele of a gene coding for said protein affects the expression of the protein.
  • quantitative analysis of the mRNA expression levels may be applicable. These methods may comprise, for example, Northern blotting or quantitative Polymerase Chain Reaction, which are well known to those skilled in the art.
  • the above-metioned mutations may be readily identified, for example, by an animal model (see Example 1).
  • the above-mentioned mutations may also be identified by any of the aforementioned methods of screening for disease-relevant alleles of said proteins.
  • the invention further comprises a kit for identifying a predisposition of a mammalian subject, particularly a human subject, for developing a neurodegenerative disease, or for identifying an association of a neurodegenerative disease of said subject with a mutation in an allele coding for a protein, which is a subunit of the dynactin/dynein complex.
  • the kit according to the invention comprises one or more of the above-mentioned oligonucleotides suitable for identifying a mutation in an allele of a gene coding for a protein which is a subunit of the dynactin/dynein complex.
  • the kit comprises oligonucleotide(s), the nucleotide sequence of which conespond(s) to a nucleotide sequence within said allele, which contains a mutation.
  • the oligonucleotide(s) is(are) suitable for hybridizing to the nucleic acid of said allele or a portion of its nucleic acid under stringent conditions.
  • the oligonucleotide(s) is(are) suitable as primer(s) for amplifying the nucleic acid of said allele or a portion of its nucleic acid.
  • the kit further comprises instructions to use the oligonucleotide or the oligonucleotides for identifying said predisposition in said subject or said association of the neurodegenerative disease of said subject with said mutation.
  • the invention further comprises a solid support, wherein at least two oligonucleotides are individually fixed to separate areas of the solid support to form an anay.
  • the oligonucleotides fixed on the solid support are suitable for for identifying a mutation in an allele of a gene coding for a protein, which is a subunit ofthe dynactin/dynein complex.
  • the oligonucleotides fixed on the solid support have a nucleotide sequence, which conesponds to a nucleotide sequence within said allele, which contains a mutation and. are suitable for hybridizing to the nucleic acid of said allele or a portion of its nucleic acid under stringent conditions.
  • the solid support can be a microplate or standard blotting membranes, such' as nylon or nitrocellulose membranes.
  • the solid support is a glass chip, preferably a glass chip with a modified surface.
  • the surface of the glass chip is modified as to bind nucleic acids, preferably oligonucleotides, more preferably oligonucleotides with a nucleotide sequence, which conesponds to a nucleotide sequence within said allele, which contains a mutation.
  • a further embodiment of the present invention comprises the use of the previously mentioned oligonucleotides, or the previously mentioned kit, or the above- mentioned solid support, in a method for identifying a predisposition of a mammalian subject, particularly a human subject, for developing a neurodegenerative disease or in a method for determining whether a neurodegenerative disease in a mammalian subject, particularly a human subject, is associated with a mutation in an allele of a gene coding for a protein, which is a subunit of the dynactin/dynein complex as mentioned previously.
  • EXAMPLE 1 Production of Animals of the Invention and Breeding Strategies To produce mouse mutants, a C3HeB/FeJ male mouse (The Jackson
  • Affected FI individuals were mated to wild type C3HeB/FeJ mice and the resulting F2 progeny was screened for the identical pathological phenotype as identified in FI generation.
  • C3HeB/FeJ mice Affected F2 individuals were intercrossed to produce homozygous F3 individuals, which undergo perinatal lethality: therefore fetal tissues of these animals were analyzed. Heterozygous F3 individuals were used for the histochemical, neurological and physiological characterization ofthe Cral phenotype.
  • EXAMPLE 2 First Identification of Phenotype The Cral mouse mutant was identified by the observation of hind limb cramping during manual tail suspension in the heterozygous condition (Figure 1). Adult mice were manually suspended by their tails for one minute. During this period it was observed whether the mice display cramping of their hind limbs. In all tests, the observation of hind limb cramping was invariably conelated to the heterozygous Cral/+ genotype.

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Abstract

L'invention a trait à un modèle animal non humain, en particulier à un modèle murin pour l'hyperactivité de mouvement, les troubles d'excitoxicité (par ex., secousses myocloniques) et la neurodégénération, qui exprime une mutation de la chaîne lourde 1 de dynéine cytoplasmique. L'invention concerne également des protéines et des acides nucléiques de la chaîne lourde 1 de dynéine cytoplasmique modifiée humains et murins, y compris les protéines recombinantes correspondantes. L'invention a également trait à l'utilisation du modèle animal non humain, et des protéines et acides nucléiques de la chaîne lourde 1 de dynéine cytoplasmique modifiée, en particulier pour le diagnostic et le traitement d'états pathologiques associés à la surexpression de la chaîne lourde 1 de dynéine cytoplasmique.
EP03807821A 2002-09-13 2003-09-11 Genes de la chaine lourde 1 de dyneine cytoplasmiques, produits d'expression, modele animal non humain : utilisation pour les maladies neurologiques humaines Withdrawn EP1556484A2 (fr)

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